Modeling and high-throughput experimental data uncover the mechanisms underlying Fshb gene sensitivity to gonadotropin-releasing hormone pulse frequency.

Neuroendocrine control of reproduction by brain-secreted pulses of gonadotropin-releasing hormone (GnRH) represents a longstanding puzzle about extracellular signal decoding mechanisms. GnRH regulates the pituitary gonadotropin's follicle-stimulating hormone (FSH) and luteinizing hormone (LH), both of which are heterodimers specified by unique ß subunits (FSHß/LHß). Contrary to Lhb, Fshb gene induction has a preference for low-frequency GnRH pulses. To clarify the underlying regulatory mechanisms, we developed three biologically anchored mathematical models: 1) parallel activation of Fshb inhibitory factors (e.g. inhibin α and VGF nerve growth factor-inducible), 2) activation of a signaling component with a refractory period (e.g. G protein), and 3) inactivation of a factor needed for Fshb induction (e.g. growth differentiation factor 9). Simulations with all three models recapitulated the Fshb expression levels obtained in pituitary gonadotrope cells perifused with varying GnRH pulse frequencies. Notably, simulations altering average concentration, pulse duration, and pulse frequency revealed that the apparent frequency-dependent pattern of Fshb expression in model 1 actually resulted from variations in average GnRH concentration. In contrast, models 2 and 3 showed "true" pulse frequency sensing. To resolve which components of this GnRH signal induce Fshb, we developed a high-throughput parallel experimental system. We analyzed over 4,000 samples in experiments with varying near-physiological GnRH concentrations and pulse patterns. Whereas Egr1 and Fos genes responded only to variations in average GnRH concentration, Fshb levels were sensitive to both average concentration and true pulse frequency. These results provide a foundation for understanding the role of multiple regulatory factors in modulating Fshb gene activity.

The forkhead transcription factor, FOXP3, is required for normal pituitary gonadotropin expression in mice.

The hypothalamic-pituitary-gonadal axis is central to normal reproductive function. This pathway begins with the release of gonadotropin-releasing hormone in systematic pulses by the hypothalamus. Gonadotropin-releasing hormone is bound by receptors on gonadotroph cells in the anterior pituitary gland and stimulates the synthesis and secretion of luteinizing hormone and, to some extent, follicle-stimulating hormone. Once stimulated by these glycoprotein hormones, the gonads begin gametogenesis and the synthesis of sex hormones. In humans, mutations of the forkhead transcription factor, FOXP3, lead to an autoimmune disorder known as immunodysregulation, polyendocrinopathy, and enteropathy, X-linked syndrome. Mice with a mutation in the Foxp3 gene have a similar autoimmune syndrome and are infertile. To understand why FOXP3 is required for reproductive function, we are investigating the reproductive phenotype of Foxp3 mutant mice (Foxp3(sf/Y)). Although the gonadotroph cells appear to be intact in Foxp3(sf/Y) mice, luteinizing hormone beta (Lhb) and follicle-stimulating hormone beta (Fshb) expression are significantly decreased, demonstrating that these mice exhibit a hypogonadotropic hypogonadism. Hypothalamic expression of gonadotropin-releasing hormone is not significantly decreased in Foxp3(sf/Y) males. Treatment of Foxp3(sf/Y) males with a gonadotropin-releasing hormone receptor agonist does not rescue expression of Lhb or Fshb. Interestingly, we do not detect Foxp3 expression in the pituitary or hypothalamus, suggesting that the infertility seen in Foxp3(sf/Y) males is a secondary effect, possibly due to loss of FOXP3 in immune cells. Pituitary expression of glycoprotein hormone alpha (Cga) and prolactin (Prl) are significantly reduced in Foxp3(sf/Y) males, whereas the precursor for adrenocorticotropic hormone, pro-opiomelanocortin (Pomc), is increased. Human patients diagnosed with IPEX often exhibit thyroiditis due to destruction of the thyroid gland by autoimmune cells. We find that Foxp3(sf/Y) mice have elevated expression of thyroid-stimulating hormone beta (Tshb), suggesting that they may suffer from thyroiditis as well. Expression of the pituitary transcription factors, Pitx1, Pitx2, Lhx3, and Egr1, is normal; however, expression of Foxl2 and Gata2 is elevated. These data are the first to demonstrate a defect at the pituitary level in the absence of FOXP3, which contributes to the infertility observed in mice with Foxp3 loss of function mutations.

