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The Notch pathway plays diverse and complex roles in cell signaling during development. In the mammalian ovary, Notch is important for the initial formation and growth of follicles, and for regulating the proliferation and differentiation of follicular granulosa cells during the periovulatory period. This study seeks to determine the contribution of female germ cells toward the initial activation and subsequent maintenance of Notch signaling within somatic granulosa cells of the ovary. To address this issue, transgenic Notch reporter (TNR) mice were crossed with Sohlh1-mCherry (S1CF) transgenic mice to visualize Notch-active cells (EGFP) and germ cells (mCherry) simultaneously in the neonatal ovary. To test the involvement of oocytes in activation of Notch signaling in ovarian somatic cells, we ablated germ cells using busulfan, a chemotherapeutic alkylating agent, or investigated KitWv/Wv (viable dominant white-spotting) mice that lack most germ cells. The data reveal that Notch pathway activation in granulosa cells is significantly suppressed when germ cells are reduced. We further demonstrate that disruption of the gene for the Notch ligand Jag1 in oocytes similarly impacts Notch activation and that recombinant JAG1 enhances Notch target gene expression in granulosa cells. These data are consistent with the hypothesis that germ cells provide a ligand, such as Jag1, that is necessary for activation of Notch signaling in the developing ovary.

The follicle is the functional unit of the mammalian ovary and serves as a tightly controlled microenvironment where granulosa cells nurture the growth and maturation of the oocyte. Early events in follicular development are autonomous of the hypothalamic-pituitary-gonadal axis and are instead controlled by local factors that are used in bidirectional communication between oocytes and somatic cells. While gonadotropins will eventually become the principle regulators of follicular growth, maturation, and ovulation, it is becoming appreciated that their actions are achieved through interactions with local intraovarian signals. The use of genetic tools developed in murine models has led to the discovery of vast networks of oocyte-, granulosa cell-, thecal cell-, and stromal cell-derived factors that govern gonadotropin-independent follicular development. Many of these discoveries from rodent models have proved broadly applicable to other mammalian systems. We summarize current knowledge of how local juxtacrine and paracrine signaling pathways control follicular development and interact with gonadotropin signaling to ensure female fertility and reproductive health.

Originally, activins were identified as stimulators of FSH release in reproduction. Other activities, including secondary axis formation in development, have since been revealed. Here, we investigated the influence of activin βA on the body, including the gastro-intestinal (GI) tract. Initially, the activin βA protein was detected in the serum proportional to the amount of pCMV-rAct plasmid injected. The induced level of activin βA in muscle was higher in female than male mice. Subsequent results revealed that stomach and intestine were severely damaged in pCMV-rAct-injected mice. At the cellular level, loss of parietal cells was observed, resulting in increased pH within the stomach. This phenomenon was more severe in male than female mice. Consistent with damage of the stomach and intestine, activin βA often led to necrosis in the tip of the tail or foot, and loss of body weight was observed in pCMV-rAct-injected male but not female mice. Finally, in pCMV-rAct-injected mice, circulating activin βA led to death at supraphysiological doses, and this was dependent on the strain of mice used. Taken together, these results indicate that activin βA has an important role outside of reproduction and development, specifically in digestion. These data also indicate that activin βA must be controlled within a narrow range because of latent lethal activity. In addition, our approach can be used effectively for functional analysis of secreted proteins.

The Notch pathway is a highly conserved juxtacrine signaling mechanism that is important for many cellular processes during development, including differentiation and proliferation. Although Notch is important during ovarian follicle formation and early development, its functions during the gonadotropin-dependent stages of follicle development are largely unexplored. We observed positive regulation of Notch activity and expression of Notch ligands and receptors following activation of the luteinizing hormone-receptor in prepubertal mouse ovary. JAG1, the most abundantly expressed Notch ligand in mouse ovary, revealed a striking shift in localization from oocytes to somatic cells following hormone stimulation. Using primary cultures of granulosa cells, we investigated the functions of Jag1 using small interfering RNA knockdown. The loss of JAG1 led to suppression of granulosa cell differentiation as marked by reduced expression of enzymes and factors involved in steroid biosynthesis, and in steroid secretion. Jag1 knockdown also resulted in enhanced cell proliferation. These phenotypes were replicated, although less robustly, following knockdown of the obligate canonical Notch transcription factor RBPJ. Intracellular signaling analysis revealed increased activation of the mitogenic phosphatidylinositol 3-kinase/protein kinase B and mitogen-activated protein kinase/extracellular signal-regulated kinase pathways following Notch knockdown, with a mitogen-activated protein kinase kinase inhibitor blocking the enhanced proliferation observed in Jag1 knockdown granulosa cells. Activation of YB-1, a known regulator of granulosa cell differentiation genes, was suppressed by Jag1 knockdown. Overall, this study reveals a role of Notch signaling in promoting the differentiation of preovulatory granulosa cells, adding to the diverse functions of Notch in the mammalian ovary.

The Notch pathway is a contact-dependent, or juxtacrine, signaling system that is conserved in metazoan organisms and is important in many developmental processes. Recent investigations have demonstrated that the Notch pathway is active in both the embryonic and postnatal ovary and plays important roles in events including follicle assembly and growth, meiotic maturation, ovarian vasculogenesis and steroid hormone production. In mice, disruption of the Notch pathway results in ovarian pathologies affecting meiotic spindle assembly, follicle histogenesis, granulosa cell proliferation and survival, corpora luteal function and ovarian neovascularization. These aberrations result in abnormal folliculogenesis and reduced fertility. The knowledge of the cellular interactions facilitated by the Notch pathway is an important area for continuing research, and future studies are expected to enhance our understanding of ovarian function and provide critical insights for improving reproductive health. This review focuses on the expression of Notch pathway components in the ovary, and on the multiple functions of Notch signaling in follicle assembly, maturation and development. We focus on the mouse, where genetic investigations are possible, and relate this information to the human ovary.

