A new experimental model for the investigation of sequential hermaphroditism.

The stunning sexual transformation commonly triggered by age, size or social context in some fishes is one of the best examples of phenotypic plasticity thus far described. To date our understanding of this process is dominated by studies on a handful of subtropical and tropical teleosts, often in wild settings. Here we have established the protogynous New Zealand spotty wrasse, Notolabrus celidotus, as a temperate model for the experimental investigation of sex change. Captive fish were induced to change sex using aromatase inhibition or manipulation of social groups. Complete female-to-male transition occurred over 60 days in both cases and time-series sampling was used to quantify changes in hormone production, gene expression and gonadal cellular anatomy. Early-stage decreases in plasma 17ß-estradiol (E2) concentrations or gonadal aromatase (cyp19a1a) expression were not detected in spotty wrasse, despite these being commonly associated with the onset of sex change in subtropical and tropical protogynous (female-to-male) hermaphrodites. In contrast, expression of the masculinising factor amh (anti-Müllerian hormone) increased during early sex change, implying a potential role as a proximate trigger for masculinisation. Collectively, these data provide a foundation for the spotty wrasse as a temperate teleost model to study sex change and cell fate in vertebrates.

A threshold dosage of estrogen for male-to-female sex reversal in the Glandirana rugosa frog.

Steroid hormones play very important roles in gonadal differentiation in many vertebrate species. Previously, we have determined a threshold dosage of testosterone (T) to induce female-to-male sex reversal in Glandirana rugosa frogs. Genetic females formed a mixture of testis and ovary, the so-called ovotestis, when tadpoles of G. rugosa were reared in water containing the dosage of T, which enabled us to detect primary changes in the histology of the masculinizing gonads. In this study, we determined a threshold dosage of estradiol-17ß (E2) to cause male-to-female sex reversal in this frog. We observed first signs of histological changes in the ovotestes, when tadpoles were reared in water containing the dosage of E2. Ovotestes were significantly larger than wild-type testes in size. By E2 treatment, male germ cells degenerated in the feminizing testis leading to their final disappearance. In parallel, oocytes appeared in the medulla of the ovotestis and later in the cortex as well. Quantitative polymerase chain reaction analysis revealed that the expression of sex-related genes involved in testis formation was significantly decreased in the ovotestis. In addition, immuno-positive signals of CYP17 that is involved in testis differentiation in this frog disappeared in the medulla first and then in the cortex. These results suggested that oocytes expanded in the feminizing gonad (ovary) contemporaneously with male germ cell disappearance. Primary changes in the histology of the gonads during male-to-female sex reversal occurred in the medulla and later in the cortex. This direction was opposite to that observed during female-to-male sex reversal in the G. rugosa frog.

Transgenic Medaka Identify Embryonic Periods Sensitive to Disruption of Sex Determination.

Gonadal development in medaka (Oryzias latipes) is dependent on the synergy between estrogens and androgens. Disruption of steroid hormone levels can lead to ovo-testis. To determine the sensitive windows for hormonally induced sex reversal in medaka, we developed a novel 42sp50-GFP_ChgH-GFP transgenic medaka line, allowing the identification of female gonadal tissue by fluorescence present in developing oocytes. Germinal transgenesis resulted in a stable line exhibiting a strong green fluorescent protein signal constitutively in the ovaries and in the liver in response to estrogens. The sensitivity of this line to disruption of sex determination following 16-d chronic exposures was in the nanograms per liter range. To identify the developmental period sensitive to exogenous agents, fry were exposed to 24-h pulses of high concentrations of 17ß-estradiol (E2) or 5α-dihydrotestosterone (DHT) at various time points between days postfertilization (dpf) 0 and 12. Evaluation of phenotype followed by genotyping at 16 dpf revealed sensitivity to E2 between 1 and 8 dpf as well as 2 periods of susceptibility to DHT between 0 and 1 dpf and 4 and 8 dpf. No phenotypic sex reversal was detected after exposure to DHT or E2 on 11 or 12 dpf. The observed effects persisted to at least 24 dpf. The identified sensitive embryonic time periods for disruption of sex determination will aid future research on sex determination and the development of screening assays using early embryonic life stages. Environ Toxicol Chem 2020;39:842-851. © 2020 SETAC.

