FSH Receptor mRNA Expression Research Articles

Potential detrimental effect of soy isoflavones on testis sertoli cells

To determine the effect of soy isoflavones on cell proliferation and the transcription levels of follicle-stimulating hormone receptor (FSHR), inhibin α (INHα), INHβB, androgen binding protein (ABP), transferrin (Tf) and vimentin in testis sertoli cells in SD rats. Sertoli cells were cultured in vitro, exposed to daidzein at 0.03, 0.3, 3, and 30 μmol/L and genistein at 0.05, 0.5, 5 and 50 μmol/L, respectively. MTT was used to detect the proliferation of sertoli cells. Real-time PCR was used to detect the relative mRNA expressions of FSHR, INHα, INHβB, ABP, Tf and vimentin. Compared with control groups, cell proliferation and the relative mRNA expression levels of INHβB and ABP in the treated cells showed no significant alternation. The INHα mRNA expression levels were increased in 0.3 and 3 μmol/L Dai and 0.05 μmol/L Gen, while the mRNA expression levels of FSHR were downregulated in 30 μmol/L Dai and Gen at all concentrations. Tf mRNA expression levels were downregulated in 30 μmol/L Dai and 5 μmol/L and 50 μmol/L Gen, and the mRNA expression levels of vimentin were downregulated in 3 and 30 μmol/L Dai and 50 μmol/L Gen. Soy Isoflavones may have potential detrimental effect on the male reproductive system, as they may impact the function of sertoli cells by downregulating the transcription levels of some important proteins.

Effect of adenovirus-mediated up-regulation of α-enolase gene products on follicle-stimulating hormone receptor mRNA and luteinizing hormone receptor mRNA of granular cells from goose F1 follicles

Enolases are glycolytic enzymes in the glycolytic pathway. In order to evaluate the effect of ENO1 on follicle-stimulating hormone receptor (FSHR) mRNA and luteinizing hormone receptor (LHR) mRNA of primary granular cell from goose F1 follicles, the recombinant plasmid adenovirus carrying ENO1 were constructed and infected the primary culture granular cells. The granular cells were randomly divided into three groups: recombinant adenovirus infected (pAd-CMV-ENO1), empty vector infected (pAd-CMV-Null) and no virus (mock control). The expression levels of FSHR mRNA and LHR mRNA of granular cells were examined by qRT-PCR. The results showed the group pAd-CMV-ENO1 had significantly higher FSHR mRNA expression levels than the other two groups (P < 0.05), but had significantly lower LHR mRNA expression levels than the other two groups (P < 0.05). The results suggested that ENO1 could improve the combination rate between FSH and FSHR to accelerate the proliferation and differentiation and steroidogenesis in poultry gonadal tissues.

Morfometría ovárica y expresión del ARN mensajero de hormona antimülleriana (AMH), receptor de FSH (FSHR) y factor nuclear kappa B (NFkB) en folículos en crecimiento de borregas expuestas prenatalmente a testosterona

SUMMARY Antenatal exposure to testosterone (T) has a series of consequences on postnatal reproductive parameters in females of several animal species. In sheep born under this condition the ovaries are characterized by an increased number of growing follicles. If such disruptions are manifested early in life or involve changes in follicular recruitment and ovary paracrine environment, it remains unclear. This study addressed the impact of prenatal T on ovarian morphometry and the mRNA expression of ovarian anti-mullerian hormone (AMH), FSH receptor (FSHR) and nuclear factor kappa B (NFkB, a transcription factor for AMH), factors that are involved in the growth and selection of the dominant follicle. At 4 weeks of age, female lambs born to dams treated with 30 mg of T propionate twice weekly from day 30 to day 90, followed by 40 mg of T propionate from day 90 to day 120 of pregnancy (T females), showed similar number of primordial, primary, secondary and antral follicles than control females (C females) born to dams treated with the vehicle. In the pool of secondary follicles (< 0.5 mm) the expression of the FSHR was slightly higher in the T females, while AMH and NFkB expression were lower than C females. Instead, the pool of small antral follicles (between 0.5 and 1 mm) showed a higher expression of FSHR and a slight increase in AMH in T females, while NFkB expression remained lower than C females. These findings provide evidence that the prenatal exposure to T impacts the expression of key local factors governing the folliculogenesis before histological alterations occur.