MicroRNAs regulate pituitary development, and microRNA 26b specifically targets lymphoid enhancer factor 1 (Lef-1), which modulates pituitary transcription factor 1 (Pit-1) expression.

To understand the role of microRNAs (miRNAs) in pituitary development, a group of pituitary-specific miRNAs were identified, and Dicer1 was then conditionally knocked out using the Pitx2-Cre mouse, resulting in the loss of mature miRNAs in the anterior pituitary. The Pitx2-Cre/Dicer1 mutant mice demonstrate growth retardation, and the pituitaries are hypoplastic with an abnormal branching of the anterior lobe, revealing a role for microRNAs in pituitary development. Growth hormone, prolactin, and thyroid-stimulating hormone ß-subunit expression were decreased in the Dicer1 mutant mouse, whereas proopiomelanocortin and luteinizing hormone ß-subunit expression were normal in the mutant pituitary. Further analyses revealed decreased Pit-1 and increased Lef-1 expression in the mutant mouse pituitary, consistent with the repression of the Pit-1 promoter by Lef-1. Lef-1 directly targets and represses the Pit-1 promoter. miRNA-26b (miR-26b) was identified as targeting Lef-1 expression, and miR-26b represses Lef-1 in pituitary and non-pituitary cell lines. Furthermore, miR-26b up-regulates Pit-1 and growth hormone expression by attenuating Lef-1 expression in GH3 cells. This study demonstrates that microRNAs are critical for anterior pituitary development and that miR-26b regulates Pit-1 expression by inhibiting Lef-1 expression and may promote Pit-1 lineage differentiation during pituitary development.

Effects of castration and androgen-treatment on the expression of FSH-beta and LH-beta in the three-spine stickleback, gasterosteus aculeatus--feedback differences mediating the photoperiodic maturation response?

In many animals, including the three-spine stickleback (Gasterosteus aculeatus), photoperiod strongly influences reproduction. The aim of this study was to investigate if feedback mechanisms on the brain-pituitary-gonadal axis play a role in mediating the photoperiodic response in the stickleback. To that end, stickleback males, exposed to either non-stimulatory short photoperiod (light/dark 8:16) or under stimulatory long photoperiod (LD 16:8), were subjected to either sham-operation, castration, castration combined with treatment with the androgens 11-ketoandrostenedione (11KA) and testosterone (T), and the effects on levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH)-beta mRNA were analyzed. During breeding season the kidney of the stickleback male hypertrophies and produces a glue used for building nests. Kidney weight and expression of both LH-beta and FSH-beta were higher in sham-operated fish kept under long than under short photoperiod. Under both photoperiods, LH-beta mRNA levels were lower in castrated males compared to sham-operated males and treatment with 11KA and T increased expression, indicating a positive feedback. A positive feedback was also found on FSH-beta expression under long photoperiod, where castration decreased, and androgen replacement restored FSH-beta mRNA expression. On the contrary, castration under short photoperiod instead increased FSH-beta levels whereas treatment with 11KA and T decreased FSH-beta expression, indicating a negative feedback on FSH-beta under these conditions. The positive feedback on FSH-beta expression under stimulatory photoperiod may accelerate maturation, whereas the negative feedback under inhibitory photoperiod may suppress maturation. This could be part of the mechanisms by which photoperiod controls maturation.

Bu-shen-zhu-yun decoction promotes synthesis and secretion of FSHß and LHß in anterior pituitary cells in vitro.

