Estrogen rescues masculinization of genetically female medaka by exposure to cortisol or high temperature

Medaka (Oryzias latipes) is a teleost fish with an XX/XY sex determination system. Recently, it was reported that XX medaka can be sex‐reversed into phenotypic males by exposure to high water temperature (HT) during gonadal sex differentiation, possibly by elevation of cortisol, the major glucocorticoid produced by the interrenal cells in teleosts. Yet, it remains unclear how the elevation of cortisol levels by HT causes female‐to‐male sex reversal. This paper reports that exposure to cortisol or HT after hatching inhibited both the proliferation of female‐type germ cells and the expression of ovarian‐type aromatase (cyp19a1), which encodes a steroidogenic enzyme responsible for the conversion of androgens to estrogens, and induced the expression of gonadal soma‐derived growth factor (gsdf) in XX gonads during gonadal sex differentiation. In contrast, exposure to either cortisol or HT in combination with 17β‐estradiol (E2) did not produce these effects. Moreover, E2 completely rescued cortisol‐ and HT‐induced masculinization of XX medaka. These results strongly suggest that cortisol and HT cause female‐to‐male sex reversal in medaka by suppression of cyp19a1 expression, with a resultant inhibition of estrogen biosynthesis. This mechanism may be common among animals with temperature‐dependent sex determination. Mol. Reprod. Dev. 79: 719–726, 2012. © 2012 Wiley Periodicals, Inc.     

High temperature causes masculinization of genetically female medaka by elevation of cortisol

In poikilothermic vertebrates, sex determination is sometimes influenced by environmental factors such as temperature. However, little is known about the molecular mechanisms underlying environmental sex determination. The medaka (Oryzias latipes) is a teleost fish with an XX/XY sex determination system. Recently, it was reported that XX medaka can be sex‐reversed into phenotypic males by high water temperature (HT; 32–34°C) treatment during the sex differentiation period. Here we report that cortisol caused female‐to‐male sex reversal and that metyrapone (an inhibitor of cortisol synthesis) inhibited HT‐induced masculinization of XX medaka. HT treatment caused elevation of whole‐body levels of cortisol, while metyrapone suppressed the elevation by HT treatment during sexual differentiation. Moreover, cortisol and 33°C treatments inhibited female‐type proliferation of germ cells as well as expression of follicle‐stimulating hormone receptor (fshr) mRNA in XX medaka during sexual differentiation. These results strongly suggest that HT induces masculinization of XX medaka by elevation of cortisol level, which, in turn, causes suppression of germ cell proliferation and of fshr mRNA expression. Mol. Reprod. Dev. 77: 679–686, 2010. © 2010 Wiley‐Liss, Inc.     

高温和皮质醇对黄颡鱼性别分化的影响

研究以性染色体类型已确定且已有性别特异分子标记的黄颡鱼为研究对象, 开展高温与皮质醇诱导黄颡鱼(Tachysurus fulvidraco Richardson)XX个体雄性化组织学进程研究, 以期为环境应激诱导鱼类雄性化提供研究基础。通过对每尾鱼采用性别特异性标记鉴定遗传性别(XX或XY)及组织学鉴定生理型性别, 仅经过24d的处理(12—35日龄), 高温或皮质醇便能诱导XX遗传型个体雄性化。在此过程中, 部分XX遗传型个体卵母细胞受到抑制, 之后发育成带有卵巢腔的精巢结构。62日龄时, XX伪雄鱼性腺较正常XY雄鱼大, XX伪雄鱼体重与正常XY雄鱼相近, 而显著大于未发生性逆转的XX雌鱼。122日龄时, XX伪雄鱼从62日龄带有卵巢腔的精巢结构发育成具有典型的精小叶结构样精巢, 且都具有生理性雄鱼特有的生殖突, 推测这些雄鱼可能具有与正常雄鱼类似的生殖能力。部分XX个体对高温处理不敏感, 没有发生性逆转, 温度处理反而加快了卵巢发育的进程, 这些个体对高温的耐受性和另外一些发生性逆转的个体对温度的敏感性值得进一步研究。     

Tamoxifen induces masculinization of genetic females and regulates P450 aromatase and Müllerian inhibiting substance mRNA expression in Japanese flounder (Paralichthys olivaceus)