Bone Morphogenetic Protein 4 Supports the Initial Differentiation of Hen (Gallus gallus) Granulosa Cells

In the hen ovary, selection of a follicle into the preovulatory hierarchy occurs from a small cohort of prehierarchal (6-8 mm) follicles. Prior to follicle selection the granulosa layer remains in an undifferentiated state despite elevated follicle-stimulating hormone receptor (FSHR) expression. The present studies describe a role for bone morphogenetic protein 4 (BMP4) in supporting FSHR mRNA expression in granulosa cells from prehierarchal follicles and promoting differentiation at follicle selection. Culture of undifferentiated granulosa cells in culture medium alone resulted in a significant decline in levels of FSHR mRNA (by ~80% compared to freshly collected cells). By comparison, granulosa cultured with BMP4 (10-100 ng/ml) maintained FSHR and expression at approximately in vivo levels. Because both granulosa and theca tissues from prehierarchal follicles express BMP4, it is suggested that BMP4 acts in a paracrine and/or autocrine fashion to support elevated FSHR expression prior to follicle selection. Granulosa cells cultured with BMP4 for 24 h also initiated FSH-induced cAMP production and indirectly initiated anti-Mullerian hormone (AMH), CYP11A, and STAR expression plus progesterone production. However, pretreatment with the BMP antagonist NOGGIN or the mitogen-activated protein kinase (MAPK) agonist transforming growth factor alpha attenuated or blocked each action promoted by BMP4. We conclude that prior to and immediately after selection, BMP4 serves to support FSHR expression within the granulosa layer, yet prior to selection, multiple factors (including inhibitory MAPK signaling, AMH, and BMP antagonists) can modulate FSHR expression and suppress FSH-mediated cell signaling to prevent granulosa cell differentiation prior to follicle selection.

GRK-6 mediates FSH action synergistically enhanced by estrogen and the oocyte in rat granulosa cells

Estrogen is known to play a pivotal role in granulosa cell responses to follicle-stimulating hormone (FSH) that is critical for the establishment of dominant follicles and subsequent ovulation in mammals. Thus, elucidating the cellular and molecular mechanisms that regulate FSH activity is important to understand female fertility. We previously discovered that the oocyte is required for estrogen to exert its positive effects on FSH activity in rat granulosa cells. This finding supports the new concept that estrogen action in granulosa cells is mediated by the oocyte. In the current study, we explored the underlying mechanism. In the presence of oocytes, estrogens enhanced FSH-induced increases in aromatase, steroidogenic acute regulatory protein and FSH receptor mRNA expression as well as cAMP production. However, as forskolin did not mimic FSH activity this indicated that coexistence of estrogen/oocytes increases FSH activity at a site upstream of adenylate cyclase in granulosa cells. We therefore sought a possible involvement of the autoregulatory molecules for FSH receptor, G protein-coupled receptor kinases (GRKs) and ß-arrestins in enhancing FSH activity in response to the estrogen/oocyte co-treatment in granulosa cells. Among the seven known GRK and two ß-arrestin molecules, we found that estrogens with oocytes suppressed FSH-induced GRK-6 mRNA expression. Consistent with this finding, transfecting granulosa cells with small interfering RNA of GRK-6 significantly increased FSH induction of aromatase mRNA, suggesting that endogenous GRK-6 plays an inhibitory role in FSH-induced aromatase mRNA expression. Consequently, these findings strongly suggest that GRK-6 is involved in the mechanism by which estrogen and oocytes synergistically augment FSH activity in granulosa cells.

Effects of growth differentiation factor-9 and FSH on in vitro development, viability and mRNA expression in bovine preantral follicles

