Scientific Data Surah 92 · Ayah 3
Untitled
Phase 2 -
Scientific Data -
By He who created the male and the female -
The following scientific articles explore the gender determination of humans as well as plants and animals |
Gender and Genetics
Genetic Components of Sex and Gender
Humans are born with 46 chromosomes in 23 pairs. The X and Y chromosomes determine a person’s sex. Most women are 46XX and most men are 46XY. Research suggests, however, that in a few births per thousand some individuals will be born with a single sex chromosome (45X or 45Y) (sex monosomies) and some with three or more sex chromosomes (47XXX, 47XYY or 47XXY, etc.) (sex polysomies). In addition, some males are born 46XX due to the translocation of a tiny section of the sex determining region of the Y chromosome. Similarly some females are also born 46XY due to mutations in the Y chromosome. Clearly, there are not only females who are XX and males who are XY, but rather, there is a range of chromosome complements, hormone balances, and phenotypic variations that determine sex.
The biological differences between men and women result from two processes: sex determination and differentiation.(3) The biological process of sex determination controls whether the male or female sexual differentiation pathway will be followed. The process of biological sex differentiation (development of a given sex) involves many genetically regulated, hierarchical developmental steps. More than 95% of the Y chromosome is male-specific (4) and a single copy of the Y chromosome is able to induce testicular differentiation of the embryonic gonad. The Y chromosome acts as a dominant inducer of male phenotype and individuals having four X chromosomes and one Y chromosome (49XXXXY) are phenotypically male. (5) When a Y chromosome is present, early embryonic testes develop around the 10th week of pregnancy. In the absence of both a Y chromosome and the influence of a testis-determining factor (TDF), ovaries develop.
Gender, typically described in terms of masculinity and femininity, is a social construction that varies across different cultures and over time. (6) There are a number of cultures, for example, in which greater gender diversity exists and sex and gender are not always neatly divided along binary lines such as male and female or homosexual and heterosexual. The Berdache in North America, the fa’afafine (Samoan for “the way of a woman”) in the Pacific, and the kathoey in Thailand are all examples of different gender categories that differ from the traditional Western division of people into males and females. Further, among certain North American native communities, gender is seen more in terms of a continuum than categories, with special acknowledgement of “two-spirited” people who encompass both masculine and feminine qualities and characteristics. It is apparent, then, that different cultures have taken different approaches to creating gender distinctions, with more or less recognition of fluidity and complexity of gender.
The Sex of Offspring Is Determined by Particular Chromosomes
In humans and many other animal species, sex is determined by specific chromosomes. How did researchers discover these so-called sex chromosomes? The path from the initial discovery of sex chromosomes in 1891 to an understanding of their true function was paved by the diligent efforts of multiple scientists over the course of many years. As often happens during a lengthy course of discovery, scientists observed and described sex chromosomes long before they knew their function.
An idea inspired by the "X element"
By the 1880s, scientists had established methods for staining chromosomes so that they could be easily visualized using a simple light microscope. With this staining method, scientists were able to observe cell division and to identify the steps that occurred during both mitosis and meiosis (Figure 1).
Figure 1: Cell division observed through the microscope (left) is redrawn to show the action of chromosomes (right). Arrows indicate the axis along which the cell divides.
The first indication that sex chromosomes were distinct from other chromosomes came from experiments conducted by German biologist Hermann Henking in 1891. While using a light microscope to study sperm formation in wasps, Henking noticed that some wasp sperm cells had 12 chromosomes, while others had only 11 chromosomes. Also, during his observation of the stages of meiosis leading up to the formation of these sperm cells, Henking noticed that the mysterious twelfth chromosome looked and behaved differently than the other 11 chromosomes. Accordingly, he named the twelfth chromosome the "X element" to represent its unknown nature. Interestingly, when Henking used a light microscope to study egg formation in female grasshoppers, he was unable to spot the X element.
Based on his observations, Henking hypothesized that this extra chromosome, the X element, must play some role in determining the sex of insects. However, he was unable to gather any direct evidence to support his hypothesis.
Before Her Time
• The Life of Nettie Stevens
Figure 2: The darkling beetle, Tenebrio molitor.
More than a decade after Henking's work, Nettie Stevens surveyed multiple beetle species and examined the inheritance patterns of their chromosomes. In 1905, while studying the gametes of the beetle Tenebrio molitor(Figure 2), Stevens noted an unusual-looking pair of chromosomes that separated to form sperm cells in the male beetles. Based on her comparisons of chromosome appearance in cells from male and female beetles, Stevens proposed that these accessory chromosomes were related to the inheritance of sex.
Over time, other scientists studied the appearance of chromosomes in a wide variety of animal species, and it became clear that there was a relationship between the physical appearance and number of chromosomes in gametes and somatic cells from males and females of a given species.
The variety of sex determination systems
Figure 3: Example set of male human chromosomes. In the image, the X and Y chromosomes are indicated by arrows.
In humans, females inherit an X chromosome from each parent, whereas males always inherit their X chromosome from their mother and their Y chromosome from their father. Consequently, all of the somatic cells in human females contain two X chromosomes, and all of the somatic cells in human males contain one X and one Y chromosome (Figure 3). The same is true of all other placental mammals — males produce X and Y gametes, and females produce only X gametes (Figure 4). In this system, referred to as the XX-XY system, maleness is determined by sperm cells that carry the Y chromosome.
Figure 4: Sex determination in humans.
Figure Detail
Figure 5: Sex determination in insects.
Figure Detail
Many people do not realize, however, that the XX-XY sex determination system is only one of a variety of such systems within the animal kingdom. In fact, sex determination can be very different between different organisms. For example, in the XX-XO system found in crickets, grasshoppers, and some other insects, sperm cells that lack an X chromosome (referred to as O) determine maleness. Here, females carry two X chromosomes (XX) and only produce gametes with X chromosomes. Males, on the other hand, carry only one X chromosome (XO) and produce some gametes with X chromosomes and some gametes with no sex chromosomes at all (Figure 5).
Figure 6: Sex determination in birds.
Figure Detail
Despite the previous examples, males are not always the sex with the mismatched chromosome pair. For example, the ZZ-ZW sex determination system used in birds, snakes, and some insects relies upon females to carry the mismatched chromosome pair (ZW) and males to carry the identical pair (ZZ) (Figure 6).
If the three systems discussed above are compared in side-by-side Punnett squares (Figure 7), it is easy to see that sex determination is simply a matter of gamete assortment. Determinations of male and female character arise from a variety of different gamete combination patterns, all of which are the result of gender coding in sexually reproducing organisms.
Figure 7: A side-by-side comparison of sex determination systems in humans, insects, and birds.
Figure Detail
More on sex determination
• In some animals, sex can be determined by environmental conditions
• Sex Determination in Honeybees
• Scientists report sex reversal in a transgenic mous
The variety of inheritance patterns described in this article illustrate that sex determination is a complex and varied feature among organisms. The XX-XY, XX-XO, and ZZ-ZW systems are only a sample of the wide variety of sex determination systems that scientists have documented in the wide world of living beings, however.
The cry of "It's a boy" or "It's a girl" marks the newborn child's first and most basic label of personal identity. But researchers' understanding of sex is undergoing profound and surprising changes due to new insights gained from sociology, biology, and medicine. The differences between females and males, once believed black and white--or pink and blue--now appear like a blurred rainbow of confusion. Researchers are learning, for example, that the Y chromosome has degenerated over the centuries. They have found that, in mice, some genes involved in early stages of sperm production are on the female X chromosome; and they have identified the gene that can produce ambiguous genitalia.
Genetic studies are revealing that men and women are more similar than distinct. So far, of the approximately 31,000 genes in the human genome, men and women differ only in the two sex chromosomes, X and Y, and only a few dozen genes seem to be involved. Moreover, it's now known that the Y has only about 30 genes and many of those are involved in basic housekeeping duties or in regulating sperm production. The X has hundreds of genes with a vast array of roles.
Strong evidence exists that these two chromosomes were once a matching pair of Xs, says Jennifer Graves, a genetics researcher at La Trobe University, in Australia. According to Graves, it's unclear why the male sex chromosome, the Y, shrunk and shed most of its genes over time. Humans are not alone in this. The Y chromosome's degeneration is well documented in fruit flies and "is clearly an ongoing process in all animals," says Sherman Silber, a medical doctor and director of the Infertility Center of St Louis.1
Past assumptions regarding these sex chromosomes are being challenged: It's recently been discovered that in mice, nearly half of all genes involved in the earliest stages of sperm production are found on the X. "Scientists and non-scientists alike are comfortable thinking about the Y chromosome as a specialist in male characteristics," says David Page, who headed the discovery team at the Whitehead Institute for Biomedical Research in Boston.2 "By default, we've traditionally thought of the X chromosome as sexually neutral or as a specialist in female characteristics," Page says. "Our findings indicate that the X chromosome has a specialty in sperm production, much like the Y chromosome does."
Sex-Determining Genes
Detailed molecular and embryological studies are revealing how genes determine the anatomical sex of a fetus and how that process can and does go awry. Andrew Sinclair, who now heads the Centre for Hormone Research, University of Melbourne, was part of the British-based team led by Peter Goodfellow, of the Imperial Cancer Research Fund, which in 1990 discovered a crucial gene, known as SRY.3 It usually occurs only on the male Y chromosome. Using this discovery, researchers at Britain's National Institute for Medical Research then showed that a fertilized female mouse egg will become male when injected with SRY. The animal in question, named Randy, was the first sex-reversed mouse ever produced in this way, Sinclair says. The testes were small but Randy otherwise grew up male in every discernible way, despite being conceived as a genetic female. Placed in a cage with some females, Randy behaved in a typically male-mouse way, mating up to six times a night. "He thought he was male, they thought he was male, and we thought that was pretty good evidence," Sinclair says. "It tells you that SRY is the only gene you need on the Y chromosome to develop testes and become male."
But other genes complicate the process of sex determination. One known as DAX1, for example, is thought to act as an anti-testis gene, promoting ovary development. Another, called SOX9, combines with SRY to promote the formation of testicular cells in a male embryo. A third gene known as WNT4 and found on chromosome 1, seems to prevent the development of Leydig cells in male testes.4 Researchers led by Eric Vilain, assistant professor of human genetics, University of California, Los Angeles, recently found that when WNT4 occurs twice it can convert an embryo from male to female, often resulting in ambiguous genitalia.5
Neutral Starting Position
It is often argued that being female is the default state for mammals; that an embryo will develop in a female way unless male genes impose themselves on the process. It now seems just as possible, however, that the default state is merely a sexless one, a case of dual potential. In the first six weeks after conception, a human embryo develops a simple gonad that is neither a testis nor an ovary, but more of a neutral sex bud that can bloom either way. The bud has two parts, the medulla and cortex. At about seven weeks, when the fetus is thumbnail size, the SRY gene, if it is present, starts to work its magic. It switches on and the cells in the medulla start to multiply and form into a testis, while the cells in the cortex regress and virtually disappear.
Every significant distinction between men and women apparently stems from that event. As Graves puts it: "Of all the differences between male and female mammals, the primary one seems to be the development of the testis in males. Early in development the mammalian embryo is ready for anything. Male and female embryos are morphologically indistinguishable ... and the embryo is equipped with both male and female internal ducting." The development of the testis triggers a cascade of hormone-controlled changes, so once this decision--testis or no testis--has been taken, the sex of the embryo is determined. Within a week the new testes soon start producing their lifelong supply of testosterone, which floods the embryo and kicks off the development of typical masculine physical traits. If no SRY gene is present, the embryonic gonad waits until the 13th week of gestation to commit itself to femaleness. The medulla shrinks away and the cortex develops into an ovary, which starts to produce estrogen and feminizes the rest of the body.
The differentiation process in the womb can be affected, however, by genetic problems, infections, or exposure to toxins, drugs, or maternal hormones. In that context, it's remarkable to note that SRY's discovery was made possible with the help of French scientists who identified four unusual men who had sought treatment for infertility, Sinclair says. Chromosome tests revealed that they were in fact genetic females, with an XX female sex-chromosome pattern. Yet all four were anatomically male and had testes. Further studies showed that they had a small fragment of Y chromosome containing the SRY gene, tacked onto one of their X chromosomes. It was just a tiny glitch, enough to reverse their sexual anatomy but not endow them with other male genes to enable their testes to make sperm.
Many other such variants are now being discovered, along with true hermaphrodites and pseudo-hermaphrodites. There are people with missing sex chromosomes, extra sex chromosomes, or tiny genetic fragments tacked onto or missing from chromosomes that seem to have nothing to do with sex determination.
Delving into the very fundamentals of sexual genetics and biochemistry and making personal contact with people whose sex and gender are ambiguous has made Sinclair see the whole issue in a fresh light. "I think humans like things to be ordered, and they get bothered about gray areas and when things become less clear-cut," he says. "But these days I don't think so much in black and white about male and female. Now I think of it all as being on a spectrum."
Genders of plants -
Angiosperms, or flowering plants as they are commonly known, dominate the plant kingdom with over a whopping 3.5 lakh species. Unlike the bisexual plants that are predominant, where both the reproductive structures are present in the same flower of the plant, some have the male and female flowers in different plants. Such plants are called dioecious plants, and there are over 15,600 species of them across the world. Many of these plants have been used traditionally as food, medicine and timber for over thousands of years. In a recent study, researchers from the University of Oslo, Norway, and The Institute of Trans-Disciplinary Health Sciences and Technology, Bangalore, have explored some aspects of the bond between folk medicine and the sex of the plants used for the same.
