Changes in androgen-producing cell size and circulating 11-ketotestosterone level during female-male sex change of honeycomb grouper Epinephelus merra

11-ketotestosterone (11-KT), a potent male-specific androgen in fish, has important roles on spermatogenesis, male behavior, and nuptial coloration. The site of 11-KT synthesis and its role on male germ cell development during protogynous sex change is not clearly understood. We examined the dynamics of steroidogenic enzymes immunolocalization, viz cholesterol side-chain cleavage (P450scc), biomarker of steroids and cytochrome P45011beta-hydroxylase (P45011beta), downstream to 11-KT production, throughout the process of sex change in honeycomb grouper (Epinephelus merra). In female, P450scc immunoreactivity (-ir) was observed in the theca layer and tunica near blood vessels (BV). During the onset of sex change, P450scc reactive cells were observed in the remaining follicle layer of degenerated oocyte of the ovo-testis in early transitional (ET) and late transitional (LT). In male, P450scc-ir was localized in the interstitial Legdig cells of testis. P45011beta reactive cells were observed in the tunica near BV in female but not in theca layer. In ET and LT phases gonads, P45011beta localized in remaining follicle layer of degenerated oocyte and tunica near BV. On the other hand, in male, both interstices and tunica near BV showed strong signals against P45011beta. Moreover, in vivo and in vitro levels of 11-KT related with the changes in the nuclei diameter of P45011beta-positive cells in both tunica near BV and remaining follicle layer of degenerated oocyte to interstices during the progress of sex change. The present results suggest that 11-KT produced in the tunica near BV may provide the stimulus for female to degenerate oocytes and initiate sex change. However, 11-KT produced both in tunica near BV and remaining follicle layer of degenerated oocyte possibly plays critical role during testicular differentiation as well as gonadal restructuring at mid to late phases (ET to LT) of sex change in honeycomb grouper.     

Differentiation of ambisexual gonads and immunohistochemical localization of P450 cholesterol side-chain cleavage enzyme during gonadal sex differentiation in the protandrous anemonefish, Amphiprion clarkii

To clarify the relationship between steroid hormones and sex differentiation of the protandrous anemonefish Amphiprion clarkii, we histologically examined its gonadal differentiation. From hatching to 30 days post hatching (dph), all of the gonads surveyed were sexually undifferentiated. The gonads of all fish first differentiated into ovaries at 60 dph, and the oocytes gradually developed and increased in number as the ovaries grew up until 183 dph. Some cysts of differentiated spermatogenic germ cells appeared in the ovaries at 214 dph, and ambisexual gonads with both ovarian and testicular tissues formed by 273 dph. Using immunohistochemistry, we then investigated the expression of cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), during gonadal sex differentiation. P450scc-immunopositive reactions first appeared in sexually undifferentiated gonads at 30 dph. Beginning at 60 dph, the number of strongly positive cells increased throughout the differentiation of the ovaries and continued to increase during the testicular differentiation until 210 dph. Immunopositive cells were observed more frequently in ovarian tissue than in testicular tissue in the ambisexual gonads at 270 dph. These results suggest that endogenous steroid hormones are important for the sex differentiation, including the primary sex differentiation and subsequent testicular differentiation, of the anemonefish.     

Differentiation of steroid-producing cells during ovarian differentiation in the protogynous Malabar grouper, Epinephelus malabaricus

To understand the mechanism of sex differentiation in the protogynous Malabar grouper Epinephelus malabaricus, we performed an immunohistochemical investigation of the expression of three steroidogenic enzymes, cholesterol-side-chain-cleavage enzyme (CYP11a), aromatase (CYP19a1a), and cytochrome P45011beta-hydroxylase (CYP11b), in the gonads during ovarian differentiation. Strong positive immunoreactivity against CYP11a, the key enzyme of steroidogenesis, and CYP19a1a which is essential for estrogen (17beta-estradiol) production, appeared first in the somatic cells surrounding gonial germ cells in undifferentiated gonads and throughout ovarian differentiation. However, positive immunoreactivity against CYP11b, which is important for androgen (11-ketotestosterone) production, first appeared in the cluster of somatic cells in the ovary tunica near the dorsal blood vessel after differentiation. CYP19a1a and CYP11b did not co-localize in any cells. These results indicate that there are two types of steroid-producing cells, estrogen-producing cells and androgen-producing cells, in the gonads of this fish, and they are distributed differently, suggesting that these cells are derived from different somatic cells. Estrogen-producing cells appeared prior to ovarian differentiation, while androgen-producing cells were first detected after ovarian differentiation. These results suggest that endogenous estrogen is involved in ovarian differentiation.     