The present study investigated the role of growth differentiation factor (GDF)-9 and FSH, alone or in combination, on the growth, viability and mRNA expression of FSH receptor, proliferating cell nuclear antigen (PCNA) and proteoglycan-related factors (i.e., hyaluronan synthase (HAS) 1, HAS2, versican, perlecan) in bovine secondary follicles before and after in vitro culture. After 12 days culture, sequential FSH (100 ng mL⁻¹) from Days 0 to 6 and 500 ng mL⁻¹ from Days 7 to 12) increased follicular diameter and resulted in increased antrum formation (P<0.05). Alone, 200 ng mL⁻¹ GDF-9 significantly reduced HAS1 mRNA levels, but increased versican and perlecan mRNA levels in whole follicles, which included the oocyte, theca and granulosa cells. Together, FSH and GDF-9 increased HAS2 and versican (VCAN) mRNA levels, but decreased PCNA mRNA expression, compared with levels in follicles cultured in α-minimum essential medium supplemented with 3.0 mg mL⁻¹ bovine serum albumin, 10 µg mL⁻¹ insulin, 5.5 µg mL⁻¹ transferrin, 5 ng mL⁻¹ selenium, 2 mM glutamine, 2mM hypoxanthine and 50 μg mL⁻¹ ascorbic acid (α-MEM⁺). Comparisons of uncultured (0.2 mm) and α-MEM⁺ cultured follicles revealed that HAS1 mRNA expression was higher, whereas VCAN expression was lower, in cultured follicles (P<0.05). Expression of HAS1, VCAN and perlecan (HSPG2) was higher in cultured than in vivo-grown (0.3 mm) follicles. In conclusion, FSH and/or GDF-9 promote follicular growth and antrum formation. Moreover, GDF-9 stimulates expression of versican and perlecan and interacts positively with FSH to increase HAS2 expression.

Assessment of theca cell function: a prerequisite to androgen or luteinizing hormone supplementation in poor responders

Poor responders are a heterogeneous population, with some patients displaying a diminished ovarian reserve and others a poor ovarian reserve with preserved granulosa cell function. Androgen and LH/hCG supplementation has been advocated for poor responders, mainly those >40 years old. Although androgens synergistically act with FSH to support folliculogenesis, and ovarian androgen secretion declines with age, there is still no evidence that androgen therapy is actually effective to improve ovarian FSH sensitivity. The main reason seems to be that theca cell function has not been appropriately assessed in patients at risk of poor response. The definition of theca insufficiency is hampered by methodologic shortcomings in routine bioassays. Provocative tests for theca cells might help to identify those patients who could benefit from androgen supplementation. The lack of data regarding theca cells in these patients might contribute to explaining the absence of evidence for a positive effect of androgen therapy.

FSH receptor in vitro modulation by testosterone and hCG in human luteinized granulosa cells

To investigate the effect of testosterone and hCG on FSH receptor (FSHR) protein and mRNA expression in human granulosa cells (GC) in vitro. Experimental in vitro cell culture obtained from healthy women undergoing IVF/ICSI due to male factor infertility. Human follicular fluid samples were obtained and after cumulus-oocyte complexes were identified, fluids were pipetted onto Ficoll gradients and centrifuged for 15 min at 400 × g at room temperature. Cells at the interface were removed and plated in 24-well plates for 3 days in M-199 with 10% FBS. Cells were treated with different concentrations of testosterone and hCG. After purification, cells were labeled with specific antibodies and the protein expression of the FSHR was evaluated by flow cytometry in the GC population. Also, total RNA was extracted from confluent GC and the FSHR gene expression was evaluated by RT-PCR. FSHR expression was modulated by treating GC in vitro at different testosterone/hCG concentrations. When compared with untreated GC, we observed a significant effect of testosterone and hCG on the expression of the FSHR at the protein level. Time course experiments confirmed that the gene expression of the FSHR peaked at 12-24h when testosterone or hCG was used as a stimulus. Both testosterone and hCG are able to positively modulate FSHR expression at gene and protein level in human GC in vitro.

Stimulation of the Canonical WNT-Signaling Pathway Inhibits Follicle-Stimulating Hormone Responsive Genes in Rat Granulosa Cells.

