Protogynous Orange-spotted Grouper Research Articles

Potential role of DNA methylation of cyp19a1a promoter during sex change in protogynous orange-spotted grouper, Epinephelus coioides

Estrogen has a pivotal role in early female differentiation and further ovarian development. Aromatase (Cyp19a) is responsible for the conversion of androgens to estrogens in vertebrates. In teleosts, cyp19a1a and it paralog cyp19a1b are mainly expressed in the ovary and hypothalamus, respectively. Decreased plasma estrogen levels and lower cyp19a1a expression are associated with the initiation of female-to-male sex change in protogynous grouper. However, an 17α-methyltestosterone (MT)-induced the sex change from a female to a precocious male is a transient phase, and a reversible sex change (induced male-to-female) occurs after chemical withdrawal. Thus, we used this characteristic to study the epigenetic modification of cyp19a1a promoter in orange-spotted grouper. CpG-rich region with a CpG island is located on the putative regulatory region of distal cyp19a1a promoter. Our results showed that cyp19a1a promoter exhibited tissue-specific methylation status. Low methylation levels of distal cyp19a1a promoter and hypomethylated (0–40%) clones of cyp19a1a promoter region were widely observed in the ovary but not shown in testis and other tissues. In femaleness, higher numbers of hypomethylated clones of cyp19a1a promoter region were observed in the vitellogenic oocyte stage compared to the primary oocyte stage. Furthermore, decreased numbers of hypomethylated clones of cyp19a1a promoter region were associated with the maleness during the female-to-male sex change. DNA methylation inhibitor (5-aza-2′-deoxycytidine) delayed the spermatogenesis process (according to germ cell stage and numbers: by decrease of sperm and increase of spermatocytes) but did not influence the reversed sex change in MT-induced bi-directional sex change. These results suggest that epigenetic modification of cyp19a1a promoter may play an important role during the sex change in orange-spotted grouper.

The characteristics and expression profile of gonadal soma-derived factor (gsdf/Gsdf) during androgen-induced reversible sex change in the protogynous orange-spotted grouper, Epinephelus coioides

Gonadal soma-derived factor (gsdf) is a teleost- and gonad-specific growth factor involved in gonadal differentiation and early germline cell development. In protogynous grouper, the androgen-induced precocious male phase is transient, and a reversible sex change (induced male-to-female change) occurs after chemical withdrawal. In this study, we used MT (17α-methyltestosterone)-induced bi-directional sex change to understand the role of Gsdf signaling in the sexual differentiation of orange-spotted grouper, Epinephelus coioides. Our results showed that the expression of gsdf was higher in the MT-induced male (functional male) than in the control female (primary oocyte stage). Conversely, high gsdf expression was also found in the transient phase of reversible induced male-to-female sex change (dormant gonad). Furthermore, gsdf/Gsdf was expressed in the Sertoli cells surrounding the spermatogonia and the somatic cells surrounding the oogonia. gsdf/Gsdf expression was also found in the somatic cells surrounding the spermatogonia-like cells in the transient phase of induced male-to-female sex change. Recombinant Gsdf (rGsdf) was produced and specific anti-Gsdf antiserum was obtained. Moreover, the proliferation activities of the oogonia were decreased by rGsdf in in vitro ovarian tissue culture (equivalent to 1.125 mg crude protein extracted from 37.5 mg testicular tissue/ml). Conversely, the treatment of ovarian tissue culture with rGsdf did not affect the expression levels of female and male sex-related genes (equivalent to 1.125 mg crude protein extracted from 37.5 mg testicular tissue/ml). Taken together, our data suggest that Gsdf signaling is related to the arrest of proliferation activity in early-stage germline cells but is not directly related to sexual phase determination.

