Expression and regulation of high mobility group AT-hook 1 (HMGA1) during ovulation and luteinisation in rat ovary

2019 ◽  
Vol 31 (4) ◽  
pp. 698 ◽  
Author(s):  
Hao-ran Li ◽  
Yan Li ◽  
Yu Liu ◽  
Jiao-jiao Yu ◽  
Fei-xue Li
Keyword(s):  
Mrna Expression ◽  
Granulosa Cell ◽  
Cell Cultures ◽  
Granulosa Cells ◽  
In Situ Hybridisation ◽  
High Mobility ◽  
High Mobility Group ◽  
Female Rats ◽  
Corpora Lutea ◽  
Hmga1 Expression

High mobility group AT-hook 1 (HMGA1) is able to regulate gene expression and function as a tumour suppressor. The spatiotemporal expression pattern of HMGA1 was investigated in this study. Immature female rats (22–23 days old) were treated with 10IU, s.c., pregnant mare’s serum gonadotrophin to stimulate follicular development, followed 48h later by injection with 5IU, s.c., human chorionic gonadotrophin (hCG). Whole ovaries or granulosa cells were collected at various times after hCG administration (n=3 per time point). Real-time polymerase chain reaction and western blot analysis revealed that HMGA1 was highly stimulated in the ovary by 4–12h after hCG treatment. In situ hybridisation analysis demonstrated that Hmga1 mRNA expression was induced in granulosa cells between 8 and 12h after hCG treatment. There was negligible Hmga1 mRNA signal observed in newly forming corpora lutea. In addition, the data indicated that both the protein kinase (PK) A and PKC pathways regulated Hmga1 expression in rat granulosa cells. In rat granulosa cell cultures, upregulation of Hmga1 was dependent on new protein synthesis because Hmga1 was inhibited by cycloheximide. Furthermore, Hmga1 mRNA expression in rat granulosa cell cultures was inhibited by AG1478, whereas NS398 and RU486 had no effect, suggesting that Hmga1 expression was regulated, in part, by the epidermal growth factor pathway. In summary, the findings of this study suggest that induction of Hmga1 may be important for theca and granulosa cell differentiation into luteal cells.

scholarly journals Cloning and Characterization of Granulosa Cell High-Mobility Group (HMG)-Box Protein-1, a Novel HMG-Box Transcriptional Regulator Strongly Expressed in Rat Ovarian Granulosa Cells

Endocrinology ◽  
2004 ◽  
Vol 145 (5) ◽  
pp. 2307-2318 ◽  
Author(s):  
Takashi Kajitani ◽  
Tetsuya Mizutani ◽  
Kazuya Yamada ◽  
Takashi Yazawa ◽  
Toshio Sekiguchi ◽  
...  
Keyword(s):  
Granulosa Cell ◽  
Granulosa Cells ◽  
High Mobility ◽  
Transcriptional Regulator ◽  
High Mobility Group ◽  
Hmg Box ◽  
Ovarian Granulosa Cells

scholarly journals The B Cell Translocation Gene (BTG) Family in the Rat Ovary: Hormonal Induction, Regulation, and Impact on Cell Cycle Kinetics

Endocrinology ◽  
2009 ◽  
Vol 150 (8) ◽  
pp. 3894-3902 ◽  
Author(s):  
Feixue Li ◽  
Jing Liu ◽  
Eun-Sil Park ◽  
Misung Jo ◽  
Thomas E. Curry
Keyword(s):  
Cell Cycle ◽  
B Cell ◽  
Mrna Expression ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Expression Patterns ◽  
Female Rats ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Rat Ovary

