Mitsuo Nyuji

Identification, characterization, and expression profiles of two subtypes of kisspeptin receptors in a scombroid fish (chub mackerel)

The kisspeptin receptor (Kiss1R) is a cognate receptor for kisspeptin (Kiss), and this Kiss-Kiss1R system has been shown to regulate seasonal reproduction in vertebrates. Our previous study found the chub mackerel (Scomber japonicus) brain expresses both kiss1 and kiss2 and exhibits sexually dimorphic changes during the seasonal reproductive cycle. The present study cloned two subtypes of kissr from the chub mackerel brain, and their signal transduction pathways to Kiss1 and Kiss2 were characterized in a mammalian cell line. Results of identification showed that kissr1 and kissr2 mRNAs encode 369 and 378 deduced amino acids, respectively, and share 52% similarity in amino acid sequences. In vitro functional analysis revealed that chub mackerel Kiss receptor signals are also preferentially transduced via the protein kinase C (PKC) rather than protein kinase A (PKA) pathway. Synthetic chub mackerel Kiss1-15 and Kiss2-12 peptides showed the highest potency for the activation of KissR1 and KissR2, respectively, stronger than their corresponding Kiss-10 peptides. Tissue distribution analyses indicated that both genes are highly expressed in the brain and that only kissr2 mRNA is expressed in the pituitary of both sexes. Unexpectedly, both kissr1 and kissr2 mRNAs were detected only in the testes. Seasonal expression changes showed higher expression levels of both kissr1 and kissr2 mRNAs in the brain of females during the early vitellogenic period; however, no significant differences were found in the brain of males. Pituitary kissr2 mRNA levels showed no significant variations. In the testes, the kissr1 mRNA expression level increased dramatically at spermiation compared with the immature and post-spawning periods. However, kissr2 mRNA levels in the testes did not vary significantly at different testicular stages. These results suggest that both kissr1 and kissr2 likely participate in the seasonal ovarian development of females, and thus in males, we propose a paracrine or autocrine role for kissr1 in testicular development.

Changes in the expression of pituitary gonadotropin subunits during reproductive cycle of multiple spawning female chub mackerel Scomber japonicus.

The endocrine regulation of reproduction in a multiple spawning fish with an asynchronous-type ovary remains largely unknown. The objectives of this study were to monitor changes in the mRNA expression of three gonadotropin (GtH) subunits (GPα, FSHβ, and LHβ) during the reproductive cycle of the female chub mackerel Scomber japonicus. Cloning and subsequent sequence analysis revealed that the cDNAs of chub mackerel GPα, FSHβ, and LHβ were 658, 535, and 599 nucleotides in length and encoded 117, 115, and 147 amino acids, respectively. We applied a quantitative real-time PCR assay to quantify the mRNA expression levels of these GtH subunits. During the seasonal reproductive cycle, FSHβ mRNA levels remained high during the vitellogenic stages, while GPα and LHβ mRNA levels peaked at the end of vitellogenesis. The expression of all three GtH subunits decreased during the post-spawning period. These results suggest that follicle-stimulating hormone (FSH) is involved in vitellogenesis, while luteinizing hormone (LH) functions during final oocyte maturation (FOM). Both GPα and FSHβ mRNA levels remained high during the FOM stages of the spawning cycle and increased further just after spawning. Thus, FSH synthesis may be strongly activated just after spawning to accelerate vitellogenesis in preparation for the next spawning. Alternatively, LHβ mRNA levels declined during hydration and then increased after ovulation. This study demonstrates that chub mackerel are a good model for investigating GtH functions in multiple spawning fish.

