FSH Receptor mRNA Research Articles

Nerve growth factor induces the expression of functional FSH receptors in newly formed follicles of the rat ovary.

The neurotrophin nerve growth factor (NGF) and its two membrane-anchored receptors are expressed in the developing ovary before the organization of the first primordial follicles. In the absence of NGF, the growth of primordial follicles is retarded, indicating that NGF contributes to facilitating early follicular development. The present experiments were undertaken to determine whether NGF can also be involved in the differentiation process by which ovarian follicles become responsive to gonadotropins. Treatment of 2-d-old rat ovaries in organ culture with NGF increased FSH receptor (FSHR) mRNA within 8 h of exposure. This effect was cAMP-independent but additive to the cAMP-mediated increase in FSHR gene expression induced by either forskolin or vasoactive intestinal peptide, a neurotransmitter previously shown to induce FSHR formation in neonatal rat ovaries. After NGF treatment, the ovary acquired the capacity of responding to FSH with cAMP formation and preantral follicular growth, indicating that exposure to the neurotrophin resulted in the formation of biologically active FSHRs. Quantitative measurement of FSHR mRNA demonstrated that the content of FSHR mRNA is reduced in the ovaries of mice carrying a null mutation of the NGF gene. These results indicate that one of the functions of NGF in the developing ovary is to facilitate the differentiation process by which early growing follicles become gonadotropin-dependent during postnatal life, and that it does so by increasing the synthesis of FSHRs.

Functional characterization of the human FSH receptor with an inactivating Ala189Val mutation.

An Ala189Val mutation of the human FSH receptor (FSHR) has been found to cause hypergonadotrophic ovarian failure with arrest of follicular maturation in women, and suppressed spermatogenesis in men. We have now characterized the molecular mechanisms of the receptor inactivation. Wild-type and mutant FSHR cDNAs were expressed in monkey kidney (COS-7) cells and murine granulosa tumour (KK-1) cells. Similar steady-state levels of FSHR mRNA were found in COS-7 and KK-1 cells transfected with both types of FSHR cDNA. Conspicuously, immunofluorescence and confocal microscopy studies revealed that whereas the wild-type receptor could be readily detected on the plasma membrane, most of the mutated protein was intracellularly sequestered. Ligand binding studies confirmed the greatly reduced cell surface expression of the mutant FSHR. A low level of mutated receptors were expressed at the cell surface, as shown by ligand binding and cAMP response. The capacity of these receptors to evoke another second messenger response, that of inositol trisphosphate (IP3), was almost totally lost. This finding may be related to the clinical picture of the patients, i.e. blockade of follicular maturation. There is a highly conserved stretch of five amino acids (Ala-Phe-Asn-Gly-Thr) in the region of the mutation in all glycoprotein hormone receptors. We therefore created the same Ala to Val transition in the human LHR and studied its functional consequences. Similar functional alterations, i.e. intracellular sequestration and attenuated signal transduction, were found, as with mutated FSHR. Hence, this particular mutation in the conserved extracellular region of glycoprotein hormone receptors induces a conformational change that suppresses cell membrane targeting of the mutated receptor, probably through altered intracellular folding.

Androgen receptor and follicle-stimulating hormone receptor in the pig ovary during the follicular phase of the estrous cycle.

Follicle-stimulating hormone (FSH) is an important regulator of follicular development. Some effects of FSH on ovarian follicles might be enhanced by androgens. The main objectives of the present study were to examine expression of the androgen receptor (AR) and FSH receptor (FSHR) in late developing follicles in pigs. Ovaries were collected from gilts on days 13, 15, 17, and 19 of the estrous cycle (day 0 = first day of estrus, n = 4 gilts/day), a period coincident with the follicular phase. One ovary was processed for immunohistochemistry (IHC) of AR. Samples of surface wall from the largest follicles (4-5 per gilt) were dissected from the other ovary, pooled and processed for determination of AR and FSHR mRNAs using reverse transcription-polymerase chain reaction (RT-PCR). Intense AR immunostaining was present in nuclei of granulosa cells of preantral and antral follicles. AR immunoreactivity was also present in the nuclei of oocytes. Weak staining for AR was observed in cells of the theca interna, ovarian surface epithelium, and in most cells of the ovarian stroma. Relative amounts of immunoreactive AR in granulosa cells of late developing follicles, or small antral follicles (< 2 mm), did not differ between days 13, 15, 17, and 19. However, amounts of AR in granulosa cells of small antral follicles was greater (P < 0.05) than in the largest follicles present in the same ovary. The relative amounts of AR mRNA in tissue from the largest follicles on days 13, 15, 17, and 19 did not differ; however, amounts of FSHR mRNA in the same follicles were not different between days 13, 15, and 17, but decreased (P < 0.05) by day 19. Results indicate that during the follicular phase in gilts, the AR protein is mainly present in granulosa cells. Relative amounts of AR protein in granulosa cells and mRNA in walls of late developing follicles did not significantly change from day 13 to 19; however, amounts of FSHR mRNA decreased in preovulatory follicles by day 19 of the estrous cycle.