An estradiol-17ß/miRNA-26a/cyp19a1a regulatory feedback loop in the protogynous hermaphroditic fish, Epinephelus coioides.

Cyp19a1a is a key gene responsible for the production of estradiol-17ß (E2), the main functional estrogen and a major downstream regulator of reproduction in teleost fish. It is widely known that CYP19 gene expression, aromatase activity, and E2 production can influence gonadal differentiation and sex reversal in teleost fish, but the feedback mechanisms whereby E2 regulates cyp19a1a remain poorly understood, especially regarding the potential roles of endogenous small RNA molecules (miRNAs). Here, we identified miR-26a-5p as a regulatory factor of its predicted target gene (cyp19a1a). In vitro and in vivo studies showed that miR-26a-5p can decrease cyp19a1a expression. Furthermore, high doses of E2 act as a repressor of miR-26a-5p. This study proposes a regulatory feedback loop whereby E2 regulates cyp19a1a through miR-26a-5p, and suggests that this positive feedback is an important aspect of the control of E2 production.

Estrogen-induced ovarian development is time-limited during the temperature-dependent sex determination of the American alligator.

Many reptiles, including the American alligator, exhibit temperature-dependent sex determination (TSD), whose thermo-sensitive period for the female alligator begins at stages-15 and ends at stage-24. Estrogen signaling plays a central role in TSD, which can be overridden by an estrogen-exposure during the thermo-sensitive period. As some environmental contaminants are estrogenic, there is growing concern about their effects on the sex ratio and reproductive health of TSD-species. It is crucial to identify the timing of gonadal commitment to either ovary or testis for a better understanding of TSD and estrogen-signals. In the current study, eggs were exposed to 5 µg/g egg of 17ß-estradiol (E2) or vehicle ethanol alone at three developmental stages-22, 24, and 26 at a male-promoting temperature, which produced 81% testis in all controls. E2-exposure at stages-22 and 24 induced more ovaries than the control group, whereas the exposure at stage-26 did not induce the same outcome. These results indicated that there is a critical commitment in the testicular development between the developmental stage 24 (100% ovary in E2 Exposure) and 26 (39% ovary with E2). Based on these results, we estimated a pivotal stage as stage-25.28. Thus, a gonadal commitment to testis could be later than a known temperature-sensitive period for promoting male in TSD.

Induction of Gonadal Development in Protogynous Grouper with Orally Delivered FSH DNA.

The availability of sexually mature fish often dictates the success of its captive breeding. In this study, we induced reproductive development in juvenile protogynous tiger grouper through oral administration of a plasmid (p) containing an engineered follicle-stimulating hormone (FSH). An expression construct (pcDNA3.1) was designed to express a single-chain FSH consisting of giant grouper FSH ß-subunit and glycoprotein subunit-α (CGα), linked by the carboxy-terminal peptide (CTP) sequence from the human chorionic gonadotropin (hCG). Single oral delivery of pFSH encapsulated in liposome and chitosan to tiger grouper yielded a significant increase in plasma FSH protein level after 4 days. Weekly pFSH feeding of juvenile tiger groupers for 8 weeks stimulated ovarian development as indicated by a significant increase in oocyte diameter and progression of oocytes to cortical alveolar stage. As the pFSH treatment progressed from 20 to 38 weeks, female to male sex change was initiated, characterized by oocyte regression, proliferation of spermatogonial cells, and occurrence of spermatogenic cysts. It was also associated with significantly lower mRNA expression of steroidogenic genes (cyp11b, cyp19a1a, and foxl2) and basal plasma levels of sex steroid hormones 17ß-estradiol (E2), testosterone (T), and 11-ketotestosterone (11KT). Results suggest that pFSH stimulates ovarian development up to cortical alveolar stage and then initiates sex change in tiger grouper. These findings significantly contribute to our knowledge on the role of FSH in the development of protogynous hermaphroditic fish. This study is the first to demonstrate induction of reproductive development in fish through oral delivery of plasmid gonadotropin.

Inter- and Intraspecific Variation in Sex Hormone-Induced Sex-Reversal in Medaka, Oryzias latipes and Oryzias sakaizumii.

