scholarly journals ATF6 knockdown decreases apoptosis, arrests the S phase of the cell cycle, and increases steroid hormone production in mouse granulosa cells

2017 ◽  
Vol 312 (3) ◽  
pp. C341-C353 ◽  
Author(s):  
Yongjie Xiong ◽  
Huatao Chen ◽  
Pengfei Lin ◽  
Aihua Wang ◽  
Lei Wang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Er Stress ◽  
Granulosa Cells ◽  
Physiological Function ◽  
Steroid Hormone ◽  
Follicular Development ◽  
S Phase ◽  
Mrna Levels ◽  
Hormone Production ◽  
Protein Levels

Activating transcription factor 6 (ATF6), a sensor protein located in the endoplasmic reticulum (ER) membrane, is an important factor in the ER stress signaling pathway. ER stress is known to be involved in folliculogenesis, follicular growth, and ovulation; however, the physiological function of ATF6 in mouse granulosa cells remains largely unknown. The aim of this study was to assess the role of ATF6 in mouse granulosa cells with respect to apoptosis, the cell cycle, and steroid hormone production, as well as several key genes related to follicular development, via RNA interference, immunohistochemical staining, real-time quantitative PCR, Western blotting, flow cytometry, terminal deoxynucleotidyltransferase-mediated deoxy-UTP nick end labeling (TUNEL) assay, and ELISA. Immunohistochemical staining revealed that ATF6 was extensively distributed in the granulosa cells of various ovarian follicles and oocytes in adult female mice. FSH or LH treatment significantly increased ATF6 protein levels in mouse granulosa cells. In the meantime, a recombinant plasmid was used to deplete ATF6 successfully using short hairpin RNA-mediated interference technology, which was verified at both the mRNA and protein levels. Flow cytometry and TUNEL assay analysis indicated that ATF6 depletion decreased apoptosis and arrested the S phase of the cell cycle in mouse granulosa cells. Consistent with these results, p53, caspase-3, B cell lymphoma 2 (Bcl-2)-associated X protein, CCAAT-enhancer-binding protein homologous protein, cyclin A1, cyclin B1, and cyclin D2 mRNA expression decreased, whereas Bcl-2 and glucose-regulated protein 78 kDa mRNA expression increased. Interestingly, ATF6 knockdown obviously increased progesterone and estradiol production in mouse granulosa cells. Cytochrome P450 1b1 ( Cyp1b1) mRNA levels were downregulated, whereas Cyp11a1, steroidogenic acute regulatory, and Cyp19a1 mRNA levels were upregulated, in keeping with the changes in steroid hormones. Furthermore, ATF6 disruption remarkably increased insulin-like growth factor binding protein 4 ( Igfbp4) expression and decreased hyaluronan synthase 2 ( Has2), prostaglandin-endoperoxide synthase 2 ( Ptgs2), and prostaglandin F receptor ( Ptgfr) expression in mouse granulosa cells, which are proteins crucial for follicular development. But, after treating with tunicamycin, the levels of Has2, Ptgs2, and Ptgfr increased relatively, whereas Igfbp4 expression decreased. Collectively, these results imply that ATF6, as a key player in ER stress signaling, may regulate apoptosis, the cell cycle, steroid hormone synthesis, and other modulators related to folliculogenesis in mouse granulosa cells, which may indirectly be involved in the development, ovulation, and atresia of ovarian follicles by affecting the physiological function of granulosa cells. The present study extends our understanding and provides new insights into the physiological significance of ATF6, a key signal transducer of ER stress, in ovarian granulosa cells.

scholarly journals Induction of Heparanase in Bovine Granulosa Cells by Luteinizing Hormone: Possible Role during the Ovulatory Process

Endocrinology ◽  
2008 ◽  
Vol 150 (1) ◽  
pp. 413-421 ◽  
Author(s):  
Eyal Klipper ◽  
Ehud Tatz ◽  
Tatiana Kisliouk ◽  
Israel Vlodavsky ◽  
Uzi Moallem ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Luteinizing Hormone ◽  
Granulosa Cells ◽  
Prostaglandin F2α ◽  
Follicular Development ◽  
Mrna Levels ◽  
Ru 486 ◽  
Protein Levels ◽  
Gonadotropin Surge ◽  
Ovulatory Process

