scholarly journals FSH Promotes Rat Follicle Development Through Inhibition of the Hippo Signalling Pathway

2021 ◽  
Author(s):  
Tairen Chen ◽  
Mengjing Wu ◽  
Yuting Dong ◽  
Bin Kong ◽  
Yufang Cai ◽  
...  
Keyword(s):  
Western Blot ◽  
In Vitro Culture ◽  
Granulosa Cells ◽  
Signalling Pathway ◽  
Control Group ◽  
Follicle Development ◽  
Follicle Growth ◽  
Expression Levels ◽  
Hippo Signalling

Abstract Purpose: Whether FSH promotes follicle growth by inhibiting the Hippo signalling pathway.METHODS: Ovaries were cultured in vitro into a control group (no intervention), an FSH group (0.3 IU/mL FSH), and a VP group (10 µg/mL vetiporfin). HE staining and follicle counts were performed at each stage after 3 hours of in vitro culture. Immunohistochemistry was performed to study the expression levels of LATS2, YAP, PLATS2, and PYAP, and their expression levels in each group were also analysed by Western blot.The number of secondary follicles was significantly increased in the FSH group, the arrangement of granulosa cells was neater, the nuclear fixation was reduced, and the number of atretic follicles was decreased in the VP group. The number of secondary follicles was significantly increased, the number of atretic follicles was reduced, and granulosa cell nuclear consolidation was reduced in the VP+FSH group. Immunohistochemistry showed that LATS2 and YAP expression levels were significantly increased and PLATS2 and PYAP expression levels were relatively decreased in the FSH group, PYAP and PLATS2 expression levels were significantly increased and YAP expression was significantly decreased in the VP group, and YAP and LATS2 expression levels were significantly increased and PYAP and PLATS2 expression levels were significantly decreased in the VP+FSH group. By Western blot, LATS2 and YAP were elevated and PYAP and PLAT2 were decreased in the FSH group, LATS2 and YAP were decreased and PYAP and PLATS were significantly elevated in the VP group, and LATS2 and YAP were elevated and PYAP and PLATS2 were decreased in the VP+FSH group.CONCLUSION: FSH promotes follicle development by inhibiting the Hippo signalling pathway.

Supplementation of c-type natriuretic peptide during in vitro growth period benefits the development of murine preantral follicles

Zygote ◽  
2020 ◽  
pp. 1-5
Author(s):  
Li Ang ◽  
Cao Haixia ◽  
Li Hongxia ◽  
Li Ruijiao ◽  
Guo Xingping ◽  
...  
Keyword(s):  
Natriuretic Peptide ◽  
Granulosa Cells ◽  
Culture System ◽  
Early Stage ◽  
Developmental Competence ◽  
Control Group ◽  
Follicle Development ◽  
Control Groups ◽  
Preantral Follicles

Summary The present study investigated the effects of c-type natriuretic peptide (CNP) on the development of murine preantral follicles during in vitro growth (IVG). Preantral follicles isolated from ovaries of Kunming mice were cultured in vitro. In the culture system, CNP was supplemented in the experimental groups and omitted in the control groups. In Experiment 1, CNP was only supplemented at the early stage and follicle development was evaluated. In Experiments 2 and 3, CNP was supplemented during the whole period of in vitro culture. In Experiment 2, follicle development and oocyte maturity were evaluated. In Experiment 3, follicle development and embryo cleavage after in vitro fertilization (IVF) were assessed. The results showed that in the control groups in all three experiments, granulosa cells migrated from within the follicle and the follicles could not reach the antral stage. In the experimental groups in all three experiments, no migration of granulosa cells was observed and follicle development was assessed as attaining the antral stage, which was significantly superior to that of the control group (P < 0.0001). Oocyte meiotic arrest was effectively maintained, hence giving good developmental competence. In conclusion, CNP supplementation in the culture system during IVG benefited the development of murine preantral follicles.

