Effects of androgens on sex differentiation of the urodele Pleurodeles waltl

In nonmammalian vertebrates, steroids have been hypothesized to induce somatic sex differentiation, since manipulations of the steroidal environment of gonads have led to various degrees of sex reversal. Whereas the critical role of estrogens in ovarian differentiation is well documented, studies on androgens have produced a perplexing variety of results depending upon species variations and nature of androgens used. In this way, testosterone induces masculinization of females in some species but provokes paradoxical feminization of males in many other species such as the urodelan Pleurodeles waltl. In reptiles this phenomenon could be interpreted by conversion of exogenous testosterone to estradiol by aromatase. Treatments of Pleurodeles larvae with nonaromatizable androgens bring support to this hypothesis and suggest a role of androgens in sex differentiation. Dihydrotestosterone (DHT) could not induce the paradoxical feminization of ZZ larvae. In addition, DHT as well as 11beta-hydroxy-androstenedione could drive a functional male differentiation of ZW larvae. Moreover, other 5alpha reduced androgens also induced sex reversal of female larvae. Yet, the 5alpha reductase inhibitor CGP 53133 and antiandrogens such as flutamide or cyproterone acetate did not exert any effect on male sex differentiation of ZZ larvae. Though the precise role of androgens is still unknown, especially for 11-oxygenated androgens, our results suggest an implication in male sex differentiation. In this way, testosterone could play a pivotal role in being metabolized either into other androgens during testis differentiation or into estradiol during ovarian differentiation.     

Differential expression of P450 aromatase during gonadal sex differentiation and sex reversal of the newt Pleurodeles waltl

A better understanding of vertebrate sexual differentiation could be provided by a study of models in which genetic sex determination (GSD) of gonads can be reversed by temperature. In the newt Pleurodeles waltl, a P450 aromatase cDNA was isolated from adult gonads, and the nucleotide or deduced amino acid sequences showed a high level of identity with various vertebrate species. In adults, aromatase expression was found in gonads and brain. In developing gonads, the expression was found to fit with the thermo-sensitive period (TSP) and was detected in both ZZ and ZW larvae, as well as in ZW submitted during the whole TSP to a masculinizing temperature. In the latter individuals, in situ hybridization and semi quantitative RT-PCR showed that, at the end of TSP, aromatase expression was at the same level than in normal ZZ larvae and was significantly lower than in normal ZW ones. Furthermore, temperature-induced down regulation did not occur when heating was performed at the end of TSP. Our results confirm the importance of aromatase regulation in female versus male differentiation and demonstrate that a down regulation of aromatase expression is involved in the process of sex reversal.     

Freemartin in the amphibian Pleurodeles waltl: parabiosis between individuals from opposite sex triggers both germ and somatic cells alterations during female gonad development

Wild type embryos of the newt Pleurodeles waltl were used to realize parabiosis, a useful model to study the effect of endogenous circulating hormones on gonad development. The genotypic sex of each parabiont (ZZ male or ZW female) was determined early from the analysis of the sex chromosome borne marker peptidase-1. In ZZ/ZZ and ZW/ZW associations, gonads develop according to genetic sex. In ZZ/ZW associations, the ZZ gonads differentiate as normal testes while ZW gonads development shows numerous alterations. At the beginning of sex differentiation, these ZW gonads possess a reduced number of germ cells and a reduced expression of steroidogenic factor 1 and P450-aromatase mRNAs when compared to gonads from ZW/ZW associations. During gonad differentiation, conversely to the control situation, these germ cells do not enter meiosis as corroborated by chromatin status and absence of the meiosis entry marker DMC1; the activity of the estradiol-producing enzyme P450-aromatase is as low as in ZZ gonads. At adulthood, no germ cells are observed on histological sections, consistently with the absence of VASA expression. At this stage, the testis-specific marker DMRT1 is expressed only in ZZ gonads, suggesting that the somatic compartment of the ZW gonad is not masculinized. So, when exposed to ZZ hormones, ZW gonads reach the undifferentiated status but the ovary differentiation does not occur. This gonad is inhibited by a process affecting both somatic and germ cells. Additionally, the ZW gonad inhibition does not occur in the case of an exogenous estradiol treatment of larvae.     

