Effect of Threonine on the Proliferation and Viability of Porcine Granulosa Cells

2017 ◽  
Vol 17 (1) ◽  
pp. 1-6
Author(s):  
Zhou Ke-Rou ◽  
Zhang Qian ◽  
Jin Er-Hui ◽  
Hu Qian-Qian ◽  
Gu You-Fang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Cell Proliferation ◽  
Cell Viability ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Cell Morphology ◽  
S Phase ◽  
Control Group ◽  
Porcine Granulosa Cells

The proliferation and viability of granulosa cells directly affects the development and function of follicles. The current study examined the effect of threonine on cell morphology and cell cycle of porcine granulosa cells. It would help to understand hormone secretion, reproductive health, and disease prevention and treatment in females. Granulosa cells were isolated from pig ovaries, and different passages of cells were stained and identified by FSHR antibody. Threonine was added to the culture medium, and cells were stained with HE to examine the changes in granulosa cell morphology. Cell viability was measured using the CCK-8 assay, and cell cycle phase was determined by flow cytometry. Immunofluorescence staining showed >98% FSHR-positive cells demonstrating that the isolated granulosa cells were >95% pure. HE-staining of different passages of granulosa cells showed that cells from the second to fourth passage have stable morphology, whereas those from the fifth and subsequent passages began to degenerate and degrade. CCK-8 results showed that =0.5 mM threonine can significantly enhance granulosa cell viability after 0.5, 1 and 2 h of treatment, and cells treated with 1 mM threonine for 4 h had the highest cell viability. In contrast, 5 and 10 mM threonine significantly reduced cell viability, indicating that only a certain amount of threonine can enhance granulosa cell viability. Flow cytometry results demonstrated that the percentage of G1 phase cells was higher in the 0.1, 0.5 and 1 mM groups than in the control group, but lower in the 10 mM group than in control group. While the 5 mM group had the highest percentage of S phase cells, the 10 mM group had no S phase cells. In contrast, the percentage of G2 phase cells was much higher in the 10 mM group than in the control group and other dose groups, indicating that an appropriate amount of threonine can promote granulosa cell proliferation, but excessive threonine can inhibit cell proliferation.

scholarly journals MicroRNA-183-96-182 Cluster Regulates Bovine Granulosa Cell Proliferation and Cell Cycle Transition by Coordinately Targeting FOXO11

2016 ◽  
Vol 94 (6) ◽  
Author(s):  
Samuel Gebremedhn ◽  
Dessie Salilew-Wondim ◽  
Michael Hoelker ◽  
Franca Rings ◽  
Christiane Neuhoff ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Cell Function ◽  
S Phase ◽  
Locked Nucleic Acid ◽  
G1 Arrest ◽  
Mirna Cluster ◽  
Foxo1 Gene

Abstract Large-scale expression profiling of micro-RNAs (miRNAs) in bovine granulosa cells from dominant and subordinate follicles on Day 19 of the estrous cycle revealed enriched micro-RNA-183-96-182 cluster miRNAs in preovulatory dominant follicles that coordinately regulate the forkhead box protein O1 (FOXO1) gene. However, little is known about the role of this cluster in bovine granulosa cell function. We used an in vitro granulosa cell culture model to investigate this role. Granulosa cells aspirated from small growing follicles (3–5 mm in diameter) were cultured in Dulbecco modified Eagle medium/F-12 medium supplemented with fetal bovine serum and transfected with locked nucleic acid-based miRNA mimics, inhibitors, and corresponding negative controls. Overexpression of the miRNA cluster resulted in suppression of FOXO1 mRNA and protein, whereas inhibition of the cluster increased expression of FOXO1 mRNA. Overexpression also increased the relative rate of cell proliferation, whereas inhibition slowed it down. Similarly, the proportion of cells under G0/G1 arrest declined, whereas the ratio of cells in S phase increased in response to miR-183-96-182 overexpression. Selective knockdown of FOXO1 mRNA using anti-FOXO1 small interfering RNA increased the rate of granulosa cell proliferation, decreased the proportion of cells under G0/G1 arrest, and increased the proportion of cells in the S phase of cell cycle. Our data suggest that miR-183-96-182 cluster miRNAs promote proliferation and G1/S transition of bovine granulosa cells by coordinately targeting FOXO1, suggesting a critical role in granulosa cell function. MicroRNA-183-96-182 cluster regulates bovine granulosa cell function by targeting FOXO1 gene.

