The Effects of Leptin and Irisin on Steroidogenic Enzyme Gene Expression in Human Ovarian Granulosa Cells - Initial Studies

Abstract Fertility and energy metabolism are closely associated, and the cytokines produced by the adipose and muscle tissue play a role in this association. Leptin, predominantly produced by the white adipose tissue, and irisin, produced by the brown adipose and skeletal muscle tissues, are cytokines that are important in balancing energy metabolism. This study aimed to investigate the effects of leptin and irisin on steroidogenic enzyme gene expression in human ovarian granulosa cells in vitro. Granulosa cells were retrieved and isolated from ovarian follicular fluid during in vitro fertilization (IVF) procedures. Cells were placed in primary in vitro cultures and treated with increasing concentrations of leptin (25, 50, 100, 200, and 400 ng/ml) or irisin (125, 250, 500, 1,000, and 2,000 ng/ml) for 24, 48, and 72 hours. mRNA expression levels of CYP11A1, CYP19A1, CYP21A2, HSD3B1, and HSD17B3 were measured by qRT-PCR analysis. Leptin treatment of granulosa cells resulted in significant upregulation of CYP21A2 mRNA levels, while irisin significantly downregulated mRNA levels of CYP11A1, CYP19A1, and HSD3B1. Taken together, these early experiments demonstrate that leptin and irisin may affect steroid hormone production in the ovary by targeting the gene expression of key steroidogenic enzymes. Additional experiments are in progress.

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Trans-resveratrol modulates the catalytic activity and mRNA expression of the procarcinogen-activating human cytochrome P450 1B1

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y00-067 ◽

2000 ◽

Vol 78 (11) ◽

pp. 874-881 ◽

Cited By ~ 29

Author(s):

Thomas KH Chang ◽

Wendy BK Lee ◽

Hin Hin Ko

Keyword(s):

Gene Expression ◽

Cytochrome P450 ◽

Catalytic Activity ◽

Pcr Analysis ◽

Mrna Levels ◽

Toxic Response ◽

Cytochrome P450 1B1 ◽

Cyp1b1 Mrna ◽

Mcf 7

The present study was performed to determine if trans-resveratrol (3,5,4'-trihydroxy-trans-stilbene) modulates the catalytic activity and gene expression of cytochrome P450 1B1 (CYP1B1). In vitro, trans-resveratrol decreased human recombinant CYP1B1-catalyzed 7-ethoxyresorufin O-dealkylation activity, with an IC50 value of 1.4 ± 0.2 µM (mean ± SEM). Enzyme kinetic analysis indicated that trans-resveratrol inhibited CYP1B1 enzyme activity by a mixed-type inhibition and the apparent Ki was 0.75 ± 0.06 µM. To determine if trans-resveratrol modulates constitutive CYP1B1 gene expression, cultured MCF-7 human breast carcinoma cells were treated with trans-resveratrol. As indicated by RT-PCR analysis, treatment of MCF-7 cells with 10 µM trans-resveratrol decreased relative CYP1B1 mRNA levels after 5 h, but not after 1.5 or 3 h, of exposure. trans-Resveratrol treatment at 5, 7.5, 10, or 20 µM for 5 h produced a concentration-dependent decrease in CYP1B1 mRNA levels. The extent of suppression was ~50% at 20 µM concentration. The suppressive effect was not a consequence of a toxic response to the compound as assessed by a cell proliferation assay. Overall, our novel finding that trans-resveratrol inhibits the catalytic activity and suppresses the constitutive gene expression of CYP1B1 leads to the possibility that this nutraceutical confers protection against toxicity and carcinogenicity induced by compounds that undergo CYP1B1-catalyzed bioactivation.Key words: cytochrome P450, CYP1B1, 7-ethoxyresorufin, nutraceutical, trans-resveratrol.

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Insulin and IGF-I are necessary for FSH-induced cytochrome P450 aromatase but not cytochrome P450 side-chain cleavage gene expression in oestrogenic bovine granulosa cells in vitro

Journal of Endocrinology ◽

10.1677/joe.0.1740499 ◽

2002 ◽

Vol 174 (3) ◽

pp. 499-507 ◽

Cited By ~ 36

Author(s):

JM Silva ◽

CA Price

Keyword(s):

