Mitochondrial humanin peptide acts as a cytoprotective factor in granulosa cell survival

Reproduction ◽  
2021 ◽  
Vol 161 (5) ◽  
pp. 581-591
Author(s):  
Carolina Marvaldi ◽  
Daniel Martin ◽  
Julia G Conte ◽  
María Florencia Gottardo ◽  
Matías L Pidre ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Cell Survival ◽  
Cell Fate ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Recombinant Baculovirus ◽  
Tumor Cell Line ◽  
Hairpin Rna ◽  
Tumor Cell Behavior ◽  
Critical Process

Humanin (HN) is a short peptide involved in many biological processes such as apoptosis, cell survival, inflammatory response, and reaction to stressors like oxidative stress, between others. In the ovary, a correct balance between pro- and anti-apoptotic factors is crucial for folliculogenesis. In the follicular atresia, survival or death of granulosa cells is a critical process. The goal of this study was to evaluate the action of HN on granulosa cell fate. To explore endogenous HN function in the ovary, we used a recombinant baculovirus (BV) encoding a short-hairpin RNA targeted to silence HN (shHN). HN downregulation modified ovarian histoarchitecture and increased apoptosis of granulosa cells. HN was also detected in a granulosa tumor cell line (KGN). Transduction of KGN cells with BV-shHN resulted in HN downregulation and increased apoptosis. On the other hand, treatment of KGN cells with exogenous HN increased cell viability and decreased apoptosis. In summary, these findings indicate that HN is a cytoprotective factor in granulosa cells of antral follicles, suggesting that this peptide would be involved in the regulation of folliculogenesis. Also, this peptide is a cytoprotective factor in KGN cells, and therefore, it could be involved in granulosa tumor cell behavior.

Effect of in vitro copper supplementation on granulosa cell estradiol synthesis and associated genes

2018 ◽  
Author(s):  
Ravi, P.S.P. Gupta, S. Nandi, S. Mondal, Kumar Soni­ ◽  
P.S.P. Gupta ◽  
S. Nandi ◽  
S. Mondal, J.R. Ippala, Avantika Mor, A Mondal ◽  
J.R. Ippala ◽  
...  
Keyword(s):  
Cell Survival ◽  
Mrna Expression ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Culture Medium ◽  
Estrus Synchronization ◽  
Ovarian Granulosa Cells ◽  
Effect Of Copper ◽  
Estradiol Synthesis

The study was conducted by supplementing cupric chloride dihydrate to modulate the estradiol synthesis in granulosa cells with a hypothesis of possible use of copper to potentiate or partially replace the hormones for estrus induction / estrus synchronization in future studies. In present study copper at three doses (0.1, 0.5 and 1 mM level in culture medium) were tested to deserve see their effects on in vitro granulosa cell survival, estradiol synthesis and their associated genes of ovarian granulosa cells of goat.There was no effect of copper on the ovarian granulosa cell survival rate. There was a considerable increase in the estradiol level per ml culture medium basis by 6th day of in vitro culture with the second dose of copper i.e. 0.5 mM, but the increase was non-significant (P greator than 0.05). There was no significant effect of copper on estradiol synthesis when expressed on per 30000 cell basis. Effect of copper (0.1 mM and 0.5 mM) on the mRNA expression of genes of aromatase (CYP19A1) and cyclin D2 (CCND2) was estimated. Copper had significantly (P less than 0.05) increased the mRNA expression of CCND2 and CYP19A1in ovarian granulosa cells with only one of the two doses tested i.e. 0.5 mM. Hence, copper can be considered as a potential mineral to supplement along with hormones in estrus induction or estrus synchronization protocols to minimize the use of hormones.

scholarly journals The Chromatin State during Gonadal Sex Determination

2021 ◽  
pp. 1-9
Author(s):  
Shannon Dupont ◽  
Blanche Capel
Keyword(s):  
Sex Determination ◽  
Cell Fate ◽  
Granulosa Cell ◽  
Histone Modifications ◽  
Granulosa Cells ◽  
Gonad Development ◽  
Chromatin State ◽  
Supporting Cells ◽  
Direct Role ◽  
Specific Factors