Differential germ cell proliferation in the salamander pleurodeles waltl: controls by sexual genotype and by thermal epigenetic factor before differentiation of sexual phenotype of gonads

In the acceptance that, during early gonadogenesis, variations of germ cell (GC) proliferation express interactions between germ and somatic cells, early events occurring before histological differentiation of gonadal sex has been detected and timed through GC counts on larvae of Pleurodeles waltl (urodele amphibia) issued from male ZZ or female ZW monosexual offspring. Gonads differentiate in accordance with sexual genotype in ZZ and ZW larvae at room temperature and in ZZ larvae at 32 degrees C whereas they are sex-reversed at 32 degrees C in ZW larvae, becoming phenotypic neomales. At both the rearing temperatures, in genital ridges, GCs do not proliferate during a period called P0 period ending earlier in ZZ than in ZW larvae. The time when proliferation starts depends on sexual genotypes and determines a ZZP0 period shorter than ZWP0 period. After P0 period, at room temperature, a moderate increase in GC number determining a P1 period is observed in both ZZ and ZW larvae, whereas a strong proliferation, determining a P2 period, occurs on a differential pattern in ZZ and ZW larvae; thus, before sexual differentiation of gonads, ZW females have more GCs than ZZ males. At 32 degrees C, GC proliferation is moderate during P1 period and does not accelerate during P2 period in ZW larvae differentiating neotestes; they have a lower GC number than ZZ larvae reared at 32 degrees C. Thus, during P2 period, at both room temperature and at 32 degrees C, GC number correlates with future phenotype of gonads. Results suggest that differential molecular events arise during early gonadogenesis and that testes may differentiate in different ways according to whether phenotype conforms to genotype or sex reversion occurs.     

Temperature sex-reversal in amphibians and reptiles

The sexual differentiation of gonads has been shown to be temperature-sensitive in many species of amphibians and reptiles. In two close species of salamanders, Pleurodeles poireti and P. waltl, both displaying a ZZ/ZW mechanism of genotypic sex determination (GSD), the rearing of larvae at high temperatures (30 degrees-32 degrees C) produces opposite effects: ZZ genotypic males of Pleurodeles poireti become phenotypic females whereas ZW genotypic females of P. waltl become phenotypic males. Sex-reversal of these individuals has been irrefutably demonstrated through genetic, cytogenetic, enzymatic and immunological studies. In many turtles, both sexes differentiate only within a critical range of temperature: above this range, all the individuals become phenotypic females, whereas below it, 100% become phenotypic males. The inverse occurs in some crocodiles and lizards. In many species of these three orders of reptiles, females are obtained at low and high temperatures, and males at intermediate ones. Preliminary studies in turtles (Emys orbicularis) indicate that within the critical range of temperature, sexual phenotype conforms with GSD, but that above and below this range, GSD is overriden. Temperature shifts during larval development in salamanders and during embryonic development in reptiles allowed the determination of thermosensitive stages for gonadal differentiation. Estrogens synthesized in the gonads at these stages appear to be involved in their sexual differentiation, higher levels being produced at feminizing temperatures than at masculinizing ones. The phenomenon of temperature sensitivity of gonadal differentiation occurs in species showing a very early stage in the evolution of sex chromosomes. Its adaptive value, chiefly in reptiles, remains an open question.     

