RESUMEN
17ß-hydroxysteroid dehydrogenases (Hsd17bs) play a critical role in sex steroid biosynthesis. Although multiple types of Hsd17b have been found in fish, there is limited research on their expression and function. Recently, we succeeded in identifying eight types of Hsd17b (types 3, 4, 7, 8, 10, 12a, 12b, and 14) by RNA sequencing in the Japanese sardine Sardinops melanostictus, a commercially important clupeoid fish; however, a homologous sequence of Hsd17b1, which catalyzes the key reaction of estradiol-17ß (E2) synthesis, was absent. Here, we aimed to identify the Hsd17b type that plays a major role in E2 synthesis during ovarian development in Japanese sardine. The cDNAs encoding those eight types of Hsd17b were cloned and sequenced. The expressions of hsd17b3, hsd17b12a, and hsd17b12b were higher in ovary than in testis. In particular, hsd17b12a was predominantly expressed in the ovary. Expression of hsd17b3, hsd17b4, hsd17b12a, and hsd17b12b in the ovary increased during ovarian development. The enzymatic activities of Hsd17b3, Hsd17b12a, and Hsd17b12b were evaluated by expressing their recombinants in human embryonic kidney 293T cells. Hsd17b12a and Hsd17b12b catalyzed the conversion of androstenedione (AD) to testosterone (T) and estrone (E1) to E2. The results of in vitro bioassays using sardine ovaries indicated that E2 is synthesized from pregnenolone via AD and T, but not E1. These results suggest that Hsd17b12a plays a major role in E2 synthesis in sardine ovary by catalyzing the conversion of AD to T.
Asunto(s)
Estradiol , Ovario , Masculino , Femenino , Animales , Humanos , Ovario/metabolismo , Estradiol/metabolismo , Testículo/metabolismo , Testosterona/metabolismo , 17-Hidroxiesteroide Deshidrogenasas/genética , 17-Hidroxiesteroide Deshidrogenasas/metabolismo , Androstenodiona/metabolismo , Peces/genética , Peces/metabolismoRESUMEN
Unlike mammals, teleost fish have high aromatase activity (AA) in the pituitary. However, the cells responsible for oestradiol synthesis and the local physiological roles of this hormone remain unclear. Hence, we investigated the effects of age and development on steroidogenic activity, mRNA expression, and cyp19a1b localization in the pituitary gland of the Japanese pufferfish Takifugu rubripes. Under aquaculture conditions, AA was highest after puberty, and the mRNA expression levels of cyp19a1b and the oestrogen receptors esr1 and 2b and the level of serum testosterone (T) were significantly increased after puberty compared with the other developmental stages in male and female pufferfish. Immunohistochemistry using multiple antibodies and in situ hybridization analysis revealed that Cyp19a1b colocalizes with luteinizing hormone (LH) in pituitary cells. Furthermore, Esr1 was localized in the nuclei of all hormone-producing cells, whereas Esr2b was localized only in the nuclei of Cyp19- and LH-positive cells. The administration of an aromatizable androgen (T) or oestrogen (E2) to reproductively inactive females induced LH synthesis in vivo. We prepared spheroids from pituitary cells to investigate the role of local E2 in LH synthesis in vitro. Immunohistochemical analysis of spheroids showed that T-induced LH synthesis could be blocked by an aromatase inhibitor and/or an ER antagonist but not an AR antagonist. Taken together, these findings suggest that LH synthesis is initiated in cyp19a1b-, esr1-, and esr2b-expressing cells at the onset of puberty under the control of steroidal feedback, and both feedback and local oestrogen may be involved in controlling LH synthesis in these cells.
