Reproductive deficiencies in transgenic mice expressing the rat inhibin alpha-subunit gene.

Inhibin is an important modulator of reproductive function at both the endocrine level, through its regulation of pituitary FSH biosynthesis, and at the paracrine and autocrine levels, as an intragonadal regulatory factor. To investigate the in vivo actions of inhibin in FSH regulation and gonadal function, transgenic mice that overexpress the rat inhibin alpha-subunit gene were generated. A transgene that includes the mouse metallothionein-I gene promoter (MT-alpha) fused to the rat inhibin alpha-subunit precursor coding sequences was used to produce three lines of transgenic mice. Transgene mRNA is expressed in numerous tissues, including the pituitary, liver, testis, ovary, and kidney. Inhibin alpha-subunit protein was also increased in transgenic pituitary and ovary. Serum inhibin alpha-subunit levels are highly increased compared with control mice. Inhibin beta(A)- and beta(B)-subunit protein amounts are lower in transgenic ovaries compared with wild type, although serum levels of activin A are not significantly reduced in transgenic female mice. FSH levels are reduced in both male and female transgenic mice, whereas LH levels are increased in MT-alpha female mice. MT-alpha transgenic females are subfertile and exhibit a 52% reduction in litter size compared with wild-type females. The smaller litter size of MT-alpha female mice was correlated with a reduction in the number of oocytes ovulated during a normal cycle. Treatment of the transgenic females with exogenous gonadotropins resulted in an ovulation rate similar to that of stimulated wild-type animals, suggesting that altered gonadotropin levels may be responsible for the decreased ovulation rates. MT-alpha transgenic male mice are fertile and sire litters of equivalent size to those sired by wild-type males, despite an approximately 50% reduction in sperm numbers. These results indicate that overexpression of the rat inhibin alpha-subunit gene in mice leads to a disruption of the normal inhibin-to-activin ratio and to reproductive deficiencies, and they support the hypothesis that inhibin and activin act to regulate FSH secretion in vivo and are essential for normal gonadal function.

Gonadal pathologies in transgenic mice expressing the rat inhibin alpha-subunit.

Inhibin and activin are structurally related dimeric peptide hormones and are members of the TGF-beta superfamily of proteins. In the accompanying paper, we describe transgenic mice that overexpress the inhibin alpha-subunit gene from a metallothionein-I promoter (MT-alpha) and examine the effects of the MT-alpha transgene on gonadotropin levels and fertility. To characterize the effects of increased inhibin alpha-subunit on gonadal morphology and function, in this report we investigate gonadal histology, steroid hormone levels, and the basis of ovarian cyst formation in MT-alpha transgenic mice. MT-alpha transgenic female mice develop large fluid-filled ovarian cysts of follicular origin as early as 3 months of age. By 12 months of age, more than 92% of female MT-alpha transgenic mice develop ovarian cysts compared with less than 25% of wild-type littermates. Ovarian cysts form unilaterally or bilaterally, and cystic ovaries often have a greatly expanded bursal sac. Additionally, the ovaries of MT-alpha transgenic mice contain polyovular follicles and have fewer mature antral follicles and corpora lutea. MT-alpha female mice exhibit abnormal steroid hormone production, with increased serum T levels and reductions in serum E with corresponding reductions in uterine mass. In the MT-alpha transgenic males, testis size was decreased by 20-40% compared with control males, and there is a corresponding reduction in seminiferous tubule volume. After a chronic treatment with a GnRH antagonist, MT-alpha female mice continued to develop ovarian cysts and bursal sac expansions, although the cysts were markedly reduced in size. These results indicate that the expression of the rat inhibin alpha-subunit in mice results in significant ovarian pathology, reduced testicular size, and altered ovarian steroidogenesis. The antagonist studies are consistent with a direct ovarian effect of the alpha-subunit transgene product mediated by changes in the inhibin-to-activin ratio in these mice.

Absence of constitutively activating mutations in the GHRH receptor in GH-producing pituitary tumors.