Gene Expression ◽

Cytochrome P450 ◽

Granulosa Cells ◽

Side Chain ◽

Mrna Levels ◽

Side Chain Cleavage ◽

P450 Aromatase ◽

Chain Cleavage ◽

Igf I

The earliest biochemical indicators of ovarian follicle deviation in cattle include lower oestradiol and free IGF concentrations in subordinate compared with dominant follicles. We determined if decreases in FSH, IGF-I or insulin cause decreased P450 aromatase (P450arom) or P450 cholesterol side-chain cleavage (P450scc) mRNA expression in oestrogenic bovine granulosa cells in vitro. In the first experiment, cells obtained from small follicles (2-5 mm diameter) were cultured in serum-free medium supplemented with physiological concentrations of FSH, IGF-I and insulin for 4 days. A decrease in specific hormone concentration was produced by replacing 70% of spent medium with medium devoid of FSH, insulin, or insulin and IGF-I on day 4 and again on day 5 of culture. Cultures were terminated on day 7. A reduction in FSH concentrations during the last 3 days of culture decreased P450arom and P450scc mRNA levels. A reduction in insulin reduced P450arom but not P450scc mRNA levels, and a reduction of both insulin and IGF-I concentrations further decreased P450arom mRNA levels and decreased P450scc mRNA levels. In a second experiment, cells obtained from small follicles (2-5 mm diameter) were cultured with insulin (100 ng/ml) without FSH for 4 days, and then insulin was withdrawn from the culture and FSH added for a further 3 days. The withdrawal of insulin decreased (P<0.02) oestradiol accumulation and reduced P450arom mRNA to below detectable levels, but did not affect P450scc mRNA levels. The addition of FSH transiently increased oestradiol secretion and P450arom mRNA levels, but P450arom mRNA levels were undetectable at the end of the culture period. The addition of FSH significantly enhanced P450scc mRNA levels and progesterone accumulation. These data demonstrated that a reduction of insulin-like activity reduced aromatase gene expression in bovine follicles without necessarily affecting progesterone synthetic capability, and thus may initiate follicle regression in cattle at the time of follicle divergence.

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Differentiation of sow and mouse ovarian granulosa cells exposed to zearalenone in vitro using RNA-seq gene expression

Toxicology and Applied Pharmacology ◽

10.1016/j.taap.2018.05.003 ◽

2018 ◽

Vol 350 ◽

pp. 78-90 ◽

Cited By ~ 4

Author(s):

Guo-Liang Zhang ◽

Jun-Lin Song ◽

Yi Zhou ◽

Rui-Qian Zhang ◽

Shun-Feng Cheng ◽

...

Keyword(s):

Gene Expression ◽

Granulosa Cells ◽

Rna Seq ◽

Ovarian Granulosa Cells

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Arachidonic Acid Regulation of Intracellular Signaling Pathways and Target Gene Expression in Bovine Ovarian Granulosa Cells

Animals ◽

10.3390/ani9060374 ◽

2019 ◽

Vol 9 (6) ◽

pp. 374 ◽

Cited By ~ 6

Author(s):

Nina Zhang ◽

Liqiang Wang ◽

Guoya Luo ◽

Xiaorong Tang ◽

Lizhu Ma ◽

...

Keyword(s):

Gene Expression ◽

Signaling Pathways ◽

Granulosa Cells ◽

Intracellular Signaling ◽

Hormone Secretion ◽

Ovarian Granulosa Cells ◽

Genes Expression ◽

Higher Doses ◽

Gene Expression Levels

In the present study, AA was used to challenge bovine ovarian granulosa cells in vitro and the related parameters of cellular and molecular biology were measured. The results indicated that lower doses of AA increased survival of bovine granulosa cells whereas higher doses of AA suppressed survival. While lower doses of AA induced accumulation of lipid droplet in granulosa cells, the higher dose of AA inhibited lipid accumulation, and AA increased abundance of FABP3, CD36 and SLC27A1 mRNA. Higher doses of AA decreased the secretion of E2 and increased the secretion of P4 accompanied by down-regulation of the mRNA abundance of CYP19A1, FSHR, HSD3B1 and STAR in granulosa cells. The signaling pathways employed by AA in the stimulation of genes expression included both ERK1/2 and Akt. Together, AA specifically affects physiological features, gene expression levels and steroid hormone secretion, and thus altering the functionality of granulosa cells of cattle.

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Apoptosis-related genes expression in primary in vitro culture of human ovarian granulosa cells

Medical Journal of Cell Biology ◽

10.2478/acb-2020-0023 ◽

2020 ◽

Vol 8 (4) ◽

pp. 176-182

Author(s):

Ievgeniia Kocherova ◽

Katarzyna Stefańska ◽

Rut Bryl ◽

Joanna Perek ◽

Wojciech Pieńkowski ◽

...

