In most vertebrates, hermaphroditism results in infertility. However, hermaphroditism occurs in 6% of teleosts, which primarily undergo protogyny. Here, to elucidate the transient stage from gonochorism to hermaphroditism, juvenile black porgies as a model animal were fed a diet containing estradiol (E2) for 3 mo, followed by withdrawal of E2 treatment. The E2-terminated fish had ectopically located oocytes in the regenerated testes. Antimüllerian hormone (amh) was strongly expressed in the Sertoli cells with type A spermatogonia and follicle cells with vitellogenic oocytes. Amh was robustly expressed in the ectopic oocytes-bordering region of regenerated testes and in testes with nonsynchronous spermatogenesis. This Amh was released by Sertoli cells and aggregated in the area containing type A spermatogonia in the ectopic oocytes-bordering region. Our in vitro results show that exogenous recombinant Amh (rAmh) can inhibit type A spermatogonia proliferation in the testis but not oogonia proliferation in the ovary. We suggest that Amh-arrested spermatogonia A may act as a boundary to block intercellular communication (i.e., prevent peptide factors released from female tissue to alter the sexual fate of type A spermatogonia) and further inhibit female growth. These results suggest that black porgy can prevent ectopic female growth in the testis and maintain male function of the digonic gonad (testes and ovary separated by the connective tissue) through Amh action. This function of amh might shed light on why the majority of syngonic fish undergo protogyny (female-to-male sex change).

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female growth results in a transient and immature female, and the sexual phase reverts from female to male after E2 is withdrawn.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.

The accessory nidamental gland (ANG) is a female reproductive organ found in most squid and cuttlefish that contains a consortium of bacteria. These symbiotic bacteria are transmitted from the marine environment and selected by the host through an unknown mechanism. In animals, a common antimicrobial mechanism of innate immunity is iron sequestration, which is based on the development of transferrin (TF)-like proteins. To understand this mechanism of host-microbe interaction, we attempted to characterize the role of transferrin in bigfin reef squid (Sepioteuthis lessoniana) during bacterial transmission. qPCR analysis showed that Tf was exclusively expressed in the outer layer of ANG,and this was confirmed by in situ hybridization, which showed that Tf was localized in the outer epithelial cell layer of the ANG. Western blot analysis indicated that TF is a soluble glycoprotein. Immunohistochemical staining also showed that TF is localized in the outer epithelial cell layer of the ANG and that it is mainly expressed in the outer layer during ANG growth. These results suggest that robust Tf mRNA and TF protein expression in the outer layer of the ANG plays an important role in microbe selection by the host during bacterial transmission.

Unlike vitellegenin, which is the sole major precursor of yolk protein in all oviparous vertebrates, a variety of major precursor of yolk proteins are found among oviparous invertebrates. Sea urchin have a transferrin-like yolk protein, while all other major precursor of yolk proteins in oviparous invertebrates belong to the superfamily of large lipid-transfer proteins (LLTPs). However, a comprehensive understanding of vitellogenesis is absent in cephalopods. To understand control of vitellogenesis by the LLTPs gene, two vitellogenins (VTG1 and VTG2), two apolipophorins (APOLP2A and APOLP2B), and a cytosolic large subunit of microsomal triglyceride-transfer protein (MTTP) found in the bigfin reef squid. Only the two VTGs showed high levels of expression in mature females compared to males. We further analyzed the expression profile and localization of both VTGs/VTGs during ovarian development. Our data showed that VTGs/VTGs expressions were correlated to the female reproductive cycle. Ovarian VTG1 and VTG2 were localized in the follicle cells but not in oocytes. In addition, VTG1 and VTG2 were represented in follicle cells and oocytes. Thus, our results showed that both VTGs were synthesized by follicle cells and are then delivered to oocytes. In addition, we demonstrated that VTGs were the major precursor of yolk protein in bigfin reef squid. We also found differential proteolytic cleavage processes of VTG1 and VTG2 during VTGs accumulation in oocytes. Therefore, our data shed light on the molecular mechanism of the yolk accumulation pathway in cephalopods.

Gonadal differentiation is tightly regulated by the initial sex determining gene and the downstream sex-related genes in vertebrates. However, sex change in fish can alter the sexual fate from one sex to the other. Chemical-induced maleness in the protogynous orange-spotted grouper is transient, and a reversible sex change occurs after the chemical treatment is withdrawn. We used these characteristics to study Amh signaling during bi-directional sex change in the grouper. We successfully induced the female-to-male sex change by chemical (aromatase inhibitor, AI, or methyltestosterone, MT) treatment. A dormant gonad (a low proliferation rate of early germ cells and no characteristics of both sexes) was found during the transient phase of reversible male-to-female sex change after the withdrawal of chemical administration. Our results showed that amh (anti-mullerian hormone) and its receptor amhr2 (anti-mullerian hormone receptor type 2) were significantly increased in the gonads during the process of female-to-male sex change. Amh is expressed in the Sertoli cells surrounding the type A spermatogonia in the female-to-male grouper. Male-related gene (dmrt1 and sox9) expression was immediately decreased in MT-terminated males during the reversible male-to-female sex change. However, Amh expression was found in the surrounding cells of type A spermatogonia-like cells during the transient phase of reversible male-to-female sex change. This phenomenon is correlated with the dormancy of type A spermatogonia-like cells. Thus, Amh signaling is suggested to play roles in regulating male differentiation during the female-to-male sex change and in inhibiting type-A spermatogonia-like cell proliferation/differentiation during the reversible male-to-female sex change. We suggest that Amh signaling might play dual roles during bi-directional sex change in grouper.