RNAi-Mediated Silencing of Catalase Gene Promotes Apoptosis and Impairs Proliferation of Bovine Granulosa Cells under Heat Stress

Heat stress in dairy cattle is recognized to compromise fertility by altering the functions of ovarian follicle-enclosed cells, e.g., oocyte and granulosa cells (GCs). Catalase is an antioxidant enzyme that plays a significant role in cellular protection against oxidative damage by the degradation of hydrogen peroxide to oxygen and water. In this study, the role and mechanism of CAT on the heat stress (HS)-induced apoptosis and altered proliferation of bovine GCs were studied. The catalase gene was knocked-down successfully in bovine GCs at both the transcriptional and translational levels. After a successful knockdown using siRNA, GCs were divided into HS (40 °C + NC and 40 °C + CAT siRNA) and 38 °C + NC (NC) groups. The GCs were then examined for ROS, viability, mitochondrial membrane potential (MMP), cell cycle, and biosynthesis of progesterone (P4) and estrogen (E2) hormones. The results indicated that CAT silencing promoted ROS production and apoptosis by up-regulating the Bcl-2-associated X protein (BAX) and Caspase-3 genes both at the transcriptional and translational levels. Furthermore, the knockdown of CAT markedly disrupted the MMP, impaired the production of P4 and E2, altered the progression of the G1 phase of the cell cycle, and decreased the number of cells in the S phase. This was further verified by the down-regulation of proliferating cell nuclear antigen (PCNA), CyclinB1, steroidogenic acute regulatory protein (STAR), and cytochrome P450 family 11 subfamily A member 1 (Cyp11A1) genes. Our study presented a novel strategy to characterize how CAT can regulate cell proliferation and apoptosis in GCs under HS. We concluded that CAT is a broad regulatory marker in GCs by regulating apoptosis, cellular progression, and simultaneously by vital fluctuations in hormonal signaling. Our findings infer a crucial evidence of how to boost the fertility of heat-stressed cows.

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Regulation of Steroidogenesis by p53 in Macaque Granulosa Cells and H295R Human Adrenocortical Cells

Endocrinology ◽

10.1210/en.2004-0253 ◽

2004 ◽

Vol 145 (12) ◽

pp. 5734-5744 ◽

Cited By ~ 11

Author(s):

Mary Cherian-Shaw ◽

Rituparna Das ◽

Catherine A. VandeVoort ◽

Charles L. Chaffin

Keyword(s):

Cell Cycle ◽

Granulosa Cells ◽

P53 Protein ◽

Ovarian Follicle ◽

Regulatory Protein ◽

Nuclear Antigen ◽

General Function ◽

Progesterone Synthesis ◽

Before And After

Abstract Ovulation and formation of a functional corpus luteum in primates involve cascades of events, including increased progesterone synthesis and changes in granulosa cell proliferation. However, critical gaps remain in our understanding of how an ovulatory gonadotropin surge initiates these processes. To more fully elucidate changes in the cell cycle during luteal formation, the actions of the tumor suppressor p53 were examined. Rhesus macaque granulosa cells were isolated during controlled ovarian stimulation protocols before (nonluteinized) or after (luteinized) an ovulatory gonadotropin stimulus. Phosphorylated p53 protein was detected in the cytoplasm of granulosa cells before and after human chorionic gonadotropin (hCG) treatment, whereas granulosa cells from hormonally controlled rats did not express p53 before or after hCG. Treatment of nonluteinized macaque granulosa cells with hCG and the p53 inhibitor pifithrin-α (PFT) in vitro did not alter markers of the cell cycle, including proliferating cell nuclear antigen, p21, and human double minute (HDM)-2 expression compared with hCG alone. Levels of pregnenolone and progesterone increased 2- and 4-fold, respectively, within 6 h of hCG treatment, whereas PFT completely blocked this hCG-induced effect. Estradiol was increased transiently (>10-fold) by hCG plus PFT relative to levels after hCG alone. PFT also inhibited hCG-induced increases in steroidogenic acute regulatory protein and 3β-hydroxysteroid dehydrogenase mRNAs. Similar results were obtained using the human adrenocortical cell line H295R, suggesting that p53 may have a general function in primate steroidogenesis. These data indicate that p53 plays a key role in luteinization of the primate ovarian follicle though the regulation of steroidogenic enzymes leading to progesterone synthesis.