The molecular events leading to the development of GH-producing pituitary tumors remain largely unknown. We hypothesized that activating mutations of the GHRH receptor might occur in a subset of GH-producing pituitary tumors. Genomic DNA samples from 54 GH-producing pituitary tumor tissues were screened for mutations of the GHRH receptor. Eleven homozygous or heterozygous nucleotide substitutions [169G > A (A57T), 338C > T (P113L), 363G > T (E121D), 409C > T (H137Y), 547G > A (D183N), 673G > A (V225I), 749G > A (W250X), 760G > A (V254M), 785G > A (S262N), 880G > A (G294R), 1268G > A (C423Y)] were found in 12 patients (22.2%). The 169G > A substitution (A57T) appears to be a polymorphism (4 patients, 7.4%). E121D and V225I were each found in 2 patients. In 1 patient with the V225I sequence, the substitution was not found in genomic DNA from peripheral leukocytes, suggesting a somatic mutation. A patient with a heterozygous W250X mutation was homozygous for the C423Y substitution. These variant GHRH receptors were studied in transfected TSA-201 cells to evaluate the functional consequences of the amino acid changes. None of the GHRH receptor variants was associated with basal elevation of intracellular cAMP. GHRH induced variable cAMP responses. With the W250X and G294R variants, there was no cAMP stimulation by GHRH, indicating that the mutations are inactivating. Expression of the W250X GHRH receptor on the cell membrane was severely decreased and GHRH binding to the G294R GHRH receptor was impaired. Although GHRH receptor variants are common in GH- producing pituitary adenomas, constitutively activating mutations, as a mechanism for GH-producing pituitary tumors appear to be rare.

Characterization of prohibitin in a newly established rat ovarian granulosa cell line.

Prohibitin is an evolutionary conserved protein that is associated with cellular differentiation, atresia, and luteolysis in the rat ovary. However, the specific cellular location and function of prohibitin in ovarian cells has not been clearly elucidated. To characterize the expression of prohibitin during cell proliferation, differentiation, and cell death, we have successfully established a temperature-sensitive granulosa cell line, designated RGA-1. At a permissive temperature of 33 C, RGA-1 cells proliferate, but revert to a differentiated phenotype at a nonpermissive temperature of 39 C. Significant inductions of prohibitin mRNA and protein expression were observed in the differentiated phenotype when compared with proliferating cells. Differentiated RGA-1 cells were found to express inhibin alpha- and beta-transcripts, as well as steroidogenic acute regulatory protein and peripheral-type benzodiazepine receptor proteins in a manner reminiscent of steroidogenic functional responses observed in primary differentiated granulosa cells. Prohibitin expression correlated well with the expression of these steroidogenic proteins. At 39 C, RGA-1 cells also displayed increases in p53 protein levels, indicative of growth arrest in the nonproliferating cells. Confocal and electron microscopic examinations revealed increased prohibitin localization to the mitochondria at 39 C, along with changes in mitochondrial size and shape. These changes were accompanied by marked reductions in cytochrome c oxidase subunit II levels and in unit mitochondrial transmembrane potential. In addition, cell fractionation studies demonstrated that the prohibitin protein was mainly localized to the mitochondrial membrane. Collectively, these findings suggest a role for prohibitin in mitochondrial structure and function during growth and differentiation in ovarian granulosa cells. Prohibitin expression may also be indicative of mitochondrial destabilization during apoptosis-related events.

Developmental regulation of mitogen-activated protein kinase-activated kinases-2 and -3 (MAPKAPK-2/-3) in vivo during corpus luteum formation in the rat.

The current study investigates the activation in vivo and regulation of the expression of components of the p38 mitogen-activated protein kinase (MAPK) pathway during gonadotropin-induced formation and development of the rat corpus luteum, employing a sequential PMSG/human CG (hCG) treatment paradigm. We postulated that the p38 MAPK pathway could serve to promote phosphorylation of key substrates during luteal maturation, since maturing luteal cells, thought to be cAMP-nonresponsive, nevertheless maintain critical phosphoproteins. Both p38 MAPK and its upstream activator MAPK kinase-6 (MKK6) were found to be chronically activated during the luteal maturation phase, with activation detected by 24 h post hCG and maintained through 4 days post hCG. The p38 MAPK downstream protein kinase target termed MAPK-activated protein kinase-3 (MAPKAPK-3) was newly induced at both mRNA and protein levels during luteal formation and maturation, while mRNA and protein expression of the closely related MAPKAPK-2 diminished. Two potential substrates for MAPKAPKs, the small heat shock protein HSP-27 and the cAMP regulatory element binding protein CREB, were monitored in vivo for phosphorylation. HSP-27 phosphorylation was not modulated during luteal maturation. In contrast, we observed sustained luteal-phase CREB phosphorylation in vivo, consistent with upstream MKK6/p38 MAPK activation and MAPKAPK-3 induction. MAPKAPK-3-specific immune complex kinase assays provided direct evidence that MAPKAPK-3 was in an activated state during luteal maturation in vivo. Cellular inhibitor studies indicated that an intact p38 MAPK path was required for CREB phosphorylation in a cellular model of luteinization, as treatment of luteinized granulosa cells with the p38 MAPK inhibitor SB 203580 strongly inhibited CREB phosphorylation. Transient transfection studies provided direct evidence that MAPKAPK-3 was capable of signaling to activate CREB transcriptional activity, as assessed by means of GAL4-CREB fusion protein construct coexpressed with GAL4-luciferase reporter construct. Introduction of wild-type, but not kinase-dead mutant, MAPKAPK-3 cDNA, into a mouse ovarian cell line stimulated GAL4-CREB- dependent transcriptional activity approximately 3-fold. Thus MAPKAPK-3 is indeed uniquely poised to support luteal maturation through the phosphorylation and activation of the nuclear transcription factor CREB.

