scholarly journals Integrative Proteomic and Phosphoproteomic Analyses of Granulosa Cells During Follicular Atresia in Porcine

2021 ◽  
Vol 8 ◽  
Author(s):  
Feng Yang ◽  
Qiang Liu ◽  
Yanhong Chen ◽  
Huizhen Ye ◽  
Han Wang ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Beta Catenin ◽  
Phosphorylation Sites ◽  
Differentially Expressed Proteins ◽  
Follicular Atresia ◽  
Dynamic Changes ◽  
Phosphorylated Proteins ◽  
Apoptosis Regulation ◽  
Quality Categories ◽  
Regulation Of Actin Cytoskeleton

Ovarian follicular atresia is a natural physiological process; however, the mechanism is not fully understood. In this study, quantitative proteomic and phosphoproteomic analyses of granulosa cells (GCs) in healthy (H), slightly atretic (SA), and atretic follicles (A) of porcine were performed by TMT labeling, enrichment of phosphopeptides, and liquid chromatography with tandem mass spectrometry (LC–MS/MS) analysis. In total, 6,201 proteins were quantified, and 4,723 phosphorylation sites of 1,760 proteins were quantified. In total, 24 (11 up, 13 down) and 50 (29 up, 21 down) proteins with a fold change (FC) > 5 were identified in H/SA and H/A, respectively. In addition, there were 20 (H/SA, up) and 39 (H/A, up) phosphosites with an FC > 7 that could serve as potential biomarkers for distinguishing different quality categories of follicles. Western blotting and immunofluorescence confirmed the reliability of the proteomic analysis. Some key proteins (e.g., MIF, beta catenin, integrin β2), phosphosites (e.g., S76 of caspase6, S22 and S636 of lamin A/C), pathways (e.g., apoptosis, regulation of actin cytoskeleton pathway), transcription factors (e.g., STAT5A, FOXO1, and BCLAF1), and kinases (e.g., PBK, CDK5, CDK12, and AKT3) involved in the atresia process were revealed via further analysis of the differentially expressed proteins (DEPs) and phosphorylated proteins (DEPPs). Further study showed that mutant caspase6 Ser76 to Ala increased the ratios of cleaved caspase6/caspase6 and cleaved caspase3/caspase3 and dephosphorylation of caspase6 at Ser76 increased cell apoptotic rate, a new potential pathway of follicular atresia. Collectively, the proteomic and phosphoproteomic profiling and functional research in the current study comprehensively analyzed the dynamic changes in protein expression and phosphorylation during follicular atresia and provided some new explanations regarding the regulation of this process.

scholarly journals Integrative proteomic and phosphoproteomic analysis of granulosa cells during follicular atresia in porcine

2020 ◽  
Author(s):  
Feng Yang ◽  
Qiang Liu ◽  
Yanhong Chen ◽  
Huizhen Ye ◽  
Han Wang ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Beta Catenin ◽  
Phosphorylation Sites ◽  
Differentially Expressed Proteins ◽  
Follicular Atresia ◽  
Dynamic Changes ◽  
Phosphorylated Proteins ◽  
Apoptosis Regulation ◽  
Quality Categories ◽  
Regulation Of Actin Cytoskeleton

AbstractOvarian follicular atresia is a natural physiological process; however, the mechanism is not fully understood. In this study, quantitative proteomic and phosphoproteomic analyses of granulosa cells (GC) in healthy (H), slightly atretic (SA), and atretic follicles (A) of porcine were performed by TMT labeling, enrichment of phosphopeptides and LC-MS/MS analysis. In total, 6,201 proteins were quantified and 4,723 phosphorylation sites of 1,760 proteins were quantified. In total, 24 (11 up, 13 down) and 50 (29 up, 21 down) proteins with a fold change (FC) > 5 were identified in H/SA and H/A, respectively. In addition, there were 20 (H/SA, up) and 39 (H/A, up) phosphosites with an FC > 7, that could serve as potential biomarkers for distinguishing different quality categories of follicles. Western blotting and immunofluorescence confirmed the reliability of the proteomic analysis. Some key proteins (e.g., MIF, beta catenin, integrin β2), phosphosites (e.g., S76 of caspase6, S22 and S636 of lamin A/C), pathways (e.g., apoptosis, regulation of actin cytoskeleton pathway), transcription factors (e.g., STAT5A, FOXO1, and BCLAF1), and kinases (e.g., PBK, CDK5, CDK12, AKT3) involved in atresia process were revealed via further analysis of the differentially expressed proteins (DEPs) and phosphorylated proteins (DEPPs). Collectively, the proteomic and phosphoproteomic profiling and functional research in the current study comprehensively analyzed the dynamic changes in protein expression and phosphorylation during follicular atresia and provided some new explanations regarding the regulation of this process.