Role of FSH, numbers of FSH receptors and testosterone in the regulation of inhibin secretion during the seasonal testicular cycle of adult rams

The regulation of inhibin secretion has not been elucidated fully in male ruminants. The aim of this study was to determine the relative importance of FSH and testosterone concentrations, and FSH receptors, in the control of secretion of immunoactive inhibin in rams. In Expt 1, temporal changes in hormone concentrations and testicular FSH binding were determined for two groups of rams (n = 4) kept under opposite, alternating 4 month periods of long (16 h light:8 h dark) and short (8 h light:16 h dark) days. Testicular biopsies (1-2 g) were collected when the testes were regressed, redeveloping, redeveloped and regressing. In Expt 2, separate groups of rams (n = 4) kept under natural photoperiod (latitude 45 degrees 48 minutes N) were designated as controls or passively immunized (for 3 weeks) with sufficient oestradiol antiserum to increase testosterone secretion without altering LH and FSH; this was done when the testes were regressed (non-breeding season) and redeveloped (breeding season). In both groups of rams (Expt 1), 'seasonal' increases in FSH concentrations began a few weeks earlier than did increases in inhibin concentrations. FSH reached maximum concentrations during testicular recrudescence, whereas numbers of FSH receptors in the testis and circulatory inhibin concentrations did not reach peak values until the testes were fully developed. Numbers of FSH receptors per testis, but not FSH concentration, were positively correlated (r = 0.65) with inhibin concentrations across the four stages of the testicular cycle. Near the end of testicular recrudescence early in the breeding season (Expt 2), relatively high FSH concentration was associated with increased abundance of FSH receptor mRNA (90%) and number of receptors (45%) in the testis and increased inhibin concentrations (50%), compared with when the testes were regressed. Moderate, physiological increases in testosterone secretion in immunized rams did not affect inhibin in either season. These results indicate that: (i) FSH stimulation of immunoactive inhibin secretion by Sertoli cells as testes recrudesce is via increases in secretion (early) and cognate receptors (late); (ii) FSH upregulates the synthesis of its own receptor late in recrudescence; and (iii) the positive correlation (r = 0.70) observed between circulatory testosterone and immunoactive inhibin does not reflect a causal relationship.

Role of FSH, numbers of FSH receptors and testosterone in the regulation of inhibin secretion during the seasonal testicular cycle of adult rams