The transition from follicle to corpus luteum after ovulation is associated with profound morphological and functional changes and is accompanied by corresponding changes in gene expression. The gene encoding the α subunit of the dimeric reproductive hormone inhibin is maximally expressed in the granulosa cells of the preovulatory follicle, is rapidly repressed by the ovulatory LH surge, and is expressed at only very low levels in the corpus luteum. Although previous studies have identified transient repressors of inhibin α gene transcription, little is known about how this repression is maintained in the corpus luteum. This study examines the role of epigenetic changes, including DNA methylation and histone modification, in silencing of inhibin α gene expression. Bisulfite sequencing reveals that methylation of the inhibin α proximal promoter is low in preovulatory and ovulatory follicles but is elevated in the corpus luteum. Increased methylation during luteinization is observed within the cAMP response element in the promoter, and EMSA demonstrate that methylation of this site inhibits cAMP response element binding protein binding in vitro. Chromatin immunoprecipitation reveals that repressive histone marks H3K9 and H3K27 trimethylation are increased on the inhibin α promoter in primary luteal cells, whereas the activation mark H3K4 trimethylation is decreased. The changes in histone modification precede the alterations in DNA methylation, suggesting that they facilitate the recruitment of DNA methyltransferases. We show that the DNA methyltransferase DNMT3a is present in the ovary and in luteal cells when the inhibin α promoter becomes methylated and observe recruitment of DNMT3a to the inhibin promoter during luteinization.

Nuclear receptor transcriptional activity is enhanced by interaction with coactivators. The highly related nuclear receptor 5A (NR5A) subfamily members liver receptor homolog 1 and steroidogenic factor 1 bind to and activate several of the same genes, many of which are important for reproductive function. To better understand transcriptional activation by these nuclear receptors, we sought to identify interacting proteins that might function as coactivators. The LIM domain protein four and a half LIM domain 2 (FHL2) was identified as interacting with the NR5A receptors in a yeast two-hybrid screen of a human ovary cDNA library. FHL2, and the closely related FHL1, are both expressed in the rodent ovary and in granulosa cells. Small interfering RNA-mediated knockdown of FHL1 and FHL2 in primary mouse granulosa cells reduced expression of the NR5A target genes encoding inhibin-α and P450scc. In vitro assays confirmed the interaction between the FHL and NR5A proteins and revealed that a single LIM domain of FHL2 is sufficient for this interaction, whereas determinants in both the ligand binding domain and DNA binding domain of NR5A proteins are important. FHL2 enhances the ability of both liver receptor homolog 1 and steroidogenic factor 1 to activate the inhibin-α subunit gene promoter in granulosa cells and thus functions as a transcriptional coactivator. FHL2 also interacts with cAMP response element-binding protein and substantially augments activation of inhibin gene expression by the combination of NR5A receptors and forskolin, suggesting that FHL2 may facilitate integration of these two signals. Collectively these results identify FHL2 as a novel coactivator of NR5A nuclear receptors in ovarian granulosa cells and suggest its involvement in regulating target genes important for mammalian reproduction.

Activin, a member of the TGF-β superfamily, is an important modulator of FSH synthesis and secretion and is involved in reproductive dysfunctions and cancers. It also regulates ovarian follicle development. To understand the mechanisms and pathways by which activin regulates follicle function, we performed a microarray study and identified 240 activin regulated genes in mouse granulosa cells. The gene most strongly inhibited by activin was Cyp26b1, which encodes a P450 cytochrome enzyme that degrades retinoic acid (RA). Cyp26b1 has been shown to play an important role in male germ cell meiosis, but its expression is largely lost in the ovary around embryonic d 12.5. This study demonstrated that Cyp26b1 mRNA was expressed in granulosa cells of follicles at all postnatal developmental stages. A striking inverse spatial and temporal correlation between Cyp26b1 and activin-βA mRNA expression was observed. Cyp26b1 expression was also elevated in a transgenic mouse model that has decreased activin expression. The Cyp26 inhibitor R115866 stimulated the proliferation of primary cultured mouse granulosa cells, and a similar effect was observed with RA and activin. A pan-RA receptor inhibitor, AGN194310, abolished the stimulatory effect of either RA or activin on granulosa cell proliferation, indicating an involvement of RA receptor-mediated signaling. Overall, this study provides new insights into the mechanisms of activin action in the ovary. We conclude that Cyp26b1 is expressed in the postnatal mouse ovary, regulated by activin, and involved in the control of granulosa cell proliferation.

Notch signaling directs cell fate during embryogenesis by influencing cell proliferation, differentiation, and apoptosis. Notch genes are expressed in the adult mouse ovary, and roles for Notch in regulating folliculogenesis are beginning to emerge from mouse genetic models. We investigated how Notch signaling might influence the formation of primordial follicles. Follicle assembly takes place when germ cell syncytia within the ovary break down and germ cells are encapsulated by pregranulosa cells. In the mouse, this occurs during the first 4–5 d of postnatal life. The expression of Notch family genes in the neonatal mouse ovary was determined through RT-PCR measurements. Jagged1, Notch2, and Hes1 transcripts were the most abundantly expressed ligand, receptor, and target gene, respectively. Jagged1 and Hey2 mRNAs were up-regulated over the period of follicle formation. Localization studies demonstrated that JAGGED1 is expressed in germ cells prior to follicle assembly and in the oocytes of primordial follicles. Pregranulosa cells that surround germ cell nests express HES1. In addition, pregranulosa cells of primordial follicles expressed NOTCH2 and Hey2 mRNA. We used an ex vivo ovary culture system to assess the requirement for Notch signaling during early follicle development. Newborn ovaries cultured in the presence of γ-secretase inhibitors, compounds that attenuate Notch signaling, had a marked reduction in primordial follicles compared with vehicle-treated ovaries, and there was a corresponding increase in germ cells that remained within nests. These data support a functional role for Notch signaling in regulating primordial follicle formation.Gamma secretase inhibitor treatment suppresses germ cell nest breakdown in the neonatal mouse ovary, supporting a role for Notch signaling in promoting primordial follicle formation.

Activins are pleiotropic members of the TGFβ superfamily and were initially characterized based on their abilities to stimulate FSH synthesis and secretion by gonadotrope cells of the anterior pituitary gland. Here, we identified the gene encoding the steroidogenic enzyme, 17β-hydroxysteroid dehydrogenase type I (17β-HSD1; Hsd17b1), as an activin-responsive gene in immortalized gonadotrope cells, LβT2. 17β-HSD1 catalyzes the conversion of estrone to the more active 17β-estradiol, and activin A stimulated an increase in this enzymatic activity in these cells. We demonstrated that activins signaled via the type I receptor, activin receptor-like kinase (ALK4), and the intracellular signaling protein, SMAD2, to regulate Hsd17b1 transcription in immediate-early fashion. Critical cis-elements, including a minimal SMAD-binding element, were mapped to within 100 bp of the start of transcription. Activin/ALK4 signaling also regulated Hsd17b1 transcription in both immortalized and primary cultured murine granulosa cells. The promoter regions mediating basal and activin/ALK4-regulated promoter activity were generally conserved across the different cell types. The data show that activin A rapidly regulates Hsd17b1 transcription in gonadotrope and granulosa cells and may thereby regulate local 17β-estradiol synthesis.