P–648 In-vitro Evaluation of SCF and StxBP1 in Polycystic Ovary Syndrome (PCOS)

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
S Erol ◽  
S Zırh ◽  
L Karako. Sokmensuer ◽  
G Bozdag ◽  
S F Muftuoglu
Keyword(s):  
Granulosa Cells ◽  
Polycystic Ovary ◽  
Critical Role ◽  
Vesicle Fusion ◽  
Snare Complex ◽  
Control Group ◽  
Signal Molecules ◽  
Expression Levels ◽  
Before And After

Abstract Study question Is the interaction between intrafollicular cells in PCOS, impaired by the change of vesicular fusion and/or exocytosis in granulosa cells (GCs)? Summary answer StxBP1 expression leves impared in GCs of PCOS. What is known already PCOS characterised as follicular arrest on antral follicles, cystic follicle formation, and follicular development failure. GCs secretes wide variety of factors via exocytosis, and plays critical role during folliculogenesis. Secretory vesicles are transported to cellular membrane. This process requires local concentrations of SNAREs consisting of tSNARE, vSNARE, and other vesicle fusion associated proteins. SNARE proteins are involved in vesicle fusion, exocytosis, and intracellular trafficking. GCs secretes KITL which provides follicular activation and growth. Syntaxins are one of the members of SNARE complex. StxBP1 is a protein which has a crucial role in secretory vesicle fusion that provides fusion of syntaxins. Study design, size, duration Granulosa cells (GCs) were collected for primary cell culture, since 2019 from both PCOS (n = 10) and healthy (male factor infertility) (n = 10) women undergoing ART. Each GCs from participant divided into two groups as in-vitro stimulated group and in-vitro nonstimulated group. Participants/materials, setting, methods GCs have been isolated from follicular fluid taken from patients during oocyte pick-up at Hacettepe University In-Vitro Fertilization Unit. nGCs were cultured at most secod subcultures after the isolation. The stimulated groups of both PCOS and control groups were stimulated by hCG(10IU/ml) ve FSH(0.5IU/ml) for 24 hours. Vesicle fusion proteins (Stx6, StxBP1, and SNAP25), KITL, and FSHr expressions were analyzed on granulosa cells from each group via immunofluorescent (IF) labeling and cyto-ELISA. Main results and the role of chance FSHr were compared in both control and PCOS before and after stimulation. There was no difference between FSHr expression levels in both groups. Indirect IF is widely considered for SNAP25, Stx6, StxBP1 proteins in all groups of GCs screening with/without stimulation. Expression of SNAP25, StxBP1 mainly scattered through all cytoplasmic area,s and membranous localization was observed. Stx6 expressions were particularly distinguished at perinuclear area of cytoplasm. However, stimulated cells of control appeared more peripherally Stx6 expression. This pattern caused by stimulation wasn’t observed in PCOS. Expressions of SNAP25, Stx6, StxBP1 were observed with less expression in PCOS. Also, the response to stimulation was lower than the control group. The differences in Stx6, SNAP25, StxBP1 and KITL levels before and after stimulation was evaluated for both control and PCOS in Cyto-ELISA. However, both SNAP25 and Stx6 expressions in GCs of both groups were similar in response to stimulation. The expression levels of StxBP1 in response to stimulation were significantly lesser than control at PCOS. KITL expressions were lower in the PCOS as expected furthermore similar to StxBP1 in response to stimulation. According to our findings, the highest response to stimulation in GCs occurred for StxBP1 and KITL in the control. Limitations, reasons for caution Since human cells were used in the study and the cells of each patient do not exhibit the same characteristics, the lowest number of patient samples identified in the statistical power analysis were included in the study. Wider implications of the findings: Our view to the disruption in the secretion of signal molecules in terms of vesicle dynamics will offer a new perspective in female infertility or cross-talks in folliclar cells of the ovary. Trial registration number TSA–2019–18196

scholarly journals Expression of FOXL2 and RSPO1 in Hen Ovarian Follicles and Implication of Exogenous Leptin in Modulating Their mRNA Expression in In Vitro Cultured Granulosa Cells

Animals ◽  
2019 ◽  
Vol 9 (12) ◽  
pp. 1083 ◽  
Author(s):  
Weihe Niu ◽  
Izhar Hyder Qazi ◽  
Sichen Li ◽  
Xiaoling Zhao ◽  
Huadong Yin ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Granulosa Cells ◽  
Ovarian Follicles ◽  
Control Group ◽  
Follicle Development ◽  
Theca Cells ◽  
Increasing Trend ◽  
Group A ◽  
Ovulatory Follicles

