scholarly journals Loss of GATA-6 and GATA-4 in Granulosa Cells Blocks Folliculogenesis, Ovulation, and Follicle Stimulating Hormone Receptor Expression Leading to Female Infertility

Endocrinology ◽  
2012 ◽  
Vol 153 (5) ◽  
pp. 2474-2485 ◽  
Author(s):  
Jill Bennett ◽  
Yan-Guang Wu ◽  
Jan Gossen ◽  
Ping Zhou ◽  
Carlos Stocco
Keyword(s):  
Granulosa Cells ◽  
Ovarian Function ◽  
Follicular Development ◽  
Receptor Expression ◽  
Double Knockout ◽  
Estrous Cycles ◽  
Fshr Gene ◽  
Reporter Assays ◽  
Hormone Receptor Expression

Single GATA-6 (G6gcko), GATA-4 (G4gcko), and double GATA-4/6 (G4/6gcko) granulosa cell-specific knockout mice were generated to further investigate the role of GATA transcription factors in ovarian function in vivo. No reproductive defects were found in G6gcko animals. G4gcko animals were subfertile as indicated by the reduced number of pups per litter and the release of significantly fewer oocytes at ovulation. In marked contrast, G4/6gcko females fail to ovulate and are infertile. Furthermore, G4/6gcko females had irregular estrous cycles, which correlate with the abnormal ovarian histology found in unstimulated adult G4/6gcko females showing lack of follicular development and increased follicular atresia. Moreover, treatment with exogenous gonadotropins did not rescue folliculogenesis or ovulation in double-knockout G4/6gcko mice. In addition, ovary weight and estradiol levels were significantly reduced in G4gcko and G4/6gcko animals when compared with control and G6gcko mice. Aromatase, P450scc, and LH receptor expression was significantly lower in G4gcko and G4/6gcko mice when compared with control animals. Most prominently, FSH receptor (FSHR) protein was undetectable in granulosa cells of G4gcko and G4/6gcko. Accordingly, gel shift and reporter assays revealed that GATA-4 binds and stimulates the activity of the FSHR promoter. These results demonstrate that GATA-4 and GATA-6 are needed for normal ovarian function. Our data are consistent with a role for GATA-4 in the regulation of the FSHR gene and provide a possible molecular mechanism to explain the fertility defects observed in animals with deficient GATA expression in the ovary.

scholarly journals The corticosterone-glucocorticoid receptor-AP1/CREB axis inhibits luteinizing hormone receptor expression in mouse granulosa cells

2020 ◽  
Author(s):  
Yinghui Wei ◽  
Ming Shen
Keyword(s):  
Luteinizing Hormone ◽  
Glucocorticoid Receptor ◽  
Granulosa Cells ◽  
Hormone Receptor ◽  
Follicular Development ◽  
The Body ◽  
Receptor Expression ◽  
Luteinizing Hormone Receptor ◽  
Control Group ◽  
Hormone Receptor Expression

Abstract Background Under stress conditions, luteinizing hormone (LH)-mediated ovulation is inhibited, resulting in insufficient oocyte production and excretion during follicular development. When the body is stressed, a large amount of corticosterone (CORT) is generated, which will lead to disorder of the body's endocrine system and damage to the body. Our previous work showed that corticosterone can block follicular development in mice. Since LH acts through binding with the LH receptor (LHR), the present study aimed to investigate whether and how CORT influence luteinizing hormone receptor expression in mouse ovarian granulosa cells (GCs). Methods For this purpose, 3-week-old ICR female mice were injected intraperitoneally with pregnant horse serum gonadotropin (PMSG). Meanwhile, the treatment group were injected with corticosterone corticosterone (1 mg/mouse) at intervals of 8 h; while the control group was injected with same volume of methyl sulfoxide (DMSO). Granulosa cells were collected at 24 h, 48 h, and 55 h after PMSG injection. For in vitro experiments, mice granulosa cells obtained from healthy follicles were treated with corticosterone alone, or together with inhibitors against glucocorticoid receptor (GR). Results The results showed that corticosterone caused down-regulation of luteinizing hormone receptor expression in granulosa cells, which was accompanied by impaired cell viability. Moreover, the effects of corticosterone was mediated by binding to its receptor in granulosa cells. Further investigations revealed that glucocorticoid receptor might regulated the transcription of luteinizing hormone receptor through inhibiting the expression of luteinizing hormone receptor transcription factors, including AP1 and CREB. Conclusions Our findings suggested a possible mechanism of corticosterone-induced anovulation involving the inhibition of luteinizing hormone receptor expression in granulosa cells by corticosterone-glucocorticoid receptor-AP1 / CREB axis.

