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 [22,23].
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 [24,25].
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 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) [26,27].
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 .
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 . 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 .
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 . (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 .
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 [3,31,32].
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 [3,33].
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 . 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 .
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 [3,33].
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 13) .
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 [33,36].
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 [38,39].
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 [40,41].
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 . 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) [39-41].
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 [40,42].