In this study, using a laying hen model, we determined the expression of FOXL2 and RSPO1 in different central and peripheral tissue and ovarian follicles at different stages of development. At the same time, mRNA expression of both genes in granulosa and theca cells harvested from follicles at different stages of folliculogenesis was also evaluated. Finally, we assessed the effect of leptin treatment on expression of FOXL2 and RSPO1 in in vitro cultured granulosa cells harvested from 1–5 mm to F3–F1 follicles. Our RT-qPCR results revealed that a comparatively higher expression of FOXL2 and RSPO1 was observed in ovary, hypothalamus, and pituitary. Abundant mRNA expression of FOXL2 was observed in small prehierarchical follicles (1–1.9 and 2–2.9 mm follicles; p < 0.05), whereas mRNA expression of RSPO1 showed an increasing trend in large hierarchical follicles (F5–F1), and its abundant expression was observed in post-ovulatory follicles. FOXL2 mRNA expression was stable in granulosa cells harvested from 3–5 mm to F4 follicles, and exhibited a significantly higher expression in large hierarchical follicles. Conversely, relatively low mRNA expression of FOXL2 was observed in theca cells. RSPO1 mRNA expression was relatively lower in granulosa cells; however, theca cells exhibited a significantly higher mRNA expression of RSPO1 in F4 to F1 follicles. In the next experiment, we treated the in vitro cultured granulosa cells with different concentrations (1, 10, 100, and 1000 ng/mL) of exogenous leptin. Compared to the control group, a significant increase in the expression of FOXL2 was observed in groups treated with 1, 10, and 100 ng/mL leptin, whereas expression of RSPO1 was increased in all leptin-treated groups. When treated with 100 ng/mL leptin, FOXL2 and RSPO1 expression was upregulated in cultured granulosa cells harvested from both large hierarchical (F3–F1) and small prehierarchical follicles (1–5 mm). Based on these findings and evidence from mainstream literature, we envisage that FOXL2 and RSPO1 genes (in connection with hypothalamic-hypophysis axis) and leptin (via modulation of FOXL2 and RSPO1 expression) might have significant physiological roles, at least in part, in modulating the ovarian mechanisms, such as follicle development, selection, and steroidogenesis in laying hens.

scholarly journals Immune Regulation of RAW264.7 Cells In Vitro by Flavonoids from Astragalus complanatus via Activating the NF-κB Signalling Pathway

2018 ◽  
Vol 2018 ◽  
pp. 1-9 ◽  
Author(s):  
Yu Li ◽  
Ning Hao ◽  
Suping Zou ◽  
Tingting Meng ◽  
Huanqing Tao ◽  
...  
Keyword(s):  
Food Industry ◽  
Macrophage Activation ◽  
Drug Research ◽  
Signalling Pathway ◽  
Raw264.7 Cells ◽  
Control Group ◽  
Expression Levels ◽  
Mapk Signalling ◽  
Gene Expression Levels

The current study aimed at investigating the effects of flavonoids from Astragalus complanatus (FAC) on the proliferation, the contents, and gene expression levels of cytokines, secretion of surface stimulating factors, cell cycle, and the expression level of the NF-κB signalling pathway in RAW264.7 cells. Our results revealed that compared with control group, the contents of IL-6, IL-1β, TNF-α, and NO and the mRNA expression levels of IL-6, IL-1β, TNF-α, and iNOS in FAC-treated groups significantly increased (p<0.01). Moreover, FAC induced macrophage activation to release the above-mentioned mediators partly involved in NF-κB/MAPK signalling pathways. Therefore, FAC regulates immune function in RAW264.7 cells via activating the NF-κB signalling pathway. FAC could be applicable for agriculture, drug research, and food industry as a potent immune-modulatory agent.

scholarly journals Lack of WWC2 Protein Leads to Aberrant Angiogenesis in Postnatal Mice

2021 ◽  
Vol 22 (10) ◽  
pp. 5321
Author(s):  
Viktoria Constanze Brücher ◽  
Charlotte Egbring ◽  
Tanja Plagemann ◽  
Pavel I. Nedvetsky ◽  
Verena Höffken ◽  
...  
Keyword(s):  
Signalling Pathway ◽  
Control Group ◽  
Knock Out ◽  
Ocular Angiogenesis ◽  
Sprouting Angiogenesis ◽  
Adult Mice ◽  
Upstream Regulator ◽  
Knock Out Mice ◽  
Hippo Signalling