The earliest record of medicinal herbs such as opium come from the Sumerian civilisation, followed by the Chinese who used ginseng and cinnamon bark as food and in the preparation of medicines. Excavations in the Gangetic region in central India present evidence for the use of medicinal plants since 4000 years. The Vedas too have a record of spices such as nutmeg, cloves and pepper used as medicinal plants.
In the study published in the Journal of Ethnopharmacology, the researchers have documented the knowledge and use of dioecious plants by Malayalis—a hill-dwelling ethnic group living in the Eastern Ghats of Tamil Nadu. They have pondered on questions like—do folk healers have a preference for plant genders; if so, how are these gender differences in plants perceived; and how the Indian systems of medicine have documented the concept of plant gender and preferences for it.
“The Indian Systems of medicine (ISM) is one among the codified systems of medicine in the world. We need to validate the traditional knowledge of medicinal plants and its formulation via modern scientific tools”, says Mr Gopalakrishnan Saroja Seethapathy, a research scholar at the University of Oslo and an author of the study. “The world herbal trade is expected to reach USD HYPERLINK "https://www.sciencedirect.com/science/article/pii/S221080331100042X"7 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S221080331100042X"trillion by the year HYPERLINK "https://www.sciencedirect.com/science/article/pii/S221080331100042X"2050. Although China has outperformed India in this, in HYPERLINK "HYPERLINK%20%22https://economictimes.indiatimes.com/news/politics-and-nation/chinas-export-of-herbal-products-is-a-challenge-for-us-narendra-modi/articleshow/30995997.cms%222014"HYPERLINK "https://economictimes.indiatimes.com/news/politics-and-nation/chinas-export-of-herbal-products-is-a-challenge-for-us-narendra-modi/articleshow/30995997.cms" HYPERLINK "HYPERLINK%20%22https://economictimes.indiatimes.com/news/politics-and-nation/chinas-export-of-herbal-products-is-a-challenge-for-us-narendra-modi/articleshow/30995997.cms%222014"2014, the Prime Minister of India admitted this as a challenge and emphasized the need to find ways to promote Ayurvedic medicines”, he adds, describing the current surge in the interest in alternative systems of medicines.
The researchers started off by compiling a comprehensive list of dioecious species used in traditional Indian medicine with data from the Indian Medicinal Plant Database. They then held interviews with folk healers—unlicensed practitioners of healing using conventional practices—and found that they have a gender preference for 13 species of plants including ten trees, two woody vines and one shrub. They differentiated the genders by looking at the plant size, and the presence and size of fruits. The interactions between the researchers and Ayurvedic doctors revealed that although Ayurvedic literature does show evidence of gender preference for medicine, the concept of gender among plants is mentioned in classical works like Charaka Samhita, Vriksh Ayurveda, and Raja Nighantu.
In several instances, the researchers noted that the use of the plants determined the preference of a particular gender. For example, in the case of toddy (palm wine), folk healers prefer female plants as they yield more toddy than male plants. Similarly, the timber of male palm trees like Borassus flabellifer (Palmyra palm) and Drypetes sepiaria (Hedge boxwood) is preferred for constructing houses, huts and making furniture because the wood is of the required size and is more durable than the timber from female trees. On the contrary, the wood of female Diospyros ebenum (Ceylon ebony) is preferred as carving the wood from the male plant is tough.
However, this sexual preference comes at a cost, say the researchers, who point out the threat to the existence of such dioecious species in a region. Also, selective logging of plants of a particular gender owing to demand can result in over-exploitation. As an attempt to conserve dioecious species that are heavily used, several conservation efforts are in place. “There are numerous schemes where Government of India supports the cultivation of medicinal plants. Cultivation is one way, and the other is creating awareness among plant collectors/harvesters about sustainable harvesting”, says Mr Seethapathy.
The study of plant diversity and the practical uses of plants through the knowledge of local people (ethnobotany) contribute to the fields of drug discovery and drug development immensely. These studies highlight the rate at which this valued knowledge is being forgotten and lost. As a boost to the field of ethnobotany, scientists around the world are attempting detailed molecular studies of male and female plants. The current research is a step in the same direction.
“One aspect is to devise measurements of selective logging by people and conservation of dioecious plants. The other is a metabolomics approach for dioecious plants to understand if there is any phytochemical variation between male and female plants. If true, it would be interesting to know the active compounds that vary between male and female plants and study its bioactivity”, remarks Mr Seethapathy before signing off.
https://researchmatters.in/news/study-shows-gender-matters-good-plants
Sex-determining mechanisms in animals
Abstract -
Biological mechanisms leading to the development of males and females are extremely varied. In the XX/XY system, the male has an unequal pair of chromosomes, while in the ZZ/ZW system, the unequal pair is in the female. Sex can also be determined by the temperature of incubation. Recent research has focused on the identification of sex-determining genes, culminating in the demonstration that the Sry gene on the Y chromosome of mice can induce male development in genetically female XX mouse embryos. Nevertheless, the occurrence of phenotypes in apparent contrast to the genotype suggests that the genetic male/female switch is not simple, and there may be common features linking different sex-determining mechanisms. There is increasing evidence that such a link may be provided by the induction of growth differences, and that the primary sex difference may result from the distinction between fast versus slow growth.
Molecular patterns of sex determination in the animal kingdom: a comparative study of the biology of reproduction
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Abstract
Determining sexual fate is an integral part of reproduction, used as a means to enrich the genome. A variety of such regulatory mechanisms have been described so far and some of the more extensively studied ones are being discussed.
For the insect order of Hymenoptera, the choice lies between uniparental haploid males and biparental diploid females, originating from unfertilized and fertilized eggs accordingly. This mechanism is also known as single-locus complementary sex determination (slCSD). On the other hand, for Dipterans and Drosophila melanogaster, sex is determined by the ratio of X chromosomes to autosomes and the sex switching gene, sxl. Another model organism whose sex depends on the X:A ratio, Caenorhabditis elegans, has furthermore to provide for the brief period of spermatogenesis in hermaphrodites (XX) without the benefit of the "male" genes of the sex determination pathway.
Many reptiles have no discernible sex determining genes. Their sexual fate is determined by the temperature of the environment during the thermosensitive period (TSP) of incubation, which regulates aromatase activity. Variable patterns of sex determination apply in fish and amphibians. In birds, while sex chromosomes do exist, females are the heterogametic (ZW) and males the homogametic sex (ZZ). However, we have yet to decipher which of the two (Z or W) is responsible for the choice between males and females.
In mammals, sex determination is based on the presence of two identical (XX) or distinct (XY) gonosomes. This is believed to be the result of a lengthy evolutionary process, emerging from a common ancestral autosomal pair. Indeed, X and Y present different levels of homology in various mammals, supporting the argument of a gradual structural differentiation starting around the SRY region. The latter initiates a gene cascade that results in the formation of a male. Regulation of sex steroid production is also a major result of these genetic interactions. Similar observations have been described not only in mammals, but also in other vertebrates, emphasizing the need for further study of both normal hormonal regulators of sexual phenotype and patterns of epigenetic/environmental disruption.
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Background
Sex determination is an integral part of reproduction and an essential process for the evolvement and enrichment of the genome. It has thus been the subject of many studies in reference to species across the entire animal kingdom. From insects to mammals, there is much to learn from the many mechanisms employed to determine sexual fate. This is no lost cause, since the study of sex determination and differentiation is only the natural expansion of comparative biology and reproductive physiology in the modern, molecular Era. Interestingly, data so far accumulated by a variety of model organisms has shown a relative economy in the molecular regulation of sex determination. More specifically, sex determination has so far proven to be a result of one of the following three mechanisms:
a) Environmental action on the embryo at a crucial stage of development. To the extent that this interaction is associated with temperature alterations, the process is also described as temperature-dependent sex determination and the developmental stage of sex determination is referred to as the thermosensitive period (TSP). This mechanism is mainly observed in reptiles and fish.
b) Genetic action, when at least one specific gene is considered to be the central regulator in a cascade of events leading to the determination of sexual phenotype. This mechanism is already known to apply in the case of several animals, including invertebrates (insects, worms) and amphibians. Moreover, it is a proposed regulatory mechanism for several species, whose study has so far been limited or led to inconclusive data as to the attempt to detect a single, specific, sex-determining gene.
c) The presence of distinct sex chromosomes or gonosomes. The identical pair may be present in both males (birds) and females (mammals) and their major sex-determining gene may be either known (e.g. mammalian SRY) or still suspected [1- HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B3"3"HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B3" HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B3"3"3].
Although sex determination has been suggested to promote specific functions at a universal level, such as selective cell proliferation (Mittwoch) or steroid hormone accumulation (Howard), this issue remains debatable [1,4]. What is even more intriguing is the fact that the conservation of relatively limited regulatory patterns in sex determination may suggest the presence of a single general regulatory scheme, at least in vertebrates, potentially involving or incorporating both hormonal elements and dosage compensation epigenetic regulatory phenomena, whenever necessary [ HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B5"5"HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B5" HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B5"5"5]. Such a discovery would bear great implications for comparative biology studies and might also allow important applications in the field of reproductive endocrinology. The study of more model organisms is a necessity to investigate this hypothesis and the consolidation of both recent and classic data from the relevant research work may significantly facilitate this discussion. This essay is dedicated to the brief and yet compact presentation of some of the better studied animal models of sex determination, in an attempt to approach that knowledge.
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Invertebrates
Hymenoptera
One such interesting mechanism is the haplodiploid genetic system we encounter in the insect order of Hymenoptera. More than 200,000 species of ants, bees and wasps are capable of laying both unfertilized eggs, that typically develop into uniparental (originating from one single female parent) haploid males, and fertilized eggs that can give us biparental (originating from two parents, male and female) diploid females (see Figure Figure HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/figure/F1/"11).
Figure 1
Haplodiploid reproduction. In Hymenoptera, unfertilized eggs develop into uniparental haploid males whereas fertilized eggs into biparental diploid females.
That can be accomplished with several strategies. One of the best understood seems to be single-locus complementary sex determination (sl-CSD), in which sex is determined by multiple alleles at a single locus. Heterozygotes at that sex locus develop as females whereas hemizygotes, and the odd case of homozygous diploids (i.e. through matched matings or faulty meiosis), develop as males (see Figure Figure HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/figure/F2/"2 HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/figure/F2/") HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/figure/F2/"2) thus providing us with the pattern presented above (see Figure Figure HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/figure/F1/"11).
Figure 2
Single-locus complementary sex determination (sl-CSD). In single-locus complementary sex determination (sl-CSD), heterozygotes at a single sex locus develop as females whereas hemizygotes and homozygous diploids develop as males. However, homozygous diploid males are generally sterile, unable to mate or not viable.
In honeybees, for example, the sex locus has recently been identified as the csd (complementary sex determiner) gene that encodes an SR protein (Arginine-Serine rich protein) [6]. The initial observation that csd function was required only in females and that its product is nonfunctional when derived from only one allele [7] was followed by the suggestion of three possible models. First, that different allelic CSD proteins form active heterodimers. Second, that CSD proteins derived from the same allele form homomers, with two homomer species in females and one in males. And third, that merely the existence of different alleles is required in females for csd to complete its function [ HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B8"8"HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B8" HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B8"8"8].
However, it should be noted that sl-CSD has been known to exhibit an evolutionary pressure against species with higher rates of inbreeding, due to one of its major faults. In most cases, mating leads to the creation of offspring with two different alleles at the sex locus (diploid females). However, a mating in such populations has higher probability of a union between a male and a female that share the same allele, a condition also known as a matched mating[9]. In matched matings half the diploid offspring are predicted to turn out homozygous at the sex locus and develop as males rather than females, whereas diploid males in species with sl-CSD are generally sterile, unable to mate or not viable (see Figure Figure HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/figure/F3/"3 HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/figure/F3/") HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/figure/F3/"3) [10]. Such is the example of the honeybee, where homozygous diploid males created from inbreeding are eaten by the workers [ HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B7"7"HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B7" HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B7"7"7].
Figure 3
Matched matings in sl-CSD. In matched matings, half the diploid offspring are homozygous at the sex locus and turn into diploid males, which are unable to contribute to reproduction.
Dipterans (Drosophila melanogaster)
Taking things a step further, we enter the realm of Dipterans and Drosophila melanogaster, one of the model organisms in which the sex determination pathway has been elucidated in the greatest detail. Here the choice between male and female development is made by one single switch gene by the name of sex-lethal (sxl) in response to the ratio of X chromosomes to autosomes (X:A ratio) [11]. The latter is communicated early in development through the delicate balance between the dose-sensitive X chromosome numerator elements (those include genes such as sis-a, sis-b, runt and less so sis-c) and the autosomal denominators (such as dpn) in conjunction with the maternally derived products of the da gene and the more recently studied emc, groucho, her and snf (see Figure Figure HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/figure/F4/"4 HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/figure/F4/") HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/figure/F4/"4) [ HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B12"12"HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B12" HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B12"12"12]. We are not yet completely certain of the logistics of it, but it seems that the feminizing effect of the numerator elements is measured against the masculinising denominators, with the maternally derived products of the rest of the genes acting as point of reference.
Figure 4
The X:A ratio determines sex in Drosophila melanogaster. In Drosophila melanogaster sex is determined by the X:A ratio, which is communicated through the balance between the X numerator elements and the autosomal denominators in the presence of several maternally derived proteins. An X:A ratio of 0.5 leads to a non-functional SXL and male development, whereas an X:A ratio of 1 maintains SXL in its active state and is conducive to female development.
