Process Of Gonadal Sex Differentiation Research Articles

Gonadal sex differentiation and early ovarian/testicular development in cultured Pacific bluefin tuna, Thunnus orientalis (Temminck et Schlegel)

Pacific bluefin tuna (PBT), Thunnus orientalis, is one of the most important species for aquaculture in Japan. Recently, the reduction in muscle fat content associated with sexual maturation in farmed PBT has become a serious problem. To develop technologies for inducing sterility, detailed and reliable data on gonadal development in PBT are needed. Here, we demonstrated the process of gonadal sex differentiation, and of early ovarian and testicular development during the immature stages in PBT. Gonadal sex differentiation was first characterized by the formation of the ovarian cavity in female and of the efferent ducts in male 57 days post hatching (dph). The gonads then differentiated into ovaries or testes according to the genotypic sex until 83 dph. During this period, primordial germ cells, oogonia, and type-A spermatogonia were solitarily distributed in the gonads, and the number of germ cells did not differ between sexes. After gonadal sex differentiation, gonads of PBTs developed in a sexually dimorphic manner: proliferation and differentiation of germ cells occurred earlier in the ovaries than in the testes. The oogonia in ovaries formed cysts at 185 dph, but the type-A spermatogonia were solitarily distributed in testes at this stage, and cysts of type-A spermatogonia were first observed at 247 dph. Moreover, the oogonia entered meiosis and differentiated into chromatin-nucleolus stage oocytes until 247 dph, and subsequently into peri-nucleolus stage oocytes until 285 dph, whereas the type-A spermatogonia differentiated into type-B spermatogonia, spermatocytes, spermatids, and spermatozoa from 446 dph onwards. We believe the results of this study provide the necessary basis for future studies on sterile PBT production.

Comprehensive transcriptomic analysis of the process of gonadal sex differentiation in the turbot (Scophthalmus maximus)

The turbot is a flatfish with a ZW/ZZ sex determination system but with a still unknown sex determining gene(s), and with a marked sexual growth dimorphism in favor of females. To better understand sexual development in turbot we sampled young turbot encompassing the whole process of gonadal differentiation and conducted a comprehensive transcriptomic study on its sex differentiation using a validated custom oligomicroarray. Also, the expression profiles of 18 canonical reproduction-related genes were studied along gonad development. The expression levels of gonadal aromatase cyp19a1a alone at three months of age allowed the accurate and early identification of sex before the first signs of histological differentiation. A total of 56 differentially expressed genes (DEG) that had not previously been related to sex differentiation in fish were identified within the first three months of age, of which 44 were associated with ovarian differentiation (e.g., cd98, gpd1 and cry2), and 12 with testicular differentiation (e.g., ace, capn8 and nxph1). To identify putative sex determining genes, ∼4.000 DEG in juvenile gonads were mapped and their positions compared with that of previously identified sex- and growth-related quantitative trait loci (QTL). Although no genes mapped to the previously identified sex-related QTLs, two genes (foxl2 and 17βhsd) of the canonical reproduction-related genes mapped to growth-QTLs in linkage group (LG) 15 and LG6, respectively, suggesting that these genes are related to the growth dimorphism in this species.

Gonadal sex differentiation in the Senegalese sole (Solea senegalensis) and first data on the experimental manipulation of its sex ratios

Like other flatfishes, the Senegalese sole exhibits sexual growth dimorphism in favor of females and thus all-female stocks would be desirable. However, reproduction-associated problems persist and basic aspects of its biology such as sex determination and differentiation are still unknown. In this study, we histologically examined the process of gonadal sex differentiation in the Senegalese sole and tested the effects of treatment with sex steroids and an aromatase inhibitor on sex ratios. Sex differentiation in females started before 98days post fertilization (dpf), when fish had an average total length (TL) of ~33mm. In contrast, males began sex differentiation by 127dpf with a mean TL of ~44mm. At this time, an incipient growth advantage in favor of females was already observed. All fish were sexually differentiated by ~48mm TL (~170dpf). Treatment between 98 and 169 dpf with the synthetic androgen 17α-methyldihydrotestosterone (10mg/kg) or the aromatase inhibitor fadrozole (100mg/kg) significantly increased the proportion of males up to ~95% while the natural estrogen, estradiol-17β (10mg/kg), increased the proportion of females by about 50% but reduced growth with respect to the control values. Together, these results show that the Senegalese sole is a differentiated gonochoristic species in which gonads start to form within the first three months and sex differentiation is completed after animals reach their fifth month of age, with females differentiating earlier than males. Results also indicate that the labile period starts at <98dpf and lasts until ~125dpf. These results show that sex control can be achieved in the Senegalese sole, an important step towards the establishment of all-female stocks to improve the currently compromised production of this economically important species.