Identification of Gαs messenger ribonucleic acid splice variants in human granulosa cells

1997 ◽  
Vol 18 (1) ◽  
pp. 27-35 ◽  
Author(s):  
G N Europe-Finner ◽  
E Cartwright ◽  
J Bellinger ◽  
H J Mardon ◽  
D H Barlow ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Ovarian Function ◽  
Splice Variants ◽  
Consensus Sequence ◽  
Phosphorylation Site ◽  
Follicular Development ◽  
Protein Isoforms ◽  
Mrna Isoforms ◽  
Messenger Ribonucleic Acid

ABSTRACT Granulosa cells are essential for follicular development and corpus luteum formation and their functions are regulated by gonadotrophins through G protein-coupled receptors. The dominant second messenger pathway involves the stimulation of cyclic AMP formation by Gαs-linked receptors. In this paper we have investigated the expression of Gαs mRNA splice variants in relation to expression of Gαs protein isoforms in granulosa cells obtained from patients undergoing in vitro fertilization. We have carried out ribonuclease protection assays using cRNA riboprobes which are capable of detecting all Gαs mRNA isoforms as well as quantifying total amounts of Gαs mRNA. Granulosa cells express the message for Gαs-Large and Gαs-Small and the presence of two distinct protein products was confirmed by immunoblotting using the antibody RM/1. Moreover, the data show that a significant fraction of Gαs-Large and Gαs-Small mRNAs contain an extra CAG codon. This should generate proteins with an extra serine residue, resulting in Gαs variants with the consensus sequence of a protein kinase C phosphorylation site. These results highlight the possible interaction between different signalling pathways in the control of cAMP production and the need to investigate the relationship between Gαs variants and different adenylyl cyclase isozymes in patients with normal and abnormal ovarian function.

scholarly journals Heat stress diminishes gonadotropin receptor expression and enhances susceptibility to apoptosis of rat granulosa cells

Reproduction ◽  
2005 ◽  
Vol 129 (4) ◽  
pp. 463-472 ◽  
Author(s):  
Takashi Shimizu ◽  
Izumi Ohshima ◽  
Manabu Ozawa ◽  
Satoko Takahashi ◽  
Atsushi Tajima ◽  
...  
Keyword(s):  
Heat Stress ◽  
Granulosa Cells ◽  
Follicular Fluid ◽  
Follicular Development ◽  
Receptor Expression ◽  
Female Rats ◽  
Aromatase Activity ◽  
Mrna Levels ◽  
Gonadotropin Receptor ◽  
Estradiol Levels

Heat stress inhibits ovarian follicular development in mammalian species. We hypothesized that heat stress inhibits the function of follicular granulosa cells and suppresses follicular development. To test this, immature female rats were injected with pregnant mare serum gonadotropin (PMSG) at 48 h after the start of temperature treatment (control: 25 °C, 50% RH; heat stress: 35 °C, 70% Relative Humidity). The ovaries and granulosa cells of follicles at different developmental stages were analyzed for gonadotropin receptor levels and aromatase activity; estradiol levels were measured in follicular fluid. Before injection, heat stress diminished only the amount of FSH receptor on granulosa cells of antral follicles. During PMSG-stimulated follicular development, heat stress strongly inhibited gonadotropin receptor levels and aromatase activity in granulosa cells, and estradiol levels in the follicular fluid of early antral, antral and preovulatory follicles. To examine apoptosis and mRNA levels of bcl-2 and bax in granulosa cells, follicles harvested 48 h after PMSG injection were cultured in serum-free conditions. Heat-stressed granulosa cells showed a time-dependent increase in apoptosis. The bcl-2 mRNA levels were similar in control and heat-stressed granulosa cells; bax mRNA levels were increased in heat-stressed granulosa cells. According to these results, heat stress inhibits expression of gonadotropin receptors in granulosa cells and attenuates estrogenic activity of growing follicles, granulosa cells of heat-stressed follicles are susceptible to apoptosis, and the bcl2/bax system is not associated with heat-stress-induced apoptosis of granulosa cells. Our study suggests that decreased numbers and function of granulosa cells may cause ovarian dysfunction in domestic animals in summer.

