Human Granulosa Cell Differentiation Research Articles

Terminal differentiation of human granulosa cells as luteinization is reversed by activin-A through silencing of Jnk pathway

Molecular mechanisms underlying luteinization (terminal differentiation of granulosa and theca cells after ovulation) and luteolysis (demise of corpus luteum) are poorly understood in human ovary. Here we report that activin-A, after binding to its cognate receptors induces a functional luteolytic state and reverses luteinization phenotype by downregulating the expression of the steroidogenic enzymes, LH receptor and VEGF and reducing estradiol (E2) progesterone (P4) production and upregulating FSH receptor and cyclin D1 expression in human primary luteinized granulosa cells. Further, this action of activin-A involves downregulation of JNK signaling pathway and is opposite to that of human chorionic gonadotropin (hCG), which acts as a luteotropic hormone and improves luteal function through the activation of JNK pathway in the same cell type. Reversal of luteinization phenotype in luteal granulosa cells by activin-A potentially makes this hormone an attractive candidate for use under certain clinical situations, where induction of luteolysis and rapid reduction of endogenous sex steroid levels are beneficial such as ovarian hyperstimulation syndrome (OHSS), in which the ovaries hyper-respond to gonadotropin stimulation by producing too many growing follicles along with development of ascites, pleural effusion, and hemo-concentrations as a result of increased vascular permeability and leakage of intravascular volume into third spaces. Our work unveils a previously undefined role for activin-A and JNK signaling pathway in human corpus luteum biology, that might have a direct clinical impact in assisted reproductive technologies.

Human mural granulosa cells cultured in serum-free conditions can be used to study molecular mechanisms of human granulosa cell differentiation in vitro

Human mural granulosa cells cultured in serum-free conditions can be used to study molecular mechanisms of human granulosa cell differentiation in vitro

Folliclestimulating hormone (FSH) regulates cytoskeletal protein expression and cell structure during human granulosa cell differentiation

Folliclestimulating hormone (FSH) regulates cytoskeletal protein expression and cell structure during human granulosa cell differentiation

Genome-wide interactions between FSH and insulin-like growth factors in the regulation of human granulosa cell differentiation.

Is the genome-wide response of human cumulus cells to FSH and insulin-like growth factors (IGFs) comparable to the response observed in undifferentiated granulosa cells (GCs)? FSH actions in human cumulus cells mimic those observed in preantral undifferentiated GCs from laboratory animals, and approximately half of the regulated genes are dependent on the simultaneous activation of the IGF1 receptor (IGF1R). Animal studies have shown that FSH and the IGFs system are required for follicle growth and maturation. In humans, IGF levels in the follicular fluid correlate with patients' responses toIVF protocols. The main targets of FSH and IGFs in the ovary are the GCs; however, the genomic mechanisms involved in the response of GCs to these hormones are unknown. Human cumulus cells isolated from IVF patients were cultured for 48 h in serum-free media in the presence of vehicle, FSH, IGF1R inhibitor or their combination. Discarded cumulus cells were donated to research by reproductive-aged women undergoing IVF due to non-ovarian etiologies of infertility at a university-affiliated clinic. The effect of FSH and/or IGF1R inhibition on cumulus cell function was evaluated using Affymetrix microarrays, quantitative PCR, western blot, promoter assays and hormone level measurements. The findings demonstrate that human cumulus cells from IVF patients respond to FSH with the expression of genes known to be markers of the preantral to preovulatory differentiation of GCs. These results also demonstrate that ~50% of FSH-regulated genes require IGF1R activity and suggest that several aspects of follicle growth are coordinately regulated by FSH and IGFs in humans. This novel approach will allow for future mechanistic and molecular studies on the regulation of human follicle maturation. Data set can be accessed at Gene Expression Omnibus number GSE86427. Experiments were performed using primary human cumulus cells. This may not represent the response of intact follicles. Understanding the mechanisms involved in the regulation of GC differentiation by FSH and IGF in humans will contribute to improving treatments for infertility. The project was financed by the National Instituted of Health grant number R56HD086054 and R01HD057110 (C.S.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. We have no competing interests to declare.