Characterization, Localization, and Stage-Dependent Gene Expression of Gonadotropin Receptors in Chub Mackerel (Scomber japonicus) Ovarian Follicles

ABSTRACT The pituitary gonadotropins (GtHs), follicle-stimulating hormone (FSH) and luteinizing hormone (LH), are key regulators of gametogenesis in teleosts. However, little is known about the physiological mechanisms by which GtHs regulate asynchronous oocyte development in multiple-spawning marine fishes. We cloned cDNAs encoding GtH receptors (FSHR and LHR) from chub mackerel (Scomber japonicus). FSH and LH were purified by anion-exchange chromatography, gel filtration, and concanavalinA-agarose. When expressed in mammalian cells, FSHR and LHR responded strongly to their own ligands. By separating LH into two subunits by the use of reverse-phase chromatography, we found that the beta-subunit is responsible for signal transduction and the alpha-subunit may be important for holding hormone-receptor complex. In situ hybridization showed that only fshr was expressed in prefollicle and granulosa cells in oocytes at the perinucleolus and cortical alveolus stages, suggesting that FSH is involved in the primary and early secondary growth of oocytes. In ovarian follicles during vitellogenesis, both fshr and lhr were expressed in granulosa and thecal cells, and lhr was strongly expressed during germinal vesicle migration (GVM). Real-time PCR analysis of stage-dependent fshr and lhr expression showed that fshr expression was high in ovarian follicles throughout vitellogenesis and decreased during GVM, whereas lhr expression was low in early vitellogenesis, but increased markedly in the late phase of vitellogenesis, remaining high during GVM. These findings suggest that switching of the expression of FSHR to LHR controls the effects of FSH and/or LH on vitellogenesis and final oocyte maturation via steroid production in granulosa and thecal cells.

Subcutaneous administration of Kiss1 pentadecapeptide accelerates spermatogenesis in prepubertal male chub mackerel (Scomber japonicus)

Kisspeptins, encoded by kiss genes, have emerged as critical regulator of reproductive function in vertebrates. Our previous studies demonstrated that the chub mackerel (Scomber japonicus) brain expresses kiss1 and kiss2 and peripheral administration of synthetic Kiss1 pentadecapeptide (Kiss1-15) but not Kiss2 dodecapeptide (Kiss2-12) induces spermiation in sexually immature adult chub mackerel. In the present study, we evaluated the potency of Kiss1-15, Kiss2-12, and GnRH analogue (GnRHa) to induce pubertal onset in prepubertal chub mackerel. Peptides were administered through subcutaneous injection for three times (bi-weekly) over 6weeks. Interestingly, gonadosomatic index (GSI) of Kiss1-15 treated fish increased significantly in comparison to other treatments. Histologically, 66.7% of Kiss1-15 treated fish exhibited presence of spermatozoa (SPZ) in the testes with only 28.6% of GnRHa treated fish. However, Kiss2-12 treated fish showed only spermatocytes (SC) as the advanced germ cells in the testes. In contrast, only spermatogonia (SPG) were observed in the testes of control fish. Changes in the number of testicular germ cells among treatments revealed a significantly higher number of SC, spermatids and SPZ in the Kiss1-15 treated fish. Gene expression analyses revealed no significant changes in gnrh1 in the telencephalon-preoptic region of the brain, including fshβ and lhβ in the pituitary of experimental fish. However, GnRHa treated fish showed significantly higher lhβ expression. Levels of sex steroids, 11-ketotestosterone and estradiol-17β were significantly higher in Kiss1-15 treated fish. These results indicate application of Kiss1-15 peptides for accelerating pubertal onset in chub mackerel.

Time course of final oocyte maturation and ovulation in chub mackerel Scomber japonicus induced by hCG and GnRHa

The question of whether the ovulation and spawning time in chub mackerel Scomber japonicus is entrained by a circadian rhythm was raised by our previous experiments. Further questions were also raised about whether the time course of human chorionic gonadotropin (hCG)-induced final oocyte maturation (FOM) and ovulation reflected the natural time course induced by endogeneous pituitary gonadotropin (GtH). To address these questions, hCG and gonadotropin-releasing hormone analog (GnRHa) were administered at two ‘opposite’ times, 14:00 and 02:00 hours, and the time courses of FOM and ovulation were compared. When hCG was injected, ovulation occurred 33 h post-injection in both groups, regardless of the timing of the hCG injection. The timing of ovulation in chub mackerel depends on the timing of hCG injection, but apparently not on circadian rhythms. When GnRHa was injected, ovulation began at 36 h post-injection of GnRHa, regardless of the timing of injection. These results indicate that the time course of FOM and ovulation in the chub mackerel followed a similar pattern whether stimulated by hCG injection or spontaneous luteinizing hormone (LH) surge because GnRHa induces the secretion of endogenous GtH (primarily LH) from the fish pituitary. Thus, it is concluded that the time course of hCG-induced FOM and ovulation in chub mackerel follows the natural time course induced by endogenous pituitary LH.