Keyword(s):

Gene Expression ◽

Oocyte Maturation ◽

Granulosa Cells ◽

Oocyte Quality ◽

Individual Gene ◽

Time Points ◽

Ovarian Granulosa Cells ◽

Genes Expression ◽

Qpcr Method

AbstractOvarian granulosa cells (GCs) play a crucial role in oocyte maturation, creating a favorable microenvironment around the oocyte. Therefore, enhanced apoptosis and GCs loss may negatively affect the intra-follicular milieu and compromise the oocyte quality, reducing pregnancy chances. Based on the RT-qPCR method, the present research revealed the differential expression of apoptosis-related genes (BCL2, BAX, p53, CASP9) during the seven days of primary in vitro culture of GCs isolated from patients undergoing in vitro fertilization (IVF) procedure. Individual gene expression changes may reflect the GCs survival and/or apoptotic status at different time points.Running title: Apoptosis-related genes expression in granulosa cells in vitro

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RNA-seq based gene expression analysis of ovarian granulosa cells exposed to zearalenone in vitro: significance to steroidogenesis

Oncotarget ◽

10.18632/oncotarget.19699 ◽

2017 ◽

Vol 8 (38) ◽

pp. 64001-64014 ◽

Cited By ~ 9

Author(s):

Guo-Liang Zhang ◽

Rui-Qian Zhang ◽

Xiao-Feng Sun ◽

Shun-Feng Cheng ◽

Yu-Feng Wang ◽

...

Keyword(s):

Gene Expression ◽

Expression Analysis ◽

Granulosa Cells ◽

Gene Expression Analysis ◽

Rna Seq ◽

Ovarian Granulosa Cells

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Amphetamine-Decreased Progesterone and Estradiol Release in Rat Granulosa Cells: The Regulatory Role of cAMP- and Ca2+-Mediated Signaling Pathways

Biomedicines ◽

10.3390/biomedicines9050493 ◽

2021 ◽

Vol 9 (5) ◽

pp. 493

Author(s):

Chung-Yu Chen ◽

Chien-Rung Chen ◽

Chiao-Nan Chen ◽

Paulus S. Wang ◽

Toby Mündel ◽

...

Keyword(s):

Granulosa Cells ◽

Prostaglandin F2α ◽

Inhibitory Effect ◽

Inhibitory Effects ◽

Steroidogenic Enzyme ◽

Estradiol Secretion ◽

Basal Release ◽

Ca2 Channels

The purpose of this study is to evaluate the amphetamine effects on progesterone and estradiol production in rat granulosa cells and the underlying cellular regulatory mechanisms. Freshly dispersed rat granulosa cells were cultured with various test drugs in the presence of amphetamine, and the estradiol/progesterone production and the cytosolic cAMP level were measured. Additionally, the cytosolic-free Ca2+ concentrations ([Ca2+]i) were measured to examine the role of Ca2+ influx in the presence of amphetamine. Amphetamine in vitro inhibited both basal and porcine follicle-stimulating hormone-stimulated estradiol/progesterone release, and amphetamine significantly decreased steroidogenic enzyme activities. Adding 8-Bromo-cAMP did not recover the inhibitory effects of amphetamine on progesterone and estradiol release. H89 significantly decreased progesterone and estradiol basal release but failed to enhance a further amphetamine inhibitory effect. Amphetamine was capable of further suppressing the release of estradiol release under the presence of nifedipine. Pretreatment with the amphetamine for 2 h decreased the basal [Ca2+]i and prostaglandin F2α-stimulated increase of [Ca2+]i. Amphetamine inhibits progesterone and estradiol secretion in rat granulosa cells through a mechanism involving decreased PKA-downstream steroidogenic enzyme activity and L-type Ca2+ channels. Our current findings show that it is necessary to study the possibility of amphetamine perturbing reproduction in females.

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Age-related decline in the expression of GDF9 and BMP15 genes in follicle fluid and granulosa cells derived from poor ovarian responders

Journal of Ovarian Research ◽

10.1186/s13048-020-00757-x ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Yan Gong ◽

Jesse Li-Ling ◽

Dongsheng Xiong ◽

Jiajing Wei ◽

Taiqing Zhong ◽

...

