Differential regulation of pituitary gonadotropin subunit messenger ribonucleic acid levels in photostimulated Siberian hamsters

FSH levels begin to rise 3-5 days after male Siberian hamsters are transferred from inhibitory short photoperiods to stimulatory long photoperiods. In contrast, LH levels do not increase for several weeks. This differential pattern of FSH and LH secretion represents one of the most profound in vivo examples of differential regulation of the gonadotropins. The present study was undertaken to characterize the molecular mechanisms controlling differential FSH and LH synthesis and secretion in photostimulated Siberian hamsters. First, we cloned species-specific cDNAs for the three gonadotropin subunits: the common alpha subunit and the unique FSHbeta and LHbeta subunits. All three subunits share high nucleotide and predicted amino acid sequence identity with the orthologous cDNAs from rats. We then used these new molecular probes to examine the gonadotropin subunit mRNA levels from pituitaries of short-day male hamsters transferred to long days for 2, 5, 7, 10, 15, or 20 days. Short-day (SD) and long-day (LD) controls remained in short and long days, respectively, from the time of weaning. We measured serum FSH and LH levels by RIA. FSHbeta, LHbeta, and alpha subunit mRNA levels were measured from individual pituitaries using a microlysate ribonuclease protection assay. Serum FSH and pituitary FSHbeta mRNA levels changed similarly following long-day transfer. Both were significantly elevated after five long days (2.3- and 3.6-fold, respectively; P < 0.02) and declined thereafter, but they remained above SD control values through 20 long days. Alpha subunit mRNA levels also increased significantly relative to SD control values (maximum 2-fold increase after seven long days; P < 0.03), although to a lesser extent than FSHbeta. Neither serum LH nor pituitary LHbeta mRNA levels changed significantly following long-day transfer. The results indicate that long-day-associated increases in serum FSH levels in Siberian hamsters reflect an underlying increase in pituitary FSHbeta and alpha subunit mRNA accumulation.     

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.     

Pituitary glycoprotein hormone beta subunits in the Australian lungfish and estimation of the relative evolution rate of these subunits within vertebrates

The beta subunits of the two pituitary gonadotropins LH and FSH and of thyroid-stimulating hormone (TSH) were cloned from Australian lungfish (Neoceratodus forsteri) pituitary glands. These three glycoprotein hormone beta subunits possess the main characteristics common to their counterparts in other vertebrates. Taking advantage of the phylogenetic position of the lungfish, close to the root of tetrapods, a maximum parsimony tree was inferred from these new sequences and sequences from representatives of the diversity of vertebrates. The topology of the tree was imposed so that it reflected as closely as possible the real evolutionary history of the subunits. This tree was used to estimate the relative evolution rate of the three subunits in vertebrates. Cumulated amino acid substitutions from the basal subunit node (ancestral subunit sequence) to the species node were calculated and compared. It showed that a burst in evolutionary rate occurred for the LHbeta subunit in the tetrapod lineage sometime after the emergence of amphibians. The rate of evolution of the LHbeta subunit was particularly high throughout the radiation of mammals while FSH and TSHbeta subunits kept quite stable in this lineage. A burst in evolutionary rate was also observed for the FSHbeta subunit in the lineage leading to teleosts sometime after the emergence of chondrosteans and the dynamic of evolution was high throughout the radiation of teleosts. These results were consistent with data obtained from pairwise comparisons.     

Luteinizing hormone and follicle-stimulating hormone exhibit different secretion patterns from cultured Madin-Darby canine kidney cells

LH, FSH, and chorionic gonadotropin (CG) are comprised of a common alpha subunit and a hormone-specific beta subunit. Using Madin-Darby canine kidney (MDCK) epithelial cells to examine the polarized secretion of human CG/LH, we previously reported that CG and LH were detected in the apical and basolateral compartments, respectively, and the carboxyl terminal end of the CGbeta subunit contains a strong apical signal. Here we show that the carboxyl seven amino acids in the LHbeta subunit contribute to the basolateral secretion of LH, and an LH chimera bearing the CGbeta apical signal is redirected from the basolateral to the apical compartments. Because LH and FSH are synthesized in the same cell, we also compared the secretion polarity of LH with FSH. MDCK cells expressing the FSH dimer displayed an almost equal distribution of protein into the apical and basolateral compartments. Given that the LHbeta and CGbeta carboxy terminal sequences, which differ from that in the FSHbeta subunit, occupy a pivotal role in their polarized behavior, the results support the hypothesis that pituitary exit of LH and FSH occur via different secretion pathways, and are released spatially from the pituitary via different circulatory routes.     