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MiR-214-3p promotes proliferation and inhibits estradiol synthesis in porcine granulosa cells

Journal of Animal Science and Biotechnology ◽

10.1186/s40104-020-00500-y ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Shengjie Shi ◽

Xiaoge Zhou ◽

Jingjing Li ◽

Lutong Zhang ◽

Yamei Hu ◽

...

Keyword(s):

Cell Cycle ◽

Cytochrome P450 ◽

Granulosa Cells ◽

Target Genes ◽

Regulatory Protein ◽

Follicular Development ◽

Luciferase Reporter ◽

Cell Cycle Protein ◽

Cytochrome P450 Family ◽

Estradiol Synthesis

Abstract Background Granulosa cells (GCs) proliferation and estradiol synthesis significantly affect follicular development. The miR-214-3p expression in the ovarian tissues of high-yielding sows is higher than that in low-yielding sows, indicating that miR-214-3p may be involved in sow fertility. However, the functions and mechanisms of miR-214-3p on GCs are unclear. This study focuses on miR-214-3p in terms of the effects on GCs proliferation and estradiol synthesis. Results Our findings revealed that miR-214-3p promotes proliferation and inhibits estradiol synthesis in porcine GCs. MiR-214-3p can increase the percentage of S-phase cells, the number of EdU labeled positive cells, and cell viability. However, E2 concentration was reduced after miR-214-3p agomir treatment. We also found that miR-214-3p up-regulates the expression of cell cycle genes including cell cycle protein B (Cyclin B), cell cycle protein D (Cyclin D), cell cycle protein E (Cyclin E), and cyclin-dependent kinase 4 (CDK4) at the transcription and translation levels, but down-regulates the mRNA and protein levels of cytochrome P450 family 11 subfamily A member 1 (CYP11A1), cytochrome P450 family 19 subfamily A member 1 (CYP19A1), and steroidogenic acute regulatory protein (StAR) (i.e., the key enzymes in estradiol synthesis). On-line prediction, bioinformatics analysis, a luciferase reporter assay, RT-qPCR, and Western blot results showed that the target genes of miR-214-3p in proliferation and estradiol synthesis are Mfn2 and NR5A1, respectively. Conclusions Our findings suggest that miR-214-3p plays an important role in the functional regulation of porcine GCs and therefore may be a target gene for regulating follicular development.

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The Cell-Cycle Regulatory Protein p21CIP1/WAF1 Is Required for Cytolethal Distending Toxin (Cdt)-Induced Apoptosis

Pathogens ◽

10.3390/pathogens9010038 ◽

2020 ◽

Vol 9 (1) ◽

pp. 38 ◽

Cited By ~ 2

Author(s):

Bruce J. Shenker ◽

Lisa M. Walker ◽

Ali Zekavat ◽

Robert H. Weiss ◽

Kathleen Boesze-Battaglia

Keyword(s):