Primordial follicle formation and the subsequent transition of follicles to the primary and secondary stages encompass the early events during folliculogenesis in mammals. These processes establish the ovarian follicle pool and prime follicles for entry into subsequent growth phases during the reproductive cycle. Perturbations during follicle formation can affect the size of the primordial follicle pool significantly, and alterations in follicle transition can cause follicles to arrest at immature stages or result in premature depletion of the follicle reserve. Determining the molecular events that regulate primordial follicle formation and early follicle growth may lead to the development of new fertility treatments. Over the last decade, many of the growth factors and signaling proteins that mediate the early stages of folliculogenesis have been identified using mouse genetic models, in vivo injection studies, and ex vivo organ culture approaches. These studies reveal important roles for the transforming growth factor beta (TGF-beta) superfamily of proteins in the ovary. This article reviews these roles for TGF-beta family proteins and focuses in particular on work from our laboratories on the functions of activin in early folliculogenesis.

Activin, a member of the transforming growth factor-beta superfamily, is an important modulator of follicle-stimulating hormone synthesis and secretion in the pituitary and plays autocrine/paracrine roles in the regulation of ovarian follicle development. From a microarray study on mouse ovarian granulosa cells, we discovered that the estrogen receptor beta (ERbeta) is inducible by activin. We previously demonstrated that estrogen suppresses activin gene expression, suggesting a feedback relationship between these two follicle-regulating hormones. The purpose of this study was to investigate fully activin A regulation of ER expression. Real time reverse transcription-PCR assays on cultured granulosa cells showed that both ERalpha and ERbeta mRNAs were induced by activin A at 4, 12, and 24 h in a dose-responsive manner. Western blots confirmed an increase in their protein levels. Consistent with increased ERalpha and ERbeta expression, activin A stimulated estradiol-induced estrogen response element promoter activity. Activin A stimulation of ER expression was a direct effect at the level of gene transcription, as it was not abolished by cycloheximide but was abolished by actinomycin D, and in transfected granulosa cells activin A stimulated ERalpha promoter activity. To investigate the effect of activin in vivo and, thus, its biological significance, we examined ER expression in inhibin transgenic mice that have decreased activin expression and discovered that these mice had decreased ERalpha and ERbeta expression in the ovary. We also found that ER mRNA levels were decreased in Müllerian inhibiting substance promoter (MIS)-Smad2 dominant negative mice that have impaired activin signaling through Smad2, and small interfering RNAs targeting Smad2 or Smad3 suppressed ERalpha promoter activation, suggesting that Smad2 and Smad3 are involved in regulating ER levels. Therefore, this study reveals an important role for activin in inducing the expression of ERs in the mouse ovary and suggests important interplay between activin and estrogen signaling.

In the ovary, the steroid hormone estrogen and the TGF-beta superfamily member activin are both produced by granulosa cells and they both have intraovarian functions. Emerging evidence has indicated an interaction of these two signaling pathways. Based on the fact that estrogen and activin can impact early follicle formation and development, we hypothesize that estrogen treatment may alter activin signaling in the neonatal ovary. Therefore, this study was designed to examine the effect of neonatal diethylstilbestrol (DES) and estradiol (E(2)) exposure on the mRNA and protein levels of the key factors involved in activin signaling in the mouse ovary. CD-1 mouse pups were given daily injections of DES, E(2), or oil on postnatal d 1-5, and ovaries and sera were collected on d 19. Neonatal DES or E(2) exposure decreased the number of small antral follicles, induced multioocytic follicle formation, and decreased activin beta-subunit mRNA and protein levels. Consistent with local loss of beta-subunit expression, the phosphorylation of Smad 2, a marker of activin-dependent signaling, was decreased in the estrogen-treated ovaries. The decreased beta-subunit expression resulted in a decrease in serum inhibin levels, with a corresponding increase in FSH. Estrogen also suppressed activin subunit gene promoter activities, suggesting a direct transcriptional effect. Overall, this study demonstrates that activin subunits are targets of estrogen action in the early mouse ovary.

The mechanisms and physiology of reproductive function have fascinated scientists throughout time. Recent cellular and molecular level structural studies have provided unprecedented insights into reproductive systems and signaling networks. This ‘cutting edge’ editorial provides a recent example in each of these areas, namely, the anatomical integrity of the follicle, the molecular structure of activin with its binding partners and the molecular regulation of inhibin. These three examples of structure informing function help explain reproductive health and may provide solutions to reproductive disease.

Mammalian females enter puberty with follicular reserves that exceed the number needed for ovulation during a single lifetime. Follicular depletion occurs throughout reproductive life and ends in menopause, or reproductive senescence, when the follicle pool is exhausted. The mechanisms regulating the production of a species-specific initial follicle pool are not well understood. However, the establishment of a follicular reserve is critical to defining the length of reproductive cyclicity. Here we show that activin A (rh-ActA), a known regulator of follicle formation and growth in vitro, increased the number of postnatal mouse primordial follicles by 30% when administered to neonatal animals during the time of germline cyst breakdown and follicle assembly. This expansion in the initial follicle pool was characterized by a significant increase in both germ cell and granulosa cell proliferation. However, the excess follicles formed shortly after birth did not persist into puberty and both adult rh-ActA- and vehicle-treated animals demonstrated normal fertility. A follicle atresia kinetic constant (kA) was modeled for the two groups of animals, and consistent with the empirical data, the kA for rh-ActA-treated was twice that of vehicle-treated animals. Kinetic constants for follicle formation, follicle loss and follicle expansion from birth to postnatal day 19 were also derived for vehicle and rh-ActA treatment conditions. Importantly, introduction of exogenous rh-ActA revealed an intrinsic ovarian quorum sensing mechanism that controls the number of follicles available at puberty. We propose that there is an optimal number of oocytes present at puberty, and when the follicle number is exceeded, it occurs at the expense of oocyte quality. The proposed mechanism provides a means by which the ovary eliminates excess follicles containing oocytes of poor quality prior to puberty, thus maintaining fertility in the face of abnormal hormonal stimuli in the prepubertal period.

