A new experimental model for the investigation of sequential hermaphroditism.

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

The Testis Is a Primary Factor That Contributes to Epigenetic Modifications in the Ovaries of the Protandrous Black Porgy, Acanthopagrus schlegelii.

In most hermaphroditic fish, the sexual phase of the gonad responds to external stimuli so that only one sex remains functional while the other sex becomes dormant. However, protandrous black porgy are male during their first two reproductive cycles. Estradiol (E2)-induced female growth results in a transient and immature female, and the sexual phase reverts from female to male after E2 is withdrawn. Conversely, excising the testis results in a precocious female when performed during the second reproductive cycle. We used these characteristics to study epigenetic modifications of cyp19a1a promoter in black porgy. Our results showed that higher levels of gonadotropins receptors were observed in testis than in ovary during the alteration of sexual phase from induced femaleness to maleness, and hCG treatment did not stimulate ovarian gene expression in male (1-yr-old maleness) and female phase (testis excision-induced femaleness) fish. The cyp19a1a promoter exhibited tissue- and lineage-specific methylation patterns. The follicle cells in the ovary had a hypomethylated (0%-20%) cyp19a1a promoter region. In the ovary, the first sign of female phase decision was decreased methylation levels and increased numbers of hypomethylated clones of cyp19a1a promoter during the natural sex change process. Similar methylation patterns were observed in the testis-removed ovary 1 mo after surgery, with no histological difference between the sham and the testis-removed fish. Conversely, there was no increase in methylation levels of cyp19a1a promoter in E2-fed fish. These results suggest that in the digonic gonad of black porgy, the testis is the primary tissue that affects epigenetics of the ovary.

Cloning and pattern of gsdf mRNA during gonadal development in the protogynous Epinephelus akaara.

Gonadal soma-derived factor (gsdf) is a teleost- and gonad-specific growth factor involved in early germ cell development. The red spotted grouper, Epinephelus akaara, as a protogynous hermaphrodite, provides a novel model for understanding the mechanisms of sex determination and differentiation in teleosts. In the present study, a 2307-bp long gsdf gene was cloned from E. akaara and there was further analysis of its tissue distribution and gonadal patterns of gene expression during the female phase and sex change developmental stages. The cellular localization of gsdf at the late transitional developmental stage was also analyzed. In addition, the concentrations of serum sex steroid hormones (E2, 11-KT and DHP) were determined. The gsdf transcripts were exclusively localized in the gonad. During the female phase at an early developmental stage, when the ovotestis contained mainly oogonia and primary growth oocytes, the gsdf mRNA was relatively more abundant. The relative abundance of gsdf decreased, however, and the lesser amount was sustained with the advancement of oocyte development. During the transitional phase, the relative abundance of gsdf mRNA increased slightly at the early developmental stage and there were further increases in relative abundance in the late developmental stage, and the gsdf transcripts were observed in the Sertoli cells surrounding early developing spermatogonia. Among the sex steroids, 11-KT concentrations were positively correlated with amount of gsdf mRNA during sex change. These results suggest that gsdf could have roles in regulating pre-meiotic germ cell proliferation and be involved in sex change in E. akaara.

Identification and expression of GnRH2 and GnRH3 in the black sea bass (Centropristis striata), a hermaphroditic teleost.

We cloned two cDNAs for two gonadotropin-releasing hormones, GnRH2 (chicken GnRH-II) and GnRH3 (salmon GnRH), respectively, from the black sea bass (Centropristis striata). Black sea bass are protogynous hermaphroditic teleosts that change from females to males between 2 and 5 years of age. Similar to other GnRH precursors, the precursors of black sea bass GnRH2 and GnRH23 consisted of a signal peptide, decapeptide, a downstream processing site, and a GnRH-associated peptide. Our analyses failed to identify GnRH1. GnRH3 precursor transcript was more widely distributed in a variety of tissues compared with GnRH2. Further examination of GnRH expression and gonadal histology was done in black sea bass from three different size groups: small (11.4-44.1 g), medium (179.4-352.2 g) and large (393.8-607.3 g). Interestingly, GnRH3 expression occurred only in the pituitaries of males in the small and medium groups compared with expression of GnRH2. Future functional studies of the sea bass GnRHs will be valuable in elucidating the potential underlying neuroendocrine mechanisms of black sea bass reproduction and may ultimately contribute to management advances in this commercially important fish.