Characterization of gonadal glucocorticoid-induced leucine zipper (GILZ) protein expression during sex change in the protogynous orange-spotted grouper, Epinephelus coioides

Steroid hormones have been proven as a key drive of sex change in sequentially hermaphroditic organisms. However, the upstream mechanism of sex steroid hormones regulation that affect sex change remain unknown. The main glucocorticoid in teleost fish is cortisol, which both regulates steroidogenesis and has antistress action. Thus, cortisol might be one of the prime factors in sex change. In this study, the glucocorticoid-induced leucine zipper (GILZ) gene, was proven to have a dramatic effect in orange-spotted groupers (Epinephelus coioides) during sex change at the early stage of gonadal transition. The specific action of the GILZ protein is at the pouch-shaped proliferative spermatogonia instead of the degenerative oocyte at the onset of sex change. Immunohistochemical (IHC) evidence revealed that GILZ performs intensively at undifferentiated spermatogonia in the early testis stage. These results imply that cortisol provokes a rise of GILZ through regulation caused by steroid hormones leading to sex change.

The Administration of Cortisol Induces Female-to-Male Sex Change in the Protogynous Orange-Spotted Grouper, Epinephelus coioides.

In this study, we injected cortisol into the protogynous orange-spotted grouper (Epinephelus coioides) to investigate the role of this hormone in sex change. Following injection, we evaluated gonadal changes, serum levels of steroid hormones, and sex-related gene expression during the processes of cortisol-induced sex change and cortisol withdrawal in the orange-spotted grouper. Cortisol treatment caused the degeneration of oocytes and induced sex change in a dose-dependent manner. Over the long-term, we observed a significant increase in serum 11-ketotestosterone (11-KT) levels in all cortisol-treated groups, although levels of 17β-estradiol did not change significantly. Consistent with the elevation of serum 11-KT levels, the expression of genes related to testicular development was also significantly up-regulated in the cortisol-treated groups. Based on our results, we propose that cortisol may trigger masculinization by inducing the synthesis of 11-KT and by directly activating the expression of sex-related genes. Furthermore, we found that cortisol-induced sex change was not permanent and could be reversed after the withdrawal of cortisol treatment.

A highly efficient method of inducing sex change using social control in the protogynous orange-spotted grouper (Epinephelus coioides)

In this study, a highly efficient method of inducing sex change using social control in the protogynous orange-spotted grouper (Epinephelus coioides) was developed. A series of experiments were carried out to investigate the influence of social unit size and seasonality on the rate of sex change rate in the orange-spotted grouper. The results revealed that the removal of male fish in social units with three females during the pre-breeding and breeding seasons was the fastest method ever recorded for inducing an artificial sex change in orange-spotted grouper. The data indicated that a sex change generally occurred in the largest female in the social unit. A sharp increase in levels of androgen and cortisol in serum are proposed as a marker of the onset of a female-to-male sex change. Using the method described in this study, a sex change from female to male occurred within two to four weeks of removing males from the social unit. To examine sperm fertility, the fertilization rate of males obtained by induced sex change was analyzed. The results revealed that males obtained by this method can successfully fertilize mature eggs. This method could address the scarcity of male groupers required for spawning in captivity, and would promote the development of grouper aquaculture.

New Insights Into the Role of Follicle-Stimulating Hormone in Sex Differentiation of the Protogynous Orange-Spotted Grouper, Epinephelus coioides.

Follicle-stimulating hormone (FSH) signaling is considered to be essential for early gametogenesis in teleosts, but its functional roles during sex differentiation are largely unknown. In this study, we investigated the effects of long-term and short-term FSH injection on sex differentiation in the protogynous orange-spotted grouper (Epinephelus coioides). Long-term FSH treatment initially promoted the formation of ovaries but subsequently induced a male fate. The expression of female pathway genes was initially increased but then decreased, whereas the expression of male pathway genes was up-regulated only during long-term FSH treatment. The genes related to the synthesis of sex steroid hormones, as well as serum 11-ketotestosterone and estradiol, were also up-regulated during long-term FSH treatment. Short-term FSH treatment activated genes in the female pathway (especially cyp19a1a) at low doses but caused inhibition at high doses. Genes in the male pathway were up-regulated by high concentrations of FSH over the short term. Finally, we found that low, but not high, concentrations of FSH treatment activated cyp19a1a promoter activities in human embryonic kidney (HEK) 293 cells. Overall, our data suggested that FSH may induce ovarian differentiation or a change to a male sex fate in the protogynous orange-spotted grouper, and that these processes occurred in an FSH concentration-dependent manner.