Luteal phase defects (LPD) are an important etiology of infertility which has increased in recent years. Studies have shown that bu-shen-zhu-yun decoction (BSZY-D) can lower the expression of estrogen receptor and progesterone receptor, in rats endometrium of embryonic implantation period, which upregulated by mifepristone, and improve uterine receptivity. The aim of present study was to determine the effect of BSZY-D on the synthesis and secretion of gonadotropic hormones in the anterior pituitary cells of rats. Rats were treated with saline (control) or BSZY-D two times/day for three estrous cycles by gavage. The cerebrospinal fluid (CSF) were collected for further cell treatment. The components in BSZY-D, serum and CSF were analysed by High Performance Liquid Chromatography (HPLC). Cells were either pretreated with normal CSF or BSZY-D/CSF before being stimulated with or without cetrorelix. The mRNA and proteins levels of receptors, hormones, and transcription factors were detected by RT-PCR, western blot analysis and immunostaining. We show that non-toxic concentrations of cetrorelix, a GnRH antagonist, can reduce the mRNA and protein levels of GnRHR, LH, and FSH. This effect could be reversed by the addition of BSZY-D/CSF. We also show decreased mRNA and protein expression of transcription factors, such as CREB, and Egr-1 and secretory vescicles, including SNAP-25 and Munc-18 upon treatment with cetrorelix could be reversed post co-treatment with BSZY-D/CSF. These results indicate that BSZY-D/CSF treatment led to increased levels of GnRHR, transcription factors, and secretory vesicles leading to increased secretion of FSH and LH. Thus, BSZY-D presents a promising candidate to treat luteal phase defects and infertility.

Production of recombinant channel catfish (Ictalurus punctatus) FSH and LH in S2 Drosophila cell line and an indication of their different actions.

Due to the lack of purified, native gonadotropins (GtH) for almost all species of fish, we designed a system for the production of recombinant bioactive luteinizing hormone (LH) and follicle stimulating hormone (FSH) using the channel catfish (Ictalurus punctatus) as a model animal. The strategy was to produce the three subunits composing FSH and LH, i.e. the common alpha-subunit (alpha-glycoprotein hormone (alpha-GP)), beta-FSH, and beta-LH subunit, individually in stable recombinant insect cells (S2) with C-terminal His-tag. This expression system was also used to co-express the alpha-subunit without the His-tag with each of the His-tagged beta-subunits. The recombinant S2 cells were capable of secreting FSH and LH heterodimers and alpha-GP in abundance; however, expression of the individual beta-subunits was much less successful. The recombinant GtHs were partially purified from the cell medium by immobilized metal affinity chromatography to ~15% purity with a yield of 7 and 4 mg per liter of medium for FSH and LH respectively. These recombinant GtHs activated their receptors in vitro, enhanced estrogen secretion, up-regulated several steroidogenic enzyme genes in channel catfish ovarian follicles, and increased androgen secretion from African catfish testis. Interestingly, the FSH and LH dose-response curves for each of these biological activities clearly demonstrate differences in their cellular action and physiological roles. This expression system may be an important development for the production of species-specific GtHs so that FSH- and LH-specific mechanisms of actions within the reproductive endocrine processes can finally be examined with homologous, albeit recombinant, hormones.

GNRHR mutations in a woman with idiopathic hypogonadotropic hypogonadism highlight the differential sensitivity of luteinizing hormone and follicle-stimulating hormone to gonadotropin-releasing hormone.