scholarly journals Dynamics and Regulations of BimEL Ser65 and Thr112 Phosphorylation in Porcine Granulosa Cells during Follicular Atresia

Cells ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 402
Author(s):  
Feng Yang ◽  
Yanhong Chen ◽  
Qiang Liu ◽  
Shizhen Dai ◽  
Shenming Zeng
Keyword(s):  
Heat Stress ◽  
Granulosa Cells ◽  
Kinase Inhibitors ◽  
Phosphorylation Sites ◽  
Follicular Atresia ◽  
Dynamic Changes ◽  
Jnk Pathway ◽  
Regulatory Pathways ◽  
Porcine Granulosa Cells ◽  
Specific Inhibitors

BimEL protein is involved in follicular atresia by regulating granulosa cell apoptosis, but the dynamic changes of BimEL phosphorylation during follicular atresia are poorly understood. The aim of this study was to explore the changes of key BimEL phosphorylation sites and their upstream regulatory pathways. First, the levels of BimEL-Ser65 and BimEL-Thr112 phosphorylation (p-BimEL-S65, p-BimEL-T112) in granulosa cells (GC) from healthy (H), slightly-atretic (SA), and atretic (A) follicles and in cultured GC after different treatments were detected by Western blotting. Next, the effects of the corresponding site mutations of BIM on apoptosis of GC were investigated. Finally, the pathways of two phosphorylation sites were investigated by kinase inhibitors. The results revealed that p-BimEL-S65 levels were higher in GC from H than SA and A, whereas p-BimEL-T112 was reversed. The prosurvival factors like FSH and IGF-1 upregulated the level of p-BimEL-S65, while the proapoptotic factor, heat stress, increased the level of p-BimEL-T112 in cultured GC. Compared with the overexpression of wild BimEL, the apoptotic rate of the GC overexpressed BimEL-S65A (replace Ser65 with Ala) mutant was significantly higher, but the apoptotic rate of the cells overexpressing BimEL-T112A did not differ. In addition, inhibition of the ERK1/2 or JNK pathway by specific inhibitors reduced the levels of p-BimEL-S65 and p-BimEL-T112. In conclusion, the levels of p-BimEL-S65 and p-BimEL-T112 were reversed during follicular atresia. Prosurvival factors promote p-BimEL-S65 levels via ERK1/2 to inhibit GC apoptosis, whereas proapoptotic factor upregulates the level of p-BimEL-T112 via JNK to induce GC apoptosis.

E-cadherin-catenin complex in the rat ovary: cell-specific expression during folliculogenesis and luteal formation

Reproduction ◽  
2000 ◽  
pp. 375-385 ◽  
Author(s):  
K Sundfeldt ◽  
Y Piontkewitz ◽  
H Billig ◽  
L Hedin
Keyword(s):  
Granulosa Cells ◽  
Interstitial Cells ◽  
Ovary Cell ◽  
Beta Catenin ◽  
Oestrogen Treatment ◽  
Altered Expression ◽  
Preantral Follicles ◽  
Rat Ovary ◽  
E Cadherin ◽  
Cell Cell