The WWC protein family is an upstream regulator of the Hippo signalling pathway that is involved in many cellular processes. We examined the effect of an endothelium-specific WWC1 and/or WWC2 knock-out on ocular angiogenesis. Knock-outs were induced in C57BL/6 mice at the age of one day (P1) and evaluated at P6 (postnatal mice) or induced at the age of five weeks and evaluated at three months of age (adult mice). We analysed morphology of retinal vasculature in retinal flat mounts. In addition, in vivo imaging and functional testing by electroretinography were performed in adult mice. Adult WWC1/2 double knock-out mice differed neither functionally nor morphologically from the control group. In contrast, the retinas of the postnatal WWC knock-out mice showed a hyperproliferative phenotype with significantly enlarged areas of sprouting angiogenesis and a higher number of tip cells. The branching and end points in the peripheral plexus were significantly increased compared to the control group. The deletion of the WWC2 gene was decisive for these effects; while knocking out WWC1 showed no significant differences. The results hint strongly that WWC2 is an essential regulator of ocular angiogenesis in mice. As an activator of the Hippo signalling pathway, it prevents excessive proliferation during physiological angiogenesis. In adult animals, WWC proteins do not seem to be important for the maintenance of the mature vascular plexus.

scholarly journals Effects of Naringin on Proliferation and Osteogenic Differentiation of Human Periodontal Ligament Stem Cells In Vitro and In Vivo

2015 ◽  
Vol 2015 ◽  
pp. 1-9 ◽  
Author(s):  
Lihua Yin ◽  
Wenxiao Cheng ◽  
Zishun Qin ◽  
Hongdou Yu ◽  
Zhanhai Yu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Osteogenic Differentiation ◽  
Periodontal Ligament ◽  
Trabecular Structure ◽  
Control Group ◽  
Periodontal Ligament Stem Cells ◽  
Expression Levels ◽  
Proliferation And Differentiation

This study is to explore the osteogenesis potential of the human periodontal ligament stem cells (hPDLSCs) induced by naringin in vitro and in vitro. The results confirmed that 1 μM naringin performs the best effect and a collection of bone-related genes (RUNX2,COL1A2, OPN, and OCN) had significantly higher expression levels compared to the control group. Furthermore, a typical trabecular structure was observed in vivo, surrounded by a large amount of osteoblasts. These results demonstrated that naringin, at a concentration of 1 μM, can efficiently promote the proliferation and differentiation of hPDLSCs both in vitro and in vivo.

scholarly journals Expression of mRNAs for Pro-and Anti-Apoptotic Factors in Granulosa Cells and Follicular Fluid of Women Undergoing in Vitro Fertilization. A Pilot Study

2021 ◽  
Author(s):  
Jozsef Bodis ◽  
Endre Sulyok ◽  
Akos Varnagy ◽  
Viktória Prémusz ◽  
Krisztina Godony ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Vitro Fertilization ◽  
Mrna Expression ◽  
Granulosa Cells ◽  
Follicular Fluid ◽  
Vitro Fertilization ◽  
Expression Levels ◽  
The Impact ◽  
Apoptotic Factors

Abstract BackgroundThis observational clinical study evaluated the expression levels and predictive values of some apoptosis-related genes in granulosa cells (GCs) and follicular fluid (FF) of women undergoing in vitro fertilization (IVF).Methods GCs and FF were obtained at oocyte retrieval from 31 consecutive patients with heterogeneous infertility diagnosis (age: 34.3±5.8 years, body mass index: 24.02±3.12 kg/m2, duration of infertility: 4.2±2.1 years). mRNA expression of pro-apoptotic (BAX, CASP3, CASP8) and anti-apoptotic (BCL2, AMH, AMHR, FSHR, LHR, CYP19A1) factors was determined by quantitative RT-PCR using ROCHE LightCycler 480. Results No significant difference in GC or FF mRNA expression of pro- and anti-apoptotic factors could be demonstrated between IVF patients with (9 patients) or without (22 patients) clinical pregnancy. Each transcript investigated was detected in FF, but their levels were markedly reduced and independent of those in GCs. The number of retrieved oocytes was positively associated with GC AMHR (r=0.393, p=0.029), but the day of embryo transfer was negatively associated with GC LHR (r=-0.414, p=0.020) and GC FSHR transcripts (r=-0.535, p=0.002). When pregnancy positive group was analysed separately the impact of apoptosis- related gene expressions on some selected measures of IVF success could be observed. Strong positive relationship was found between gene expression levels of pro- and anti-apoptotic factors in GCs.ConclusionOur study provides only marginal evidences for the apoptosis dependence of IVF outcome and suggests that the apoptosis process induces adaptive increases of the anti-apoptotic gene expression to attenuate apoptosis and to protect cell survival.

scholarly journals Identification and Validation of Serum Autoantibodies in Children with B-cell Acute Lymphoblastic Leukemia by Serological Proteome Analysis

