The effect of gonadal hormones on the gene expression of brain-pituitary in protandrous black porgy, Acanthopagrus schlegelii.

In black porgy (Acanthopagrus schlegelii), the brain-pituitary-testis (Gnrh-Gths-Dmrt1) axis plays a vital role in male fate determination and maintenance, and then inhibiting female development in further (puberty). However, the feedback of gonadal hormones on regulating brain signaling remains unclear. In this study, we conducted short-term sex steroid treatment and surgery of gonadectomy to evaluate the feedback regulation between the gonads and the brain. The qPCR results show that male phase had the highest gths transcripts; treatment with estradiol-17ß (E2) or 17α-methyltestosterone (MT) resulted in the increased pituitary lhb transcripts. After surgery, apart from gnrh1, there is no difference in brain signaling genes between gonadectomy and sham fish. In the diencephalon/mesencephalon transcriptome, de novo assembly generated 283,528 unigenes; however, only 443 (0.16%) genes showed differentially expressed between sham and gonadectomy fish. In the present study, we found that exogenous sex steroids affect the gths transcription; this feedback control is related to the gonadal stage. Furthermore, gonadectomy may not affect gene expression of brain signaling (Gnrh-Gths axis). Our results support the communication between ovotestis and brain signaling (Gnrh-Gths-testicular Dmrt1) for the male fate.

The Ovarian Transcriptome at the Early Stage of Testis Removal-Induced Male-To-Female Sex Change in the Protandrous Black Porgy Acanthopagrus schlegelii.

Unlike gonochoristic fishes, sex is fixed after gonadal differentiation (primary sex determination), and sex can be altered in adults (secondary sex determination) of hermaphroditic fish species. The secondary sex determination of hermaphroditic fish has focused on the differences between testicular tissue and ovarian tissue during the sex change process. However, comprehensive studies analyzing ovarian tissue or testicular tissue independently have not been performed. Hermaphroditic black porgy shows a digonic gonad (ovarian tissue with testicular tissue separated by connective tissue). Protandrous black porgy has stable maleness during the first two reproductive cycles (<2 years old), and approximately 50% enter femaleness (natural sex change) during the third reproductive cycle. Precocious femaleness is rarely observed in the estradiol-17ß (E2)-induced female phase (oocytes maintained at the primary oocyte stage), and a reversible female-to-male sex change is found after E2 is withdrawn in <2-year-old fish. However, precocious femaleness (oocytes entering the vitellogenic oocyte stage) is observed in testis-removed fish in <2-year-old fish. We used this characteristic to study secondary sex determination (femaleness) in ovarian tissue via transcriptomic analysis. Cell proliferation analysis showed that BrdU (5-bromo-2'-deoxyuridine)-incorporated germline cells were significantly increased in the testis-removed fish (female) compared to the control (sham) fish (male) during the nonspawning season (2 months after surgery). qPCR analysis showed that there were no differences in pituitary-releasing hormones (lhb and gtha) in pituitary and ovarian steroidogenesis-related factors (star, cyp11a1, hsd3b1, and cyp19a1a) or female-related genes (wnt4a, bmp15, gdf9, figla, and foxl2) in ovarian tissues between intact and testis-removed fish (2 months after surgery). Low expression of pituitary fshb and ovarian cyp17a1 was found after 2 months of surgery. However, we did find small numbers of genes (289 genes) showing sexual fate dimorphic expression in both groups by transcriptomic analysis (1 month after surgery). The expression profiles of these differentially expressed genes were further examined by qPCR. Our present work identified several candidate genes in ovarian tissue that may be involved in the early period of secondary sex determination (femaleness) in black porgy. The data confirmed our previous suggestion that testicular tissue plays an important role in secondary sex determination in protandrous black porgy.

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.