MT-Feeding-Induced Impermanent Sex Reversal in the Orange-Spotted Grouper during Sex Differentiation.

In this study, we systematically investigated the process of sex reversal induced by 17-methyltestosterone (MT) feeding and MT-feeding withdrawal at the ovary differentiation stage in orange-spotted groupers, Epinephelus coioides. Gonadal histology showed that MT feeding induced a precocious sex reversal from immature ovaries to testes, bypassing the formation of an ovarian cavity, and MT-feeding withdrawal led to an ovarian fate. In both the MT feeding and MT-feeding withdrawal phases, cytochrome P450 family 11 subfamily B (cyp11b) gene expression and serum 11-KT levels were not significantly changed, suggesting that the MT-treated fish did not generate endogenous steroids, even though active spermatogenesis occurred. Finally, by tracing doublesex-expressing and Mab-3-related transcription factor 1 (dmrt1)-expressing cells and TUNEL (terminal deoxynucleotidyl transferase 2-deoxyuridine, 5-triphosphate nick end labeling) assays, we found that the efferent duct formed first, and then, the germ cells and somatic cells of the testicular tissue were generated around the efferent duct during MT-feeding-induced precocious sex reversal. Collectively, our findings provide insights into the molecular and cellular mechanisms underlying sex reversal induced by exogenous hormones during sex differentiation in the protogynous orange-spotted grouper.

Expression profile of amh/Amh during bi-directional sex change in the protogynous orange-spotted grouper Epinephelus coioides.

Gonadal differentiation is tightly regulated by the initial sex determining gene and the downstream sex-related genes in vertebrates. However, sex change in fish can alter the sexual fate from one sex to the other. Chemical-induced maleness in the protogynous orange-spotted grouper is transient, and a reversible sex change occurs after the chemical treatment is withdrawn. We used these characteristics to study Amh signaling during bi-directional sex change in the grouper. We successfully induced the female-to-male sex change by chemical (aromatase inhibitor, AI, or methyltestosterone, MT) treatment. A dormant gonad (a low proliferation rate of early germ cells and no characteristics of both sexes) was found during the transient phase of reversible male-to-female sex change after the withdrawal of chemical administration. Our results showed that amh (anti-mullerian hormone) and its receptor amhr2 (anti-mullerian hormone receptor type 2) were significantly increased in the gonads during the process of female-to-male sex change. Amh is expressed in the Sertoli cells surrounding the type A spermatogonia in the female-to-male grouper. Male-related gene (dmrt1 and sox9) expression was immediately decreased in MT-terminated males during the reversible male-to-female sex change. However, Amh expression was found in the surrounding cells of type A spermatogonia-like cells during the transient phase of reversible male-to-female sex change. This phenomenon is correlated with the dormancy of type A spermatogonia-like cells. Thus, Amh signaling is suggested to play roles in regulating male differentiation during the female-to-male sex change and in inhibiting type-A spermatogonia-like cell proliferation/differentiation during the reversible male-to-female sex change. We suggest that Amh signaling might play dual roles during bi-directional sex change in grouper.

Molecular regulation of sex change induced by methyltestosterone -feeding and methyltestosterone -feeding withdrawal in the protogynous orange-spotted grouper†

The sex identity of fish can be easily manipulated by exogenous hormones. Treatment with 17-methyltestosterone (MT) has been widely used to induce a male fate, but the molecular and cellular processes underlying sex changes induced by MT treatments and the withdrawal of MT are not well studied. In this study, we systematically investigated gonadal histology, gene expression profiles, sex steroid hormone levels, and cellular changes during sex changes induced by MT-feeding and MT-feeding withdrawal in the protogynous orange-spotted grouper, Epinephelus coioides. Based on gonadal histology, we demonstrated that MT-feeding-induced sex reversal can be divided into early and late phases: in the early phase, male and female germ cells coexist, and MT-feeding withdrawal leads to a female fate; in the late phase, only male germ cells are observed, and MT-feeding withdrawal does not reverse the process, leading to a male fate. In both the early and late phases, cytochrome P450 family19 subfamily A member 1 (cyp19a1a) gene expression increased in response to MT-feeding withdrawal. Finally, by tracing doublesex- and Mab-3-related transcription factor 1 (dmrt1)-expressing cells, we found that gonia-like cells in the germinal epithelium might be the major germ cell sources for developing testes during sex reversal. Collectively, our findings provide insights into the molecular and cellular mechanisms underlying sex changes induced by exogenous hormones.