All this takes place early in development, leading to the activation of the sxl gene through an "early" promoter in females. This early form of the SXL protein, absent in males, then orchestrates a specific splicing of the mRNA produced through the activation of the "mature" promoter in females. In males, standard splicing of the sxl mRNA leads to a non-functional protein. It is only in females, through an autoregulatory feedback loop, that sxl manages to keep itself in an active state through this sex-specific splicing [11,13] (see Figure Figure HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/figure/F5/"55).
Figure 5
Sex-specific splicing of the sxl mRNA. Early activation of the sxl gene through a different primer (PE) in females allows the appearance of an early SXL protein that guides the splicing of the mRNA originating from the 'standard' primer (PM). This alternative splicing leads to a functional 'mature' SXL protein that then takes up the role of retaining its active state. In males, where no early transcripts can be found, a male-specific exon is included which contains many early stop codons thus leading to the creation of a truncated and non functional protein.
Once the SXL active state has been established, it then goes on to regulate a series of other proteins that control female development, once again through the process of alternative splicing, leading finally to the two alternative products of the doublesex gene (dsx), DSXF and DSXM [14,15] (see Figure Figure HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/figure/F6/"6 HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/figure/F6/"). HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/figure/F6/"6). The end-result? A series of intricate gene interactions that can take it from there and establish the development of the appropriate sex. Still, it is interesting to note, that the Y chromosome, present in males, takes no part in this entire process, and that its sole use is to help in the successful completion of the process of spermatogenesis later on in the differentiation of the male germline.
Figure 6
Genes involved in sex determination in Drosophila melanogaster. The SXL protein regulates the female-specific splicing of the tra mRNA. The TRA protein then forms dimers with TRA-2 which regulate the sex-specific splicing of dsx mRNA. DSXF is the result of said sex-specific splicing in females, whereas DSXM is present in males. All of the above also interact with other genes in turn, in order to mediate sexual development.
Nematodes (Caenorhabditis elegans)
Another model organism that uses a single gene switch and the subsequent hierarchy of gene pathways to determine sex is the nematode C.elegans. Here again the animal's sexual fate depends on the X:A ratio, and there isn't even a Y chromosome present in males to later on interfere with the germline. However, C.elegans worms are special in that the choice lies between males with one X chromosome and hermaphrodites with two.
As before, the X:A ratio is communicated with the help of several "X-signal elements", such as the SEX-1 (signal element on X) protein that acts on the level of transcription and the FOX-1 (feminizing locus on X) protein that acts post-transcriptionally [16]. These two, among others that have yet to be deciphered, manage to suppress the levels of the XOL-1 (XO lethal) key protein, or what we could call the C.elegans sex switching gene [17] (see Figure Figure HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/figure/F7/"7 HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/figure/F7/"). HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/figure/F7/"7). From there on, it is a matter of tracking down a pathway of inhibitory genes, to result at the TRA-1 (transformer) protein, free to act in hermaphrodites and regulate several other genes [18,19]. This pathway in fact involves several groups of gene products, some of which retain their active state in males and others in hermaphrodites (see Figure Figure HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/figure/F8/"8 HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/figure/F8/"). HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/figure/F8/"8). One possible model incorporating these interactions is depicted in Figure Figure HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/figure/F9/"9,9, and includes the interaction between HER-1 and the TRA-2 receptor in males, which allows the FEM proteins to inhibit TRA-1 from acting as a transcription factor (see Figure Figure HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/figure/F9/"99).
Figure 7
The currently known X-signal elements in C.elegans. In C.elegans, the X-signal elements, such as the SEX-1 and FOX-1 proteins, control the levels of XOL-1 and help determine sex.
Figure 8
Sex gene pathway in C.elegans. A simple depiction of the sex determination gene pathway as it is known today in the soma of C.elegans. The interactions between several groups of gene products that have been observed to have an inhibitory effect on each other follow the switch of xol-1. The result is that several of these proteins remain active only in males only and others only in hermaphrodites.
Figure 9
Sex determining interactions on a cellular level. Suggested protein interactions in the later stages of the sex determination pathway in C.elegans. While the SDC proteins have also been known to serve as part of the dosage compensation mechanism in C.elegans, HER-1 has been pictured as capable of binding to the TRA-2 receptor, which then releases the FEM molecules in males. Those in turn bind to the TRA-1 transcription factors rendering them inactive. In hermaphrodites, the TRA-2 receptors retain their hold on FEM, and TRA-1 is free to act as a transcription factor on the genome.
However, the C.elegans hermaphrodites pose an interesting issue. These are specialized females which in the fourth and final larval stage (L4) produce around 300 sperm, to use for self-fertilization when there are no males available [20]. This requires a careful regulation of the switching between the male and female differentiation of the same germ cells without the benefit of the usual sex determination pathway, since the "male" genes that normally regulate spermatogenesis are inactive anyway. Instead, a new series of genes take over in a specific stage of development and act in place of the HER-1 protein to inhibit tra-2 and allow spermatogenesis to take place till the end of the L4 stage (see Figure Figure HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/figure/F10/"10 HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/figure/F10/"). HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/figure/F10/"10). Once this is over and at the onset of adult life, a new series of genes take their place, tra-2 is once again active, and the adult hermaphrodite is free to continue with oogenesis for the rest of its life [19,21] (see Figure Figure HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/figure/F10/"1010).
Open in a separate window
Figure 10
Gene interactions that allow spermatogenesis and oogenesis in the hermaphrodite C.elegans, as opposed to the gene pathway in the soma. The top half of each frame displays the gene pathway for sex determination in the C.elegans soma and the bottom half the changes that concern the hermaphrodite germline. During the fourth larval stage (L4), a special set of genes expressed in the germline (fog-2, gld-1, laf-1) allows spermatogenesis to occur in hermaphrodites by interfering with the original sex determination pathway (inhibition of tra-2 that leads to the activation of the fem gene products and others such as fog-1 and fog-3). Once spermatogenesis is over and the hermaphrodite enters its mature stage (M), the original sex determination pathway is re-established (tra-2 becomes active again) in the germline of adult hermaphrodites and makes the switch to oogenesis (by inactivating the genes fem, fog-1 and fog-3 gene products that allowed spermatogenesis).
Go to:
Vertebrates
Reptiles
The different species of reptiles present a considerable variety of sex determination patterns. For instance, most snakes possess a ZZ/ZW pattern of sex chromosomes, similar to that discussed later as the model mechanism for sex determination in birds. The study of lizards has led to more complex findings, with different species having either a ZZ/ZW sex chromosome pair or a XX/XY system, similar to that observed in mammals [22, HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B23"23"HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B23" HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B23"23"23].
On the other hand, many species of reptiles, including most terrestrial turtles and all crocodilians and sea turtles examined to this date, have no discernible sex chromosomes, nor is their sex determined by the presence or absence of specific genes. In these organisms, it is the temperature of the environment in a specific period of incubation that can determine whether the animal in question will turn into a male or a female [24, HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B25"25"HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B25" HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B25"25"25].
Indeed, studies have shown that there seem to be no significant differences in the expression of sex-related genes. Instead, there is a specific period of incubation, which is generally considered to lie in the middle third of development, during which the temperature of the eggs controls quite accurately their sexual fate. This particular period is also known as the thermosensitive period (TSP).
It is during this period that a very specific enzyme enters into the equation. Aromatase, a cyt450 enzyme responsible for the conversion of androgens into estrogens is common among many organisms (see Figure Figure HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/figure/F11/"11 HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/figure/F11/"). HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/figure/F11/"11). In reptiles, while steroidogenesis begins very early, prior even to the thermosensitive period, aromatase activity remains universally low. With the onset of the thermosensitive period however, aromatase activity seems to increase in certain temperatures, which vary for each species. For example, in marine and freshwater turtles, higher temperatures cause an exponential increase of aromatase activity, whereas in lower temperatures aromatase activity remains low. The different levels of aromatase activity then guide the differentiation of the indifferent gonad into an ovary or testis. Once the thermosensitive period is over and the fate of the gonad has been established, further changes in temperature seem to have no effects (see Figure Figure HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/figure/F12/"12 HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/figure/F12/") HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/figure/F12/"12) [26, HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B27"27"HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B27" HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B27"27"27].
Figure 11
Aromatase. Aromatase is a cytP450 enzyme that allows the conversion of androgens into estrogens.
Figure 12
Temperature-dependent sex determination. Aromatase activity levels during the thermosensitive period (TSP) are regulated by the temperature of the environment and control gonadal differentiation. Changes in the environment temperature before and after TSP do not seem to affect sex.
Interestingly, a number of genes originally described as part of the genetic regulation of sex development in men and other mammals have also been detected in reptiles. For instance, in the sea turtle Lepidochelys olivacea, several genes so far related to mammalian sex determination are expressed, including DAX1 (dosage-sensitive sex reversal 1), DMRT1 (doublesex- and mab-3-related transcription factor 1) and SOX9 (SRY related HMG box 9). In particular, DAX1 is a known regulator of gonadal development in mice and other mammals, considered to be an "anti-testis" gene, although this may approach may prove to be too simplified. In reptiles, the gene is not differentially expressed in response to temperature variation during the TSP, therefore, its role in reptile sex determination is unclear. The gene is also expressed in crocodilians with temperature-dependent sex determination, such as Alligator mississippiensis. Whether this gene could indeed be a target for androgen or estrogen-related actions following the TSP remains unknown. As far as DMRT1 is concerned, the gene was initially related to sex determination in D. melanogaster, due to the presence of a domain compatible to the sex determinant gene DSX. Subsequent research, however, has proven the gene's expression in several other species as well, including birds, fish and reptiles. In alligators, such as A. mississippiensis, the gene is expressed exclusively in the gonads of males. Moreover, its expression appears to precede that of SOX9, another testis-specific gene conserved in a vast number of species, ranging from reptiles to mammals. The latter gene is originally expressed in the bipotential gonad of reptile embryos, but following the TSP, it remains active only in males, making it a candidate gene for sex steroid-induced regulation. In alligators, SOX9 is also related to increased AMH (Anti-Müllerian Hormone) levels, but, contrary to mammals, AMH induction chronologically precedes that of SOX9 [3,23]. In the case of lizards, an attempt has also been made to examine sexual dimorphism in the brain. The first results from these experimental series show distinct differences in estrogen receptor expression and progesterone concentrations in specific areas of the central nervous system, a finding that may imply that aromatase regulation is only the first step in a sequence of several more complex sex-specific/dimorphic genetic phenomena that still remain to be examined [ HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B28"28"HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B28" HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B28"28"28].
Finally, it has recently been suggested that aromatase may also be regulated by secondary parameters, other than temperature. This has been described for instace, in the case of Prostaglandin E2, which appears to be associated with increased aromatase action [29]. Immunological reactions and cytokine levels may also be important. The latter has led to clinical applications in humans, with the attempt to treat oncological patients with hormone-sensitive cancer, with selective Interleukin-6 pharmaceutical modulators, thus indirectily aiming at aromatase suppression [ HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B30"30"HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B30" HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B30"30"30].
Amphibians
The thermosensitivity of the gonads has been demonstrated not only in reptiles, but also in several fish and some amphibians. These tend to combine a genotypic sex determination mechanism -either male heterogamety, female heterogamety or polygenic- with the mechanism demonstrated above. The result is a phenomenon known as sex reversal, where the effects of temperature may go against the genotypic directions, allowing the existence of animals in genotypic and phenotypic sex discordance [27]. (Table (Table HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/table/T1/"11)
Table 1
Sex reversal
| Female-producing temperatures | Male-producing temperatures |
XX (ZW) | Female (in accordance with genotype) | Female OR male (in discordance with genotype) |
XY (ZZ) | Male OR female (in discordance with genotype) | Female (in accordance with genotype) |
The combination of genotypic and temperature-dependent sex determination allows a phenomenon known as sex reversal, where the phenotypic sex does not always agree with the genotypic directions.
In particular, male or female heterogamety has been described in various species of anurans and urodeles. Sex chromosomes of various types may be present, following both the XY/XX and WZ/ZZ pattern that usually apply to mammals and birds, respectively. The exact mechanism by which temperature regulates sex determination in amphibians is not yet deciphered, but it doesn't seem to apply to the TSP-aromatase regulation model of reptiles. Hormonal action may also act in the process of acquisition of sexual phenotype, either independently or in conjunction with temperature variation [ HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B3"3"HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B3" HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B3"3"3].
Gene studies in amphibian sex determination are not as extensive as in other animal models. Of the various genes so far associated with sex determination in other species, amphibians appear to express DMRT1. However, it is not yet clear whether this is a downstream product in the sex differentiation cascade or a factor with a more central role in sex determination [3,31, HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B32"32"HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B32" HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B32"32"32].
Fish
There are numerous species of fish in the animal kingdom, with estimations as to their current number reaching a mean price of 25.000. As one may easily perceive, among such a variety of living organisms, research has been focused on relatively few, specific model organisms, each of which has been considered representative of the reproductive physiology of several other closely related species. Among the mechanisms observed, one may refer to a) the presence of true hermaphrodites, a strategy usually associated with lower evolutionary levels (e.g. the previously described model of invertebrates-nematodes) b)temperature-dependent sex determination, with a process similar to the one known to be characteristic of most reptiles and c) sex chromosomes. The latter may follow either the XY/XX or the ZW/ZZ pattern [3, HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B33"33"HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B33" HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B33"33"33].