We compared sex-reversal ratios induced by 17α-methyltestosterone (MT) and 17ß-estradiol (E2) exposure in two inbred medaka strains: Hd-rR derived from Oryzias latipes and HNI-II from O. sakaizumii. All MT exposures (0.2-25 ng mL-1) induced complete XX sex-reversal in HNI-II. Although MT exposure at 0.2 ng mL-1 induced XX sex-reversal at > 95% in Hd-rR, other concentrations tested caused XX sex-reversal at lower frequencies (<50%). MT exposure at 1, 5, and 25 ng mL-1 induced XY sex-reversal in Hd-rR, but not in HNI-II. In Hd-rR, E2 exposure induced XY sex-reversal at > 10 ng mL-1, and in all fish feminization occurred 500 ng mL-1. In HNI-II, E2 induced XY sex-reversal at 50 and 250 ng mL-1, but only at rates below 20%. To clarify whether the strain differences in sex hormone-induced sex-reversal are characteristic of each species, we examined the effects of MT and E2 exposure on sex differentiation in five and two additional strains or wild stocks/populations of O. latipes and O. sakaizumii, respectively. MT exposure induced low XX and high XY sex-reversal rates in O. latipes, except in the Shizuoka population, but the trend was reversed in O. sakaizumii. Furthermore, E2-induced XY sex-reversal rates varied intraspecifically in O. latipes. Our results demonstrated that sensitivity to MT and E2 varied within O. latipes species. To evaluate the ecological impacts of environmental chemicals using medaka, it is important to define not only the species, but the strains, stocks, and populations to obtain accurate results.

The co-administration of estradiol/17α-methyltestosterone leads to male fate in the protogynous orange-spotted grouper, Epinephelus coioides.

Estrogen plays a pivotal role in the sex differentiation of teleosts, whereas the precise function of androgens is more controversial. In this study, orange-spotted grouper (Epinephelus coioides) fry were treated with letrozole (an aromatase inhibitor, AI), 17α-methyltestosterone (MT), or MT and 17ß-estradiol (E2) simultaneously, during the period of gonadal formation and sex differentiation. MT feeding at 50 days after hatching resulted in gonadal dysgenesis, which could be rescued by E2 supplementation. Different doses of AI treatment led to different phenotypes: undifferentiated gonads were maintained in the AI group fed a low dose (5 mg/kg diet), whereas female-to-male sex reversal was observed in the AI group fed a high dose (100 mg/kg diet). MT and MT + E2 treatment could induce female-to-male sex reversal during sex differentiation (90 days after hatching). The expression of female pathway genes was suppressed, while the expression of genes in the male pathway was up-regulated in the MT + E2 group. Consistent with the expression of sex-related genes, the serum 11- ketotestosterone level was also upregulated in MT and MT + E2 group. Finally, we examined the expression of male-specific mark (DMRT1) and proliferating cell nuclear antigen in MT and MT + E2 induced sex reversal, and the result indicated that male germ cells and somatic cells may origin from the gonium and proliferative somatic cells surrounding the efferent duct, respectively. Overall, our data suggested that estrogen acts as a natural inducer of female differentiation, and that the co-administration of estrogen and androgen during sex differentiation leads to a male sex fate in the protogynous orange-spotted grouper.

Potential Interference of Oil Vehicles on Genital Tubercle Development during the Fetal Period in ICR Mice.

Corn oil, sesame oil, and 10% ethanol in corn oil are commonly used as dosing vehicles in toxicology studies. Since these vegetable oils contain bioactive compounds, it is important for toxicology studies to characterize the toxicities of the dosing vehicles themselves. It has been recently proposed that the width of the genital tubercle (GT), the dorsal-ventral length (D-V length) of the GT, and urethral tube closure in mouse fetuses can be used as novel markers for monitoring sexual development in mice. However, how these parameters are influenced by the dosing vehicles themselves remains unclear. Therefore, we evaluated the effects of corn oil, sesame oil, and 10% ethanol in corn oil on GT width, D-V length, and GT morphology in ICR mice. Our results showed that all three vehicles influenced GT width and D-V length, but not GT morphology, suggesting that the effects of dosing vehicles themselves might need to be considered when GT width or D-V length is used as a parameter to evaluate the effects of chemicals on GT development.

Temperature- vs. estrogen-induced sex determination in Caiman latirostris embryos: Both females, but with different expression patterns of key molecules involved in ovarian development.