Mutations in the GnRH receptor gene (GNRHR) are a cause of idiopathic hypogonadotropic hypogonadism. We describe a normosmic female subject with congenital idiopathic hypogonadotropic hypogonadism in whom treatment with pulsatile GnRH resulted in an unusual response. The subject not only required an increased dose of pulsatile GnRH for ovarian follicular development, but LH secretion did not increase appropriately, estradiol levels remained low, and she did not ovulate spontaneously. Sequencing of the GNRHR coding sequence revealed compound heterozygous mutations leading to amino acid substitutions [N10K+Q11K] and P320L. The introduction of the P320L mutation into the GnRH receptor led to failure of detectable ligand binding and failure of stimulation of inositol phosphate production and gonadotropin subunit gene promoter activity in response to GnRH in transiently transfected cells. The [N10K+Q11K] mutation resulted in reduced binding of a GnRH agonist to 25% of the wild-type receptor. In addition, the EC(50) value for GnRH stimulation of inositol phosphate production was significantly increased, and the dose-response curves for stimulation of alpha gonadotropin subunit, LHbeta, and FSHbeta gene transcription by GnRH were similarly shifted to the right. Stimulation of FSHbeta gene transcription was more sensitive to GnRH than LHbeta for both wild-type and [N10K+Q11K] GnRH receptors, resulting in a greater loss of LHbeta stimulation than FSHbeta by the [N10K+Q11K] mutant at any given submaximal GnRH concentration. We propose that the mutations in the GnRH receptor result in a rightward shift of the dose-response curves of gonadotropin responses to pulsatile GnRH in the subject and unmask the differential sensitivities of LH and FSH to GnRH, resulting in low LH and estradiol levels despite appropriate FSH secretion and follicular growth.

In vitro induction of luteinizing hormone synthesis by estrogen in organ-cultured pituitary glands of the Japanese eel, Anguilla japonica.

An organ culture method for pituitary glands isolated from immature Japanese eels (Anguilla japonica) was developed. This method could conserve the histological features of the pituitary glands for at least 21 days. The ability to synthesize gonadotropic hormone (GTH) in cultured eel pituitary glands was examined by detecting luteinizing hormone (LH) beta protein immunohistochemically. In a basal medium (Leibovitz L-15), LH beta-immunoreactive cells were very scarce, but after addition of estradiol-17beta (E2) a large number of immunoreactive cells appeared, particularly in the proximal pars distalis. The stimulatory effects of E2 on LH beta synthesis were dose (1-100 ng/ml)- and time (1.5-7 days)-dependent. Thus, in contrast with previous reports of the lack of a direct effect of E2 on GTH synthesis in primary cultured eel pituitary cells, the present results clearly indicate that E2 can stimulate GTH synthesis in immature eel pituitary glands. This organ culture method is useful to examine the actions of steroids and also other endocrine factors on the eel pituitary gland.

betaFSH, betaLH and growth hormone gene expression in blue gourami (Trichogaster trichopterus, Pallas 1770) during spermatogenesis and male sexual behavior.

The relationship between gonadal development (histological evidence for spermiogenesis and/or spermatogenesis), sexual behavior (nest-building) and mRNA levels of gonadotropins (betaFSH and betaLH) and growth hormone (GH) in the male pituitary was investigated. Amplification of betaFSH cDNA showed a significantly higher mRNA level in mature males (whether sexually active or not) than in juveniles. However, following PCR amplification of betaLH cDNA, a significantly higher mRNA level was found in the sexually active group compared to the sexually inactive group. These results suggest that FSH may participate in spermatogenesis, whereas LH is more involved in spermiogenesis. The GH mRNA level increased slightly during the maturation process but no significant differences were found between the groups studied.

GnRH increases c-Fos half-life contributing to higher FSHß induction.

GnRH is a potent hypothalamic regulator of gonadotropin hormones, LH and FSH, which are both expressed within the pituitary gonadotrope and are necessary for the stimulation of gametogenesis and steroidogenesis in the gonads. Differential regulation of LH and FSH, which is essential for reproductive fitness, is achieved, in part, through the varying of GnRH pulse frequency. However, the mechanism controlling the increase in FSH during the periods of low GnRH has not been elucidated. Here, we uncover another level of regulation by GnRH that contributes to differential expression of the gonadotropins and may play an important role for the generation of the secondary rise of FSH that stimulates folliculogenesis. GnRH stimulates LHß and FSHß subunit transcription via induction of the immediate early genes, Egr1 and c-Fos, respectively. Here, we determined that GnRH induces rapidly both Egr1 and c-Fos, but specifically decreases the rate of c-Fos degradation. In particular, GnRH modulates the rate of c-Fos protein turnover by inducing c-Fos phosphorylation through the ERK1/2 pathway. This extends the half-life of c-Fos, which is normally rapidly degraded. Confirming the role of phosphorylation in promoting increased protein activity, we show that a c-Fos mutant that cannot be phosphorylated by GnRH induces lower expression of the FHSß promoter than wild-type c-Fos. Our studies expand upon the role of GnRH in the regulation of gonadotropin gene expression by highlighting the role of c-Fos posttranslational modification that may cause higher levels of FSH during the time of low GnRH pulse frequency to stimulate follicular growth.

