MON-012 The Direct Effect of Kisspeptin on Human Ovarian Granulosa Cells to Regulate Steroidogenesis

Abstract Kisspeptin has a central role to stimulate the hypothalamic-pituitary-gonadal (HPG) axis. Furthermore, a previous study has suggested that kisspeptin might have a peripheral role in follicular development (1). This study aimed to 1) explore the effect of kisspeptin on CYP19A1 (aromatase) mRNA expression in human granulosa cells and aromatase concentrations in the supernatant; and 2) investigate the effect of kisspeptin on FSHR mRNA expression in human granulosa cells. In this study, human granulosa-like tumor cell line (KGN) (n=3) was incubated for 24 hours with FSH (10-8 M); FSH with IGF-1 (10-8 M); different doses of kisspeptin including 1, 10, 100, 1,000, and 10,000 nM; FSH with different doses of kisspeptin; and FSH with IGF-1 together with different doses of kisspeptin. FSH treatment alone or FSH with IGF-1 did not increase CYP19A1 mRNA expression when compared to control. Interestingly, kisspeptin treatment at the doses of 100 nM (P=0.028), 1,000 nM (P=0.005), and 10,000 nM (P=0.009) in the presence of FSH together with IGF-1 enhanced CYP19A1 mRNA expression when compared with control. Furthermore, FSH or FSH with IGF-1 or FSH with all doses of kisspeptin or FSH with IGF-1 together with all doses of kisspeptin increased aromatase concentrations in the supernatant when compared to control (P<0.01 all). Surprisingly, kisspeptin at the dose of 10,000 nM with FSH or FSH together with IGF-1 statistically increased aromatase concentrations in the supernatant when compared with FSH treatment alone or FSH with IGF-1 treatment (P<0.01 all). FSHR mRNA expression was comparable between control and all treatments. As a result, kisspeptin combined with FSH and IGF-1 could enhance CYP19A1 mRNA expression in human granulosa cells and the high dose of kisspeptin (10,000 nM) might be able to augment aromatase secretion in the supernatant. These results suggest that kisspeptin might enhance aromatase expression and secretion, which probably leads to enhance estrogen synthesis. Further studies regarding kisspeptin treatment on estrogen synthesis or secretion in human granulosa cells should be confirmed. Reference: (1) Fernandois D, et al. J Endocrinol. 2016;228(3):161-70.

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Rosiglitazone and Pioglitazone Alter Aromatase Kinetic Properties in Human Granulosa Cells

PPAR Research ◽

10.1155/2011/926438 ◽

2011 ◽

Vol 2011 ◽

pp. 1-5 ◽

Cited By ~ 4

Author(s):

Takako Araki ◽

Miroslava Varadinova ◽

Michael Goldman ◽

Zev Rosenwaks ◽

Leonid Poretsky ◽

...

Keyword(s):

Tissue Culture ◽

Granulosa Cells ◽

Culture Medium ◽

Kinetic Properties ◽

Enzyme Kinetic ◽

Human Granulosa Cells ◽

Estrogen Synthesis ◽

Aromatase Mrna ◽

Androgen Binding ◽

Noncompetitive Inhibitors

We have previously reported that, in human granulosa cells, thiazolidinediones rosiglitazone and pioglitazone inhibit estrogen synthesis by interfering with androgen binding to aromatase, without an effect on aromatase mRNA or protein expression. In the current paper, we explore the effects of rosiglitazone and pioglitazone on the aromatase enzyme kinetic properties in human granulosa cells. The cells were incubated with various concentrations of testosterone or androstenedione, with or without rosiglitazone or pioglitazone. Estradiol and estrone concentrations in the conditioned tissue culture medium were measured by radioimmunoassay or immunosorbent assay. When testosterone was used as substrate, rosiglitazone or pioglitazone inhibited theVmaxby 35% (P<0.001) and 24% (P<0.001), respectively. When androstenedione was used as substrate, both rosiglitazone or pioglitazone inhibitedVmaxby 13% (P<0.007). We conclude that rosiglitazone or pioglitazone has no effect onKmbut inhibitsVmaxof aromatase in human granulosa cells, therefore, acting as noncompetitive inhibitors.