Caiman latirostris is a species with temperature dependent sex determination (TSD), which implies that the incubation temperature of the eggs is the main factor that determines the sex during a thermo-sensitive period (TSP). However, estrogens play a critical role in this process. The administration of 17ß-estradiol (E2) previous to TSP overrides the effects of male incubation temperature, producing phenotypic females. This effect has been defined as sex reversal or estrogen-induced sex determination (E2SD). The aim of the present study is to describe similarities and differences in the effects of TSD and E2SD treatment conditions on ovary development. Our results show that the two treatment conditions studied are able to produce different ovaries. Treatment with E2 modified the expression pattern of estrogen receptor alpha and progesterone receptor, and expression of the enzyme aromatase. Moreover, in E2SD females, the proliferation/apoptosis dynamic was also altered and high expression of TAp63 was observed suggesting the presence of greater DNA damage in germ cells. To the best of our knowledge, this is the first report that describes the morphology of the female gonad of C. latirostris in three stages of embryonic development and shows the expression of TAp63 during the gonad development of a reptile. It is important to emphasize that the changes demonstrated in E2SD female gonads of embryos show that environmental compounds with proven estrogenic activity alter the follicular dynamics of C. latirostris in neonatal as much as in juvenile animals, endangering their reproductive health and possibly bringing consequences to ecology and evolution.

Feminizing effects of exposure to Corexit-enhanced water-accommodated fraction of crude oil in vitro on sex determination in Alligator mississippiensis.

Deepwater Horizon spilled over 200 million gallons of oil into the waters of the Gulf of Mexico in 2010. In an effort to contain the spill, chemical dispersants were applied to minimize the amount of oil reaching coastal shorelines. However, the biological impacts of chemically-dispersed oil are not well characterized, and there is a particular lack of knowledge concerning sublethal long-term effects of exposure. This study examined potential estrogenic effects of CWAF, Corexit 9500-enhanced water-accommodated fraction of oil, by examining its effect on estrogen receptors and sex determination in the American alligator, Alligator mississippiensis. The alligator exhibits temperature-dependent sex determination which is modulated by estrogen signals, and exposure to 17ß-estradiol (E2) and estrogenic compounds in ovo during the thermosensitive period of embryonic development can induce ovarian development at a male-producing temperature (MPT). CWAF induced transactivation up to 50% of the maximum induction by E2 via alligator estrogen receptors in vitro. To determine potential endocrine-disrupting effects of exposure directly on the gonad, gonad-adrenal-mesonephric (GAM) organ complexes were isolated from embryos one day prior to the thermosensitive period and exposed to E2, CWAF, or medium alone in vitro for 8-16 days at MPT. Both CWAF and E2 exposure induced a significant increase in female ratios. CWAF exposure suppressed GAM mRNA abundances of anti-Müllerian hormone (AMH), sex determining region Y-box 9, and aromatase, whereas E2 exposure suppressed AMH and increased Forkhead box protein L2 mRNA abundances in GAM. These results indicate that the observed endocrine-disrupting effects of CWAF are not solely estrogenically mediated, and further investigations are required.

Endocrine disruption of phenanthrene in the protogynous dusky grouper Epinephelus marginatus (Serranidae: Perciformes).

The dusky grouper Epinephelus marginatus is a protogynous hermaphrodite fish, that maintains high levels of plasma steroids as juveniles, as substrates for sex inversion. These fish are exposed to marine pollution from oil spills during cargo handling. Polycyclic aromatic hydrocarbons (PAHs), such as phenanthrene (Phe), are the main crude oil components and are toxic to fish, acting as endocrine disruptors (ED). This is the first study that investigated impacts of Phe as an ED in E. marginatus juveniles. An in vivo sublethal exposure (96h) to Phe was carried out at two concentrations (0.1mg/L and 1mg/L); exposure to the vehicle (ethanol; ETOH) was also performed. Plasma levels of 17ß-estradiol (E2), testosterone (T) and 11-ketotestosterone (11-KT) were measured by ELISA. Gonads, liver and spleen were processed for histological analysis. In an in vitro bioassay, gonad fragments were incubated with Phe (8.91mg/L) or ETOH. Steroid levels in the culture media were measured by ELISA. The in vivo exposure to Phe triggered an increase of the area of the hepatocytes, increased number of melanomacrophagic centers and hemosiderosis in the spleen; ETOH induced similar effects on spleen. E2 and T levels did not change in plasma or in vitro media. In plasma, ETOH decreased 11-KT levels. Phenanthrene sharply reduced 11-KT levels in vitro. Although in vivo bioassay results were not unequivocal owing to ethanol effects, Phe might disrupt steroidogenesis in juvenile grouper, possibly causing dysfunctions during sex change and gonadal maturity, considering the importance of 11-KT in developing ovaries.