Kisspeptin regulates gonadotropin genes via immediate early gene induction in pituitary gonadotropes.

Kisspeptin signaling through its receptor, Kiss1R, is crucial for many reproductive functions including puberty, sex steroid feedback, and overall fertility. Although the importance of Kiss1R in the brain is firmly established, its role in regulating reproduction at the level of the pituitary is not well understood. This study presents molecular analysis of the role of kisspeptin and Kiss1R signaling in the transcriptional regulation of the gonadotropin gene ß-subunits, LHß and FSHß, using LßT2 gonadotrope cells and murine primary pituitary cells. We show that kisspeptin induces LHß and FSHß gene expression, and this induction is protein kinase C dependent and mediated by the immediate early genes, early growth response factor 1 and cFos, respectively. Additionally, kisspeptin induces transcription of the early growth response factor 1 and cFos promoters in LßT2 cells. Kisspeptin also increases gonadotropin gene expression in mouse primary pituitary cells in culture. Furthermore, we find that Kiss1r expression is enhanced in the pituitary of female mice during the estradiol-induced LH surge, a critical component of the reproductive cycle. Overall, our findings indicate that kisspeptin regulates gonadotropin gene expression through the activation of Kiss1R signaling through protein kinase C, inducing immediate early genes in vitro, and responds to physiologically relevant cues in vivo, suggesting that kisspeptin affects pituitary gene expression to regulate reproductive function.

Effect of phytoestrogens on basal and GnRH-induced gonadotropin secretion.

Plant-derived estrogens (phytoestrogens, PEs), like endogenous estrogens, affect a diverse array of tissues, including the bone, uterus, mammary gland, and components of the neural and cardiovascular systems. We hypothesized that PEs act directly at pituitary loci to attenuate basal FSH secretion and increase gonadotrope sensitivity to GnRH. To examine the effect of PEs on basal secretion and total production of FSH, ovine pituitary cells were incubated with PEs for 48 h. Conditioned media and cell extract were collected and assayed for FSH. Estradiol (E2) and some PEs significantly decreased basal secretion of FSH. The most potent PEs in this regard were coumestrol (CM), zearalenone (ZR), and genistein (GN). The specificity of PE-induced suppression of basal FSH was indicated by the absence of suppression in cells coincubated with PEs and an estrogen receptor (ER) blocker (ICI 182 780; ICI). Secretion of LH during stimulation by a GnRH agonist (GnRH-A) was used as a measure of gonadotrope responsiveness. Incubation of cells for 12 h with E2, CM, ZR, GN, or daidzein (DZ) enhanced the magnitude and sensitivity of LH secretion during subsequent exposure to graded levels of a GnRH-A. The E2- and PE-dependent augmentation of gonadotrope responsiveness was nearly fully blocked during coincubation with ICI. Collectively, these data demonstrate that selected PEs (CM, ZR, and GN), like E2, decrease basal secretion of FSH, reduce total FSH production, and enhance GnRH-A-induced LH secretion in a manner that is dependent on the ER.

Effects of the phytoestrogen genistein on the porcine anterior pituitary insulin-like growth factor system.