The regulation of inhibin secretion has not been elucidated fully in male ruminants. The aim of this study was to determine the relative importance of FSH and testosterone concentrations, and FSH receptors, in the control of secretion of immunoactive inhibin in rams. In Expt 1, temporal changes in hormone concentrations and testicular FSH binding were determined for two groups of rams (n = 4) kept under opposite, alternating 4 month periods of long (16 h light:8 h dark) and short (8 h light:16 h dark) days. Testicular biopsies (1-2 g) were collected when the testes were regressed, redeveloping, redeveloped and regressing. In Expt 2, separate groups of rams (n = 4) kept under natural photoperiod (latitude 45 degrees 48 minutes N) were designated as controls or passively immunized (for 3 weeks) with sufficient oestradiol antiserum to increase testosterone secretion without altering LH and FSH; this was done when the testes were regressed (non-breeding season) and redeveloped (breeding season). In both groups of rams (Expt 1), 'seasonal' increases in FSH concentrations began a few weeks earlier than did increases in inhibin concentrations. FSH reached maximum concentrations during testicular recrudescence, whereas numbers of FSH receptors in the testis and circulatory inhibin concentrations did not reach peak values until the testes were fully developed. Numbers of FSH receptors per testis, but not FSH concentration, were positively correlated (r = 0.65) with inhibin concentrations across the four stages of the testicular cycle. Near the end of testicular recrudescence early in the breeding season (Expt 2), relatively high FSH concentration was associated with increased abundance of FSH receptor mRNA (90%) and number of receptors (45%) in the testis and increased inhibin concentrations (50%), compared with when the testes were regressed. Moderate, physiological increases in testosterone secretion in immunized rams did not affect inhibin in either season. These results indicate that: (i) FSH stimulation of immunoactive inhibin secretion by Sertoli cells as testes recrudesce is via increases in secretion (early) and cognate receptors (late); (ii) FSH upregulates the synthesis of its own receptor late in recrudescence; and (iii) the positive correlation (r = 0.70) observed between circulatory testosterone and immunoactive inhibin does not reflect a causal relationship.

Relative expression of insulin like growth factor I (IGFI) and follicle stimulating hormone receptor (FSHR) in follicles and ovarian tissue from Bos primigenius indicus (Nelore)

The improvement of techniques to maximize reproductive potential of females needs the whole comprehension of the controlling mechanisms of follicular development. An alternative for this purpose is the quantification of relative gene expression from those genes involved in recruitment, selection and follicular development, using reverse transcriptase - polymerase chain reaction (RT - PCR). The present study aimed to quantify relative gene expression from insulin-like growth factor I (IGF-I) and follicle stimulating hormone receptor (FSHR) in cattle (Bos primigenius indicus), using as internal control the gliceraldheyde 3 phosphate dehydrogenase (GAPDH) gene. Ovaries in different estral cycle stages were obtained from slaughtered animals. Total RNA from follicles and ovarian tissue was purified and the RT-PCR conditions were standardized. PCR products were analyzed in ethydium bromide agarose gels and the bands submitted to densitometric analysis. Exponential amplification curves were constructed and the method's validation was performed using regression analysis to determine the amplification coefficient (E) for each of the three genes. Relative expression for each gene was calculated using the formula described by Prelle et al.12. In every sample there was gene expression detected for each gene showing differences related to cycle temperature. Amplification coefficient (E) was similar between control gene (GAPDH) and IGFI, independently of the class analyzed. IGFI linear amplification could be related to the constitutive characteristic of this protein since the transcripts are not dependents of FSH levels. It was observed difference in FSHR mRNA expression between the classes analyzed. It could be due to the variation of the receptor number in granulosa cells for each different phase of estral cycle. Semi-quantitative RT-PCR has a large application in biotechnology since it is useful to help us the better understanding of follicular dynamics. However these studies must be conducted using other genes in order to provide new clues for the physiology of folliculogenesis.

Follistatin Inhibits the Function of the Oocyte-Derived Factor BMP-15

Recent studies have highlighted the importance of a novel oocyte-derived growth factor, bone morphogenetic protein-15 (BMP-15) in the regulation of proliferation and differentiation of granulosa cells in the ovary. Namely, BMP-15 stimulates granulosa cell mitosis and inhibits follicle-stimulating hormone (FSH) receptor mRNA expression in granulosa cell, thereby playing a critical role in the elaborate mechanism controlling ovarian folliculogenesis. At present, however, nothing is known about molecules which may regulate the biological activity of BMP-15. Here we demonstrate evidence that follistatin can form an inactive complex with BMP-15, through which follistatin inhibits BMP-15 bioactivities. The binding of follistatin to BMP-15 was directly demonstrated by a surface plasmon resonance biosensor, and the ability of follistatin to inhibit BMP-15 functions was determined by established BMP-15 bioassays using primary rat granulosa cells. Specifically, follistatin attenuated BMP-15 stimulation of granulosa cell proliferation and reversed BMP-15 inhibition of FSH receptor mRNA expression leading to the suppression of FSH-induced progesterone synthesis. This is the first demonstration of the biochemical interaction and biological antagonism of follistatin and BMP-15, which may be involved in the complex yet well-controlled mechanism of the regulation of follicle growth and development.