The importance of the initial follicle pool in fertility in female adult mammals has recently been debated. Utilizing a mathematical model of the dynamics of follicle progression (primordial to primary to secondary), we examined whether the initial follicle pool is sufficient for adult fertility through reproductive senescence in CD1 mice. Follicles in each stage were counted from postnatal day 6 through 12 months and data were fit to a series of first-order differential equations representing two mechanisms: an initial pool of primordial follicles as the only follicle source (fixed pool model), or an initial primordial follicle pool supplemented by germline stem cells (stem cell model). The fixed pool model fit the experimental data, accurately representing the maximum observed primary follicle number reached by 4–6 months of age. Although no germline stem cells could be identified by SSEA-1 immunostaining, the stem cell model was tested using a range of de novo primordial follicle production rates. The stem cell model failed to describe the observed decreases in follicles over time and did not parallel the accumulation and subsequent reduction in primary follicles during the early fertile lifespan of the mouse. Our results agree with established dogma that the initial endowment of ovarian follicles is not supplemented by an appreciable number of stem cells; rather, it is sufficient to ensure the fertility needs of the adult mouse.

GHRH is a hypothalamic peptide that stimulates the synthesis and secretion of GH from pituitary somatotroph cells. The GHRH receptor is a seven-transmembrane G protein-coupled receptor that localizes to the surface of somatotroph cells and binds GHRH. Alternative splicing of the GHRH receptor primary transcript at the intron/exon boundary 3' of exon 11 results in inclusion of sequence that is normally intronic. In the human, this inclusion has an in-frame premature stop codon, and this variant mRNA encodes a protein truncated just before the sixth transmembrane domain. To identify the effects of the truncated receptor on signaling of the wild-type receptor and the mechanisms by which its effects are produced, the full-length and truncated receptor constructs were epitope tagged and transfected into HeLa T4 cells to examine signaling and expression. Results show that the truncated GHRH receptor cannot signal through the cAMP pathway and acts as a dominant inhibitor of wild-type receptor signaling. The wild-type and truncated GHRH receptor proteins form a complex. Stably transfected cell lines were generated to examine the mechanism of signal inhibition by the truncated receptor. The data show that receptor cell surface expression is not altered when the wild-type and truncated receptors are cotransfected, but that truncated receptor coexpression substantially reduces GHRH binding by the wild-type receptor. The results support an important role for alternative splicing in mediating the effects of G protein-coupled receptors in general, and suggest that the GHRH receptor can form multimers, which may be important to its signaling properties.

Steroidogenic factor 1 (SF1) is a member of the NR5A subfamily of nuclear hormone receptors and is considered a master regulator of reproduction because it regulates a number of genes encoding reproductive hormones and enzymes involved in steroid hormone biosynthesis. Like other NR5A members, SF1 harbors a highly conserved approximately 30-residue segment called the FTZ-F1 box C-terminal to the core DNA binding domain (DBD) common to all nuclear receptors and binds to 9-bp DNA sequences as a monomer. Here we describe the solution structure of the SF1 DBD in complex with an atypical sequence in the proximal promoter region of the inhibin-α gene that encodes a subunit of a reproductive hormone. SF1 forms a specific complex with the DNA through a bipartite motif binding to the major and minor grooves through the core DBD and the N-terminal segment of the FTZ-F1 box, respectively, in a manner previously described for two other monomeric receptors, nerve growth factor-induced-B and estrogen-related receptor 2. However, unlike these receptors, SF1 harbors a helix in the C-terminal segment of the FTZ-F1 box that interacts with both the core DBD and DNA and serves as an important determinant of stability of the complex. We propose that the FTZ-F1 helix along with the core DBD serves as a platform for interactions with coactivators and other DNA-bound factors in the vicinity.

Peptide hormones within the secretin-glucagon family are expressed in endocrine cells of the pancreas and gastrointestinal epithelium and in specialized neurons in the brain, and subserve multiple biological functions, including regulation of growth, nutrient intake, and transit within the gut, and digestion, energy absorption, and energy assimilation. Glucagon, glucagon-like peptide-1, glucagon-like peptide-2, glucose-dependent insulinotropic peptide, growth hormone-releasing hormone and secretin are structurally related peptides that exert their actions through unique members of a structurally related G protein-coupled receptor class 2 family. This review discusses advances in our understanding of how these peptides exert their biological activities, with a focus on the biological actions and structural features of the cognate receptors. The receptors have been named after their parent and only physiologically relevant ligand, in line with the recommendations of the International Union of Pharmacology Committee on Receptor Nomenclature and Drug Classification (NC-IUPHAR).

Ovarian follicles form through a process in which somatic pregranulosa cells encapsulate individual germ cells from germ cell syncytia. Complementary expression of the Notch ligand, Jagged1, in germ cells and the Notch receptor, Notch2, in pregranulosa cells suggests a role for Notch signaling in mediating cellular interactions during follicle assembly. Using a Notch reporter mouse, we demonstrate that Notch signaling is active within somatic cells of the embryonic ovary, and these cells undergo dramatic reorganization during follicle histogenesis. This coincides with a significant increase in the expression of the ligands, Jagged1 and Jagged2; the receptor, Notch2; and the target genes, Hes1 and Hey2. Histological examination of ovaries from mice with conditional deletion of Jagged1 within germ cells (J1 knockout [J1KO]) or Notch2 within granulosa cells (N2 knockout [N2KO]) reveals changes in follicle dynamics, including perturbations in the primordial follicle pool and antral follicle development. J1KO and N2KO ovaries also contain multi-oocytic follicles, which represent a failure to resolve germ cell syncytia, and follicles with enlarged oocytes but lacking somatic cell growth, signifying a potential role of Notch signaling in follicle activation and the coordination of follicle development. We also observed decreased cell proliferation and increased apoptosis in the somatic cells of both conditional knockout lines. As a consequence of these defects, J1KO female mice are subfertile; however, N2KO female mice remain fertile. This study demonstrates important functions for Jagged1 and Notch2 in the resolution of germ cell syncytia and the coordination of somatic and germ cell growth within follicles of the mouse ovary.

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.

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 [169GA (A57T), 338CT (P113L), 363GT (E121D), 409CT (H137Y), 547GA (D183N), 673GA (V225I), 749GA (W250X), 760GA (V254M), 785GA (S262N), 880GA (G294R), 1268GA (C423Y)] were found in 12 patients (22.2%). The 169GA 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.

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 (P0.05 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 ofor =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 P0.05). 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.

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 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 59 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 cAMPmediated 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. (Endocrinology 140: 4152‐ 4165, 1999)

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.

