Shinji Adachi’s research while affiliated with Hokkaido University and other places

The Japanese eel, Anguilla japonica is one of the most important species in the Japanese and East Asian aquaculture industries. Although the supply of Japanese eels to aquaculture farms is fully dependent on wild-caught glass eels, their stocks are rapidly declining. Therefore, it is necessary to establish artificial seedling production technology; however, Japanese eels do not mature in captivity. Because the pituitary gland of captive eels secretes gonadotropins, artificial hormone treatment is essential to induce sexual maturation (Ijiri et al. 2011). In this chapter, we review previous studies on artificial maturation in Japanese eels together with our results.

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.

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.

Most cultured Japanese eels (Anguilla japonica) show male sex differentiation; however, natural gonadal sex differentiation has not been evaluated. In this study, this process was characterized in wild eels. Differentiated ovaries and testes were observed after the eels grew to 320 and 300 mm in total length, respectively. The youngest ovary and testis appeared at 3 and 4 years old, respectively; however, undifferentiated gonads were found up to 7 years, suggesting that sex differentiation was triggered by growth rather than aging. gsdf, amh, foxl2b and foxl3b were highly expressed in the testes, whereas figla, sox3, foxn5, zar1, and zp3 were highly expressed in the ovaries. The expression of cyp19a1a and foxl2a did not differ significantly between the testis and ovary. In the ovaries, the cyp19a1a and foxl2a levels were highest in the early stages, suggesting that their function is limited to early ovarian differentiation. The foxn5, zar1 and zp3 levels tended to increase in the later stages, suggesting that they function after the initiation of ovarian differentiation. In undifferentiated gonads, dimorphic gene expression was not observed, suggesting that the molecular sex differentiation phase is short and difficult to detect. These findings provide the first demonstration of the whole course of natural gonadal sex differentiation in eels at molecular and morphological levels.

In several teleosts, 17α, 20β-dihydroxy-4-pregnen-3-one (DHP) has been identified as a maturation-inducing steroid. DHP is synthesized from 17α-hydroxyprogesterone (17OHP) by 17β-hydroxysteroid dehydrogenase type 12-like (hsd17b12L). Along with 3β-hydroxysteroid dehydrogenase/Δ5-4 isomerase (3β-HSD), 17α-hydroxylase and C17-20 lyase are associated with 17OHP production. This study aimed to determine the roles of Amur sturgeon hsd3b, P450c17-I (cyp17a1), and P450c17-II (cyp17a2) in 17OHP production and to examine their enzyme activity and mRNA expression pattern during oocyte maturation. In the sturgeons used in this study, hsd3b encoded 3β-HSD, cyp17a1 catalyzed 17α-hydroxylase production with C17-20 lyase activity, and cyp17a2 processed 17α-hydroxylase activity alone. In the ovarian follicles of individuals that underwent induced ovulation, hsd3b mRNA levels increased rapidly, cyp17a1 expression was downregulated, and cyp17a2 expression was upregulated during oocyte maturation. Finally, an in vitro study revealed that salmon pituitary extract (SPE) stimulation rapidly induced hsd3b expression, whereas cyp17a1 expression was downregulated. In vitro, cyp17a2 expression did not rapidly increase with SPE stimulation. This rapid upregulation of hsd3b during oocyte maturation was first observed in teleosts. It was suggested that hsd17b12L expression is upregulated after 17OHP production, which is regulated by hsd3b, cyp17a1, and cyp17a2, resulting in DHP production.

To clarify the role of estrogens in the onset of sex change in fish, estrogen levels were artificially reduced in hermaphroditic and gonochoristic female fish via the treatment with aromatase inhibitors (AIs). AI treatments caused depletion in blood estrogen levels and induced complete sex change from a female to a male in protogynous three-spotted wrasse and the honeycomb grouper. Opposite-direction sex change of the protandrous yellowtail anemonefish was also induced by AI treatments. Not only in hermaphrodites, AI treatments induced testicular differentiation and, in certain circumstances, a complete sex change in the developing ovaries of the gonochoristic fish: tilapia, medaka, zebrafish, carp and golden rabbitfish. We demonstrated that estrogen depletion induces the female to male sex change in both hermaphroditic and gonochoristic fish. Results suggested that some germ cells in the ovaries of both hermaphroditic and gonochoristic fish maintain sexual bipotentiality, which is the ability to differentiate into both female and male germ cells, throughout the life and that the fate of these germ cells' differentiation depends on the level of endogenous estrogen. Higher levels of circulating estrogen maintain femaleness, while lower levels force their differentiation into males. These studies contribute to the progress of aquaculture.