At embryonic day (E) 10.5, prior to gonadal sex determination, XX and XY gonads are bipotential and able to differentiate into either a testis or an ovary. At this point, they are transcriptionally and morphologically indistinguishable. Sex determination begins around E11.5 in the mouse when the supporting cell lineage commits to either Sertoli or granulosa cell fate. Testis-specific factors such as SRY and SOX9 drive differentiation of bipotential-supporting cells into the Sertoli cell pathway, whereas ovary-specific factors like WNT4 and FOXL2 guide differentiation into granulosa cells. It is known that these 2 pathways are mutually antagonistic, and repression of the alternative fate is critical for maintenance of the testis or ovary programs. While we understand much about the transcription factor networks guiding the process of sex determination, it is only more recently that we have begun to understand how this process is epigenetically controlled. Studies in the past decade have demonstrated the importance of the chromatin state for gene expression and cell fate commitment, with histone modifications and DNA accessibility having a direct role in gene regulation. It is now clear that the chromatin state during sex determination is dynamic and likely critical for the establishment and/or maintenance of the transcriptional programs. Prior to sex determination, supporting cells have similar chromatin structure and histone modification profiles, reflecting the bipotential nature of these cells. After differentiation to Sertoli or granulosa cells, the chromatin state acquires sex-specific profiles. The proteins that regulate the deposition of histone modifications or the opening of compact chromatin likely play an important role in Sertoli and granulosa cell fate commitment and gonad development. Here, we describe studies profiling the chromatin state during gonadal sex determination and one example in which depletion of <i>Cbx2</i>, a member of the Polycomb Repressive Complex 1 (PRC1), causes male-to-female sex reversal due to a failure to repress the ovarian pathway.

scholarly journals GATA-4 Regulates Bcl-2 Expression in Ovarian Granulosa Cell Tumors

Endocrinology ◽  
2008 ◽  
Vol 149 (11) ◽  
pp. 5635-5642 ◽  
Author(s):  
Antti Kyrönlahti ◽  
Maarit Rämö ◽  
Maija Tamminen ◽  
Leila Unkila-Kallio ◽  
Ralf Butzow ◽  
...  
Keyword(s):  
Cell Fate ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Dominant Negative ◽  
Rt Pcr ◽  
Cyclin D2 ◽  
Granulosa Cell Tumors ◽  
Ovarian Granulosa Cell ◽  
Cell Cycle Regulator Cyclin

Excessive cell proliferation and decreased apoptosis have been implicated in the pathogenesis of ovarian granulosa cell tumors (GCTs). We hypothesized that transcription factor GATA-4 controls expression of the antiapoptotic factor Bcl-2 and the cell cycle regulator cyclin D2 in normal and neoplastic granulosa cells. To test this hypothesis, a tissue microarray based on 80 GCTs was subjected to immunohistochemistry for GATA-4, Bcl-2, and cyclin D2, and the data were correlated to clinical and histopathological parameters. In addition, quantitative RT-PCR for GATA-4, Bcl-2, and cyclin D2 was performed on 21 human GCTs. A mouse GCT model was used to complement these studies. The role of GATA-4 in the regulation of Bcl2 and ccdn2 (coding for cyclin D2) was studied by transactivation assays, and by disrupting GATA-4 function with dominant negative approaches in mouse and human GCT cell lines. We found that GATA-4 expression correlated with Bcl-2 and cyclin D2 expression in human and murine GCTs. Moreover, GATA-4 enhanced Bcl-2 and cyclin D2 promoter activity in murine GCT cells. Whereas GATA-4 overexpression up-regulated and dominant negative GATA-4 suppressed Bcl-2 expression in human GCT cells, the effects on cyclin D2 were negligible. Our results reveal a previously unknown relationship between GATA-4 and Bcl-2 in mammalian granulosa cells and GCTs, and suggest that GATA-4 influences granulosa cell fate by transactivating Bcl-2.

scholarly journals Saturated FFAs, Palmitic Acid and Stearic Acid, Induce Apoptosis in Human Granulosa Cells

Endocrinology ◽  
2001 ◽  
Vol 142 (8) ◽  
pp. 3590-3597 ◽  
Author(s):  
Yi-Ming Mu ◽  
Toshihiko Yanase ◽  
Yoshihiro Nishi ◽  
Atsushi Tanaka ◽  
Masayuki Saito ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Arachidonic Acid ◽  
Stearic Acid ◽  
Palmitic Acid ◽  
Cell Survival ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Critical Role ◽  
Dependent Manner ◽  
Human Granulosa Cells