scholarly journals TNF-α Suppressed FSH-Induced LH Receptor Expression Through Transcriptional Regulation in Rat Granulosa Cells

Endocrinology ◽  
2015 ◽  
Vol 156 (9) ◽  
pp. 3192-3202 ◽  
Author(s):  
Kohshiro Nakao ◽  
Hiroshi Kishi ◽  
Fumiharu Imai ◽  
Hiroto Suwa ◽  
Takashi Hirakawa ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Granulosa Cells ◽  
Promoter Region ◽  
Ovarian Function ◽  
Receptor Expression ◽  
Luciferase Assay ◽  
Pelvic Cavity ◽  
Transcriptional Level ◽  
Lh Receptor ◽  
Tnf Α

Several inflammatory cytokines regulate ovarian function. TNF-α is produced in granulosa cells under physiological conditions and has a reciprocal action on follicle development. In contrast, in pelvic inflammatory diseases, TNF-α is excessively produced in the pelvic cavity and has an adverse effect on reproductive functions. The objective of this study was to elucidate the mechanism of action of TNF-α on the expression of LH receptor (LHR) in immature rat granulosa cells. TNF-α suppressed FSH-induced LHR mRNA and protein expression and was not associated with cAMP accumulation. By using a luciferase assay, the construct containing base pairs −1389 to −1 of the rat Lhcgr promoter revealed that TNF-α decreased FSH-induced promoter activity. In response to TNF-α, nuclear factor (NF)-κB p65 was translocated to the nucleus, and the suppressive effect of TNF-α on LHR mRNA expression was abrogated by an NF-κB inhibitor. In a chromatin immunoprecipitation assay, TNF-α induced the association of NF-κB p65 with the rat Lhcgr transcriptional promoter region. NF-κB p65 and histone deacetylase (HDAC) interact to mediate expression of several genes at a transcriptional level. HDAC activity is thought to induce tight connections within local chromatin structures and repress gene transcription. Furthermore, the TNF-α–induced suppression of LHR mRNA expression was blocked by an HDAC inhibitor. Taken together, these results suggest that the interaction of NF-κB p65 with HDAC in the promoter region of rat Lhcgr might be responsible for TNF-α action on the regulation of LHR.

scholarly journals GnRH Receptor Expression and Reproductive Function Depend on JUN in GnRH Receptor‒Expressing Cells

Endocrinology ◽  
2018 ◽  
Vol 159 (3) ◽  
pp. 1496-1510 ◽  
Author(s):  
Carrie R Jonak ◽  
Nancy M Lainez ◽  
Ulrich Boehm ◽  
Djurdjica Coss
Keyword(s):  
Target Genes ◽  
Reproductive Function ◽  
Gnrh Receptor ◽  
Receptor Expression ◽  
Specific Expression ◽  
Α Subunit ◽  
Estrous Cycles ◽  
Hormone Synthesis

Abstract Gonadotropin-releasing hormone (GnRH) from the hypothalamus regulates synthesis and secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the anterior pituitary gonadotropes. LH and FSH are heterodimers composed of a common α-subunit and unique β-subunits, which provide biological specificity and are limiting components of mature hormone synthesis. Gonadotrope cells respond to GnRH via specific expression of the GnRH receptor (Gnrhr). GnRH induces the expression of gonadotropin genes and of the Gnrhr by activation of specific transcription factors. The JUN (c-Jun) transcription factor binds to AP-1 sites in the promoters of target genes and mediates induction of the FSHβ gene and of the Gnrhr in gonadotrope-derived cell lines. To analyze the role of JUN in reproductive function in vivo, we generated a mouse model that lacks JUN specifically in GnRH receptor‒expressing cells (conditional JUN knockout; JUN-cKO). JUN-cKO mice displayed profound reproductive anomalies such as reduced LH levels resulting in lower gonadal steroid levels, longer estrous cycles in females, and diminished sperm numbers in males. Unexpectedly, FSH levels were unchanged in these animals, whereas Gnrhr expression in the pituitary was reduced. Steroidogenic enzyme expression was reduced in the gonads of JUN-cKO mice, likely as a consequence of reduced LH levels. GnRH receptor‒driven Cre activity was detected in the hypothalamus but not in the GnRH neuron. Female, but not male, JUN-cKO mice exhibited reduced GnRH expression. Taken together, our results demonstrate that GnRH receptor‒expression levels depend on JUN and are critical for reproductive function.

