Regulation of gonadotropin subunit genes in tilapia

A steroidogenic tilapia gonadotropin (taGtH=LH) was purified from pituitaries of hybrid tilapia (Oreochromis niloticus x O. aureus) and a homologous RIA was established. This RIA enabled the study of the endocrine regulation of GtH release, the transduction pathways involved in its secretion and its profile during the spawning cycle. Discrepancies between steroid and taGtH peaks during the cycle led to the conclusion that an additional gonadotropin similar to salmonid FSH operates early in the cycle. In order to identify this hormone and to study the endocrine control of synthesis of all gonadotropin (GtH) subunits, a molecular approach was taken. The cDNA sequences and the entire gene sequences encoding the FSHbeta and LHbeta subunits, as well as an incomplete sequence of the glycoprotein hormone alpha subunit (GPalpha), were cloned. Salmon gonadotropin-releasing hormone (sGnRH) elevated mRNA steady-state levels of all three GtH subunits in cultured pituitary cells. Pituitary adenylate cyclase-activating polypeptide (PACAP) and neuropeptide Y (NPY) also stimulated the expression of these subunits and potentiated the effect of GnRH, except that NPY did not affect FSHbeta. The GnRH and NPY effects were found to be mediated mainly through protein kinase C (PKC), while protein kinase A (PKA) cascade was involved to a lesser extent. Mitogen-activated protein kinase (MAPK) cascade takes part in mediating GnRH effects, possibly via PKC. Testosterone (T) and estradiol (E2), but not 11-ketotestosterone (KT), are able to elevate GPalpha and LHbeta mRNAs in pituitary cells of early maturing or regressing males. Low levels of T exposure are associated with elevated FSHbeta mRNA in cells of mature fish, while higher levels suppress it, but elevate LHbeta mRNA. In vivo observations also showed the association of low T levels with increased FSHbeta mRNA and high T levels with elevated LHbeta mRNA. In accordance with these findings, analysis of LHbeta and FSHbeta 5' gene-flanking regions revealed on both gene promoters a GtH-specific element (GSE), half site estrogen response elements (ERE), cAMP response element (CRE) and AP1. In vitro experiments showed that recombinant human activin-A leads to higher levels of GPalpha, FSHbeta and LHbeta mRNAs in pituitary cell culture. Porcine inhibin marginally decreased the mRNA levels of GPalpha and FSHbeta, but at a low level (1 ng/ml) it stimulated that of LHbeta. These results shed some light on certain hypothalamic and gonadal hormones regulating the expression of GtH subunit genes in tilapia. In addition, they provide evidence for their differential regulation, and insight into their mode of action.     

Regulation of gonadotropin beta subunit gene expression by testosterone and gonadotropin-releasing hormones in the goldfish, Carassius auratus

Sex steroids differentially regulate gonadotropin (GTH) beta subunits (FSHbeta and LHbeta) gene expression in the pituitary of goldfish: a strong in vivo inhibitory effect on FSHbeta mRNA production, but a weak stimulatory effect on LHbeta in sexually immature and recrudescent fish. In the present study, to examine a direct effect of testosterone (T) and gonadotropin-releasing hormone (GnRH) on the mRNA levels of FSHbeta and LHbeta subunits in the pituitary, in vitro experiments were performed using dispersed pituitary cells of sexually immature, recrudescent, mature and regressed goldfish. T treatment in vitro did not significantly decrease FSHbeta mRNA levels, but increased that of LHbeta only in the cells of immature fish. Salmon-type GnRH increased FSHbeta mRNA levels in cells of mature fish, but decreased the levels in cells of sexually regressed fish. From these results, it was suggested that: (1) in vivo effect of sex steroids on gene expression of GTH beta subunits is not always exerted on the pituitary; and (2) the different responses of GTH beta subunits by sex steroids between in vivo and in vitro are partly due to a complex pathway through hypothalamic factors, such as GnRH, in the case of in vivo.     

Changes along the pituitary-gonadal axis during maturation of the black carp, Mylopharyngodon piceus