Gonadal restructuring and correlative steroid hormone profiles during natural sex change in protogynous honeycomb grouper (Epinephelus merra)

The honeycomb grouper shows protogynous hermaphroditism. The endocrine mechanisms involved in gonadal restructuring throughout protogynous sex change are largely unknown. In the present study, we investigated changes in the gonadal structures and levels of serum sex steroid hormones during female to male sex change in the honeycomb grouper. On the basis of histological changes, entire process of sex change was assigned into four developmental phases: female, early transition (ET), late transition (LT), and male phase. At the female phase, the oocytes of several developmental stages were observed including gonial germ cells in the periphery of ovigerous lamellae. At the beginning of ET phase, perinucleolar and previtellogenic oocytes began degenerating, followed by proliferation of spermatogonia toward the center of lamella. The LT phase was characterized by further degeneration of oocytes and rapid proliferation of spermatogenic germ cells throughout the gonad. At the male phase, no ovarian cells were observed and testis had germ cells undergoing active spermatogenesis. Serum levels of estradiol-17beta (E2) were high in females in the breeding season, but low in the non-breeding female, transitional and male phase, and those of 11-ketotestosterone (11-KT) and testosterone (T) were low in females and gradually increased in the transitional and male phase. The present results suggest that low serum E2 levels and degeneration of oocytes accompanied by concomitant increase in the 11-KT levels and proliferation of spermatogenic germ cells are probably the events mediating protogynous sex change in the honeycomb grouper.     

Steroid enzyme gene expressions during natural and androgen-induced gonadal differentiation in the rainbow trout, Oncorhynchus mykiss.

In fish, according to Yamamoto's model, androgens would drive testis differentiation and estrogens ovarian differentiation. In order to study the implication of steroid enzymes in rainbow trout gonadal differentiation, we examined the expression of some steroid enzyme genes during natural differentiation (cholesterol side chain cleavage = P450scc, 17-hydroxylase/lyase = P450c17, 3beta-hydroxysteroid dehydrogenase = 3betaHSD) and androgen-induced differentiation (P450scc, P450c17, 3betaHSD, aromatase = P450aro, and 11beta-hydroxylase = P45011beta). Expressions of P450scc, 3betaHSD, and P450c17 were all detected in male and female gonads at 55 days post-fertilization (dpf), i.e., two weeks before histological differentiation. There were no differences in their expression level respective to the sex. The androgen treatment was carried out by administration of 11beta-hydroxyandrostenedione (11betaOHDelta4) in genetic all-female populations and the resulting sex ratios were found to be 100% male even at a low dosage of 1 mg/kg of food. Following 11betaOHDelta4 treatment, only the expression of P450c17 was found to be sustained when compared with the female untreated control. In contrast, P450scc was clearly up-regulated and 3betaHSD and P450aro down-regulated by the androgen treatment. P45011beta gene expression remained low in gonads of androgen-treated females, as it did in control untreated females. These results together demonstrate that steroidogenesis in rainbow trout is potentially active in pre-differentiating gonads of both sexes, and that one of the masculinizing actions of androgens in the species may be to down-regulate the female-specific gonadal P450aro gene expression. However, in vivo androgen treatment in genetic females does not induce the same pattern of steroid gene expression as in genetic males. These data suggest that exogenous androgens might induce a male differentiation process with P450aro inhibition being one of the steps required. However, this process would not involve endogenously produced 11-oxygenated androgens.     

Efferent duct differentiation during female-to-male sex change in honeycomb grouper Epinephelus merra

Efferent duct (ED) differentiation was examined histologically during female-to-male sex change in the honeycomb grouper Epinephelus merra . During natural sex change, ED differentiation began with the appearance of slit-like structures between the stromal tissue and the tunica ovary and small oval-shaped spaces within the wall of the ovarian cavity, accompanied by oocyte degeneration and the initiation of spermatogenesis, i.e. the early transitional phase. In the late transitional phase, ED structure formation expanded and further ED differentiation occurred, including the rapid multiplication of spermatogonial germ cells. In sex-changed males, the slit-like structures increased in size, fused with each other and finally formed a well-developed ED. The oval-shaped spaces also increased in size and fused to form an ED. In contrast, during artificial sex change, induced by aromatase inhibitor (AI, 1 mg kg⁻¹), ED differentiation in E. merra was first observed as the appearance of slit-like structures and small oval-shaped spaces in the restructuring gonads in the third week after AI treatment. These were accompanied by oocyte degeneration and the proliferation of gonial germ cells into spermatogonia. In the fifth week, the rapid multiplication of spermatogonial germ cells, increases in 11-ketotestosterone (11-KT), and further differentiation of EDs were observed. Sex-changed males had testes containing sperm in the completely differentiated EDs; the significantly highest levels of 11-KT were observed in the sixth week. Simultaneous increases in 11-KT and initiation of ED differentiation were observed, suggesting a role of 11-KT in ED differentiation during sex change. There were no basic differences in the mechanisms of natural and artificially induced ED differentiation. Two types of structure led to the formation of EDs in two different areas of the newly formed testis during sex change.     