The cadherins and their cytoplasmic counterparts, the catenins, form the adherens junctions, which are of importance for tissue integrity and barrier functions. The development and maturation of the ovarian follicle is characterized by structural changes, which require altered expression or function of the components involved in cell-cell contacts. The present study examined the cell-specific localization and temporal expression of epithelial cadherin (E-cadherin) and alpha- and beta-catenin during follicular development, ovulation and corpus luteum formation in the immature gonadotrophin- and oestrogen-stimulated rat ovary. Immunohistochemistry and immunoblotting demonstrated the expression of E-cadherin in theca and interstitial cells of immature ovaries before and after injection of equine chorionic gonadotrophin (eCG). E-cadherin was not detected in granulosa cells, except in the preantral follicles located to the inner region of the ovary. The content of E-cadherin in theca and interstitial cells decreased after an ovulatory dose of hCG. Granulosa cells of apoptotic follicles did not express E-cadherin. Oestrogen treatment (diethylstilboestrol) of immature rats for up to 3 days did not result in a measurable expression of E-cadherin in granulosa cells. alpha- and beta-catenin were expressed in all ovarian compartments. The concentration of beta-catenin was constant during the follicular phase, whereas the content of alpha-catenin decreased in granulosa cells after treatment with diethylstilboestrol or hCG. The expression of alpha-catenin was also reduced in theca and interstitial cells after hCG. alpha- and beta-catenin were present in most ovarian cells at all stages of folliculogenesis. Therefore, the catenins have the potential to associate with different members of the cadherin family and to participate in the regulation of cytoskeletal structures and intracellular signalling. The restricted expression of E-cadherin in granulosa cells of preantral follicles indicates a role in the recruitment of these follicles to subsequent cycles. The specific decrease of alpha-catenin in granulosa cells and the reduction of both alpha-catenin and E-cadherin in theca cells of ovulatory follicles might reflect some of the molecular changes in cell-cell adhesion associated with ovulation and luteinization.

scholarly journals Expression and Function of Apoptosis Initiator FOXO3 in Granulosa Cells During Follicular Atresia in Pig Ovaries

2011 ◽  
Vol 57 (1) ◽  
pp. 151-158 ◽  
Author(s):  
Fuko MATSUDA ◽  
Naoko INOUE ◽  
Akihisa MAEDA ◽  
Yuan CHENG ◽  
Takafumi SAI ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Follicular Atresia ◽  
And Function

scholarly journals EGF receptor–mediated FUS phosphorylation promotes its nuclear translocation and fibrotic signaling

2020 ◽  
Vol 219 (9) ◽  
Author(s):  
Manuel Chiusa ◽  
Wen Hu ◽  
Jozef Zienkiewicz ◽  
Xiwu Chen ◽  
Ming-Zhi Zhang ◽  
...  
Keyword(s):  
Egf Receptor ◽  
Nuclear Translocation ◽  
Collagen Iv ◽  
Phosphorylation Sites ◽  
Kidney Fibrosis ◽  
Antifibrotic Therapy ◽  
Phosphorylated Proteins ◽  
Fused In Sarcoma ◽  
A Cell ◽  
Excessive Accumulation

Excessive accumulation of collagen leads to fibrosis. Integrin α1β1 (Itgα1β1) prevents kidney fibrosis by reducing collagen production through inhibition of the EGF receptor (EGFR) that phosphorylates cytoplasmic and nuclear proteins. To elucidate how the Itgα1β1/EGFR axis controls collagen synthesis, we analyzed the levels of nuclear tyrosine phosphorylated proteins in WT and Itgα1-null kidney cells. We show that the phosphorylation of the RNA-DNA binding protein fused in sarcoma (FUS) is higher in Itgα1-null cells. FUS contains EGFR-targeted phosphorylation sites and, in Itgα1-null cells, activated EGFR promotes FUS phosphorylation and nuclear translocation. Nuclear FUS binds to the collagen IV promoter, commencing gene transcription that is reduced by inhibiting EGFR, down-regulating FUS, or expressing FUS mutated in the EGFR-targeted phosphorylation sites. Finally, a cell-penetrating peptide that inhibits FUS nuclear translocation reduces FUS nuclear content and collagen IV transcription. Thus, EGFR-mediated FUS phosphorylation regulates FUS nuclear translocation and transcription of a major profibrotic collagen gene. Targeting FUS nuclear translocation offers a new antifibrotic therapy.

scholarly journals Investigation of the Relation between Follicular Atresia and Granulosa Cells in Terms of Cell Death Mechanisms in Premature Ovarian Failure Model