Role of aromatase and androgen receptor expression in gonadal sex differentiation of ZW/ZZ-type frogs,Rana rugosa

To elucidate the mechanisms of amphibian gonadal sex differentiation, we examined the expression of aromatase and androgen receptor (AR) mRNAs for days 17-31 after fertilization. The effects of inhibitors and sex steroid hormones were also examined. In ZZ males, expression of AR decreased after day 19, while aromatase expression was low throughout the sampling period. Males treated with 17beta-estradiol (E2) showed increasing aromatase expression after day 21, and formed ovaries. AR antagonist treatment also induced high-level aromatase expression and ovarian differentiation. In males co-treated with an aromatase inhibitor and E2, the undifferentiated gonads developed into testes despite high-level aromatase expression. Males treated with androgen and E2 before and during an estrogen sensitive period, respectively, also formed testes. In ZW females, AR expression persisted at a low-level, while aromatase expression increased after day 18. Short-term treatment with an aromatase inhibitor was ineffective in preventing ovarian differentiation, whereas long-term treatment resulted in testes developing from ovarian structure. Compared with the ZZ males and ZW females, WW females did not exhibit detectable expression of AR, suggesting that the active AR gene(s) itself, or a putative gene regulating AR gene expression, is located on Z chromosomes. From the time lag of aromatase expression between ZW females and ZZ males treated with E2 and the effect of AR antagonist, it was found that in males elevated AR expression suppresses aromatase expression directly or indirectly. Consequently, endogenous androgens, accumulated by blocking estrogen biosynthesis, induced testicular differentiation. The gonadogenesis of males is dependent on sex hormone, whereas that of females has evolved to hormone-independence.     

Sex determination: a hypothesis based on steroid ratios

This paper presents a hypothesis for sex determination based on the ratio of androgen to estrogen in the gonad during sexual differentiation. In vertebrates the ratio of these steroids, and therefore, the sex of an individual is controlled by the quantity of the enzyme aromatase. For animals with a ZZ, ZW sex determining mechanism, such as birds, in which the heterogametic sex is female, an inducer for the aromatase gene is postulated to be on the W chromosome. In animals with an XX, XY system in which the heterogametic sex is male, such as mammals, the Y chromosome is postulated to code for a repressor of the aromatase gene. This hypothesis can account for naturally occurring sex reversal such as seen in some fish and amphibians, experimentally induced sex reversal by administration of steroids in birds, reptiles, fish and amphibians, and temperature-dependent sex determination as in reptiles. For invertebrates the same hypothetical model applies though the specific androgenic and estrogenic steroids differ. Both the X-to-autosome ratio method of sex determination typified by fruit flies and the haplodiploid method of bees as well as hormonal control of sexual differentiation in crustaceans are accounted for by this hypothesis.     

鸟类性别决定候选基因在性反转鸡胚中的表达

DMRT1、PKCIW和FET1是鸟类性别决定过程中重要的候选基因。以芳香化酶抑制剂处理的鸡胚为实验材料, 对这3个基因的表达变化进行了研究。结果表明, 在整个性别决定关键时期(E4.5 ~ E10.5), DMRT1在雄性的表达量显著高于雌性, 并且在ZW性反转鸡胚中表达大幅上升, 表明DMRT1的上调表达是与睾丸形成相关的。PKCIW基因在雌性特异表达并在性反转鸡胚表达上升, 这可能与其特殊作用模式有关, 即使性反转鸡胚PKCIW代偿性的表达升高, 却也未能阻止睾丸的形成。此外, FET1为雌性特异表达, 但在性反转鸡胚中表达无变化。综上, 实验结果支持了DMRT1是鸟类睾丸发育决定因子的假说。     