The B cell translocation gene (BTG) family regulates gene transcription and cellular differentiation and inhibits proliferation. The present study investigated the spatiotemporal expression pattern of BTG members and their potential role in the rat ovary during the periovulatory period. Immature female rats (22–23 d old) were injected with pregnant mare serum gonadotropin to stimulate follicular development. Ovaries or granulosa cells were collected at various times after hCG administration (n = 3 per time point). Real-time PCR analysis revealed that mRNA for Btg1, Btg2, and Btg3 were highly induced both in intact ovaries and granulosa cells by 4–8 h after hCG treatment, although their temporal expression patterns differed. In situ hybridization analysis demonstrated that Btg1 mRNA expression was highly induced in theca cells at 4 h after hCG, primarily localized to granulosa cells at 8 h, and decreased at 24 h. Btg2 and Btg3 mRNA was also induced in granulosa cells; however, Btg2 mRNA was observed in newly forming corpora lutea. Inhibition of progesterone action and the epidermal growth factor pathway did not change Btg1 and Btg2 mRNA expression, whereas inhibition of prostaglandin synthesis or RUNX activity diminished Btg2 mRNA levels. Overexpression of BTG1 or BTG2 arrested granulosa cells at the G0/G1 phase of the cell cycle and decreased cell apoptosis. In summary, hCG induced Btg1, Btg2, and Btg3 mRNA expression predominantly in the granulosa cell compartment. Our findings suggest that the induction of the BTG family may be important for theca and granulosa cell differentiation into luteal cells by arresting cell cycle progression.

The Fine Structure of Rat Granulosa Cell Cultures Correlated with Progestin Secretion

1973 ◽  
Vol 31 ◽  
pp. 552-553
Author(s):  
T. M. Crisp ◽  
F.R. Denys
Keyword(s):  
Fine Structure ◽  
Granulosa Cell ◽  
Cell Cultures ◽  
Single Injection ◽  
Surrounding Medium ◽  
Petri Dish ◽  
Female Rats ◽  
Vaginal Smear ◽  
Immature Female ◽  
Serum Gonadotropin

The purpose of this paper is to present observations on the fine structure of rat granulosa cell cultures grown in the presence of an adenohypophyseal explant and to correlate the morphology of these cells with progestin secretion. Twenty-six day old immature female rats were given a single injection of 5 IU pregnant mares serum gonadotropin (PMS) in order to obtain ovaries with large vesicular follicles. At 66 hrs. post-PMS administration (estrus indicated by vaginal smear cytology), the ovaries were removed and placed in a petri dish containing medium 199 and 100 U penicillin/streptomycin (P/S)/ml. Under a 20X magnification dissecting microscope, some 5-8 vesicular follicles/ovary were punctured and the granulosa cells were expressed into the surrounding medium. The cells were transferred to centrifuge tubes and spun down at 1000 rpm for 5 mins.

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.

P–229 Oxidative stress and Metformin; an in-vitro study on serum and primary human granulosa cell cultures

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
F Alam ◽  
R Rehman ◽  
N Farooqui ◽  
F Jehan ◽  
S H Abidi
Keyword(s):  
Oxidative Stress ◽  
Sample Size ◽  
Granulosa Cell ◽  
Cell Cultures ◽  
Granulosa Cells ◽  
Metformin Treatment ◽  
Serum Samples ◽  
Human Granulosa Cells ◽  
Human Granulosa Cell ◽  
Infertile Patients