Thermal modulation of anthropogenic estrogen exposure on a freshwater fish at two life stages.

Human-mediated environmental change can induce changes in the expression of complex behaviors within individuals and alter the outcomes of interactions between individuals. Although the independent effects of numerous stressors on aquatic biota are well documented (e.g., exposure to environmental contaminants), fewer studies have examined how natural variation in the ambient environment modulates these effects. In this study, we exposed reproductively mature and larval fathead minnows (Pimephales promelas) to three environmentally relevant concentrations (14, 22, and 65ng/L) of a common environmental estrogen, estrone (E1), at four water temperatures (15, 18, 21, and 24°C) reflecting natural spring and summer variation. We then conducted a series of behavioral experiments to assess the independent and interactive effects of temperature and estrogen exposure on intra- and interspecific interactions in three contexts with important fitness consequences; reproduction, foraging, and predator evasion. Our data demonstrated significant independent effects of temperature and/or estrogen exposure on the physiology, survival, and behavior of larval and adult fish. We also found evidence suggesting that thermal regime can modulate the effects of exposure on larval survival and predator-prey interactions, even within a relatively narrow range of seasonally fluctuating temperatures. These findings improve our understanding of the outcomes of interactions between anthropogenic stressors and natural abiotic environmental factors, and suggest that such interactions can have ecological and evolutionary implications for freshwater populations and communities.

Neonatal Inhibition of DNA Methylation Alters Cell Phenotype in Sexually Dimorphic Regions of the Mouse Brain.

Many of the best-studied neural sex differences relate to differences in cell number and are due to the hormonal control of developmental cell death. However, several prominent neural sex differences persist even if cell death is eliminated. We hypothesized that these may reflect cell phenotype "decisions" that depend on epigenetic mechanisms, such as DNA methylation. To test this, we treated newborn mice with the DNA methyltransferase (DNMT) inhibitor zebularine, or vehicle, and examined two sexually dimorphic markers at weaning. As expected, control males had more cells immunoreactive for calbindin-D28k (CALB) in the medial preoptic area (mPOA) and fewer cells immunoreactive for estrogen receptor α (ERα) in the ventrolateral portion of the ventromedial nucleus of the hypothalamus (VMHvl) and the mPOA than did females. Neonatal DNMT inhibition markedly increased CALB cell number in both sexes and ERα cell density in males; as a result, the sex differences in ERα in the VMHvl and mPOA were completely eliminated in zebularine-treated animals. Zebularine treatment did not affect developmental cell death or the total density of Nissl-stained cells at weaning. Thus, a neonatal disruption of DNA methylation apparently has long-term effects on the proportion of cells expressing CALB and ERα, and some of these effects are sex specific. We also found that sex differences in CALB in the mPOA and ERα in the VMHvl persist in mice with a neuron-specific depletion of either Dnmt1 or Dnmt3b, indicating that neither DNMT alone is likely to be required for the sexually dimorphic expression of these markers.

TRPV4 associates environmental temperature and sex determination in the American alligator.

Temperature-dependent sex determination (TSD), commonly found among reptiles, is a sex determination mode in which the incubation temperature during a critical temperature sensitive period (TSP) determines sexual fate of the individual rather than the individual's genotypic background. In the American alligator (Alligator mississippiensis), eggs incubated during the TSP at 33 °C (male producing temperature: MPT) yields male offspring, whereas incubation temperatures below 30 °C (female producing temperature: FPT) lead to female offspring. However, many of the details of the underlying molecular mechanism remains elusive, and the molecular link between environmental temperature and sex determination pathway is yet to be elucidated. Here we show the alligator TRPV4 ortholog (AmTRPV4) to be activated at temperatures proximate to the TSD-related temperature in alligators, and using pharmacological exposure, we show that AmTRPV4 channel activity affects gene expression patterns associated with male differentiation. This is the first experimental demonstration of a link between a well-described thermo-sensory mechanism, TRPV4 channel, and its potential role in regulation of TSD in vertebrates, shedding unique new light on the elusive TSD molecular mechanism.