Change of the heterogametic sex from male to female in the frog

Two different types of sex chromosomes, XX/XY and ZZ/ZW, exist in the Japanese frog Rana rugosa. They are separated in two local forms that share a common origin in hybridization between the other two forms (West Japan and Kanto) with male heterogametic sex determination and homomorphic sex chromosomes. In this study, to find out how the different types of sex chromosomes differentiated, particularly the evolutionary reason for the heterogametic sex change from male to female, we performed artificial crossings between the West Japan and Kanto forms and mitochondrial 12S rRNA gene sequence analysis. The crossing results showed male bias using mother frogs with West Japan cytoplasm and female bias using those with Kanto cytoplasm. The mitochondrial genes of ZZ/ZW and XX/XY forms, respectively, were similar in sequence to those of the West Japan and Kanto forms. These results suggest that in the primary ZZ/ZW form, the West Japan strain was maternal and thus male bias was caused by the introgression of the Kanto strain while in the primary XX/XY form and vice versa. We therefore hypothesize that sex ratio bias according to the maternal origin of the hybrid population was a trigger for the sex chromosome differentiation and the change of heterogametic sex.     

Sex determination and sexual differentiation in the avian model

The sex of birds is determined by the inheritance of sex chromosomes (ZZ male and ZW female). Genes carried on one or both of these sex chromosomes control sexual differentiation during embryonic life, producing testes in males (ZZ) and ovaries in females (ZW). This minireview summarizes our current understanding of avian sex determination and gonadal development. Most recently, it has been shown that sex is cell autonomous in birds. Evidence from gynandromorphic chickens (male on one side, female on the other) points to the likelihood that sex is determined directly in each cell of the body, independently of, or in addition to, hormonal signalling. Hence, sex-determining genes may operate not only in the gonads, to produce testes or ovaries, but also throughout cells of the body. In the chicken, as in other birds, the gonads develop into ovaries or testes during embryonic life, a process that must be triggered by sex-determining genes. This process involves the Z-linked DMRT1 gene. If DMRT1 gene activity is experimentally reduced, the gonads of male embryos (ZZ) are feminized, with ovarian-type structure, downregulation of male markers and activation of female markers. DMRT1 is currently the best candidate gene thought to regulate gonadal sex differentiation. However, if sex is cell autonomous, DMRT1 cannot be the master regulator, as its expression is confined to the urogenital system. Female development in the avian model appears to be shared with mammals; both the FOXL2 and RSPO1/WNT4 pathways are implicated in ovarian differentiation.     

Primordial germ cell proliferation in the salamander Pleurodeles waltl: genetic control before gonadal differentiation

PGC counts were carried out on larvae of Pleurodeles waltl (urodele amphibia) issued from standard, monosexual male and monosexual female offspring while the genital ridges were settling. During this period, which is characterized by a zero mitotic index (and is therefore called the Po period), and which lasts from stage 35 to stage 41, no PGC proliferation occurs. A statistical analysis indicated that PGC counts per larva are sex genotype independent and that offspring may be divided into three groups with average PGC counts of 96.9, 51.0 and 31.1, respectively. A fourth group with an average of 18.3 PGCs has been identified using experimental larvae reared at 30 degrees C from stage 30. The PGC count of 96.9 would result from at least three mitotic cycles. Before the Po period, germ cells are not identifiable. A hypothesis concerning genetic control of PGC proliferation before Po was deduced from this analysis.     

Estrogen-Independent Ovary Formation in the Medaka Fish, Oryzias latipes

To investigate whether a female sex steroid, estrogen, acts as a natural inducer of female gonadal sex determination (or ovary formation) in the medaka fish, Oryzias latipes, the effects of an aromatase inhibitor and anti-estrogens on sexual differentiation of gonads were examined. We found that both drugs did not show any discernible effects on the genetically determined sex differentiation in both sexes. However, the aromatase inhibitor impaired the paradoxical effects of androgen (a male sex steroid), and the anti-estrogens inhibited the male-to-female sex reversal caused by estrogen. Treatments of the fertilized eggs with androgen disturbed the gonadal sex developments in both sexes, suggesting that sex steroid synthesis is detrimental to the gonadal sex developments in the medaka embryos. These results are consistent with the previous observation that sex steroids are not synthesized before the onset of gonadal sex differentiation, and suggest that ovary formation in the genetic females of the medaka fish is not dependent on estrogen.