Gonadal sex differentiation in the Malabar grouper, Epinephelus malabaricus

To obtain basic information on sex differentiation in groupers, the process of gonadal sex differentiation in the Malabar grouper, Epinephelus malabaricus, was followed using histology for one year from hatching. Gonads of fish less than 39 days post-hatch (DPH) were undifferentiated and consisted of a few germ cells surrounded by somatic cells. There were no morphological indications of sex differentiation at this stage. By approximately 47 DPH, however, two elongations of somatic cells had formed in the gonads on the sides facing the gonad lateral walls. These elongations correspond morphologically to the initiation of ovarian cavity formation, a characteristic of ovarian differentiation in other teleost fish. By 74 DPH, the elongations had developed further and were evident in the ovaries of all fish. However, by 144 DPH, ovarian germ cells had not yet begun active division. Oocytes were first evident in the ovaries at 243 DPH, distributed mainly within the somatic tissue on the distal sides of the ovaries facing the ovarian cavity. The oocytes increased in number and in size from 243 to 360 DPH. These observations demonstrate that morphological changes associated with ovarian differentiation in the Malabar grouper begins at approximately 47 DPH and that in all individuals the gonads differentiate directly into ovaries.

Early aspects of gonadal sex differentiation in Xenopus tropicalis with reference to an antero‐posterior gradient

In an effort to contribute to the development of Xenopus tropicalis as an amphibian model system, we carried out a detailed histological analysis of the process of gonadal sex differentiation and were able to find evidence that gonadal differentiation in X. tropicalis follows an antero-posterior gradient. Although the main reason for the presence of a gradient of sex differentiation is still unknown, this gradient enabled us to define the early events that signal ovarian and testicular differentiation and to identify the undifferentiated gonad structure. Given the various advantages of this emerging model, our work paves the way for experiments that should contribute to our understanding of the dynamics and mechanisms of gonadal sex differentiation in amphibians.

Where does gonadal sex differentiation begin? Gradient of histological sex differentiation in the gonads of pejerrey, Odontesthes bonariensis (Pisces, Atherinidae)

This study investigated the possibility that the histological process of gonadal sex differentiation in pejerrey (Odontesthes bonariensis), a fish with marked temperature-dependent sex determination (TSD), occurs through a predictable gradient of differentiation as opposed to simultaneous or random differentiation throughout the gonad. For this purpose, fish reared at 17 degrees, 24 degrees, and 29 degrees C from hatching were sampled weekly for 11 weeks, fixed, and prepared for histological observation of serial cross-sections of the gonads. The thermal manipulation and sampling procedure ensured the availability of males and females at various degrees of gonadal sex differentiation. The location of the differentiated area(s) was estimated in the right and left gonads of 17 females and 14 males selected among the available specimens so as to represent increasing degrees of differentiation. The analysis revealed that sex differentiation followed a gradient from the anterior to posterior areas of the gonads regardless of sex. Furthermore, plotting of the degree of sex differentiation in the right gonad as a function of the degree of differentiation of the left gonad clearly showed that sex differentiation only begins in the right gonad when 10-30% of the length of the left gonad has already differentiated. The mean rostral edge of the differentiated areas in females was 9% and 10.8% for the left and right gonads, respectively, while for males these values were 7.3% and 7.0%, respectively. Thus, it was established that ovarian and testicular differentiation in pejerrey follow both a cephalocaudal and a left-to-right gradient. Possible explanations for this gradient and its relevance for TSD in pejerrey, that is, as a mechanism to prevent discrepant differentiation of male and female features within the same gonad, are discussed.