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 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.     

Effects of starvation on gonadotropin and thyrotropin subunit mRNA levels and plasma hormone levels in the male Japanese quail (Coturnix coturnix japonica)

Contents of mRNAs encoding LHbeta-, FSHbeta-, TSHbeta- and common a-subunit precursor molecules were measured in male Japanese quail deprived of food for three days. Plasma LH, FSH, thyroxine and triiodothyronine levels were also measured in the same birds. Plasma LH levels declined during the period of food deprivation. Levels in starved birds were not different from those in control birds after one day of starvation but were significantly lower after three days. Plasma FSH levels showed a similar decline, although the changes were not significant. Plasma thyroxine levels did not decrease during starvation, whilst plasma triiodothyronine levels decreased drastically and significantly soon after the start of starvation. All the hormone subunit mRNA contents in starved birds also decreased, with differences from control birds significant 3 days after the start of starvation. Plasma FSH levels showed a strong positive correlation with pituitary FSHbeta mRNA levels, while plasma LH levels had a strong positive correlation with common a mRNA levels and practically no correlation or even a negative correlation with LHbeta mRNA levels. These results suggest that starvation suppresses not only gonadotropin and thyrotropin secretion but also their synthesis in the pituitary gland. Furthermore, these results showed that FSH and LH have different synthesis and secretion dynamics in the Japanese quail. Contradicting results with TSHbeta mRNA and thyroid hormones lead us to assume that starvation affects thyroid hormone metabolism in peripheral tissue, presumably in the liver.     

Expression of alpha subunit and luteinizing hormone beta genes in the ovine anterior pituitary. Estradiol suppresses accumulation of mRNAS for both alpha subunit and luteinizing hormone beta

Bovine cDNA clones containing coding sequences for growth hormone, prolactin, alpha subunit, and luteinizing hormone beta (LH beta) have been used to quantitate their respective mRNA concentrations in anterior pituitary glands isolated from ovariectomized ewes, or from ovariectomized ewes treated for three weeks with estradiol. Concentrations of mRNAs for prolactin or growth hormone remained unchanged in either physiological state. In contrast, treatment with estradiol resulted in a 98% decrease of mRNA for LH beta, relative to untreated animals. This change in mRNA was associated with a similar decrease in the concentrations of pituitary and serum LH. Administration of estradiol also led to a reduction (86%) of alpha subunit mRNA. These results suggest that estrogen regulates the expression of the genes encoding both the alpha and LH beta subunit prior to translation. Furthermore, the pronounced effect of estradiol on the concentrations of mRNAs for alpha subunit and LH beta suggest that the assembly of mature glycoprotein hormones may not be limited solely by the rate of accumulation of the beta subunit.     

Follicle-stimulating hormone and its alpha and beta subunits in rainbow trout (Oncorhynchus mykiss): purification, characterization, development of specific radioimmunoassays, and their seasonal plasma and pituitary concentrations in females.

Gonad development in fish, as in mammals, is regulated by two gonadotropins (GTHs), FSH and LH. The function of LH in fish has been clearly established; however, the function(s) of FSH is less certain. The lack of specific and sensitive assays to quantify FSH and its alpha and beta subunits has hindered studies to assess physiological function. In this study, gel filtration chromatography, ion exchange chromatography, and HPLC were employed to purify FSH and its subunits from pituitary glands of rainbow trout (Oncorhynchus mykiss), and the identities of the isolates were confirmed by amino acid analysis. Polyclonal antibodies were raised against the free GTHalpha2 and free FSHbeta subunits to develop specific RIAs. The sensitivities of the intact FSH, GTHalpha2, and FSHbeta assays were 1 ng/ml, 0.2 ng/ml, and 0.1 ng/ml, respectively, and the cross-reaction of these molecules with each other and with intact LH in the heterologous assays was <10.4% throughout. Pituitary and plasma samples diluted in parallel with the standards in all three assays and spiked sample recoveries were >90% throughout. Measurement of plasma and pituitary concentrations of intact FSH in female rainbow trout confirmed the established seasonal profiles. Concentrations of free GTHalpha2 subunit were elevated both in the plasma and in the pituitary in females at ovulation (maximum concentrations: 34.93 +/- 6.3 ng/ml in plasma; 37.63 +/- 5.79 microg/pituitary). In both the plasma and the pituitary, free FSHbeta subunit was present throughout the reproductive cycle but at very low concentrations when compared with both free GTHalpha2 and intact FSH. The presence of free GTHalpha2 subunit in the plasma similarly occurs in mammals, but its functional significance in fish has yet to be established.     