Peripheral Administration of Kiss1 Pentadecapeptide Induces Gonadal Development in Sexually Immature Adult Scombroid Fish

Kisspeptins have emerged as potent regulators of the reproductive brain-pituitary-gonad (BPG) axis. Our previous study demonstrated that the brain of the chub mackerel (Scomber japonicus), a scombroid fish, expresses two kisspeptin-encoding genes, kiss1 and kiss2, and exhibits sexually dimorphic expression profiles. Recent studies strongly suggest that teleost Kiss1 and Kiss2 precursors produce mature Kiss1-pentadecapeptides (Kiss1–15) and Kiss2-dodecapeptides (Kiss2–12), respectively. In light of the above, the present study evaluated the potency of synthetic peptides of Kiss1–15, Kiss2–12, and a GnRH analog (GnRHa) on inducing gonadal development in sexually immature adult chub mackerel. Synthetic peptides were administered subcutaneously through mini-osmotic pumps. On day 45 post-administration, gonadosomatic index (GSI) values (%) of male fish treated with Kiss1–15 (1.82) significantly increased in comparison to initial control (0.33), final control (0.49), Kiss2–12 (0.24), and GnRHa (1.13)-treated fish. Interestingly, the testis of all Kiss1–15 treated fish revealed spermiation, and were full of spermatozoa. These fish showed significantly higher levels of pituitary fsh&bgr; and Ih&bgr; mRNAs and circulating 11-ketotestosterone. GnRHa treated fish also revealed the presence of few spermatozoa in the testis. In females, no significant changes in GSI values were found between treatments; however, Kiss1–15- and GnRHa-treated fish showed prominent signs of vitellogenic onset, with many early yolk oocytes in their ovaries. Interestingly, Kiss1–15-treated fish exhibited higher levels of pituitary fsh&bgr; and circulating estradiol-17&bgr;. These results indicate that peripheral administration of Kiss1–15 and GnRHa can induce gonadal development in sexually immature chub mackerel.

Functional analysis of kisspeptin peptides in adult immature chub mackerel (Scomber japonicus) using an intracerebroventricular administration method.

In vertebrates (including teleosts), the pivotal hierarchical factor in the control of gonadotropin secretion is the hypothalamic gonadotropin-releasing hormone (GnRH) decapeptide, which regulates the release of pituitary follicle-stimulating hormone (FSH) and luteinizing hormone (LH). Recently, kisspeptins encoded by the Kiss1 gene have been shown to act as upstream endogenous regulators of GnRH neurons in mammals. The chub mackerel (Scomber japonicus) brain expresses two kiss genes (kiss1 and kiss2) that show sexually dimorphic expression profiles during the seasonal gonadal cycle. In the present study, we evaluated the biological potency of kisspeptin peptides to induce transcriptional changes in gnrh1 (hypophysiotropic GnRH form in this species), fshβ and lhβ during the immature stage of adult chub mackerel (2+ years old). Synthetic Kiss1 pentadecapeptide (Kiss1-15) or Kiss2 dodecapeptide (Kiss2-12) at a dose of 100 ng were administered into the intracerebroventricular (ICV) region, and brains were sampled at 6 and 12 h post-injection. In female fish, gnrh1 levels decreased in the presence of both kisspeptin peptides at 12 h post-injection. No significant variation was observed in male fish. In contrast, ICV administration of Kiss2-12 (but not Kiss1-15) significantly increased fshβ and lhβ mRNAs at 12 h post-injection compared to a saline injected control in both sexes. These results suggested that synthetic Kiss2-12 could induce transcriptional changes in gnrh1 and gths.