Japanese flounder, Paralichthys olivaceus, provides an excellent model to elucidate the roles of sex steroid hormones in gonadal sex differentiation because the sex is easily altered by sex steroid treatments or water temperature control during the sex differentiation. We have previously shown that high water temperature, an aromatase inhibitor (fadrozole), or 17alpha-methyltestosterone treatment causes the sex-reversal from genetic females to phenotypic males and suppression of mRNA expression of ovary-type P450 aromatase (P450arom), which is a steroidogenic enzyme responsible for the conversion of androgens to estrogens, in Japanese flounder. In the present study, we demonstrate that treatment of the genetic females with anti-estrogen (tamoxifen) leads to their masculinization, suppresses P450arom mRNA expression, and induces mRNA expression of Müllerian inhibiting substance (MIS), a member of the transforming growth factor-beta (TGF-beta) superfamily, while it has no effect on mRNAs expression of estrogen receptor-alpha (ERalpha) and ERbeta. In contrast, 17beta-estradiol counteracted masculinization of the genetic females by tamoxifen or high water temperature treatment, up-regulated P450arom mRNA expression, and down-regulated MIS mRNA expression. These results strongly suggest that estrogen signaling through ERs dramatically influences the gonadal sex differentiation by regulating P450arom and MIS mRNA expression.     

Differential expression of foxl2 and cyp19a1a mRNA during gonad developmental stages in great sturgeon Huso huso

This study aimed to determine the sex specificity and expression pattern of foxl2 and cyp19a1a genes in great sturgeon Huso huso gonads during gonadal sex differentiation and development. The results revealed that foxl2 and cyp19a1a mainly expressed in female gonads and during gonad development the foxl2 and cyp19a1a mRNA expression is required for ovarian development.     

Sexually dimorphic expression of a teleost homologue of Müllerian inhibiting substance during gonadal sex differentiation in Japanese flounder, Paralichthys olivaceus

Müllerian inhibiting substance (MIS), also known as anti-Müllerian hormone, is a glycoprotein belonging to transforming growth factor beta superfamily. In mammals, MIS is responsible for regression of Müllerian ducts, anlagen of the female reproductive ducts, in the male fetus. However, the role of MIS in gonadal sex differentiation of teleost fishes, which do not have the Müllerian ducts, has yet to be clarified. To address the role of MIS on gonadal sex differentiation in fishes, we isolated a MIS cDNA from the Japanese flounder testis and examined the expression pattern of MIS mRNA in gonads of both sexes during sex differentiation period. In this study, we present the first demonstration of sexually dimorphic expression of MIS mRNA during sex differentiation in teleost fishes, similarly to amniote vertebrates which possess the Müllerian ducts.     

Induction of XY sex reversal by estrogen involves altered gene expression in a teleost, tilapia

To clarify the importance of endogenous estrogens during sex differentiation in a teleost fish, the Nile tilapia, we examined the target events for endogenous estrogens and their role during gonadal sex differentiation. The expression of CYP19a (P450arom) precedes any morphological gonadal sex differentiation. Further to these findings, the treatment of XX fry with non-steroidal aromatase inhibitor (AI), Fadrozole, from seven to 14 days after hatching caused complete sex reversal to functional males. The XX sex reversal induced by AI was rescued completely with simultaneous estrogen treatment. We also found that XY fry treated with estrogen, before the appearance of morphological sex differences, caused complete sex reversal from males to females. Taken together, these results suggest that endogenous estrogens are required for ovarian differentiation. To identify the down-stream gene products of estrogen during ovarian differentiation, we performed subtractive hybridization using mRNA derived from normal and estrogen treated XY gonads. Two out of ten gene products were expressed in germ cells, whereas the others were expressed in somatic cells.     

Cortisol-Induced Masculinization: Does Thermal Stress Affect Gonadal Fate in Pejerrey,a Teleost Fish with Temperature-Dependent Sex Determination?

Background

Gonadal fate in many reptiles, fish, and amphibians is modulated by the temperature experienced during a critical period early in life (temperature-dependent sex determination; TSD). Several molecular processes involved in TSD have been described but how the animals “sense” environmental temperature remains unknown. We examined whether the stress-related hormone cortisol mediates between temperature and sex differentiation of pejerrey, a gonochoristic teleost fish with marked TSD, and the possibility that it involves glucocorticoid receptor- and/or steroid biosynthesis-modulation.

Methodology/Principal Findings

Larvae maintained during the period of gonadal sex differentiation at a masculinizing temperature (29°C; 100% males) consistently had higher cortisol, 11-ketotestoterone (11-KT), and testosterone (T) titres than those at a feminizing temperature (17°C; 100% females). Cortisol-treated animals had elevated 11-KT and T, and showed a typical molecular signature of masculinization including amh upregulation, cyp19a1a downregulation, and higher incidence of gonadal apoptosis during sex differentiation. Administration of cortisol and a non-metabolizable glucocorticoid receptor (GR) agonist (Dexamethasone) to larvae at a “sexually neutral” temperature (24°C) caused significant increases in the proportion of males.