GH mRNA levels are elevated by forskolin but not GH releasing hormone in GHRH receptor-expressing MtT/S somatotroph cell line.

The MtT/S somatotroph cell line should be a growth hormone-releasing hormone (GHRH)-responsive model system for the study of physiological control of growth hormone (GH) transcription because GH secretion from these cells is stimulated by GHRH. To examine the GH transcriptional activity of these cells, endogenous GH mRNA levels were measured using a ribonuclease protection assay following treatment under a variety of hormonal conditions. While omission of serum led to reduction of GH mRNA to 22% of control levels by 2 days and to 8% by 5 days (P<0.05 for both), GH mRNA levels were maintained at control values in serum-free medium containing 5 nM dexamethasone and 30 pM triiodothyronine (TDM). However, the addition of 10 nM GHRH under any treatment condition did not significantly alter GH mRNA levels. Characterization of the MtT/S cells showed that GHRH-receptor (GHRH-R) mRNA was detectable by reverse transcription-polymerase chain reaction (RT-PCR) amplification. Measurement of extracellular cAMP showed that the MtT/S cells have basal levels of > or =20 nmol/10(6) cells per h in both serum-containing and serum-free media, and that GHRH had no effect on cAMP levels, suggesting constitutive activation. To rule out the possibility of autocrine stimulation by GHRH produced endogenously, GHRH mRNA was not detectable in MtT/S cells using RT-PCR amplification. The stimulatory G-protein alpha subunit, mutations of which are known to activate adenylate cyclase constitutively in acromegaly, was sequenced but found not to differ from normal pituitary in the regions most commonly mutated. Finally, treatment with 10 microM forskolin, to directly activate adenylate cyclase, increased GH mRNA to 140% of controls in TDM, and to 163% in serum-free medium after 2 days, and to 166% in TDM-treated cells and 174% in serum-free culture after 5 days (all P<0.05). Taken together, these data indicate that although MtT/S cells express the GHRH-R, GHRH cannot stimulate adenylate cyclase to increase GH transcription due to constitutive elevation of cAMP levels, by a means that may be similar to that in cases of acromegaly not caused by oncogenic gsp mutations.

Regulation of the pituitary somatotroph cell by GHRH and its receptor.

Hormones from the hypothalamus mediate interactions between the nervous and endocrine systems by controlling the activity of specific target cells in the anterior pituitary gland. The hypothalamic peptide, growth hormone-releasing hormone (GHRH), acts on pituitary somatotroph cells to stimulate their proliferation during development and to regulate their ability to produce and secrete growth hormone (GH). These actions are mediated by a recently identified receptor for GHRH that belongs to family B-III of the G protein-coupled receptor superfamily. The rat GHRH receptor is expressed predominantly in the pituitary gland and in somatotroph cells. To investigate this tissue- and cell-specific expression, the receptor gene has been cloned and characterized. The receptor gene promoter is selectively expressed in pituitary cells and is regulated by the pituitary-specific transcription factor Pit-1. There is a sexual dimorphism in GHRH receptor expression in the rat pituitary, suggesting regulation by gonadal steroids. In addition, glucocorticoids are potent positive regulators of GHRH receptor gene expression. Substantial evidence points to an important role for GHRH in regulating the proliferation and functional activity of the somatotroph cell. This is best observed in the dwarf little mouse, which harbors a mutation in the extracellular domain of the GHRH receptor that abolishes the receptor's hormone-binding and signaling properties, resulting in severe somatotroph hypoplasia. Complementary studies in transgenic mice overexpressing the ligand GHRH reveal corresponding somatotroph hyperplasia. Consistent with these observations, GHRH potently activates the MAP kinase pathway in pituitary somatotroph cells. To better understand the hormone-binding and signaling properties of the GHRH receptor, mutant and chimeric receptors have been analyzed to define domains important for GHRH interaction. The GHRH receptor signals predominantly through cAMP-dependent pathways; however, a variant form of the GHRH receptor with an insertion into the third intracellular domain, generated through alternative RNA processing, binds GHRH but fails to signal, suggesting potential modulation of receptor function at a post-transcriptional level. This chapter will integrate these basic investigations of GHRH and its receptor with current information on the involvement of the GHRH signaling system in human diseases of GH secretion and growth.