Asunto(s)
Aromatasa , Takifugu , Animales , Aromatasa/genética , Estradiol/farmacología , Estrógenos , Femenino , Hormona Folículo Estimulante , Hormona Luteinizante , Masculino , Mamíferos/metabolismo , Hipófisis/metabolismo , Pubertad , ARN Mensajero/genética , Takifugu/genética , Testosterona/metabolismoRESUMEN
The pituitary gonadotropins (Gths), follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh), play critical roles in regulating gonadal development and sexual maturation in vertebrates. We developed non-competitive enzyme-linked immunosorbent assays (ELISAs) to measure Fsh and Lh in chub mackerel Scomber japonicus, which is a commercially important scombrid species. Mouse monoclonal antibodies specific for Fsh and Lh, and a rabbit polyclonal antibody against both Gths were produced by immunization with hormones purified from chub mackerel pituitaries. These monoclonal and polyclonal antibodies were used as capture and detection antibodies in the developed sandwich ELISAs. The ELISAs were reproducible, sensitive, and specific for chub mackerel Fsh and Lh. Parallelism between the standard curve and serial dilutions of chub mackerel serum and pituitary extract was observed for both Fsh and Lh ELISAs. Comparison between vitellogenic and immature females revealed that Fsh is secreted during vitellogenesis and Lh is barely released during immaturity. After gonadotropin-releasing hormone analog (GnRHa) injection, vitellogenic females showed increases in serum Lh, whereas serum levels of Fsh did not vary. Moreover, the serum steroid profiles revealed that estradiol-17ß was continuously produced after GnRHa treatment, whereas 17,20ß-dihydroxy-4-pregnen-3-one secretion was transiently induced. These results indicate that, in vitellogenic females, GnRHa stimulates the release of Lh, but not Fsh, which results in acceleration of vitellogenesis and induction of oocyte maturation via steroid production.
Asunto(s)
Cyprinidae , Perciformes , Animales , Anticuerpos Monoclonales , Ensayo de Inmunoadsorción Enzimática/métodos , Estradiol , Femenino , Hormona Folículo Estimulante , Hormona Liberadora de Gonadotropina , Gonadotropinas , Gonadotropinas Hipofisarias , Hormona Luteinizante , Ratones , Perciformes/fisiología , Conejos , VitelogénesisRESUMEN
BACKGROUND: The clupeoid fishes are ecologically and commercially important fish species worldwide that exhibit a high level of population fluctuation, accompanied by alteration of reproductive traits. However, knowledge about their reproductive physiology in order to understand mechanisms underlying such population dynamics is limited. The endocrine system along with the brain-pituitary-gonadal (BPG) axis is critical for regulating reproduction. The aims of this study were to provide transcript data and genes related to the BPG axis, and to characterize the expression profiles of ovarian steroidogenesis-related genes in the Japanese sardine (Sardinops melanostictus, Clupeidae). RESULTS: RNA sequencing was performed using the sardine brain, pituitary, and gonad in both sexes. A total of 290,119 contigs were obtained and 115,173 non-redundant ORFs were annotated. The genes differentially expressed between ovary and testis were strongly associated with GO terms related to gamete production. The tissue-specific profile of the abundance of transcripts was characterized for the major regulators in the BPG axis, such as gonadotropin-releasing hormone, gonadotropin, and steroidogenic enzyme. By comparing between ovary and testis, out of eight different 17ß-hydroxysteroid dehydrogenase (Hsd17b) genes identified, higher hsd17b7 expression was found in testis, whereas higher expression of hsd17b8, hsd17b10, hsd17b12a, and hsd17b12b was found in ovary. The cDNAs encoding key endocrine factors in the ovarian steroidogenic pathway were cloned, sequenced, and quantitatively assayed. In the pituitary, follicle-stimulating hormone beta peaked during vitellogenesis, while luteinizing hormone beta peaked at the completion of vitellogenesis. In the ovary, follicle-stimulating hormone receptor and luteinizing hormone receptor were upregulated from mid- to late phase of vitellogenesis. Furthermore, three steroidogenic enzyme genes (cyp11a1, cyp17a1, and cyp19a1a) gradually increased their expression during ovarian development, accompanying a rise in serum estradiol-17ß, while 3ß-hydroxysteroid dehydrogenase and steroidogenic acute regulatory protein did not change significantly. CONCLUSIONS: This is the first report of deep RNA sequencing analysis of Japanese sardine, in which many key genes involved in the BPG axis were identified. Expression profiles of ovarian steroidogenesis-related genes provide a molecular basis of the physiological processes underlying ovarian development in the sardine. Our study will be a valuable resource for clarifying the molecular biology of clupeoid fishes.
