Long-day stimulation increases thyroid-stimulating hormone expression and affects gonadal development in chub mackerel.

For seasonal breeders, photoperiodic changes are important signals that mark the start of the breeding season. Thyroid-stimulating hormone (TSH) is a glycoprotein hormone that not only promotes the secretion of thyroid hormone but also plays a key role in regulating seasonal reproduction in birds and mammals. However, whether TSH activation has been implicated as a seasonal indicator in fish breeding has not been fully investigated. In this study, we isolated tshb as a starting point to elucidate the effect of photoperiodic changes on the activation of the reproductive axis of chub mackerel. The isolated tshb was classified as tshba, which is widely conserved in vertebrates. The quantitative PCR results showed that tshb was strongly expressed in the pituitary. When female and male chub mackerel with immature gonads were reared for six weeks under different photoperiodic conditions, the gonads developed substantially in the long-day (LD) reared fish compared to those in the short-day reared fish. Real-time PCR results showed that the expression level of tshb in the pituitary gland was significantly elevated in the LD group. Although there was no difference in the gonadotropin-releasing hormone 1 gene expression level in the preoptic area of the brain, follicle-stimulating hormone and luteinizing hormone gene expression levels in the pituitary were also significantly elevated in the LD group. In conclusion, TSH is a potential mediator of seasonal information in the reproductive endocrine axis and may induce gonadal development during the breeding season of chub mackerel.

Effects of LPXRFamide peptides on chub mackerel gonadotropin secretion†.

Gonadotropin-inhibitory hormone (GnIH), a neuropeptide, suppresses gonadotropin (GTH) secretion in birds and mammals. In fish, the GnIH homolog LPXRFamide (LPXRFa) produces mature peptides with species-dependent effects on sexual reproduction. Here, we investigated the effects of LPXRFa on GTH secretion in the chub mackerel (cm; Scomber japonicus). We cloned cmlpxrfa (603 bp) and cmlpxrfa-r (1416 bp). Additionally, we isolated lpxrfa from the bluefin tuna (Thunnus orientalis) to confirm the conservation of the LPXRFa mature sequence. Phylogenetic analysis showed that the LPXRFa precursor protein produces three mature peptides, LPXRFa-1, -2, and -3, in both species. Reverse transcription-quantitative polymerase chain reaction revealed that cmlpxrfa is expressed in the hypothalamus and thalamus and midbrain (T.MB), and sexual differences were observed. Receptor expression was observed in the preoptic area, hypothalamus, T.MB, and pituitary. Female hypothalamic lpxrfa expression did not change during puberty. Reporter gene assay showed that LPXRFa induced receptor activation via the CRE and SRE signaling pathways. However, in the presence of forskolin, an intracellular cyclic AMP enhancer, none of the LPXRFa could suppress receptor activity. The in vitro bioassay results showed that gonadotropin-releasing hormone-1 (GnRH1) had no effect on follicle-stimulating hormone (FSH) secretion, whereas the three LPXRFa significantly increased FSH secretion in pituitary cells from male chub mackerel. Contrarily, GnRH1 and three LPXRFa significantly increased luteinizing hormone secretion. The in vivo administration of LPXRFa had no effect on fshb and lhb expression in prepubertal and mature male chub mackerel. Overall, cmLPXRFa lacks the ability to suppress GTH secretion but can promote GTH secretion.

In vitro action of leptin on gonadotropin secretion in pre-pubertal male chub mackerel.

Leptin directly influences gonadotropin (GTH) secretion from female pituitary cells in vitro and is a key signal at the onset of puberty in female chub mackerel (Scomber japonicus). Here, we investigated whether leptin also influences GTH secretion in male chub mackerel. The addition of 1 nM homologous recombinant leptin to pre-pubertal male pituitary cells stimulated follicle-stimulating hormone secretion after 1 and 2 h of culture. Therefore, leptin signaling could also directly facilitate GTH secretion in male chub mackerel.

Alanine scanning and characterization of core peptides in Scombridae fish family for construction of Kiss1 super analog.