scholarly journals miR-3188 Regulates proliferation and apoptosis of granulosa cells by targeting KCNA5 in the polycystic ovary syndrome

2021 ◽  
Author(s):  
Shan Zhou ◽  
Liang Xia ◽  
Yuanyuan Chen ◽  
Weiying Guo ◽  
Jinxing Hu
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Polycystic Ovary Syndrome ◽  
Cell Viability ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Polycystic Ovary ◽  
Healthy Controls ◽  
Ovarian Dysfunction ◽  
Ovary Syndrome

Abnormal proliferation of granulosa cells is implicated in ovarian dysfunction and dysregulated folliculogenesis in the polycystic ovary syndrome (PCOS). Aberrant microRNA (miRNA) expression might contribute to disordered folliculogenesis and granulosa cell proliferation in PCOS. This study aimed to investigate the roles of miR-3188 in ovarian dysfunction, as well as the mechanism involved in granulosa cell proliferation in PCOS. Firstly, peripheral blood samples were isolated from PCOS patients and healthy controls, and qRT-PCR analysis demonstrated a dramatic increase in miR-3188 in PCOS patients when compared to the healthy controls. Secondly, miR-3188 overexpression increased cell viability of the granulosa-like tumor cell line (KGN). However, cell viability of KGN was repressed by interference with miR-3188. MiR-3188 promoted cell cycle of KGN through increasing cyclinD1 and decreasing p21 levels. Moreover, cell apoptosis was suppressed by miR-3188 in KGN, indicated by enhanced Bcl-2, and reduced Bax and cleaved caspase-3 levels, whereas knockdown of miR-3188 resulted in opposite effects. Lastly, potassium voltage-gated channel subfamily A member 5 (KCNA5) was verified as a target of miR-3188. KCNA5 expression was decreased and displayed negative correlation with miR-3188 levels in PCOS patients. Overexpression of KCNA5 attenuated the promotive effects of miR-3188 on cell viability and cell cycle in KGN. In conclusion, miR-3188, a key miRNA enhanced in PCOS, promoted granulosa cell proliferation through down-regulation of KCNA5, providing a new therapeutic target for PCOS.

scholarly journals Follicle-Stimulating Hormone Inhibits Adenosine 5′-Monophosphate-Activated Protein Kinase Activation and Promotes Cell Proliferation of Primary Granulosa Cells in Culture through an Akt-Dependent Pathway

Endocrinology ◽  
2009 ◽  
Vol 150 (2) ◽  
pp. 929-935 ◽  
Author(s):  
Pradeep P. Kayampilly ◽  
K. M. J. Menon
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Protein Kinase ◽  
Mrna Expression ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Ampk Activation ◽  
Cyclin D2 ◽  
Cell Cycle Inhibitor ◽  
Dependent Pathway

FSH, acting through multiple signaling pathways, regulates the proliferation and growth of granulosa cells, which are critical for ovulation. The present study investigated whether AMP-activated protein kinase (AMPK), which controls the energy balance of the cell, plays a role in FSH-mediated increase in granulosa cell proliferation. Cells isolated from immature rat ovaries were grown in serum-free, phenol red free DMEM-F12 and were treated with FSH (50 ng/ml) for 0, 5, and 15 min. Western blot analysis showed a significant reduction in AMPK activation as observed by a reduction of phosphorylation at thr 172 in response to FSH treatment at all time points tested. FSH also reduced AMPK phosphorylation in a dose-dependent manner with maximum inhibition at 100 ng/ml. The chemical activator of AMPK (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside, 0.5 mm) increased the cell cycle inhibitor p27 kip expression significantly, whereas the AMPK inhibitor (compound C, 20 μm) and FSH reduced p27kip expression significantly compared with control. FSH treatment resulted in an increase in the phosphorylation of AMPK at ser 485/491 and a reduction in thr 172 phosphorylation. Inhibition of Akt phosphorylation using Akt inhibitor VIII reversed the inhibitory effect of FSH on thr 172 phosphorylation of AMPK, whereas ERK inhibitor U0126 had no effect. These results show that FSH, through an Akt-dependent pathway, phosphorylates AMPK at ser 481/495 and inhibits its activation by reducing thr 172 phosphorylation. AMPK activation by 5-amino-imidazole-4-carboxamide-1-β-d-ribofuranoside treatment resulted in a reduction of cell cycle regulatory protein cyclin D2 mRNA expression, whereas FSH increased the expression by 2-fold. These results suggest that FSH promotes granulosa cell proliferation by increasing cyclin D2 mRNA expression and by reducing p27 kip expression by inhibiting AMPK activation through an Akt-dependent pathway. FSH stimulates granulosa cell proliferation by reducing cell cycle inhibitor p27 kip through AMP kinase inhibition.