Estrogens have profound effects on the serum and anterior pituitary (AP) insulin-like growth factor (IGF) system in pigs. In this study we determined whether administration of the phytoestrogen genistein increased serum and AP concentrations of IGF-I and relative amounts of serum and AP insulin-like growth factor-binding protein (IGFBP). Twenty barrows of similar age (190 d) and weight (110 kg) were stratified by litter into one of four treatments: controls (C), estradiol (E), 200 mg genistein (G200), and 400 mg genistein (G400). Estradiol-treated pigs were injected daily with 2 mg of estradiol-17ß intramuscularly (i.m.), whereas the G200 and G400 groups were injected daily with either 200 or 400 mg of genistein i.m., respectively, beginning on d 0 and continuing through d 15. Blood was collected on d 0, 3, 6, 9, and 13. Blood and AP were collected at slaughter on d 16. Serum and AP concentrations of IGF-I and luteinizing hormone (LH) were determined by radioimmunoassay. Relative amounts of serum IGFBP were determined by Western ligand blot analysis. Relative expression of AP IGF-I, IGF-I receptor (IGF-IR), gonadotropin-releasing hormone receptor, and LHß subunit was determined by real-time reverse transcriptase polymerase chain reaction. Anterior pituitary concentrations of IGF-I were greater (P > 0.05) in E and G400 pigs compared with controls, whereas AP concentrations of LH were greater (P < 0.05) in G400 pigs compared with C and G200 pigs. Relative expression of LHß was greater in G200 pigs compared with C pigs but did not differ from that in G400 pigs. Relative expression of AP IGF-IR was greater (P < 0.05) in E pigs compared with all other treatments; however, relative expression of AP IGF-IR was greater (P < 0.05) in both G200 and G400 pigs vs C pigs. No differences were detected (P > 0.05) in serum concentrations of IGF-I or relative amounts of serum and AP IGFBP among treatments. These data provide evidence that genistein is capable of modulating components of the AP IGF system that could affect the synthesis and release of LH.

Production of recombinant orange-spotted grouper (Epinephelus coioides) luteinizing hormone in insect cells by the baculovirus expression system and its biological effect.

The cDNA sequence encoding orange-spotted grouper lhb (LHbeta) and cga (GTHalpha) subunits were cocloned into baculovirus transfer vectors and expressed in insect Sf9 cells. The results showed that two bands of 15.6 kDa and 11.4 kDa could be detected by SDS-PAGE and a band of 30 kDa could be detected by native PAGE. The recombinant grouper Lh (rgLh) could stimulate the secretion of testosterone (T) and estradiol-17beta (E2) from the gonad in a static incubation system in a time-dependent, but not a dose-dependent, manner. Using in vivo bioassay, the mRNA levels of two aromatases (cyp19a1a [P450aromA] and cyp19a1b [P450aromB]), gnrh (GnRH), lhb, and cga in the pituitary, gonad, and hypothalamus were determined in different groups of orange-spotted groupers treated respectively with rgLh, human chorionic gonadotropin (hCG), and a culture medium of insect cells transformed with an expression vector without lhb and cga subunits. The mRNA levels of cyp19a1a and cyp19a1b rose dramatically after injecting rgLh intraperitoneally, which was consistent with the secretion of sex steroid hormones. Interestingly, the mRNA levels of gnrh dropped in the pituitary, hypothalamus, and gonad, and the mRNA levels of lhb and cga in the pituitary of the experimental group expressed at a higher level than that of the hCG group. These results are in accord with the long positive feedback loop of Lh on gonad sex steroid hormones and the short negative feedback loop of Lh on gnrh mRNA levels. These results indicate that the rgLh is successfully expressed by the baculovirus-insect expression system and that the rgLh has biological activity.

A systematic immunohistochemical survey of the distribution patterns of GH, prolactin, somatolactin, beta-TSH, beta-FSH, beta-LH, ACTH, and alpha-MSH in the adenohypophysis of Oreochromis niloticus, the Nile tilapia.