Activin A-induced HepG2 liver cell apoptosis: involvement of activin receptors and smad proteins.

A balance between cell proliferation and apoptosis is important for regulating normal liver function. Proteins of the transforming growth factor-beta superfamily are known to be important mediators of apoptosis in the liver. In this study we demonstrate that activin A potently induces apoptotic cell death in a hepatoma cell line, HepG2 cells. To determine the roles of activin receptors and downstream signaling proteins in activin A-induced apoptosis in these cells, the activin signaling pathway was analyzed using the transcription of an activin-responsive reporter gene, p3TP-Lux, as an assay. Although individual activin receptors had little effect on transcriptional activity, coexpression of an activin type I receptor and a type II receptor significantly increased both basal and activin-induced transcriptional activation, with the combination ofreceptors IB and IIB being the most potent. Similarly, expression of individual Smad proteins had only a modest effect on reporter gene activity, but the combination of Smad2 and Smad4 strongly stimulated transcription. Activin signaling induced a rapid relocation of Smad2 to the nucleus, as determined using a green fluorescence protein-Smad2 fusion protein. In contrast, green fluorescence protein-Smad4 remained localized to the cytoplasm unless it was coexpressed with Smad2. In agreement with the transcriptional response assays, overexpression or suppression of activin signaling components in HepG2 cells altered apoptosis. Overexpression of receptors IB and IIB or Smad proteins 2 and 4 stimulated apoptosis, whereas dominant negative mutant forms of the activin type IIB receptor or Smad2 blocked activin-stimulated apoptosis. These studies suggest that signaling from the cell surface to the nucleus through Smad proteins is a required component of the activin A-induced cell death process in liver cells.

Synergistic activation of the inhibin alpha-promoter by steroidogenic factor-1 and cyclic adenosine 3',5'-monophosphate.

The inhibin alpha-subunit gene is expressed in the ovary, testis, adrenal, and pituitary. Because this pattern of expression corresponds to that of the orphan nuclear receptor, steroidogenic factor-1 (SF-1), we hypothesized that the inhibin alpha promoter might be regulated by SF-1. Expression of exogenous SF-1, in an SF-1 deficient cell line, caused modest stimulation of the inhibin alpha promoter. However, activation of the cAMP pathway, which is known to regulate inhibin alpha expression, greatly enhanced the actions of SF-1. Coexpression of SF-1 with the catalytic subunit of cAMP-dependent protein kinase A caused greater than 250-fold stimulation, whereas only 4- or 7-fold stimulation was seen by the SF-1 or protein kinase A pathway alone. Synergistic stimulation by SF-1 and the cAMP pathway was also seen in GRMO2 granulosa cells, which express endogenous SF-1. Deletion and site-directed mutagenesis localized a novel SF-1 regulatory element (TCA GGGCCA; -137 to -129) adjacent to a variant cAMP-response element (CRE; -120 to -114). The synergistic property of SF-1 and cAMP stimulation was inherent within this composite inhibin alpha fragment (-146 and -112), as it was transferable to heterologous promoters. Mutations in either the CRE or the SF-1 regulatory element completely eliminated synergistic activation by these pathways. The binding of SF-1 and CRE binding protein (CREB) to the inhibin alpha regulatory elements was relatively weak in gel mobility shift assays, consistent with their deviation from consensus binding sites. However, SF-1 was found to interact with CREB using an assay in which epitope-tagged SF-1 was expressed in cells and used to pull down in vitro translated CREB. Expression of CREB binding protein (CBP), a coactivator that interacts with SF-1 and CREB, further enhanced transcription by these pathways. Stimulation by the SF-1 and cAMP pathways was associated with increased histone H4 acetylation, suggesting that chromatin remodeling accompanies their actions. We propose a model in which direct interactions of SF-1, CREB, and associated coactivators like CBP induce strongly cooperative transactivation by pathways that individually have relatively weak effects on transcription.