Intra-follicular activin availability is altered in prenatally-androgenized lambs

Prenatal exposure of sheep to testosterone (T) disrupts ovarian cyclicity and leads to anovulation in adulthood. We propose that the disruption of ovarian function in prenatally-androgenized sheep is mediated via follicular defects stemming from reduced intrafollicular activin availability/action. The intra-follicular activin availability/action that facilitates follicular development is dictated by the relative proportions of activins, inhibins (antagonists of activin action) and follistatins (FS; binding proteins of activin and negator of activin action). Inhibin α, βA, βB, and FS mRNA expression were determined by in situ hybridization in 5 week-old ovaries from control (C) lambs or those exposed to testosterone (T) or DHT from 30–90 days of gestation. In utero exposure to T, but not DHT, increased total ovarian weight (0.4±0.1, 1.5±0.5 and 0.3±0.1 g, C, T and DHT, respectively) and total number of follicles (16.5±2.8, 37.8±7.9, and 18.8±3.0). With the exception of two follicles in T animals, all follicles were ≤2 mm in diameter. All follicles ≤2 mm in all groups expressed FSH receptor mRNA in the granulosa cells and LH receptor only in the thecal cells. The percentage of follicles expressing FS mRNA was increased ( P<0.05) in sheep prenatally-androgenized with either T (80.4±8) or DHT (80.3±5.5) as compared to C (50.8±8.2). In contrast, the percentage of follicles expressing activin βB mRNA tended to be lower ( P=0.06) in the T (30.9±7.1) and DHT (40.5±3.3) groups as compared to C (66.1±15.6). Increased expression of FS along with the reduced expression of activin βB mRNA provides evidence for compromised intra-follicular activin availability in the majority of follicles in the androgenized groups. The increase in ovarian weight and follicular number in the T, but not in the DHT group, suggests that the effects of T are mediated through the action of estrogen. We speculate that the decrease in relative abundance of activin may contribute to the selection defects in prenatally-androgenized sheep. If true, this may be a useful model to understand the etiology of polycystic ovarian syndrome.

Molecular cloning, sequence and expression of follicle-stimulating hormone receptor in the lizard Podarcis sicula

The present paper reports the full nucleotide sequence of a cloned cDNA prepared from RNA of lizard ovaries. The open reading frame consists of 2019 nucleotides, which encodes a protein of 673 amino acids belonging to the G protein-coupled receptor superfamily with a large extracellular N-terminal domain involved in hormone recognition. The transmembrane domain ends with a short intracytoplasmic COOH-terminal domain involved in effector activation. Phylogenetic analysis showed that the lizard receptor belongs to the family of follicle-stimulating hormone (FSH) receptors. The hydrophobicity profile is similar to that observed for mammalian and avian FSH receptors. Northern blot analysis of total RNA revealed that the FSH receptor is expressed at high levels in the ovary. In situ hybridization experiments demonstrate that FSH receptor mRNA is specifically localized within the small cells of the follicular epithelium surrounding the oocyte.

Concentrations of steroids and expression of messenger RNA for steroidogenic enzymes and gonadotropin receptors in bovine ovarian follicles of first and second waves and changes in second wave follicles after pulsatile LH infusion