Asunto(s)
Encéfalo/metabolismo , Peces/genética , Hormonas Esteroides Gonadales/genética , Ovario/metabolismo , Hipófisis/metabolismo , Transcriptoma , 11-beta-Hidroxiesteroide Deshidrogenasas/genética , 11-beta-Hidroxiesteroide Deshidrogenasas/metabolismo , Animales , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Femenino , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Hormona Folículo Estimulante/genética , Hormona Folículo Estimulante/metabolismo , Hormonas Esteroides Gonadales/metabolismo , Hormona Luteinizante/genética , Hormona Luteinizante/metabolismoRESUMEN
The effects of gonadotropin-releasing hormone agonist (GnRHa) on plasma levels of follicle-stimulating hormone (Fsh) and luteinising hormone (Lh) are reported for female greater amberjack Seriola dumerili with post-vitellogenic ovarian oocytes. Five females were implanted with pellets containing GnRHa (600 µg kg-1 body weight), while five other females were injected with saline. All females implanted with GnRHa-containing pellets ovulated 36-42 h post-implantation. The GnRHa implants elevated Lh, but not Fsh plasma levels within 42 h of GnRHa administration.
Asunto(s)
Hormona Folículo Estimulante/sangre , Hormona Liberadora de Gonadotropina/agonistas , Hormona Luteinizante/sangre , Ovulación/efectos de los fármacos , Perciformes/fisiología , Animales , Femenino , Hormona Liberadora de Gonadotropina/farmacología , Oocitos/efectos de los fármacos , Ovario/efectos de los fármacosRESUMEN
In Seriola species, exposure to a long photoperiod regime is known to induce ovarian development. This study examined photoperiodic effects on pituitary gene expression and plasma levels of follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) in previtellogenic greater amberjack (Seriola dumerili). The fish were exposed to short (8L:16D) or long (18L:6D) photoperiod. The water temperature was maintained at 22⯰C. Compared with the short-photoperiod group, plasma Fsh levels were higher on days 10 and 30 in the long-photoperiod group, but plasma Lh levels did not significantly differ. On day 30, pituitary Fsh- and Lh-ß subunit gene expressions were also higher in the long-photoperiod group than the short-photoperiod group, whereas α-subunit gene expressions were higher on days 20 and 30. Throughout the experiment, average gonadosomatic index and plasma E2 levels did not significantly differ between the two groups. This study clearly demonstrated that a long photoperiod induced Fsh release in the previtellogenic fish followed by upregulation of pituitary Fsh and Lh subunit gene expressions. An increase in plasma Fsh levels may be a key factor that mediates the photoperiodic effect on the initiation of ovarian development.
Asunto(s)
Gonadotropinas/sangre , Perciformes/sangre , Perciformes/fisiología , Fotoperiodo , Vitelogénesis , Animales , Estradiol/sangre , Femenino , Hormona Folículo Estimulante/sangre , Hormona Folículo Estimulante/genética , Hormona Folículo Estimulante/metabolismo , Hormonas Glicoproteicas de Subunidad alfa/genética , Hormonas Glicoproteicas de Subunidad alfa/metabolismo , Hormona Luteinizante/sangre , Hormona Luteinizante/genética , Hormona Luteinizante/metabolismo , Hormona Luteinizante de Subunidad beta/genética , Hormona Luteinizante de Subunidad beta/metabolismo , Ovario/crecimiento & desarrollo , Perciformes/crecimiento & desarrollo , Perciformes/metabolismo , Hipófisis/citología , Hipófisis/metabolismo , Temperatura , AguaRESUMEN
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.