Chronic Kiss1 administration strongly promotes gonadal development in immature chub mackerel (cm) (Scomber japonicus). Here, we performed an Alanine scanning (Ala-scanning) of Kiss1 to determine its key residues. Additionally, we examined functional peptides from 16 Scombridae species to develop maturation-inducing super-analogs that can be used universally in Scombridae species. In the Ala-scanning of Kiss1-15 (QDMSSYNFNSFGLRY), substitution of Gln1 and Asp2 did not affect agonistic activity. This suggests that peptides could be downsized. Furthermore, it is possible that Phe8 can be substituted by unnatural amino acids that are difficult to degrade. In molecular cloning, only Scomber showed a 16-residue form as a putative mature peptide. The other genera, did not have a His residue at the N-terminal, which indicated that the functional peptide was 15 residues and the second and third residues from the N-terminal showed variation between interspecies. Next, we examined the binding affinity of various synthetic Kiss1 core peptides in Scombridae interspecies using an SRE-Luc reporter system. We cloned Kiss1 receptors (KissR1) from bluefin tuna (bft) (Thunnus orientalis) and Japanese Spanish mackerel (jsm) (Scomberomorus niphonius) for the first time. In binding affinity with cmKissR1, bftKissR1, and jsmKissR1, the species specificity of the second residue from the N-terminus in each ligand could be ignored, but the difference in the third residue strongly affected receptor binding. Scombridae species possess the same Kiss1 system but the structure of the functional peptide might be species-specific.

Leptin stimulates gonadotropin release and ovarian development in marine teleost chub mackerel.

Leptin transmits information about energy stored in the periphery to the reproductive axis and is an essential signal for puberty initiation in mammals; however, to date, few studies have focused on the direct effects of leptin stimulation on reproductive factors in fish. This study demonstrated the effect of leptin stimulation on important reproductive factors and ovarian development in the marine teleost chub mackerel (Scomber japonicus). We prepared recombinant leptin and conducted functional analyses through in vitro bioassays using primary pituitary cells, long-term leptin treatment administered to pre-pubertal females, and intracerebroventricular (ICV) administration. The results showed that leptin stimulation strongly induced gonadotropin (follicle-stimulating hormone: FSH and luteinizing hormone: LH) secretion from pituitary cells collected from pre-pubertal females, and that long-term leptin treatment significantly promoted ovarian development and triggered pubertal onset. Furthermore, ICV administration of leptin did not affect kisspeptin gene expression but significantly upregulated gonadotropin-releasing hormone 1 (gnrh1), fshb and lhb gene expression in sexually immature females. These results strongly suggest leptin as an important signal for reproductive-axis activation in chub mackerel.

Kiss1 hexadecapeptide directly regulates gonadotropin-releasing hormone 1 in the scombroid fish, chub mackerel.

Here we report that the Kiss1 hexadecapeptide (Kiss1-16) directly regulates the functional form of gonadotropin-releasing hormone (GnRH) in the preoptic area (POA) of a scombroid fish model. In this study, we analyzed the localization of two kisspeptin (kiss1 and kiss2) neurons and two kisspeptin receptors (kissr1 and kissr2) in the brain of adult chub mackerel using in situ hybridization to determine whether the kisspeptin receptors co-localize with GnRH1 neurons. The kiss1- and kiss2-expressing neurons were mainly localized in the nucleus recessus lateralis (NRL) and the nucleus of the posterior recess (NRP) in the hypothalamus. Kissr1 was present in the anterior POA and the habenular nucleus. Kissr2 was widely distributed, including in the POA, lateral tuberal nucleus, NRL, and NRP. Notably, GnRH1 was expressed in neurons in the POA, and these neurons co-expressed kissr1. In contrast, kissr2 was expressed abundantly in the vicinity of GnRH1 neurons, but their co-expression did not seem to occur. We also characterized the endogenous mature form of the Kiss1 peptide. An in vitro reporter gene assay clearly showed that Kiss1-16 (HQDMSSYNFNSFGLRY-NH2) was more potent at receptor activation than Kiss1 pentadecapeptide (Kiss1-15), which is the form of Kiss1 found in other fish species. This study strongly suggests that kisspeptin signaling, especially Kiss1 signaling, is important for regulating reproduction in scombroid fish.

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.

Possible role of the leptin system in controlling puberty in the male chub mackerel, Scomber japonicus.

Leptin directly regulates kisspeptin neurons in the hypothalamus and gonadotropin secretion from the pituitary, making it a central player in the onset of mammalian puberty. Recently, we identified two leptin genes (lepa and lepb) and a single leptin receptor (lepr) in the marine perciform fish chub mackerel; however, the expression of these genes did not correlate with the expression of important reproductive genes or ovarian stage during female puberty. Here, we expand upon these initial observations by evaluating the expression of lepa, lepb, and lepr during pubertal transition and under differential feeding conditions in the male chub mackerel. Reverse transcription-polymerase chain reaction (RT-PCR) showed that lepa was primarily expressed in the liver of pubertal and gonadal recrudescence adults, as well as in the brain of adult fish; lepb was primarily expressed in the brain of all fish tested; and lepr was widely expressed in a variety of tissues. qRT-PCR analyses revealed significant increases in the hepatic expression of lepa in accordance with testicular stage, whereas pituitary follicle-stimulating hormone (fshß) expression increased in unison with hepatic lepa. In contrast, expression of both brain lepa and lepb dramatically decreased during pubertal transition, with brain kisspeptin 1 (kiss1) expression strongly correlating with leptin expression patterns. In pre-pubertal males, lepa, lepb, and lper gene expression in the brain, pituitary gland, and liver decreased in fish given a high feed diet, relative to the controlled feeding group. Taken together, these results indicate high sexual specificity of leptin expression, suggesting a possible role for leptin signaling in endocrine and neuroendocrine functions during spermatogenesis in the pubertal male chub mackerel.