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KISS1 Suppresses Apoptosis and Stimulates the Synthesis of E2 in Porcine Ovarian Granulosa Cells

Animals ◽

10.3390/ani9020054 ◽

2019 ◽

Vol 9 (2) ◽

pp. 54 ◽

Cited By ~ 5

Author(s):

Xiaoping Xin ◽

Zhonghui Li ◽

Yuyi Zhong ◽

Qingqing Li ◽

Jiaying Wang ◽

...

Keyword(s):

Cell Cycle ◽

Mrna Expression ◽

Signaling Pathway ◽

Granulosa Cells ◽

Cell Apoptosis ◽

Pi3k Signaling ◽

Mrna Levels ◽

Ovarian Granulosa Cells ◽

Estrogen Synthesis ◽

Pi3k Signaling Pathway

Previous studies have strongly recommended that KISS-1 metastasis suppressor (KISS1) plays an essential gatekeeper of the initiation of reproductive maturation in mammals. However, KISS1 has been recently reported to highly express in ovarian granulosa cells (GCs). But the biological functionalities of KISS1 on cell apoptosis, cell cycle, and synthesis of estradiol-17β (E2) have not been explored in GCs. In this study, using porcine GCs as a cellular model, the overexpression plasmid of KISS1 was built to explore the biological effects of KISS1 on the PI3K signaling pathway, estrogen signaling pathway, cell apoptosis, cell cycle, and E2 secretion. We found that mRNA of KISS1 highly expressed in the ovary and significantly increased from immature to mature follicles in gilts. Overexpression of KISS1 could significantly increase the mRNA expression of PIK3CG, PIK3C1, and PDK1, and significantly decreased the mRNA levels of FOXO3, TSC2, and BAD of PI3K signaling pathway. Furthermore, results of the flow cytometry showed that overexpression of KISS1 significantly inhibited the apoptosis of GCs and decreased the percentage of GCs at G0/G1 phase of the cell cycle. Additionally, overexpression of KISS1 could increase the mRNA levels of Star, CYP17, 3B-HSD, 17B-HSD of estrogen synthesis signaling pathway, significantly increase the concentration of E2 in the supernatant of the cultured GCs, and up-regulate the mRNA expression levels of ESR1 and ESR2. These results suggested that KISS1 might suppress cell apoptosis through activating the PI3K signaling pathway and stimulate synthesis of E2 via boosting the estrogen synthesis signaling pathway. This study would be of great interests for exploring the biological functionalities of KISS1 in the folliculogenesis and sex steroid production of the ovaries in mammals.

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Metformin Inhibits Aromatase via an Extracellular Signal-Regulated Kinase-Mediated Pathway

Endocrinology ◽

10.1210/en.2009-0540 ◽

2009 ◽

Vol 150 (10) ◽

pp. 4794-4801 ◽

Cited By ~ 57

Author(s):

Suman Rice ◽

Laura Pellatt ◽

Kumaran Ramanathan ◽

Saffron Anne Whitehead ◽

Helen Diane Mason

Keyword(s):

Polycystic Ovary Syndrome ◽

Mrna Expression ◽

Granulosa Cells ◽

Insulin Treatment ◽

Polycystic Ovary ◽

Aromatase Activity ◽

Erk Pathway ◽

Ovary Syndrome ◽

Human Granulosa Cells ◽

Aromatase Mrna

Abstract Metformin treatment, now widely prescribed in polycystic ovary syndrome, is aimed at correcting the associated insulin resistance, but it has also been shown to directly inhibit ovarian steroidogenesis. The mechanisms, however, by which metformin inhibits estradiol production in human granulosa cells remains unknown. Granulosa luteal cells were incubted with metformin, insulin, or combined metformin and insulin treatment, and aromatase mRNA expression was quantified using real-time RT-PCR. Enzyme activity was assessed by the conversion of 3H-androstenedione to estrone and estradiol. Metformin’s effect on the expression of specific untranslated first exon aromatase promoters was analyzed using semiquantitative PCR. The involvement of MAPK kinase (MEK)/ERK pathway was investigated by immunoblotting for aromatase, phosphorylated, and total ERK-1,2 from cells cultured as above with/without the MEK inhibitor PD98059. Metformin significantly inhibited basal and insulin-stimulated aromatase mRNA expression, with parallel results from the aromatase activity assay and protein assessment. This suppression was via down-regulation of aromatase promoter II, I.3, and 1.4 expression and was reversed by the addition of PD98059. Involvement of the ERK signaling pathway was demonstrated by the significant increase in phosphorylated ERK-1,2 with the combined metformin and insulin treatment. We have shown for the first time in human granulosa cells that metformin signficantly attenuated basal and insulin-stimulated P450 aromatase mRNA expression and activity, via silencing of key promoters. This occurred by activation of MEK/ERK pathway, which negatively regulated aromatase production. This is an important consideration given metformin’s widespread use in polycystic ovary syndrome and may further support a possible therapeutic indication in estrogen-dependent breast tumors.