Beta-catenin, a key component of WNT signaling, participates in FSH mediated regulation of estrogen production. The purpose of these studies was to determine if β-catenin's contribution to FSH-mediated steroidogenesis in primary rat granulosa cells was due in part to extracellular stimulation of the canonical WNT signaling pathway. To achieve this purpose, primary cultures of rat granulosa cells were exposed to vehicle or recombinant mouse WNT3A (500 ng/ml) for 24 h before treatment with vehicle or FSH (100 ng/ml) for an additional 24 h (n = 5). Steroidogenic enzymes and ovarian differentiation factor mRNAs were quantified using real-time PCR. Expression of steroidogenic enzyme mRNAs aromatase (Cyp19a1), P450 side chain cleavage (Cyp11a1), and steroidogenic acute regulatory protein (Star) were increased (P < 0.05) following FSH treatment. Co-incubation of FSH and WNT3A reduced the ability of FSH to stimulate Cyp19a1 mRNA expression (P < 0.01) and Cyp11a1 and Star expression (P = 0.07). Subsequent steroid production demonstrated a parallel effect as FSH-induced estradiol was reduced from 559 pg/ml to 75.5 (± 131) pg/ml (P = 0.06) and progesterone was reduced from 1.51 ng/ml to 0.12 ng/ml (± 0.22) ng/ml (P < 0.01) with addition of WNT3A. Concomitant activation of FSH and WNT pathways results in marked reduction of LH receptor (P = 0.02) but not inhibin-alpha (P = 0.28). Additionally, WNT3A alone down regulated FSH receptor mRNA expression compared to controls (P < 0.01). To ascertain the contribution of WNT3A to suppression of the steroidogenic enzymes and differentiation factors, we used a known inhibitor of the WNT pathway, Dickkopf (DKK). Primary cultures of rat granulosa cells were pretreated with vehicle or recombinant human DKK4 (5μg/ml) before treatment with WNT3A (n = 2). Pretreatment of DKK4 preceding activation of the WNT signaling pathway with WNT3A, resulted in a restored ability of FSH to stimulate expression of the steroidogenic enzymes. Consequent production of estradiol and progesterone concentrations in cell culture media were also reestablished from 27.5 pg/ml to 430 (± 113) pg/ml (P < 0.01) and 0.02 ng/ml to 1 (± 0.26) ng/ml (P < 0.05), respectively following disruption of the WNT pathway by DKK4 when WNT3A and FSH were co-incubated when compared to vehicle treated controls. Expression of ovarian derived differentiation factor target genes, LH receptor, and inhibin-alpha returned to levels greater than controls in granulosa cells exposed to DKK before co-incubation with WNT3A and FSH (P = 0.01). FSH receptor mRNA expression returned to control levels when DKK disrupted WNT signaling before treatment of WNT3A. Forkhead box protein O1 (FoxO1) mRNA expression was analyzed as a potential mechanism for suppressing steroidogenic enzymes and differentiation factor mRNAs. FoxO1 mRNA expression was increased by WNT3A alone (P < 0.05) and in combination with FSH (P = 0.07). Disruption of WNT signaling resulted in a reduction of FoxO1 mRNA. Data suggest WNT signaling inhibits FSH target genes associated with maturation and differentiation of the ovarian follicle. A likely mechanism for WNT3A inhibition of FSH signaling is reduction of FSH receptor occurring through increased FOXO1, a repressor of FSH target genes. Supported in part by NIH (R15065668 to JAHG) and Oklahoma Center for the Advancement of Science and Technology (HR10-030S to JAHG).

Insufficient androgen and FSH signaling may be responsible for the azoospermia of the infantile primate testes despite exposure to an adult-like hormonal milieu

In humans, as well as in other higher primates, the infantile testis is exposed to an adult-like hormonal milieu, but spermatogenesis is not initiated at this stage of primate development. In the present study, we examined the molecular basis of this intriguing infertile state of the primate testis. The integrity of androgen receptor (AR) and FSH receptor (FSHR) signaling pathways in primary cultures of Sertoli cells (Scs) harvested from azoospermic infant and spermatogenic pubertal monkey testes were investigated under identical in vitro hormonal conditions. In order to synchronously harvest Scs from early pubertal testis, the activation of testicular puberty was timed experimentally by prematurely initiating gonadotrophin secretion in juvenile animals with an intermittent infusion of gonadotrophin-releasing hormone. While qRT-PCR demonstrated that AR and FSHR mRNA expression in Scs from infant and pubertal testes were comparable, androgen-binding and FSH-mediated cAMP production by infant Scs was extremely low. Compromised AR and FSHR signaling in infant Scs was further supported by the finding that testosterone (T) and FSH failed to augment the expression of the T responsive gene, claudin 11, and the FSH responsive genes, inhibin-βB, stem cell factor (SCF) and glial cell line-derived neurotrophic factor (GDNF) in Scs harvested at this stage of development. These results indicate that compromised AR and FSHR signaling pathways in Scs underlie the inability of the infant primate testis to respond to an endogenous hormonal milieu that later in development, at the time puberty, stimulates the initiation of spermatogenesis. This finding may have relevance to some forms of idiopathic infertility in men.