Cell Cycle ◽

Human Lymphocytes ◽

Regulatory Protein ◽

Cytolethal Distending Toxin ◽

The Novel ◽

Cell Cycle Regulatory Protein ◽

Cycle Arrest ◽

Induced Apoptosis ◽

Hsp 90 ◽

Lymphoid Cell Lines

The Aggregatibacter actinomycetemcomitans cytolethal distending toxin (Cdt) induces lymphocytes to undergo cell-cycle arrest and apoptosis; toxicity is dependent upon the active Cdt subunit, CdtB. We now demonstrate that p21CIP1/WAF1 is critical to Cdt-induced apoptosis. Cdt induces increases in the levels of p21CIP1/WAF1 in lymphoid cell lines, Jurkat and MyLa, and in primary human lymphocytes. These increases were dependent upon CdtB’s ability to function as a phosphatidylinositol (PI) 3,4,5-triphosphate (PIP3) phosphatase. It is noteworthy that Cdt-induced increases in the levels of p21CIP1/WAF1 were accompanied by a significant decline in the levels of phosphorylated p21CIP1/WAF1. The significance of Cdt-induced p21CIP1/WAF1 increase was assessed by preventing these changes with a two-pronged approach; pre-incubation with the novel p21CIP1/WAF1 inhibitor, UC2288, and development of a p21CIP1/WAF1-deficient cell line (Jurkatp21−) using clustered regularly interspaced short palindromic repeats (CRISPR)/cas9 gene editing. UC2288 blocked toxin-induced increases in p21CIP1/WAF1, and JurkatWT cells treated with this inhibitor exhibited reduced susceptibility to Cdt-induced apoptosis. Likewise, Jurkatp21− cells failed to undergo toxin-induced apoptosis. The linkage between Cdt, p21CIP1/WAF1, and apoptosis was further established by demonstrating that Cdt-induced increases in levels of the pro-apoptotic proteins Bid, Bax, and Bak were dependent upon p21CIP1/WAF1 as these changes were not observed in Jurkatp21− cells. Finally, we determined that the p21CIP1/WAF1 increases were dependent upon toxin-induced increases in the level and activity of the chaperone heat shock protein (HSP) 90. We propose that p21CIP1/WAF1 plays a key pro-apoptotic role in mediating Cdt-induced toxicity.

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Effects of Osthole on Progesterone Secretion in Chicken Preovulatory Follicles Granulosa Cells

Animals ◽

10.3390/ani10112027 ◽

2020 ◽

Vol 10 (11) ◽

pp. 2027

Author(s):

Na Sun ◽

Yutong Zhang ◽

Yaxin Hou ◽

Yanyan Yi ◽

Jianhua Guo ◽

...

Keyword(s):

Granulosa Cells ◽

Regulatory Protein ◽

Adenosine Monophosphate ◽

Nuclear Antigen ◽

Enzyme Linked Immunosorbent Assay ◽

Active Constituent ◽

Progesterone Secretion ◽

Corticosterone Secretion ◽

Significant Difference ◽

Preovulatory Follicles

Osthole (Ost) is an active constituent of Cnidium monnieri (L.) Cusson which possesses anti-inflammatory and anti-oxidative properties. It also has estrogen-like activity and can stimulate corticosterone secretion. The present study was aimed to check the role of Ost on progesterone (P4) secretion in cultured granulosa cells obtained from hen preovulatory follicles. Different concentrations (5, 2.5, and 1.25 µg/mL) of Ost was added to granulosa cells for 6, 12, 18, and 24 h to investigate the level of progesterone secretions using enzyme linked immunosorbent assay (ELISA). The results showed that progesterone secretion was significantly increased in cells treated with Ost at 2.5 μg/mL. Also, qRT-PCR showed that mRNA expression of steroidogenic acute regulatory protein (StAR) was significantly up-regulated by Ost at 2.5 μg/mL concentration. Cytochrome P450 side-chain cleavage (P450scc) and 3β-hydroxysteroid dehydrogenase (3β-HSD) was significantly up-regulated by Ost. However, no significant differences were observed for the expression of proliferating cell nuclear antigen (PCNA). The protein expression of StAR, P450scc and 3β-HSD were significantly up-regulated by Ost treatment. The concentration of cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) in cell lysates showed no change with Ost treatment at 2.5 μg/mL by ELISA. An ROS kit showed non-significant difference in the level of reactive oxygen species (ROS). In conclusion, Ost treatment at a concentration of 2.5 μg/mL for 24 h had significantly up-regulated P4 secretion by elevating P450scc, 3β-HSD and StAR at both gene and protein level in granulosa cells obtained from hen preovulatory follicles.

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Development of an experimental ovarian tumor: immunocytochemical analysis

Acta Endocrinologica ◽

10.1530/eje.0.1470387 ◽

2002 ◽

pp. 387-395 ◽

Cited By ~ 3

Author(s):

E Sorianello ◽

S Fritz ◽

C Beyer ◽

DB Hales ◽

A Mayerhofer ◽

...