Contrary to mammals, the sex determining genes have not yet been described in fish, although some candidacies have been proposed. It might also be possible that, instead of a common, uniform gene pattern for all fish, different genes will be proven to be the major sex determinants in every species. According to some researchers, it might also be possible to assume a number of competing genes in every species, with environmental and/or hormonal parameters regulating their relative priority in sex determination in every birth [ HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B33"33"HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B33" HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B33"33"33]. Of the various model organisms available for study, we will limit our reference to four characteristic examples, namely the atlantic salmon, the platyfish, the medaka and the zebra fish.
The atlantic salmon (Salmo salar) was, until recently, an organism within unknown genetic sex determinants. However, recent data has detected the candidate sex-determining locus of this species as part of chromosome 2. For this reason, this large metacentric chromosome is now regarded as the sex chromosome of this species. Research has now turned to the detailed study of the region, in an attempt to identify the exact position and structure of the single sex-determining gene, which has been proposed to exist within the aforementioned locus [ HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B34"34"HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B34" HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B34"34"34].
The platyfish (Xiphophorus maculatus)'s genome may contain any of three sex chromosomes, namely X, Y and W. This allows significantly more combinations in the population than those observed in other species, applying to the "traditional" principle of only two sex chromosome types available (ZW and XY pairs, respectively). Of all the combinations, WX, WY and XX develop as females, while XY and YY become males. No specific sex-determining gene has been described so far, although the W chromosome is considered a major candidate for its position, since its presence coincides with female phenotype regardless of the type of the second sex chromosome. However, some genes, previously described in other species and associated to reproductive physiology and development, are also found in this and other fish species. These include SOX family members, such as SOX9 and DMRT1. On the other hand, classical hormonal regulators of sex differentiation, such as AMH have not yet been identified in fish [3, HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B33"33"HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B33" HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B33"33"33].
DMRT1 has been been shown to be particularly important for sex determination in the teleost medaka, Oryzias latipes. The sex determining system of the medaka is male heterogametic, i.e. it follows the XX/XY principle known from mammalian reproduction. Although some similarities with genes of the mammalian sex chromosomes may exist, the major sex determinant of mammals, i.e. SRY (sex determining region of the Y chromosome) is missing. Consequently, another, previously unknown, sex-determining gene must be present in the medaka genome. Indeed, in the Y chromosome of the fish a new gene has been detected, bearing six exons and a DM domain. The latter is a major characteristic of genes involved in sex determination in invertebrates, such as doublesex and mab3 in D.melanogaster and C.elegans, respectively. This new gene was named DMY (DM domain of the Y chromosome) and it is homologous to DMRT1 gene, which is conserved in various species. Although a lot of information is still missing, it appears that in the male, DMY and DMRT1 operate in procession as strong determinants of gonadal development. In the female, the role of aromatase is once again central, although its induction, in this case, may be a genetic rather than temperature-related event. Other genes' expression has also been detected exclusively in females, such as FIGa (factor in the germ line a), but their correlation with aromatase induction remains to be proven (see Figure Figure HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/figure/F13/"13 HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/figure/F13/") HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/figure/F13/"13) [ HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B35"35"HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B35" HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B35"35"35].
Figure 13
Sex determination in the medaka. Although many details for the molecular model of sex determination in the medaka are still missing, the mechanism is known to be based on the presence of XX/XY sex chromosomes. Following a stage of undifferentiated gonads, males exclusively express DMY, a gene bearing a DM domain, which is a genetic feature that considered central in sex determination pathways of various species. Among the genes induced downstream is DMRT1, which participates in gonadal development and differentiation in fish, birds and mammals. In XX females, the exact genetic cascade triggered in the absence of DMY is unclear, but it supposed to involve sex-specific gene expression, such as FIGa and sex steroid/aromatase regulation.
Finally, sex determination in the zebra fish is considered to be a genetic phenomenon, but the details of the process are still under examination. Of particular interest are recent data, proving the expression of two sex-related genes in the zebra fish [33,36,37]. These are a) vasa, a gene family expressed exclusively in the gonads of several species, including D.melanogaster, mice and fish and b) FtzF1 (fushi tarazu factor 1), a gene originally described in Drosophila and nkown to encode the steroidogenic factor 1 (SF1) in mammals, thus regulating sex steroid production [33, HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B36"36"HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B36" HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B36"36"36].
Birds
Next, approaching birds, we begin to tread on more familiar ground, as once again we return to sex chromosomes. In birds however, females are the heterogametic sex, carrying one copy of each of the so called Z and W sex chromosomes, whereas males are homogametic ZZ. The Z and W chromosomes have no relation to the mammalian X and Y, and in fact seem to have evolved from different pairs of autosomes. And this is part of the reason we are not yet certain which of the two carries the genetic trigger for sex determination [38, HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B39"39"HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B39" HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B39"39"39].
To this day, there are two major theories under investigation. Sex may depend on Z chromosome dosage, according to the example of Drosophila melanogaster and C.elegans. One candidate gene for this theory is the DMRT1, which is located on Z chromosomes, escapes dosage compensation and is expressed specifically in the gonads, and is thus capable of linking the number of Z chromosomes with gonadal differentiation [40, HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B41"41"HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B41" HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B41"41"41].
On the other hand, sex may be determined by the feminizing presence of the W chromosome, following the example of Y in eutherian mammals. There are two different mechanisms that are being studied and can support this theory. One includes the FET1 gene, which is located on W, does not have a Z homologue and is expressed almost exclusively in the female urogenital system [18]. The other includes the ASW gene, also known as WPKCI, and its Z homologue ZPKCI, since it has been proposed that the products of those two genes are capable of dimerisation, with a ZPKCI homodimer acting as a testis factor and a WPKCI/ZPKCI heterodimer preventing this effect (see Figure Figure HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/figure/F14/"14 HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/figure/F14/") HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/figure/F14/"14) [39- HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B41"41"HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B41" HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B41"41"41].
Open in a separate window
Figure 14
The role of ZPKCI and ASW (WPKCI) in ZW sex determination. According to one theory, the ZPKCI proteins form homodimers in ZZ males that stimulate a factor required for the differentiation of the testes. Whereas in ZW females, the ASW (also known as WPKCI) proteins form heterodimers with ZPKCI that may prevent the activation of that factor or stimulate directly the differentiation of ovaries.
One way to discern between the two theories would be to look into different combinations of Z and W chromosomes. Indeed, scientists have studied ZW aneuploidy in an effort to better understand how things work. It turns out that ZZZ animals develop testes but are infertile, ZWW animals die early in embryonic development, but ZZW combinations manifest as intersexual: the animals appear female on hatching, but slowly turn into males at sexual maturity. It is still possible, thus, that a combination of the above is in fact applied [40, HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B42"42"HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B42" HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B42"42"42].
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Mammals
Marsupials
That final idea was borrowed by the mechanism applied in marsupials. Here we have a set of X and Y chromosomes related to those found in eutherian mammals. The basic marsupial Y chromosome is the smallest of any mammal but retains its ability to turn the undifferentiated gonads into testes [22]. However, the differentiation of the embryonic testis does not also control all aspects of sex differentiation. The formation of the mammary glands and scrotum develops before gonadal differentiation takes place and is independent of gonadal hormones [43]. In fact, it appears to be under the control of genes located on the X chromosome. So it happens that XXY animals have testes, but a pouch with mammary glands has replaced their scrotum, whereas XO animals have no testes, but an empty scrotum in place of a pouch (Table (Table HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/table/T2/"2 HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/table/T2/") HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/table/T2/"2) [43,44]. These X-linked genes have yet to be identified, but already the autosomal SOX9 has been reported of being expressed in the scrotum and mammary primordial before birth [ HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B45"45"HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B45" HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B45"45"45].
Table 2
Different aspects of sex differentiation in marsupials
| Y chromosome (testes) | No Y chromosome (absence of testes) |
One X chromosome (scrotum) | XY | XO |
| Male animals with scrotum and testes | Animals without testes and an empty scrotum instead of a pouch |
Two X chromosomes (pouch and mammary glands) | XXY | XX |
| Animals with testes and a pouch with mammary glands instead of scrotum | Female animals with a pouch and mammary glands |
The marsupial Y has the ability to turn the undifferentiated gonads into testes, but other aspects of sex differentiation seem to rely on the number of X chromosomes present. This is how we come across the above combinations in sex chromosome aneuploidies in marsupials.
From monotremes to eutherian mammals
Intriguing as the marsupial X and Y chromosomes may be, it appears that they also exhibit close similarities to those encountered in man, as well as practically every other mammalian species. This observation has lead to the hypothesis of a common origin for the gonosomes of all current mammals. In an attempt to verify this theory, recent research related to sex determination in man has shifted its focus on the application of comparative genetics for different sex-specific sequences, both codal and non-codal, aiming to unravel the mystery of X and Y evolution [46,47]. It is important to note that the concept of a common ancestry for sex chromosomes has been originally proposed by S.Ohno as early as the 1960s and it was based on comparative observations on mammalian reproductive biology and gonadal physiology [48]. In fact, one of the authors (R.A.) has also had the fortune to contribute to the latter research as a member of the College de France research group, under the direct supervision of the late Professor Alfred Jost [ HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B49"49"HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B49" HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B49"49"49].
According to a number of recent publications made by the research team of Dr D. Page and his colleagues (Jegalian and Page 1998, Jegalian and Lahn 2001) [50] the comparison of the nucleotide sequence of the X and Y chromosomes and their extend of homology in different mammals has lead to the conclusion that the development of discrete X and Y gonosomes is the result of, at least four, independent and recurrent, major stages of genomic evolution. In every stage, a failure in recombination between the homologous ancestors of the modern X and Y has been transferred to the next generations, leading to the continuous destabilization of their structure, permanent deletions in one of the chromosomes (the future Y) and a steady differentiation of their content, up to the current state, where homology remains only in the two remote pseudoautosomal areas in the telomeres of human gonosomes (see Figure Figure HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/figure/F15/"1515).
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Figure 15
The multistage model of sex chromosome evolution. The mammalian X and Y chromosomes are thought to derive from a common initial autosomal pair. By a gradual process of genetic instability, which may have been related to failure in the recombination process, the chromosomes have begun to differ from each other. The first area to acquire a sex-specific role is considered to be the locus around the major sex determinant gene, i.e. SRY. Thus, in evolutionary lower mammals with a more conserved chromosomal content, such as monotremes, X and Y retain homology in all their length but for the SRY region. Subsequent stages of X-Y recombination failure have led to other, transient forms of X-Y structure, such as those observed in marsupials and primates. The greatest level of heterogeny is considered to be that found in modern humans.
The evolutionary model proposed by Page, Jegalian and Lahn in 1999 [ HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B51"51"HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B51" HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B51"51"51] suggests that more than 400 million years ago (i.e. before the evolution of the mammalian common ancestral line, roughly 300 million years ago), the common ancestor of modern reptiles and mammals retained an intact pair of autosomal, homologous genes instead of the X and Y. At the time, the two chromosomes where identical, sharing the same content and participating in autosomal recombination during the first meiotic division.
In the following evolutionary stage, it seems that failure in recombination during meiosis resulted in the reversal of part of one of the chromosomes (the future Y). This process inhibited further recombination attempts in the inverted region, since the presence of homologous sequences in opposite positions of the two chromosomes is a prerequisite for recombination. In future generations, this mistake wasn't repaired, thus instituting a permanent non-recombining area in the genome. As further failures in recombination followed, with a constant nucleotide loss in the unstable chromosome (Y), homology between the two chromosomes gradually decreased, until finally it was limited in the distant tips of the X and Y [ HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B52"52"HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B52" HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B52"52"52].
The first region where recombination failure must have occurred was the region of the SRY gene, which has since initiated a new role for genes of the Y chromosome, i.e. sex determination and sexual trait differentiation. This shift in gene function was the result of a long process of sequence variation, both in the encoding area and its regulatory elements. This evolutionary stage is placed 240–320 million years ago, an era consistent with the appearance of the ancestor of monotreme mammals (300 million years ago), animals known indeed to carry the SRY gene and only a limited area around it, where recombination between X and Y is not possible, contrary to the rest of their sequences [ HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B53"53"HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B53" HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B53"53"53].
A second stage in the formation of the X and Y is also attributed to a recombination failure, about 130–170 million years ago. At this time, the united mammalian line was separated in two others, namely the marsupial and placental lines.
As a result of the first two waves of X and Y differentiation discussed so far, the ability to estimate and regulate the level of gene expression in mammals was significantly hindered, since the processes of failed recombination, genomic reversal and deletion had already resulted in a significant loss of the sequences of the initial Y chromosome, contrary to the relatively intact X. In an attempt for proper dosage compensation between the two sexes, mammals developed the process of X chromosome inactivation (XCI) [54]. In effect, this process evolved in the course of time and in parallel with the continuous alteration in X and Y morphology and structure [55, HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B56"56"HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B56" HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B56"56"56]. It is also possible that, initially, simpler mechanisms of dosage compensation where applied, such as the hyper-expression of the genes in the single X chromosome of males, as is the case in the modern fruitfly, Drosophila melanogaster.