The black carp, Mylopharyngodon piceus, is a late-maturing cyprinid reaching sexual maturity at the age of 6-7 years. The present work attempted to define nonfunctional sites along the pituitary-gonadal axis in immature fish utilizing in vivo and in vitro challenge experiments. Two- and 3-year old fish injected with salmon gonadotropin-releasing hormone analog (sGnRHa; 10 microg/kg) and metoclopramide (20 mg/kg) did not reveal any increase in circulating gonadotropin (cGtH) or estradiol (E(2)) level. Furthermore, cGtH release from cultured pituitary cells of fish at these ages did not increase in response to sGnRH (0.1 nM - 1 microM) but was augmented when exposed to TPA (12.5 nM). However, 4-year old female fish did respond to the above treatments both in vivo and in vitro. These results suggest the existence of nonfunctional site(s) proximal to the activation of PKC in the immature black carp gonadotrophs, probably at the level of GnRH receptors. These site(s) start to become functional in 4-year old fish. Two- and 3-year old fish injected with common carp pituitary extract (CPE) containing 350 microg cGtH/kg did not show any increase in circulating E(2). In addition, the estrogen secretion from fragments of the rudimentary gonads did not increase after exposure to CPE containing cGtH (0.5-4 microg/ml) but was elevated dose-dependently by exposure to dbcAMP (0.3-3 mM). However, the ovaries of 4-year old fish did respond to the gonadotropic stimulation, both in vivo and in vitro. These results suggest the existence of other non-functional site(s) in the immature black carp, proximal to the formation of cAMP in the gonads, probably at the level of GtH receptors. These site(s) start to become functional in 4-year old females. Another source of E(2) was discovered in the immature black carp: namely, the fat pad adjacent to the gonads. In contrast to the visceral adipose tissue, the fat pad secretes estrogen in response to cAMP elevation in 2- and 3-year old fish while in 4-year old fish it also responds to gonadotropic stimulation. Due to its large mass and high steroidogenic potency, it is assumed that the gonadal fat pad is involved in the process of puberty in the black carp. J. Exp. Zool. 286:405-413, 2000.     

Regulation of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) gene expression by gonadotropin-releasing hormone (GnRH) and sexual steroids in the Mediterranean Sea bass

The secretion of gonadotropins, the key reproductive hormones in vertebrates, is controlled from the brain by the gonadotropin-releasing hormone (GnRH), but also by complex steroid feedback mechanisms. In this study, after the recent cloning of the three gonadotropin subunits of sea bass (Dicentrarchus labrax), we aimed at investigating the effects of GnRH and sexual steroids on pituitary gonadotropin mRNA levels, in this valuable aquaculture fish species. Implantation of sea bass, in the period of sexual resting, for 12 days with estradiol (E2), testosterone (T) or the non-aromatizable androgen dihydrotestosterone (DHT), almost suppressed basal expression of FSHbeta (four to 15-fold inhibition from control levels), while slightly increasing that of alpha (1.5-fold) and LHbeta (approx. twofold) subunits. Further injection with a GnRH analogue (15 microg/kg BW; [D-Ala6, Pro9-Net]-mGnRH), had no effect on FSHbeta mRNA levels, but stimulated (twofold) pituitary alpha and LHbeta mRNA levels in sham- and T-implanted fish, and slightly in E2- and DHT-implanted fish (approx. 1.5-fold). The GnRHa injection, as expected, elevated plasma LH levels with a parallel decrease on LH pituitary content, with no differences between implanted fish. In conclusion, high circulating steroid levels seems to exert different action on gonadotropin secretion, inhibiting FSH while stimulating LH synthesis. In these experimental conditions, the GnRHa stimulate LH synthesis and release, but have no effect on FSH synthesis.     

The reproductive cycle of yellowfin bream, Acanthopagms australis (Günther), with particular reference to protandrous sex inversion

Yellowfin bream, Acanthopagrus australis , of all age classes were collected from Moreton Bay, Australia. The species possessed typical sparid ovotestes in which the testis and ovary occur in separate zones. During the spawning period (June-August) juveniles, functional males and functional females could be distinguished by the macroscopic appearance of the gonad. The sex ratio of males to females decreases with age, indicating protandrous sex inversion.
Histological and structural study of the ovotestis showed all fish have previtellogenic cells in the ovarian zone but only juvenile and male fish have developing spermatogenic cells in the testis. Most juveniles become functional males by the age of two years but a small proportion of juveniles develop directly into functional females (primary females). Protandrous sex inversion commences after the spawning period when male fish appear with spermatozoa and no other spermatogenic cells in the testis. During the period November-January male fish with no spermatogenic cells are common and a reduction in size of the testis occurs so that by March-April the ovotestis becomes structurally and histologically similar to the female ovotestis. Some fish remain functional males during their whole life-history (primary males). In functional females vitellogenic cells are present in the ovary only during the spawning period and the testis remains very small in size.     

