Onset Of Sex Change Research Articles

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, Notolabruscelidotus, 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 17β-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.

Bulk and single-cell RNA-seq reveal the sexually dimorphic expression pattern of dmrtb1 in zig-zag eel (Mastacembelus armatus)

Sequential hermaphroditism, whereby the sex of one individual changes during its development, has been described in many fish species. Yet, the genetic regulation of this dynamic process of sexual differentiation is not well understood. Here, we report the characterization of intersexual differences in an aquacultural fish, the zig-zag eel (Mastacembelus armatus), based on 4 years of artificial breeding. All M. armatus individuals initially developed as females, with approximately half of them then becoming males after going through an intersex phase. A DM-domain candidate gene, dmrtb1, was identified from gonadal transcriptome data screening. The expression of dmrtb1 was restricted to testis, similar to dmrt1, which is one of the conserved sex-determining genes in vertebrates. Interestingly, dmrtb1 was expressed at the onset of sex change (early stage), and gradually increased in expression in the intersex gonads until the late stage of differentiation. Dmrtb1 was also expressed in the seminiferous lobules containing cysts with germ cells. A mixed cell population from ovotestis was identified by single-cell RNA sequencing, and included mitotic fetal germ cells, meiotic prophase fetal germ cells, and gonad endothelial cells. Spermatocyte and oocyte marker genes were both enriched in the same gonadal cell population, indicating this cell population might have the potential of bisexual differentiation. The correlation between dmrtb1 expression and sex-change development suggests M. armatus as an emerging model to study the molecular mechanism of sexual differentiation in fish. Such information will be vital for promoting the conservation of this species in the wild as well as for developing a sustainable aquacultural industry.

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 Genetics and Epigenetics of Sex Change in Fish.

Fish show extraordinary sexual plasticity, changing sex naturally as part of their life cycle or reversing sex because of environmental stressors. This plasticity shows that sexual fate is not an irreversible process but the result of an ongoing tug-of-war for supremacy between male and female signaling networks. The behavioral, gonadal, and morphological changes involved in this process are well described, yet the molecular events that underpin those changes remain poorly understood. Epigenetic modifications emerge as a critical link between environmental stimuli, the onset of sex change, and subsequent maintenance of sexual phenotype. Here we synthesize current knowledge of sex change, focusing on the genetic and epigenetic processes that are likely involved in the initiation and regulation of sex change. We anticipate that better understanding of sex change in fish will shed new light on sex determination and development in vertebrates and on how environmental perturbations affect sexual fate.

Stress and sex: does cortisol mediate sex change in fish?

Cortisol is the main glucocorticoid (GC) in fish and the hormone most directly associated with stress. Recent research suggests that this hormone may act as a key factor linking social environmental stimuli and the onset of sex change by initiating a shift in steroidogenesis from estrogens to androgens. For many teleost fish, sex change occurs as a usual part of the life cycle. Changing sex is known to enhance the lifetime reproductive success of these fish and the modifications involved (behavioral, gonadal and morphological) are well studied. However, the exact mechanism behind the transduction of the environmental signals into the molecular cascade that underlies this singular process remains largely unknown. We here synthesize current knowledge regarding the role of cortisol in teleost sex change with a focus on two well-described transformations: temperature-induced masculinization and socially regulated sex change. Three non-mutually exclusive pathways are considered when describing the potential role of cortisol in mediating teleost sex change: cross-talk between GC and androgen pathways, inhibition of aromatase expression and upregulation of amh (the gene encoding anti-Müllerian hormone). We anticipate that understanding the role of cortisol in the initial stages of sex change will further improve our understanding of sex determination and differentiation across vertebrates, and may lead to new tools to control fish sex ratios in aquaculture.

Ovarian degeneration during the female-to-male sex change in the protogynous hermaphroditic fish, Halichoeres poecilopterus

Halichoeres poecilopterus (Labridae) is a protogynous, hermaphroditic fish common in temperate waters around Japan. Although oocyte degeneration is an established event at the onset of sex change, there is little information on the process. We examined cell numbers and proportional ratios of ovarian germ cells in sex-changing gonads of H. poecilopterus. This confirmed a lack of YV (yolk vesicle stage) and Y (yolk stage) oocytes, a rapid decrease of P (perinucleolus stage) oocytes, and a slight increase of CN (before/at chromatin nucleolus stage) germ cells. We discuss gonadal conditions necessary for the protogynous sex change of H. poecilopterus.

Short term treatment with aromatase inhibitor induces sex change in the protogynous wrasse, Halichoeres trimaculatus

The purpose of this study was to specify the time when individuals are committed to female to male sex change in the protogynous wrasse, Halichoeres trimaculatus, induced by treatment with the nonsteroidal aromatase inhibitor (AI) Fadrozole. In this study, treatment with AI was carried out by providing adult females with a diet containing 500 μg AI/g food for 3 (AI-3), 5 (AI-5), and 10 days (AI-10). We examined the gonadal structure of the fishes histologically at the end of the AI treatment and 30 days after the start of the experiment. At the end of the AI treatment, all individuals in the AI-3 treated group had gonads with degeneration of yolky oocytes, indicating the onset of sex change. Most individuals in the AI-5 treated group had gonads with atretic vitellogenic oocytes, like those in AI-3 treated group, whereas most individuals in the AI-10 treated group had gonads with testicular tissue. At 30 days after the onset of the experiment, approximately 70% of the individuals in the AI-3 treated group had mature ovaries, whereas all fishes in AI-5 and AI-10 treated groups had mature testes, indicating sex change. Therefore, treatment with AI for only 5 days resulted in complete sex change. Our results also indicate that crucial events for testicular differentiation occur within 5 days from the start of AI treatment. Thus, we conclude that females are committed to change into males after 5 days of AI treatment.