Cloning and sequence analysis of FSH and LH in the giant panda (Ailuropoda melanoleuca)

The giant panda (Ailuropoda melanoleuca) is an endangered species and indigenous to China. It has been proposed that it has a highly specialized reproductive pattern with low fecundity, but little is known about its basic reproductive biology at the molecular level. In this report the genes encoding gonadotropin subunits alpha, follicle-stimulating hormone (FSH) beta and luteinizing hormone (LH) beta of the giant panda were amplified for the first time by RT-PCR from pituitary total RNA, and were cloned, sequenced and analyzed. The results revealed that the open reading region (ORF) of gonadotropin subunits alpha, FSH beta and LH beta are 363, 390 and 426 bp long, respectively. They displayed a reasonably high degree (74-94, 85-93, 75-91%, for alpha, FSH beta and LH beta subunits, respectively) of identity when deduced amino acids were compared with homologous sequences from partial available mammals including human, cattle, sheep, pig, rat, mouse. Three distinct differences were found at the site of 59 aa of the alpha subunit and 55 aa, 68 aa of FSH beta subunit. Our results provide an insight into understanding the mechanism of reproduction regulation and genetic characteristics of giant panda which will make an actual contribution to its conservation. In addition they lay a foundation for a further study towards producing recombinant panda FSH and LH which can be used in artificial breeding aimed to increase its captive reproductive efficiency.     

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.     

A carboxyl-terminal sequence in the lutropin beta subunit contributes to the sorting of lutropin to the regulated pathway

Although synthesized in the same pituitary gonadotropes, the secretion profiles of lutropin (LH) and follitropin (FSH) differ. LH is secreted through a regulated pathway and associated with a bolus release at mid-estrous cycle. In contrast, the majority of FSH is secreted constitutively with an incremental increase until ovulation. Both share an identicalalpha subunit, and thus thebeta subunit contains determinants for sorting into the regulated pathway. Previously, we demonstrated that a hydrophobic carboxyl-terminal heptapeptide of the LHbeta subunit (Leu-Ser-Gly-Leu-Leu-Phe-Leu), not found in the FSHbeta subunit, influences the intracellular behavior of the LH dimer. To test the hypothesis that the peptide contributes to differential sorting, we monitored the fates of LH and LHDeltaT (LHbeta subunit lacking the carboxyl-terminal seven amino acids) dimers in the rat somatotrope-derived GH(3) cell line in which both the regulated and constitutive secretory pathways operate. Pulse-chase labeling demonstrated that the LHDeltaT dimer was diverted to the constitutive pathway, resulting in a significant decrease in the corresponding intracellular pool. Forskolin stimulated LH dimer release 3-fold, which was accompanied by a parallel decrease of intracellular LH; only marginal forskolin stimulation of LHDeltaT was seen. Immunofluorescence after cycloheximide treatment demonstrated decreased retention of LHDeltaT compared with LH, consistent with increased constitutive secretion of LHDeltaT. We also demonstrated that fusing the heptapeptide to the carboxyl terminus of the FSHbeta subunit resulted in an increased regulated secretion of this FSH analog compared with wild-type FSH. These data are the first to identify a novel structural determinant responsible for the sorting of a member of the glycoprotein hormone family into the regulated secretory pathway.     