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ID: 1039 The antidepressant fluoxetine inhibits adenylate cyclase stimulation by FSH or Forskolin in the COV434 human ovarian granulosa tumor cell line

Biomedical Research and Therapy ◽

10.15419/bmrat.v4is.315 ◽

2017 ◽

Vol 4 (S) ◽

pp. 117

Author(s):

Thi Mong Diep Nguyen ◽

Danièle Klett ◽

Minh Thu Vo ◽

Yves Combarnous

Keyword(s):

Signaling Pathways ◽

Granulosa Cells ◽

Molecular Mechanisms ◽

Follicular Development ◽

Tumor Cell Line ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Cell Types ◽

Intracellular Camp ◽

Human Granulosa Cells

Fluoxetine (Prozac), a selective Serotonin Reuptake Inhibitor antidepressant, exhibits other mechanisms of action in various cell types and has been shown to induce cell death in cancer cells, paving the way for its potential use in cancer therapy. The ovary is a complex endocrine organ responsible for steroidogenesis and folliculogenesis, and human granulosa cells are essential for scientific research to improve the understanding of these two processes. However, little is known about fundamental signaling pathways in human granulosa cells. In this study, we investigated the dynamics of intracellular cyclic adenosine monophosphate AMP, a conserved signaling messenger that can regulate virtually every physiological process. We show that incubating COV434 human ovarian granulosa cells with fluoxetine induces a decrease in intracellular cAMP response to Follicle-stimulating hormone (FSH) and forskolin (FSK). In order to study the intracellular cAMP kinetic responses of COV434 cells to FSH or FSK, we used COV434 cells transiently expressing a chimeric cAMP-responsive luciferase so that real-time variations of intracellular cAMP concentration could be monitored, by using oxiluciferin luminescence produced from catalyzed luciferin oxidation. Our data show that fluoxetine induces an increase in the extracellular Ca2+ entry and reduces ATP concentration as well as cell viability. Targeting these signaling pathways with fluoxetine could permit to get better knowledge in the molecular mechanisms involved in ovarian follicular development

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Toll-Like Receptor 4 Inhibits Estradiol Secretion via NF-κB Signaling in Human Granulosa Cells

Frontiers in Endocrinology ◽

10.3389/fendo.2021.629554 ◽

2021 ◽

Vol 12 ◽

Author(s):

Hai-Yun Guan ◽

He-Xia Xia ◽

Xiu-Ying Chen ◽

Lu Wang ◽

Zhi-Jing Tang ◽

...

Keyword(s):