Aromatase activity in the European sea bass ( Dicentrarchus labrax L.) brain. Distribution and changes in relation to age, sex, and the annual reproductive cycle

Cytochrome P450 aromatase activity (AA) was measured in different tissues of the sea bass ( Dicentrarchus labrax L.) using a tritiated water release assay that was previously optimized and validated for this species. In adult fish entering the reproductive season, AA was highest on a per mg protein basis, in the brain (2.04±0.4 pmol/mg prot/h; mean ± SEM), followed by the ovary (0.59±0.1) and was detectable in visceral fat (0.21±0.05), liver (0.08±0.009), and head kidney (0.03±0.004). However, AA was negligible in the rest of the tissues tested: heart, testis, muscle, and spleen. Consistent with results obtained in other species, dissection of the brain into its major constitutive parts revealed that AA was concentrated in areas implicated in the control of reproduction, including the olfactory bulb, telencephalon, and hypothalamus (range: 2.6–16.2 pmol/mg prot/h), as well as the pituitary gland (6.2–9.3 pmol/mg prot/h). Lower AA was noted in the optic bulb, cerebellum, and medulla. However, in contrast to some previously published reports concerning the content and distribution of neural aromatase in fish, males consistently exhibited higher AA than females. In one-year-old juvenile fish completing the process of gonadal sex differentiation, brain AA (0.63 pmol/mg prot/h) was similar in both sexes and ten times lower than that measured in the brain of first time spawners (6.52 pmol/mg prot/h), in this case with males showing an overall higher (24%) activity than females. When surveyed throughout the year, brain AA dramatically changed during the reproductive cycle. Maximum average values of ∼7 pmol/mg prot/h were obtained that coincided with the spawning season. The peak in brain AA was preceded by two and one months by the peak of plasma testosterone and the peak of the gonadosomatic index, respectively. This is the first measurement of the distribution of the activity of a steroidogenic enzyme in the sea bass, an established model in comparative endocrinology. Together, these results demonstrate sex- and seasonally-related variations in AA and establish the basis for further comparative studies of certain androgen-mediated actions through locally formed estrogen in both central and peripheral targets.

Gonadal sex differentiation in Atlantic halibut

The process of gonadal sex differentiation in 338 Atlantic halibut Hippoglossus hippoglossus larvae, ranging in size from 10 mm Ls to 230 mm LF, is described histologically. Gonadal sex differentiation occurred by 38.0 mm LF, which coincided with the weaned, post‐metamorphic, settled stage in the life cycle. This was a gradual process that coincided with other organogenesis in the developing larvae.

Morphology, endocrinology, and environmental modulation of gonadal sex differentiation in teleost fishes

Successful reproduction by an adult depends on the normal ontogenesis of the gonads, a complex process of cellular and histological differentiation that starts early in life. This process is theoretically predetermined by genetic factors and includes sensitisation of the bipotential gonads to endogenous endocrine factors prior to, during and even after commitment to maleness or femaleness. However, young fish are relatively vulnerable to a host of environmental (physical and chemical) factors that can affect this endogenous endocrine axis, disturbing or even overriding the putative developmental pathway. This sexually lability can be exploited to our advantage for the production of monosex fish populations of the most valuable sex for food production or aquarium fish trade. On the other hand, it represents also a potential path for undesirable influences from endocrine-disrupting chemicals and climatic factors, particularly environmental temperature. This paper provides a detailed account of the early histological process of gonadal sex differentiation, with special reference to gonochoristic species, and reviews the criteria employed to positively identify ovarian and testicular differentiation. It also reviews the development of endocrine competence and sensitivity of the differentiating gonads to exogenous influences in the context of the relative stability of genotypic sex determination in various fish species. Sex differentiation in some species seems to be under strong genetic control and may not require endogenous sex steroid production. Conversely, reliance on endogenous sex steroids for gonadal differentiation is observed in other species and this phenomenon is apparently associated with a higher incidence of environment (mainly temperature)-labile sex differentiation.

Histological observations of gonadal sex differentiation in the F2 hybrid sturgeon, the bester

: The process of gonadal sex differentiation in the F2 hybrid sturgeon, the bester (Huso huso female ×Acipenser ruthenus male), was investigated histologically. In fish more than 6 months old, two types of gonads were distinguishable. In the putative ovary, the germinal epithelium had invaginations in the stroma of the gonad, and a few germ cells were localized immediately below the epithelium. In putative testis, invaginations were lacking and germ cells were observed deep in the stroma. However, active mitotic and meiotic germ cells were not observable even by 16 months of age in ovaries, and only some of the testes showed initiation of spermatogenesis by 21 months of age. The invaginations in the stroma of putative ovaries developed into ovarian lamella-like structures, whereas the gonadal surface of putative testes remained smooth. These results suggest that the formation of invaginations in the stroma of gonads is the morphological index of ovarian differentiation and that it is necessary to start hormone treatment for functional sex reversal before 6 months of age in these fish.