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。     

Progesterone does not affect the amount of mRNA for gonadotropins in the anterior pituitary gland of ovariectomized ewes

To evaluate the effect of progesterone on the synthesis and secretion of gonadotropins, ovariectomized ewes either were treated with progesterone (n = 5) for 3 wk or served as controls (n = 5) during the anestrous season. After treatment for 3 wk, blood samples were collected from progesterone-treated and ovariectomized ewes. After collection of blood samples, hypothalamic and hypophyseal tissues were collected from all ewes. Half of each pituitary was used to determine the content of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), and the number of receptors for gonadotropin-releasing hormone (GnRH). The amounts of mRNA for LH beta subunit, FSH beta subunit, alpha subunit, growth hormone, and prolactin were measured in the other half of each pituitary. Treatment with progesterone reduced mean serum concentrations of LH (p less than 0.001) but ot FSH (p greater than 0.05). Further, progesterone decreased (p less than 0.05) the total number of pulses of LH. We were unable to detect pulsatile release of FSH. Hypothalamic content of GnRH, number of receptors for GnRH, pituitary content of gonadotropins and mRNA for LH beta subunit, FSH beta subunit, alpha subunit, growth hormone, and prolactin were not affected (p greater than 0.05) by treatment with progesterone. Thus, after treatment with progesterone, serum concentrations of LH (but not FSH) are decreased. This effect, however, is not due to a decrease in the steady-state amount of mRNA for LH beta or alpha subunits.     

Rat inhibin: molecular cloning of alpha- and beta-subunit complementary deoxyribonucleic acids and expression in the ovary

Inhibin is a gonadal protein hormone that suppresses the secretion of FSH from pituitary gonadotrophs. It has previously been characterized as a heterodimer of two dissimilar subunits (alpha, 18 kilodaltons and beta, 14 kilodaltons) the smaller of which exists in two forms (beta A and beta B) and can form dimers that stimulate the secretion of FSH. In the present work, cDNA clones encoding the inhibin alpha- and beta A-subunits have been isolated from rat ovary and characterized. The alpha-inhibin cDNA predicts a precursor protein of 366 amino acids containing the 133 amino acid mature alpha-subunit at its COOH-terminus. The beta A-inhibin cDNA predicts a precursor protein of 424 amino acids containing the 116 amino acid beta A-subunit at its COOH-terminus. Analysis of rat ovarian RNA indicates that alpha-inhibin mRNA levels are stimulated by PMSG treatment in vivo. In cultured granulosa cells, FSH also stimulates alpha-inhibin mRNA, and the FSH effect is suppressed by cotreatment with GnRH. Hybridization in situ to rat ovarian tissue demonstrates that both the alpha-inhibin and beta A-inhibin mRNAs are specifically expressed in granulosa cells of the developing follicles.     

Genetic fusion of an alpha-subunit gene to the follicle-stimulating hormone and chorionic gonadotropin-beta subunit genes: production of a bifunctional protein.

The human glycoprotein hormones, hCG, TSH, LH, and FSH, are composed of a common alpha-subunit assembled to a hormone-specific beta-subunit. The subunits combine noncovalently early in the secretory pathway and exist as heterodimers but not as multimers. LH/FSH are synthesized in the pituitary gonadotrophs, and several of the alpha-subunit sequences required for association with either the LHbeta or FSHbeta subunits are different. Thus, it is intriguing that no ternary complexes are observed for LH and FSH in vivo (e.g. two different beta-assembled to a single alpha-subunit). To examine whether the alpha-subunit can interact with more than one beta-subunit, and to study the conformational relationships between the ligand and the receptor, we constructed a vector encoding two tandemly arranged beta-subunits fused to a single alpha-subunit gene (FSHbeta-CGbeta-alpha). This approach permitted structure-function analyses of alpha/beta domain complexes without the possibility of subunit dissociation. We reported previously that the CGbeta or FSHbeta subunit gene can be genetically fused to the alpha-gene and the resulting single chains (CGbetaalpha and FSHbetaalpha, respectively) were biologically active. Here we demonstrate that a triple-domain single chain bearing the configuration FSHbeta-CGbeta-alpha is efficiently secreted from transfected Chinese hamster ovary (CHO) cells and exhibits high-affinity receptor binding to both FSH and LH/hCG receptors, comparable to the native heterodimers. These results indicate that the alpha-subunit can interact with each beta-subunit in the same complex and that an alpha-domain fused to a beta-domain can still interact with an additional beta-subunit. The data also demonstrate the remarkable flexibility of the receptor to accommodate the increased bulkiness of the triple-domain ligand. In addition, the formation of intrachain FSH- and CG-like complexes observed in a triple-domain single chain suggests that the alpha-subunit can resonate, i.e. shuttle between alpha-beta heterodimeric intermediates during the early stages of synthesis and accumulation in the endoplasmic reticulum. Such model compounds could be useful as substrates to generate a new class of analogs in which the ratio of the LH/FSH activity is varied. This could aid in the design of analogs that could be used to mimic the in vivo hormonal profiles.     