Keyword(s):

Granulosa Cells ◽

Regulatory Protein ◽

Tumor Development ◽

Female Rats ◽

Nuclear Antigen ◽

Normal Ovary ◽

Rt Pcr ◽

Thecal Cells ◽

Junction Protein ◽

Steroidogenic Acute Regulatory

OBJECTIVE: The aim of the present work was to study whether immunocytochemical parameters present in the normal ovary were altered after tumor development under high gonadotropin levels. METHODS: Ovarian tumors (luteoma): castrated female rats had an ovary grafted into the spleen; tumors were left to develop for 1, 2, 3 or 7 months. The presence of apoptotic cells (TUNEL method) and the expression of proliferating cell nuclear antigen (PCNA), gap junction protein (Cx43), steroidogenic acute regulatory protein (StAR), aromatase and synaptosome-associated protein of 25 kDa (SNAP-25) were determined by immunocytochemistry. Some of these findings were confirmed by RT-PCR (Cx43, StAR, SNAP-25). Inhibin subunit mRNAs were investigated by Northern blot. RESULTS: PCNA staining of tumors was mainly found in granulosa cells of transforming follicles and was absent from luteinized follicles. A nearly complete absence of apoptosis was observed. Cx43 was mainly found in follicles, while it was very weakly expressed or absent in luteinized follicles. StAR protein expression, indicating active steroidogenesis, was demonstrated only in luteinized follicles and in thecal cells, but was absent from granulosa cells. Aromatase immunoreactivity was very intense in granulosa and also present in luteal cells. Membrane-associated and cytoplasmic SNAP-25 immunostaining was determined in patches of endocrine cells in the follicles, as well as in the luteinized follicles. The expression of mRNAs for Cx43, StAR and SNAP-25 (RT-PCR) and inhibin subunits (Northern blots) were confirmed in 1-, 3- and 7-month-old tumors. CONCLUSIONS: These results indicated that luteoma most likely develop from unruptured follicles by hypertrophy and proliferation of follicular cells. Circulating gonadotropins seem to play a fundamental role in maintaining the expression of proteins typically expressed in normal ovary, while avoiding apoptosis in this tissue.

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SMAD3 regulates the diverse functions of rat granulosa cells relating to the FSHR/PKA signaling pathway

Reproduction ◽

10.1530/rep-12-0325 ◽

2013 ◽

Vol 146 (2) ◽

pp. 169-179 ◽

Cited By ~ 15

Author(s):

Yexia Li ◽

Yujie Jin ◽

Yuxia Liu ◽

Chunyan Shen ◽

Jingxia Dong ◽

...

Keyword(s):

Granulosa Cells ◽

Transforming Growth Factor ◽

Ovarian Follicle ◽

Transforming Growth Factor Β ◽

Female Rats ◽

Nuclear Antigen ◽

Follicle Development ◽

Sprague Dawley ◽

Ovarian Follicle Development ◽

Estrogen Production

The function of Smad3, a downstream signaling protein of the transforming growth factor β (TGFβ) pathway, in ovarian follicle development remains to be elucidated. The effects of Smad3 on ovarian granulosa cells (GCs) in rat were studied. Female rats (21 days of age Sprague–Dawley) received i.p. injections of pregnant mare serum gonadotropin, and GCs were harvested for primary culture 48 h later. These cells were engineered to overexpress or knockdown Smad3, which were validated by immunohistochemistry and western blot. The expression of proliferating cell nuclear antigen (PCNA), cyclin D2, TGFβ receptor II (TGFβRII), protein kinase A (PKA), and FSH receptor (FSHR) was also detected by western blotting. Cell cycle and apoptosis of GCs were assayed by flow cytometry. The level of estrogen secreted by GCs was detected by ELISA. Smad3 overexpression promoted estrogen production and proliferation while inhibiting apoptosis of GCs. Reduction in Smad3 by RNAi resulted in reduced estrogen production and proliferation and increased apoptosis of GCs. Manipulation of Smad3 expression also resulted in changes in FSHR and PKA expression, suggesting that the effects of Smad3 on follicle development are related to FSHR-mediated cAMP signaling.

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HO-1 reduces heat stress-induced apoptosis in bovine granulosa cells by suppressing oxidative stress

Aging ◽

10.18632/aging.102136 ◽

2019 ◽

Vol 11 (15) ◽

pp. 5535-5547 ◽

Cited By ~ 6

Author(s):

Yiru Wang ◽

Caixia Yang ◽

Nahla Abdalla Hassan Elsheikh ◽

Chengmin Li ◽

Fangxiao Yang ◽

...