The objectives were to compare expression of mRNA for cytochrome P450 cholesterol side-chain cleavage (P450scc), cytochrome P450 17α-hydroxylase (P450c17), cytochrome P450 aromatase (P450arom), 3β-hydroxysteroid dehydrogenase Δ 4, Δ 5 isomerase (3β-HSD), FSH receptor (FSHr) and LH receptor (LHr) in bovine ovarian follicles of the first and second waves of the bovine oestrous cycle and to determine if LH infusion changes growth, steroidogenesis and gene expression in second wave follicles. Transrectal ultrasonography was used to examine follicular size changes during the oestrous cycle in non-lactating Holstein cows ( n=31). Saline or purified bovine LH was infused intravenously into cows at emergence of follicular waves for 2 or 4 days using a computer-controlled syringe pump ( n=5–6 per treatment). Treatments were: wave 1, saline (W1S); wave 2, saline (W2S) or LH (25 μg/h; W2LH). During infusion, blood samples were collected at 12 min intervals for 8 h via i.v. catheters for measurement of serum LH concentrations. Ovaries were removed from cows on days 2 or 4 after emergence of follicular waves. Follicles were frozen and stored at −80°C. Follicular fluid (FF, 50 μl) was collected for determination of progesterone (P 4), oestradiol-17β (E 2) and androstenedione (A 4) concentrations. Frozen sections (14 μm) were used for in situ hybridization to measure expression of mRNA (% pixel intensity) for P450scc, P450c17, P450arom, 3β-HSD, FSHr, and LHr. LH infusion resulted in a serum LH pattern (high frequency) similar to the early luteal phase. There were no significant differences in size of follicles among the three treatment groups. Follicular fluid concentrations of E 2 and A 4 in W2S were lower than those of W1S on day 2 of a follicular wave. LH infusion into cows during the midluteal phase increased follicular fluid E 2 and A 4 concentrations in second wave follicles on day 2 of a follicular wave (W2LH) compared to those of W2S. The increase in follicular fluid E 2 on day 2 in wave 2 follicles after LH infusion occurred possibly through an increase in mRNA expression of P450c17 and 3β-HSD. In conclusion, follicular fluid concentrations of E 2 and A 4 were lower in W2S than in W1S and E 2 and A 4 concentrations were restored by infusion of LH in W2LH with an increase in mRNA expression of P450c17 and 3β-HSD.

Biological Function and Cellular Mechanism of Bone Morphogenetic Protein-6 in the Ovary

The process of ovarian folliculogenesis is composed of proliferation and differentiation of the constitutive cells in developing follicles. Growth factors emitted by oocytes integrate and promote this process. Growth differentiation factor-9 (GDF-9), bone morphogenetic protein (BMP)-15, and BMP-6 are oocyte-derived members of the transforming growth factor-beta superfamily. In contrast to the recent studies on GDF-9 and BMP-15, nothing is known about the biological function of BMP-6 in the ovary. Here we show that, unlike BMP-15 and GDF-9, BMP-6 lacks mitogenic activity on rat granulosa cells (GCs) and produces a marked decrease in follicle-stimulating hormone (FSH)-induced progesterone (P(4)) but not estradiol (E(2)) production, demonstrating not only the first identification of GCs as BMP-6 targets in the ovary but also its selective modulation of FSH action in steroidogenesis. This BMP-6 activity resembles BMP-15 but differs from GDF-9 activities. BMP-6 also exhibited similar action to BMP-15 by attenuating the steady state mRNA levels of FSH-induced steroidogenic acute regulatory protein (StAR) and P450 side-chain cleavage enzyme (P450scc), without affecting P450 aromatase mRNA level, supporting its differential function on FSH-regulated P(4) and E(2) production. However, unlike BMP-15, BMP-6 inhibited forskolin- but not 8-bromo-cAMP-induced P(4) production and StAR and P450scc mRNA expression. BMP-6 also decreased FSH- and forskolin-stimulated cAMP production, suggesting that the underlying mechanism by which BMP-6 inhibits FSH action most likely involves the down-regulation of adenylate cyclase activity. This is clearly distinct from the mechanism of BMP-15 action, which causes the suppression of basal FSH receptor (FSH-R) expression, without affecting adenylate cyclase activity. As assumed, BMP-6 did not alter basal FSH-R mRNA levels, whereas it inhibited FSH- and forskolin- but not 8-bromo-cAMP-induced FSH-R mRNA accumulation. These studies provide the first insight into the biological function of BMP-6 in the ovary and demonstrate its unique mechanism of regulating FSH action.

Analysis of ovarian gene expression in follicle-stimulating hormone beta knockout mice.