Conclusions/Significance

Our results suggest a role of cortisol in the masculinization of pejerrey and provide a possible link between stress and testicular differentiation in this gonochoristic TSD species. Cortisol role or roles during TSD of pejerrey seem(s) to involve both androgen biosynthesis- and GR-mediated processes. These findings and recent reports of cortisol effects on sex determination of sequential hermaphroditic fishes, TSD reptiles, and birds provide support to the notion that stress responses might be involved in various forms of environmental sex determination.     

Real-time PCR analysis of ovary- and brain-type aromatase gene expression during Atlantic halibut (Hippoglossus hippoglossus) development

Two forms of cytochrome P450 aromatase, acting in both the brain and the ovary, have been implicated in controlling ovarian development in fish. To better understand the expression of these two enzymes during sexual differentiation in Atlantic halibut (Hippoglossus hippoglossus), real-time PCR was used to quantify the mRNA levels of ovary- (cyp19a) and brain-type cytochrome P450 aromatase (cyp19b) genes in the gonad and brain during gonadal development. Both enzymes showed high levels of expression in both tissues in developmental stages prior to histologically detectable ovarian differentiation (38 mm fork length), with increased expression occurring slightly earlier in the brain than the gonad. Cyp19a showed a second peak of expression in later stages (> 48 mm) in the gonad, but not the brain. Cyp19b expression was generally higher in the brain than the gonad. These results suggest that sexual differentiation may begin in the brain prior to gonadal differentiation, supporting the idea that steroid hormone expression in the brain is a key determinant of phenotypic sex in fish. In an examination of sexually immature adults, cyp19a was highly expressed in female gonad while cyp19b was very highly expressed in the pituitary of both sexes. The ratio of cyp19a to cyp19b expression was much higher in ovaries than in testes in the adult fish, so this ratio was analyzed in the developing gonads of juvenile halibut in an attempt to infer their sex. This was only partially successful, with about half the fish in later developmental stages showing apparently sex-specific differences in aromatase expression.     

Sox9b/sox9a2-EGFP transgenic medaka reveals the morphological reorganization of the gonads and a common precursor of both the female and male supporting cells

We have established an enhanced green fluorescent protein (EGFP) transgenic medaka line that mimics the expression of sox9b/sox9a2 to analyze the morphological reorganization of the gonads and characterize the sox9b-expressing cells during gonadal formation in this fish. After the germ cells have migrated into the gonadal areas, a cluster of EGFP-expressing cells in the single gonadal primordium was found to be separated by the somatic cells along the rostrocaudal axis and form the bilateral lobes. We observed in these transgenic fish that EGFP expression persists only in the somatic cells directly surrounding the germ cells. As sex differentiation proceeds, dmrt1 and foxl2 begin to be expressed in the EGFP-expressing cells in the XY and the XX gonads, respectively. This indicates that the sox9b-expressing cells reorganize into two lobes of the gonad and then differentiate into Sertoli or granulosa cells, as common precursors of the supporting cells. Hence, our sox9b-EGFP medaka system will be useful in future studies of gonadal development.     

Epigenetic control of cyp19a1a expression is critical for high temperature induced Nile tilapia masculinization

In fish species with temperature-dependent sex determination (TSD) or genotypic sex determination plus temperature effects (GSD + TE), temperature can either affect sex differentiation or determine the sex. However, it is unknown if epigenetic control of cyp19a1a expression is critical for high temperature induced masculinization in the freshwater fish Nile tilapia. We analyzed the cyp19a1a DNA methylation levels in three age groups and found that they were lower in females than in males. At 8 months of age, males had DNA methylation levels of the cyp19a1a promoter that were almost twice as high as those of females. Exposure to high temperatures increased the cyp19a1a promoter DNA methylation levels from 30.87 ± 4.56% to 48.34 ± 0.92% (P = 0.035) in females and from 50.33 ± 7.38% to 51.66 ± 4.75% in males (P = 0.867). The increases in the cyp19a1a promoter DNA methylation levels were associated with the mRNA expression levels and might play a role in promoting gonadal differentiation in high temperature induced group females toward the male pathway. Western blot analysis revealed that the cyp19a1a protein expression levels in females significantly declined after high temperature treatment; only a slight decline was recorded in male fish. These results reveal that epigenetic control of cyp19a1a mRNA and protein expression is related to the environmental temperature and sex ratios in fish with TSD or GSD + TE.     