TO assess the in vivo regulation of ovarian insulin-like growth factor binding protein-4 (IGFBP-4) mRNA expression by gonadotropins and estrogen. Whole ovarian RNA, obtained from two models of follicular development, was extracted and analyzed by Northern blotting. Immature rats were treated with pregnant mare serum gonadotropin (PMSC) followed 48 hours later with hCG, or alternatively were hypophyseciomized and treated with FSH and/or diethylstilbestrol (DES). Localization ojTGFBP-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. The ovarian content of IGFBP-4 transcripis increased threefold (P

Aging is associated with a variety of alterations in circadian rhythms, including changes in the response to environmental stimuli. The underlying causes for these age-related changes in the circadian system remain unknown. Recent studies have demonstrated that light induces the expression of Fos and phosphorylation of the cyclic-AMP response element-binding protein in the rodent suprachiasmatic nuclei, the location of a master circadian pacemaker in mammals, suggesting that these transcription factors may mediate the effects of light on the circadian clock. The purpose of this study was to determine the effects of aging upon light-induced phase-shifting of circadian locomotor activity rhythms, Fos protein expression and cyclic-AMP response element-binding protein phosphorylation in the suprachiasmatic nuclei. Young (three to four months) and old (18-22 months) male golden hamsters free-running in constant darkness were exposed to 5-min monochromatic light pulses of different irradiance levels, at circadian time 19, after which either steady-state phase shifts of locomotor activity rhythms were measured, or else immunocytochemistry for Fos or for phospho-cyclic-AMP response element-binding protein was performed. Old hamsters were approximately 20 times less sensitive to the phase-shifting effects of light on the activity rhythm, and the photic irradiance threshold for Fos-like immunoreactivity induction in the suprachiasmatic nuclei was elevated when compared to young animals. Aging was also associated with a deficit in cyclic-AMP response element-binding protein phosphorylation by light. These data indicate that there are dramatic changes in light-activated molecular responses in the suprachiasmatic nuclei of old hamsters, and suggest that these molecular changes may underlie age-related changes in the effects of light on the circadian clock system.

Basic leucine zipper (bZip) transcription factors regulate cellular gene expression in response to a variety of extracellular signals and nutrient cues. Although the bZip domain is widely known to play significant roles in DNA binding and dimerization, recent studies point to an additional role for this motif in the recruitment of the transcriptional apparatus. For example, the cAMP response element binding protein (CREB)-regulated transcriptional coactivator (CRTC) family of transcriptional coactivators has been proposed to promote the expression of calcium and cAMP responsive genes, by binding to the CREB bZip in response to extracellular signals. Here we show that the CREB-binding domain (CBD) of CRTC2 folds into a single isolated 28-residue helix that seems to be critical for its interaction with the CREB bZip. The interaction is of micromolar affinity on palindromic and variant half-site cAMP response elements (CREs). The CBD and CREB assemble on the CRE with 2:2:1 stoichiometry, consistent with the presence of one CRTC binding site on each CREB monomer. Indeed, the CBD helix and the solvent-exposed residues in the dimeric CREB bZip coiled-coil form an extended protein–protein interface. Because mutation of relevant bZip residues in this interface disrupts the CRTC interaction without affecting DNA binding, our results illustrate that distinct DNA binding and transactivation functions are encoded within the structural constraints of a canonical bZip domain.

Despite the potential importance of insulin-like growth factor binding protein-5 (IGFBP-5) to follicular development, the hormonal regulation of tfris antgonadotropic IGFBP has not been investigated. Therefore, it was the objective of this study to eludicate the role of gonadotropins and estrogen in the in vivo regulation of IGFBP-5 mRNA expression. Two models of follicular development in immature rats were used. Specifically, rats were hypophysectomized and treated with FSH and/or diethylstilbestrol (DES). Alternatively, terminal follicular development was induced in intact immature rats by pregnant mare serum gonadotropin (PMSG) and hCG. The IGFBP-5 mRNA in whole ovarian RNA was assayed by Northern blot hybridization. Localization of expression in PMSG mid HCG-stimulated (ovaries was further assessed by in situ hybridization. Expression of IGFBP-5 mRNA was increased in ovaries from hypophysectomized rats. Treatmet with FSH and/or DES did not alter the abundance of this mRNA. Treatment with PMSG induced a transient increase in IGFBP-5 expression that was localized in a subset of α-inhibin-negative follicles. At later times after PMSG, IGFBP-5 expression persisted in the surface epithelium but was not detected in large preovulatory follicles. In vitro studies affirmed the antigonadotropic action of IGFBP-5. In vivo expression oj IGFBP-5 in the rat ovary is moderated by hormonal treatment both in terms of total expression and follicular localization.

Inhibins and activins exhibit a broad spectrum of biologic activities, many of which affect the reproductive axis. Within the ovary and testis, the synthesis of the inhibin and activin alpha and beta subunits is regulated by circulating hormones, such as the pituitary gonadotropins, and by diverse paracrine factors. Considerable progress has been made in establishing patterns of inhibin and activin subunit gene expression in the gonads and in cultured gonadal cells. Analysis of the inhibin and activin subunit genes is now providing insight into the signaling pathways and molecular mechanisms by which inhibin and activin subunit gene expression is modulated in the ovary and testis. Genetic manipulation of the inhibin and activin subunit genes promises additional revelations on the biologic functions of these intriguing hormones.

Inhibin and activin gene expression in the ovary and in cultured granulosa cells is modulated by the pituitary gonadotropins FSH and LH. In granulosa cells, two messenger RNAs (mRNAs) of 4.3 and 3.3 kilobases (kb) encoding the common beta B-chain of inhibin and activin are observed. We demonstrate here that FSH or pharmacological agents that elevate intracellular levels of cAMP induce expression of the 4.3-kb mRNA to a greater extent than that of the 3.3-kb mRNA. To elucidate the mechanism(s) by which the two beta B transcripts are generated, we characterized the rat beta B gene and used probes from various regions of the gene to examine the structures of the beta B mRNAs. RNA blot analysis using probes from the 5'-nontranslated and flanking regions of the beta B gene revealed that the 4.3- and 3.3-kb transcripts differ in the length of their 5'-nontranslated regions. S1 nuclease protection and primer extension assays were used to map two adjacent transcriptional start sites 1051 and 1052 basepairs (bp) up-stream of the most 3'-start site previously reported for the beta B gene. These novel start sites are used to generate the 4.3-kb mRNA, whereas transcription of the 3.3-kb mRNA initiates at down-stream start sites. DNA sequence analysis did not reveal any consensus CCAAT or TATA box elements up-stream of the novel start sites. Multiple potential binding sites for the transcription factors SP1 and activator protein-2 are present throughout a region of the beta B gene from -1461 to 82 bp. Fusion genes were constructed that contain beta B sequences from -1460 to 14 bp, -1460 to -914 bp, and -913 to 14 bp controlling luciferase reporter gene expression. Analysis of beta B promoter activity in transiently transfected primary granulosa cells demonstrated that sequences flanking both the up- and down-stream transcriptional start sites have substantial basal promoter activity; however, forskolin did not affect expression of the beta B/luciferase fusion genes.