The mutant growth hormone-releasing hormone (GHRH) receptor of the little mouse does not bind GHRH.

The little mouse is a dwarf strain characterized by low levels of GH, pituitary hypoplasia, and an unresponsiveness to treatment with exogenous GHRH. The defect has been mapped to a missense mutation in the GHRH receptor gene that abolishes the function of the receptor, but the mechanism of this inactivation is unknown. Receptor function might be affected at the level of protein expression, maturation and processing, localization to the cell surface, ligand binding, or signaling. In this study, Western blots, using antiserum raised against the GHRH receptor and immunoprecipitation analysis of epitope-tagged receptors, demonstrate that both wild-type and mutant receptor proteins are expressed at equivalent levels in transfected cells. Immunofluorescence analysis of intact and permeabilized cells expressing the epitope-tagged receptors suggests that wild-type and little mouse receptors are similarly localized to the cell surface. A species homologous binding assay was developed and used to show that 125I-mouse GHRH binds with high affinity to the wild-type mouse receptor but not to the little mutant receptor. Consistent with this, the mutant receptor fails to stimulate intracellular cAMP accumulation. Our results demonstrate that the little mutation does not dramatically affect the expression level, glycosylation, or cellular localization of the receptor protein but that it blocks specific GHRH binding, and therefore, signaling does not take place.

The rat growth hormone-releasing hormone receptor gene: structure, regulation, and generation of receptor isoforms with different signaling properties.

The interaction of GHRH with membrane-bound receptors on somatotroph cells of the anterior pituitary is an important step in the regulation of GH synthesis and secretion. The identification of a G protein-coupled receptor for GHRH has made it possible to investigate the pathway by which GHRH regulates pituitary somatotroph cell function. To initiate an analysis of the mechanisms regulating expression and function of the GHRH receptor, the structure of the gene and its promoter region were analyzed. The coding sequence of the rat GHRH receptor gene is contained within 14 exons spanning approximately 15 kb of genomic DNA. Four transcription start sites are located within 286 bp upstream of the initiation codon. The 5' flanking region of the GHRH receptor gene acts as a functional promoter in rat pituitary tumor GH3 cells, and basal promoter activity is enhanced in GH3 and COS7 cells by cotransfection of an expression construct encoding the pituitary-specific transcription factor Pit-1. The rat GHRH receptor gene is subject to at least 1 alternative RNA processing event that generates 2 receptor isoforms differing by 41 amino acids within the third intracellular loop (IL) of the protein. The short isoform of the GHRH receptor is predominant in pituitary cells. The MtT/S pituitary tumor cell line was found to express the GHRH receptor, and different populations of these cells produce predominantly the long or short isoforms of the receptor messenger RNA, suggesting that the alternative splicing can be regulated. Functional analysis of the two GHRH receptor isoforms demonstrates that both bind GHRH, but only the short isoform signals through a cAMP-mediated pathway. Neither receptor isoform is able to stimulate calcium mobilization from internal stores after GHRH treatment. Our findings indicate that the pituitary-specific transcription factor Pit-1 is involved in the somatotroph-specific expression of the GHRH receptor gene and that functionally distinct receptor proteins are generated by an alternative RNA processing mechanism.

Targeted disruption of the estrogen receptor-alpha gene in female mice: characterization of ovarian responses and phenotype in the adult.