Fish pituitary plays a central role in the control of growth, development, reproduction and adaptation to the environment. Several types of hormone-secreting adenohypophyseal cells have been characterised and localised in diverse teleost species. The results suggest a similar distribution pattern among the species investigated. However, most studies deal with a single hormone or hormone family. Thus, we studied adjacent sections of the pituitary of Oreochromis niloticus, the tilapia, by conventional staining and immunohistochemistry with specific antisera directed against growth hormone (GH), prolactin (PRL), somatolactin (SL), thyrotropin (beta-TSH), follicle-stimulating hormone (beta-FSH), luteinising hormone (beta-LH), adrenocorticotropic hormone (ACTH) and melanocyte-stimulating hormone (alpha-MSH). The pituitary was characterised by a close interdigitating neighbourhood of neurohypophysis (PN) and adenohypophysis. PRL-immunoreactive and ACTH-immunoreactive cells were detected in the rostral pars distalis. GH-immunoreactive cells were present in the proximal pars distalis (PPD). A small region of the PPD contained beta-TSH-immunoreactive cells, and beta-LH-immunoreactive cells covered approximately the remaining parts. Centrally, beta-FSH-immunoreactive cells were detected in the vicinity of the GH-containing cells. Some of these cells also displayed beta-LH immunoreactivity. The pars intermedia was characterised by branches of the PN surrounded by SL-containing and alpha-MSH-immunoreactive cells. The ACTH and alpha-MSH antisera were observed to cross-react with the respective antigens. This cross-reactivity was abolished by pre-absorption. We present a complete map of the distinct localisation sites for the classical pituitary hormones, thereby providing a solid basis for future research on teleost pituitary.

Differentiation of mouse embryonic stem cells into gonadotrope-like cells in vitro.

OBJECTIVE: This research was conducted to investigate the potential of mouse embryonic stem (ES) cells to differentiate in vitro into gonadotropes. METHODS: Undifferentiated ES cells were maintained on mitomycin C-inactivated fibroblasts in the presence of leukemia inhibitory factor (LIF). By a 5-day hanging drop culture devoid of them, ES cells were induced to form multidifferentiated structures called embryoid bodies (EBs). Reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting, and immunocytochemistry were used to analyze gene expression of gonadotrope markers in EBs at different time points during the culture. RESULTS: Homeo box gene expressed in ES cells (Hesx1), LIM homeobox protein 3 (Lhx3), paired like homeodomain factor 1 (Prop1), GATA binding protein 2 (GATA2), follicle-stimulating hormone beta (FSHbeta), and luteinizing hormone beta (LHbeta) mRNAs were detected at day 6 EBs and maintained throughout the culture to day 56. FSHbeta and LHbeta proteins were expressed in EBs from day 6 onward. Immunofluorescent labeling of FSHbeta and LHbeta showed that specific staining was restricted to the cytoplasm of some differentiated EB cells. With the prolongation of EB culture, the number of positive cells increased significantly. Both monohormonal and bihormonal cells were present, mainly in clusters within EBs and sparsely distributed among the outermost cells surrounding the EBs. CONCLUSION: These results indicate that mouse ES cells can give rise to mature gonadotrope-like cells in EBs. It also shows that EBs may serve as a novel model system to study the development and function of gonadotropes.

Differential regulation of gonadotropins and glycoprotein hormone alpha-subunit by IGF-I in anterior pituitary cells from male rats.

IGF-I has been demonstrated to stimulate basal and GnRH-induced gonadotropin release. IGF-I also elicites alpha-subunit secretion in human pituitary tumor cells. The aims of this study were to evaluate both the effect of IGF-I on gonadotropin LH-beta and FSH-beta mRNA levels and glycoprotein alpha-subunit gene expression in cultured rat anterior pituitary cells. The exposure of pituitary cells to recombinant human IGF-I (rhlGF-I; 2 microg/ml) for 72 h markedly stimulated basal LH and FSH release whereas their mRNA levels remained unmodified. IGF-I elicited a-subunit release from pituitary cells (p < 0.01) and augmented its mRNA levels. Exposure to IGF-I consistently reduced GH release from pituitary cells. This study shows that the gonadotropin-releasing effects of IGF-I are not paralleled by changes in their mRNAs whereas IGF-I stimulates not only alpha-subunit release but also its mRNA levels. This study provides the first observation of alpha-subunit regulation by IGF-I in normal pituitary cells, where a differential regulation between release and synthesis for gonadotropin a-and 1-subunits is also shown.

Changes in LHbeta-gene and FSHbeta-gene expression in the ram pars tuberalis according to season and castration.