Keyword(s):

Granulosa Cells ◽

Enzyme Linked Immunosorbent Assay ◽

Mrna Levels ◽

Clinical Trial Registry ◽

Altered Expression ◽

Vitro Fertilization ◽

Age Related ◽

Tertiary Teaching ◽

Poor Ovarian Responders

Abstract Background Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) genes play important roles in folliculogenesis. Altered expression of the two have been found among patients with poor ovarian response (POR). In this prospective cohort study, we have determined the expression of the GDF9 and BMP15 genes in follicle fluid (FF) and granulosa cells (GCs) derived from poor ovarian responders grouped by age, and explored its correlation with the outcome of in vitro fertilization and embryo transfer (IVF-ET) treatment. Methods A total of 196 patients with POR were enrolled from a tertiary teaching hospital. The patients were diagnosed by the Bologna criteria and sub-divided into group A (< 35 year old), group B (35–40 year old), and group C (> 40 year old). A GnRH antagonist protocol was conducted for all patients, and FF and GCs were collected after oocyte retrieval. Expression of the GDF9 and BMP15 genes in the FF and GCs was determined with enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. Results Compared with group C, groups A and B had significantly more two pronuclei (2PN) oocytes and transplantable embryos, in addition with higher rates of implantation and clinical pregnancy (P < 0.05). The expression level of GDF9 and BMP15 genes in the FF and GCs differed significantly among the three groups (P < 0.05), showing a trend of decline along with age. The ratio of GDF9/BMP15 mRNA levels were similar among the three groups (P > 0.05). The relative levels of GDF9 and BMP15 proteins in GCs have correlated with the relative mRNA levels in GCs and protein concentrations in FF (P < 0.05). Conclusions For poor ovarian responders, in particular those over 40, the expression of GDF9 and BMP15 is declined along with increased age and in accompany with poorer oocyte quality and IVF outcome, whilst the ratio of GDF9/BMP15 mRNA levels remained relatively constant. Trial registration Chinese Clinical Trial Registry Center (ChiCTR1800016107). Registered on 11 May 2018.

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Differential Effects of Gonadotropin-Releasing Hormone (GnRH) Pulse Frequency on Gonadotropin Subunit and GnRH Receptor Messenger Ribonucleic Acid Levels in Vitro*

Endocrinology ◽

10.1210/endo.138.3.4968 ◽

1997 ◽

Vol 138 (3) ◽

pp. 1224-1231 ◽

Cited By ~ 112

Author(s):

Ursula B. Kaiser ◽

Andrzej Jakubowiak ◽

Anna Steinberger ◽

William W. Chin

Keyword(s):

Gene Expression ◽

Messenger Rna ◽

Pulse Frequency ◽

Gnrh Receptor ◽

Pituitary Cells ◽

Mrna Levels ◽

Subunit Gene ◽

Differential Effects ◽

Messenger Ribonucleic Acid

Abstract The hypothalamic hormone, GnRH, is released and transported to the anterior pituitary in a pulsatile manner, where it binds to specific high-affinity receptors and regulates gonadotropin biosynthesis and secretion. The frequency of GnRH pulses changes under various physiological conditions, and varying GnRH pulse frequencies have been shown to regulate differentially the secretion of LH and FSH and the expression of the gonadotropin α, LHβ, and FSHβ subunit genes in vivo. We demonstrate differential effects of varying GnRH pulse frequency in vitro in superfused primary monolayer cultures of rat pituitary cells. Cells were treated with 10 nm GnRH pulses for 24 h at a frequency of every 0.5, 1, 2, or 4 h. α, LHβ, and FSHβ messenger RNA (mRNA) levels were increased by GnRH at all pulse frequencies. α and LHβ mRNA levels and LH secretion were stimulated to the greatest extent at a GnRH pulse frequency of every 30 min, whereas FSHβ mRNA levels and FSH secretion were stimulated maximally at a lower GnRH pulse frequency, every 2 h. GnRH receptor (GnRHR) mRNA levels also were increased by GnRH at all pulse frequencies and were stimulated maximally at a GnRH pulse frequency of every 30 min. Similar results were obtained when the dose of each pulse of GnRH was adjusted to maintain a constant total cumulative dose of GnRH over 24 h. These data show that gonadotropin subunit gene expression is regulated differentially by varying GnRH pulse frequencies in vitro, suggesting that the differential effects of varying GnRH pulse frequencies on gonadotropin subunit gene expression occur directly at the level of the pituitary. The pattern of regulation of GnRHR mRNA levels correlated with that of α and LHβ but was different from that of FSHβ. This suggests that α and LHβ mRNA levels are maximally stimulated when GnRHR levels are relatively high, whereas FSHβ mRNA levels are maximally stimulated at lower levels of GnRHR expression, and that the mechanism for differential regulation of the gonadotropins by varying pulse frequencies of GnRH may involve levels of GnRHR. Furthermore, these data suggest that the mechanisms whereby varying GnRH pulse frequencies stimulate α, LHβ, and GnRHR gene expression are similar, whereas the stimulation of FSHβ mRNA levels may be different.

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