Bone morphogenetic protein 6 promotes FSH receptor and anti-Müllerian hormone mRNA expression in granulosa cells from hen prehierarchal follicles

A growing body of literature provides evidence of a prominent role for bone morphogenetic proteins (BMPs) in regulating various stages of ovarian follicle development. Several actions for BMP6 have been previously reported in the hen ovary, yet only within postselection (preovulatory) follicles. The initial hypothesis tested herein is that BMP6 increases FSH receptor (FSHR) mRNA expression within the granulosa layer of prehierarchal (6-8 mm) follicles (6-8 GC). BMP6 mRNA is expressed at higher levels within undifferentiated (1-8 mm) follicles compared with selected (≥9 mm) follicles. Recombinant human (rh) BMP6 initiates SMAD1, 5, 8 signaling in cultured 6-8 GC and promotes FSHR mRNA expression in a dose-related fashion. In addition, a 21 h preculture with rhBMP6 followed by a 3 h challenge with FSH increases cAMP accumulation, STAR (StAR) expression, and progesterone production. Interestingly, rhBMP6 also increases expression of anti-Müllerian hormone (AMH) mRNA in cultured 6-8 GC. This related BMP family member has previously been implicated in negatively regulating FSH responsiveness during follicle development. Considering these data, we propose that among the paracrine and/or autocrine actions of BMP6 within prehierarchal follicles is the maintenance of both FSHR and AMH mRNA expression. We predict that before follicle selection, one action of AMH within granulosa cells from 6 to 8 mm follicles is to help suppress FSHR signaling and prevent premature granulosa cell differentiation. At the time of selection, we speculate that the yet undefined signal directly responsible for selection initiates FSH responsiveness. As a result, FSH signaling suppresses AMH expression and initiates the differentiation of granulosa within the selected follicle.

Effect of a GnRH agonist on the FSH receptors in prepubertal ewes

The present study was aimed at investigating the effects of the GnRH agonist, alarelin, on the expression of the follicle-stimulating hormone receptor (FSHR) in the pituitary gland. Also to evaluate the presence and immunolocalization of FSHR in the ovary, as well as to confirm the efficacy on the development of the follicles in the ovaries of ewes. Twenty-eight prepubertal ewes (5–6months of age) were randomly assigned to four experimental groups (EG, n=7 per group). The animals in experimental group EG-I, EG-II and EG-III were subcutaneously injected with 200μg, 300μg or 400μg alarelin antigens twice (day 0 and 14), respectively. Animals in the control group (CG) were twice subcutaneously injected with 2.0mL of a solvent (day 0 and 14). Samples of the pituitary gland and ovaries were collected aseptically on day 70 following treatment. Blood samples were taken from the jugular vein on day 0, 7, 14, 21, 28, 35, 50, 60 and 70 after the first alarelin antigen injection and ELISA used to measure the serum concentration of the GnRH antibody and FSH. Fluorescence quantitative RT-PCR was implemented to detect the gene expression of FSHR mRNA in the pituitary. Immunohistochemistry was performed to localize the FSHR in the ovary. GnRH antibody concentrations in the EG-I, EG-II and EG-III treatment groups increased gradually and were higher than that of the CG (P<0.05) or control from day 14 to day 60. Pituitary FSHR mRNA levels were significantly reduced (P<0.05) 1.38, 7.33 and 10.11 times in the EG-I, EG-II and EG-III groups, respectively. Immunohistochemistry identified that the immunostained cells of the FSHR were present in the ewes’ ovaries, which predominantly concentrated in the cytomembrane, cytoplasm and nuclei of the follicle cells. The oocytes had different immunostaining intensities at different developing stages. The gray values of microscopy images in EG-I, EG-II and EG-III groups increased, when compared to the CG. Ovaries in alarelin treated ewes of groups EG-II and EG-III were significantly higher (P<0.05), when compared to ewes in group EG-I and CG. The follicle vertical diameter (FVD), follicle transverse diameter (FTD), follicle-wall thickness (FWT), follicle externatheca thickness (FET) and follicle internatheca thickness (FIT) in the alarelin immunity ewes were greater than those in the CG. The serum FSH concentrations of the treated ewes remained higher than that in the CG (P<0.05). In conclusion, active immunity with alarelin stimulated the production of the GnRH antibody, inhibited the expression of FSHR mRNA in the pituitary gland, improved the distribution of FSHR in the ovary, increased the FSH secretion and thereby promoted the development of the ovaries and follicles in the ewes. This has important potential for developing a novel technique in the superovulation and regulation of reproductive functions in ewes.