Follicular development, follicular rupture, and corpus luteum (CL) formation are accompanied by extensive tissue remodeling. We examined whether heparanase (HPSE), which cleaves heparan sulfate glycosaminoglycans, is induced during these processes. Prostaglandin F2α injection, which initiated luteolysis and the development of a preovulatory follicle, moderately increased HPSE mRNA in bovine granulosa cells (GCs). GnRH, used to induce gonadotropin surge, markedly augmented HPSE mRNA levels 12 h after its injection. The temporal pattern of HPSE gene expression in follicular-luteal transition was further examined in follicles collected before, and 4, 10, 20, 25, and 60 h after GnRH injection. HPSE mRNA increased transiently 10–20 h after GnRH injection to levels 10-fold higher than in untreated heifers. HPSE protein levels were similarly elevated 20 h after GnRH injection in GCs, but not in the theca layer. Cyclooxygenase-2 (PTGS2) mRNA peaked before ovulation when HPSE levels returned to baseline levels. HPSE mRNA abundance also remained low in the CLs. The antiprogesterone, RU-486, elevated HPSE levels in GC culture, suggesting that progesterone secreted by CLs may inhibit HPSE. HPSE immunostaining was more abundant in GCs than thecae. In cultured GCs, LH induced a transient increase in HPSE mRNA 3–6 h after its addition, but not at 24 h. However, PTGS2 mRNA was clearly induced at this time. These findings suggest that: 1) HPSE may play a role in ovulation but much less so during CL development, and 2) GC-derived HSPE may be a novel member of the LH-induced extracellular matrix-degrading enzyme family and may contribute to follicular rupture. Granulosa-derived heparanase is a novel member of the luteinizing hormone-induced extracellular matrix-degrading enzymes contributing to follicular rupture and ovulation.

scholarly journals Influence of mutations affecting gonadotropin production or responsiveness on expression of inhibin subunit mRNA and protein in the mouse ovary

Reproduction ◽  
2004 ◽  
Vol 128 (1) ◽  
pp. 43-52 ◽  
Author(s):  
Rachel C Hirst ◽  
Margaret H Abel ◽  
Vivienne Wilkins ◽  
Christine Simpson ◽  
Phil G Knight ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Follicular Development ◽  
Inhibin B ◽  
Mrna Levels ◽  
Gene Encoding ◽  
Α Subunit ◽  
Lh Receptor ◽  
Protein Levels ◽  
Subunit Mrna ◽  
Major Cell Type

Measurement of inhibins A and B in the serum of normal cyclic rodents has implicated FSH in the regulation of these peptides within the ovary. To extend these observations we have used a panel of mutant mice carrying mutations which affect either the production of, or the ability to respond to, FSH and LH. As a consequence, the females are infertile and show different degrees of follicular development. The aim of this study was to measure inhibin gene transcription in the ovaries of these mutant females together with inhibin protein levels in ovaries and serum and to relate these to follicular development within the ovary. Comparison was made with a pool of normal/heterozygous females. In hpg females where lack of GnRH production results in the absence of gonadotropin synthesis, in FSHβ knockout (FSHβKO) females where disruption of the gene encoding FSHβ results in the absence of FSH production, and in FSH receptor knockout (FSHRKO) females which are unable to respond to circulating FSH, follicular development remains at the pre-antral stage in these three mutants. Only in the hpg females were common inhibin α subunit mRNA levels significantly lower than normal. In these three mutants, however, mRNA levels for both the βA and βB subunits were extremely low compared with normal mice. At the protein level, neither inhibin A nor B was detected in the serum of these three mutants; however inhibin B, albeit at very low levels, was detectable within the ovaries. These observations confirm a major role for FSH in the control of transcription of the βA and βB genes but suggest that the constitutive transcription of the alpha subunit is less dependent on FSH. In contrast, in LH receptor knockout (LuRKO) female mice inhibin βA subunit mRNA levels were similar to those measured in normal/heterozygous females but levels of inhibin α and βB subunit mRNAs were significantly higher than in the normal group. This was reflected in significantly higher inhibin B protein levels in ovaries and serum. An inability to respond to LH combined with high circulating levels of FSH leads to a high proportion of antral follicles in LuRKO females, with granulosa cells constituting the major cell type within the ovary. The high percentage of antral granulosa cells is likely to account for the significantly higher levels of inhibin B production in these ovaries.