FSH is a heterodimeric glycoprotein hormone that is produced in the gonadotroph cells of the anterior pituitary. It acts on Sertoli cells of the testis and granulosa cells of the ovary. We previously demonstrated that FSHbeta knockout female mice are infertile due to a block in folliculogenesis preceding antral stage development. To investigate aberrations of ovarian gene regulation in the absence of FSH, we analyzed the expression of several important marker genes using Northern blot and in situ hybridization techniques. Key findings are as follows: 1) Follicles of FSHbeta knockout mice develop a well organized thecal layer, which is positive for P450 17alpha-hydroxylase and LH receptor messenger RNAs (mRNAs). This indicates that theca recruitment is completed autonomously with respect to FSH. 2) Granulosa cells in FSH-deficient mice demonstrate an increase in FSH receptor mRNA, and decreases in P450 aromatase, serum/glucocorticoid-induced kinase, and inhibin/activin subunit mRNAs. These data support studies that implicate FSH signaling cascades in the expression of these genes. 3) In contrast to the thecal layer, granulosa cell populations in FSHbeta knockout mice do not accumulate LH receptor mRNA. This suggests that although the granulosa cells have a block in proliferation at the antral follicle stage in the absence of FSH, they do not initiate programs of terminal differentiation as seen in luteinizing cells of wild-type ovaries. 4) Ovaries of FSH-deficient mice demonstrate a modest decrease in cyclin D2 mRNA, without up-regulation of cell cycle inhibitor mRNAs associated with luteinization (i.e. p15, p27, and p21). Although components of the FSH null phenotype may be caused by partial cyclin D2 loss of function, these findings indicate that the mechanisms of granulosa cell cycle arrest in FSHbeta knockout mice are distinct from those of cycle withdrawal at luteinization. Underscoring the usefulness of the FSH-deficient mouse model, this study clarifies aspects of gonadotropin-dependent folliculogenesis, thecal layer development, cycle control in granulosa cells, and luteinization.

Discrepancy Between Molecular Structure and Ligand Selectivity of a Testicular Follicle-Stimulating Hormone Receptor of the African Catfish (Clarias gariepinus)1

A putative FSH receptor (FSH-R) cDNA was cloned from African catfish testis. Alignment of the deduced amino acid sequence with other (putative) glycoprotein hormone receptors and analysis of the African catfish gene indicated that the cloned receptor belonged to the FSH receptor subfamily. Catfish FSH-R (cfFSH-R) mRNA expression was observed in testis and ovary; abundant mRNA expression was also detected in seminal vesicles. The isolated cDNA encoded a functional receptor since its transient expression in human embryonic kidney (HEK-T) 293 cells resulted in ligand-dependent cAMP production. Remarkably, African catfish LH (cfLH; the catfish FSH-like gonadotropin has not been purified yet) had the highest potency in this system. From the other ligands tested, only human recombinant FSH (hrFSH) was active, showing a fourfold lower potency than cfLH, while hCG and human TSH (hTSH) were inactive. Human CG (as well as cfLH, hrFSH, eCG, but not hTSH) stimulated testicular androgen secretion in vitro but seemed to be unable to bind to the cfFSH-R. However, it was known that hCG is biologically active in African catfish (e.g., induction of ovulation). This indicated that an LH receptor is also expressed in African catfish testis. We conclude that we have cloned a cDNA encoding a functional FSH-R from African catfish testis. The cfFSH-R appears to be less discriminatory for its species-specific LH than its avian and mammalian counterparts.

Expression of messenger RNA for gonadotropin receptor in the granulosa layer during the ovulatory cycle of hens

The present experiments were conducted to evaluate the mRNA levels of luteinizing hormone receptor (LHR) and follicle-stimulating hormone receptor (FSHR) in granulosa layers during the ovulatory cycle of hens, in relation to the release of LH and steroid hormones. After the release of LH, progesterone (P4) and estradiol-17β (E2), found 4–5 h before ovulation, LHR and FSHR mRNA levels were observed to decrease in the granulosa layers of the largest (F1) and second largest (F2) preovulatory follicles, with the greatest in the LHR mRNA level of F1. P4 concentrations in the granulosa layers of F1 and F2 increased 4–5 h before ovulation, with greater in F1 than in F2. F2 concentrations in the theca layers were greater in F2 than in F1 throughout the ovulatory cycle. Also, the injection of ovine LH caused decreases in the mRNA levels of LHR and FSHR in the granulosa layers. However, these decreases were abolished by the injection of aminoglutethimide, an inhibitor of steroid synthesis. These results suggest that in hen granulosa cells, the mRNA levels of not only LHR but also FSHR are down-regulated by LH and the down-regulation may be mediated steroid hormones.

