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 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 [45].
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 [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 15).
The evolutionary model proposed by Page, Jegalian and Lahn in 1999 [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 [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 [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 [55,56]. 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) [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 [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 [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 [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 [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 [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 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 [64].
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 [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 [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 [69,70].
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 [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) [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 [5].
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