Molecular mechanism of feedback regulation of 17β-estradiol on two kiss genes in the protogynous orange-spotted grouper (Epinephelus coioides).

Kisspeptins are considered critical regulators in the hypothalamic-pituitary-gonadal axis because they can stimulate secretion of Gonadotropin-releasing hormone in mammals, and may also mediate the feedback regulation of sex steroids in the hypothalamus. Two kiss1 paralogues (kiss1 and kiss2) were identified in teleosts, hinting at their increased complexity of signaling for sex-steroid feedback regulation. In the present study, molecular pathways by which 17β-estradiol (E2 ) exerted feedback regulation on two kiss genes, via three types of estrogen receptors, were investigated in the protogynous orange-spotted grouper (Epinephelus coioides). kiss2 expression in the brain significantly increased in ovariectomized orange-spotted groupers, while E2 replacement in ovariectomized fish reversed these changes to levels in the sham-surgery group; conversely, kiss1 expression did not change. Dual-label in situ hybridization showed that kiss1 and kiss2 neurons express erα, erβ1, and erβ2, indicating that E2 may directly regulate kiss1 and kiss2. Indeed, E2 treatment of transiently transfected HEK293T cells decreased the activity of both kiss promoters in the presence of erβ1 and erβ2 rather than erα. Further deletion and site-directed mutagenesis of the kiss promoters indicated that kiss1 is regulated by E2 via an estrogen-responsive element (ERE)-dependent, classical pathway utilized by Erβ1, as well as via an Activator protein 1 (Ap1)-dependent, non-classical pathway utilized by Erβ2. kiss2 was also differently regulated by E2 through the Creb transcription factor, utilized by Erβ1 as well as a half-ERE-dependent, classical pathway utilized by Erβ2. Taken together, multiple signaling pathways in orange-spotted grouper are clearly involved in the feedback regulation of E2 on kiss genes via different estrogen receptors.

Molecular identification of StAR and 3βHSD1 and characterization in response to GnIH stimulation in protogynous hermaphroditic grouper (Epinephelus coioides).

Gonadal steroids are critical factors in reproduction and sex reverse process. StAR (steroidogenic acute regulatory protein), transferring the cholesterol from the outer mitochondrial membrane to the inner membrane, is the rate-limiting factor of steroidogenesis. 3βHSD (3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase), converting Δ5-steroids into Δ4-steroids, is an important oxidoreductase in steroidogenesis. In the present study, StAR and 3βHSD1 were cloned and characterized from protogynous orange-spotted grouper. StAR cDNA contains an 861bp open reading frame (ORF), encoding a predicted protein of 286 amino acids, and the ORF of 3βHSD1 was 1125bp, encoding a predicted protein of 374 amino acids. The transcript of StAR was mainly expressed in gonad, while 3βHSD1 mRNA was predominantly detected in brain and gonad. In the previous study, we found the expression of GnIH mRNA level in male, as well as in 17 alpha-methyltestosterone (MT)-induced male fish was significantly higher than in female fish, this indicating that GnIH/GnIHR signaling might be involved in the regulation of sex reversal and male maintenance. In order to figure out the function of GnIH in steroidogenesis, the expression of StAR and 3βHSD1 regulated by GnIH was examined. In vitro study showed that treatment of cultured ovary fragments with gGnIH peptides significantly stimulated the expression of StAR and 3βHSD1. In addition, the mRNA levels of StAR and 3βHSD1 were significantly increased after intraperitoneal injection (i.p.) with gGnIH peptides. Moreover, during MT-induced sex change from female to male, the levels of StAR mRNA significantly increased by 5.2, 24.8 and 353.5 folds, and that of 3βHSD1 mRNA by 3.5, 32.5 and 55.4 folds at the 2nd, 4th and 6th week after MT implantation, respectively. Collectively, our results indicate that GnIH may be involved in the regulation of sex reversal or male maintenance by stimulating the expression of StAR and 3βHSD1 in protogynous grouper.