A classic example of this gradual evolution of the dosage compensation strategies and, particularly, X inactivation, refers to the origin of LINE1 (long interspersed nuclear elements 1) sequences. These sequences interact with XIST (X inactivation specific transcript) RNA and, possibly other transitory proteins, forming a three-dimensional pattern that promotes gene silencing in the spreading stage of XCI. In placental mammals, LINE1 sequences have multiplied and spread throughout the Y chromosome, about 100-60 million years ago. This estimation suggests that the LINE1 sequences, an element necessary for stable XCI have evolved a long time after the separation of marsupials and placental mammals. Indeed, experimental data suggests that only the latter are capable of stable XCI, while the first possess an imprinted transient mechanism, lacking maintenance processes (e.g. methylation) [ HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B57"57"HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B57" HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B57"57"57].
Finally, a third stage in X-Y evolution is placed 80 to 130 million years ago and a final one 30 to 50 million years ago, coinciding with primate initial appearance. As in previous stages, failure in recombination is once again considered the promoter for these steps in the formation of the current X and Y.
A number of researchers focusing in comparative genomic studies and, especially, point mutations, attempt to clarify the exact evolutionary pattern for the gonosomes of every mammalian species. The addition of experimental data improves the estimation of the exact separation time for the ancestors of the major types of species on the earth, allowing the recognition of further sub-stages in the main pattern that has already been described. For instance, in the case of the X and Y, the initial recombination failure stage has been challenged, with some researchers proposing its substitution by two distinct phases, 350-290 and 290-230 million years ago [ HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B58"58"HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B58" HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B58"58"58]. In this case, it would be possible to assume a common origin for the gonosomes of mammals, reptiles and birds, preceding all other steps in the X-Y genomic evolution. However, no such procedure has been proven to this day. On the other hand, the classic concept of a common history for all mammal gonosomes and a completely discrete pattern for Z/W evolution in birds remains the most widely accepted in current evolutionary genetics.
The analysis of chromosome Y nucleotide sequence was an especially difficult task for the research teams involved in the Human Genome Project. According to a recent report (Skaletsky et al 2003), about 95% of the Y chromosome is now defined as the male specific region of the Y chromosome or MSY, for short. This area coincides with the previously described as non-recombining region of the Y chromosome or NRY. This change in terminology is not only aiming to emphasize the importance of the region for male sex determination, as it includes the SRY gene and its regulatory and downstream acting agents, but also attempting to correct a chronic misunderstanding, since this area is in fact participating in recombination. Interestingly, the latter doesn't involve the X, since no homology is present, but different parts of the MSY, in the form of a unique Y-Y internal recombination [ HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B59"59"HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B59" HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B59"59"59]. On the other hand, X-Y recombination is limited to the two pseudoautosomal regions, i.e. PAR1 and PAR2, thus leaving no part of the Y without the ability to participate in some form of recombination, as the term NRY would obviously suggest.
A further study of the sequences in the MSY allows a classification in three categories, each including areas of distinct structure, function and origin:
1. X-degenerate genes. This category includes genes deriving from the various stages of X-Y gradual differentiation proposed by Page and described so far. The term degenerate is used to emphasize their origin from the former ancestral autosome, which was equivalent in size to the X, before it gradually degenerated. One of the genes in this group is the SRY gene. In total, the category includes single copies of 14 pseudogenes and 13 genes, all having a homologue allele in the X. Most of these genes aren't expressed exclusively in a single, specific tissue. Their products are proteins produced in a variety of cells of the body, mediating non-sexual functions.
2. X-transposed genes [ HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B60"60"HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B60" HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B60"60"60]. These loci include a minimal number of genes and a large proportion of LINE1 sequences and other examples of non-coding DNA. Their homology with regions of the X chromosome leads to the conclusion that they must be a result of a distinct evolutionary process, significantly more recent than the stages proposed by Page. It is possible that these regions were directly translocated from X to Y, a process involving the parallel transfer of several intact genes.
3. Ampliconic genes. These genes exist in multiple copies on the Y, resulting from the replication of an initial copy. Apart from multiple copies per gene, this category also includes eight large palindromes. These genomic areas are characterized by inverted repeat sequences in their edges, while their centre appears to protect "hidden" genes and repeat, non-coding sequences [ HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B61"61"HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B61" HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B61"61"61]. In attempt to explain the creation of the palindromes the following pattern has been proposed:
1) An initial failure in recombination leads to the transfer of genes from autosomes to the ancestor of the modern Y
2) A series of amplification circles resulted in the presence of multiple copies for each one
3) The reversal of some of the copies has promoted the creation of the palindromes, trapping parts of the Y in between.
It is interesting to note that the formation of the palindromes increased the inherent stability of the Y, raising the question of its possible settlement in its current form, after thousands of years of degeneration and decay [62]. If this is indeed so, the whole theory of continuous Y deterioration as a cause of an increase in male infertility, due to the constant removal of genes essential for effective spermatogenesis, is seriously challenged [ HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B63"63"HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B63" HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B63"63"63].
Sex determination in mammals has been more extensively studied than in any other species, most probably due to its direct relevance to human physiology and pathophysiology. A large number of genes have already been described and many more are expected to be added in the process, since the relevant research constantly reveals new players in the complex network of reactions related to sex determination (see Figure Figure HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/figure/F16/"16 HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/figure/F16/"). HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/figure/F16/"16). Even in the common, bipotential gonad, the expression of several genes is considered crucial for subsequent development and normal sexual dimorphism. These include, among others, WT1 (Wilm's tumor 1), FtzF1/SF1 (Fushi tarazu factor 1/steroidogenic factor 1) and Lim1. Absence of any of these products at this stage, especially WT1, is inconsistent with further gonadal development and may also cause other malformations, e.g. affecting the adrenal gland and renal buds. Genes of the wnt family, such as wnt4, may also participate in the regulation of epithelial organization and epithelial-mesenchymal interactions in the area of the gonadal primordium [ HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B64"64"HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B64" HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B64"64"64].
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Figure 16
Genetic model of sex determination in humans. The formation of the undifferentiated/bipotential gonad is controlled by several genes acting simultaneously, such as WT1, SF1 and Lim1. Primary sex determination is based on the presence of the Y chromosome and its main sex-determining gene, SRY. In this case, SOX9, FtzF1/SF1 and AMH expression divert the gonad and the reproductive tract towards the male phenotype. This differentiation process is regulated by several other genes, including DAX1, GATA4, FOXL2 and, possibly, DMRT1 and 2 (not shown in the figure). In females, SRY absence allows gonadal development towards a female phenotype, mediated by genes such as DAX1, Wnt4 and SF1, resulting in aromatase upregulation. The exact role of stra8 (not shown) in this process remains to be clarified.
SRY expression is the major sex-determining signal, since it is prerequisite for normal testis formation. Its role mimicks that of a molecular switch, since its peak expression is limited in a specific time period that is still considered sufficient to induce male-type differentiation of the reproductive system, via downstream gene action. The latter refers to several genes, including sox family members, SF1 (sex steroid regulation) and transcriptional factors, such as GATA4. Sox family genes share a common HMG box, similar to that observed in SRY, which is considered necessary for their action at a molecular level. The fact that members of the group have been detected in various species of vertebrates, such as fish (sox9) and all mammals (e.g. sox2 and sox 14 in monotremes) further emphasizes their importance for genetic sex determination [65]. The observation of this gene family's evolutionary conservation adds further credit to the multistage model of sex chromosome evolution described above, since sox3 has been proposed as the autosomal ancestor of SRY, which places it among the chronologically first sex-related genes in the common evolutionary history of all vertebrates [ HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B66"66"HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B66" HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B66"66"66].
In the female embryo, the Y chromosome is not present and, therefore, SRY is not expressed. The genetic cascade regulating female reproductive system differentiation is not as extensively studied as in men, but DAX1 (and its regulatory system, including genes such as Wnt4 and SF1) is generally considered as a significant player in this process, which is how it came to acquire the rather simplistic description of the "antitestis gene". Sex steroid production regulation is also important for the establishment of a normal female phenotype and it is mediated via SF1 expression and aromatase enzyme complex induction [ HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B64"64"HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B64" HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B64"64"64].
Two relatively recently described genes with a potential role in sex determination and differentiation are DMRT1 and Stra8 (stimulated by retinoic acid gene 8). The first has been already discussed in previous units as a conserved sex-related gene, bearing a DM domain originally studied in nematodes [67]. In humans, XY sex reversal in cases of 9p chromosome deletions have been attributed to impaired action of DMRT1 or its homologue, DMRT2. Still, their exact involvement in the sex determination circuit has not been clarified [68]. Stra8, on the other hand, is exclusively expressed in female germ cells and its presence signals their sexual gradual differentiation, in an anterior to posterior direction. However, it has not yet been established whether the gene's product directly induces sex determination towards the female pathway, or rather acts a simple marker of this phenomenon, without active participation in the process per se [69, HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B70"70"HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B70" HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B70"70"70].
Hormonal and epigenetic regulation of sex determination
Hormonal regulation of sex determination is a vast research field in modern reproductive endocrinology. In fact, recent advances have resulted in a more generalized study of sexual dimorphism, with the discovery that differences expand to far more than the reproductive organs, including visceral tissues and the brain. The study of sex steroid concentrations and the presence of their receptors in various parts of the CNS has already been attempted in various species, including mammals and reptiles. After all, the role of androgens and estrogens in sexual differentiation in vertebrates is a classic concept that modern research data has only supported and expanded, rather than criticize [5,28]. For instance, aromatase regulation appears to be the final target in the sex determination circuit of several turtles. This has been proven by the experimental work of C.Pieau and colleagues, using aromatase inhibitors to effectively block feminization of the embryos [ HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B26"26"HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B26" HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B26"26"26].
Other scientists have even attempted to suggest sex steroids as a driving force in X-Y evolution. According to JM Howard (2002), androgens may be a major regulator of X-Y differentiation [4]. Although increased testosterone may be beneficial for fertility, constant exposure to high quantities may result in spermatogenic arrest. The DAZ gene of the Y is believed to have appeared 30–40 million years ago as a means to maintain spermatogenesis. [71] In females, increased testosterone levels caused evolutionary pressure and limited the total population, as only few of them survived and transferred their DNA in next generations, a process detected by mitochondrial DNA comparative studies. This is an example of the bottleneck phenomenon, and due to its reference to females, it has been described as the mitochondrial Eve hypothesis [4,71,72]. A number of studies in comparative genomic support this theory, such as the results of the research team lead by Hammer (1995) [73]. Increased testosterone levels acting in descendants of these women has resulted in a second wave of evolutionary pressure, surpassed by the maintenance of spermatogenesis by a duplication of the DAZ gene, about 50.000–200.000 years ago [74]. These stages of evolutionary pressure and limitation of the total population may explain the large-scale homology of the regions of the Y chromosome among all modern males (Adam phenomenon) [ HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B75"75"HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B75" HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B75"75"75]. Failure to provide sufficient evidence, such as the description of all androgen gene targets, their exact importance for male fertility and the degree of their conservation among modern men has not allowed to adequately verify the validity of this theory to this date.
Moreover, sex determination may be related to other, non-hormonal phenomena as well. For instance, immunological parameters and paracrine messages/cytokines may be involved in aromatase regulation, as some relevant initial data indicate [29,30]. In addition, sex has been proposed to be associated with selective cell proliferation. This view is supported by U. Mittwoch and is largely based on the comparative observation of male and female gonadal development in different successive stages and for a number of different model organisms [1]. If this is indeed so, it could be the result of sex steroid regulation, thus sharing some common ground with the abovementioned theories. Alternatively, there could be a completely independent pathway of mitotic induction, implicating a number of growth factors. The description of several sex-related genes conserved in various species may support this view, since sex steroids alone may not be sufficient to explain these genes' action, especially in the case of invertebrates. On the other hand, epigenetic regulation of the sexual phenotype has been proposed, which means that the products of these genes (or their downstream aftermath) could influence DNA replication and/or transcription by direct contact within the nucleus. This mechanism may be evaluated by the analytical description of all epigenetic changes occuring at a chromatin level during the various stages of normal sex differentiation and their comparison with observations made in individuals with sex distortions [ HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B5"5"HYPERLINK "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B5" HYPERLINK "HYPERLINK%20%22https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1660543/#B5"5"5].
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Conclusion
Sex determination is a crucial process in developmental biology. Its accurate regulation is a prerequisite for reproductive success and, therefore, the continued survival of a species. Since reproduction is also the function that determines the categorization of specific populations in the same or different species, the analysis of the specific molecular patterns that this process may follow is crucial for the comprehension of the detailed biochemical background mediating and maintaining the phenotypical variety observed at a macroscopical level. This is also useful for the explanation of the mechanism of infertility, since in many cases the disorder is caused by a genetic default.
Contemplating the above mechanisms as a whole, it is clear that they exhibit many differences (e.g. environmental contribution, number of genes involved, known primary sex-determinant or simultaneous action of different genes), but intriguing similarities as well. Among the latter one may briefly point at: a) the central role of aromatase regulation for female vertebrates. This similarity could also be generalized to include all sex steroids and their regulators, such as SF1. One should not fail to detect the homology of SF1 encoding gene, FtzF1 to Drosophila's fushi tarazu, which has not yet been adequately explained in terms of either evolutionary origin or gene function.
b) the action of sox family proteins in all mammals and some other vertebrates, such as fish. This category includes the primary sex determinant of all mammals, namely SRY, which might justify its proposed evolutionary history from an original autosome homologue, namely sox3.
c) the conservation of genes bearing a DM or LIM domain. This is a relatively new finding, but the fact that these products spread from dipterans and nematodes to humans must imply some degree of coherence in their regulatory mechanisms.