We have examined the expression and function of heat shock transcription factor 2 (HSF2) in spermatogenic cells of mouse testis. The results of in situ RNA hybridization analysis, RNA filter hybridization, and reverse transcription-polymerase chain reaction (RT-PCR) analysis indicate that HSF2 mRNA expression in testis is subject to developmental and cell type-dependent, as well as stage-dependent, regulation. Localized expression of HSF2 mRNA in testis first appears between Day 14 and Day 21 of postnatal development. In adult testis, HSF2 mRNA is found at highest levels in spermatocytes and round spermatids. Immunocytochemical staining and gel mobility shift analysis demonstrate that HSF2 protein is localized to the nuclei of spermatocytes and round spermatids and that this transcription factor exists in testis in a constitutively active DNA-binding state. We further demonstrate that the constitutive HSF2 DNA-binding activity present in testis is able to interact with promoter sequences of the hsp70.2 gene, a testis-specific member of the hsp70 gene family. Taken together, our results show that the expression and functional properties of HSF2 are regulated in spermatogenic cell types of the mouse testis, supporting a role for this transcription factor as a regulator of hsp gene expression during spermatogenesis.

The molecular mechanisms specifying patterns of gene expression in the vertebrate brain, which in turn determine the developmental fates of specific neurons, are yet to be clearly defined. Individual members of a recently identified family of transcriptional regulatory proteins, the GATA factors, are required for the differentiation of certain hematopoietic cell lineages. We show here that two of the members of this gene family, GATA-2 and GATA-3, are expressed within discrete cell populations of the chicken optic tectum during embryogenesis, and that they have highly restricted patterns of expression in the developing chicken brain. Furthermore, the induction of GATA factor expression within specific cell layers parallels the well established spatial (rostral to caudal) and temporal pattern of optic tectum development. The observation that both the timing of appearance and the localization of expression of GATA-2 and GATA-3 are correlated with optic tectum development suggest that these transcription factors may be associated with the initiation of gene transcription required for the determination of specific neuronal fates within visual areas of the vertebrate brain.

We have examined the effects of the pituitary gonadotropins on expression of the progesterone receptor (PR) gene in rat ovary and in granulosa cell cultures. Both gonadotropins (LH/hCG and FSH) rapidly induce PR mRNA expression in the granulosa cells of preovulatory follicles in vivo. Gonadotropins also effectively induce PR mRNA expression in primary cultures of rat granulosa cells, and this action can be mimicked by agents that elevate intracellular cAMP (forskolin or 8-bromo-cAMP). Estrogen does not induce PR mRNA expression in these cells. The cAMP-induced PR mRNA expression in rat granulosa cells is blocked by an inhibitor of transcription, but not by an inhibitor of protein synthesis. Structural characterization of the rat PR gene 5'-flanking region indicates that the proximal promoter does not contain a consensus cAMP response element. However, when luciferase fusion genes containing a 1375-basepair rat PR gene promoter were transiently transfected into rat granulosa cells, luciferase activity could be stimulated several-fold by hCG or forskolin, but not by estrogen. These results indicate that gonadotropins, most likely acting through a pathway mediated by cAMP, are able to stimulate transcription of the PR gene in rat granulosa cells and suggest a mechanism for regulating the intraovarian actions of progesterone.

The growth hormone-releasing hormone receptor (GHRHR) is a member of the family of G protein-coupled receptors that is expressed on pituitary somatotrope cells and mediates the actions of GHRH in stimulating growth hormone (GH) synthesis and secretion. We report that the Ghrhr gene is located in the middle of mouse chromosome 6 in the same region as the little mutation. Mice homozygous for this mutation have reduced GH secretion and a dwarf phenotype. A missense mutation was identified in the extracellular domain of the little GHRHR that disrupts receptor function, suggesting that the growth deficit in these mice results from a defect in the GHRHR. Similar alterations in GHRHR might explain some isolated GH deficiencies in humans.

The human placenta produces several hypophysiotropic factors that participate in the control of local hormone secretion. In particular, previous reports showed that inhibin and activin modulate both GnRH release and hormonal effect in cultured human placental cells. The present study evaluated the possible correlations among the synthesis and cellular distribution of inhibin, activin, and GnRH in placental villi at term. mRNA coding for inhibin alpha- and beta A-subunits and GnRH were localized in human placenta at term by in situ hybridization using the corresponding cDNA probes. Autoradiograms revealed that some placental cells express inhibin alpha- and beta A-subunit and GnRH mRNAs. A common localization of the three hormonal mRNAs has been found in the inner part of the villi. Using affinity-purified polyclonal antisera, immunocytochemical studies confirmed that in placental villi at term, immunoreactive inhibin alpha- and beta A-subunits and GnRH had a distribution that was superimposable in several areas. Both the outer layer and the inner trophoblasts contained immunoreactive hormonal products. The present data show that some placental cells at term can produce and release inhibin, activin, and/or GnRH. This complement of hypophysiotropic factors may, thus, represent a local paracrine/autocrine control mechanism within the placenta.

The suprachiasmatic nuclei (SCN) of the hypothalamus comprise the primary pacemaker responsible for generation of circadian rhythms in mammals. Light stimuli that synchronize this circadian clock induce expression of the c-fos gene in rodent SCN, which suggests a possible role for Fos in circadian entrainment. Appropriate light stimuli also induce the expression of jun-B messenger RNA in the SCN of golden hamsters but only slightly elevate c-jun messenger RNA levels. In addition, light increases the amount of a protein complex in the SCN that binds specifically to sites on DNA known to mediate regulation by the AP-1 transcription factor. The photic regulation of both jun-B messenger RNA expression and AP-1 binding activity is dependent on circadian phase: only light stimuli that shift behavioral rhythms induce jun-B and AP-1 expression. Thus, light and the circadian pacemaker interact to regulate a specific set of immediate-early genes in the SCN that may participate in entrainment of the circadian clock.