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.

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 LHβ 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.

Neurosteroidogenic enzymes and their regulation in the early brain of the protogynous grouper Epinephelus coioides during gonadal sex differentiation

The regulatory role of neurosteroids in the early brain during gonadal sex differentiation is unclear. The aim of this study was to investigate the expression and cellular localization of key steroidogenic enzymes in the early brain of the protogynous orange-spotted grouper Epinephelus coioides and the temporal expressions has been correlated with gonadal sex differentiation. In this study, we showed that peak neurosteroidogenesis occurs in the early brain during gonadal sex differentiation. The temporal expressions of key enzymes, cyp11a1 (cytochrome P450 side chain cleavage), hsd3b1 (3β-hydroxysteroid dehydrogenase) and cyp17a1 (cytochrome P450c17) were studied at different developmental ages (from 90- to 150-dah: days after hatching) using quantitative real-time PCR (q-PCR). q-PCR analysis indicated that the transcript expressions of cyp11a1, hsd3b1 and cyp17a1 were increased in the brain around the period of gonadal sex differentiation. Further, in situ hybridization (ISH) analysis showed that cyp11a1, hsd3b1 and cyp17a1 transcripts were widely expressed in several discrete brain regions, especially the intense expression in the forebrain, with an overall similar expression pattern. High density in the cyp19a1b/Cyp19a1b expression was detected in radial glial cells. Thus, the expression of grouper cyp19a1b/Cyp19a1b is restricted to radial glial cells, suggesting estrogens can modulate their activity. Next, by combining Cyp19a1b immunohistochemistry (IHC) with florescence ISH (FISH) of cyp11a1, hsd3b1 and cyp17a1, we showed that sub-cellular localization of cyp11a1, hsd3b1 and cyp17a1 transcripts, in partial, appeared to be in Cyp19a1b radial glial cell soma. Further, exogenous estradiol (E2) increased the expression of cyp17a1 and cyp19a1b/Cyp19a1b in the brain of grouper. Consequently, our results illustrated that the locally synthesized E2 upregulated neurosteroidogenic enzymes in the early brain and suggest a role for these enzymes in the neurogenic process during gonadal sex differentiation.

Increase in estrogen signaling in the early brain of orange-spotted grouper Epinephelus coioides: a mini-review

Despite neurosteroidogenic enzymes are playing important roles in the regulation of brain development and function, the potential link between brain and gonad by the action of steroid hormones during gonadal sex differentiation is still not clear in teleosts. In this mini-review, we summarized our understanding on the early brain development related to the synthesis of neurosteroids and receptor signaling during gonadal sex differentiation in protogynous orange-spotted grouper, Epinephelus coioides (functional females for the first 6years of life and start to sex change around the age of 7years) and protandrous black porgy (functional males for the first 2years of life but begin to change sex during the third year). We found a similar profile in the increased expression of brain aromatase gene (aromatatse B or cyp19a1b), aromatase activity, estradiol (E(2)), and estrogen signaling in the brain of both grouper and black porgy fish during gonadal sex differentiation. In contrast to mammals, teleost fish Cyp19a1b expressed in a unique cell type, a radial glial cell, which is acted as progenitors in the brain of developing and adult fish. In agreement with these pioneer studies, we demonstrated that the grouper cyp19a1b/Cyp19a1b was expressed in radial glial cells. Further, in vivo data in the grouper brain showed that exogenous E(2) upregulated Cyp19a1b immunoreactivity (ir) in radial glial cells. These data suggest the possible roles of Cyp19a1b and E(2) in early brain development which is presumably related to gonadal sex differentiation.