scholarly journals FGF21 Promotes Proliferation and Estradiol Synthesis in Porcine Granulosa Cells

2021 ◽  
Author(s):  
Yamei Hu ◽  
Xiaoge Zhou ◽  
Shengjie Shi ◽  
Yankun Li ◽  
Liang Huang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Viability ◽  
Granulosa Cells ◽  
Ovarian Tissue ◽  
Follicular Development ◽  
Cell Number ◽  
Cell Cycle Protein ◽  
Protein Levels ◽  
Porcine Granulosa Cells ◽  
Estradiol Synthesis

Abstract Background: The proliferation and estradiol synthesis in granulosa cells (GCs) directly promotes follicular development. Previous studies had found that FGF21 regulated the hypothalamic-pituitary-gonad axis in response to the control of fertility. However, the functions and mechanisms of FGF21 in GCs are unclear.Results: Here, we found that the mRNA and protein levels of FGF21 in the ovarian tissue of high-yielding sows (Sus scrofa) was higher than that in low-yielding sows. Moreover, FGF21 was predominantly expressed in porcine GCs. Additionally, ELISA assay showed estradiol was significantly increased when overexpression of FGF21 in porcine GCs. Meanwhile, overexpressed FGF21 up-regulated both the mRNA and protein levels of key estradiol synthesis-related genes in porcine GCs, including StAR, CYP11A1 and CYP19A1. Corresponsingly, knockdown FGF21 inhibited estradiol levels and its synthesis-related genes expression. Besides, overexpression of FGF21 promoted the proliferation of porcine GCs, displayed as increasing the percentage of S-phase cells in cell cycle and EdU positive cells, including cell viability, and upregulated cell cycle genes, including cell cycle protein B (Cyclin B) and protein E (Cyclin E). Corresponsingly, knockdown FGF21 in porcine GCs suppressed the cell cycle and cell viability, as well as EdU positive cell number.Conclusions: These findings highlight that FGF21 is associated with the development of GCs and may be a novel underlying regulator of porcine follicular development.

scholarly journals Maternal obesity during pregnancy leads to adipose tissue ER stress in mice via miR-126-mediated reduction in Lunapark

Diabetologia ◽  
2021 ◽  
Author(s):  
Juliana de Almeida-Faria ◽  
Daniella E. Duque-Guimarães ◽  
Thomas P. Ong ◽  
Lucas C. Pantaleão ◽  
Asha A. Carpenter ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Glucose Tolerance ◽  
Er Stress ◽  
Maternal Obesity ◽  
Luciferase Assay ◽  
Whole Body ◽  
Mrna Levels ◽  
Protein Levels ◽  
Novel Targets