Abstract Obesity is associated with insulin resistance and some reproductive abnormalities. Circulating FFAs are often elevated in obese subjects and are also closely linked to insulin resistance. In this study, we demonstrated that saturated FFAs, such as palmitic acid and stearic acid, markedly suppressed the granulosa cell survival in a time- and dose-dependent manner. Polyunsaturated FFA, arachidonic acid, had no effect on the cell survival, even at supraphysiological concentrations. The suppressive effect of saturated FFAs on cell survival was caused by apoptosis, as evidenced by DNA ladder formation and annexin V-EGFP/propidium iodide staining of the cells. The apoptotic effects of palmitic acid and stearic acid were unrelated to the increase of ceramide generation or nitric oxide production and were also completely blocked by Triacsin C, an inhibitor of acylcoenzyme A synthetase. In addition, acylcoenzyme A, pamitoylcoenzyme A, and stearylcoenzyme A markedly suppressed granulosa cell survival, whereas arachidonoylcoenzyme A had no such effect, and this finding was consistent with the effect of the respective FFA form. Surprisingly, arachidonic acid instead showed a protective effect on palmitic acid- and stearic acid-induced cell apoptosis. A Western blot analysis showed the apoptosis of the granulosa cells induced by palmitic acid to be accompanied by the down-regulation of an apoptosis inhibitor, Bcl-2, and the up-regulation of an apoptosis effector, Bax. These results indicate that saturated FFAs induce apoptosis in human granulosa cells caused by the metabolism of the respective acylcoenzyme A form, and the actual composition of circulating FFAs may thus play a critical role in the apoptotic events of human granulosa cells. These effects of FFAs on granulosa cell survival may be a possible mechanism for reproductive abnormalities, such as amenorrhea, which is frequently observed in obese women.

scholarly journals Granulosa Cell Survival and Proliferation Are Altered in Polycystic Ovary Syndrome

2008 ◽  
Vol 93 (3) ◽  
pp. 881-887 ◽  
Author(s):  
M. Das ◽  
O. Djahanbakhch ◽  
B. Hacihanefioglu ◽  
E. Saridogan ◽  
M. Ikram ◽  
...  
Keyword(s):  
Gene Expression ◽  
Polycystic Ovary Syndrome ◽  
Cell Survival ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Polycystic Ovary ◽  
Reproductive Age ◽  
Ovary Syndrome ◽  
Survival Factor ◽  
Effector Caspase

Abstract Context: Polycystic ovary syndrome (PCOS) represents the most common endocrine abnormality in women of reproductive age. The cause of PCOS remains largely unknown, but studies suggest an intrinsic ovarian abnormality. Objective: The objective of the study was to test our hypothesis that differences in granulosa cell proliferation and apoptosis may underlie abnormalities that affect follicular development. Design: Granulosa cells were prepared from follicular fluid aspirated from 4- to 8-mm follicles of unstimulated ovaries during routine laparoscopy or laparotomy from women with anovulatory PCOS and those with regular ovulatory cycles. Setting: The study was conducted at a university hospital. Patients: Fourteen women with anovulatory PCOS and nine women with regular ovulatory cycles participated in the study. Main Outcome Measures: Immunocytochemistry on granulosa cells to investigate apoptotic and proliferation rates, together with real-time RT-PCR to analyze gene expression profiles of apoptotic regulators, was measured. Results: Significantly lower apoptotic rates were found in granulosa cells from patients with PCOS, compared with women with regular ovulatory cycles (P = 0.004). Lower apoptotic rates were associated with decreased levels of the apoptotic effector caspase-3 (P = 0.001) and increased levels of the anti-apoptotic survival factor cellular inhibitor of apoptosis proteins-2 in the PCOS group that were coupled to higher proliferation rates (P = 0.032). Gene expression profiling confirmed the immunocytochemical findings. Conclusions: Our findings indicate that there are significant differences in the rate of cell death and proliferation in granulosa cell populations in PCOS patients. These are associated with decreased expression of apoptotic effectors and increased expression of a cell survival factor. These results provide new insights that may be useful in developing specific therapeutic intervention strategies in PCOS.

scholarly journals FOXO1/3 and PTEN Depletion in Granulosa Cells Promotes Ovarian Granulosa Cell Tumor Development

2015 ◽  
Vol 29 (7) ◽  
pp. 1006-1024 ◽  
Author(s):  
Zhilin Liu ◽  
Yi A. Ren ◽  
Stephanie A. Pangas ◽  
Jaye Adams ◽  
Wei Zhou ◽  
...  
Keyword(s):  
Cell Fate ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Mammalian Cells ◽  
Granulosa Cell Tumor ◽  
Cell Tumor ◽  
Pten Gene ◽  
Cell Fate Decisions ◽  
Activin Signaling ◽  
Human Adult