Stimulatory effects of insulin-like growth factor 1 on expression of gonadotropin subunit genes and release of follicle-stimulating hormone and luteinizing hormone in masu salmon pituitary cells early in gametogenesis

Insulin-like growth factor-I (IGF-I) has been shown to be involved in pubertal activation of gonadotropin (GTH) secretion. The aim of this study was to determine if IGF-I directly stimulates synthesis and release of GTH at an early stage of gametogenesis. The effects of IGF-I on expression of genes encoding glycoprotein alpha (GPalpha), follicle-stimulating hormone (FSH) beta, and luteinizing hormone (LH) beta subunits and release of FSH and LH were examined using primary pituitary cells of masu salmon at three reproductive stages: early gametogenesis, maturing stage, and spawning. IGF-I alone or IGF-I + salmon GnRH (sGnRH) were added to the primary pituitary cell cultures. Amounts of GPalpha, FSHbeta, and LHbeta mRNAs were determined by real-time PCR. Plasma and medium levels of FSH and LH were determined by RIA. In males, IGF-I increased the amounts of all three subunit mRNAs early in gametogenesis in a dose-dependent manner, but not in the later stages. In females, IGF-I stimulated release of FSH and LH early in gametogenesis, whereas no stimulatory effects on the subunit mRNA levels were observed at any stage. IGF-I + sGnRH stimulated release of FSH and LH at all stages in both sexes, but had different effects on the subunit mRNA levels depending on subunit and stage. The present results suggest that IGF-I itself directly stimulates synthesis and release of GTH early in gametogenesis in masu salmon, possibly acting as a metabolic signal that triggers the onset of puberty.     

Photoperiod effects on the expression of kisspeptin and gonadotropin genes in Atlantic cod, Gadus morhua, during first maturation

In order to investigate the potential role of the kisspeptin system in the entrainment of reproduction in Atlantic cod, qPCR assays were developed for kiss2 and its receptor kissr4. mRNA expression was monitored in the brain over a full reproductive cycle in 2 populations of males and females: 1) a maturing population (exposed to simulated natural photoperiod, SNP) and 2) a maturation inhibited population (exposed to constant light, LL). Pituitary expression of gonadotropin subunit mRNA (fshβ and lhβ) was also measured. Results from this study indicated no clear temporal pattern in expression of kiss2 or kissr4 mRNAs in either population of cod, however acute elevations were apparent in maturing (SNP) individuals, namely an elevation in kiss2 in vitellogenic females and spermiating males and spikes in kissr4 during early vitellogenesis in females and spermatogenesis in males. Gonadotropin mRNA expression displayed strong amplitudinal changes over time with fshβ and lhβ mRNA expression increasing towards spawning in maturing individuals. No firm conclusions on the role of the kisspeptin system in cod puberty can be drawn at this stage, however mRNA increases in kiss2 and kissr4 may elude to conserved kisspeptin functions in cod and opens up interesting avenues on potential gender specific functions.     

促黄体素β基因表达中的转导通路及转录因子

促性腺激素释放激素 (GnRH)为下丘脑促垂体激素 ,其脉冲式地释放调节垂体促卵泡素(FSH)和促黄体素 (LH)的合成与释放 ,进而调节动物的生殖活动。LH是由α亚基和 β亚基组成的异二聚体糖蛋白激素 ,其中 β亚基决定激素的特异性。LHβ基因的表达是由GnRH诱发的 ,此过程主要依靠PKC和Ca2 两类信号通路 ,并调节LHβ基因的表达。目前已经发现 ,多种转录因子 ,如早期生长反应基因 (Egr 1)、核受体SF 1基因、Ptx1基因和Sp1基因等 ,通过与LHβ亚基基因的启动子区直接结合 ,而对该基因的表达进行调控。     

Gonadotrope and thyrotrope development in the human and mouse anterior pituitary gland

Genes and orthologous intrinsic and extrinsic factors critical for embryonic pituitary gonadotrope and thyrotrope cell differentiation have been identified mainly in rodents, but data on the human are very limited. In human fetal pituitaries examined between 14 and 19 weeks of gestation using immunofluorescent confocal microscopy, we found that most fetal gonadotropes expressed alpha-GSU, LHbeta, and FSHbeta gonadotropin subunits while almost no cells expressed alpha-GSU and LHbeta alone. Gonadotropes expressing alpha-GSU and FSHbeta only were detected in both male and female pituitaries, increasing in proportion to total gonadotropes in both males and females from 14 (approximately 4.5%) to 19 weeks (approximately 16.5%) with a peak in males of 45.5% compared with females of 16.5% at 17 weeks of gestation. When FSHbeta or LHbeta genes were expressed, gonadotropes were non-dividing. This profile of human fetal gonadotrope development differs from the current mouse model. Furthermore, while expression of alpha-GSU appears to be the lead protein in gonadotropes, in thyrotropes which ultimately express alpha-GSU with TSHbeta, we observed that most if not all thyrotropes were TSHbeta-positive but alpha-GSU-negative until around 19 weeks in human, and e15 in mouse, fetal pituitaries. Furthermore, the TSHbeta-only thyrotropes were dividing, and TSHbeta rather than alpha-GSU was the lead protein in thyrotrope development. Thus, while biologically active dimeric FSH and LH can be produced by the human fetal pituitary by 14 weeks, dimeric biologically active TSH will only be produced from around 17 weeks of gestation. The mechanism(s) responsible for the different molecular regulation of alpha-GSU gene expression in gonadotropes and thyrotropes in the developing human fetal pituitary now requires investigation.     

