Sethu Selvaraj

Molecular characterization, tissue distribution, and mRNA expression profiles of two Kiss genes in the adult male and female chub mackerel (Scomber japonicus) during different gonadal stages

Kisspeptins, encoded by the Kiss1 gene, have emerged as key modulators of reproduction in mammals. In contrast to the placental mammals, some teleosts express two Kiss genes, Kiss1 and Kiss2. In the present study, full-length cDNAs of Kiss1 and Kiss2 in the chub mackerel were cloned and sequenced. Chub mackerel Kiss1 and Kiss2 cDNAs encode 105 and 123 amino acids, respectively. A comparison of the deduced amino acid sequences of chub mackerel Kiss1 and Kiss2 with those of other vertebrate species showed a high degree of conservation only in the kisspeptin-10 region (Kp-10). The Kp-10 of chub mackerel Kiss1 (YNFNSFGLRY) and Kiss2 (FNFNPFGLRF) showed variations at three amino acids. Tissue distribution analysis using quantitative real-time PCR (qRT-PCR) revealed that the Kiss1 and Kiss2 transcripts were expressed in different tissues of adult chub mackerel. In addition, their levels in the adipose tissue exhibited sexually dimorphic expression. Further, to have a basic understanding on the involvement of Kiss1 and Kiss2 in the seasonal gonadal development, their relative mRNA expression profiles in the brain, pituitary, and gonads at different gonadal stages were analyzed using qRT-PCR. Kiss1 and Kiss2 levels in the brain showed a differential expression profile between male and female fish. In males, Kiss1 and Kiss2 levels gradually decreased from the immature stage to spermiation and reached a minimal level during the post-spawning period. In contrast, Kiss1 levels in the brain of females did not vary significantly among the different gonadal stages. However, Kiss2 levels fluctuated as that of males, gradually declining from the immature stage to the post-spawning period. The pituitary Kiss1 levels did not show significant fluctuations. However, Kiss1 levels in the gonads were highly elevated during spermiation and late vitellogenesis compared to the immature and post-spawning period. These results suggest the possible involvement of two Kiss genes in the brain and Kiss1 in the gonads of chub mackerel during seasonal gonadal development.

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.

Immunological Characterization and Distribution of Three GnRH Forms in the Brain and Pituitary Gland of Chub Mackerel (Scomber japonicus)

The presence of three gonadotropin-releasing hormone (GnRH) forms in the brain of the chub mackerel, Scomber japonicus, namely, salmon GnRH (sGnRH), chicken GnRH-II (cGnRH-II), and seabream GnRH (sbGnRH), was confirmed by combined high performance liquid chromatography (HPLC) and time-resolved fluoroimmunoassay (TR-FIA). Immunocytochemical localization of the three GnRH forms in the brain was Investigated by using specific antisera, to elucidate possible roles of each GnRH form in reproduction in this species, and double immunolabeling was used to localize GnRH-ir (immunoreactive) fibers Innervating the pituitary. sGnRH-ir neurons were localized in the ventral olfactory bulb and terminal nerve ganglion region. Further, sGnRH-ir fibers were found in different regions of the brain, with prominent fibers running in parallel in the preoptic area (POA) without entering the pituitary. cGnRH-II-ir cell bodies were observed only in the midbrain tegmentum region, with a wide distribution of fibers, which were dense in the midbrain tegmentum and spinal cord. SbGnRH-ir cell bodies were localized in the nucleus preopticus of the POA, with fibers in the olfactory bulb, POA, and hypothalamus. Among the three GnRH forms, only SbGnRH-ir fibers innervated the pituitary gland from the preoptic-hypothalamic region, targeting follicle stimulating hormone (FSH) and luteinizing hormone (LH)-producing cells in the proximal pars distalis, as demonstrated by double immunocytochemistry. The localization of the GnRH-ir system was similar in male and female fish. These results demonstrate that multiple GnRH forms exist in the brain of the chub mackerel and suggest that they serve different functions, with SbGnRH having a significant role in reproduction in stimulating FSH- and LH-producing cells, and sGnRH and cGnRH-II serving as neurotransmitters or neuromodulators.

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.

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.

Increased expression of kisspeptin and GnRH forms in the brain of scombroid fish during final ovarian maturation and ovulation

BackgroundKisspeptins (Kiss) are prime players in the control of reproductive function through their regulation of gonadotropin-releasing hormone (GnRH) expression in the brain. The experimental scombroid fish, chub mackerel (Scomber japonicus) expresses two kiss (kiss1 and kiss2) and three gnrh (gnrh1, gnrh2, and gnrh3) forms in the brain. In the present study, we analyzed expression changes of kiss and gnrh mRNAs in the brain and corresponding GnRH peptides in the brain and pituitary during final ovarian maturation (FOM) and ovulation.MethodsFemale fish possessing late vitellogenic oocytes were injected with GnRH analogue to induce FOM and ovulation. Fish were observed for daily spawning activities and sampled one week post-injection at germinal vesicle migration (GVM), oocyte hydration, ovulation, and post-ovulatory time periods. Changes in relative mRNA levels of kiss and gnrh forms in the brain were determined using quantitative real-time PCR. Changes in GnRH peptides in the brain and pituitary were analyzed using time-resolved fluoroimmunoassay.ResultsBoth kiss1 and kiss2 mRNA levels in the brain were low at late vitellogenic stage and increased significantly during the GVM period. However, kiss1 mRNA levels decreased during oocyte hydration before increasing again at ovulatory and post-ovulatory periods. In contrast, kiss2 mRNA levels decreased at ovulatory and post-ovulatory periods. Levels of gnrh1 mRNA in the brain increased only during post-ovulatory period. However, levels of gnrh2 and gnrh3 mRNAs were elevated during GVM and then, decreased during oocyte hydration before increasing again at ovulatory period. During post-ovulatory period, both gnrh2 and gnrh3 mRNA levels declined. Peptide levels of all three GnRH forms in the brain were elevated during GVM and oocyte hydration; their levels were significantly lower during late vitellogenic, ovulatory, and post-ovulatory periods. In contrast, pituitary GnRH peptide levels did not show any significant fluctuations, with the GnRH1 peptide levels being many-fold higher than the GnRH2 and GnRH3 forms.ConclusionThe results indicate increased expression of multiple Kiss and GnRH forms in the brain and suggest their possible involvement in the regulation of FOM and ovulation in captive female 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.