Targeted disruption of the mouse estrogen receptor-alpha gene (estrogen receptor-alpha knockout; ERKO) results in a highly novel ovarian phenotype in the adult. The ERKO mouse model was used to characterize ER alpha-dependent processes in the ovary. Visualization of the ovaries of 10-, 20-, and 50-day-old wild-type (WT) and ERKO mice showed that the ERKO phenotype developed between 20 and 50 days of age. Developmental progression through the primordial, primary, and antral follicle stages appeared normal, but functional maturation of preovulatory follicles was arrested resulting in atresia or in anovulatory follicles, which in many cases formed large, hemorrhagic cysts. Corpora lutea were absent, which also indicates that the normal biochemical and mechanical processes that accomplish ovulation were compromised. Northern and ribonuclease protection analyses indicated that ERKO ovary FSH receptor (FSHR) messenger RNA (mRNA) expression was approximately 4-fold greater than in WT controls. Ovarian LH receptor (LHR) mRNA expression was also higher in the ERKO animals. Cellular localization studies by in situ hybridization analysis of ERKO ovaries showed a high level of LHR mRNA expression in the granulosa and thecal layers of virtually all the antral follicles. Ribonuclease protection analyses showed that ovarian progesterone receptor and androgen receptor mRNA expression were similar in the two groups. These results indicated that ER alpha action was not a prerequisite for LHR mRNA expression by thecal or granulosa cells or for ovarian expression of progesterone receptor mRNA. Ovarian estrogen receptor beta (ER beta) was detected immunohistochemically, was sharply compartmentalized to the granulosa cells, and was expressed approximately equally in the ERKO animals and the WT controls. In contrast, ER alpha staining was present in the thecal cells but not the granulosa cells of the WT animals. The summary findings indicate that in the adult the major cause of the ERKO phenotype is high circulating LH interacting with functional LHR of the theca and granulosa cells. These features result in a failure of the normal maturational events leading to successful ovulation and luteinization and presumably involve both hypothalamic-pituitary and intraovarian mechanisms dependent upon ER alpha action. The presence of ER beta in the granulosa cells did not rescue the phenotype of the ovary.

Structure of the rat inhibin and activin betaA-subunit gene and regulation in an ovarian granulosa cell line.

We have isolated the rat inhibin and activin betaA-subunit gene, which is composed of three exons, and have characterized a 571-bp region upstream from the transcriptional start site that functions as a promoter in transient transfection studies in an ovarian granulosa cell line, GRMO2. Deletion analysis of the 571-bp promoter region has identified DNA sequences between -362 bp and -110 bp to be essential in mediating basal promoter activity and activation by forskolin (FSK) and/or 12-O-tetradecanoylphorbol-13-acetate (TPA). Within this region, a variant CRE (cAMP response element) has been identified at -120 bp. Point mutations in the variant CRE substantially reduce the ability of FSK and/or TPA to induce promoter activity in GRMO2 cells. A single nucleotide change in the variant CRE, which converts it to a consensus CRE, does not enhance promoter activity in response to FSK and/or TPA, but rather reduces promoter activity to the same extent as the other inactivating mutation in the variant CRE, suggesting that this element does not act as a classical CRE. Consistent with this, electrophoretic mobility shift assays performed using antibodies to a variety of cAMP and phorbol ester-responsive transcription factors indicate that the AP-1 family proteins jun-B and fos-B are present in the protein complex binding to the variant CRE. Overexpression of jun-B and fos-B in GRMO2 cells resulted in a robust activation of the betaA-subunit promoter. Our results suggest that this novel variant CRE sequence mediates both cAMP and phorbol ester regulation through its interactions with AP-1family proteins.

Gonadotropins regulate inducible cyclic adenosine 3',5'-monophosphate early repressor in the rat ovary: implications for inhibin alpha subunit gene expression.

Many hormones that stimulate intracellular signaling pathways utilizing the second messenger cAMP affect gene expression in target cells through the activation of cAMP-responsive transcriptional regulatory proteins. Two of the best characterized of these are the cAMP-response element (CRE)-binding protein (CREB) and the CRE-modulatory protein (CREM). CREB and CREM are expressed as a family of proteins that have diverse activities in either stimulating or repressing gene transcription. In this study we examined the expression and regulation of the CREM gene in the rat ovary and in granulosa cells, to determine whether repressor isoforms of CREM might have a role in the LH-mediated suppression of inhibin alpha-subunit gene expression that occurs just before ovulation. We found that the predominant CREM mRNAs in the ovary correspond to previously described internal transcripts of the CREM gene that encode the inducible cAMP early repressor (ICER). ICER mRNAs are strongly induced in the ovary by exogenous gonadotropins in immature rats and are transiently expressed in the ovary immediately after the preovulatory LH surge in adult cycling rats. Although ICER is expressed in multiple ovarian cell types, expression in granulosa cells is observed only in response to LH stimulation. ICER mRNAs are also induced by the activation of cAMP-signaling pathways in cultured primary granulosa cells. To determine whether ICER can act as a functional repressor to modulate potential target genes such as the inhibin alpha-subunit gene, an ICER expression construct was transiently co-transfected into a granulosa cell line along with an inhibin alpha-subunit promoter-luciferase reporter gene. Both basal and cAMP-induced expression of the inhibin alpha-subunit promoter were suppressed by ICER. These studies reveal that CREM, a tissue-specific factor, is expressed and regulated by gonadotropins in the ovary, that the predominant CREM transcripts encode the repressor protein ICER, and that ICER is capable of inhibiting cAMP-induced expression of the inhibin alpha-subunit gene. Our findings are consistent with a role for repressors such as ICER in mediating the suppression of inhibin alpha-subunit gene expression that occurs in the ovary at the time of the preovulatory LH surge.