Keyword(s):

Oxidative Stress ◽

Heat Stress ◽

Granulosa Cells ◽

Induced Apoptosis

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The susceptibility of granulosa cells to apoptosis is influenced by oestradiol and the cell cycle

Journal of Endocrinology ◽

10.1677/joe.1.06549 ◽

2006 ◽

Vol 189 (3) ◽

pp. 441-453 ◽

Cited By ~ 59

Author(s):

Susan M Quirk ◽

Robert G Cowan ◽

Rebecca M Harman

Keyword(s):

Cell Cycle ◽

Granulosa Cells ◽

Cell Cycle Progression ◽

Fas Ligand ◽

S Phase ◽

G1 Phase ◽

Cycle Progression ◽

Induced Apoptosis ◽

Cell Proliferation Marker ◽

D2 Protein

Experiments were conducted to test whether oestradiol (E2) protects granulosa cells from Fas ligand (FasL)-induced apoptosis and whether protection involves modulation of the cell cycle of proliferation. Treatment of cultured bovine granulosa cells with E2 decreased susceptibility to FasL-induced apoptosis. The effects of E2 were mediated through oestrogen receptor and were not mediated by stimulation of IGF production. E2 also increased the percentage of cells progressing from G1 to S phase of the cell cycle, and increased expression of cyclin D2 protein and the cell proliferation marker Ki67. Progression from G1 to S phase of the cell cycle was necessary for the protective effect of E2; blocking progression from G1 to S phase with the cdk2 inhibitor roscovitine, or blocking cells in S phase with hydroxyurea, prevented protection by E2. The stages of the cell cycle during which granulosa cells are susceptible to apoptosis were assessed. First, treatment with the G1 phase blocker, mimosine, protected cells from FasL-induced apoptosis, indicating that cells in G0 or early- to mid-G1 phase are relatively resistant to apoptosis. Secondly, examination of recent DNA synthesis by cells that became apoptotic indicated that apoptosis did not occur in S, G2 or M phases. Taken together, the experiments indicate that cells may be most susceptible to apoptosis at the transition from G1 to S phase. E2 stimulates transition from G1 to S phase and protects against apoptosis only when cell cycle progression is unperturbed.

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2 TRANSCRIPTOME ANALYSIS OF GRANULOSA CELLS FROM GROWING DOMINANT FOLLICLE REVEALS AGE-ASSOCIATED CHANGES AT THE TIME OF FOLLICLE SELECTION IN AGED BEEF CATTLE

Reproduction Fertility and Development ◽

10.1071/rdv25n1ab2 ◽

2013 ◽

Vol 25 (1) ◽

pp. 148 ◽

Cited By ~ 3

Author(s):

M. I. R. Khan ◽

F. C. F. Dias ◽

M. A. Sirard ◽

G. P. Adams ◽

J. Singh

Keyword(s):