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.

Two leptin genes and a leptin receptor gene of female chub mackerel (Scomber japonicus): Molecular cloning, tissue distribution and expression in different obesity indices and pubertal stages.

Leptin is a hormone produced by fat cells that regulates the amount of fat stored in the body and conveys nutritional status to the reproductive axis in mammals. In the present study we identified two subtypes of leptin genes (lepa and lepb) and a leptin receptor gene (lepr) from chub mackerel (Scomber japonicus) and there gene expression under different feeding conditions (control and high-feed) and pubertal development stages was analyzed using quantitative real-time PCR. The protein lengths of LepA, LepB and LepR were 161 amino acids (aa), 163 aa and 1149 aa, respectively and both leptin subtypes shared only 15% similarity in aa sequences. In pubertal females, lepa was expressed in the brain, pituitary gland, liver, adipose tissue and ovary; however, in adult (gonadal maturation after the second in the life) females, lepa was expressed only in the liver. lepb was expressed primarily in the brain of all fish tested and was expressed strongly in the adipose tissue of adults. lepr was characterized by expression in the pituitary. The high-feed group showed a high conditioning factor level; unexpectedly, hepatic lepa and brain lepr were significantly more weakly expressed compared with the control-feed group. Furthermore, the expression levels of lepa, lepb and lepr genes showed no significant differences between pre-pubertal and post-pubertal fish. On the other hand, pituitary fshß and lhß showed no significant differences between different feeding groups of pre-pubertal fish. In contrast, fshß and lhß expressed abundantly in the post-pubertal fish of control feed group. Based on these results, whether leptin plays an important role in the nutritional status and pubertal onset of chub mackerel remains unknown.

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.

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.

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.

Development of a chub mackerel with less-aggressive fry stage by genome editing of arginine vasotocin receptor V1a2.

Genome editing is a technology that can remarkably accelerate crop and animal breeding via artificial induction of desired traits with high accuracy. This study aimed to develop a chub mackerel variety with reduced aggression using an experimental system that enables efficient egg collection and genome editing. Sexual maturation and control of spawning season and time were technologically facilitated by controlling the photoperiod and water temperature of the rearing tank. In addition, appropriate low-temperature treatment conditions for delaying cleavage, shape of the glass capillary, and injection site were examined in detail in order to develop an efficient and robust microinjection system for the study. An arginine vasotocin receptor V1a2 (V1a2) knockout (KO) strain of chub mackerel was developed in order to reduce the frequency of cannibalistic behavior at the fry stage. Video data analysis using bioimage informatics quantified the frequency of aggressive behavior, indicating a significant 46% reduction (P = 0.0229) in the frequency of cannibalistic behavior than in wild type. Furthermore, in the V1a2 KO strain, the frequency of collisions with the wall and oxygen consumption also decreased. Overall, the manageable and calm phenotype reported here can potentially contribute to the development of a stable and sustainable marine product.

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.

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.

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.

Leptin Is an Important Endocrine Player That Directly Activates Gonadotropic Cells in Teleost Fish, Chub Mackerel.

Leptin, secreted by adipocytes, directly influences the onset of puberty in mammals. Our previous study showed that leptin stimulation could promote the secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from pituitary cells in primary culture and ovarian development in chub mackerel. This study aimed to elucidate the detailed mechanism of leptin-induced effects on gonadotropin hormone-producing cells. We produced recombinant leptin using silkworm pupae and investigated the effects of leptin on FSH and LH secretion and gene expression in the primary culture of pituitary cells from chub mackerel. The presence or absence of co-expression of lepr mRNA, FSH and LH b-subunit mRNA in gonadotropic cells was examined by double-labeled in situ hybridization. The addition of leptin significantly increased the secretion and gene expression of FSH and LH from male and female pituitary cells in primary culture. In contrast, gonadotropin-releasing hormone 1 affected neither FSH secretion in cells from females nor fshb and lhb expression in cells from males and females. The expression of lepr was observed in FSH- and LH-producing cells of both males and females. The results indicate that leptin directly regulates gonadotropin synthesis and secretion and plays an important role in the induction of puberty in teleost fish.