Proceedings ◽  
2018 ◽  
Vol 2 (25) ◽  
pp. 1529 ◽  
Author(s):  
Damla Akogullari ◽  
Elgin Turkoz Uluer ◽  
H. Seda Vatansever
Keyword(s):  
Cell Death ◽  
Granulosa Cells ◽  
Premature Ovarian Failure ◽  
Ovarian Failure ◽  
Blood Levels ◽  
Failure Model ◽  
Follicular Atresia ◽  
Treatment Protocols ◽  
New Treatment ◽  
Cell Death Mechanisms

Premature Ovarian Failure; is characterized by the dysfunction or early depletion of ovarian reserves due to follicular loss in the ovary in women under age of 40. POF is the important cause of infertility and its etiology is still not clearly understood. Investigation of cell death mechanisms (CDM) that play a role in the follicular atresia (FA) triggered by excessive loss of granulosa cells (GCs) that provide metabolic support for oocyte and follicle development in the ovary will help to understand POF etiology. It was known that apoptosis and autophagy play a role in FA. Recent studies have shown that paraptosis, associated with endoplasmic reticulum stress (ERS), also exist in FA. POF model was established in C57BL/6 female mice by CTX and it was confirmed by increased follicle stimulating hormone (FSH), luteal hormone (LH) and decreased estradiol (E2) blood levels and follicle count. According to the results of the immunohistochemistry (IHC) cell death markers were significantly more expressed than control (C) and sham (S) groups in the POF model. In addition, more apoptotic cells were observed in the POF group compared to C and S in the TUNEL analysis. In consequence of this study apoptosis and autophagy as well as paraptosis play a role in the FA leading to POF, will help to develop new treatment protocols.

scholarly journals MicroRNA-204-5p regulates apoptosis by targeting Bcl2 in rat ovarian granulosa cells exposed to cadmium†

2020 ◽  
Vol 103 (3) ◽  
pp. 608-619
Author(s):  
Ping Zhong ◽  
Jin Liu ◽  
Hong Li ◽  
Senbin Lin ◽  
Lingfeng Zeng ◽  
...  
Keyword(s):  
Protein Expression ◽  
Granulosa Cells ◽  
B Cell Lymphoma ◽  
Experimental Conditions ◽  
Expression Levels ◽  
Ovarian Granulosa Cells ◽  
Mrna And Protein Expression ◽  
Induced Apoptosis ◽  
Apoptosis Regulation

Abstract This study aimed to investigate whether cadmium (Cd) cytotoxicity in rat ovarian granulosa cells (OGCs) is mediated through apoptosis or autophagy and to determine the role of microRNAs (miRNAs) in Cd cytotoxicity. To test this hypothesis, rat OGCs were exposed to 0, 10, and 20 μM CdCl2 in vitro. As the Cd concentration increased, OGC apoptosis increased. In addition, Cd promoted apoptosis by decreasing the mRNA and protein expression levels of inhibition of B-cell lymphoma 2 (Bcl2). However, under our experimental conditions, no autophagic changes in rat OGCs were observed, and the mRNA and protein expression levels of the autophagic markers microtubule-associated protein 1 light chain 3 alpha (Map1lc3b) and Beclin1 (Becn1) were not changed. Microarray chip analysis, miRNA screening, and bioinformatics approaches were used to further explore the roles of apoptosis regulation-related miRNAs. In total, 19 miRNAs putatively related to Cd-induced apoptosis in rat OGCs were identified. Notably, miR-204-5p, which may target Bcl2, was identified. Then, rat OGCs were cultured in vitro and used to construct the miR-204-5p-knockdown cell line LV2-short hairpin RNA (shRNA). LV2-shRNA cells were exposed to 20 μM Cd for 12 h, and the mRNA and protein expression levels of Bcl2 were increased. Our findings suggest that Cd is cytotoxic to rat OGCs, and mitochondrial apoptosis rather than autophagy mediates Cd-induced damage to OGCs. Cd also affects apoptosis-related miRNAs, and the underlying apoptotic mechanism may involve the Bcl2 gene.

scholarly journals Proteomic determination of the lysine acetylome and phosphoproteome in the rat native inner medullary collecting duct