Rainbow trout gonadal masculinization induced by inhibition of estrogen synthesis is more physiological than masculinization induced by androgen supplementation

The present study was designed to obtain new insights into fish gonadal sex differentiation by comparing the effects of two different masculinizing treatments on some candidate gene expression profiles. Masculinization was induced in rainbow trout, Oncorhynchus mykiss, genetic all-female populations using either an active fish androgen (11betaAnd, 11beta-hydroxyandrostenedione) or an aromatase inhibitor (ATD, 1,4,6-androstatriene-3,17-dione). The expression profiles of 100 candidate genes were obtained by real-time RT-PCR, and 46 profiles displayed a significant differential expression between control populations (males and females) and ATD/11betaAnd-treated populations. These expression profiles were grouped in four temporally correlated expression clusters. Among the common responses shared by the two masculinizing treatments, the inhibition of some early female differentiating genes (cyp19a1, foxl2a, fst, and fshb) appears to be crucial for effective masculinization, suggesting that these genes act together via a short regulation loop to maintain high sex-specific ovarian expression of cyp19a1. This simultaneous down-regulation of female-specific genes could be triggered by some testicular genes, such as dmrt1, nr0b1 (also known as dax1), and pdgfra, which are quickly up-regulated by the two masculinizing treatments. In contrast to 11betaAnd, ATD quickly restored the expression levels of steroidogenesis related genes (cyp11b2.1, cyp11b2.2, hsd3b1, cyp17a, star, and nr5a1) and some Sertoli cell markers (sox9a2 and amh) to the expression levels observed during control testicular differentiation. This demonstrates that these genes are probably not needed for active masculinization and that the inhibition of endogenous estrogen synthesis produces a much more complete and specific testicular pattern of gene expression than that observed following androgen-induced masculinization.     

Sexual Fate Reprogramming in the Steroid-Induced Bi-Directional Sex Change in the Protogynous Orange-Spotted Grouper,Epinephelus coioides

Androgen administration has been widely used for masculinization in fish. The mechanism of the sex change in sexual fate regulation is not clear. Oral administration or pellet implantation was applied. We orally applied an aromatase inhibitor (AI, to decrease estrogen levels) and 17α-methyltestosterone (MT, to increase androgen levels) to induce masculinization to clarify the mechanism of the sex change in the protogynous orange-spotted grouper. After 3 mo of AI/MT administration, male characteristics were observed in the female-to-male sex change fish. These male characteristics included increased plasma 11-ketotestosterone (11-KT), decreased estradiol (E2) levels, increased male-related gene (dmrt1, sox9, and cyp11b2) expression, and decreased female-related gene (figla, foxl2, and cyp19a1a) expression. However, the reduced male characteristics and male-to-female sex change occurred after AI/MT-termination in the AI- and MT-induced maleness. Furthermore, the MT-induced oocyte-depleted follicle cells (from MT-implantation) had increased proliferating activity, and the sexual fate in a portion of female gonadal soma cells was altered to male function during the female-to-male sex change. In contrast, the gonadal soma cells were not proliferative during the early process of the male-to-female sex change. Additionally, the male gonadal soma cells did not alter to female function during the male-to-female sex change in the AI/MT-terminated fish. After MT termination in the male-to-female sex-changed fish, the differentiated male germ cells showed increased proliferating activities together with dormancy and did not show characteristics of both sexes in the early germ cells. In conclusion, these findings indicate for the first time in a single species that the mechanism involved in the replacement of soma cells is different between the female-to-male and male-to-female sex change processes in grouper. These results also demonstrate that sexual fate determination (secondary sex determination) is regulated by endogenous sex steroid levels.     

High Temperature Increases the Masculinization Rate of the All-Female (XX) Rainbow Trout “Mal” Population

Salmonids are generally considered to have a robust genetic sex determination system with a simple male heterogamety (XX/XY). However, spontaneous masculinization of XX females has been found in a rainbow trout population of gynogenetic doubled haploid individuals. The analysis of this masculinization phenotype transmission supported the hypothesis of the involvement of a recessive mutation (termed mal). As temperature effect on sex differentiation has been reported in some salmonid species, in this study we investigated in detail the potential implication of temperature on masculinization in this XX mal-carrying population. Seven families issued from XX mal-carrying parents were exposed from the time of hatching to different rearing water temperatures ((8, 12 and 18°C), and the resulting sex-ratios were confirmed by histological analysis of both gonads. Our results demonstrate that masculinization rates are strongly increased (up to nearly two fold) at the highest temperature treatment (18°C). Interestingly, we also found clear differences between temperatures on the masculinization of the left versus the right gonads with the right gonad consistently more often masculinized than the left one at lower temperatures (8 and 12°C). However, the masculinization rate is also strongly dependent on the genetic background of the XX mal-carrying families. Thus, masculinization in XX mal-carrying rainbow trout is potentially triggered by an interaction between the temperature treatment and a complex genetic background potentially involving some part of the genetic sex differentiation regulatory cascade along with some minor sex-influencing loci. These results indicate that despite its rather strict genetic sex determinism system, rainbow trout sex differentiation can be modulated by temperature, as described in many other fish species.