Asunto(s)
Perciformes/metabolismo , Hipófisis/metabolismo , Receptores LHRH/metabolismo , Animales , Encéfalo/metabolismo , ADN Complementario/genética , Femenino , Ligandos , Masculino , Filogenia , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores LHRH/genética , Análisis de Secuencia de ADN , Distribución TisularRESUMEN
Using a recombinant chimeric single-chain follicle stimulating hormone (FSH), we established a radioimmunoassay (RIA) for red seabream (Pagrus major) FSH (pmFSH) which became a powerful tool for studying reproductive physiology. We studied the profiles in plasma and pituitary concentrations of FSH and luteinizing hormone (LH) during sexual maturation. A pre-established RIA for red seabream LH was used for the LH measurements. The regulation of FSH and LH secretion from the pituitary was investigated using a gonadotropin-releasing hormone analog (GnRHa) in vivo and in vitro. Marked differences in plasma and pituitary FSH levels were observed between males and females; pituitary FSH content in males was much higher than that in females during all seasons, and plasma FSH levels in males were high during the spawning season, whereas those in females were unchanged. In contrast, plasma and pituitary levels of LH were elevated before and during the spawning season in males and females. Injecting or implanting (cholesterol pellet) a GnRHa into adult and juvenile red seabream resulted in significant increases in plasma LH concentrations; however, no significant change was observed in plasma FSH. Moreover, GnRHa stimulated only LH secretion in an in vitro experiment using dispersed pituitary cells. The discrete FSH and LH secretion profiles revealed suggest differential roles for the two gonadotropins during red seabream gametogenesis. In addition, the marked difference in pituitary FSH levels in males and females suggests the relative significance of FSH in male reproduction.
Asunto(s)
Hormona Folículo Estimulante/análisis , Hormona Liberadora de Gonadotropina/fisiología , Gonadotropinas/análisis , Gonadotropinas/metabolismo , Dorada/metabolismo , Animales , Femenino , Hormona Folículo Estimulante/sangre , Gametogénesis/fisiología , Hormona Luteinizante/análisis , Hormona Luteinizante/sangre , Masculino , Hipófisis/metabolismo , Radioinmunoensayo/métodos , Dorada/fisiología , Estaciones del Año , Maduración Sexual/fisiologíaRESUMEN
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 (Fshß, Lhß, 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 Fshß (or Lhß) 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.
Asunto(s)
Hormona Folículo Estimulante/metabolismo , Hormona Luteinizante/metabolismo , Ovario/crecimiento & desarrollo , Receptores de HFE/metabolismo , Receptores de HL/metabolismo , Animales , Ensayo de Inmunoadsorción Enzimática , Femenino , Hormona Folículo Estimulante/genética , Hormona Luteinizante/genética , Oogénesis/fisiología , Ovario/metabolismo , Ovulación/metabolismo , Perciformes/metabolismo , Hipófisis/metabolismo , ARN Mensajero/genética , Receptores de HFE/genética , Receptores de HL/genética , Diferenciación Sexual/fisiologíaRESUMEN
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.
Asunto(s)
Encéfalo/metabolismo , Hormona Liberadora de Gonadotropina/genética , Kisspeptinas/genética , Perciformes/genética , Receptores Acoplados a Proteínas G/genética , Maduración Sexual/genética , Animales , Femenino , Regulación del Desarrollo de la Expresión Génica , Hormona Liberadora de Gonadotropina/metabolismo , Gónadas/crecimiento & desarrollo , Gónadas/metabolismo , Kisspeptinas/metabolismo , Masculino , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
Understanding puberty is important for establishing aquaculture in fish. In this study, we analyzed the timing and completion of pubertal development along with changes in pituitary gonadotropin genes (fshb and lhb) in cultured chub mackerel (Scomber japonicus). At 45 days post-hatching (dph), gonadal sex differentiation was observed. The onset of puberty occurred at 192 dph in females with the start of vitellogenesis, whereas it occurred at 164 dph in males, with the beginning of spermatogenesis (proliferation and differentiation of germ cells). The completion of puberty was at 326 dph in females when vitellogenesis completed, and it was at 338 dph in males during spermiation. All fish sampled during the spawning season completed pubertal development. In the pituitary of female fish, fshb expression was activated during early secondary growth and was maintained high throughout vitellogenesis, whereas lhb expression was highest at the completion of vitellogenesis. In male fish, fshb and lhb expression were activated from the onset of spermatogenesis and further activated during late pubertal development; fshb remained high between late spermatogenesis and spermiation, whereas lhb was highest during spermiation.