Abstract Aims/hypothesis Levels of the microRNA (miRNA) miR-126-3p are programmed cell-autonomously in visceral adipose tissue of adult offspring born to obese female C57BL/6J mice. The spectrum of miR-126-3p targets and thus the consequences of its dysregulation for adipocyte metabolism are unknown. Therefore, the aim of the current study was to identify novel targets of miR-126-3p in vitro and then establish the outcomes of their dysregulation on adipocyte metabolism in vivo using a well-established maternal obesity mouse model. Methods miR-126-3p overexpression in 3T3-L1 pre-adipocytes followed by pulsed stable isotope labelling by amino acids in culture (pSILAC) was performed to identify novel targets of the miRNA. Well-established bioinformatics algorithms and luciferase assays were then employed to confirm those that were direct targets of miR-126-3p. Selected knockdown experiments were performed in vitro to define the consequences of target dysregulation. Quantitative real-time PCR, immunoblotting, histology, euglycaemic–hyperinsulinaemic clamps and glucose tolerance tests were performed to determine the phenotypic and functional outcomes of maternal programmed miR-126-3p levels in offspring adipose tissue. Results The proteomic approach confirmed the identity of known targets of miR-126-3p (including IRS-1) and identified Lunapark, an endoplasmic reticulum (ER) protein, as a novel one. We confirmed by luciferase assay that Lunapark was a direct target of miR-126-3p. Overexpression of miR-126-3p in vitro led to a reduction in Lunapark protein levels and increased Perk (also known as Eif2ak3) mRNA levels and small interference-RNA mediated knockdown of Lunapark led to increased Xbp1, spliced Xbp1, Chop (also known as Ddit3) and Perk mRNA levels and an ER stress transcriptional response in 3T3-L1 pre-adipocytes. Consistent with the results found in vitro, increased miR-126-3p expression in adipose tissue from adult mouse offspring born to obese dams was accompanied by decreased Lunapark and IRS-1 protein levels and increased markers of ER stress. At the whole-body level the animals displayed glucose intolerance. Conclusions/interpretation Concurrently targeting IRS-1 and Lunapark, a nutritionally programmed increase in miR-126-3p causes adipose tissue insulin resistance and an ER stress response, both of which may contribute to impaired glucose tolerance. These findings provide a novel mechanism by which obesity during pregnancy leads to increased risk of type 2 diabetes in the offspring and therefore identify miR-126-3p as a potential therapeutic target. Graphical abstract

In vitro evidence that LHRH stimulates the recruitment of prolactin mRNA-expressing cells during the postnatal period in the rat

1994 ◽  
Vol 12 (1) ◽  
pp. 107-118 ◽  
Author(s):  
A Van Bael ◽  
R Huygen ◽  
B Himpens ◽  
C Denef
Keyword(s):  
Cell Cycle ◽  
Cell Population ◽  
Blot Hybridization ◽  
Postnatal Period ◽  
S Phase ◽  
Female Rats ◽  
Mrna Levels ◽  
Dot Blot ◽  
Total Cell Population

ABSTRACT We have studied the effect of LHRH and neuropeptide Y (NPY) on prolactin (PRL) mRNA levels in pituitary reaggregate cell cultures from 14-day-old female rats, by means of in situ hybridization and Northern blot analysis. As estimated by computer-image analysis, addition of LHRH on day 5 in culture for 40 h resulted in a 37% increase in the total cytoplasmic areas of cells containing PRL mRNA, visualized using a digoxigenin-labelled PRL cRNA. The size of individual PRL-expressing cells was not influenced, nor was the content of PRL mRNA per cell. A similar effect of LHRH was found by dot blot hybridization of extracted RNA. PRL mRNA levels were not affected by NPY. LHRH induced a 29% increase in the number of PRL mRNA-expressing cells processing through the S phase of the cell cycle, visualized by the incorporation of [3H]thymidine ([3H]T) into DNA over 16 h. The fraction of [3H]T-labelled cells was 10–12% of the total cell population. NPY did not influence the number of [3H]T-positive cells expressing PRL mRNA, but completely blocked the effect of LHRH on the latter population. The present data suggest that LHRH, probably via a paracrine action of gonadotrophs, stimulates the recruitment of new lactotrophs, an action which is negatively modulated by NPY. Since the magnitude of this effect was the same in the total pituitary cell population as in cells processing through the S phase of the cell cycle and presumably mitosis, recruitment of lactotrophs seems to be based on differentiation of progenitor or immature cells into PRL-expressing cells, rather than on a mitogenic action on pre-existing lactotrophs alone.