Abstract The forkhead box (FOX), FOXO1 and FOXO3, transcription factors regulate multiple functions in mammalian cells. Selective inactivation of the Foxo1 and Foxo3 genes in murine ovarian granulosa cells severely impairs follicular development and apoptosis causing infertility, and as shown here, granulosa cell tumor (GCT) formation. Coordinate depletion of the tumor suppressor Pten gene in the Foxo1/3 strain enhanced the penetrance and onset of GCT formation. Immunostaining and Western blot analyses confirmed FOXO1 and phosphatase and tensin homolog (PTEN) depletion, maintenance of globin transcription factor (GATA) 4 and nuclear localization of FOXL2 and phosphorylated small mothers against decapentaplegic (SMAD) 2/3 in the tumor cells, recapitulating results we observed in human adult GCTs. Microarray and quantitative PCR analyses of mouse GCTs further confirmed expression of specific genes (Foxl2, Gata4, and Wnt4) controlling granulosa cell fate specification and proliferation, whereas others (Emx2, Nr0b1, Rspo1, and Wt1) were suppressed. Key genes (Amh, Bmp2, and Fshr) controlling follicle growth, apoptosis, and differentiation were also suppressed. Inhbb and Grem1 were selectively elevated, whereas reduction of Inha provided additional evidence that activin signaling and small mothers against decapentaplegic (SMAD) 2/3 phosphorylation impact GCT formation. Unexpectedly, markers of Sertoli/epithelial cells (SRY [sex determining region Y]-box 9/keratin 8) and alternatively activated macrophages (chitinase 3-like 3) were elevated in discrete subpopulations within the mouse GCTs, indicating that Foxo1/3/Pten depletion not only leads to GCTs but also to altered granulosa cell fate decisions and immune responses. Thus, analyses of the Foxo1/3/Pten mouse GCTs and human adult GCTs provide strong evidence that impaired functions of the FOXO1/3/PTEN pathways lead to dramatic changes in the molecular program within granulosa cells, chronic activin signaling in the presence of FOXL2 and GATA4, and tumor formation.

Morroniside suppresses hydrogen peroxide-stimulated autophagy and apoptosis in rat ovarian granulosa cells through the PI3K/AKT/mTOR pathway

2020 ◽  
pp. 096032712096076
Author(s):  
D Deng ◽  
J Yan ◽  
Y Wu ◽  
K Wu ◽  
W Li
Keyword(s):  
Oxidative Stress ◽  
Hydrogen Peroxide ◽  
Cell Survival ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Cell Apoptosis ◽  
Critical Role ◽  
Mtor Pathway ◽  
Dependent Manner ◽  
Ovarian Granulosa Cells

Previous evidences have indicated that granulosa cells play a critical role in follicular growth. Hydrogen peroxide (H2O2)-induced oxidative stress has been associated with ovarian granulosa cell apoptosis and ovarian function. Recently, a study highlighted the protective role of morroniside against H2O2-induced damage. In this study, we aimed to investigate the effects of morroniside on H2O2-stimulated rat ovarian granulosa cells and its underlying molecular mechanisms. Our results showed that H2O2 treatment suppressed cell survival and increased apoptosis in rat granulosa cells, while treatment with morroniside markedly increased H2O2-induced granulosa cell survival in a dose-dependent manner (0, 10, 50 and 100 µM). Moreover, treatment with 50 µM morroniside impeded H2O2-induced cell apoptosis. An elevation in intracellular ROS, MDA, SOD, GSH-Px, and CAT level was observed in H2O2-induced granulosa cells; however, this effect was abrogated by morroniside treatment. Further studies suggested that administration of morroniside inhibited H2O2-induced granulosa cell apoptosis and caspase-3 activity. In addition, after morroniside treatment of H2O2-stimulated granulosa cells, autophagy-related protein (LC3-II/LC3-I ratio) and beclin-1 expression was decreased and p62 level was increased. Interestingly, we found that morroniside treatment activated the PI3K/AKT/mTOR pathway in H2O2-stimulated granulosa cells. Finally, we showed that treatment with PI3K and mTOR inhibitors reversed the protective effects of morroniside on H2O2-induced granulosa cells. Taken together, our data suggest that treatment with morroniside decreased apoptosis, autophagy, and oxidative stress in rat granulosa cells through the PI3K/AKT/mTOR pathway.

scholarly journals Ovulatory signals alter granulosa cell behavior through YAP1 signaling

2019 ◽  
Vol 17 (1) ◽  
Author(s):  
Tianyanxin Sun ◽  
Francisco J. Diaz
Keyword(s):  
Cell Proliferation ◽  
Cell Survival ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Ovarian Function ◽  
Hippo Pathway ◽  
Cell Viability Assay ◽  
Post Hoc