The Relationship Between the Regulation of TOB1 Gene with Cell Proliferation, Apoptosis and Cell Cycle in BCR-ABL Positive Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 5125-5125
Author(s):  
Cintia Do Couto Mascarenhas ◽  
Anderson Ferreira Cunha ◽  
Ana Flavia Brugnerotto ◽  
Sheley Gambero ◽  
Joao Machado-Neto ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Cell Proliferation ◽  
K562 Cells ◽  
S Phase ◽  
Tumour Suppressor ◽  
Signalling Pathways ◽  
G2 Phase ◽  
Induction Of Apoptosis ◽  
The Relationship

Abstract Abstract 5125 The TOB1 gene is a transcription factor responsible for the transduction of the gene ERBB2. It is a member of a family of cell suppressor proliferation proteins called TOB/BTG1 family; also, this gene operates on the inhibition of neoplastic transformation. The TOB1 gene presents a decreased expression in several types of cancer such as lung, breast, thyroid and stomach cancer. However, the function of this gene in chronic myeloid leukemia (CML) remains unknown. Aiming to evaluate the inhibition of gene TOB1 into BCR-ABL positive cells and trying to elucidate the molecular mechanisms associated with the inhibition of this gene in the CML we proceed to a more detailed study of this gene. The inhibition of this gene in K562 cells was performed using specific lentivirus. The effect of silencing TOB1 in the proliferation of K562 cells was assessed by the MTT assay after 48 hours of culture; in shTOB1 the proliferation was increased in comparison with shControl cells. To evaluate the synergistic effect between the inhibition of kinase tyrosine activity of BCR-ABL and the inhibition of TOB1 we performed a treatment with different concentrations of imatinib (0. 1, 0. 5 and 1μM), but we observed the decrease in cell proliferation of shTOB1 cells to similar levels of shControl cells only at the 1μM concentration. Therefore, the TOB1 silencing increased the proliferation of K562 cells without an additional effect of a treatment with Imatinib. To analyze the clonogenicity, we performed a formation of colonies assay, in methylcellulose, to determine whether silencing TOB1 could cause a change in the clonal growth of positive BCR-ABL cells. There was no significant change in the number of colonies that grew in cell culture shTOB1 compared to shControl cells. These results suggest that silencing TOB1 in K562 cells may not change the clonogenicity. In the assessment of cell cycle, the flow cytometry analysis revealed a significant accumulation of K562 cells in S phase, with consequent reduction of cells in the G2 phase of the cell cycle in cells shTOB1 compared to cells shControl. The TOB1 gene silencing in K562 cells kept the cells in the S phase and prevented the entry of cells in the G2 phase showing that the inhibition of gene TOB1 induced an increase in proliferation of K562 BCR-ABL cells. The level of apoptosis was assessed by flow cytometry after labeling the cells with anexin-V/PI. The Imatinib treatment presented dose-response in the induction of apoptosis as expected. However, a cumulative effect with TOB1 silencing was not observed. Furthermore, the apoptosis was also assessed by assays of caspases 3, 8 and 9, which showed an increase of the caspase activity of shControl cells in relation of the shTOB1 cells, showing that inhibition of this gene also changes the level of apoptosis. These results corroborate the literature data that report the relationship of this tumour suppressor gene in signalling pathways related to angiogenesis, carcinogenesis, apoptosis and metastasis. When we relate the results obtained with the LMC, we can consider the possibility of TOB1 regulation changes be related to modification of important signalling pathways such as AKT, PI3K, STAT3 and STAT5, among others. Furthermore, the inhibition of TOB1 may be related with an increase on the number of BCR-ABL positive cells and subsequent disease progression. In conclusion, this study confirmed literature data showing that TOB1 gene works as a tumour suppressor protein in cells of many types of cancer. From this work we can infer that in CML the expression of this gene is transformed, resulting in changing of the capacity of induction of apoptosis, decrease tumour necrosis and increase cell proliferation. This work was supported by FAPESP and INCT. Disclosures: No relevant conflicts of interest to declare.