Bone Morphogenetic Protein-15 Inhibits Follicle-stimulating Hormone (FSH) Action by Suppressing FSH Receptor Expression

We have recently reported that oocyte-derived bone morphogenetic protein-15 (BMP-15) can directly modulate follicle-stimulating hormone (FSH) action in rat granulosa cells. Here, we investigate underlying mechanisms of this BMP-15 effect. Treatment with BMP-15 alone exerted no significant effect on the basal expression of mRNAs encoding steroidogenic acute regulatory protein, P450 side chain cleavage enzyme, P450 aromatase, 3beta-hydroxysteroid dehydrogenase, luteinization hormone receptor, and inhibin/activin subunits. However, BMP-15 markedly inhibited the FSH-induced increases in these messages. In striking contrast, BMP-15 did not change the forskolin-induced levels of these transcripts. Thus, the inhibitory effect of BMP-15 on FSH action must be upstream of cAMP signaling. We next examined changes in FSH receptor mRNA expression. Interestingly, BMP-15 severely reduced the levels of FSH receptor mRNA in both basal and FSH-stimulated cells. To determine whether this effect was at the level of FSH function, we investigated the effect of BMP-15 on FSH bioactivity. Consistent with the mRNA data, BMP-15 inhibited the biological response of FSH, but not that of forskolin. Based on these results, we propose that BMP-15 is an important determinant of FSH action through its ability to inhibit FSH receptor expression. Because FSH plays an essential role in follicle growth and development, our findings could have new implications for understanding how oocyte growth factors contribute to folliculogenesis.

Stability of human follicle-stimulating hormone receptor mRNA in stably transfected cells.

In order to assess the impact of mRNA degradation on steady state levels of follicle-stimulating hormone receptor (FSHR) mRNA and on regulation of FSHR gene expression, the stability and half-life of FSHR mRNA were determined in transfected cells expressing recombinant FSHR. Time-dependent changes in FSHR mRNA content were determined by nuclease protection-solution hybridization assay (NPA) or by qualitative reverse transcription-competitive polymerase chain reaction (RT-PCR) in cultured hFSHR-YI cells, cell lines stably transfected with a human FSHR cDNA. FSHR mRNA content remained constant during 8 h control incubations of hFSHR-Y1 cells (NPA, 2.9 +/- 0.3 micrograms/mg RNA; RT-PCR, 2.7 +/- 0.3 micrograms/mg RNA). Actinomycin D (ActD, 5 micrograms/ml) inhibited mRNA synthesis, as assessed by incorporation of [3 H]uridine into total RNA, by 90% within 1 h in hFSHR-Y1 cells. No effect of ActD on cellular morphology or viability was observed. ActD caused a time-dependent decrease in FSHR mRNA content in hFSHR-Y1 cell lines with a lag time of 1 h. There were no significant differences in the rate of FSHR mRNA degradation between the two methods of mRNA quantification. The half-life of hFSHR mRNA was 3.6 +/- 0.2 h by NPA and 3.1 +/- 0.1 h by RT-PCR. The results indicated that degradation of mRNA was an important process in maintenance of steady state expression of the FSHR gene in cells stably expressing recombinant receptor.

Expression of mRNA for the LH and FSH receptors in mouse oocytes and preimplantation embryos.

The gonadotrophins LH and FSH are known to regulate gonadal growth, and differentiation, endocrine function and gametogenesis. The LH receptor is expressed in ovarian theca, granulosa and luteal cells, and in testicular Leydig cells. The FSH receptor is expressed only in ovarian granulosa cells and in testicular Sertoli cells. The expression of the FSH and LH receptors was analysed by RT-PCR to study the role of these receptors in early mouse development. After reverse transcription, strategically designed nested primers were used for amplification from cDNA. Transcripts for the receptors were present in mouse oocytes and preimplantation embryos. The presence of mRNA for FSH and LH receptors in oocytes, zygotes and preimplantation embryos indicates a potential role for the gonadotrophins in the modulation of meiotic resumption and completion of oocyte maturation, as well as a beneficial effect on early embryonic development in mice.