Abstract Study question What is the effect of administration of Metformin on the oxidative stress (OS) levels in serum and primary human granulosa cell cultures of infertile females? Summary answer Metformin suppresses oxidative stress in serum and human granulosa cells and increases the expression of SIRT1 in OS induced environment. What is known already Oxidative stress (OS) is a resultant of mitochondrial dysfunction when it either fails to fight against the oxidants or the expression of the antioxidants is not sufficient. Cellular damage including DNA damage is a common resultant of oxidative stress. OS effects the oocyte maturation and moreover, the cleavage phase in the early embryonic stage. The raised levels of OS makers are hypothesized to compromise the nuclear maturation and the mitotic spindles of the maturing oocytes. Metformin seemed to decrease oxidative stress and improve insulin resistance, dyslipidaemia and endothelial dysfunction in PCOS patients Study design, size, duration This cross-sectional study was conducted from August 2017 – July 2019, at Aga Khan Hospital in collaboration with Australian Concept Infertility Medical Centre (ACIMC) on ten infertile patients undergoing egg retrieval after ethical approval from of Aga Khan Hospital (AKU-ERC–2018–0557–601). Participants/materials, setting, methods Serum samples were obtained and analysed. Follicular fluid of these subjects was collected for establishment of primary cell culture model of normal human granulosa cells (hGCs). Serum and hGC cultures were grouped as; a) control: treatment, b) Test1: H2O2 induced OS, and c) Test2: H2O2 induced OS treated with metformin. OS was estimated in all groups by Mishra method. The two Test groups were assessed for SIRT1 levels using quantitative PCR employing SIRT1 specific primers Main results and the role of chance With mean age of 32.04 ± 2.29 years the mean BMI was 27.61 ± 2.15 kg/m2. OS was induced and measured by an increase in optical density (OD) in hGC Test samples which showed 0.28 (0.16–0.40) OD when compared with control hGC samples 0.153 (0.09–0.23). There was a significant reduction in ODs after metformin treatment in the stress induced cells 0.182 (0.05–0.30). A similar pattern was observed in the serum samples in ODs; control: 0.105 (0.09–0.15), stress induced samples: 0.199 (0.19–0.20). and stress induced serum sample with metformin treatment: 0.1415 (0.06–0.18). The Ct values obtained to express the effect of metformin on SIRT1 levels, for OS induced (Test1) and OS induced metformin treated (Test2) cells were found to be 29.12 and 26.42, respectively. We also observed a significant (85%) difference in the fold change of SIRT1 expression between metformin treated and untreated cells. Limitations, reasons for caution Small sample size is the limitation of this study. The impact of metformin on cell cultures due to different causes of infertility could not be ascertained Wider implications of the findings: Metformin suppresses oxidative stress in serum and human granulosa cells and increases the expression of SIRT1 in OS induced environment, therefore, metformin may be considered as a treatment of oxidative stress in infertile patients. Randomized control trial with large sample size is recommended to confirm the cause and effect relationship. Trial registration number Not applicable

scholarly journals Stimulatory Effect of Insulin on 5α-Reductase Type 1 (SRD5A1) Expression through an Akt-Dependent Pathway in Ovarian Granulosa Cells

Endocrinology ◽  
2010 ◽  
Vol 151 (10) ◽  
pp. 5030-5037 ◽  
Author(s):  
Pradeep P. Kayampilly ◽  
Brett L. Wanamaker ◽  
James A. Stewart ◽  
Carrie L. Wagner ◽  
K. M. J. Menon
Keyword(s):  
Catalytic Activity ◽  
Mrna Expression ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Insulin Treatment ◽  
Reductase Mrna ◽  
Dose Dependent Increase ◽  
Dose Dependent ◽  
Dependent Pathway