Exogenous estradiol alters gonadal growth and timing of temperature sex determination in gonads of sea turtle.

Temperature sex determining species offer a model for investigating how environmental cues become integrated to the regulation of patterning genes and growth, among bipotential gonads. Manipulation of steroid hormones has revealed the important role of aromatase in the regulation of the estrogen levels involved in temperature-dependent sex determination. Estradiol treatment counteracts the effect of male-promoting temperature, but the resulting ovarian developmental pattern differs from that manifested with the female-promoting temperature. Hypoplastic gonads have been reported among estradiol-treated turtles; however the estradiol effect on gonadal size has not been examined. Here we focused on the sea turtle Lepidochelys olivacea, which develops hypoplastic gonads with estradiol treatment. We studied the effect of estradiol on cell proliferation and on candidate genes involved in ovarian pattern. We found this effect is organ specific, causing a dramatic reduction in gonadal cell proliferation during the temperature-sensitive period. Although the incipient gonads resembled tiny ovaries, remodeling of the medullary cords and down-regulation of testicular factor Sox9 were considerably delayed. Contrastingly, with ovarian promoting temperature as a cue, exogenous estradiol induced the up-regulation of the ovary factor FoxL2, prior to the expression of aromatase. The strong expression of estrogen receptor alpha at the time of treatment suggests that it mediates estradiol effects. Overall results indicate that estradiol levels required for gonadal growth and to establish the female genetic network are delicately regulated by temperature.

Adaptation of a Bioinformatics Microarray Analysis Workflow for a Toxicogenomic Study in Rainbow Trout.

Sex steroids play a key role in triggering sex differentiation in fish, the use of exogenous hormone treatment leading to partial or complete sex reversal. This phenomenon has attracted attention since the discovery that even low environmental doses of exogenous steroids can adversely affect gonad morphology (ovotestis development) and induce reproductive failure. Modern genomic-based technologies have enhanced opportunities to find out mechanisms of actions (MOA) and identify biomarkers related to the toxic action of a compound. However, high throughput data interpretation relies on statistical analysis, species genomic resources, and bioinformatics tools. The goals of this study are to improve the knowledge of feminisation in fish, by the analysis of molecular responses in the gonads of rainbow trout fry after chronic exposure to several doses (0.01, 0.1, 1 and 10 µg/L) of ethynylestradiol (EE2) and to offer target genes as potential biomarkers of ovotestis development. We successfully adapted a bioinformatics microarray analysis workflow elaborated on human data to a toxicogenomic study using rainbow trout, a fish species lacking accurate functional annotation and genomic resources. The workflow allowed to obtain lists of genes supposed to be enriched in true positive differentially expressed genes (DEGs), which were subjected to over-representation analysis methods (ORA). Several pathways and ontologies, mostly related to cell division and metabolism, sexual reproduction and steroid production, were found significantly enriched in our analyses. Moreover, two sets of potential ovotestis biomarkers were selected using several criteria. The first group displayed specific potential biomarkers belonging to pathways/ontologies highlighted in the experiment. Among them, the early ovarian differentiation gene foxl2a was overexpressed. The second group, which was highly sensitive but not specific, included the DEGs presenting the highest fold change and lowest p-value of the statistical workflow output. The methodology can be generalized to other (non-model) species and various types of microarray platforms.

Retinoic acid homeostasis through aldh1a2 and cyp26a1 mediates meiotic entry in Nile tilapia (Oreochromis niloticus).