Identification of binding domains of the growth hormone-releasing hormone receptor by analysis of mutant and chimeric receptor proteins.

The hypothalamic peptide GH-releasing hormone (GHRH) stimulates the release of GH from the pituitary through binding and activation of the GHRH receptor, which belongs to the family of G protein-coupled receptors. The objective of this study was to identify regions of the receptor critical for interaction with the ligand by expressing and analyzing truncated and chimeric epitope-tagged GHRH receptors. Two truncated receptors, GHRHdeltaN, in which part of the N-terminal domain between the putative signal sequence and the first transmembrane domain was deleted, and GHRHdeltaC, which was truncated downstream of the first intracellular loop, were generated. Both the receptors were deficient in ligand binding, indicating that neither the N-terminal extracellular domain (N terminus) nor the membrane-spanning domains with the associated extracellular loops (C terminus) are alone sufficient for interaction with GHRH. In subsequent studies, chimeric proteins between the receptors for GHRH and vasoactive intestinal peptide (VIP) or secretin were generated, using the predicted start of the first transmembrane domain as the junction for the exchange of the N terminus between receptors. The chimeras having the N terminus of the GHRH receptor and the C terminus of either the VIP or secretin receptor (GNVC and GNSC) did not bind GHRH or activate adenylate cyclase after GHRH treatment. The reciprocal chimeras having the N terminus of either the VIP or secretin receptors and the C terminus of the GHRH receptor (VNGC and SNGC) bound GHRH and stimulated cAMP accumulation after GHRH treatment. These results suggest that although the N-terminal extracellular domain is essential for ligand binding, the transmembrane domains and associated extracellular loop regions of the GHRH receptor provide critical information necessary for specific interaction with GHRH.

Glucocorticoids regulate pituitary growth hormone-releasing hormone receptor messenger ribonucleic acid expression.

Glucocorticoids regulate GH synthesis and secretion by influencing both hypothalamic and pituitary function. With respect to GH-releasing hormone (GHRH), an important GH secretagogue, glucocorticoids are reported not only to suppress hypothalamic GHRH expression but also to augment pituitary responsiveness to GHRH. To investigate further this latter observation, we have determined the effects of this steroid on expression of the GHRH receptor (GHRH-R) gene in the rat pituitary in vivo and in pituitary cells in vitro. Adult male rats were adrenalectomized or sham operated and treated with s.c. implants of cholesterol or corticosterone. Adrenalectomized animals showed substantially reduced pituitary GHRH-R mRNA levels, when compared with untreated sham-operated animals. Conversely, administration of corticosterone increased pituitary GHRH-R mRNA levels in intact, as well as adrenalectomized rats. We also analyzed the effects of the synthetic glucocorticoid, dexamethasone, on GHRH-R mRNA expression in cultured rat anterior pituitary cells. GHRH-R mRNA was significantly increased by dexamethasone, with a maximal response observed in the presence of 100 nM hormone. This dose of dexamethasone substantially elevated GHRH-R mRNA after 6 h, 12 h, and 24 h of treatment. Dexamethasone did not increase GHRH-R mRNA in the presence of the transcriptional inhibitor actinomycin D, indicating that the predominant effect of the hormone is to increase transcription of the GHRH-R gene. These data demonstrate that GHRH-R mRNA levels are directly stimulated by glucocorticoids, both in the presence and absence of hypothalamic influences, providing a probable explanation for the ability of this steroid to alter pituitary responsiveness to GHRH.