Epigenetic modifications during sex change repress gonadotropin stimulation of cyp19a1a in a teleost ricefield eel (Monopterus albus).

In vertebrates, cytochrome P450 aromatase, encoded by cyp19a1, converts androgens to estrogens and plays important roles in gonadal differentiation and development. The present study examines whether epigenetic mechanisms are involved in cyp19a1a expression and subsequent gonadal development in the hermaphroditic ricefield eel. The expression of the ricefield eel cyp19a1a was stimulated by gonadotropin via the cAMP pathway in the ovary but not the ovotestis or testis. The CpG within the cAMP response element (CRE) of the cyp19a1a promoter was hypermethylated in the ovotestis and testis compared with the ovary. The methylation levels of CpG sites around CRE in the distal region (region II) and around steroidogenic factor 1/adrenal 4 binding protein sites and TATA box in the proximal region (region I) were inversely correlated with cyp19a1a expression during the natural sex change from female to male. In vitro DNA methylation decreased the basal and forskolin-induced activities of cyp19a1a promoter. Chromatin immunoprecipitation assays indicated that histone 3 (Lys9) in both regions I and II of the cyp19a1a promoter were deacetylated and trimethylated in the testis, and in contrast to the ovary, phosphorylated CRE-binding protein failed to bind to these regions. Lastly, the DNA methylation inhibitor 5-aza-2'-deoxycytidine reversed the natural sex change of ricefield eels. These results suggested that epigenetic mechanisms involving DNA methylation and histone deacetylation and methylation may abrogate the stimulation of cyp19a1a by gonadotropins in a male-specific fashion. This may be a mechanism widely used to drive natural sex change in teleosts as well as gonadal differentiation in other vertebrates.

GnRH isoforms expression in relation to the gonadal cycle and to dominance rank in the gilthead seabream, Sparus aurata.

The manner in which behavior influences the gonadotropin-releasing hormone (GnRH) axis in hermaphroditic fishes is not understood. The Gilthead seabream, Sparus aurata, is a protandrous hermaphrodite with a complex gonadal cycle consisting of a quiescent, pre-spawning, spawning, and post-spawning stage. On two separate experiments, I used real-time quantitative PCR to measure the mRNA expression of three GnRH isoforms in homogenized seabream whole-brain extracts. In the first experiment, I measured the levels of GnRH-1, GnRH-2, and GnRH-3 mRNA throughout the gonad cycle. All three GnRH mRNAs increase around the peak of the spawning season (December). GnRH-3 mRNA expression is also elevated in August, which coincides with the beginning of gonad differentiation. All three GnRH mRNAs have the lowest expression levels in the month of September. There was no difference between males and females in the expression levels of any of the three GnRH mRNA. In the second experiment, I measured individual dominance ranks in six groups of fish, three during quiescence and three during spawning. GnRH-1 mRNA expression was positively correlated with dominance rank only during the quiescent period. The more dominant fish tended to have higher GnRH-1 mRNA expression. The existence of a quiescent-only correlation between GnRH-1 mRNA and dominance rank suggests a mechanism by which activation of gonad maturation could occur first in the most dominant ambisexual fish.

Isolation and characterization of cyp19a1a and cyp19a1b promoters in the protogynous hermaphrodite orange-spotted grouper (Epinephelus coioides).