Rapamycin Induced Autophagy and Suppression in Human Leukemia Cells Proliferation

2014 ◽  
Vol 926-930 ◽  
pp. 1112-1115
Author(s):  
Yi Ju Hou ◽  
Zhong Hai Yuan ◽  
Xiao Dong Liu ◽  
Ke Xin Sun ◽  
Chen Zhao ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Cell Proliferation ◽  
Mtt Assay ◽  
Leukemia Cells ◽  
Control Group ◽  
Human Leukemia ◽  
Hl60 Cells ◽  
Human Leukemia Cells ◽  
Autophagic Activity

To investigate the effect of autophagy induced by rapamycin (Rap) on proliferation of Human Leukemia Cells (HL60). HL60 cells are treated with rapamycin to induce autophagy. Then, Western blot is performed to examine the expression of Beclin1 and LC3. MTT assay is used to evaluate cell proliferation. The cell cycle is analyzed by flow cytometry (FCM). After treatment with rapamycin for 24 hours, LC3 is dramatically increased at protein level and autophagic activity is significantly increased. And MTT assay indicated that cell proliferation is inhibited by rapamycin. Compared with control group, more cells are arrested at G0/ G1 phases. We conclude that rapamycin can induce autophagy and suppress proliferation in HL60 cells.

The effects of gemcitabine on cell apoptosis and cycle of gastric cancer

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 15181-15181
Author(s):  
L. Wang
Keyword(s):  
Gastric Cancer ◽  
Cell Cycle ◽  
Cell Proliferation ◽  
Cell Apoptosis ◽  
Morphological Changes ◽  
S Phase ◽  
Control Group ◽  
Control Groups ◽  
Gastric Cancer Cells ◽  
M Phase

15181 Background: To study the effects of gemcitabine on cell apoptosis and cell cycle of gastric cancer Methods: Gastric cancer cells were cultured with different concentrations of gemcitabine (0.001, 0.01 and 0.1μM). MTT test was performed to evaluate the cell proliferation. The cells were divided into three groups: control group (cultured in RPMI-1640) and 5-FU group ( cultured in RPMI-1640 with 5- FU) and gemcitabine group ( cultured in RPMI-1640 with Gemcitabine). Flow cytometry was performed to determine the apoptotic rate and the cell cycle phases. Morphological changes were observed by phasecontrast microscope. Results: The cell proliferation was inhibited in experiment groups treated with gemcitabine and 5-FU, compared with control groups(P<0.01). Gemcitabine can induce cell apoptosis. 0.01μM and 0.1μM gemcitabine were much more effective than 0.001μM. On the third day, S phase cells accounted for 24.5% and G2-M phase cells 0.08% in the control group, while 24.6% and 0.06%, respectively in the gemcitabine group. However, on the seventh day, those came to 20.8% and 0.41% in the control group, and 18.2% and 1. 55% in the gemcitabine group, indicating a significant change in the cell cycle ( P<0.01). Conclusions: Gemcitabine can inhibit the cell proliferation, and it maybe related to cell apoptosis. No significant financial relationships to disclose.

scholarly journals R-spondin2 signaling is required for oocyte-driven intercellular communication and follicular growth

