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Nile tilapia, as an ideal model for studying sex differentiation, is a popular farmed fish worldwide with a stable XX/XY sex-determination system. In tilapia, ovarian differentiation is triggered by estradiol-17β (E2) production in undifferentiated gonads. In a previous study, we suggested that follicle-stimulating hormone (FSH) signaling might be involved in ovarian differentiation in Nile tilapia. In this study, we further investigated the role of FSH signaling in ovarian differentiation via aromatase expression, which converts testosterone to E2. Masculinization of XX fry by aromatase inhibitor or 17α-methyltestosterone leads to suppression of fshr expression. Feminization of XY fry by E2 treatment increased fshr expression from 15 days after hatching, when E2 treatment was terminated. XX tilapia developed ovaries harboring aromatase expression if fsh and fshr were double knockdowns by morpholino-oligo injections. Finally, the transcriptional activity in the upstream region of the aromatase gene (cyp19a1a) was further increased by FSH stimulation when HEK293T cells were co-transfected with foxl2 and ad4bp/sf1. Collectively, this study suggests that the role of FSH signaling is not critical in tilapia ovarian differentiation; however, FSH signaling may have a compensatory role in ovarian differentiation by increasing cyp19a1a transcription in cooperation with foxl2 and ad4bp/sf1 in Nile tilapia.

The luteinizing hormone (LH) and maturation-inducing steroids (MIS), such as 17α,20β-dihydroxy-4-pregnen-3-one, regulate the final oocyte maturation in teleosts. Oocyte maturational competence (OMC) and ovulatory competence measure the sensitivity to MIS for oocyte maturation and ovulation, respectively. However, the molecular mechanisms underlying the acquisition of ovulatory competence remain unknown. Sturgeons are an excellent research model for investigating these mechanisms. We examined the seasonal profiles of OMC and ovulatory competence in vitro and the expression of 17 ovulation-related gene candidates using quantitative PCR in Amur sturgeon ovarian follicles. The ovulatory competence was induced by the LH-releasing hormone analog (LHRHa) priming injection after acquiring the OMC, which was spontaneously induced in spring or autumn. Seven genes, including the tissue-type plasminogen activator (plat), were enhanced following the LHRHa priming injection in ovarian follicles sampled from anovulated and ovulated fish. The activin receptor type 1 (acvr1) and prostaglandin G/H synthase 2 (ptgs2) were only upregulated in ovulated fish. Our results suggest that plat/plasmin and prostaglandin (PG)/PG receptor systems are essential for sturgeon ovulation, similar to other vertebrates. Notably, successful ovulation depends on a sufficient PG synthesis, and mediators activating the PG/PG receptor system are essential for acquiring the ovulatory competence. We provide the first report of ovulation-related gene alterations in the ovarian follicles of Amur sturgeons.

Estradiol-17β (E2) production is the key event for the initiation of ovarian differentiation in Nile tilapia. The enzyme aromatase, encoded by the cyp19a1a , converts testosterone into E2. Along with the transcription factors foxl2 and ad4bp / sf1 , follicle-stimulating hormone (FSH) is also suggested to be involved in cyp19a1a expression. Although fsh is expressed equally in genetic female (XX) and genetic male (XY) pituitaries, expression of the FSH receptor ( fshr ) is significantly higher in XX undifferentiated gonads than in XY undifferentiated gonads during early sex differentiation, possibly indicating that FSH may play a role in ovarian differentiation via cyp19a1a expression. To investigate whether FSH signaling influences E2 production through the expression of steroidogenic enzymes, we injected recombinant FSH (rFsh) into XX larvae during early sex differentiation and then assessed the expression of various sex differentiation–related genes. We found that after the injections, hsd3b levels were significantly higher in the rFsh injection group than in the control groups, and that the levels of cyp11a1 , cyp17a1 , and cyp19a1a tended to be higher in the rFsh injection group. These results suggest that FSH may induce the expression of cyp11a1 , hsd3b , cyp17a1 , and cyp19a1a , thereby contributing to E2 production. However, FSH stimulation of the expression of these enzymes does not appear to be strong, indicating that the role of FSH in E2 production may be limited and suggesting a compensatory role in ovarian differentiation.

In sturgeon aquaculture, oocyte maturation and ovulation are induced by the administration of hormones such as luteinizing hormone–releasing hormone analog (LHRHa). However, ovulation often fails to occur, probably as a result of inappropriate hormone injection timing. It is thus necessary to understand the molecular mechanisms of sturgeon ovulation responsible for producing good-quality eggs. In this study, we investigated the proteolytic genes and their inhibitory genes in a de novo transcriptome constructed using RNA samples of ovarian follicles from Amur sturgeon (Acipenser schrenckii). We subsequently identified 14 mmp, nine adam, ten adamts, nine cathepsins, and three timp contigs by a homology search with BLASTx. We compared the messenger RNA (mRNA) levels of these genes in the ovarian follicles of anovulated and ovulated Amur sturgeons using RNA sequencing and quantitative PCR analysis. The results showed that the mRNA levels of mmp16, adam23, adamts9, and timp2 were significantly increased by LHRHa injection only in ovulated sturgeons. We also examined the expression patterns in sterlet (Acipenser ruthenus) ovarian follicles in in vitro culture. adamts9 mRNA levels were drastically upregulated by the administration of 17α-hydroxyprogesterone, suggesting that Adamts9 is a key factor in successfully inducing ovulation in sturgeons. Clarifying the functions of these four genes during ovulation will lead to improved sturgeon seedling production technologies.