Immunohistochemical evidence identifying the site of androgen production in the ovary of the protogynous grouper <Emphasis Type="Italic">Epinephelus merra</Emphasis>

Androgen plays an important role in the developing ovaries of female fish. However, little is known regarding either the sites of production of androgen or its functional roles. In the present study, we investigated immunohistochemically the localization of cholesterol-side-chain-cleavage (P450scc) and cytochrome P45011-hydroxylase (P45011) with antibodies P450scc and P45011 in the ovary of the female honeycomb grouper Epinephelus merra during its reproductive cycle. Clusters of strongly immunopositive cells, with 100–1000 cells in each cluster, against both P450scc and P45011, were observed throughout the annual reproductive cycle in tissue near blood vessels in the tunica ovary surrounding the outer periphery of the ovary. The ultrastructural characteristics of these cells showed that they were steroid-producing cells. In contrast, immunopositive cells against P450scc but not against P45011 were localized in the theca layer surrounding the outer periphery of oocytes. These results suggest that two distinct steroid biosynthesis sites exist in the ovary and that cells at the two sites differ functionally. The only cells that biosynthesize 11-ketotestosterone are found in clusters in the vicinity of blood vessels; they possibly play a physiological role in oocyte growth and gonadal restructuring during the sex change of individuals of this species. CREST, JST (Japan Science and Technology Corporation), and the Takeda Science Foundation provided grants supporting this work. This study was also partially supported by a grant for the 21st century COE project entitled, The Comprehensive Analyses of Biodiversity in Coral Reef and Island Ecosystems in Asian and Pacific Regions. We also gratefully acknowledge a Japanese Government (Monbukagakusho) Scholarship.     

Gonadal morphogenesis and sex differentiation in intraovarian embryos of the viviparous fish Zoarces viviparus (Teleostei, Perciformes, Zoarcidae): a histological and ultrastructural study

It is essential to know the timing and process of normal gonadal differentiation and development in the specific species being investigated in order to evaluate the effect of exposure to endocrine-disrupting chemicals on these processes. In the present study gonadal sex differentiation and development were investigated in embryos of a viviparous species of marine fish, the eelpout, Zoarces viviparus, during their intraovarian development (early September to January) using light and electron microscopy. In both sexes of the embryos at the time of hatching (September 20) the initially undifferentiated paired bilobed gonad contains primordial germ cells. In the female embryos, ovarian differentiation, initiated 14 days posthatch (dph), is characterized by the initial formation of the endoovarian cavity of the single ovary as well as by the presence of some early meiotic oocytes in a chromatin-nucleolus stage. By 30 dph, the endoovarian cavity has formed. By 44 dph and onward, the ovary and the oocytes grow in size and at 134 dph, just prior to birth, the majority of the oocytes are at the perinucleolar stage of primary growth and definitive follicles have formed. In the presumptive bilobed testis of the male embryos, the germ cells (spermatogonia), in contrast to the germ cells of the ovary, remain quiescent and do not enter meiosis during intraovarian development. However, other structural (somatic) changes, such as the initial formation of the sperm duct (30 dph), the presence of blood vessels in the stromal areas of the testis (30 dph), and the appearance of developing testicular lobules (102 dph), indicate testicular differentiation. Ultrastructually, the features of the primordial germ cells, oogonia, and spermatogonia are similar, including nuage, mitochondria, endoplasmic reticulum, and Golgi complexes.     