Cytochrome P450 ◽

Granulosa Cells ◽

Prostaglandin F2α ◽

Differentially Expressed ◽

Nuclear Antigen ◽

Dominant Follicle ◽

Age Related ◽

Junction Protein ◽

Cytochrome P450 Family ◽

Follicle Selection

Growing dominant follicles at the time of selection from aged (n = 3; 15 ± 1.5 years) and young (n = 3; 6 ± 1.1 years) Hereford cows were compared using bovine-specific microarrays containing 40 000 targets. The objective of the study was to determine age-associated changes in transcriptome of granulosa cells at the time of dominant follicle selection. Cows were given prostaglandin F2α to cause ovulation (Day 0) and granulosa cells from dominant follicles were collected on Day 3 either by ultrasound-guided follicle aspiration or after ovariectomy. The mRNA was extracted, analyzed for quality, converted into antisense RNA, amplified, labelled with red and green florescent dyes, and hybridized with microarrays. Feature intensities were measured using Array-Pro software (Media Cybernetics Inc., Rockville, MD), and differentially expressed genes were obtained using FlexArray 1.6. A total of 169 transcripts were differentially expressed with a fold change of ≥2 (P ≤ 0.05) in aged cows v. young cows. Ingenuity System Pathway (IPA; Ingenuity Systems Inc., Redwood City, CA) analysis of these transcripts revealed that granulosa cells of aged cows exhibit (1) reduced capability to regulate gonadotropins [↓follistatin (FST), ↓inhibin beta A (INHBA), ↓inhibin beta B (INHBB)] and reduced responsiveness to gonadotropin-induced changes in cytoskeleton [↑tropomyosin 2 (TPM2), ↑actin gamma 2 (ACTG2), ↓tubulin beta] and extracellular matrix [↓tumor necrosis factor alpha-induced protein 6 (TNFAIP6), ↓versican (VCAN)], (2) inefficiency in processing lipids [↓low-density lipoprotein receptor (LDLR), ↓stearoyl-coenzyme A desaturase (SCD), ↑cluster of differentiation 36 (CD36), ↓sterol-C4-methyl oxidase-like (SC4MOL)] and synthesizing steroids [↓cytochrome P450, family 19, subfamily A, polypeptide 1 (CYP19A1), ↓cytochrome P450, family 51, subfamily A, polypeptide 1 (CYP51A1)], (3) decreased proliferation [↓proliferating cell nuclear antigen (PCNA)] and control of cell cycle check points [↓checkpoint kinase 1 (CHEK1), ↓centromere-associated protein E (CENPE)] and have poor intercellular communication [↓gap junction protein alpha 1 (GJA1)], and (4) higher expression of oxidative stress responsive genes [↑vanin-1 (VNN1), ↑vanin-2 (VNN2), and ↑glutathione peroxidase 3 (GPX3)]. A total of 6 transcripts: CYP19A1 (aromatase; P ≤ 0.1), VNN1 (P ≤ 0.05), INHBA (P ≥ 0.1), PCNA (P ≤ 0.001), TPM2 (P ≤ 0.1), and GJA1 (P ≤ 0.05) were selected to validate the microarray results via quantitative real-time PCR. In conclusion, granulosa cells of growing dominant follicles exhibit age-related changes in the transcriptome at the time of selection relative to young cows; changes that may explain follicle-associated loss of oocyte competence in aged cows. Research was supported by the Natural Sciences and Engineering Research Council of Canada (Ottawa, ON, Canada).

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Progesterone Receptor and the Cell Cycle Modulate Apoptosis in Granulosa Cells

Endocrinology ◽

10.1210/en.2004-0140 ◽

2004 ◽

Vol 145 (11) ◽

pp. 5033-5043 ◽

Cited By ~ 45

Author(s):

Susan M. Quirk ◽

Robert G. Cowan ◽

Rebecca M. Harman

Keyword(s):

Cell Cycle ◽

Progesterone Receptor ◽

Granulosa Cells ◽

Fas Ligand ◽

Lh Surge ◽

Theca Cells ◽

Development Of Resistance ◽

Preovulatory Follicles ◽

Induced Apoptosis ◽

Antagonist Ru486

Abstract Our previous studies showed that exposure of bovine preovulatory follicles to the LH surge-induced resistance of granulosa cells, but not theca cells, to apoptosis. Here, the temporal development of resistance to apoptosis and potential roles of progesterone receptor (PR) and alterations in the cell cycle in mediating this effect were examined. Injection of cows with GnRH induced an LH surge within 2 h. Granulosa cells isolated 0, 6, and 10 h after GnRH were sensitive to Fas ligand-induced apoptosis, but cells isolated at 14 h were resistant. PR was first detectable in granulosa cells at 10 and 14 h and was not detectable in theca. Treatment of granulosa cells isolated 14 h after GnRH with the PR antagonist, RU486, induced susceptibility to apoptosis, an effect mediated by PR and not glucocorticoid receptor. After GnRH treatment, granulosa cells, but not theca cells, exited the cell cycle, expression of cyclin D2 was reduced, and p27Kip1 was elevated. Treatment of granulosa cells isolated from small antral follicles with the G1 phase blocker, mimosine, reduced Fas ligand-induced killing, suggesting that nonproliferating cells are resistant to apoptosis. Treatment of granulosa cells isolated 14 h after GnRH with RU486 induced reentry of some cells into the cell cycle and reversed resistance to apoptosis, suggesting that cycling cells became susceptible to apoptosis. Treatment with mimosine prevented the ability of RU486 to promote susceptibility to apoptosis. In summary, the LH surge induces expression of PR by granulosa cells and withdrawal from the cell cycle, and these events promote resistance to apoptosis.

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