Immunoreactive changes in pituitary FSH and LH cells during seasonal reproductive and spawning cycles of female chub mackerel Scomber japonicus

The physiological functions of pituitary gonadotropins (GtHs) are well established in higher vertebrates, whereas those in teleosts are still poorly understood. To describe the role of GtHs during gonadal development of female chub mackerel Scomber japonicus, changes in follicle-stimulating hormone (FSH) and luteinizing hormone (LH) cells were investigated immunohistochemically during the seasonal reproductive and spawning cycles. FSH and LH cells were identified in the different cell types of the proximal pars distalis (PPD); FSH cells were located in the central PPD, whereas LH cells were localized along the border of the pars intermedia. To examine changes in FSH and LH cells, the percentage of FSH or LH cell-occupying area in the PPD was evaluated and represented as FSHβ-immunoreactive (ir) or LHβ-ir levels, respectively. FSHβ-ir levels increased significantly from immature to the completion of vitellogenesis, whereas LHβ-ir levels were maintained at high levels from early vitellogenesis to post-spawning. During the spawning cycle, which consisted of four stages from just after spawning to the next oocyte maturation, both FSHβ-ir and LHβ-ir levels showed no significant changes among different stages; however, LHβ-ir levels remained relatively high, and FSHβ-ir levels were constantly low. These results suggest that both FSH and LH may be involved in vitellogenesis and LH may act at final oocyte maturation in female chub mackerel, although the role of FSH during the spawning cycle is still unclear.

Steroidogenic and maturation-inducing potency of native gonadotropic hormones in female chub mackerel, Scomber japonicus

BackgroundThe gonadotropins (GtHs), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are produced in the pituitary gland and regulates gametogenesis through production of gonadal steroids. However, respective roles of two GtHs in the teleosts are still incompletely characterized due to technical difficulties in the purification of native GtHs.MethodsNative FSH and LH were purified from the pituitaries of adult chub mackerel, Scomber japonicus by anion-exchange chromatography and immunoblotting using specific antisera. The steroidogenic potency of the intact chub mackerel FSH (cmFSH) and LH (cmLH) were evaluated in mid- and late-vitellogenic stage follicles by measuring the level of gonadal steroids, estradiol-17beta (Ε2) and 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P). In addition, we evaluated the maturation-inducing potency of the GtHs on same stage follicles.ResultsBoth cmFSH and cmLH significantly stimulated E2 production in mid-vitellogenic stage follicles. In contrast, only LH significantly stimulated the production of 17,20beta-P in late-vitellogenic stage follicles. Similarly, cmLH induced final oocyte maturation (FOM) in late-vitellogenic stage follicles.ConclusionsPresent results indicate that both FSH and LH may regulate vitellogenic processes, whereas only LH initiates FOM in chub mackerel.

Greater amberjack Fsh, Lh, and their receptors: Plasma and mRNA profiles during ovarian development.

To understand the endocrine regulation of ovarian development in a multiple spawning fish, the relationship between gonadotropins (Gths; follicle-stimulating hormone [Fsh] and luteinizing hormone [Lh]) and their receptors (Gthrs; Fshr and Lhr) were investigated in greater amberjack (Seriola dumerili). cDNAs encoding the Gth subunits (Fshβ, Lhβ, and glycoprotein α [Gpα]) and Gthrs were cloned. The in vitro reporter gene assay using recombinant hormones revealed that greater amberjack Fshr and Lhr responded strongly to their own ligands. Competitive enzyme-linked immunosorbent assays (ELISAs) were developed for measuring greater amberjack Fsh and Lh. Anti-Fsh and anti-Lh antibodies were raised against recombinant chimeric single-chain Gths consisting of greater amberjack Fshβ (or Lhβ) with rabbit GPα. The validation study showed that the ELISAs were precise (intra- and inter-assay coefficient of variation, <10%) and sensitive (detection limit of 0.2ng/ml for Fsh and 0.8ng/ml for Lh) with low cross-reactivity. A good parallelism between the standard curve and serial dilutions of greater amberjack plasma and pituitary extract were obtained. In female greater amberjack, pituitary fshb, ovarian fshr, and plasma E2 gradually increased during ovarian development, and plasma Fsh significantly increased during the post-spawning period. This suggests that Fsh plays a role throughout ovarian development and during the post-spawning period. Pituitary lhb, ovarian lhr, and plasma Lh were high during the spawning period, suggesting that the synthesis and secretion of Lh, and Lhr expression are upregulated to induce final oocyte maturation and ovulation.