2021 ◽  
Author(s):  
Runhong Yu ◽  
Shiwei Yang ◽  
Yufeng Liu ◽  
Zunmin Zhu
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Western Blot ◽  
B Cell ◽  
Lymphoblastic Leukemia ◽  
Immunohistochemical Analysis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Control Group ◽  
Healthy Children ◽  
Expression Levels ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Purpose: Study was by intention to screen serum autoantibodies that may contribute to the early detection of B-cell acute lymphoblastic leukemia (B-ALL) in children.Patients and methods: The total protein from three pooled B-ALL cell lines(NALM-6, REH and BALL-1 cells) was separated using two-dimensional gel electrophoresis(2-DE), which was followed by Western blot by mixed serum from B-ALL patients (n=20) or healthy children(n=20). We obtained and analyzed the images of 2-D gel and Western blot by PDQuest software,and then identify the spots of immune responses in B-ALL samples compared with those in control samples.The proteins from spots were identified using mass spectrometry (MS). The autoantibodies against α-enolase and voltage-dependent anion-selective channel protein 1(VDAC1) were further validated on the use of enzyme-linked immunosorbent assay(ELISA). The protein expression levels of the candidate antigens α-enolase and VDAC1 in B-ALL were thoroughly studied by immunohistochemical analysis.Results: Six protein dots were identified with MS as Aconitase,apoptosis-inducing factor(AIF),dihydrolipoamide dehydrogenase(DLD), α-enolase,medium-chain acyl-CoA dehydrogenase(MCAD) and VDAC 1.The frequencies of autoantibodies against α-enolase and VDAC1 in children with B-ALL were 27% and 23%, respectively, which were significantly higher than those in normal controls(4% and 0). Immunohistochemical analysis showed the expression of α-enolase and VDAC1 was positive in 95% and 85% of B-ALL patients, respectively, but negative expression levels were showed in the control group. Conclusion: This study incidates that α-enolase and VDAC1 may be the antigen associated with B-ALL .α-enolase and VDAC1 autoantibodies may develop into potential serological markers of B-ALL in children.Other proteins also need to be confirmed in a large number of serum samples.

scholarly journals 2-Hydroxy-4-Methylselenobutanoic Acid (HMSeBA) Promote Follicle Development By Antioxidant Pathway in Gilts

2021 ◽  
Author(s):  
Yanpeng Dong ◽  
Sirun Chen ◽  
Yalei Liu ◽  
Zimei Li ◽  
Xinlin Jia ◽  
...  
Keyword(s):  
Gene Expression ◽  
Antioxidant Capacity ◽  
Granulosa Cells ◽  
Body Weight Gain ◽  
Control Diet ◽  
Negative Control ◽  
Follicle Development ◽  
Selenium Content ◽  
Total Selenium

Abstract Background Dietary 2-hydroxy-4-methylselenobutanoic acid (HMSeBA) supplementation can exert antioxidant effects in poultry, pigs and weaned pigs. However, it is unknown whether HMSeBA could improve the development of follicle by anti-oxidize effects in gilt. This study was conducted to evaluate the effects of dietary HMSeBA supplementation on the follicle development in gilt. A total of 36 gilts were randomly fed the control diet (CON, negative control), Na2SeO3 diet containing 0.3 mg Se/kg (positive control) or the HMSeBA diet containing 0.3 mg Se/kg from weaning to the 19th day after the second estrus. In another study, the effect of HMSeBA on the cells viability, proliferation, release of 17βestradiol (E2 ) and antioxidant capacity were investigated in the mouse ovarian granulosa cells in vitro. Results Results showed that HMSeBA group increased the average daily body weight gain (ADG) and decreased the ratio of feed: gain during day 120 to 176 in gilts ( P < 0.05). The selenium (HMSeBA and Na 2 SeO 3 ) increased the weight of uterine at the third estrus. There was no effect of HMSeBA on the number of large follicles (diameter >5mm), but HMSeBA decreased the gene expression of growth differentiation factor-9 ( GDF-9 ) and bone morphogenetic protein-15 ( BMP-15 ) in cumulus-oocyte complexes (COCs). HMSeBA group increased the total selenium content in serum ( P < 0.05) and liver ( P < 0.01) and tended to increase the total selenium content in ovary ( P = 0.08). HMSeBA group decreased the malondialdehyde (MDA) concentration in the serum, liver and ovary ( P < 0.05), increased the total antioxidant capacity (T-AOC) in the liver, thioredoxin reductase (TrxR) in the ovary ( P < 0.05) and increased the activity of GPx in the serum, liver and ovary ( P < 0.05). Na 2 SeO 3 supplementation decreased MDA and increased the T-AOC in liver, increased the T-SOD and TrxR in the ovary compared with control. At the transcription level, HMSeBA group increased the glutathione peroxidase 2 ( GPx2 ) and TrxR1 ( P < 0.05) expression in the liver, and increased the GPx1 expression ( P < 0.05) in the ovary of gilts compared with Na2SeO3 treatment. Besides, HMSeBA group increased the expressions of superoxide dismutase 1 ( SOD1 ) and Thioredoxin l ( Trx1 ) in the liver. In vitro experiment, HMSeBA improved granulosa cells’ proliferation and E2 secretion ( P < 0.05). HMSeBA and Na 2 SeO 3 both increased the T-AOC and decreased MDA in granulosa cells in vitro. Meanwhile, HMSeBA increased T-SOD, GPx, glutathione reductase (GR) and TrxR activity in granulosa cells in vitro. In addition, HMSeBA up-regulated SOD2 and GPx1 gene expression in the granulosa cells in vitro.Conclusion These results demonstrate directly, HMSeBA was more conducive to absorption and storage of selenium in the liver and ovary in gilt, and beneficial to exert the effect of HMSeBA on the antioxidant function in the liver and ovary of gilt. Moreover, HMSeBA has stronger antioxidant capacity in granular cells in vitro , which is more conducive to promoting follicle development. Therefore, the new type of organic selenium, HMSeBA, could be potentially useful for the control of reproductive processes in gilt.