Whether to establish inter-relating patterns of evolution or simply for the sake of the knowledge that can be gleaned from understanding these vastly diffident mechanisms, these differences and similarities will definitely continue to hold the interest of the scientific community for years to come.
Differences in Male and Female Chromosomes
The main differences between males and females are the X and Y chromosomes. Among humans, two X chromosomes make a woman, and an X and a Y chromosome make a man. However, there are other differentiating features between these chromosomes. Some differences include size, number of genes and even abnormal chromosome pairings. In some species, animals have a different sex-determining system, as they use a Z and a W chromosome.
Working Genes
While the male Y chromosome and female X chromosome differ in size, they also vary in the number of working genes on the chromosome. The X chromosome contains more than 1,000 working genes, and the Y chromosome has less than 100 working genes. Although males have an X chromosome, it behaves very differently when there is another X chromosome present compared to its behavior when a Y chromosome is present. Of the working genes on the X chromosome, 200 to 300 are unique to sex, so only 700 to 800 of the working genes are shared and active in both males and females.
Size
The actual size of the chromosomes differs between males and females. Several chromosome pairs in males were found to be larger than those in females when sheep chromosomes were investigated. The differences in chromosome sizes may be key to explaining some differences between the sexes that are not explained by the X or Y chromosome.
Temperature
Some species, such as lizards, moths, birds and flatworms, have different sex-determination genes than X and Y. These genes are Z and W. The ZZ genotype produces males, and the ZW produces females. Sex determination in some of these species is directed by temperature. High temperatures have been known dictate the sex of the animal. For example, high incubation temperatures for alligator eggs promotes male, ZZ, genotypes. However, in many lizards and turtles, high incubation temperatures favor the female, ZW, genotype.
Sex Abnormalities
There are several syndromes that create sex differentiation abnormalities. Females with only one X chromosome have Turner syndrome, and if the girl survives birth, she will experience abnormal growth and be very small, with extra folds of skin on the neck. Triple X syndrome occurs in females with an additional X chromosome. These are known as super females and tend to be similar to females with two X chromosomes. Men who are born with two X chromosomes and a Y chromosome have Klinefelter syndrome. These men tend to be very feminine and can even have high-pitched voices. XYY syndrome occurs when men have an extra Y chromosome. These are known as super males and tend to produce much more testosterone than typical males.
Difference between Male and Female Gametophytes Flowering Plant -
Some of the major differences between male and female gametophytes flowering plant are as follows:
Male Gametophyte:
1. It is derived from a pollen grain or microspore.
Image Courtesy : upload.wikimedia.org/wikipedia/commons/e/ea/Ranunculus_glaberrimus_labelled.jpg
2. It does not remain permanently embedded inside the microsporangium.
3. It has two phases of growth— pre-pollination and post-pollination.
4. Only pre-pollination growth occurs inside the microsporangium. The remaining occurs over the female reproductive organs.
5. The male gametophyte comes out of the confines of the pollen grain by forming a pollen tube.
6. The male gametophyte is only 3-ceiled.
7. All the cells of the male gametophyte are functional. The tube cell is required to carry the two male gametes, both of which take part in fertilization.
8. The remains of male gametophyte disintegrate after fertilization.
Female Gametophyte:
1. It is derived from a megaspore.
2. The female gametophyte remains permanently embedded in the mega sporangium or nucellus.
3. All the cells are formed in a single phase of growth.
4. The whole growth occurs inside the mega sporangium.
5. The female gametophyte remains surrounded by the membrane of the megaspore.
6. The female gametophyte is 7-celled.
7. The antipodal cells do not seem to perform any function except absorption of nourishment from nucleolus in certain cases. Out of two synergies only one is required for receiving the pollen tube.
8. After fertilization two new structures are produced both of which show active growth.
Sex Chromosome Effects on Male–Female Differences in Mammals -
Main Text
Introduction
Men and women differ in their physical appearance, indicative of an anatomical and physiological sexual dimorphism that is widespread in the natural world [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib1"1 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib1"]. In primates, for example, males of Gorilla and Mandrillus taxa species are significantly larger than females; in contrast, females are generally larger than males in the Lorisidand Cheirogalid taxa [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib2"2 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib2"]. Ultimately such differences must be attributed to male–female variation at the genetic level, which in turn drives the development of the gonads, and production of gonadal sex hormones in utero. Prior to this point of physiological differentiation, though, the sex chromosomes have already induced sex-specific aspects of organ development in the absence of gonadal sex hormones 3, 4. Subsequently, however, as mammals and their gonads do not each exist in isolation, these two variables must be separated in order to further understand their relative contributions to sexual dimorphism in human health and disease. In this review, we seek first to highlight some of the key evidence of human sexual dimorphism. Subsequently, we use the evolution of the sex chromosomes as a paradigm with which to understand the possible sources of genetic sexual dimorphism in mammals. Finally, we summarise a small number of the model systems available for investigating the mechanisms of mammalian genetic sexual dimorphism.
Evidence for Sexual Dimorphism from Human Health and Disease
The difference in disease prevalence rates between males and females has been recognised for many years, with examples from cradle to grave. Boys are more likely to be born with pyloric stenosis or malformations of the genitourinary tract, whereas girls are more likely to have developmental dysplasia of the hip or scoliosis 5, 6. In early childhood, boys have a higher incidence of bacterial and viral infections, including meningitis, septicaemia, influenza A and respiratory syncytial viruses 7, 8, 9, 10. In adult life, autoimmune disease, depression and dementia are more common in females, whereas cardiovascular disease, schizophrenia, and Parkinson’s disease are more prevalent in males 11, 12, 13, 14, 15, HYPERLINK "HYPERLINK%20%22https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib16"16"HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib16" HYPERLINK "HYPERLINK%20%22https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib16"16"16. Although these associations have been shown to be reproducible, the underlying mechanisms are yet to be definitively elucidated. The most prominent two hypotheses attribute these sexual dimorphisms to either the gonadal sex hormones or the sex chromosomes.
Gonadal Sex Hormones
Both male and female human fetuses are exposed to high levels of maternal oestrogens in utero, in addition to hormones produced by the placenta [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib17"17 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib17"]. Furthermore, males start producing testosterone following testis determination at around eight weeks gestation [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib18"18 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib18"]. At birth, sex hormone levels drop significantly in both sexes as the feto-placental unit is separated, before rising again transiently at around two to three months during ‘mini-puberty’ [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib19"19 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib19"]. Subsequently, girls enter puberty slightly earlier than boys, and both sexes achieve maximum sex hormone levels during their mid-teens. In later life, from around the age of 50, testosterone levels in men drop gradually, whereas oestrogen levels in women fall precipitously during menopause [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib19"19 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib19"].
A number of diseases have been associated with these sex-specific patterns of hormone secretion. For example, boys have a high prevalence of asthma in the pre-pubertal years [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib20"20 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib20"]. Following puberty, when testosterone production is markedly increased, the burden of disease is significantly reduced. In contrast, the prevalence of asthma during childhood in girls is low, but this increases significantly during puberty, as does the risk of severe asthma [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib21"21 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib21"]. Interestingly, there is a subsequent drop in asthma severity in women aged 50–65, correlating with the timing of menopause and reduced oestrogen production [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib21"21 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib21"]. Post-menopausal women are also at increased risk of developing cardiovascular disease, which has similarly been attributed to reduced oestrogen levels [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib22"22 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib22"]. The endocrine system therefore appears to play a significant role in mediating some sexual dimorphisms in disease. However, with our ever-increasing understanding of sex chromosomes, it has become clear that some of the differences between males and females are due to genetics.
Genetic Causes of Sexual Dimorphism: Understanding from Evolution
Genetic testis determination triggered the evolution of the mammalian sex chromosomes, producing a pair of chromosomes fundamentally different from the autosomes in terms of gene content, regulation of gene expression, and inheritance. The extant X and Y chromosomes, and the females and males in which they exist, also differ from each other as a result of this process. We can therefore use the evolution of the sex chromosomes as a paradigm for understanding possible genetic mechanisms underlying male–female differences.
The mammalian sex chromosomes have evolved from a pair of autosomes during the past 166 million years (Figure HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#fig1"1A) 23, 24. Between 148 and 166 million years ago, mutations on the proto-Y chromosome resulted in the creation of the testis-determining gene SRY: carriers of SRY develop with testes, while non-carriers develop with ovaries 25, 26. SRY-based genetic testis determination is conserved in most eutherian mammals, and the sequence is present in metatherians [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib27"27 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib27"], though whether it retains a role in testis determination in this mammalian clade remains an open question.
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Figure 1. The evolution of the mammalian sex chromosomes and dosage compensationmechanisms.
(A) A testis-determining locus (proto-SRY, white) was acquired on an autosome around 148–166 million years ago. Sexually antagonistic alleles (orange) then evolved at nearby loci, selected for in males due to their tight linkage to SRY. Recombination suppression between the proto-X and -Y chromosomes likely followed on from chromosomal inversions (grey), which were subsequently only carried by males. Over evolutionary time, the lack of sexual recombination led to the appearance of repetitive DNA sequences and short-term expansion. In the longer term, large deletions took place. The outcome of this process is the small, relatively gene poor Y chromosome observed in most eutherian mammals today. Concurrent with this process, X upregulation (XUR) evolved to balance X gene dosage between the single X chromosome and the autosomes in males: this is depicted as the doubled surface area of the X chromosomes in (C) compared to (B). However, XUR was passed on to XX offspring, resulting in X:autosome dosage disparity between males and females. X chromosome inactivation (XCI) then evolved to repress one of the two X chromosomes in XX cells (D). This is depicted as the loss of colour of the X chromosome. Abbreviations: Xa, active X chromosome; Xi, inactive X chromosome.
After the acquisition of SRY, the proto-Y chromosome picked up a number of male-beneficial mutations. As a result of linkage with the testis-determining locus, these mutations provided the selective force to suppress recombination between proto-X and proto-Y [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib28"28 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib28"]. Mechanistically, the suppression and eventual elimination of recombination was possibly achieved by a series of local inversions [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib29"29 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib29"]. Non-recombining regions also accumulated deleterious mutations that could not be repaired. Over evolutionary time, lack of recombination led to the accrual of repetitive DNA sequences and a short-term increase in the size of the chromosome, though this eventually resulted in large deletions and explains the relatively diminutive size of the Y chromosome in many mammals 28, 30. Most genes from the ancestral autosome pair were therefore lost from the Y chromosome, whereas the X chromosome largely maintained its gene content 23, 27, 31. Taking humans as an example, the extant Y chromosome encodes fewer than 78 proteins; in contrast, the X chromosome contains around 800 genes [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib28"28 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib28"].
As a result of the evolution of XY testis determination, female mammals carry two copies of the relatively gene-rich X chromosome, whereas male mammals carry a single copy of the X chromosome and a gene-poor Y chromosome. The difference in X-linked gene dosage between males and females led to the appearance of compensation mechanisms aiming, firstly, to balance X expression with that of the autosomes and, secondly, to balance X expression between the homogametic (XX) and heterogametic (XY) sexes. Susumu Ohno hypothesised that X:autosome balance was achieved in males by X chromosome upregulation (XUR), the two-fold transcriptional upregulation of the X chromosome (Figure HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#fig1"1B,C). However, XUR alone would leave females expressing X genes at twice the level of autosomal genes. In order to correct this X:autosome imbalance, a further step is the inactivation of one X chromosome in the homogametic sex (two of the same sex chromosomes) — X chromosome inactivation (XCI, Figure HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#fig1"1D). In female human embryos, XCI is random, resulting in the silencing of either the maternally derived (Xm) or paternally derived X chromosome (Xp) in each cell 32, 33. The process of XCI is effected by the long non-coding RNAs XIST in eutherians 34, 35, 36 and RSX in metatherians [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib37"37 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib37"]. XIST RNA is expressed from and coats the future inactive X chromosome. Subsequently, a number of other mechanisms lock-in the inactive state, including the histonemodification H3K27 tri-methylation 38, 39, DNA methylation 40, 41, 42, and a shift in replication timing relative to the rest of the nucleus 43, HYPERLINK "HYPERLINK%20%22https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib44"44"HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib44" HYPERLINK "HYPERLINK%20%22https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib44"44"44.
The X chromosome has the potential to cause differences between males and females in a number of ways. Firstly, XCI can be skewed, resulting in preferential expression of either Xm or Xp. Secondly, a number of genes escape XCI and are thus expressed from both X chromosomes. These genes are therefore more highly expressed in XX females compared to XY males, resulting in further potential for cell autonomous sexual dimorphism. Thirdly, the parental origin of the X chromosome in males and females is not equivalent, and differential gene expression between the sexes could result from genomic imprinting.
X Chromosome Inactivation: Mosaicism and Skewing
As a result of XCI, XX females are mosaic, with each cell expressing either Xm- or Xp-genes. A well-known representation of this phenomenon is the tortoiseshell cat, which is a mosaic of black and orange X-linked coat colours [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib45"45 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib45"]. X chromosome mosaicism has long been recognised as a way in which individuals with two X chromosomes differ from those with a single X chromosome, both in terms of normal physiology and disease [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib46"46 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib46"]. Physiologically, XX females express paternal X alleles in 50% of cells, whereas XY males express maternal X alleles in 100% of cells. Any subtle difference in function between the two alleles could therefore manifest as sexual dimorphism (Figure HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#fig2"2). Significant differences in function present as X-linked disease. In males, the presence of a single X chromosome means that X-linked recessive mutations have a fully-penetrant phenotype, but in females this is usually mild or not clinically apparent. X-linked diseases present a range of phenotypes, from relatively benign colour blindness [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib47"47 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib47"], through life-limiting Duchenne and Becker muscular dystrophies [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib48"48 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib48"], to embryonic lethality, as in incontinentia pigmenti [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib49"49 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib49"].