Temperature effects on sex differentiation of two South American atherinids, Odontesthes argentinensis and Patagonina hatcheri

The present study investigated the effects of water temperature (18, 21, and 25 °C) on the histological process of gonadal sex differentiation of two commercially important atherinid fishes from South America, Odontesthes argentinensis (sea pejerrey) and Patagonina hatcheri (Patagonian freshwater pejerrey). In both species, female gonadal sex differentiation began with the formation of lateral stromal cell outgrowths and the appearance of meiotic oocytes. The male gonads remained quiescent for about twice as long as the female gonads, with differentiation becoming evident by the formation of the main sperm duct and of cysts of germ cells at the periphery of the gonads. Meiosis in males occurred relatively long after somatic differentiation of the testis. The ovaries of O. argentinensis differentiated at 28 days (20.3 mm) at 25 °C, 42 days (24.0 mm) at 21 °C, and 56 days (23.8 mm) at 18 °C. In the males, differentiation was observed at 98 days at 25 and 21 °C (39.4 mm and 40.4 mm, respectively), but at 112 days under 18 °C (40.7 mm). In P. hatcheri, differentiation of females occurred at 21 days (17.8 mm) at 25 °C, 28 days (20.8 mm) at 21 °C, and 35 days (23.2 mm) at 18 °C. Male differentiation became evident at 56 days under 25 and 21 °C (30.8 and 32.7 mm, respectively), and at 70 days (37.7 mm) at 18 °C. The sex-ratios of O. argentinensis reared at 18 or 21 °C were female-biased whereas those at 25 °C were not; groups reared at 18 °C had significantly more females than groups from the same progeny reared at 25 °C. In contrast, the sex-ratios in all groups of P. hatcheri did not differ significantly from 1:1 and no significant differences were found between groups of the same progeny reared at different temperatures. These results suggest the occurrence of thermolabile sex determination (TSD) in O. argentinensis whereas in P. hatcheri gonadal sex appears to be strongly genetically determined.

Sex differentiation and hormonal feminization in pejerrey Odontesthes bonariensis

An investigation was conducted on the histological process of gonadal sex differentiation and the suitability of steroid hormone administration for sex control in pejerrey, Odontesthes bonariensis. Light histological examination of the gonads of larvae and juvenile fish revealed that the primordial gonads were already formed at hatching, but remained sexually undifferentiated until 49 days post-hatch (14.9 mm standard length). Germinal (mitosis and meiosis) and somatic (cluster and outgrowth of somatic cells from the ventral and dorsal edges of the gonads) differentiation began virtually simultaneously in ovaries between 49 and 63 days (18.2 mm). Fusion of the free edges of the outgrowths and formation of the ovarian cavity occurred between 91 and 112days (27.5–37.4 mm). Somatic differentiation (formation of the main sperm duct) began concomitant with intense germ cell mitosis at 98 days (30.4 mm), whereas occasional meiotic activity was observed after 119 days (41.4 mm) in male-type gonads. Only one case of intersexuality was observed, which was characterized by the presence of a small number of diplotene-stage oocytes in an otherwise normally developing testis. This situation was deemed abnormal and did not preclude the characterization of pejerrey as a ‘differentiated’ type of gonochorist teleost. Estradiol-17β administered orally at 20 and 50 mg kg −1 diet from 28 to 105 days (10.9–33.7 mm) produced 100% females. The combined results of histological analysis and hormone administration suggest that the critical period of sex determination in pejerrey reared under gradually rising water temperatures (16–23 °C) occurs between 28 and 49 days post-hatch.

Life-history pathways in relation to gonadal sex differentiation in the anemonefish, Amphiprion clarkii, in temperate waters of Japan

The process of gonadal sex differentiation in Amphiprion clarkii was investigated for 2 years at Murote Beach, Shikoku Island, Japan. Six color phases were discriminated on the basis of the caudal fin coloration, which corresponded well to six gonadal phases. From changes of the color phases with growth, three life history pathways were detected: (1) subadult male → subadult female → adult female, (2) subadult male → adult male → adult female, (3) subadult male → adult male. Different pathways were due to the difference of timing among individuals in the development of ovarian tissues of the hermaphroditic gonads involving the atrophy of testicular tissues. Irreversible differentiation of ovarian tissues of the gonads occurred more frequently among nonbreeders (10 cases) than among breeders (4 cases). The second pathway, which has been thought the norm of tropical anemonefishes, was therefore not primary in this population. This can be attributed to ecological conditions: fish are able to move between host sea anemones and nonbreeders can escape from social suppression by adult pairs because of high population density of hosts.