Increased abundance of aromatase and follicle stimulating hormone receptor mRNA and decreased insulin-like growth factor-2 receptor mRNA in small ovarian follicles of cattle selected for twin births1,2

Cattle genetically selected for twin ovulations and births (Twinner) exhibit increased ovarian follicular development, increased ovulation rate, and greater blood and follicular fluid IGF-1 concentrations compared with contemporary cattle not selected for twins (Control). Experimental objectives were to 1) assess relationships among aromatase (CYP19A1), IGF-1 (IGF1), IGF-2 receptor (IGF2R), and FSH receptor (FSHR) mRNA expression in small (≤5 mm) antral follicles and 2) determine their association with increased numbers of developing follicles in ovaries of Twinner females. Ovaries were collected from mature, cyclic (d 3 to 6) Twinner (n = 11), and Control (n = 12) cows at slaughter and pieces of cortical tissue were fixed and embedded in paraffin. Expression of mRNA was evaluated by in situ hybridization using (35)S-UTP-labeled antisense and sense probes for CYP19A1, FSHR, IGF1, and IGF2R mRNA. Silver grain density was quantified within the granulosa and theca cells of individual follicles (2 to 7 follicles/cow) by Bioquant image analysis. Follicles of Twinners tended to be smaller in diameter than Controls (1.9 ± 0.1 vs. 2.3 ± 0.1 mm; P = 0.08), but thickness of granulosa layer did not differ (P > 0.1) by genotype. Relative abundance of CYP19A1 (P < 0.01) and FSHR (P < 0.05) mRNA was greater in granulosa cells of Twinners vs. Controls, respectively, whereas IGF2R mRNA expression was less in both granulosa (P < 0.01) and theca (P < 0.05) cells in follicles of Twinners vs. Controls, respectively. Abundance of CYP19A1 mRNA in granulosa cells was correlated negatively with IGF2R mRNA expression in both granulosa (r = -0.33; P < 0.01) and theca (r = -0.21; P = 0.05) cells. Expression of IGF1 mRNA was primarily in granulosa cells, including cumulus cells, and its expression did not differ between Twinners vs. Controls (P > 0.10). Detected increases in CYP19A1 and FSHR, but not IGF1, mRNA expression along with decreases in IGF2R mRNA expression in individual follicles of Twinners support the hypothesis that increased follicular development and steroidogenesis in Twinner females result from increased extra-ovarian IGF-1 production. Furthermore, a reduction in follicular IGF2R mRNA expression accompanied by a reduction in receptor numbers would increase availability of free IGF-2 and its stimulation of follicular development in Twinners.

Genetic effect of the follicle-stimulating hormone receptor gene on reproductive traits in Beijing You chickens

We analyzed the effects of the polymorphisms of the follicle-stimulating hormone receptor (FSHR) gene on egg production in a Beijing You chicken population divergently selected for egg number, egg weight, and BW across 3 generations. The FSHR gene encodes the receptor of follicle-stimulating hormone, which controls follicular development and recruitment in the ovary. Seven SNP of the FSHR gene were investigated in 768 pedigreed hens from the G2 generation. Association analysis revealed that g.-310A > G, g.-181A > T, and g.159C > T were associated with egg number at different weeks of age (P < 0.05) and that g.75470A > G and g.75860G > A were associated with egg weight at first egg (P < 0.05). The favorable allele of g.-181A > T and g.159C > T had increased frequencies not only in the high line but also over the 2 generations (G2 vs. G1) within the high line. To confirm the association study, we tested for FHSR mRNA expression level in the chicken ovaries. The results showed that the homozygous favorable genotypes of g.-181A > T and g.-310A > C increased the FSHR mRNA expression level compared with the other genotypes (P = 0.001 and P = 0.026, respectively). Hence, our findings implied that the SNP g.-181A > T could be a potential genetic marker for egg performance in the Beijing You chicken, but further replications of our study in other chicken populations are needed to verify such effects detected here.

