The Roles of Kisspeptin System in the Reproductive Physiology of Fish With Special Reference to Chub Mackerel Studies as Main Axis.

Kisspeptin, a novel neuropeptide product of the Kiss1 gene, activates the G protein-coupled membrane receptor G protein-coupled receptor 54 (now termed Kiss1r). Over the last 15 years, the importance of the kisspeptin system has been the subject of much debate in the mammalian research field. At the heart of the debate is whether kisspeptin is an absolute upstream regulator of gonadotropin-releasing hormone secretion, as it has been proposed to be the master molecule in reproductive events and plays a special role not only during puberty but also in adulthood. The teleostean kisspeptin system was first documented in 2004. Although there have been a number of kisspeptin studies in various fish species, the role of kisspeptin in reproduction remains a subject of controversy and has not been widely recognized. There is an extensive literature on the physiological and endocrinological bases of gametogenesis in fish, largely derived from studying small, model fish species, and reports on non-model species are limited. The reason for this discrepancy is the technical difficulty inherent in developing rigorous experimental systems in many farmed fish species. We have already established methods for the full life-cycle breeding of a commercially important marine fish, the chub mackerel (cm), and are interested in understanding the reproductive function of kisspeptins from various perspectives. Based on a series of experiments clarifying the role of the brain-pituitary-gonad axis in modulating reproduction in cm, we theorize that the kisspeptin system plays an important role in the reproduction of this scombroid species. In this review article, we provide an overview of kisspeptin studies in cm, which substantially aids in elucidating the role of kisspeptins in fish reproduction.

Molecular characterization and functional analysis of pituitary GnRH receptor in a commercial scombroid fish, chub mackerel (Scomber japonicus).

The gonadotropin-releasing hormone (GnRH) is essential during pubertal onset, for its regulation of the synthesis and release of pituitary gonadotropins. Its action is mediated by GnRH receptors (GnRHRs) in the pituitary gonadotrophs. Our previous study demonstrated that the chub mackerel brain expresses three GnRH forms (gnrh1, gnrh2, and gnrh3), and that only GnRH1 neurons innervate anterior pituitary regions. Furthermore, chub mackerel gnrh1 mRNA exhibited a significant increase at pubertal onset. The present study aimed to isolate the functional GnRHR form involved in chub mackerel puberty. The open reading frame of our cloned receptor encodes 428 amino acids and contains seven transmembrane domains. Phylogenetic analysis also indicated clustering with other teleost-type IIB GnRHRs, mainly those involved in reproduction. Reporter gene assay results showed that all four synthetic peptides (GnRH1, GnRH2, GnRH3, and GnRH analogue) bind to the cloned receptor. Three deduced GnRH ligands stimulated luteinizing hormone (LH) release from cultured pituitary cells in vitro. Receptor gene expression was mainly detected in the pituitary and showed an increasing trend in the developing gonadal stages of both sexes during the pubertal process; this process was synchronous with previous studies of follicle-stimulating hormone beta (fshß) and lhß gene expression in chub mackerel. These results suggest that the cloned receptor is likely involved in the regulation of pubertal onset in this species. Therefore, we have designated the receptor cmGnRHR1.

Expression changes of mRNAs encoding kisspeptins and their receptors and gonadotropin-releasing hormones during early development and gonadal sex differentiation periods in the brain of chub mackerel (Scomber japonicus).

In recent years, brain kisspeptin system has been shown to be involved in diverse reproductive function, including sexual differentiation in vertebrates. Our previous reports demonstrated that the chub mackerel (Scomber japonicus) brain expresses two kisspeptin (kiss1, kiss2), two kisspeptin receptor (kissr1, kissr2) and three gonadotropin-releasing hormone (gnrh1, gnrh2, gnrh3) genes. In the present study, using quantitative real-time PCR (qRT-PCR) assays, we analysed expression changes of these genes during early development (0-30dphs) and gonadal sex differentiation periods (37-60dphs). Absolute expression level of kiss-kissr-gnrh in the whole head was higher between 0 and 15dphs, in comparison to later developmental periods. Histological analyses revealed presence of sexually differentiated males and females with testicular and ovarian features at 37, 45, and 60dphs. In both males and females, kiss2, kissr1, and kissr2 levels were higher at 37dph, in comparison to 45 and 60dphs, with kiss1 showing no significant differences. Levels of all three gnrh mRNAs were higher at 45dph, in comparison to 60dph. Changes in the expression level of kiss-kissr-gnrh mRNAs in different brain regions of sexually differentiated males and females indicated differences in their regional distribution. These results suggest possible involvement of Kiss-KissR-GnRH systems during early development and gonadal sex differentiation in the chub mackerel.

mRNA levels of kisspeptins, kisspeptin receptors, and GnRH1 in the brain of chub mackerel during puberty.

