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 1).

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 2) thus providing us with the pattern presented above (see Figure 1).

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 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

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 4) 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.

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 1113 (see Figure 5).

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), DSX^F and DSX^M 1415 (see Figure 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.

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 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 1819. 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 8). One possible model incorporating these interactions is depicted in Figure 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 9).

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 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 1921 (see Figure 10).

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 2223.

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 2425.

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 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 12) 2627.

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 323. 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 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 30.

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 1)

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 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 33132.

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 333.

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 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 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 333.

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 13) 35.

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 333637. 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 3336.

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 3839.

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 4041.

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 14) 394041.

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 4042.

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) 4344. 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 4647. 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 5556. 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 6970.

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 528. 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 47172. 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 2930. 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.