Developmental expression of genes involved in neural estrogen biosynthesis and signaling in the brain of the orange-spotted grouper Epinephelus coioides during gonadal sex differentiation

In the brain, the synthesis of neurosteroids and receptor activation during gonadal sex differentiation in teleosts are poorly understood. In the present study, the protogynous orange-spotted grouper ( Epinephelus coioides) was selected as a model fish, and we hypothesized that de novo synthesis of neural estrogen may play a role in the female grouper brain during gonadal sex differentiation. We investigated the temporal expression of the genes StAR, cyp19a1b and pcna and the sex steroid nuclear receptors for estrogen ( ERα, ERβ1 and ERβ2), androgen ( AR) and the plasma membrane-associated estrogen receptor ( GPR30) in the brain during early developmental ages from 90 days after hatching (dah) to 180 dah after gonadal sex differentiation. Our results revealed that mRNA for ERs and GPR30 but not AR was significantly increased at 110 dah (a time close to gonadal sex differentiation) in the forebrain and midbrain and for cyp19a1b at 110 dah in the forebrain. Brain aromatase activity and estradiol (E2) levels, but not testosterone (T), were increased in the forebrain at 110 and 120 dah, respectively. Furthermore, exogenous E2 stimulated cyp19a1b transcripts in the forebrain and hypothalamus and immunoreactive (ir)Cyp19a1b (aromatase enzyme) in the forebrain. irCyp19a1b localized in the glial cells of the forebrain regions. Therefore, we identified a peak of functional aromatase activity and estrogen signaling in the early grouper brain during gonadal sex differentiation. Moreover, pcna transcripts (a marker for cell proliferation activity) were higher in the early brain at 110–150 dah. Thus, a peak time of development in the brain is suggested to occur during gonadal sex differentiation in the grouper.

Androgens stimulate sex change in protogynous grouper, Epinephelus coioides: spawning performance in sex-changed males

We examined the efficacy of androgens (1.0 mg/kg body mass), testosterone (T), 11-ketotestosterone (11-KT), 17α-methyltestosterone (MT), testosterone propionate (TP) or androgen mixture (T, MT and TP in an equal ratio), for induction of sex change in protogynous orange-spotted grouper, Epinephelus coioides. The spawning performance in sex-changed males was also investigated. MT and androgen mixture at a dose of 1.0 mg/kg BW induced a sex transition and completion of spermatogenesis up to the functional male phase. The androgen mixture was most effective. Significantly, higher plasma T levels were found in MT and androgen mixture groups compared to control and other androgen implantation (T, TP or 11-KT) groups. We found that plasma levels of estradiol-17β (E2) or 11-KT were not different among treated groups. Sex-changed males could successfully fertilize mature eggs. Fertilization and hatching rates were of 23.5–70.4% and 8.4–44.6%, respectively. The data demonstrated that induction of sex change by exogenous androgens in groups could apply to the aquaculture field for seed production.

The effects of exogenous androgens on ovarian development and sex change in female orange-spotted protogynous grouper, Epinephelus coioides

The efficacy of various doses of an androgen mixture, containing testosterone (T), 17α-methyltestosterone (MT), and testosterone propionate (TP) in equal ratios, for induction of sex change in protogynous orange-spotted grouper, Epinephelus coioides, was examined. The androgen mixture, with doses from 1 to 20,000 μg/kg BW, was implanted into each fish (body weight 1.7 kg) in July (post-spawning season), and gonadal stage and plasma T were monitored at various time intervals for a period of 90 days. Gonadosomatic (GSI) and hepatosomatic indices (HSI), gonadal histology, sex steroids (T, 11-ketotestosterone=11-KT, and estradiol=E2) in plasma were determined after 90 days of implantation. The implanted T was released to plasma for 60 days. All androgen mixtures at doses higher than 1000 μg/kg BW were capable of inducing a sex transition and completion of spermatogenesis up to the functional male phase. Low doses of androgens induced ovarian development and higher GSI and HSI indices than in the control and other groups. Significantly higher plasma T levels were found in the developing and spermiating males as compared to the females and intersex-transitional fish. No significantly difference of plasma levels of E2 and 11-KT was found in the control and all the androgen-treated groups (during the nonreproductive season). Therefore, it is concluded that the stimulation of sex change or ovarian development is dependent on the dose and time course of implanted androgens. Plasma T levels were correlated with the development of controlled male phase in protogynous grouper, E. coioides.