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Figure 2. Possible mechanisms underlying male–female genetic sexual dimorphism in eutherian mammals.
The organism-wide expression of an individual gene allele is represented by block colour, with XY males in the left-hand column and XX females in the right-hand column. (A) A single allele of an X-linked gene is expressed in all cells in the male, whereas due to X chromosome inactivation (XCI), the same allele is only expressed in 50% of cells in the female. (B) XCI skewing can result in a change to the percentage of cells expressing any given X allele in females. (C) As both alleles of XCI escapee genes are expressed in females, the relative expression is increased compared to males. (D) Imprinting resulting in Xp allelic expression would be absent in males due to the absence of Xp, and would be present in 50% of cells in females. Imprinting resulting in Xm allelic expression would be present in all cells in males and 50% of cells in females. (E) Ubiquitously expressed Y-linked genes are only present in males. Abbreviations: Xm, maternally derived X chromosome; Xp, paternally derived X chromosome. Gene expression is depicted in arbitrary units, taking 1 as normal expression for a single chromosome.
Occasionally females also have a typically male disease phenotype, denoted as manifesting heterozygosity. In these individuals, XCI is no longer random, and a skew is present. Such a skew can be classified as either primary, if it arose at the onset of XCI, or secondary if it arose later [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib50"50 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib50"]. There is abundant evidence for the existence of primary skewing in mouse, resulting from the influence of a locus denoted the X controlling element (Xce). Cattanach observed that certain mouse strains have stronger Xces, such that the X chromosomes carrying these Xces are more likely to remain active in F HYPERLINK "https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/f1-hybrid"1 HYPERLINK "https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/f1-hybrid"hybrid crosses [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib51"51 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib51"]. In humans, there is little evidence for either the presence of an Xce or primary skewing. Some studies have suggested there may be a genetic component to XCI choice, i.e. inferring the existence of a human XCE 52, HYPERLINK "HYPERLINK%20%22https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib53"53"HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib53" HYPERLINK "HYPERLINK%20%22https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib53"53"53; however, more work is required to definitively address this question.
Secondary skewing has been observed at a population level in humans [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib53"53 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib53"]. In a situation where no primary skew is present, two populations of cells will exist in an XX female: each expressing either the Xm or Xp allele of any given X-linked gene(Figure HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#fig2"2). Each of these alleles may differentially affect cellular growth, such that the rate of proliferation varies between the two populations, and a competition ensues [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib54"54 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib54"]. The cell population with the growth advantage will outgrow the other — usually, but not always, the normal and mutant alleles, respectively 46, 55. Based on evidence in the literature, it is likely that this secondary skewed XCI is largely tissue-specific, and is common in normal, healthy individuals 53, 56. Skewed XCI has also been proposed as one of the causes underlying the female sex bias in autoimmune disease, and in this context it is known as the loss of mosaicism hypothesis [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib57"57 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib57"].
Escape from X Chromosome Inactivation
A number of X-linked genes are not silenced by XCI, and could therefore effect male–female differences in expression (Figure HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#fig2"2). Some of these genes are located within the pseudoautosomal region (PAR), and others have been found outside this region.
The PAR is an area of sequence homology between the X and Y chromosomes [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib58"58 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib58"]. This homology enables X–Y pairing, synapsis, and recombination during meiosis 59, 60, 61. As males and females have PAR genes in equal copy number, it was expected that expression levels would be equivalent between the sexes. However, recent work indicates the existence of a male expression bias in humans [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib62"62 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib62"]. This bias likely results from XCI spreading into the PAR on the inactive X (Xi) in females, but increased expression from the Y-linked genes in males could also contribute. The PAR is perhaps one of the most poorly studied genomic regions in sequenced eutherian mammals, as the assembly quality is not equivalent to the rest of the genome. Further work may build upon the recently reported male expression bias to reveal an unexpected role for the PAR in mammalian sexual dimorphism.
Outside of the human PAR, it has been estimated that around 12% of X genes show consistent escape, and a further 8% escape variably in different individuals and different tissues 62, 63, 64, whereas in mouse, the equivalent numbers are 3% and 4%, respectively 65, 66. The disparity in constitutive escape genes between human and mouse has been attributed to the arrangement of the genes on the chromosome. In mouse, escapees are situated in blocks of only one or two genes, whereas in human these blocks contain 10–15 genes [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib67"67 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib67"]. Even within species, though, the process has inherent variability, creating the potential for sexually dimorphic effects. A recent study in humans showed that only 41% of XCI-escape genes are consistently expressed from both alleles across multiple tissues. For the remainder, inter-tissue variability in XCI-escape was observed. There was also significant variability in Xi gene expression between the two X chromosome haplotypes within an individual. Furthermore, escapee expression from the Xi was on average only one-third of the level of expression from the active X (Xa) [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib62"62 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib62"]. Importantly, 52 of the 67 non-PAR escape genes showed female-biased expression. Taken together, these data suggest that the process of XCI-escape is tightly regulated for some genes and highly variable for others. This may reflect absolute limits on gene dosage for tightly regulated genes and flexibility of expression for those showing variability. The outcomes following XCI, i.e. silencing, variable escape and consistent escape, have the potential to give rise to male–female and female–female phenotypic variation, as evidenced by the female-biased expression of those consistent escapees. More work will be required to elucidate whether expression bias at the RNA level translates into phenotypic sexual dimorphism at the organism level.
Further evidence of the role of escape genes in sexual dimorphism has emerged from studies of human cancers. Many cancers show a sex bias, including those affecting the kidney and renal pelvis, blood, and brain 68, 69, 70, 71. Recent work has associated part of this bias with mutations in genes that escape XCI, so-called escape from X inactivation tumor suppressors (EXITS) [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib72"72 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib72"]. By analysing 21 different tumor types, loss-of-function mutations in X genes ATRX, CNKSR2, DDX3X, KDM5C, KDM6A, and MAGEC3 were found more commonly in males than females [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib72"72 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib72"]. These genes could be tumor suppressors that are required in at least one copy to prevent oncogenesis. The single X gene copy in males is therefore more vulnerable to a mutation event than the two copies present in females [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib73"73 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib73"]. A number of these X genes have Y homologues, which may or may not carry out the same function as the X copy (also see next section). As loss-of-function mutations in X genes were found more commonly in males, this would argue against functional conservation between X and Y genes. The study further investigated whether the Y homologue could also act as a tumor suppressor by looking at chromosome loss instead of mutations. It was found that tumors from female patients with an EXITS gene mutation lose the second X chromosome more commonly than males with an EXITS gene mutation lose the Y chromosome 74, 75, HYPERLINK "HYPERLINK%20%22https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib76"76"HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib76" HYPERLINK "HYPERLINK%20%22https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib76"76"76. This result suggested that EXITS genes were more effective tumor suppressors than their Y-linked homologues, as male patients received a cancer diagnosis without the loss of Y-linked genes. Moreover, it supports the hypothesis that X–Y gene pairs have functionally diverged, and thus contribute to male–female sexual dimorphism.
X–Y Gene Pairs and the Y Chromosome
Males have a Y chromosome, whereas females do not: this is the most recognisable genetic difference between the mammalian sexes and, therefore, an obvious focus for the study of male–female genetic sexual dimorphism. However, 166 million years of evolution has led the Y chromosome to become specialised for reproduction, with primary roles in testis determination and spermatogenesis [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib30"30 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib30"]. These roles are reflected in gene content and expression patterns, suggesting the Y may have less of an impact on male–female differences outside of the gonad than appearances might initially suggest. For example, SRY drives testis determination and testis specification, resulting in gonadal sex hormone production, and a number of ampliconic genes are expressed exclusively in the testis and contribute to sperm production 23, 30. Nevertheless, a small number of single copy, ubiquitously expressed Y genes have been maintained across the mammalian group and through evolutionary time (Figure HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#fig2"2) 23, 27. Based on Gene Ontology annotations, these genes are involved in the regulation of transcription and translation 23, 27, functions that are acutely sensitive to haploinsufficiency [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib77"77 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib77"]. Furthermore, each of these genes has an X-linked homologue that escapes XCI. It is therefore likely that this group was initially maintained on the ancestral mammalian Y chromosome in order to balance dosage between XX females and XY males 23, 27. This newly ascribed role for the Y chromosome as a ‘balancer’ could mean that the sexes are more similar, if both X and Y genes retain ancestral homologous function [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib78"78 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib78"]. However, emerging evidence suggests a degree of functional divergence in such X–Y gene pairs, as mentioned previously for EXITS genes [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib72"72 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib72"]. This small group of widely expressed genes is therefore of considerable interest in the investigation of male–female sexual dimorphism and the development of new therapeutics. While more work is required to more comprehensively profile the functions of the Y-linked homologues, some of the X-linked genes have already been characterised. Among these, genes KDM5C, KDM6A and DDX3X are of specific relevance. All three genes are conserved across almost all eutherian species and are implicated in sexually dimorphic diseases (Figure HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#fig3"3), including the cancers described above, and X-linked intellectual disability(XLID).
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Figure 3. Genes escaping XCI are implicated in a range of sexually dimorphic diseases.
A small number of X-linked genes show ubiquitous expression, have extant Y-linked homologues, and escape XCI. These genes have roles in the regulation of gene expression and are implicated in male–female sexual dimorphism in a wide range of diseases. Specific organs affected are indicated by the gene-related colours, i.e. KDM5C in orange.
KDM5C is a histone lysine demethylase active at both di- and tri-methylated histone H3 position K4, and has a key role in transcriptional repression 79, 80, 81. Although widely expressed, KDM5C has been clinically implicated in impaired neuronal function and XLID, suggesting a key role in brain development 82, 83, 84, 85, 86, 87, 88, 89. A high proportion of reported KDM5C mutations primarily affect males and, when females are affected, the phenotype is generally less severe. The presence of the XLID phenotype in males suggests that the Y homologue, KDM5D, cannot fully compensate for loss of KDM5C, possibly because of divergence in expression or function of these two genes. The intermediate phenotype observed in heterozygous females carrying a wildtype copy of KDM5C supports the case for dosage sensitivity in this gene, implying that a single wild-type copy is unable to fully compensate for loss of the second copy.
KDM6A (UTX) is a histone lysine demethylase that catalyses removal of H3K27me2 and me3, and possibly also functions as a transcriptional activator 90, 91, 92. Similar to mutations in KDM5C, mutations in KDM6A have been reported in the context of XLID and are linked to the developmental disorder Kabuki syndrome [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib93"93 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib93"]. Males with mutations in KDM6A show a more severe phenotype than females, supporting the hypothesis that the X–Y gene pair KDM6A and KDM6D (UTY) have diverged 94, 95, 96. In mice, males with mutations in Kdm6a survive to birth, whereas females carrying homozygous mutant alleles are lost mid-way through gestation [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib97"97 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib97"]. This result suggests that Uty is able to partly compensate for the loss of Kdm6a in embryonic development. However, mutant males are born at sub-Mendelian frequency and show life-long grow deficiency relative to wild-type littermates, and heterozygous female mutants have no detectable phenotype [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib97"97 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib97"]. Collectively, these data further support the case for divergence between Kdm6a and Kdm6d and, moreover, implicate this gene pair in male–female sexual dimorphism.
DDX3X is a member of the DEAD box protein family, with diverse roles in RNA splicing and export, translation initiation, cell cycle regulation, and apoptosis 98, 99, 100, 101, 102, 103, 104, 105, 106, 107. DDX3X mutations have been described in the context of XLID, almost exclusively affecting females 108, 109, though two recent cases have been described in males with predicted hypomorphic gene variants [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib110"110 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib110"]. The absence of live males carrying loss-of-function mutations in DDX3X can be explained by an absolute requirement for the DDX3X protein product, which is present in heterozygous females but not in mutant males [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib110"110 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib110"]. Additionally, this observation implies that DDX3X has functionally diverged from its Y homologue DDX3Y, as presence of the Y homologue does not facilitate survival. A similar picture is noted in the mouse model, whereby males carrying a mutated Ddx3x allele are lost earlier in embryonic development than heterozygous mutant females [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib111"111 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib111"]. DDX3X/Y functional divergence likely contributes to phenotypic differences between males and females.
X Imprinting
Eutherian embryos derived solely from paternal genomes (androgenotes) or from maternal genomes (gynogenotes) do not survive in utero development 112, 113, 114, 115. The requirement during embryogenesis for a paternal and maternal genome is explained by genomic imprinting, in which genes are expressed monoallelically in a parent-of-origin-specific manner [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib116"116 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib116"]. As females inherit an X chromosome from both parents, whereas males inherit only a maternal X chromosome, imprinted expression of X-linked genes could result in phenotypic differences between the sexes regardless of XCI. For example, a gene expressed only from Xp would be expressed in half the cells in a female, but in no cells in a male. In contrast, a gene expressed only from Xm would be present in 100% of male cells but only half of female cells (Figure HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#fig2"2).