Aromatase (CYP19A1) catalyzes the conversion of androgens to estrogens. In teleosts, duplicated copies of cyp19a1 genes, namely cyp19a1a and cyp19a1b, were identified, however, the transcriptional regulation of these two genes remains poorly understood. In the present study, the 5'-flanking regions of the orange-spotted grouper cyp19a1a (gcyp19a1a) and cyp19a1b (gcyp19a1b) genes were isolated and characterized. The proximal promoter regions of both genes were relatively conserved when compared to those of the other teleosts. Notably, a conserved FOXO transcriptional factor binding site was firstly reported in the proximal promoter of gcyp19a1a, and deletion of the region (-112 to -60) containing this site significantly decreased the promoter activities. The deletion of the region (-246 to -112) containing the two conserved FTZ-F1 sites also dramatically decreased the transcriptional activities of gcyp19a1a promoter, and both two FTZ-F1 sites were shown to be stimulatory cis-acting elements. A FTZ-F1 homologue isolated from ricefield eel (eFTZ-F1) up-regulated gcyp19a1a promoter activities possibly via the FTZ-F1 sites, however, a previously identified orange-spotted grouper FTZ-F1 homologue (gFTZ-F1) did not activate the transcription of gcyp19a1a promoter unexpectedly. As to gcyp19a1b promoter, all the deletion constructs did not show good promoter activities in either TM4 or U251-MG cells. Estradiol (100nM) up-regulated gcyp19a1b promoter activities by about 13- and 36-fold in TM4 and U251-MG cells, respectively, via the conserved ERE motif, but did not stimulate gcyp19a1a promoter activities. These results are helpful to further elucidate the regulatory mechanisms of cyp19a1a and cyp19a1b expression in the orange-spotted grouper as well as other teleosts.

Expression profiles of gonadotropins and their receptors during 17α-methyltestosterone implantation-induced sex change in the orange-spotted grouper (Epinephelus coioides).

It is known that the hypothalamic-pituitary-gonadal axis participates in the sex change of hermaphrodite teleosts, and gonadal steroid hormones mediate this physiological process. The secretion of gonadal steroids is directly regulated by signaling pathways involving gonadotropins (GtHs) and gonadotropin receptors (GtHRs) in teleosts. To gain insight into the involvement of GtH/GtHR systems in the sex change process, cDNAs encoding follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) were firstly isolated from gonads of orange-spotted grouper (Epinephelus coioides), a protogynous hermaphrodite fish. Reverse transcription-PCR (RT-PCR) analysis demonstrated that the expression of the FSHR was confined to the brain, pituitary gland, ovary, and testis, while the LHR was expressed only in the brain, ovary, and testis. Furthermore, the expression profiles of GtH subunits (FSHß and LHß) and their receptors were analyzed in parallel with the serum levels of estradiol-17ß (E(2) ), testosterone (T), and 11-ketotestosterone (11-KT) during 17α-methyltestosterone (MT)-induced sex change. Quantitative real-time PCR determined that the abundances of FSHß and FSHR were significantly inhibited after MT treatment for 2 and 4 weeks, but subsequently returned to the control level after 6 weeks. In contrast, the mRNA levels of LHß and LHR were significantly elevated throughout the sex change process. During MT-induced sex change, serum concentrations of E(2) remained constant while T and 11-KT levels were significantly increased. Taken together, our results suggest that GtH/GtHR systems are involved in MT-induced sex change, and two signaling pathways may have distinct roles in modulating the variations of the corresponding steroid hormones in the orange-spotted grouper.

Molecular cloning, characterization and expression profiles of three estrogen receptors in protogynous hermaphroditic orange-spotted grouper (Epinephelus coioides).

Estrogen plays key roles in vertebrate reproductive system via estrogen receptors (ERs) as mediating pathways. In the present study, three full-length ERs cDNA sequences were isolated from a protogynous teleost, the orange-spotted grouper (Epinephelus coioides), and were 2235bp for gERα, 1967bp for gERß1 and 2158bp for gERß2, respectively. Phylogenetic and amino acid alignment analyses showed that each gER was clustered in the corresponding taxonomic groups of the perciformes and exhibited high evolutional conservation in functional domains. RT-PCR revealed that gERs expressed at different levels in all the obtained tissues. gERα highly expressed in mature ovaries, gERß1 mainly expressed in immature ovaries and gERß2 varied greatly during ovarian development. During female to male sex reversal induced by 17α-methyltestosterone (MT) implantation, gERα decreased gradually, gERß1 increased gradually, and gERß2 decreased firstly and recovered subsequently in male stage. The present study speculated the potential roles of gERs during female maturation and female to male sex reversal induced by MT in the protogynous grouper E. coioides.