Expression of mRNA for the LH and FSH receptors in mouse oocytes and preimplantation embryos

The gonadotrophins LH and FSH are known to regulate gonadal growth, and differentiation, endocrine function and gametogenesis. The LH receptor is expressed in ovarian theca, granulosa and luteal cells, and in testicular Leydig cells. The FSH receptor is expressed only in ovarian granulosa cells and in testicular Sertoli cells. The expression of the FSH and LH receptors was analysed by RT-PCR to study the role of these receptors in early mouse development. After reverse transcription, strategically designed nested primers were used for amplification from cDNA. Transcripts for the receptors were present in mouse oocytes and preimplantation embryos. The presence of mRNA for FSH and LH receptors in oocytes, zygotes and preimplantation embryos indicates a potential role for the gonadotrophins in the modulation of meiotic resumption and completion of oocyte maturation, as well as a beneficial effect on early embryonic development in mice.

Site-specific methylation of the promoter alters deoxyribonucleic acid-protein interactions and prevents follicle-stimulating hormone receptor gene transcription.

In the male gonad, the FSH receptor (FSHR) gene is expressed only in Sertoli cells. To date, the mechanism(s) responsible for Sertoli cell-specific expression of the FSHR gene are unknown. In this study, DNA methylation at specific sites in the promoter are shown to lead to changes in the DNA-protein interactions at those sites and, subsequently, to transcriptional repression of the gene. The extent of methylation of cytosine residues within the core promoter region of genomic DNA isolated from cells/tissues that expressed, or did not express, the FSHR gene was analyzed by the sodium bisulfite conversion technique. All seven cytosine residues in CpG dinucleotides within the core promoter region were found to be unmethylated in primary cultured rat Sertoli cells that were actively expressing FSHR mRNA. In contrast, in tissues not expressing FSHR the same region of the gene was methylated at each of the CpG dinucleotides examined. In addition, DNA-protein interactions in three primary regulatory regions of the promoter were examined by electrophoretic mobility shift assays (EMSA) with synthetic oligonucleotides containing selectively methylated cytosine residues. Methylation of a CpG sequence within a consensus E box element (CACGTG, -124/-119) decreased the binding affinity of USF1/2 transcription factors for this element. Methylation of the CpG sequence in the Inr region (CCGG, -85/-82) allowed the formation of an additional DNA-protein complex. Methylation at both cytosine residues in the E2F element ((m)CG(m)CG) generated a new methylcytosine-specific DNA-protein complex. The core FSHR promoter region of a mouse Sertoli cell line (MSC-1) that does not express FSHR was shown to be methylated at four CpG dinucleotides. The demethylation of these four sites by treatment of the MSC-1 cells with 5-aza-2'-deoxycytidine (5-azaCdR) activated the transcription of the FSHR gene. Taken together, these results suggest that cytosine methylation is a major factor in the repression of the expression of the FSHR gene.

Targeted disruption of luteinizing hormone/human chorionic gonadotropin receptor gene.

LH/hCG receptors were disrupted by gene targeting in embryonic stem cells. The disruption resulted in infertility in both sexes. The gonads contained no receptor mRNA or receptor protein. Serum LH levels were greatly elevated, and FSH levels were moderately elevated in both sexes; estradiol and progesterone levels decreased but were not totally suppressed in females; testosterone levels were dramatically decreased and estradiol levels moderately elevated in males. The external and internal genitalia were grossly underdeveloped in both sexes. Abnormalities included ambiguous vaginal opening, abdominal testes, micropenis, dramatically decreased weights of the gonads and reproductive tract, arrested follicular growth beyond antral stage, disarray of seminiferous tubules, diminished number and hypotrophy of Leydig cells, and spermatogenic arrest beyond the round spermatid stage. LH/hCG receptor gene disruption had no effect on FSH receptor mRNA levels in ovaries and testes, progesterone receptor (PR) levels in ovaries and androgen receptor (AR) levels in testes. However, it caused a dramatic decrease in StAR and estrogen receptor-alpha (ERalpha) mRNA levels and an increase in ERbeta mRNA levels in both ovaries and testes. Estradiol and progesterone replacement therapy in females and testosterone replacement in males, to determine whether phenotype and biochemical changes were a consequence of decreased gonadal steroid levels or due to a loss of LH signaling, revealed complete restoration of some and partial restoration of others. Nevertheless, the animals remained infertile. It is anticipated that the LH receptor knockout animals will increase our current understanding of gonadal and nongonadal actions of LH and hCG.