2018 ◽  
Vol 50 (9) ◽  
pp. 669-679 ◽  
Author(s):  
Kelly A. Hyndman ◽  
Chin-Rang Yang ◽  
Hyun Jun Jung ◽  
Ezigbobiara N. Umejiego ◽  
Chung-Ling Chou ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Collecting Duct ◽  
Water Channel ◽  
Receptor Activation ◽  
Phosphorylation Sites ◽  
Water Reabsorption ◽  
Inner Medullary Collecting Duct ◽  
Phosphorylated Proteins ◽  
V2 Receptor ◽  
Medullary Collecting Duct

Phosphorylation and lysine (K)-acetylation are dynamic posttranslational modifications of proteins. Previous proteomic studies have identified over 170,000 phosphorylation sites and 15,000 K-acetylation sites in mammals. We recently reported that the inner medullary collecting duct (IMCD), which functions in the regulation of water-reabsorption, via the actions of vasopressin, expresses many of the enzymes that can modulated K-acetylation. The purpose of this study was to determine the K-acetylated or phosphorylated proteins expressed in IMCD cells. Second we questioned whether vasopressin V2 receptor activation significantly affects the IMCD acetylome or phosphoproteome? K-acetylated or serine-, threonine-, or tyrosine-phosphorylated peptides were identified from native rat IMCDs by proteomic analysis with four different enzymes (trypsin, chymotrypsin, ASP-N, or Glu-C) to generate a high-resolution proteome. K-acetylation was identified in 431 unique proteins, and 64% of the K-acetylated sites were novel. The acetylated proteins were expressed in all compartments of the cell and were enriched in pathways including glycolysis and vasopressin-regulated water reabsorption. In the vasopressin-regulated water reabsorption pathway, eight proteins were acetylated, including the novel identification of the basolateral water channel, AQP3, acetylated at K282; 215 proteins were phosphorylated in this IMCD cohort, including AQP2 peptides that were phosphorylated at four serines: 256, 261, 264, and 269. Acute dDAVP did not significantly affect the IMCD acetylome; however, it did significantly affect previously known vasopressin-regulated phosphorylation sites. In conclusion, presence of K-acetylated proteins involved in metabolism, ion, and water transport in the IMCD points to multiple roles of K-acetylation beyond its canonical role in transcriptional regulation.

scholarly journals Expression and activity of Apaf1 and caspase-9 in granulosa cells during follicular atresia in pig ovaries

Reproduction ◽  
2003 ◽  
pp. 113-120 ◽  
Author(s):  
T Matsui ◽  
N Manabe ◽  
Y Goto ◽  
N Inoue ◽  
S Nishihara ◽  
...  
Keyword(s):  
Western Blot ◽  
Granulosa Cells ◽  
Blot Analysis ◽  
Crucial Role ◽  
Mitochondrial Pathway ◽  
Signalling Pathway ◽  
Follicular Atresia ◽  
Rt Pcr ◽  
Caspase 9 ◽  
Expression And Activity

Apoptosis in granulosa cells plays a crucial role in ovarian follicular atresia, but the intracellular regulating mechanism, especially the mitochondrion-dependent apoptosis signalling pathway, is still largely unknown. This study examined whether the mitochondrial pathway is associated with granulosa cell apoptosis during atresia in pig ovaries. Both mRNAs of caspase-9 and apoptotic protease-activating factor 1 (Apaf1), which are major signal transducing components in the mitochondrial pathway, were detected in granulosa cells in healthy, early atretic and progressed atretic follicles by RT-PCR. No changes in the expression of Apaf1 mRNA were seen during follicular atresia, but the expression of caspase-9 mRNA increased during atresia. Apaf1 protein was steadily detected in granulosa cells prepared from healthy, early atretic and progressed atretic follicles by western blot analysis, but high expression of the precursor of caspase-9 (procaspase-9) was detected only in granulosa cells of healthy follicles. Decreased procaspase-9 protein was demonstrated during follicular atresia. Proteolytic activity of caspase-9 increased during atresia, in agreement with the diminution of procaspase-9 protein. Intensive expression of caspase-9 mRNA was demonstrated in the granulosa cells of early atretic and progressed atretic follicles but not in those of healthy follicles. These results indicate that the mitochondrial signalling pathway, which is mediated by Apaf1 and caspase-9, plays a crucial role in determining the fate of granulosa cells during atresia in pig ovaries.

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