Mutagenesis and chimeric genes define determinants in the beta subunits of human chorionic gonadotropin and lutropin for secretion and assembly

Chorionic gonadotropin (CG) and lutropin (LH) are members of a family of glycoprotein hormones that share a common alpha subunit but differ in their hormone-specific beta subunits. The glycoprotein hormone beta subunits share a high degree of amino acid homology that is most evident for the LH beta and CG beta subunits having greater than 80% sequence similarity. However, transfection studies have shown that human CG beta and alpha can be secreted as monomers and can combine efficiently to form dimer, whereas secretion and assembly of human LH beta is less efficient. To determine which specific regions of the LH beta and CG beta subunits are responsible for these differences, mutant and chimeric LH beta-CG beta genes were constructed and transfected into CHO cells. Expression of these subunits showed that both the hydrophobic carboxy-terminal seven amino acids and amino acids Trp8, Ile15, Met42, and Asp77 together inhibit the secretion of LH beta. The carboxy-terminal amino acids, along with Trp8, Ile15, Met42, and Thr58 are implicated in the delayed assembly of LH beta. These unique features of LH beta may also play an important role in pituitary intracellular events and may be responsible for the differential glycosylation and sorting of LH and FSH in gonadotrophs.     

Androgens positively regulate follicle-stimulating hormone beta-subunit mRNA levels in rat pituitary cells

We have shown previously that androgens negatively regulate LH alpha and beta-subunit mRNA levels, but have little or no effect on FSH beta mRNA levels in rats in vivo. In contrast, estrogen negatively regulates all three gonadotropin subunit mRNA levels in vivo. We have examined the effects of these sex steroids on gonadotropin subunit synthesis directly at the level of the pituitary gland by using cultured rat pituitary cells. Adult female and male rat pituitaries were dissected, dispersed enzymatically, and maintained in culture for 2 days. At that time, cells were treated for varying lengths of time with either medium alone or sex-steroid hormone treatments (estradiol or testosterone). Dose-response and time-course experiments were performed. Cells were then harvested and total RNA was extracted. Gonadotropin subunit mRNA levels were assessed by blot hybridization techniques. Sex-steroid hormones were added to achieve final concentrations ranging from 10(-12) to 10(-6) M for dose response experiments and 10(-8) M for time-course experiments. Testosterone treatment (10(-8) M) increased FSH beta mRNA levels 3-fold in females (P less than 0.01) and males (P less than 0.05), but had no effect on alpha or LH beta mRNA levels in either sex. Dose-related increases in FSH beta mRNA levels with increasing concentrations of testosterone were observed in both female and male pituitary cell cultures. Time-course studies revealed that the testosterone-stimulated increases in FSH beta mRNA levels are statistically significant by 12 h and 6 h after hormone addition in female and male cultures, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibin, activin, and follistatin: regulation of follicle-stimulating hormone messenger ribonucleic acid levels

Primary pituitary cell cultures derived from adult male rats were used to explore the direct effects of purified porcine inhibin and follistatin, and recombinant human activin A on FSH beta, as well as LH beta and alpha-subunit mRNA levels. Subunit mRNAs were determined by blot hybridization using alpha, LH beta, and FSH beta cDNA and genomic fragments. Treatment with inhibin for 72 h significantly suppressed alpha and FSH beta mRNA levels with parallel changes in FSH secretion. No change in LH beta mRNA levels was observed. A decrease in FSH beta mRNA to undetectable levels was seen 4 h after inhibin administration. Recombinant human Activin A caused dose-dependent and parallel increases in FSH beta mRNA levels and FSH secretion. This increase was evident at 4 h after activin administration and maintained at longer times. alpha and LH beta mRNA levels remained unchanged. Follistatin addition to cultures for 72 h significantly reduced FSH beta mRNA levels. In a time-course experiment, a reduction in FSH beta mRNA to undetectable levels was observed 24 h after follistatin administration. There were no changes in alpha or LH beta mRNA levels. These data demonstrate that the actions of these gonadal peptides on FSH secretion may be accounted for, at least in part at the level of biosynthesis, by reductions in FSH beta mRNA levels directly at the level of the anterior pituitary gland.