Complementary DNAs encoding the mouse GLUT3/brain facilitative glucose transporter have been isolated and sequenced. The predicted amino acid sequence indicates that mouse GLUT3 is composed of 493 amino acids and has 83 and 89% identity and similarity, respectively, to the sequence of human GLUT3. In contrast to human GLUT3 mRNA, which can be readily detected by RNA blotting in all human tissues that have been examined, mouse GLUT3 mRNA was only present at significant levels in brain. In situ hybridization showed differential expression of GLUT3 mRNA in several regions of adult mouse brain. Specific expression was observed in the hippocampus, with GLUT3 mRNA levels being higher in areas CA1 to CA3 than in the dentate gyrus. It was also detected in the Purkinje cell layer of the cerebellum and in the cerebral cortex, with higher expression in the piriform cortex than in other regions of the cortex. Antisera to mouse GLUT3 immunoblotted a series of proteins of 45-50 kDa in mouse brain plasma membranes. These results are consistent with GLUT3 being a neuronal glucose transporter.

The murine primordial follicle pool develops largely within 3 days after birth through germline nest breakdown and enclosure of oocytes within pregranulosa cells. The mechanisms that trigger primordial follicle formation likely are influenced by a transition from the maternal to fetal hormonal milieu at the time of birth. High levels of maternal estrogen maintain intact germline nest in fetal ovary, and decrease of estrogen after birth is permissive of follicle formation. In the present study, we measured an increase in neonatal serum follicle-stimulating hormone (FSH), which corresponded to falling estradiol (E(2)) levels during the critical window of primordial follicle formation (Postnatal Days 1-3). To determine whether fetal hormones contribute in an active manner to primordial follicle formation, mouse fetal ovaries (17.5 days postcoitus) were cultured in vitro at two concentrations of E(2) (meant to reflect maternal and fetal levels of E(2)) and FSH for 6 days. High levels of E(2) (10(-6) M) inhibited germline nest breakdown, and this effect was significantly reduced when fetal ovaries were cultured in the low E(2) concentration (10(-10) M). FSH facilitated germline nest breakdown and primordial follicle formation under both high and low E(2) culture conditions. Low E(2) was identified as being more permissive for the effects of FSH on primordial follicle formation by stimulating the up-regulation of Fshr and activin beta A subunit (Inhba) expression, pregranulosa cell proliferation, and oocyte survival. The decrease of E(2) plus the presence of FSH after birth are critical for primordial follicle formation and the expression of oocyte-specific transcription factors (Figla and Nobox) in that inappropriate exposure to FSH or E(2) during follicle formation resulted in premature or delayed primordial folliculogenesis. In conclusion, with the drop of E(2) level after birth, FSH promotes primordial follicle formation in mice by stimulating local activin signaling pathways and the expression of oocyte-specific transcription factors.

Use of reporter genes provides a convenient way to study the activity and regulation of promoters and examine the rate and control of gene transcription. Many reporter genes and transfection methods can be efficiently used for this purpose. To investigate gene regulation and signaling pathway interactions during ovarian follicle development, we have examined promoter activities of several key follicle-regulating genes in the mouse ovary. In this chapter, we describe use of luciferase and beta-galactosidase genes as reporters and a cationic liposome mediated cell transfection method for studying regulation of activin subunit- and estrogen receptor alpha (ERalpha)-promoter activities. We have demonstrated that estrogen suppresses activin subunit gene promoter activity while activin increases ERalpha promoter activity and increases functional ER activity, suggesting a reciprocal regulation between activin and estrogen signaling in the ovary. We also discuss more broadly some key considerations in the use of reporter genes and cell-based transfection assays in endocrine research.

The pituitary gonadotropins FSH and LH are key hormones for regulating gametogenesis and steroidogenesis in the ovary and testis. The cell surface receptors that mediate the biological activities of these hormones are thought to be expressed in a cell-specific fashion in the ovary and are regulated as animals progress through the reproductive cycle. Using cloned receptor cDNAs, we have examined the expression and hormonal regulation of the ovarian FSH and LH receptor mRNAs in the rat. A quantitative reverse transcription-polymerase chain reaction amplification scheme was used to measure relative levels of the FSH and LH receptor mRNAs, while in situ hybridization was used to localize FSH and LH receptor transcripts. In immature animals, low levels of FSH receptor mRNA are observed in the granulosa cells of small follicles, while low levels of LH receptor mRNA are found in the thecal cells of these same follicles. After stimulation with PMSG, levels of both mRNAs increase, and the LH receptor mRNA is localized in both the granulosa and thecal cells of large follicles. Further treatment of PMSG-primed animals with hCG results in down-regulation, particularly of the LH receptor mRNA in granulosa cells. In adult animals, LH receptor mRNA levels change dramatically during the estrous cycle, particularly after the preovulatory LH surge. FSH receptor mRNA levels show a similar pattern of change, but the FSH receptor mRNA is of lower abundance and is not as highly regulated as the LH receptor mRNA. FSH receptor mRNA is confined to the granulosa cells of healthy developing follicles, whereas LH receptor mRNA is localized predominantly to thecal cells of small follicles on estrous morning, then appears in the granulosa cells of growing follicles by diestrous morning. LH receptor mRNA is also found in interstitial tissues and corpora lutea throughout much of the estrous cycle. Our results indicate that the gonadotropin receptor genes are regulated in a complex fashion during the recruitment, maturation, and ovulation of the ovarian follicle.

Chimeric genes containing either the mouse transferrin (Trf) enhancer/promoter fused to the structural sequences encoding bovine growth hormone (GH) or the mouse albumin (Alb) enhancer/promoter fused to the gene for human growth hormone-releasing factor (GRF) were microinjected into sheep zygotes. A low percentage of resulting transgenic sheep chronically expressed the respective genes, resulting in elevated plasma concentrations of circulating GH or GRF, respectively. Growth hormone-releasing factor expression induced elevated plasma levels of endogenous GH production. In addition, elevated levels of circulating insulin-like growth factor-I were observed in the bovine GH-expressing Trf transgenic sheep. Growth of these founder transgenic sheep relative to controls were not enhanced. In part, this may be due to the development of the diabetic condition exhibited by both transgenic groups. These results demonstrate that the mouse Trf and Alb enhancer/promoters are active in sheep and suggest that alternate strategies for expressing growth-related genes may be required to modulate growth in sheep.