Endocrine disrupting chemicals modulate expression of O6-methylguanine DNA methyltransferase (O6-MGMT) gene in the hermaphroditic fish, Kryptolebias marmoratus.

O6-methylguanine-DNA methyltransferase (O6-MGMT; EC 2.1.1.63) is a key repair enzyme that helps to protect the cell against alkylation on DNA by removing a methyl group from the O6-position of guanine. Here, we cloned and sequenced the full-length O6-MGMT cDNA from the hermaphroditic fish, Kryptolebias marmoratus. Complete Km-O6-MGMT cDNA was 1324 bp in length, and the open reading frame of 567 bp encoded a polypeptide of 188 amino acid residues. Phylogenetic analysis revealed that Km-O6-MGMT was clustered with those of other fish species. Embryo, juveniles, and aged secondary fish had low levels of Km-O6-MGMT mRNA than adults, indicating more susceptibility to DNA damage by alkylating agent exposure during these developmental stages. Km-O6-MGMT mRNA levels differed according to tissue type and was highest in the liver. Exposure to an alkylating agent, N-methyl-N-nitrosourea (MNU) exposure increased the mRNA expression of tumor suppressor gene such as p53 and oncogenes such as R-ras1, R-ras3, N-ras, c-fos as well as Km-O6-MGMT mRNA in a time-dependent manner. On the contrary, several (anti)estrogenic compounds (17ß-estradiol 100 ng/L, tamoxifen 10 µg/L, bisphenol A 600 µg/L, and 4-tert-octylphenol 300 µg/L) suppressed mRNA expression of Km-O6-MGMT in most tissues, especially the liver. In juvenile fish, 17ß-estradiol, bisphenol A, and 4-tert-octylphenol also decreased the expression of Km-O6-MGMT mRNA in a time-dependent manner. Overall, our finding shows that Km-O6-MGMT mRNA levels can be modulated by environmental estrogenic compounds as well as alkylating agents. This finding will be helpful to improve our knowledge of the effects of estrogenic compounds that contain the genotoxic ability to inhibit the DNA repair process in aquatic animals.

Bisphenol A modulates expression of gonadotropin subunit genes in the hermaphroditic fish, Kryptolebias marmoratus.

Pituitary gonadotropins (GTHs), follicle stimulating hormone beta (FSH-beta), and luteinizing hormone beta (LH-beta) are the key hormones in the hypothalamus-pituitary-gonad (HPG) axis, and form the heterodimers between a common alpha subunit (gonadotropin-alpha) and FSH-beta and/or LH-beta. To obtain a better understanding on the modulation of gonadotropin subunit genes expression upon bisphenol A (BPA) exposure in hermaphroditic fish, we studied differential regulation of gonadotropin subunit genes from Kryptolebias marmoratus after the exposure of several EDCs. Expression profiles of these three genes when using quantitative real-time RT-PCR revealed that brain/pituitary tissues were highly expressed in these genes compared to other tissues. At different developmental stages, expression of those genes dramatically increased over the course of development but showed a decrease in expression at the secondary male (showing atresia) stage. When adult fish were exposed to BPA (600 microg/L for 96 h), a significant upregulation of these three genes was observed in the brain/pituitary. A time course study also revealed the increased expression of gonadotropin subunit genes over 12 h with a more pronounced effect on the expression of FSH-beta and LH-beta genes, indicating that both genes were associated with the BPA exposure on the transcriptional regulation. This is the first report of gonadotropin subunit genes from K. marmoratus, with particular emphasis on the modulation of their expressions by EDCs. In addition, these findings suggest that EDCs modulate the expression of gonadotropin subunit genes and would act as potential biomarkers upon EDCs exposure.