scholarly journals CLN3 expression is sufficient to restore G1-to-S-phase progression in Saccharomyces cerevisiae mutants defective in translation initiation factor eIF4E

1999 ◽  
Vol 340 (1) ◽  
pp. 135-141 ◽  
Author(s):  
Parisa DANAIE ◽  
Michael ALTMANN ◽  
Michael N. HALL ◽  
Hans TRACHSEL ◽  
Stephen B. HELLIWELL
Keyword(s):  
Cell Cycle ◽  
S Phase ◽  
Translation Initiation Factor ◽  
Yeast Cells ◽  
Initiation Factor ◽  
G1 Phase ◽  
Mrna Levels ◽  
Wild Type ◽  
Phase Progression ◽  
Type Gene

The essential cap-binding protein (eIF4E) of Saccharomycescerevisiae is encoded by the CDC33 (wild-type) gene, originally isolated as a mutant, cdc33-1, which arrests growth in the G1 phase of the cell cycle at 37 °C. We show that other cdc33 mutants also arrest in G1. One of the first events required for G1-to-S-phase progression is the increased expression of cyclin 3. Constructs carrying the 5ʹ-untranslated region of CLN3 fused to lacZ exhibit weak reporter activity, which is significantly decreased in a cdc33-1 mutant, implying that CLN3 mRNA is an inefficiently translated mRNA that is sensitive to perturbations in the translation machinery. A cdc33-1 strain expressing either stable Cln3p (Cln3-1p) or a hybrid UBI4 5ʹ-CLN3 mRNA, whose translation displays decreased dependence on eIF4E, arrested randomly in the cell cycle. In these cells CLN2 mRNA levels remained high, indicating that Cln3p activity is maintained. Induction of a hybrid UBI4 5ʹ-CLN3 message in a cdc33-1 mutant previously arrested in G1 also caused entry into a new cell cycle. We conclude that eIF4E activity in the G1-phase is critical in allowing sufficient Cln3p activity to enable yeast cells to enter a new cell cycle.

scholarly journals BMP15 Suppresses Progesterone Production by Down-Regulating StAR via ALK3 in Human Granulosa Cells

2013 ◽  
Vol 27 (12) ◽  
pp. 2093-2104 ◽  
Author(s):  
Hsun-Ming Chang ◽  
Jung-Chien Cheng ◽  
Christian Klausen ◽  
Peter C. K. Leung
Keyword(s):  
Granulosa Cells ◽  
Critical Role ◽  
Regulatory Protein ◽  
Granulosa Cell Tumor ◽  
Type I ◽  
Mrna Levels ◽  
Protein Levels ◽  
Human Granulosa Cells ◽  
Progesterone Production ◽  
Steroidogenic Acute Regulatory

In addition to somatic cell-derived growth factors, oocyte-derived growth differentiation factor (GDF)9 and bone morphogenetic protein (BMP)15 play essential roles in female fertility. However, few studies have investigated their effects on human ovarian steroidogenesis, and fewer still have examined their differential effects or underlying molecular determinants. In the present study, we used immortalized human granulosa cells (SVOG) and human granulosa cell tumor cells (KGN) to compare the effects of GDF9 and BMP15 on steroidogenic enzyme expression and investigate potential mechanisms of action. In SVOG cells, neither GDF9 nor BMP15 affects the mRNA levels of P450 side-chain cleavage enzyme or 3β-hydroxysteroid dehydrogenase. However, treatment with BMP15, but not GDF9, significantly decreases steroidogenic acute regulatory protein (StAR) mRNA and protein levels as well as progesterone production. These suppressive effects, along with the induction of Sma and Mad-related protein (SMAD)1/5/8 phosphorylation, are attenuated by cotreatment with 2 different BMP type I receptor inhibitors (dorsomorphin and DMH-1). Furthermore, depletion of activin receptor-like kinase (ALK)3 using small interfering RNA reverses the effects of BMP15 on SMAD1/5/8 phosphorylation and StAR expression. Similarly, knockdown of ALK3 abolishes BMP15-induced SMAD1/5/8 phosphorylation in KGN cells. These results provide evidence that oocyte-derived BMP15 down-regulates StAR expression and decreases progesterone production in human granulosa cells, likely via ALK3-mediated SMAD1/5/8 signaling. Our findings suggest that oocyte may play a critical role in the regulation of progesterone to prevent premature luteinization during the late stage of follicle development.

scholarly journals The Saccharomyces cerevisiae YAK1 gene encodes a protein kinase that is induced by arrest early in the cell cycle.