Visual sensitivities of nur77 (NGFI-B) and zif268 (NGFI-A) induction in the suprachiasmatic nucleus are dissociated from c-fos induction and behavioral phase-shifting responses.

Mammalian circadian rhythms are regulated by a pacemaker in the suprachiasmatic nucleus of the hypothalamus. Recent work from several laboratories has shown that light induces the IEGs, c-fos and jun-B, in the rodent suprachiasmatic nucleus. In hamsters, there is a strong correlation between circadian entrainment and the induction of c-fos and jun-B in the suprachiasmatic nucleus by light. Previous work has shown that the IEGs, nur77 and zif268, both of which encode transcription factors, are also light-inducible in the rat suprachiasmatic nucleus [Rusak, B., McNaughton, L., Robertson, H.A. and Hunt, S.P., Circadian variation in photic regulation of IEG mRNAs in rat suprachiasmatic nucleus cells, Mol. Brain Res., 14 (1992) 124-130.; Sutin, E.L. and Kilduff, T.S., Circadian and light-induced expression of IEG mRNAs in the rat suprachiasmatic nucleus, Mol. Brain Res., 15 (1992) 281-290.]. To characterize the photic-regulation of these genes in the suprachiasmatic nucleus of golden hamsters, we used in situ hybridization to measure nur77 and zif268 mRNA levels with 33P-labeled complementary RNA probes. 5-min monochromatic light pulses at CT19 induced a dramatic increase in both nur77 and zif268 mRNA levels. Peak mRNA levels occurred 45-60 min after light onset for both nur77 and zif268. In addition, the induction of both nur77 and zif268 mRNA levels was gated by the circadian pacemaker. Light pulses during subjective day (CT3 and CT9), which do not cause behavioral phase-shifts, did not significantly alter mRNA levels of either nur77 or zif268; whereas light pulses during the subjective night (CT14 and CT19), which induce phase-shifts, dramatically increased both nur77 and zif268 mRNA levels. In contrast to c-fos induction, which has a photic threshold indistinguishable from that of the behavioral phase-shifting response, nur77 and zif268 mRNA induction were found to have visual sensitivities greater than the phase-shifting response by 1-2 log units (10-100-fold). Although light and circadian phase regulate nur77 and zif268 expression in the SCN, these results demonstrate that their induction is not rate-limiting for photic entrainment of the hamster circadian system.

Rat ovarian insulin-like growth factor binding protein-4: a hormone-dependent granulosa cell-derived antigonadotropin.

OBJECTIVE: To assess the in vivo regulation of ovarian insulin-like growth factor binding protein-4 (IGFBP-4) mRNA expression by gonadotropins and estrogen. METHODS: Whole ovarian RNA, obtained from two models of follicular development, was extracted and analyzed by Northern blotting. Immature rats were treated with pregnant more serum gonadotropin (PMSG) followed 48 hours later with hCG, or alternatively were hypophysectomized and treated with FSH and/or diethylstilbestrol (DES). Localization of IGFBP-4 expression was assessed in the former study by in situ hybridization. Finally, the ability of human IGFBP-4 to antagonize FSH-stimulated progesterone accumulation was assessed in vitro. RESULTS: The ovarian content of IGFBP-4 transcripts increased threefold (P < .05) at 12 hours after PMSG but was near baseline at 24 and 48 hours. The abundance of IGFBP-4 mRNA increased (P < .05) again at 6 and 24 hours after hCG. The expression of IGFBP-4 was localized to granulosa cells of preantral (untreated) and small antral (12 hours after PMSG) follicles. No IGFBP-4 expression was noted in large (gonadotropin-primed) antral follicles. Hypophysectomy increased (P < .05) the ovarian content of IGFBP-4 mRNA by 1.5-fold, an effect further enhanced (1.8-fold; P < .05) by the provision of FSH and DES. In vitro studies revealed the ability of increasing concentrations (0.01-1 microgram/mL) of recombinant human IGFBP-4 to inhibit the FSH-supported accumulation of progesterone. CONCLUSION: Increased expression after administration of PMSG, hCG, and FSH/DES suggests that IGFBP-4 is a dynamic and hormonally responsive component of the ovarian cycle. The lack of expression in preovulatory follicles and its antigonadotropic actions in vitro imply that the attenuated expression of IGFBP-4 may constitute a requirement for successful follicular maturation.