Characterization of a novel and testis-specific zinc finger protein during sexual development of Pacific white shrimp Litopenaeus vannamei.

Since females grow faster in penaeid shrimp, all-female aquaculture was proposed. Environmental conditions in the Pacific white shrimp did not found to affect genetic sex determination (ZZ/ZW system). The androgenic gland (AG)-secreting insulin-like androgenic gland hormone (IAG) is a key controlling factor in crustacean male differentiation. However, functional sex reversal (neo-male) in penaeid shrimp has not yet been achieved by manipulating the IAG-sexual switch. Therefore, understanding the molecular mechanisms of gonadal differentiation may help build appropriate tools to generate neo-male (ZW) for all-female breeding. This study describes the potential role of the novel penaeid-specific testicular zinc finger protein (pTZFP) in the gonads of Pacific white shrimp. First, pTZFP transcripts show a male-bias expression pattern in undifferentiated gonads, which is then exclusively expressed in the testis and absent or slightly expressed in the ovary and other tissues. Besides, the knockdown of pTZFP in undifferentiated males results in smaller testes but no sex reversal. Immunohistochemical (IHC) staining of proliferating cell nuclear antigen (PCNA) further confirmed that the smaller testes in pTZFP-deficient males are due to the lower proliferating activity of spermatogonia. These data reveal that pTZFP may be involved in testicular development but have fewer effects on gonadal differentiation. Moreover, testicular pTZFP transcription levels were not reduced with estradiol-17ß (E2) administration or AG excision. Therefore, our data suggest that pTZFP may regulate testicular development through downstream genes regulating spermatogonia proliferation. Moreover, our data provide an appropriate molecular marker for identifying the sex of undifferentiated gonads.

The germline-specific expression of Foxl3a and its paralogous Foxl3b are associated with male gonadal differentiation in the Japanese eel, Anguilla japonica.

Unlike its paralog Foxl2, which is well known for its role in ovarian development in vertebrates, the function of Foxl3 is still unclear. Foxl3 is an ancient duplicated copy of Foxl2. It is present as a single copy in ray-finned fish. But, due to repeated losses, it is absent in most tetrapods. Our transcriptomic data, however, show that two Foxl3s (Foxl3a and its paralog Foxl3b) are present in Japanese eel. Foxl3a is predominantly expressed in the pituitary, and Foxl3b is predominantly expressed in the gills. Both Foxl3s show a sex-dimorphic expression, being higher expression in testes than in ovaries. Moreover, Foxl3a and Foxl3b were exclusively expressed during gonadal differentiation in control eels (100% male). Conversely, Foxl3a and Foxl3b significantly decreased after gonadal differentiation in E2-treated eels (100% female). Furthermore, in accordance the difference in adhesive ability between somatic cells and germline cells in testes, Foxl3s showed a high expression in suspension cells (putative germline cells) and low expression in adhesive cells (putative somatic cells). In situ hybridization further showed that Foxl3a and Foxl3b were expressed in the testicular germline cells. In addition, Foxl3s expression was not changed by sex steroids in in vitro testes culture. Taken together, our results suggest that the teleost-specific Foxl3 paralog was repeatedly lost in most fish after the third round of whole genome duplication. The two germline-expressed Foxl3s had higher expression levels in males than in females during gonadal differentiation in Japanese eel. These results demonstrated that Foxl3s might play an important role in germline sexual fate determination from ancient fish to modern fish.

Two distinct vitellogenin genes are similar in function and expression in the bigfin reef squid Sepioteuthis lessoniana.

Unlike vitellogenin, 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 urchins have a transferrin-like yolk protein, while all other major precursors 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.

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.

The Potential Role of Amh to Prevent Ectopic Female Development in Testicular Tissue of the Protandrous Black Porgy, Acanthopagrus schlegelii.

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).

Duplicated paralog of sulfide: quinone oxidoreductase contributes to the adaptation to hydrogen sulfide-rich environment in the hydrothermal vent crab, Xenograpsus testudinatus.

The hydrothermal crab, Xenograpsus testudinatus (xtcrab) inhabits shallow-water, hydrogen sulfide (H2S)-rich hydrothermal vent regions. Until now, the adaptative strategy of xtcrab to this toxic environment was unknown. Herein, we investigated the sulfide tolerance and detoxification mechanisms of xtcrabs collected in their high-sulfide hydrothermal vent habitat. Experimental immersion of xtcrab in various sulfide concentrations in the field or in aquaria assessed its high sulfide tolerance. HPLC measurement of hemolymph sulfur compounds highlighted xtcrab detoxification capacity via catabolism of sulfide into much less toxic thiosulfate. We focused on a key enzyme for H2S detoxification, sulfide: quinone oxidoreductase (SQR). Cloning and phylogenetic analysis revealed two SQR paralogs in xtcrab, that we named xtSQR1 and xtSQR2. As shown by qPCR, xtSQR2 and xtSQR1 were expressed in the digestive gland, suggesting the involvement of both paralogs in the detoxification of food-related H2S. In contrast, xtSQR1 transcript was highly expressed in the gill, while xtSQR2 was not detectable, suggesting a specific role of SQR1 in gill detoxification of H2S of environmental origin. Comparison between xtcrabs in their hydrogen sulfide-rich hydrothermal habitat, and xtcrabs maintained for one month in sulfide-free seawater aquarium, showed higher transcript levels of gill xtSQR1 in sulfide-rich habitat, further supporting the specific role of xtSQR1 paralog in environmental H2S detoxification in the gill. Gill SQR protein level as measured by Western blot, and gill SQR enzyme activity were also higher in sulfide-rich habitat. Immunohistochemical staining further showed that SQR expression was co-localized with Na+/K+-ATPase-positive epithelial and pillar cells of the gill filament. This is the first evidence of duplicate SQR genes in crustaceans. Overall, our study suggests that the subfunctionalization of duplicate xtSQR genes may play an important role in sulfide detoxification to maintain the sulfide homeostasis in X. testudinatus, providing an ecophysiological basis for its adaptation to the high-sulfide hydrothermal vent environment.