Meiosis is a process unique to the differentiation of germ cells. Retinoic acid (RA) is the key factor controlling the sex-specific timing of meiotic initiation in tetrapods; however, the role of RA in meiotic initiation in teleosts has remained unclear. In this study, the genes encoding RA synthase aldh1a2, and catabolic enzyme cyp26a1 were isolated from Nile tilapia (Oreochromis niloticus), a species without stra8. The expression of aldh1a2 was up-regulated and expression of cyp26a1 was down-regulated before the meiotic initiation in ovaries and in testes. Treatment with RA synthase inhibitor or disruption of Aldh1a2 by CRISPR/Cas9 resulted in delayed meiotic initiation, with simultaneous down-regulation of cyp26a1 and up-regulation of sycp3. By contrast, treatment with an inhibitor of RA catabolic enzyme and disruption of cyp26a1 resulted in earlier meiotic initiation, with increased expression of aldh1a2 and sycp3. Additionally, treatment of XY fish with estrogen (E2) and XX fish with fadrozole led to sex reversal and reversion of meiotic initiation. These results indicate that RA is indispensable for meiotic initiation in teleosts via a stra8 independent signaling pathway where both aldh1a2 and cyp26a1 are critical. In contrast to mammals, E2 is a major regulator of sex determination and meiotic initiation in teleosts.

Molecular identification of GnIH/GnIHR signal and its reproductive function in protogynous hermaphroditic orange-spotted grouper (Epinephelus coioides).

Gonadotropin-inhibitory hormone (GnIH) and its receptor (GnIHR) play an important role in reproduction regulation in birds, mammals and some teleost species. In protogynous hermaphroditic orange-spotted grouper (Epinephelus coioides), the GnIH/GnIHR signaling pathway and its reproductive function have not been addressed yet. In this study, GnIH and GnIHR in orange-spotted grouper were characterized. gGnIH possessed three putative peptides (gGnIH-I, -II, -III), while gGnIHR showed the characteristics of G protein-coupled receptor and was clustered with GPR147. Functional assays demonstrated that three synthetic gGnIH peptides significantly decreased the forskolin-induced CRE promoter activity, but only gGnIH-I could significantly decrease SRE promoter activity in COS-7 cells transfected with gGnIHR. During the process of ovarian differentiation and development, gGnIH mRNA level in hypothalamus was low at the gonadal primordium stage with gonia, then increased significantly at the early differentiated gonad with primary growth oocytes, while decreased significantly at the developing gonads with cortical-alveolus and vitellogenic stage oocytes. During MT-induced sex reversal, gGnIH mRNA level in hypothalamus increased significantly when the fish completely reversed from female to male. However, gGnIHR mRNA level in pituitary decreased significantly in intersex and completely reversed male fish. Intraperitoneal injection (i.p.) of three gGnIH peptides significantly decreased GnRH1 mRNA levels in hypothalamus, and gGnIH-II significantly inhibited synthesis of LHß in pituitary. In summary, we firstly identified the GnIH/GnIHR signal in protogynous orange-spotted grouper, which might be involved in the regulation of the reproductive function of sex differentiation, gonadal development and sex reversal via regulating the synthesis of both GnRH and GtH.

The Potential Role of Amh to Prevent Ectopic Female Development in Testicular Tissue of the Protandrous Black Porgy, Acanthopagrus schlegelii.

In most vertebrates, hermaphroditism results in infertility. However, hermaphroditism occurs in 6% of teleosts, which primarily undergo protogyny. Here, to elucidate the transient stage from gonochorism to hermaphroditism, juvenile black porgies as a model animal were fed a diet containing estradiol (E2) for 3 mo, followed by withdrawal of E2 treatment. The E2-terminated fish had ectopically located oocytes in the regenerated testes. Antimüllerian hormone (amh) was strongly expressed in the Sertoli cells with type A spermatogonia and follicle cells with vitellogenic oocytes. Amh was robustly expressed in the ectopic oocytes-bordering region of regenerated testes and in testes with nonsynchronous spermatogenesis. This Amh was released by Sertoli cells and aggregated in the area containing type A spermatogonia in the ectopic oocytes-bordering region. Our in vitro results show that exogenous recombinant Amh (rAmh) can inhibit type A spermatogonia proliferation in the testis but not oogonia proliferation in the ovary. We suggest that Amh-arrested spermatogonia A may act as a boundary to block intercellular communication (i.e., prevent peptide factors released from female tissue to alter the sexual fate of type A spermatogonia) and further inhibit female growth. These results suggest that black porgy can prevent ectopic female growth in the testis and maintain male function of the digonic gonad (testes and ovary separated by the connective tissue) through Amh action. This function of amh might shed light on why the majority of syngonic fish undergo protogyny (female-to-male sex change).