Granulosa Cells ◽

Critical Role ◽

Follicular Development ◽

Tumor Cell Line ◽

Immunohistochemical Analysis ◽

Toll Like Receptor ◽

Toll Like Receptor 4 ◽

Specific Expression ◽

Human Granulosa Cells ◽

Tlr4 Activation

Toll-like receptor 4 (TLR4) may play a critical role in regulating follicular development. Data are scarce on the role of TLR4 in the follicle. This study investigated the effects of TLR4 on steroidogenesis in human granulosa cells. Immunohistochemical analysis revealed stage-specific expression of TLR4 in the mouse ovarian cycle, and immunofluorescence showed TLR4 expression in the human granulosa-like tumor cell line (KGN). TLR4 agonist lipopolysaccharides (LPS) significantly inhibited follicular development and synthesis of estradiol (E2) in mice. In KGN cells, TLR4 activation significantly inhibited CYP19A1, FSHR and StAR, and TLR4 inhibition reversed these effects. TLR4 activation also inhibited forskolin-induced secretion of E2 by inhibiting CYP19A1, with no effect on progesterone. Further studies showed activation of p38, JNK and NF-κB signaling after TLR4 activation. Subsequent analyses showed that an NF-κB antagonist reversed the inhibitory effects on CYP19A1 expression and E2 secretion. Together, our results suggest that TLR4 activation may suppress CYP19A1 expression and E2 secretion via NF-κB signaling in human granulosa cells, with important implications for the regulation of ovarian pathophysiology.

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The effects of IGF-I on bovine follicle development and IGFBP-2 expression are dose and stage dependent

Reproduction ◽

10.1530/rep.1.00682 ◽

2006 ◽

Vol 131 (3) ◽

pp. 515-523 ◽

Cited By ~ 30

Author(s):

Kirsty A Walters ◽

John P Binnie ◽

Bruce K Campbell ◽

David G Armstrong ◽

Evelyn E Telfer

Keyword(s):

Growth Factor ◽

Granulosa Cells ◽

Size Range ◽

Regulatory System ◽

Follicular Development ◽

High Dose ◽

Insulin Like Growth Factor ◽

High Concentration ◽

Antral Follicles ◽

Igf I

This study aimed to determine the effect of insulin-like growth factor-I (IGF-I) on early antral bovine follicular development, and the expression of insulin-like growth factor-binding protein-2 (IGFBP-2). Antral follicles separated into three different size groups were cultured for 6 days in medium supplemented with either a low (10 ng/ml) or high (1 μg/ml) dose of human recombinant IGF-I. Oestradiol production by follicles in all size ranges, cultured in the presence of the high concentration of IGF-I, significantly increased by day 6 (P < 0.05). Follicles in the smallest size range, 165–215 μm, cultured in a high dose of IGF-I, were found to be significantly increased in size (P < 0.01). Oocyte health of the largest follicles (281–380 μm) was significantly improved by the addition of IGF-I to the culture medium. mRNA expression of IGFBP-2 was decreased in the granulosa cells of follicles, size range 216–280 μm, cultured with a high dose of IGF-I (P < 0.05). Granulosa cells (P < 0.05) and oocytes (P < 0.01) of the largest follicles (281–380 μm) showed a decrease in IGFBP-2 expression (protein) when cultured in the control and low-IGF-I treatment groups. Therefore, the response of a bovine follicle to IGF-I is both dose and stage dependent. This work supports a role for IGF-I in modulating somatic and germ-cell maturation and development in early antral follicles. Furthermore, the inverse relationship between the level of IGF-I stimulation and IGFBP-2 expression suggests a local regulatory system modulating IGF-I availability.

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Estrogen synthesis and secretion during postnatal testicular development in male goats: In situ aromatase mRNA expression

Small Ruminant Research ◽

10.1016/j.smallrumres.2015.07.013 ◽

2015 ◽

Vol 130 ◽

pp. 146-152 ◽

Cited By ~ 1

Author(s):

L. Ortiz-Carrera ◽

R.A. Valdez ◽

J.A. Mondragón ◽

P. Gariglio ◽

L. Zarco ◽

...

Keyword(s):

Mrna Expression ◽

Testicular Development ◽

Estrogen Synthesis ◽

Aromatase Mrna

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Impaired follicular development and endocrine disorders in female rats by prepubertal exposure to toxic doses of cadmium

Toxicology and Industrial Health ◽

10.1177/0748233720912060 ◽

2020 ◽

Vol 36 (2) ◽

pp. 63-75

Author(s):

Saman Saedi ◽

Mohammad Reza Jafarzadeh Shirazi ◽

Mohammad Javad Zamiri ◽

Mehdi Totonchi ◽

Mohammad Dadpasand ◽

...