Expression profiling of primary cultured buffalo granulosa cells from different follicular size in comparison with their in vivo counterpart

Zygote ◽  
2020 ◽  
Vol 28 (3) ◽  
pp. 233-240
Author(s):  
Ahmed S.A. Sosa ◽  
Sally Ibrahim ◽  
Karima Gh. M. Mahmoud ◽  
Mohamed M. Ayoub ◽  
Mohamed S.S. Abdo ◽  
...  
Keyword(s):  
Relative Abundance ◽  
Granulosa Cells ◽  
Expression Patterns ◽  
Marker Gene ◽  
Follicular Development ◽  
Nuclear Antigen ◽  
Enzyme Linked Immunosorbent Assay ◽  
Suitable Model ◽  
Follicular Size

SummaryThis study aimed to: (i) characterize cultured granulosa cells (GCs) from different follicle sizes morphologically and molecularly; and (ii) select a suitable model according to follicular size that maintained GC function during culture. Buffalo ovaries were collected from a slaughterhouse and follicles were classified morphologically into: first group ≤ 4 mm, second group 5–8 mm, third group 9–15 mm and fourth group 16–20 mm diameter. GC pellets were divided into two portions. The first portion served as the control fresh pellet, and the secondwas used for 1 week for GC culture. Total RNA was isolated, and qRT-PCR was performed to test for follicle-stimulating hormone receptor (FSHR), cytochrome P450 19 (CYP19), luteinizing hormone/choriogonadotropin receptor (LHCGR), proliferating cell nuclear antigen (PCNA), apoptosis-related cysteine peptidase (CASP3), anti-Müllerian hormone (AMH), and phospholipase A2 group III (PLA2G3) mRNAs. Estradiol (E2) and progesterone (P4) levels in the culture supernatant and in follicular fluids were measured using enzyme-linked immunosorbent assay (ELISA). Basic DMEM-F12 medium maintained the morphological appearance of cultured GCs. The relative abundance of FSHR, CYP19, and LHCGR mRNAs was 0.001 ≤ P ≤ 0.01 and decreased at the end of culture compared with the fresh pellet. There was a fine balance between expression patterns of the proliferation marker gene (PCNA) and the proapoptotic marker gene (CASP3). AMH mRNA was significantly increased (P < 0.001) in cultured GCs from small follicles, while cultured GCs from other three categories (5–8 mm, 9–15 mm and 16–20 mm) showed a clear reduction (P < 0.001). Interestingly, the relative abundance of PLA2G3 mRNA was significantly (P < 0.001) increased in all cultured GCs. E2 and P4 concentrations were significantly (P < 0.001) decreased in all cultured groups. Primary cultured GCs from small follicles could be a good model for better understanding follicular development in Egyptian buffaloes.

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

2011 ◽  
Vol 96 (9) ◽  
pp. 2805-2812 ◽  
Author(s):  
Swapna S. Desai ◽  
Swati K. Achrekar ◽  
Bhakti R. Pathak ◽  
Sadhana K. Desai ◽  
Vijay S. Mangoli ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Academic Research ◽  
In Silico Analysis ◽  
Ovarian Response ◽  
Receptor Expression ◽  
Receptor Protein ◽  
Altered Level ◽  
Vitro Fertilization ◽  
Fshr Gene

Abstract Context: 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. Objective: We investigated whether the polymorphism at position −29 in the promoter of the FSHR gene may contribute in altered receptor expression. Design and patients: 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. Setting: The study was conducted at an academic research institute and private IVF clinic. Methods: 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. Results: 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. Conclusion: Poor ovarian response observed in subjects with the AA genotype at position −29 of the FSHR gene is due to reduced receptor expression.

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