2020 ◽  
Vol 27 (10) ◽  
pp. 2856-2871 ◽  
Author(s):  
Marie-Cécile De Cian ◽  
Elodie P. Gregoire ◽  
Morgane Le Rolle ◽  
Simon Lachambre ◽  
Magali Mondin ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Cell Cycle Progression ◽  
Ovarian Function ◽  
Follicular Growth ◽  
Oocyte Growth

Abstract R-spondin2 (RSPO2) is a member of the R-spondin family, which are secreted activators of the WNT/β-catenin (CTNNB1) signaling pathway. In the mouse postnatal ovary, WNT/CTNNB1 signaling is active in the oocyte and in the neighboring supporting cells, the granulosa cells. Although the role of Rspo2 has been previously studied using in vitro experiments, the results are conflicting and the in vivo ovarian function of Rspo2 remains unclear. In the present study, we found that RSPO2/Rspo2 expression is restricted to the oocyte of developing follicles in both human and mouse ovaries from the beginning of the follicular growth. In mice, genetic deletion of Rspo2 does not impair oocyte growth, but instead prevents cell cycle progression of neighboring granulosa cells, thus resulting in an arrest of follicular growth. We further show this cell cycle arrest to be independent of growth promoting GDF9 signaling, but rather associated with a downregulation of WNT/CTNNB1 signaling in granulosa cells. To confirm the contribution of WNT/CTNNB1 signaling in granulosa cell proliferation, we induced cell type specific deletion of Ctnnb1 postnatally. Strikingly, follicles lacking Ctnnb1 failed to develop beyond the primary stage. These results show that RSPO2 acts in a paracrine manner to sustain granulosa cell proliferation in early developing follicles. Taken together, our data demonstrate that the activation of WNT/CTNNB1 signaling by RSPO2 is essential for oocyte-granulosa cell interactions that drive maturation of the ovarian follicles and eventually female fertility.

scholarly journals AMPK Activation by Dihydrotestosterone Reduces FSH-Stimulated Cell Proliferation in Rat Granulosa Cells by Inhibiting ERK Signaling Pathway

Endocrinology ◽  
2012 ◽  
Vol 153 (6) ◽  
pp. 2831-2838 ◽  
Author(s):  
Pradeep P. Kayampilly ◽  
K. M. J. Menon
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Cell Cycle Progression ◽  
Maximum Effect ◽  
Ampk Activation ◽  
Cyclin D2 ◽  
Compound C ◽  
Erk Phosphorylation

We have previously reported that 5α-dihydrotestosterone (DHT) inhibits FSH-mediated granulosa cell proliferation by reducing cyclin D2 mRNA expression and blocking cell cycle progression at G1/S phase. The present study investigated the role of AMP activated protein kinase (AMPK) in DHT-mediated inhibition of granulosa cell proliferation. Granulosa cells harvested from 3-d estradiol primed immature rats were exposed to different concentrations of DHT (0, 45, and 90 ng/ml) for 24 h. Western blot analysis of immunoprecipitated AMPK showed a dose-dependent activation (P &lt; 0.05) as evidenced by the increased phosphorylation at thr 172. In addition, time-courses studies (0, 6, 12, and 24 h) using DHT (90 ng/ml) showed a time-dependent increase in AMPK activation with maximum effect at 24 h. FSH inhibited AMPK phosphorylation and promoted granulosa cell proliferation, but pretreatment with DHT (90 ng/ml) for 24 h prior to FSH treatment reduced this effect. Pharmacological activation of AMPK with 5-aminoimidazole-4-carboxamide-1-β4-ribofuranoside abolished FSH-mediated ERK phosphorylation, indicating that AMPK is a negative upstream regulator of ERK. Furthermore, inhibition of AMPK activation by compound C reversed the DHT-mediated reduction in positive cell cycle regulator, cyclin D2, and 5-bromo-2′-deoxyuridine incorporation. These results suggest that elevated levels of DHT activate AMPK, which in turn inhibits ERK phosphorylation. Thus, inhibition of ERK phosphorylation by activated AMPK in response to DHT might contribute to decreased granulosa cell mitogenesis and ovulatory dysfunction seen in hyperandrogenic states.

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.

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