17α, 20β-dihydroxy-4-pregnen-3-one (DHP) has been identified as a maturation-inducing steroid (MIS) in several teleosts. DHP is converted from 17α-hydroxyprogesterone (17OHP) by 20β-hydroxysteroid dehydrogenase (20β-HSD). A recent study reported that 17β-hydroxysteroid dehydrogenase type 12-like (hsd17b12L) is responsible for DHP production, and its expression is upregulated by gonadotropin during oocyte maturation in masu salmon and Nile tilapia. However, whether the hsd17b12L gene encodes 20β-HSD in Actinopterygii remains unknown. Sturgeons belong to ancient fish and are suggested to have diverged 200 million years ago. Currently, the MIS of sturgeons and its associated mechanism remain unknown. This study aimed to understand the evolution of Actinopterygii hsd17b12L and determine its function during sturgeon oocyte maturation. In the Amur sturgeon and sterlet, two hsd17b12L types were repeatedly isolated. Mammalian expression vectors containing Amur sturgeon hsd17b12L type I and sterlet hsd17b12L type I were transfected into HEK293T cells, which then exhibited strong 20β-oxidoreductase activity, converting 17OHP to DHP. LC/MS analyses indicated that Amur sturgeon hsd17b12L did not convert DHP, androstenedione, testosterone, estrone, estradiol-17β, adrenosterone, and 11-ketotestosterone, confirming that Amur sturgeon hsd17b12L has 20β-oxidoreductase activity, but not 20β-dehydrogenase activity toward DHP neither17β-HSD activity. Masu salmon hsd17b12L and Nile tilapia hsd17b12 expression were limited to the follicles and testes at the maturation stage, while Amur sturgeon hsd17b12L was upregulated in all tissues examined. In individuals that reached ovulation, serum DHP concentrations during oocyte maturation increased, and the Amur sturgeon hsd17b12L mRNA levels of follicles during oocyte maturation were higher than prior to maturation. Finally, sterlet and Amur sturgeon ovarian follicles were incubated with chum salmon pituitary extract (SPE, 500 μg/mL) for different times. The DHP concentration in the medium was increased by SPE. However, hsd17b12L mRNA levels were not significantly different following SPE stimulation. The present study demonstrated that sturgeon hsd17b12L encodes 20β-HSD, suggesting that the hsd17b12L gene encodes 20β-HSD in related fish species that evolved after the sturgeon. An in vivo experiment revealed that sturgeon hsd17b12L was associated with oocyte maturation and DHP production, but its mRNA expression in vitro did not exhibit a rapid increase during oocyte maturation as previously observed in masu salmon, suggesting that hsd17b12L does not solely regulate sturgeon DHP production.

During the induction of Japanese eel maturation, administering maturation-inducing steroids (MIS) or their precursors at an inappropriate maturational status is a major cause of poor egg quality. In this study, we investigated the feasibility of controlling oocyte maturation progress by rearing in cold and warm water to manipulate the timing of MIS administration. Mature females with oocytes at the migratory nucleus stage were reared for two terms (3 days to 1 day and 1 day to 0 days before MIS administration) at 20/20 °C, 20/15 °C, 15/20 °C, or 15/15 °C, and the maturational status was monitored based on their lipid droplet morphology and oocyte diameter. Oocytes matured faster at 20 °C than at 15 °C in either term. Next, the mature females were reared at 15 or 20 °C depending on the maturational status of each female 3 days and 1 day before MIS administration; the immature females were reared at 20 °C to accelerate their maturation. Consequently, the maturational status of most females was similar at MIS administration. After improvement, this method would lead most females to the optimum maturational status at MIS administration by properly rearing in cold and warm water.