Sperm production and steroidogenesis in testes of the common carp, Cyprinus carpio L., at different stages of maturation

Testes from carp, Cyprinus carpio L., at five different maturational stages from immature through to spermiation and regression were incubated with or without addition of carp hypophysial homogenate (chh) for 8 or 20 h. Concentrations of steroids and spermatozoa were measured in the medium and the residual tissue examined histologically. There was an increase in the area of the germinal cysts containing spermatozoa, the percentage of the testis which they occupied and in the production of spermatozoa as the gonadosomatic index (GSI) increased, but this was unaffected either by incubation or by pretreatment with chh. The major steroid in plasma and in in vitro testicular cultures from all of the maturing fish captured in winter was 1 I-ketotestosterone. The production rate of this steroid in virro was unaffected by GSI, while plasma levels tended to increase with GSI. 17.20β-Dihydroxy-4-pregnen-3-one was detectable in significant amounts in only a few spermiating fish in summer, but was stimulated more in incubations with chh in maturing winter than in summer spermiating or post-spawning fish. 17,20a-Dihydroxy-4-pregnen-3-one was not detectable in incubations, but plasma concentrations tended to increase towards spermiation and were positively correlated with the size of the cyst. After spawning, fish had low plasma steroid levels and failed to respond in vitro to pituitary extract, indicating a testicular post-spawning refractoriness.     

Early maturity in the male striped bass, Morone saxatilis: follicle-stimulating hormone and luteinizing hormone gene expression and their regulation by gonadotropin-releasing hormone analogue and testosterone

Striped bass are seasonal breeding fish, spawning once a year during the spring. All 3-yr-old males are sexually mature; however, 60-64% of the fish mature earlier as 1- or 2-yr-old animals. The endocrine basis underlying early maturity in 2-yr-old males was studied at the molecular level by monitoring changes in pituitary beta FSH and beta LH mRNA levels by ribonuclease protection assay, and correlating these changes to stages of testicular development. In maturing males, the mRNA levels of beta FSH were elevated during early spermatogenesis, whereas beta LH mRNA levels peaked during spermiation. The appearance of spermatozoa in the testis was associated with a decrease in beta FSH mRNA and a rise in beta LH mRNA abundance. Immature males had lower levels of beta LH mRNA than maturing males, but there were no differences in beta FSH mRNA levels between immature and maturing males. The regulation of gonadotropin gene expression in 2-yr-old males was studied by the chronic administration of GnRH analogue (GnRHa) and testosterone (T), with or without pimozide (P) supplementation. In immature males, the combination of T and GnRHa stimulated a three- to fivefold increase in beta FSH and beta LH mRNA levels, but the same treatment had no effect on gonadotropin gene expression in maturing males. In addition, the coadministration of P to immature males suppressed the stimulatory effect of GnRHa and T on beta FSH and beta LH mRNA levels, suggesting that dopamine may have a novel role in regulating gonadotropin gene expression.     

Sequence analysis and expressional regulation of messenger RNAs encoding beta subunits of follicle-stimulating hormone and luteinizing hormone in the red-bellied newt, Cynops pyrrhogaster

Two distinct cDNAs encoding beta subunits of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were cloned from the cDNA library constructed for the pituitary of the red-bellied newt, Cynops pyrrhogaster, and sequenced. The newt FSHbeta and LHbeta cDNAs encode polypeptides of 129 and 131 amino acids, including signal peptides of 20 and 19 amino acids, respectively. The number and position of cysteine and N-glycosylation in each of the beta subunits of FSH and LH, which are considered essential for assembly of the alpha subunit, are well conserved between the newt and other tetrapods. The high homology (41.6%) between the beta subunits of newt FSH and LH imply less specificity of FSH and LH in gonadal function. One cDNA encoding the common polypeptide chain alpha subunit of FSH and LH was also isolated from the newt pituitary gland. The mRNAs of FSHbeta, LHbeta, and the alpha subunit were expressed only in the pituitary gland among various newt tissues. Double-staining with in situ hybridization and immunohistochemistry revealed coexpression of FSHbeta and LHbeta in the same newt pituitary cells. Ovariectomy induced a significant increase in FSHbeta mRNA levels, but there was no significant change in LHbeta or alpha subunit mRNA levels compared with those in control animals. Taken together, these data suggest that two kinds of gonadotropins, namely FSH and LH, are expressed in the same gonadotropin-producing cells in the pars distalis of the newt as well as in other tetrapods and that the expression of FSHbeta is negatively regulated by the ovaries.