Anti-Müllerian hormone reduces follicle sensitivity to follicle-stimulating hormone in human granulosa cells

To determine that anti-Müllerian hormone (AMH) has been shown to inhibits E(2) production in rodents and in luteinized granulosa cells (GC). We determined whether this occurs in human cells most highly expressing AMH (i.e., from small antral follicles) and whether this is an effect on aromatase promoter activity. We also investigated the effects of AMH on other factors determining FSH sensitivity. Granulosa cells were exposed to AMH with and without gonadotropins for 48 hours. University laboratory. Not applicable. None. Aromatase and FSH receptor messenger RNA expression measured using real time quantitative polymerase chain reaction (PCR). Aromatase promoter II activity measured using a luciferase assay. Estradiol, inhibin A and B, and vascular endothelial growth factor production were measured in the conditioned medium. The AMH decreased gonadotropin-stimulated aromatase expression and decreased forskolin-stimulated aromatase in KGN cells and this effect was through a dose-dependent inhibition of promoter II. Surprisingly, AMH also reduced FSH receptor mRNA expression. High AMH doses had no effect on inhibin B, whereas a low dose stimulated production. There was no effect on inhibin A or vascular endothelial growth factor. The AMH inhibits factors affecting FSH sensitivity. As AMH levels decrease with follicle growth, this inhibition would be removed. The AMH overproduction in anovulatory polycystic ovaries (PCO) may therefore restrict folliculogenesis by an inhibitory effect on FSH sensitivity, thereby contributing to anovulation.

Concentration of activin A and follistatin in follicular fluid from human small antral follicles associated to gene expression of the corresponding granulosa cells

The present study correlated concentrations of activin A and follistatin in follicular fluid (FF) from human small antral follicles to FF concentrations of AMH, inhibin B, progesterone, and oestradiol and to the mRNA expression of FSH-receptor (FSHR), LH-receptor (LHR), AMH-receptor2 (AMHR2), CYP19a, and androgen-receptor (AR) in the corresponding granulosa cells (GC). FF from 144 follicles (3–12mm in diameter) was included whereas mRNA expression profiles were established in GC from 66 of the 144 follicles.Levels of follistatin remained constant in relation to follicular diameter, whereas activin A levels increased in follicles exceeding 10mm in diameter. Levels of activin A and inhibin B showed a highly significant inverse association. Follistatin showed highly significant positive associations with AMH and inhibin B levels and with FSHR and AR gene expression in GC.This study revealed unexpected associations that probably reflect the complicated regulatory mechanisms governing human folliculogenesis.

Follicle-Stimulating Hormone Receptor Polymorphism (G−29A) Is Associated with Altered Level of Receptor Expression in Granulosa Cells

Polymorphisms of the FSHR gene are associated with variable ovarian response to FSH stimulation in subjects undergoing in vitro fertilization (IVF) treatment. The type of ovarian response is correlated with the level of FSH receptor (FSHR) expression on granulosa cells. We investigated whether the polymorphism at position -29 in the promoter of the FSHR gene may contribute in altered receptor expression. FSHR polymorphism at position -29 was studied in 100 subjects undergoing IVF treatment. Association of this polymorphism with level of FSHR expression was retrospectively analyzed. The study was conducted at an academic research institute and private IVF clinic. The genotype at position -29 of the FSHR gene was studied in IVF subjects by PCR-restriction fragment length polymorphism. Total RNA and protein was extracted from granulosa cells. The relative FSHR mRNA expression was carried out by real-time PCR. The receptor protein expression was evaluated by Western blot and confocal microscopy. The clinical and endocrinological parameters revealed that almost 72% of subjects with the AA genotype at position -29 of FSHR gene were poor ovarian responders (odds ratio 8.63, 95% confidential interval 1.84-45.79; P = 0.001). The lower cleavage intensity predicted by in silico analysis for A allele as compared with the G allele suggest the difference in the DNA-protein binding affinity. The relative expression of FSHR at mRNA and protein level was significantly reduced in subjects with AA genotype as compared with the GG genotype. Poor ovarian response observed in subjects with the AA genotype at position -29 of the FSHR gene is due to reduced receptor expression.