Sex Change in the Gobiid Fish Is Mediated through Rapid Switching of Gonadotropin Receptors from Ovarian to Testicular Portion or Vice Versa

Sex-changing fish Trimma okinawae can change its sex back and forth from male to female and then to male serially, depending on the social status in the harem. T. okinawae is well equipped to respond to its social status by possessing both ovarian and testicular tissues even though only one gonad remains active at one time. Here we investigated the involvement of gonadotropins in sex change by determining the changes in gonadotropin receptor (GtHR) gene expression during the onset of sex change from female to male and male to female. The expression of the GtHR was found to be confined to the active gonad of the corresponding sexual phase. During the sex-change from female to male, initially the ovary had high levels of FSHR and LHR, which eventually went up in the testicular tissue if the fish was bigger. Changing of the gonads started with switching of GtHR expression discernible within 8-12 h of the visual cue. Further in vitro culture of the transitional gonads with a supply of exogenous gonadotropin (human chorionic gonadotropin) revealed that the to-be-active gonad acquired the ability to produce the corresponding sex hormone within 1 d of the activation of GtHR. Conversely, the to-be-regressed gonad did not respond to the exogenous gonadotropin. Our findings show that the gonads of successive sex-changing fish possess the intrinsic mechanism to respond to the social cue differentially. Additionally, this location switching of GtHR expression also could substantiate the importance of the hypothalamo-pituitary-gonadotropic axis.

Anatomical changes to the gonad during protogynous sex change in the half‐moon grouper Epinephelus rivulatus (Valenciennes)

Anatomical changes to the gonad during sex change in the protogynous grouper Epinephelus rivulatus are described from histological observations. A decrease in ovarian mass occurred soon after the onset of sex change as oocytes atrophied and were removed from the gonad. Blood supply and abundance of unidentified somatic cells increased at this time as proliferation of sperm tissue commenced. As the gonad was cleared of ovarian tissue, the rate of spermatogenesis increased and the lamellae soon became dominated by sperm and connective tissue. Putative Leydig cells, the probable sites of male steroid production, appeared in transitional gonads and were most abundant in the testes of immature males. Peripheral sperm sinuses subsequently formed within basal tissue layers of the tunica and expanded as they filled with spermatids. The process of sex change, occurring as a result of experimental manipulation of wild populations at the start of the spawning season, took c. 3 weeks. This appears rapid compared to other hermaphroditic species and may reduce the impacts of fishing on reproductive output by E. rivulatus populations.

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.

Treatment with an aromatase inhibitor induces complete sex change in the protogynous honeycomb grouper (Epinephelus merra)

To study the mechanism of protogynous sex change in honeycomb grouper, the wild fish of both sexes and transitionals were collected for one year, and changes in gonadal structures and serum levels of sex hormones in each individual were examined. The onset of sex change was associated with low serum estradiol - 17β (E2) levels. In order to clarify whether E2 deprivation causes sex change, maturing females were implanted with the non-steroidal aromatase inhibitor (Fadrozole, AI). AI caused complete sex change from female to functional males in two and half months. The present results suggest that E2 is an important endogenous factor which regulates protogynous sex change in the honeycomb grouper.

Female and male territoriality and mating system of the sand tilefish,Malacanthus plumieri

The social and reproductive biology of the sand tilefish,Malacanthus plumieri (Malacanthidae), was studied at Glover's Reef, Belize, where this species occurs in colonies over sand-rubble flats. Individuals each occupy a home burrow refuge and a surrounding home range. Home range overlap among adjacent fish of the same sex is low, and individuals defend exclusive use of much of their home range against all conspecifics except mates (i.e., territoriality). Areas defended by males overlap the territories of up to 6 females; and male territory area is positively related to the number of female residents. Males maintain dominance over females within their territories by aggression, including intervention into some female disputes. Females spawn pelagically-dispersed eggs as frequently as every day. Each female spawns near her burrow, almost exclusively with the male whose defended area encompasses her territory (harem polygyny). Tilefish colonies therefore consist of a mosaic of female territories over which adjacent male territories are superimposed. Histological evidence and observation of behavioral sex change in one female revealed thatM. plumieri is capable of protogynous sex reversal. Females did not change sex in response to removal of one male. Occurrence of small transitional fish indicates that the onset of sex change is controlled by factors other than size-related social hierarchies within harems or colonies.

Increased H-Y antigen levels associated with behaviorally induced, female-to-male sex reversal in a coral-reef fish

It has been proposed that H-Y antigen is the synthetic product of sex-determining genes, and that H-Y antigen controls ontogenetic differentiation of the heterogametic sex throughout vertebrates. The coral-reef fish Anthias squamipinnis is a protogynous hermaphrodite in which all individuals mature initially as females. Males result when adult females change sex as a consequence of alterations in behavioral interactions within social groups. Three assay methods were used to measure H-Y antigen levels in the spleens, gonads, and epidermal tissue of 16 adult females and in 16 males that had been induced to change sex from a prior female phase by the removal of a pre-existing male from each of 16 social groups. In 15 male-female pairs, the H-Y antigen levels were higher in male than in female spleen, gonad, and epidermis tissues. The precise temporal relationship between the onset of sex change and the increase in the H-Y antigen level was not examined. If, as we strongly suspect, the temporal relationship proves to be close, the inference will be that the behavioral cues inducing sex change also influence the synthetic activity of genes controlling H-Y antigen production.