Bacterial Dynamics in the Accessory Nidamental Gland of Sepioteuthis lessoniana throughout Maturation.

The accessory nidamental gland (ANG) is part of the reproduction organ in the majority of female cephalopods, including the bigfin reef squid Sepioteuthis lessoniana, an economically important fishery product. Microbes in Alphaproteobacteria, Gammaproteobacteria, and Verrucomicrobia have been suggested to play a role in the maturation of the S. lessoniana ANG and are responsible for its color. However, the bacterial composition and dynamics of the different maturation stages of the ANG remain unclear. In the present study, we surveyed ANG-associated bacterial dynamics in wild-caught S. lessoniana at various developmental stages in different populations over 3 years. The results obtained showed that the ANG bacterial community shifted gradually and decreased in diversity throughout maturation. Verrucomicrobia occupied the ANG during the early stages in large numbers, and was replaced by Bacteroidia, Alphaproteobacteria, and Gammaproteobacteria in the later stages. Flavobacteriales and Alphaproteobacteria both appeared to contribute to pigmentation, while Bacteroidia, Alphaproteobacteria, and Gammaproteobacteria may be involved in enriching the heme biosynthesis pathway in the ANG with the maturation of S. lessoniana. The present results provide an open question of whether S. lessoniana actively selects the bacterial community in the ANG to adjust to its surrounding environment.

The Characteristics and Expression Profile of Transferrin in the Accessory Nidamental Gland of the Bigfin Reef Squid during Bacteria Transmission.

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.

Expression profile of amh/Amh during bi-directional sex change in the protogynous orange-spotted grouper Epinephelus coioides.

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.

Robust gdf9 and bmp15 expression in the oocytes of ovotestes through the Figla-independent pathway in the hermaphroditic black porgy, Acanthopagrus schlegelii.

More than 1,500 fish species are hermaphroditic, but no hermaphroditic lineage appears to be evolutionarily ancient in fishes. Thus, whether more than one sex at a time was present during the evolutionary shift from gonochorism to hermaphroditism in fishes is an intriguing question. Ectopic oocytes were created in the ovotestes of protandrous black porgy via the withdrawal of estradiol (E2) administration. These ectopic oocytes reprogrammed the surrounding cells, which changed from Sertoli cells to follicle-like cells. We observed that gdf9 and bmp15 expression was localized in the primary oocytes and gradually decreased after oocytes entered a secondary oocyte stage. Robust expression of gdf9 and bmp15 in ectopic oocytes was associated with the surrounding Sertoli cells. However, blocking Cyp19a1a activity and increasing androgen levels did not stimulate the expression of gdf9 and bmp15. Thus, the robust gdf9 and bmp15 expression was not related to the inappropriate male microenvironment. Furthermore, in vitro data demonstrated that gdf9 and bmp15 were not downstream genes of Figla signaling. Therefore, our results suggest that there are two independent mechanisms, a Figla-dependent pathway and a Figla-independent pathway, by which oocyte-surrounding cells are altered from a male somatic fate to a female somatic fate. This functional switch might clarify how oocytes created an appropriate microenvironment during the transition from the ancient gonochorism to the present hermaphroditism.

Sexual Fate Reprogramming in the Steroid-Induced Bi-Directional Sex Change in the Protogynous Orange-Spotted Grouper, Epinephelus coioides.

Androgen administration has been widely used for masculinization in fish. The mechanism of the sex change in sexual fate regulation is not clear. Oral administration or pellet implantation was applied. We orally applied an aromatase inhibitor (AI, to decrease estrogen levels) and 17α-methyltestosterone (MT, to increase androgen levels) to induce masculinization to clarify the mechanism of the sex change in the protogynous orange-spotted grouper. After 3 mo of AI/MT administration, male characteristics were observed in the female-to-male sex change fish. These male characteristics included increased plasma 11-ketotestosterone (11-KT), decreased estradiol (E2) levels, increased male-related gene (dmrt1, sox9, and cyp11b2) expression, and decreased female-related gene (figla, foxl2, and cyp19a1a) expression. However, the reduced male characteristics and male-to-female sex change occurred after AI/MT-termination in the AI- and MT-induced maleness. Furthermore, the MT-induced oocyte-depleted follicle cells (from MT-implantation) had increased proliferating activity, and the sexual fate in a portion of female gonadal soma cells was altered to male function during the female-to-male sex change. In contrast, the gonadal soma cells were not proliferative during the early process of the male-to-female sex change. Additionally, the male gonadal soma cells did not alter to female function during the male-to-female sex change in the AI/MT-terminated fish. After MT termination in the male-to-female sex-changed fish, the differentiated male germ cells showed increased proliferating activities together with dormancy and did not show characteristics of both sexes in the early germ cells. In conclusion, these findings indicate for the first time in a single species that the mechanism involved in the replacement of soma cells is different between the female-to-male and male-to-female sex change processes in grouper. These results also demonstrate that sexual fate determination (secondary sex determination) is regulated by endogenous sex steroid levels.