Abstract Background The Hippo pathway plays critical roles in regulating cell proliferation, differentiation and survival among species. Hippo pathway proteins are expressed in the ovary and are involved in ovarian function. Deletion of Lats1 causes germ cell loss, ovarian stromal tumors and reduced fertility. Ovarian fragmentation induces nuclear YAP1 accumulation and increased follicular development. At ovulation, follicular cells stop proliferating and terminally differentiate, but the mechanisms controlling this transition are not completely known. Here we explore the role of Hippo signaling in mouse granulosa cells before and during ovulation. Methods To assess the effect of oocytes on Hippo transcripts in cumulus cells, cumulus granulosa cells were cultured with oocytes and cumulus oocyte complexes (COCs) were cultured with a pSMAD2/3 inhibitor. Secondly, to evaluate the criticality of YAP1 on granulosa cell proliferation, mural granulosa cells were cultured with oocytes, YAP1-TEAD inhibitor verteporfin or both, followed by cell viability assay. Next, COCs were cultured with verteporfin to reveal its role during cumulus expansion. Media progesterone levels were measured using ELISA assay and Hippo transcripts and expansion signatures from COCs were assessed. Lastly, the effects of ovulatory signals (EGF in vitro and hCG in vivo) on Hippo protein levels and phosphorylation were examined. Throughout, transcripts were quantified by qRT-PCR and proteins were quantified by immunoblotting. Data were analyzed by student’s t-test or one-way ANOVA followed by Tukey’s post-hoc test or Dunnett’s post-hoc test. Results Our data show that before ovulation oocytes inhibit expression of Hippo transcripts and promote granulosa cell survival likely through YAP1. Moreover, the YAP1 inhibitor verteporfin, triggers premature differentiation as indicated by upregulation of expansion transcripts and increased progesterone production from COCs in vitro. In vivo, ovulatory signals cause an increase in abundance of Hippo transcripts and stimulate Hippo pathway activity as indicated by increased phosphorylation of the Hippo targets YAP1 and WWTR1 in the ovary. In vitro, EGF causes a transient increase in YAP1 phosphorylation followed by decreased YAP1 protein with only modest effects on WWTR1 in COCs. Conclusions Our results support a YAP1-mediated mechanism that controls cell survival and differentiation of granulosa cells during ovulation.

CD98hc expression to predict prognosis in renal cell cancer.

2012 ◽  
Vol 30 (30_suppl) ◽  
pp. 26-26
Author(s):  
Gerald Prager ◽  
Marina Poettler
Keyword(s):  
Tumor Cell ◽  
Cell Survival ◽  
Renal Cancer ◽  
Clear Cell ◽  
Metastatic Tumor ◽  
Silent Mutation ◽  
Cell Behavior ◽  
Metastatic Tumor Cell ◽  
Early Apoptosis ◽  
Tumor Cell Behavior

26 Background: CD98, a transmembrane protein, has a heteromeric structure, consisting of a heavy subunit (CD98hc) and a light subunit, extracellular linked together via disulfid bounds. A genetic knockout of CD98hc is embryonic lethal and overexpression of CD98hc in somatic cells led to malignant transformation. CD98hc is highly expressed in low differentiated papillary, clear cell and chromophobe RCC, but not in benign tumors. Notably, CD98hc expression directly correlated with grade of differentiation. Methods: To evaluate a potential functional role of CD98hc in renal cancer cell metastatic behavior, we generated a stable low CD98hc clear cell RCC cell line (Caki2) via lentiviral shRNA infection and compared tumor cell behavior with a high expressing mock transfected control. Results: We found that tumor cell behavior such as proliferation (52 ± 3% less 3[H] thymidin – incorporation in low CD98hc/Caki2 cells), cell survival upon anoikis (46% late and 45% early apoptosis in low CD98hc/Caki2 cells compared to high/CD98hc/Caki2 cells with 18% late apoptosis and 64% early apoptosis) and invasion/transmigration (520 ± 67 cells / field after 24h in low CD98hc/ Caki2 cells and 1257 ± 346 cells / field in high CD98hc/Caki2 cells analyzed in a modified boyden chamber) were considerably impaired whenever CD98hc expression was downregulated. To examine the mechanism by which CD98hc affected metastatic tumor cell behavior, we introduced two mutations in a reconstitution (silent mutation). A truncation mutant interfered with integrin interaction and a two point mutations (Cys109Ser and Cys330Ser) mutant affected amino acid transporter activity. Whenever integrin/CD98hc interaction was impaired tumor cell behavior including cell proliferation, cell survival, invasio/transmigration, and cell spreading as well as signal transduction pathways including FAK, c-src, MEK/ERK pathways were significantly compromised resembling the low CD98hc phenotype. Conclusions: For these data we conclude that metastatic tumor cell behavior such as cell survival, invasion and proliferation are dependent on CD98hc expression in renal cancer cells.

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