Keyword(s):

Steroid Hormones ◽

Endocrine System ◽

Follicular Development ◽

Female Rats ◽

High Dose ◽

Endocrine Disorders ◽

Sprague Dawley ◽

Sprague Dawley Rats ◽

Puberty Onset ◽

Different Doses

Cadmium (Cd) has been associated with several physiological problems including reproductive and endocrine system dysfunction resulting in temporary infertility. The principal objective of this project was to investigate the effects of prepubertal exposure to toxic doses of Cd on puberty onset, the endocrine system, and follicular development. For this purpose, 16 female Sprague-Dawley rats weaned on postnatal day (PND) 21 were randomly divided into 4 groups ( n = 4 per group). The treatments were as follows: 0, 25, 50, and 75 mg/kg/day of cadmium chloride (CdCl2) by oral gavage from PND 21 to observation of first vaginal opening (VO). The results demonstrated that prepubertal exposure to different doses of CdCl2 delays the age of VO, first diestrus, and first proestrus via altering the concentrations of estradiol and progesterone. The low level of these steroid hormones contributed to lower differentiation and maturation of follicles and it finally led to reduced ovarian reservoir of follicles and impaired follicular development. The number of atretic follicles and secondary follicles with premature cavity increased in rats that received a high dose of CdCl2, whereas the number of secondary follicles and corpora luteum decreased in the same circumstances. Taken together, these data suggest that prepubertal exposure to toxic doses of Cd delays the onset of puberty via disorderliness in the concentration of steroid hormones and reduces the ovarian reservoir of follicles, as well as folliculogenesis.

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Effect of in vitro copper supplementation on granulosa cell estradiol synthesis and associated genes

Indian Journal of Animal Research ◽

10.18805/ijar.b-3396 ◽

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.

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Follistatin but not α or βA inhibin subunit mRNA is expressed in ovine fetal ovaries in late gestation

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0130001 ◽

1994 ◽

Vol 13 (1) ◽

pp. 1-9 ◽

Cited By ~ 12

Author(s):

R Braw-Tal ◽

D J Tisdall ◽

N L Hudson ◽

P Smith ◽

K P McNatty

Keyword(s):

Mrna Expression ◽

Granulosa Cells ◽

Cell Function ◽

Follicular Development ◽

Primordial Follicle ◽

Late Gestation ◽

Follicle Growth ◽

Primordial Follicles ◽

Fetal Ovary ◽

Ovine Fetal

ABSTRACT The aim of this study was to investigate the sites of follistatin and α and βA inhibin mRNA expression in the ovaries of female sheep fetuses at 90, 100, 120 and 135 days of gestation (term=day 147). At 90 and 100 days primordial follicles were formed, followed by the appearance of primary follicles at 100 days of gestation. At days 120 and 135, primordial, primary and preantral (i.e. secondary) follicles were present in the ovaries, but antral (i.e. tertiary) follicles were not observed at any of these gestational ages. Two Booroola genotypes were studied: homozygous carriers (BB) and non-carriers (++) of the fecundity gene (FecB). Irrespective of genotype no specific hybridization of the α and βA inhibin riboprobes was detected in any ovarian cells at days 90, 100, 120 or 135 of gestation. In control mature ovaries, on the other hand, strong hybridization in the granulosa cells of antral follicles was observed. In contrast to α and βA inhibin, follistatin antisense (but not sense) riboprobes hybridized specifically to the granulosa cells of preantral follicles with two or more layers of cells at days 120 and 135 of gestation. Moreover, hybridization was also evident in the cells of the ovarian rete at days 120 and 135, but not at 90 or 100 days. No follistatin mRNA expression was observed in the granulosa cells of primordial or primary follicles or in any other ovarian cell type at any of the gestational ages examined. No FecB-specific differences in follistatin expression were noted with respect to stage of preantral follicular development and there were no obvious differences in the intensity of expression. These results show that follistatin mRNA is expressed specifically in the granulosa cells and intraovarian rete. Expression of follistatin in rete cells was coincident with the increasing numbers of growing follicles within the fetal ovary, indicating that rete cell function may have a role in the ontogeny of early follicular growth. Our results suggest that follistatin and α and βA inhibin may not be important for the initiation of follicle growth in the sheep ovary, since these genes are not expressed during the transformation of a primordial follicle to a primary structure. However, the evidence for follistatin mRNA expression in the ovine fetal ovary implies that this hormone is likely to play a role during the early stages of follicle growth.

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