In sturgeon aquaculture, all-female production is desirable due to the high value of caviar. Genetic sexing and the production of WW superfemales are important steps toward achieving this. In this study, we identified the WSR and ZSR primers for amplification of W- and Z-specific regions, respectively. WSR primers were designed on the gene W-linked RT RNase H-like domain containing protein (rnhW). The polymerase chain reaction (PCR) bands were obtained with the WSR primer only in phenotypic female sturgeons, indicating that stable genetic sexing was achieved in most species, including those captured around Hokkaido. Moreover, rnhW showed female-specific expression in the gonads during early sex differentiation in kaluga and Amur sturgeon. ZSR primers were developed from the orofacial cleft 1 candidate gene 1 protein homolog. Clear and distinct gel band patterns for ZZ, ZW, and WW genotypes were obtained using WSR and ZSR primers, consistent with genotypic estimations by quantitative PCR. This consistency confirmed the presence of WW superfemales among offspring produced by fertilizing ZW females with ZW pseudomales masculinized using 17α-methyltestosterone. Our findings provide new insights into the mechanisms of sex determination and differentiation in sturgeons, bringing the establishment of an all-female production system within reach.

The natural reproductive ecology of freshwater eels remained a mystery even after some of their offshore spawning areas were discovered approximately 100 years ago. In this study, we investigate the spawning ecology of freshwater eels for the first time using collections of eggs, larvae and spawning-condition adults of two species in their shared spawning area in the Pacific. Ovaries of female Japanese eel and giant mottled eel adults were polycyclic, suggesting that freshwater eels can spawn more than once during a spawning season. The first collection of Japanese eel eggs near the West Mariana Ridge where adults and newly hatched larvae were also caught shows that spawning occurs during new moon periods throughout the spawning season. The depths where adults and newly hatched larvae were captured indicate that spawning occurs in shallower layers of 150–200 m and not at great depths. This type of spawning may reduce predation and facilitate reproductive success. Little is known about the reproductive ecology of freshwater eels. In this article, the authors describe the capture of two species of eels together with eggs and newly hatched larvae, and suggest that spawning takes place during the new moon at shallower depths than previously thought.

In the original publication [...]

In Nile tilapia, one of the most important aquaculture species, males are larger than females, and an all-male monosex culture offers significant economic benefits. Although the pituitaries of genetic female (XX) and genetic male (XY) tilapia have identical expression levels of follicle-stimulating hormone (fsh), FSH receptor (fshr) expression remains relatively low in XY-undifferentiated gonads and then increases following morphological sex differentiation. The expression patterns of genes related to androgen biosynthesis in XY-undifferentiated gonads are similar to those of fshr during testis differentiation. This might imply that FSH has a potential function in testis differentiation through regulating the expression of genes related to androgen biosynthesis. To determine whether FSH signaling regulated androgen biosynthesis, we microinjected recombinant FSH (rFsh) into XY larvae during the early sex-differentiation stage. We compared the expression of various genes related to testis differentiation after injection. The genes hsd3b, cyp17a1, dmrt1, and gsdf were found to have higher expression in the rFsh treatment group. These results suggest that FSH signaling can activate androgen biosynthesis by regulating steroidogenic enzymes, including hsd3b and cyp17a1. Moreover, injected rFsh can upregulate dmrt1, which has a positive effect on the expression of gsdf. Therefore, during testis differentiation and development, FSH plays a role in both androgen synthesis and the expression of genes related to testis differentiation in Nile tilapia.

Background The loach ( Misgurnus anguillicaudatus ), the most widely distributed species of the family Cobitidae, displays a mud-dwelling behavior and intestinal air-breathing, inhabiting the muddy bottom of extensive freshwater habitats. However, lack of high-quality reference genome seriously limits the interpretation of the genetic basis of specialized adaptations of the loach to the adverse environments including but not limited to the extreme water temperature, hypoxic and noxious mud environment. Results This study generated a 1.10-Gb high-quality, chromosome-anchored genome assembly, with a contig N50 of 3.83 Mb. Multiple comparative genomic analyses found that proto-oncogene c-Fos ( fos ), a regulator of bone development, is positively selected in loach. Knockout of fos (ID: Mis0086400.1) led to severe osteopetrosis and movement difficulties, combined with the comparison results of bone mineral density, supporting the hypothesis that fos is associated with loach mud-dwelling behavior. Based on genomic and transcriptomic analysis, we identified two key elements involved in the intestinal air-breathing of loach: a novel gene (ID: mis0158000.1) and heat shock protein beta-1 ( hspb1 ). The flavin-containing monooxygenase 5 ( fmo5 ) genes, central to xenobiotic metabolism, undergone expansion in loach and were identified as differentially expressed genes in a drug stress trial. A fmo5 −/− (ID: Mis0185930.1) loach displayed liver and intestine injury, indicating the importance of this gene to the adaptation of the loach to the noxious mud. Conclusions Our work provides valuable insights into the genetic basis of biological adaptation to adverse environments.

Under aquaculture conditions, Japanese eels (Anguilla japonica) produce a high percentage of males. However, females gain higher body weight and have better commercial value than males, and, therefore, a high female ratio is required in eel aquaculture. In this study, we examined the effects of isoflavones, genistein, and daidzein on sex differentiation and sex-specific genes of eels. To investigate the effects of these phytoestrogens on the gonadal sex, we explored the feminizing effects of soy isoflavones, genistein, and daidzein in a dose-dependent manner. The results showed that genistein induced feminization more efficiently than daidzein. To identify the molecular mechanisms of sex-specific genes, we performed a comprehensive expression analysis by quantitative real-time PCR and RNA sequencing. Phenotypic males and females were produced by feeding elvers a normal diet or an estradiol-17β- or genistein-treated diet for 45 days. The results showed that female-specific genes were up-regulated and male-specific genes were down-regulated in the gonads, suggesting that genistein induces feminization by altering the molecular pathways responsible for eel sex differentiation.