Luteinizing hormone beta (LHbeta) and follicle stimulating hormone beta (FSHbeta) subunits and their mRNAs were studied in the ram pars tuberalis following different seasonal (winter vs summer) and experimental (intact vs castrated animals) conditions. Hormone-containing cells were identified by immunohistochemistry, using homologous double-stranded 35S-cDNAs. The labelling was quantified by image analysis. Immunohistochemical staining showed that cells containing LHbeta and FSHbeta were localized mainly in the ventral part of the pars tuberalis but that, in the summer, additional LHbeta containing cells were present in the dorsal part in intact rams. On the other hand, LHbeta-mRNA labelling was found in the whole pars tuberalis in wethers but only in the ventral part in intact rams. The magnitude of LHbeta-mRNA labelling was significantly greater in summer than in winter rams, and in castrated than in intact animals (P<0.001). However, the number of labelled cells was found to be the greatest in the winter (P<0.001) and was not affected by castration. FSHbeta-mRNA expression was similar to that of LHbeta-mRNA except that the level and extent were considerably lower. Thus, our results show an increase in the magnitude of gonadotropin beta subunit-mRNA in the summer and following castration; this increase appears to involve the entire pars tuberalis.

Hypophyseal gene expression profiles of FSH-beta, LH-beta, and glycoprotein hormone-alpha subunits in Ictalurus punctatus throughout a reproductive cycle.

A determination of the seasonal changes in the expression of the genes encoding the subunits of gonadotropic hormones is an important first step in the understanding of the molecular control of the onset of puberty and the reproductive cycle in fish. In this study, the abundance of transcripts encoding the glycoprotein hormone alpha (GpH-alpha), follicle-stimulating hormone beta (FSH-beta), and luteinizing hormone beta (LH-beta) subunits in pituitaries of female channel catfish were systematically tracked throughout an annual reproductive cycle. All three genes showed a concurrent elevation coinciding with the onset of ovarian recrudescence but then each showed a second elevation at different times of the ovarian cycle. In addition to the initial peak at recrudescence, the expression of FSH-beta and GpH-alpha gene peaked again during mid- and late-vitellogenic growth, respectively. The LH-beta gene expression remained low during the phases of regression and vitellogenic growth but was moderately elevated (7-fold) at the onset of ovarian recrudescence and dramatically elevated (36-fold) just prior to spawning (June-July) when the FSH-beta levels were at their lowest. The expression patterns of FSH-beta and LH-beta are remarkably similar to the ovarian expression of their respective receptors.

Modulation of human luteinizing hormone beta gene transcription by MIP-2A.

MIP-2A was recently identified as a MBP-1 interacting cellular protein. We have shown previously that MBP-1 acts as a transcriptional repressor. Functional association between MIP-2A and MBP-1 suggests that MIP-2A can act as a cofactor and relieves MBP-1-mediated transcriptional repression. In this study, we report the tissue-specific expression of MIP-2A and its role in the regulation of gene transcription. RNA dot blot analysis of human multiple tissue expression array suggested that MIP-2A is highly abundant in right cerebellum, pituitary, adrenal, and testis but barely detectable in skeletal muscle. Predominant expression of MIP-2A in pituitary tissue led us to investigate whether MIP-2A can transcriptionally regulate luteinizing hormone beta (LHbeta), a pituitary-specific hormone synthesized and secreted from gonadotropic cells. The LHbeta promoter is regulated by the orphan nuclear receptor SF-1 and homeodomain protein Ptx1. Although each factor enhances the LHbeta promoter, coexpression of both results in a strong synergistic activation. Therefore, we examined whether MIP-2A can modulate SF-1- and Ptx1-mediated transcriptional activation. Our results suggested that MIP-2A expression inhibits SF-1- and Ptx1-mediated transactivation of LHbeta promoter. Subsequent analysis demonstrated that MIP-2A physically interacts with both SF-1 and Ptx1, thereby inhibiting transactivation of the LHbeta promoter. Taken together, our results indicate that MIP-2A preferentially expresses in certain tissues, including the pituitary gland, and negatively regulates the LHbeta gene transcription.