The Expression of FSH Receptor (FSHR) in the Neonatal Porcine Ovary and its Regulation by Flutamide

This study was designed to reveal the FSHR mRNA and protein expression in the neonatal porcine ovary and to determine whether maternal administration of antiandrogen flutamide may affect FSHR expression in the ovary of newborn piglets using real-time PCR, immunohistochemistry and Western blot analysis. Pregnant sows were injected with flutamide at a dose of 50 mg/kg body weight, given five times, every second day, starting at day 20 post-coitum (p.c.) or day 80 p.c., and ovaries were obtained from neonatal pigs. The FSHR mRNA expression was significantly decreased after flutamide administration. Furthermore, higher down-regulation was observed following exposure to antiandrogen at day 20 than at day 80 p.c. Immunohistochemistry showed the positive immunostaining for FSHR in the oocytes, granulosa cells of primary follicles and the surface epithelium of the ovaries from both control and flutamide-treated pigs. However, oocytes and granulosa cells of primary follicles in the ovaries exposed in utero to flutamide were weakly immunostained when compared to those in the control ones. The presence of FSHR protein in all investigated ovaries was confirmed by Western blot analysis. Based on our findings, we suggest that FSHR may be involved in the early follicle formation in pigs, which begins during prenatal life. Furthermore, the regulation of FSHR mRNA and protein expression in neonatal porcine ovaries after maternal exposure to flutamide confirms that androgens play a crucial role in porcine folliculogenesis at the early stages.

Regulation of Sertoli cell activin A and inhibin B by tumour necrosis factor α and interleukin 1α: Interaction with follicle-stimulating hormone/adenosine 3′,5′-cyclic phosphate signalling

Regulation of crucial events during spermatogenesis involves dynamic changes in cytokine production and interactions across the cycle of the seminiferous epithelium. Regulation of activin A and inhibin B production by the inflammatory cytokines, tumour necrosis factor α (TNFα) and interleukin 1α (IL1α), alone and in conjunction with FSH or a cAMP analogue (dibutyryl cAMP), was examined in cultures of Sertoli cells from 20-day old rats. Both TNFα and IL1α stimulated activin A secretion and expression of its subunit (βA) mRNA, and suppressed inhibin B secretion and expression of its subunit (α and βB) mRNAs. The actions of TNFα and IL1α were opposed by FSH and dibutyryl cAMP. Both cytokines inhibited FSH/dibutyryl cAMP-stimulated inhibin B secretion and mRNA expression as well as stem cell factor mRNA expression. Both cytokines also inhibited FSH-induced cAMP production, and reduced baseline FSH receptor mRNA expression. These data highlight the reciprocal relationship that exists between FSH/cAMP signalling and inflammatory cytokine signalling pathways in the control of Sertoli cell function, and production of activin A/inhibin B in particular. It is anticipated that these interactions play important roles in the fine control of events during the cycle of the seminiferous epithelium and in the inhibition of spermatogenesis during inflammation.

Prenatal testosterone excess alters Sertoli and germ cell number and testicular FSH receptor expression in rams

Exposure to excess testosterone (T) during fetal life has a profound impact on the metabolic and reproductive functions in the female's postnatal life. However, less is known about the effects of excess testosterone in males. The aim of the present study was to evaluate the impact (consequences) of an excess of T during fetal development on mature male testis. The testicular evaluation was by histological analysis and by determination of mRNA expression of the FSH receptor (FSH-R), transforming growth factor-β type I receptor (TβR-I), and two members of the TGF-β superfamily, transforming growth factor-β3 (TGFβ3) and anti-Müllerian hormone (AMH) in males born to mothers receiving an excess of T during pregnancy. At 42 wk of age, postpubertal males born to mothers treated with 30 mg of T propionate twice weekly from day 30 to 90, followed by 40 mg of T propionate from day 90 to 120 of pregnancy (T males), showed higher concentrations of FSH in response to a GnRH analog, a higher number of Sertoli cells/seminiferous tubule cross-section, and a lower number of germ cells/tubules (P < 0.05) than control males (C males) born to mothers treated with the vehicle. The mRNA expression of FSH-R and of TβR-I was higher in T males compared with C males (P < 0.05). Moreover, in T males, AMH expression level correlated negatively with the expression level of TGFβ3. In C males, this latter correlation was not observed. These results suggest that prenatal exposure to an excess of T can negatively modify some histological and molecular characteristics of the mature testis.