scholarly journals Transcriptional, Mitochondrial Activity, and Viability of Egyptian Buffalo’s Granulosa Cells In Vitro Cultured under Heat Elevation

2021 ◽  
Vol 11 (2) ◽  
pp. 193-201
Author(s):  
Nasser Ghanem ◽  
Marwa Said Faheem ◽  
Romysa Samy ◽  
Ashraf Hesham Barkawi
Keyword(s):  
Heat Stress ◽  
Granulosa Cells ◽  
Transcriptional Activity ◽  
The Other ◽  
Control Group ◽  
Mitochondrial Activity ◽  
Fluorescent Intensity ◽  
Stressed Group ◽  
Other Hand

It is documented that heat stress caused impairment on the reproductive performance of dairy animals. However, there are few reports that have focused on the molecular and intracellular responses of in vitro cultured buffalo granulosa cells during heat elevation. The present study was conducted to investigate the effect of heat elevation during in vitro culture of buffalo granulosa cells on their viability, quality, mitochondrial activity, and transcriptional activity. Granulosa cells were harvested after aspiration of cumulus-oocytes complexes that were collected from abattoir ovaries. The granulosa cells were cultured in vitro either at a normal physiological temperature suitable for oocyte maturation and embryo development (38.5°C) or exposed to the elevated temperature of 40.5°C on day 3 of culture (the first two days were for confluence) for two hours of culture then continued at 38.5°C up to day 7 of culture. The viability of granulosa cells was measured using trypan blue and quality was estimated by measuring the level of intracellular reactive oxygen species (ROS) on day 7. Moreover, metabolic activity was performed by measuring the fluorescent intensity of mitochondria. Moreover, transcriptional activity was done by profiling four selected candidate genes using quantitative real-time PCR. The results indicated that the granulosa cells viability rate significantly decreased in the heat stress group (25.1 ± 3.7), compared to the control group (36.6 ± 5.3) on confluence day (day 3). In addition, the viability rate on the last day of culture (day 7) decreased in heat stress, compared to control (83.7 ± 4.5 and 97.4 ± 0.4, respectively). On the other hand, there was a nonsignificant difference in ROS profile between the control (21.7*104 ± 1.3) and the heat-stressed group (15.7 ± 0.7) on day 7 of culture. However, the mitochondrial fluorescent intensity was higher in the control (21.9 ± 1.9) than in the heat-stressed group (15.4 ± 0.8) on day 7 of culture. The expression of cellular defense (HSF1) and apoptosis-inducing gene (P53) were significantly up-regulated in granulosa cells exposed to heat elevation, compared to the control group. On the other hand, the steroidogenesis-regulating gene (StAR) was down-regulated in granulosa cells cultured under heat shock, compared to the control group. In conclusion, heat stress reduced the viability of granulosa cells by inducing the expression of an apoptosis-related gene (P53) and compromised expression of genes regulating the steroid biosynthesis, which resulted in up-regulation of cell defense gene (HSF1) in an attempt to ameliorate the deleterious effect of heat stress on the biological activity of the granulosa cells.

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