Elevated levels of 5α-reduced androgens have been shown to be associated with hyperandrogenism and hyperinsulinemia, the leading causes of ovulatory dysfunction in women. 5α-Dihydrotestosterone reduces ovarian granulosa cell proliferation by inhibiting FSH-mediated mitogenic signaling pathways. The present study examined the effect of insulin on 5α-reductase, the enzyme that catalyses the conversion of androgens to their 5α-derivatives. Granulosa cells isolated from immature rat ovaries were cultured in serum-free, phenol red-free DMEM-F12 media and treated with different doses of insulin (0, 0.1, 1.0, and 10.0 μg/ml) for different time intervals up to 12 h. The expression of 5α-reductase type 1 mRNA, the predominant isoform found in granulosa cells, showed a significant (P < 0.05) increase in response to the insulin treatment up to 12 h compared with control. The catalytic activity of 5α-reductase enzyme was also stimulated in a dose-depended manner (P < 0.05). Inhibiting the Akt-dependent signaling pathway abolished the insulin-mediated increase in 5α-reductase mRNA expression, whereas inhibition of the ERK-dependent pathway had no effect. The dose-dependent increase in 5α-reductase mRNA expression as well as catalytic activity seen in response to insulin treatment was also demonstrated in the human granulosa cell line (KGN). In addition to increased mRNA expression, a dose-dependent increase in 5α-reductase protein expression in response to insulin was also seen in KGN cells, which corroborated well with that of mRNA expression. These results suggest that elevated levels of 5α-reduced androgens seen in hyperinsulinemic conditions might be explained on the basis of a stimulatory effect of insulin on 5α-reductase in granulosa cells. The elevated levels of these metabolites, in turn, might adversely affect growth and proliferation of granulosa cells, thereby impairing follicle growth and ovulation.

Toxic Effects of Aluminium on Female Reproductive System in Presence of Ethanol Coexposure

2021 ◽  
pp. 3809-3815
Author(s):  
Buddhadeb Ghosh ◽  
Ravi Kant Sharma ◽  
Suman Yadav ◽  
Ankita Randev
Keyword(s):  
Body Weight ◽  
Granulosa Cells ◽  
Reproductive Toxicity ◽  
Concomitant Administration ◽  
Exposure Period ◽  
Treated Group ◽  
Female Rats ◽  
Corpora Lutea ◽  
Combined Administration ◽  
Increasing Trends

Both aluminium and ethanol are pro-oxidants and toxic. Uncontrolled use of aluminium and increasing trends of ethanol consumption in India increased the chance of coexposure to aluminium and ethanol. There are possibilities, that both of them follow common mechanisms to produce reproductive toxicity. The present study was planned to identify the effects of aluminium administration on the microscopic structure of ovary and to clarify any possible protection conferred by the concomitant administration of ethanol. Sixteen female rats divided into one control and three experimental groups exposed to aluminium (4.2mg/kg body weight) and ethanol (1gm/kg body weight) for 3 months. After the exposure period, ovaries were processed for light microscopic examination. Ovary showed significant atretic follicles with degenerated ova and vacuolation. Rupture of zona pellucida in oocyte seen in aluminium treated animals. Ethanol treated group showing absence of growing follicles, increased large corpora lutea. Dilated and congested vessels were observed in the growing follicle. The effects of combined administration of aluminium and ethanol treated groups showed with acute degeneration of growing follicles, with desquamation of pyknotic granulosa cells and degenerated oocyte. Multiple vacuoles of degenerated granulosa cells with dilated congested vessels and edema seen. Hyaline material seen inside the degenerating follicles. It has been suggested that the ethanol induced augmentation of impacts of aluminium on the Ovary.

Prolactin, LH, and estradiol-17β in utilization of lipoprotein substrate by porcine granulosa cells in vitro

1988 ◽  
Vol 66 (5) ◽  
pp. 561-566 ◽  
Author(s):  
K. Rajkumar ◽  
P. Klingshorn ◽  
P. J. Chedrese ◽  
B. D. Murphy
Keyword(s):  
Granulosa Cell ◽  
Cell Cultures ◽  
Granulosa Cells ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Low Density ◽  
Porcine Granulosa Cells ◽  
Progesterone Synthesis ◽  
Serum Free Conditions

Porcine granulosa cells cultured under serum free conditions responded by increased progesterone secretion to the addition of the leuteotropic hormones, LH, prolactin, and estradiol. Provision of extracellular substrate for steroidogenesis in the form of porcine high density lipoprotein or low density lipoprotein enhanced progesterone accumulation by granulosa cell cultures. Estradiol, LH, and prolactin all greatly increased progesterone accumulation in the presence of either high or low density lipoproteins. Increases in progesterone accumulation following addition of prolactin or LH in combination with estradiol suggested the presence of a synergistic interaction among leuteotropins. Pre-exposure of granulosa cell cultures to estradiol increased the subsequent stimulatory effect of prolactin on lipoprotein utilization. It is concluded that all three leuteotropins function to enhance and may interact in the utilization of extracellular lipoprotein substrate for progesterone synthesis.