Annual changes in testicular development and plasma sex steroids in the captive male dojo loach <Emphasis Type="Italic">Misgurnus anguillicaudatus</Emphasis>

Testicular development in the captive male dojo loach Misgurnus anguillicaudatus was examined monthly in relation to the levels of plasma sex steroids [testosterone (T), 11-ketotestostrone (11-KT), and 17,20β-dihydroxy-4-pregnen-3-one (DHP)]. On the basis of testicular histology, the annual gonadal cycle was found to be divisible into 3 periods: the recovery and proliferation period, which mainly consists of early spermatogenic testis from August to November (reproductive phase I); the preparation period for the next spawning period, which mainly consists of late spermatogenic testis from December to April (reproductive phase II); and the mature period, characterized by a high proportion of mature testis from May to July (reproductive phase III). Individual variability in testicular development was high, and continuous spermatogenesis was observed throughout the year. High levels of plasma T, 11-KT, and DHP were observed during reproductive phase III. 11-KT began to increase in February, while T was present at low levels in reproductive phase II. These results suggest that the physiologically active season of testis development for breeding in the dojo loach is from May to July, although spermatogenesis occurs throughout the year.     

Aromatase immunoreactivity and the role of enzymes in steroid pathways for inducing sex change in the hermaphrodite gobiid fish Trimma okinawae

The role of aromatase (Arom) in the process of bi-directional sex change in the gobiid fish Trimma okinawae was investigated by immunohistochemical methods. Irrespective of sexual phase, gonads comprised both ovarian and testicular tissues. In each sexual phase of females, the 2nd (2DF-M) and 4th (4DF-M) days after initiation of sex change to male, males, and the 2nd (2DM-F), 4th (4DM-F) and 6th (6DM-F) days after the initiation of reversion from male to female, ovarian and testicular histological observations were made. During the female, 2DF-M, 4DF-M and 6DM-F phases, the ovary contained vitellogenic and previtellogenic oocytes, compared with previtellogenic oocytes in the other phases. Although sperm was found in the testis in every phase, sperm ducts were apparent in the male phase, but not the female phase. Arom immunoreactivity was detected in the interstitial cells between the oocytes in all phases. On the other hand, it was localized in the thecal and granulosa cells of the follicular layer enclosing the oocytes in the female, 2DF-M, 4DF-M and 6DM-F phases. Activity of Arom in the thecal and granulosa cells is thought to be important for the development of oocytes and subsequent sex change.     

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.     

Aromatase inhibitor induces complete sex change in the protogynous honeycomb grouper (Epinephelus merra)

The protogynous hermaphrodite fish change sex from female to male at the certain stages of life cycle. The endocrine mechanisms involved in gonadal restructuring throughout protogynous sex change are not clearly understood. In the present study, we implanted maturing female honeycomb groupers with nonsteroidal aromatase inhibitor (AI), Fadrozole (0, 1, and 10 mg/fish) and examined changes in gonadal structures and serum levels of sex steroid hormones 2(1/2) months after implantation. The ovaries of control females had oocytes undergoing active vitellogenesis, whereas AI caused females to develop into functional males. These males had testes, which were indistinguishable in structure from those of normal males, but bigger in size, and completed all stages of spermatogenesis including accumulation of large amount of sperm in the seminiferous tubules. AI significantly reduced the serum levels of estradiol-17beta (E2) and increased levels of testosterone (T), 11-ketotestosterone (11-KT), and 17alpha, 20beta-dihydroxy-4-pregnen-3-one (DHP). Further, AI suppressed in vitro production of E2, and stimulated the production of T and 11-KT in the ovarian fragments of mature female. In the honeycomb grouper, suppression of both in vitro and in vivo production of E2 and degeneration of oocytes by AI suggests that AI induces complete sex change through inhibition of estrogen biosynthesis, and perhaps, subsequent induction of androgen function.     

Gonadal sex reversal in triploid rainbow trout (Oncorhynchus mykiss).

Sex determination in salmonids is primarily governed by sex chromosomes; however, phenotypic expression and successful development of the gonads may be influenced by additional factors. Exposure to exogenous steroids during the critical period of gonadal differentiation will reverse the expected phenotypic sex of both female and male trout. Triploidy, a viable condition in rainbow trout (RBT), alters the degree of gonadal development in a gender-specific manner. Males produce testes with similar morphology and function as diploid fish, but females produce underdeveloped ovaries devoid of growing oocytes. One possible explanation for this observed gender difference is that the timing of meiotic initiation may influence ovarian/testicular development in triploid RBT. To determine whether the early entrance of germ cells into meiosis results in the lack of ovarian development in triploid females, the objective of this study was to sex-reverse genotypic triploid female RBT (XXX) into phenotypic males and genotypic triploid male RBT (XXY) into phenotypic females. Male fish were exposed to estradiol-17beta (E(2)) and females were exposed to the non-aromatizable androgen 17alpha-methyldihydrotestosterone (MDHT). Over 90% of the male fish treated with exogenous E(2) developed gonadal structures indistinguishable from the gonads of triploid females. Triploid female RBT treated with MDHT developed testes; however, not all fish treated with this androgen were completely sex reversed. The results of this investigation are consistent with the hypothesis that the failure of ovarian development in triploid RBT is due to the early onset of meiosis and does not appear to be due to genotypic sex. J. Exp. Zool. 284:466-472, 1999.     