The ovarian steroid progesterone affects reproductive physiology by regulating the expression of specific genes in target tissues. In an attempt to address the question of whether the ovary itself is a target tissue for progesterone action, we have examined the localization and regulation of progesterone receptor (PR) mRNA in the rat ovary. We used the polymerase chain reaction to clone the steroid-binding domain of the rat PR from uterine cDNA and used this as a probe to isolate a larger cDNA from a rat placental cDNA library. We used RNA filter hybridization, a quantitative reverse transcription-polymerase chain reaction amplification assay, and in situ hybridization to detect PR mRNA in the rat ovary. Expression of the PR gene was initially studied in an immature animal model; 23-day-old rats were treated with either PMSG or PMSG followed by hCG. We found little or no PR mRNA in the ovaries of control or PMSG-treated animals; however, the mRNA was highly expressed in the granulosa cells of large follicles in the ovaries of animals treated with PMSG followed by hCG. Other cell types, including thecal and interstitial cells, did not express detectable levels of PR mRNA. The PR mRNA was induced more than 20-fold in the immature ovary 5 h after hCG administration and was down-regulated to near-basal levels by 12 h after hCG administration. In a subsequent series of experiments, we examined PR gene expression in adult rats during the estrous cycle. The expression of PR mRNA was transient and was tightly coupled to the preovulatory LH surge on proestrous evening. PR mRNA was localized to the granulosa cells of mature ovarian follicles during the estrous cycle. In cycling animals treated with pentobarbital to block the preovulatory LH surge, no induction of PR mRNA on proestrous evening was observed. This transient, hormonally regulated, and cell-specific expression of the PR gene in the rat ovary strongly suggests an important intraovarian function for progesterone during the rat reproductive cycle.

This study investigated the regulation of Wilms' tumor gene (WT1) in the ovary by nerve growth factor and FSH to better understand signals that initiate early follicular growth. Nerve growth factor showed a direct stimulatory effect on endogenous expression of WT1, whereas FSH attenuated basal and nerve growth factor-stimulated WT1 protein expression, which most likely depended on FSH responsiveness according to the follicle growth stage.

The GHRH receptor is expressed in the somatotroph cell of the anterior pituitary, where it functions to mediate GHRH-stimulated GH release. To study pituitary and somatotroph cell-specific expression of this gene, a transgenic mouse model and complementary cell culture experiments were developed. The activity of the 1.6-kb proximal rat GHRH receptor promoter was examined in vivo by generating transgenic mice with the promoter directing expression of a luciferase reporter. The promoter directs tissue-specific expression; luciferase is highly expressed in the pituitary but absent from 14 other tissues. Immunocytochemistry experiments show that transgene expression is targeted to GH-expressing somatotroph cells. The transgene is 5-fold more highly expressed in males than females, and there is an increase in transgene expression leading up to the onset of puberty. The 1.6-kb promoter was further examined in cell culture experiments, which revealed that the promoter is selectively activated in pituitary cells and that promoter-reporter expression in nonpituitary cells can be enhanced by the pituitary-specific transcription factor Pit-1. EMSAs identified 10 short regions that specifically bind Pit-1 with highly variable relative affinities. The highest affinity site was previously identified and is required for Pit-1 activation of the promoter. Four additional sites contribute to Pit-1 regulation of the promoter and are important to achieving full activation of the gene. The results show that the 1.6-kb promoter is sufficient to direct tissue- and cell-specific expression in vivo and is regulated by Pit-1.

The inhibin α-subunit gene is transcriptionally activated by FSH in ovarian granulosa cells during follicular growth. We have investigated the roles of the NR5A family nuclear receptors steroidogenic factor 1 (SF-1) and liver receptor homolog 1 (LRH-1) in transcriptional activation of the inhibin α-subunit gene. Transfection assays using an inhibin α-subunit promoter reporter in GRMO2 granulosa cells show that LRH-1 and SF-1 act similarly to increase promoter activity, and that the activity of both transcription factors is augmented by the coactivators cAMP response element-binding protein-binding protein and steroid receptor coactivator 1. However, chromatin immunoprecipitation experiments illustrate differential dynamic association of LRH-1 and SF-1 with the α-subunit inhibin promoter in both primary cells and the GRMO2 granulosa cell line such that hormonal stimulation of transcription results in an apparent replacement of SF-1 with LRH-1. Transcriptional stimulation of the inhibin α-subunit gene is dependent on MAPK kinase activity, as is the dynamic association/disassociation of SF-1 and LRH-1 with the promoter. Inhibition of the phosphatidylinositol 3-kinase signaling pathway influences promoter occupancy and transcriptional activation by SF-1 but not LRH-1, suggesting a possible mechanistic basis for the distinct functions of these NR5A proteins in inhibin α-subunit gene regulation.

This chapter discusses signaling pathways through which follicle-stimulating hormone (FSH) promotes follicle maturation and luteinizing hormone (LH) promotes ovulation and corpus luteum formation. The gonadotropic hormones FSH and LH initiate signaling events in ovarian target cells that govern the reproductive cycle and thus the continuation of a species. The ovarian follicle is the key structure that houses the oocyte, and upon appropriate stimulation by FSH and LH, follicles produce hormones that promote the development of secondary sex characteristics and that regulate the hypothalamic–pituitary axis as well as uterine receptivity. Additionally, the follicle must extrude the oocyte at an appropriate time to allow for fertilization, and remaining cells within the follicle must differentiate into cells of the corpus luteum that produce hormones necessary to sustain pregnancy. Although there is a great deal of species variability, LH is uniformly required to initiate progesterone production by the corpus luteum, which is necessary for implantation and the maintenance of pregnancy. In the absence of FSH, follicles do not develop beyond the preantral stage, and animals are infertile. Similarly, in the absence of LH, ovulation does not ensue, corpora lutea do not form, and animals are infertile. Both gonadotropic hormones initiate their activities by binding to surface protein receptors.

The rodent ovary is regulated throughout the reproductive cycle to maintain normal cyclicity. Ovarian follicular development is controlled by changes in gene expression in response to the gonadotropins FSH and LH. The inhibin alpha-subunit gene belongs to a group of genes that is positively regulated by FSH and negatively regulated by LH. Previous studies established an important role for inducible cAMP early repressor (ICER) in repression of alpha-inhibin. These current studies investigate the mechanisms of repression by ICER. It is not clear whether all four ICER isoforms expressed in the ovary can act as repressors of the inhibin alpha-subunit gene. EMSAs demonstrate binding of all isoforms to the inhibin alpha-subunit CRE (cAMP response element), and transfection studies demonstrate that all isoforms can repress the inhibin alpha-subunit gene. Repression by ICER is dependent on its binding to DNA as demonstrated by mutations to ICER's DNA-binding domain. These mutational studies also demonstrate that repression by ICER is not dependent on heterodimerization with CREB (CRE-binding protein). Competitive EMSAs show that ICER effectively competes with CREB for binding to the inhibin alpha CRE in vitro. Chromatin immunoprecipitation assays demonstrate a replacement of CREB dimers bound to the inhibin alpha CRE by ICER dimers in ovarian granulosa cells in response to LH signaling. Thus, there is a temporal association of transcription factors bound to the inhibin alpha-CRE controlling inhibin alpha-subunit gene expression.