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 Temporal and hormonal regulation of inhibin protein and subunit mRNA expression by post-natal and immature rat ovaries

2000 ◽  
Vol 166 (2) ◽  
pp. 339-354 ◽  
Author(s):  
AE Drummond ◽  
M Dyson ◽  
E Thean ◽  
NP Groome ◽  
DM Robertson ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Cell Cultures ◽  
Granulosa Cells ◽  
Inhibin B ◽  
Alpha Subunit ◽  
Inhibin A ◽  
Preantral Follicles ◽  
Subunit Mrna ◽  
Ovarian Cells ◽  
Old Rats

The contribution of specific follicle populations to dimeric inhibin production and inhibin subunit mRNA expression by the rat ovary has been investigated in two model systems, granulosa cells isolated from 25-day-old diethylstilboestrol (DES)-treated rats and post-natal rat ovaries, dispersed in culture or whole ovaries, using specific two-site immunoassays and 'real time' PCR. Media from FSH-stimulated granulosa cell cultures fractionated by gel filtration and RP-high performance liquid chromatography revealed two predominant peaks of alpha subunit activity which were attributed to alpha subunit and 31 k dimeric inhibin-A. The corresponding inhibin-B levels were low. FSH stimulation did not alter the ratio of inhibin-A:alpha subunit produced by granulosa cells. All three inhibin subunit mRNAs were expressed by granulosa cells, with eight-fold more alpha subunit mRNA relative to either of the beta subunits. Administration of DES to immature rats prior to the isolation of granulosa cells from the ovary led to beta(A) and beta(B) mRNA expression being down-regulated in the absence of any significant change in alpha subunit expression by the granulosa cells. Inhibin-A, -B and -alpha subunit were produced by basal and stimulated cultures of ovarian cells prepared from 4-, 8- and 12-day-old rats, indicating that primary, preantral and antral follicles contribute to total inhibin production. Consistent with these results, follicles within these ovaries expressed all three inhibin subunit mRNAs, with maximal expression observed in the ovaries of 8-day-old rats. The appearance of antral follicles in the ovary at day 12 led to a decline in the mRNA levels of each of the subunits but was most evident for the beta subunits. There was a profound influence of secondary preantral follicles on dimeric inhibin-A production, with FSH stimulation increasing inhibin-A relative to alpha subunit levels in cultures of ovarian cells prepared from 8-day-old rats. Thus, preantral follicles exposed to FSH contribute significantly to beta(A) subunit production by the ovary. In contrast, primary and preantral follicles did not produce inhibin-B in response to FSH stimulation. Transforming growth factor-beta (TGF-beta) enhanced, in a time-dependent manner, the production of the inhibin forms by ovarian cells in culture, although inhibin-B production was not responsive until day 8. The simultaneous treatment of ovarian cell cultures with FSH and TGF-beta elicited the greatest increases in production of all the inhibin forms. In summary, ovaries of 4-, 8- and 12-day-old rats expressed inhibin subunit mRNAs and produced dimeric inhibin-A and -B and free alpha subunit. Preantral follicles (day-8 ovarian cell cultures) were particularly sensitive to stimulation by FSH and TGF-beta and had a substantial capacity for inhibin production. The production of oestrogen by follicles may be instrumental in regulating inhibin production given that beta subunit mRNA expression was down-regulated by DES. The mechanisms by which inhibin-A and inhibin-B are individually regulated are likely to be similar during the post-natal period, when folliculogenesis is being established, and diverge thereafter, when inhibin-A becomes the predominant form in the fully differentiated ovary.

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