1991 ◽  
Vol 11 (8) ◽  
pp. 4045-4052 ◽  
Author(s):  
S Garrett ◽  
M M Menold ◽  
J R Broach
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Kinase Activity ◽  
Specific Activity ◽  
S Phase ◽  
Protein Kinase Activity ◽  
Dependent Protein Kinase ◽  
Protein Levels ◽  
Cycle Arrest ◽  
Dependent Protein

Null mutations in the gene YAK1, which encodes a protein with sequence homology to known protein kinases, suppress the cell cycle arrest phenotype of mutants lacking the cyclic AMP-dependent protein kinase (A kinase). That is, loss of the YAK1 protein specifically compensates for loss of the A kinase. Here, we show that the protein encoded by YAK1 has protein kinase activity. Yak1 kinase activity is low during exponential growth but is induced at least 50-fold by arrest of cells prior to the completion of S phase. Induction is not observed by arrest at stages later in the cell cycle. Depending on the arrest regimen, induction can occur either by an increase in Yak1 protein levels or by an increase in Yak1 specific activity. Finally, an increase in Yak1 protein levels causes growth arrest of cells with attenuated A kinase activity. These results suggest that Yak1 acts in a pathway parallel to that of the A kinase to negatively regulate cell proliferation.

scholarly journals Mammalian ECD Protein Is a Novel Negative Regulator of the PERK Arm of the Unfolded Protein Response

2017 ◽  
Vol 37 (18) ◽  
Author(s):  
Appolinaire A. Olou ◽  
Aniruddha Sarkar ◽  
Aditya Bele ◽  
C. B. Gurumurthy ◽  
Riyaz A. Mir ◽  
...  
Keyword(s):  
Er Stress ◽  
Translation Initiation Factor ◽  
Negative Regulator ◽  
Initiation Factor ◽  
Mrna Levels ◽  
Produce Cell ◽  
Stress Induction ◽  
Content Type ◽  
Protein Levels ◽  
The Unfolded Protein Response

ABSTRACT Mammalian Ecdysoneless (ECD) is a highly conserved ortholog of the Drosophila Ecd gene product whose mutations impair the synthesis of Ecdysone and produce cell-autonomous survival defects, but the mechanisms by which ECD functions are largely unknown. Here we present evidence that ECD regulates the endoplasmic reticulum (ER) stress response. ER stress induction led to a reduced ECD protein level, but this effect was not seen in PKR-like ER kinase knockout (PERK-KO) or phosphodeficient eukaryotic translation initiation factor 2α (eIF2α) mouse embryonic fibroblasts (MEFs); moreover, ECD mRNA levels were increased, suggesting impaired ECD translation as the mechanism for reduced protein levels. ECD colocalizes and coimmunoprecipitates with PERK and GRP78. ECD depletion increased the levels of both phospho-PERK (p-PERK) and p-eIF2α, and these effects were enhanced upon ER stress induction. Reciprocally, overexpression of ECD led to marked decreases in p-PERK, p-eIF2α, and ATF4 levels but robust increases in GRP78 protein levels. However, GRP78 mRNA levels were unchanged, suggesting a posttranscriptional event. Knockdown of GRP78 reversed the attenuating effect of ECD overexpression on PERK signaling. Significantly, overexpression of ECD provided a survival advantage to cells upon ER stress induction. Taken together, our data demonstrate that ECD promotes survival upon ER stress by increasing GRP78 protein levels to enhance the adaptive folding protein in the ER to attenuate PERK signaling.

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.

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