Asunto(s)
Gonadotropinas/metabolismo , Ovario/crecimiento & desarrollo , Perciformes/crecimiento & desarrollo , Perciformes/fisiología , Maduración Sexual/fisiología , Testículo/crecimiento & desarrollo , Animales , Acuicultura , Femenino , Gonadotropinas/genética , Masculino , Ovario/metabolismo , Perciformes/metabolismo , Testículo/metabolismoRESUMEN
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.
Asunto(s)
Oogénesis/genética , Folículo Ovárico/metabolismo , Perciformes/metabolismo , Receptores de HFE/metabolismo , Receptores de HL/metabolismo , Animales , Femenino , Expresión Génica , Regulación de la Expresión Génica , Células de la Granulosa/metabolismo , Ovario/metabolismo , Perciformes/genética , Receptores de HFE/genética , Receptores de HL/genética , Células Tecales/metabolismoRESUMEN
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.
Asunto(s)
Kisspeptinas/farmacología , Perciformes/crecimiento & desarrollo , Maduración Sexual/efectos de los fármacos , Animales , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Hormonas Esteroides Gonadales/genética , Hormonas Esteroides Gonadales/metabolismo , Masculino , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
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.
Asunto(s)
Proteínas de Peces/metabolismo , Perciformes/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Encéfalo/metabolismo , Encéfalo/fisiología , Femenino , Proteínas de Peces/genética , Kisspeptinas/metabolismo , Masculino , Perciformes/fisiología , Receptores Acoplados a Proteínas G/genética , Reproducción/fisiología , Testículo/metabolismo , Testículo/fisiologíaRESUMEN
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.
Asunto(s)
Proteínas de Peces/administración & dosificación , Kisspeptinas/administración & dosificación , Fragmentos de Péptidos/administración & dosificación , Perciformes/fisiología , Espermatogénesis/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Estradiol/sangre , Proteínas de Peces/química , Inyecciones Subcutáneas , Kisspeptinas/química , Masculino , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Testículo/citología , Testículo/efectos de los fármacos , Testosterona/sangreRESUMEN
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.
Asunto(s)
Hormona Folículo Estimulante/farmacología , Hormona Luteinizante/farmacología , Perciformes/metabolismo , Hipófisis/química , Animales , Estradiol/análisis , Estradiol/biosíntesis , Femenino , Hormona Folículo Estimulante/aislamiento & purificación , Hidroxiprogesteronas/análisis , Hidroxiprogesteronas/metabolismo , Hormona Luteinizante/aislamiento & purificación , Folículo Ovárico/química , Folículo Ovárico/efectos de los fármacos , Folículo Ovárico/metabolismo , Vitelogénesis/efectos de los fármacosRESUMEN
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.
Asunto(s)
Hormona Folículo Estimulante/metabolismo , Gonadotropinas/metabolismo , Hormona Luteinizante/metabolismo , Perciformes/metabolismo , Hipófisis/fisiología , Animales , Gonadotropinas/química , Masculino , Subunidades de Proteína/fisiología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducción/fisiología , Maduración Sexual/fisiología , Testículo/crecimiento & desarrolloRESUMEN
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.
Asunto(s)
Gonadotropinas Hipofisarias/genética , Perciformes/genética , Perciformes/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Cartilla de ADN/genética , ADN Complementario/genética , Femenino , Hormona Folículo Estimulante de Subunidad beta/genética , Regulación de la Expresión Génica , Hormonas Glicoproteicas de Subunidad alfa/genética , Gonadotropinas Hipofisarias/química , Hormona Luteinizante de Subunidad beta/genética , Masculino , Datos de Secuencia Molecular , Ovario/fisiología , Perciformes/anatomía & histología , Filogenia , Subunidades de Proteína/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducción/genética , Reproducción/fisiología , Vitelogénesis/genética , Vitelogénesis/fisiologíaRESUMEN
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.