Seasonal changes of serum sex steroids concentration and aromatase activity of gonad and brain in red-spotted grouper (Epinephelus akaara)

Levels of serum sex steroids (estradiol-17beta, E2; testosterone, T; 11-ketotestosterone, 11-KT) in male, female and natural sex-reversing red-spotted grouper (Epinephelus akaara), and aromatase activity of gonad and brain in both male and female were investigated throughout an annually reproductive cycle. In females, serum E2 and T peaked during vitellogenesis, but in males and natural sex-reversing fish, 11-KT, T and E2 reached peak during spermatogenesis. In addition, in females, serum 11-KT levels (monthly means: 0.32 +/- 0.03 ng/ml) which were very low did not significantly fluctuate during the annual reproductive cycle. In breeding season, females displayed higher E2 levels than males and sex-reversing fish, while males and sex-reversing fish showed higher 11-KT levels and, to a lesser extent, higher T levels than females. Furthermore, the changing pattern of sex steroids in males was similar to that in natural sex-reversing fish, and a second peak of serum androgens 11-KT and T appeared in December both in male and natural sex-reversing fish; significantly higher serum 11-KT levels were observed in natural sex-reversing fish than that in females from December to April. In females, but not in males, aromatase activity of brain and gonad demonstrated significantly seasonal changes (exhibiting a peak in breeding season); moreover, aromatase activity in females was higher than that in males. Furthermore, significantly lower aromatase activity in testis was observed in breeding season, in contrast to that in ovary. Taken together, the present findings indicated that changes of serum sex steroids levels and aromatase activity in red-spotted grouper were closely associated with sex inversion. In addition, the present results also suggested that sex inversion in red-spotted grouper peaked mainly from December to March.     

Bi-directional sex change and gonad structure in the gobiid fish <Emphasis Type="Italic">Trimma yanagitai</Emphasis>

Bi-directional sex change in the deep-water gobiid fish Trimma yanagitai was examined. The gonads of all individuals consisted of ovarian and testicular elements, and an accessory gonadal structure. In no gonads were both testicular and ovarian parts simultaneously active. Bi-directional sex changes occurred during the rearing experiments in aquaria under conditions of which there was co-existence of two males or plural females. The sex of individuals could be determined by their relative body size or social dominance: the largest individuals acting as male and the remainder as female.     

Studies of gonadal sex differentiation

Gonadal differentiation has a determinative influence on sex development in human embryos. Disorders of sexual development (DSD) have been associated with persistent embryonal differentiation stages. Between 1998 and 2015, 139 female patients with various (DSD) underwent operations at the Scientific Center of Obstetrics, Gynaecology and Perynatology in Moscow, Russia. Clinical investigations included karyotyping, ultrasound imaging, hormonal measurement and investigations of gonadal morphology. The male characteristics in the embryo are imposed by testicular hormones. When these are absent or inactive, the fetus may be arrested at between developmental stages, or stay on indifferent stage and become phenotypically female. A systematic analysis of gonadal morphology in DSD patients and a literature review revealed some controversies and led us to formulate a new hypothesis about sex differentiation. Proliferation of the mesonephric system (tubules and corpuscles) in the gonads stimulates the masculinization of gonads to testis. Sustentacular Sertoli cells of the testes are derived from mesonephric excretory tubules, while interstitial Leydig cells are derived from the original mesenchyme of the mesonephros. According of the new hypothesis, the original mesonephric cells (tubules and corpuscles) potentially persist in the ovarian parenchyma. In female gonads, some mesonephric excretory tubules regress and lose the tubular structure, but form ovarian theca interna and externa, becoming analogous to the sustentacular Sertoli cells in the testis. The ovarian interstitial Leydig cells are derived from intertubal mesenchyme of the mesonephros, similar to what occurs in male gonads (testis). Surprisingly, the leading determinative factor in sexual differentiation of the gonads is the mesonephros, represented by the embryonic urinary system.