There is emerging evidence for X-linked imprinted genes in mammals. Any effects of X-linked imprinting may be shown more clearly in women with Turner syndrome, who have a single X chromosome (XO), than women with two X chromosomes. Women with Turner syndrome (XO) can inherit their single X chromosome paternally or maternally. Skuse and colleagues observed that XmO women had poorer social and verbal skills than XpO women [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib117"117 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib117"], based on evidence from a set of neuropsychological tests. An imprinted X locus responsible for this effect has not yet been identified. These two populations also differ in their abdominal fat accumulation: XmO women show a typically male distribution of fat, whereas XpO women have a typically female fat distribution [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib118"118 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib118"]. As all males carry a single Xm, whereas the Xp is inherited only by females, this result in XO females is consistent with X-linked imprinting.
In the mouse, a small number of X-linked imprinted genes have been reported, with the majority expressed in the placenta 119, 120, 121 and brain [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib122"122 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib122"]. Similar to the result from humans, Davies and colleagues found that XmO female mice have poorer cognitive function than XpO female mice, as assessed by a reversal learning task. X-linked imprinted genes expressed in the placenta may underlie a growth phenotype long known to affect XpO female embryos. XpO embryos are growth retarded relative to XX littermates at preimplantation [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib123"123 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib123"], egg cylinder (E7.25), and E10.5 stages of development 124, 125, 126. In contrast, XmO embryos, along with XY embryos, are developmentally advanced relative to XX littermates at E10.5 [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib126"126 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib126"]. Interestingly, the ectoplacental cone (part of the early placenta) was found to have reduced volume in XpO conceptuses compared to XX controls [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib127"127 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib127"]. Together, these data strongly suggest a role for parental origin of the X chromosome in mouse embryonic growth. However, despite the identification of a number of X imprinting candidates in other studies 119, 120, HYPERLINK "HYPERLINK%20%22https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib121"121"HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib121" HYPERLINK "HYPERLINK%20%22https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib121"121"121, no specific locus has yet been linked to the growth deficit phenotype.
X imprinting is still at the nascent stage of development as an area of study and, as such, accurate and appropriate models are not yet in place. Person-to-person variability clouds the picture in humans when looking at the phenotypic level, while at the molecular level, any effect size is likely to be subtle and difficult to detect. The inbred mouse model provides a greater degree of control over genetics and therefore phenotype, though with the caveat of evolutionary distance between mouse and human.
Paradigms for Investigating Cell-Autonomous Sexual Dimorphism
In order to identify the contribution of these mechanisms to male–female differences, relative contributions from genes and gonadal sex hormones must first be teased apart. Traditionally, linkage and association analyses have been used to look for sexual dimorphism in gene expression. Initial genome-wide association studies(GWAS) neglected sex chromosome data in their analyses: the sexually dimorphic expressed quantitative trait loci (eQTLs) they identified associated with polygenic traits such as waist HYPERLINK "https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/waist-hip-ratio"– HYPERLINK "https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/waist-hip-ratio"hip ratio 1, 128, and bone density [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib129"129 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib129"], were autosomal [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib130"130 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib130"]. More recently, X-linked sexually dimorphic eQTLs have been identified, associated with height and fasting insulin [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib131"131 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib131"], and genomic regulatory variation [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib132"132 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib132"]. While association studies are useful for unbiased, observational work at the population level in humans, there is little room for experimental manipulation, and no control for hormonal differences. Herein lies the strength of the mouse as a model organism, which is the most genetically tractable in vivo system available for recapitulating areas of human health and disease.
Mouse Models
In the study of genetic sex differences between males and females, the main confounding factor is gonadal sex hormones. An ideal experimental system would therefore facilitate the separation of these two key variables, in order to attribute phenotypic effects to either genetics or hormones. Such a system would also allow for the manipulation of sex chromosome copy number, enabling further investigation into dosage of escapees from XCI, and functional redundancy in X–Y gene pairs.
A model denoted ‘four core genotypes’ (FCG) achieves the most important aim of the ideal system. It can be used to detect XX versus XY differences that are independent of the gonad and its hormonal influence [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib133"133 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib133"]. The FCG model utilises a Y chromosome with a mutated, inactive copy of Sry (denoted Y−) and an autosomal Srytransgene [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib25"25 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib25"]. As a result, testis determination is separated from inheritance of the Y chromosome, and so XY females and XX males can be generated in addition to XX females and XY males (Figure HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#fig4"4).
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Figure 4. The Four Core Genotypes (FCG) model.
In this Punnett square, the maternal genotype is depicted on the left-hand side and the paternal genotype is depicted at the top. A gamete from each parent carries a single sex chromosome, which come together to create the two possible offspring genotypes, XX (green column) and XY−(blue column). Furthermore, the father carries Sry as a transgene, the inheritance of which determines the gonadal sex of the offspring: female above the dashed line (XX, XY−), and male below the dashed line (XXSry, XY−Sry). The FCG model can therefore be used to separate sex chromosome effects (XX, green; XY, blue) from gonadal sex hormone effects (ovarian hormones above dotted line, and testicular hormones below dotted line) in mouse.
XX and XY mice with testes have been shown to produce similar levels of gonadal sex hormones to one another, as have XX and XY mice with ovaries [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib134"134 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib134"]. Phenotypic differences are therefore more likely to be attributable to sex chromosome complement 135, HYPERLINK "HYPERLINK%20%22https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib136"136"HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib136" HYPERLINK "HYPERLINK%20%22https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib136"136"136. In a further modification, the gonads can be removed (gonadectomy), which serves to reduce gonadal sex hormone levels to zero. Following this procedure, further phenotypic differences between XX and XY mice have been identified.
Despite its utility, the FCG model does not control for X chromosome copy number or the presence of a Y chromosome. For example, the XX versus XY− comparison varies the number of X chromosomes, but the presence of a Y chromosome is a confounding factor. When looking to uncover the specific mechanism underlying a sex difference detected by the FCG model, a supplementary model can be used, in which a male with a variant Y chromosome (also known as XY∗ model) generates XX, XO, XY, and XXY offspring [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib137"137 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib137"]. The effects of X chromosome copy number can be tested either in the presence of ovaries (XO versus XX) or testes (XY versus XXY), and the effects of Y chromosome presence can be tested with one (XO versus XY) or two X chromosomes (XX versus XXY). In the latter comparison, however, gonadal sex hormones are not controlled for.
Both the FCG and Y chromosome variant mouse models have been used to great effect in a number of experiments, facilitating the identification of sex differences in cardiovascular disease, metabolism and adiposity, and behaviour. For example, it is well known that men and women differ in their susceptibility to cardiovascular disease, though any mechanism has remained elusive. The FCG and Y chromosome variant models were combined with a mouse model of ischaemia reperfusion injury to mimic myocardial infarction, and removal of the gonads was performed one month prior in order to isolate sex chromosome specific effects [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib138"138 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib138"]. Using both in vivo and ex vivomodelling to characterise infarct size and functional recovery, respectively, hearts from XX mice were found to perform significantly worse than hearts from XY mice, independent of gonadal sex [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib138"138 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib138"]. The presence of a Y chromosome, assessed using gonadectomised variant Y chromosome model mice, seemed to have no effect on outcome. The study concluded that presence of two X chromosomes burdens the organism with greater disease risk than a single X chromosome, and therefore provides a potential focus for future human work.
The FCG model has also been used to identify sex differences in gene regulation. Euchromatic genes are silenced in a proportion of cells following translocation near to regions of heterochromatin, an effect known as position effect variegation (PEV) [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib139"139 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib139"]. Using a heterochromatin-sensitive reporter transgene, Festenstein and colleagues demonstrated X chromosome dosage-dependent differences in PEV [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib140"140 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib140"]. Mice with a single X chromosome showed a greater degree of silencing than those with two X chromosomes. Utilising the FCG and an XO mouse model, this effect was found to be independent of the hormonal milieu or presence of a Y chromosome. Mechanistically, it is possible that the inactive X chromosome acts as a heterochromatic sink, reducing availability of factors required for gene silencing at other heterochromatic loci [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib140"140 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib140"].
Additional evidence for sexually dimorphic gene regulation has recently been observed during mammalian germ cell development. While female and male somatic cells show similar dosage compensation states in the form of XUR 141, 142, 143, the status of XUR in the germline was previously unknown. Sangrithi and colleagues (2017) found that both male and female germ cells initially exhibit upregulation of the active X chromosome. XX female germ cells then exhibited a period of X dosage excess, whereas XY male germ cells showed a period of X dosage decompensation. Interestingly, these patterns were conserved in human germ cells. In order to differentiate sex chromosome dosage effects from gonadal hormone effects, FCG and XO mouse models were used. These data revealed that like XX female germ cells, XX male germ cells had X dosage excess. Furthermore, XO female germ cells had X dosage decompensation, like XY male germ cells [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib144"144 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib144"]. Mammalian germ cells therefore have an X dosage compensation state that is determined by the number of X chromosomes, and not the phenotypic sex or hormonal milieu. This contrast between X dosage compensation state and phenotypic sex could contribute to the infertility widely associated with XXY and XO genotypes and represents an interesting therapeutic angle for future work [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib144"144 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib144"].
By maintaining the same autosomes and hormonal milieu, these mouse models can be used to isolate the phenotypic effects of different sex chromosome complements. However, to better understand cell autonomous differences between men and women, a human experimental system is necessary.
Human Isogenic Cell Lines
It has been known for many years that stem cells can lose sex chromosomes when cultured in vitro for long periods, even when the originating clone was karyotypically normal [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib145"145 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib145"]. Recent work has shown that cellular reprogramming, which generates induced pluripotent stem cells (iPSCs), also results in sex chromosome loss [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib146"146 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib146"]. This process can be utilised to generate iPSC lines that are autosomally isogenic but carry different sex chromosome complements. For example, reprogramming of XXY cells generates XX, XY, XXY, and XO iPSCs. These iPSCs could potentially be screened using a multi-omics approach for differences in transcription, translation and metabolism [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib147"147 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib147"] to characterise cell autonomous sexual dimorphism in the stem cell population. Additionally, iPSCs can be differentiated to study the effects of sex chromosome complement on specific cell types (i.e. as in [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib148"148 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib148"]). Furthermore, mutations of interest could be introduced via genome editing to explore sexually dimorphic gene expression in a given disease model (i.e. as in [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib149"149 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib149"]). Such a system would be excellent for isolating the specific effects of sex chromosome complement on gene expression at a cellular level, fully removing hormonal effects. Models of increasing complexity could then be built up, utilising multiple cell types to form tissues and introducing hormones to further understand the relationship between the two variables.
Summary and Outlook
In 2001, the Institute of Medicine Committee on Understanding the Biology of Sex and Gender Differences reported on why sex matters in human health and disease [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib150"150 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib150"], and a number of recommendations were made. Although seemingly aspirational at the time, significant progress has been made towards “determining the functions and effects of X-chromosome and Y-chromosome-linked genes in somatic cells as well as germ-line cells”. However, it was not until 2014 that the NIH mandated the consideration of sex as a biological variable in grant proposals [ HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib151"151 HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0960982218312132#bib151"], to partially address the seemingly simple recommendation “(to) determine and disclose the sex of origin of biological research materials”. In this review, we sought to build on this mandate and recommendation, providing an introduction to sex chromosome effects on male–female dimorphism, and describing a number of useful experimental modelsfor hypothesis testing. Through fundamental sex chromosome biology, we have highlighted potential mechanisms underlying cell autonomous differences between the sexes, drawing particular attention to the role of X–Y pairs. The difference between human males and females, at the level of the DNA, is less than 80 genes on the Y chromosome. However, in order to unpick the effects of this genetic difference from that of the gonadal sex hormones and truly understand what it is to be XX versus XY, no single model provides enough data. We must use multiple models in combination. Human population work allows us to make associations between genotype and phenotype, and cell lines provide a faithful recapitulation of human physiology, but at the most basic cellular level. Animal models currently bridge the gap, allowing organism-level investigation and facilitating genetic manipulation. In order to more completely understand genetic sexual dimorphism in the context of human health and disease, we must invest in higher level models that bring together multiple cell types into tissues, tissues into organs and, eventually, organs into organisms.
Summary
Fundamental differences exist between males and females, encompassing anatomy, physiology, behaviour, and genetics. Such differences undoubtedly play a part in the well documented, yet poorly understood, disparity in disease susceptibility between the sexes. Although traditionally attributed to gonadal sex hormone effects, recent work has begun to shed more light on the contribution of genetics — and in particular the sex chromosomes — to these sexual dimorphisms. Here, we explore the accumulating evidence for a significant genetic component to mammalian sexual dimorphism through the paradigm of sex chromosome evolution. The differences between the extant X and Y chromosomes, at both a sequence and regulatory level, arose across 166 million years. A functional result of these differences is cell autonomous sexual dimorphism. By understanding the process that changed a pair of homologous ancestral autosomes into the extant mammalian X and Y, we believe it easier to consider the mechanisms that may contribute to hormone-independent male–female differences. We highlight key roles for genes with homologues present on both sex chromosomes, where the X-linked copy escapes X chromosome inactivation. Finally, we summarise current experimental paradigms and suggest areas for developments to further increase our understanding of cell autonomous sexual dimorphism in the context of health and disease.
Speakers -