Kisspeptin (Kiss) and its cognate receptor (Kiss1R), implicated in the neuroendocrine control of GnRH secretion in mammals, have been proposed to be the key factors in regulating puberty. However, the mechanisms underlying the initiation of puberty in fish are poorly understood. The chub mackerel Scomber japonicus expresses two forms of Kiss (kiss1 and kiss2) and two Kiss receptor (kissr1 and kissr2) genes in the brain, which exhibit sexually dimorphic changes during the seasonal reproductive cycle. This indicates that the kisspeptin system plays an important role in gonadal recrudescence of chub mackerel; however, the involvement of the kisspeptin system in the pubertal process has not been identified. In the present study, we examined the mRNA expression of kiss1, kiss2, kissr1, kissr2, and gnrh1 (hypophysiotropic form) in the brain of a chub mackerel during puberty. In male fish, kiss2, kissr1 and kissr2 levels increased significantly at 14weeks post-hatch (wph), synchronously with an increase in type A spermatogonial populations in the testis; kiss2 and gnrh1 levels significantly increased at 22wph, just before the onset of meiosis in the testes. In female fish, kiss2 increased significantly at 14wph, synchronously with an increase in the number of perinucleolar oocytes in the ovary; kiss1 and kiss2 levels significantly increased concomitantly with an increase in the kissr1, kissr2, and gnrh1 levels at 24wph, just before the onset of vitellogenesis in oocytes. The present results suggest positive involvement of the kisspeptin-GnRH system in the pubertal process in the captive reared 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), fshß and lhß 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 fshß and lhß 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.

Identification and distribution of three gonadotropin-releasing hormone (GnRH) isoforms in the brain of a clupeiform fish, Engraulis japonicus.

To gain a better understanding of the reproductive endocrinology of a primitive order clupeiform fish (Japanese anchovy, Engraulis japonicus), cDNAs encoding three gonadotropin-releasing hormone (GnRH) isoforms were isolated from the brain, and their distribution was analyzed using insitu hybridization (ISH). The three GnRH isoforms include GnRH1 (herring GnRH), GnRH2 (chicken GnRH-ll) and GnRH3 (salmon GnRH), and their full-length cDNAs encode 88, 86, and 89 deduced amino acids (aa), respectively. Alignment analysis of Japanese anchovy GnRH isoforms showed lower identities with other teleost fish. The major population of GnRH1 neurons was localized in the ventral telencephalon (VT) and nucleus preopticus (NPO) of the preoptic area (POA) with minor population in the anterior olfactory bulb (OB). GnRH2 neurons were restricted to the midbrain tegmentum (MT), specific to the nucleus of the medial longitudinal fasciculus (nMLF). GnRH3 neurons were localized in the olfactory nerve (ON), ventral OB, and transitional area between OB and ON. Interestingly, GnRH1 neurons were also localized in the olfactory bulb, in addition to its major population in the preoptic area. These results indicate the differential distribution of three GnRH isoforms expressed in the brain of the Japanese anchovy.

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.

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 fshß and lhß in the pituitary of experimental fish. However, GnRHa treated fish showed significantly higher lhß expression. Levels of sex steroids, 11-ketotestosterone and estradiol-17ß 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 fshß and Ihß 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 fshß and circulating estradiol-17ß. These results indicate that peripheral administration of Kiss1-15 and GnRHa can induce gonadal development in sexually immature chub mackerel.

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

BACKGROUND: The 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. METHODS: Native 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. RESULTS: Both 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. CONCLUSIONS: Present results indicate that both FSH and LH may regulate vitellogenic processes, whereas only LH initiates FOM in chub mackerel.

Immunoreactivity of gonadotrophs (FSH and LH Cells) and gonadotropin subunit gene expression in the male chub mackerel Scomber japonicus pituitary during the reproductive cycle.

The gonadotropins (GtHs), follicle-stimulating hormone (FSH) and luteinizing hormone (LH), are heterodimers composed of a common α subunit (GPα) and a unique ß subunit (FSHß or LHß); they are synthesized in and secreted from gonadotrophs (FSH and LH cells) in the pituitary. Little is known about the roles of FSH and LH during spermatogenesis in perciform fishes. In this study, we examined immunoreactive changes in FSH and LH cells, and changes in the gene expression of the three gonadotropin subunits in the pituitary of male chub mackerel Scomber japonicus during testicular development. FSHß-immunoreactive (ir) and LHß-ir cell area were measured immuno-histochemically based on the FSH and LH cell-occupying area in the proximal pars distalis. The FSHß-ir cell area increased significantly during spermiation, while FSHß mRNA levels, already high at the beginning of spermatogenesis, increased further, peaking during spermiation. In contrast, LHß-ir cell area and LHß mRNA levels, which were low at the beginning of spermatogenesis, increased significantly during late spermatogenesis, peaking during spermiation. For both FSH and LH, GtHß-ir cell area and GtHß mRNA levels decreased until gonadal resting. GPα mRNA levels showed similar changes to LHß mRNA levels. These results suggest that in the chub mackerel, FSH may play an important role in the early and late phases of spermatogenesis, and that LH may play a role during late spermatogenesis and spermiation. Moreover, our results demonstrate that changes in GtHß-ir cell area were accompanied by similar changes in the expression of the FSHß and LHß genes, both of which increased during testicular development.

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

BACKGROUND: Kisspeptins (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. METHODS: Female 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. RESULTS: Both 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. CONCLUSION: The 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.

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, FSHß, and LHß) during the reproductive cycle of the female chub mackerel Scomber japonicus. Cloning and subsequent sequence analysis revealed that the cDNAs of chub mackerel GPα, FSHß, and LHß 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, FSHß mRNA levels remained high during the vitellogenic stages, while GPα and LHß 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 FSHß 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, LHß 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.

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.

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.