Fsh receptor (FSHR) and IGF-1 receptor (IGF-1R) coordinated activation of akt is essential for human granulosa cell differentiation

FSH is used in IVF to induce follicle maturation, which results in granulosa cell differentiation and coordination of oocyte maturation. Insulin-like growth factors (IGFs) in conjunction with FSH play an important role in folliculogenesis and steroidogenesis. Our objective was to study the molecular crosstalk between FSH and IGF-1 signaling and the regulation of human granulosa cell differentiation.

Follicle stimulating hormone (FSH) differentially regulates gene expression through AKT and ERK1/2 activation in human granulosa cells

FSH administration is routine for in vitro fertilization (IVF) patients to promote ovarian follicle development; however, the mechanisms by which FSH promotes follicle development, including granulosa cell differentiation, are not clearly defined. The objective of the study was to gain an understanding of signaling pathways activated by FSH to elicit human granulosa cell differentiation.

IGF1R signaling is necessary for FSH-induced activation of AKT and differentiation of human Cumulus granulosa cells.

FSH is routinely administered to in vitro fertilization patients to induce follicle maturation. During this process, granulosa cells differentiate and acquire specific functional characteristics that are required to coordinate ovulation and oocyte maturation. The objective of the study was to gain insight into the molecular mechanisms regulating human granulosa cell differentiation. Design, Setting, Patients, and Interventions: Cumulus and mural granulosa cells were isolated from the follicular aspirates of in vitro fertilization patients and analyzed immediately or cultured in serum-free media in the presence of FSH, IGFs, or an inhibitor of type I IGF receptor (IGF1R) activity. We quantified the mRNA and protein levels of steroidogenic enzymes, components of the IGF system, and gonadotropin receptors; measured 17β-estradiol levels; and examined the activation of intracellular signaling pathways to assess the granulosa cell differentiation as well as the FSH and IGF actions in both cumulus and mural cells. In freshly isolated cells, LH receptor (Lhr) and steroidogenic acute regulator (Star) were expressed at lower levels in cumulus than mural cells, whereas FSH receptor (Fshr) and anti-Müllerian hormone (Amh) were expressed at higher levels in cumulus than mural cells. In vitro, the expression of Igf2, the differentiation markers Lhr, Star, and Cyp19a1 (aromatase) as well as 17β-estradiol production remained low in untreated cumulus cells but increased significantly after FSH treatment. Strikingly, this stimulatory effect of FSH was abolished by the inhibition of IGF1R activity. FSH-induced activation of v-akt murine thymoma viral oncogene homolog 3 (AKT) required IGF1R activity, and overexpression of constitutively active AKT rescued the induction of differentiation markers and 17β-estradiol production by FSH in the presence of the IGF1R inhibitor. The cumulus cell response to FSH resembles the differentiation of preantral to preovulatory granulosa cells. This differentiation program requires IGF1R activity and subsequent AKT activation.

Role of reconstituted basement membrane in human granulosa cell culture.

The effects of Matrigel, a reconstituted basement membrane, on human granulosa cells were investigated. Cells were obtained from follicular aspirate in the course of oocyte retrieval for in vitro fertilization and were cultured on either a surface coated with Matrigel or uncoated plastic. Light and electron microscopy showed that granulosa cells cultured on Matrigel demonstrated three-dimensional aggregated cells with well differentiated morphology: numerous lipid droplets, microvilli, junctional complexes and lumen-like structures were seen. In contrast, cells cultured on plastic were flattened, poorly differentiated and showed apoptotic cells. Immunocytochemistry showed that the proportion of immunopositive cells for 3beta-hydroxysteroid dehydrogenase was increased in cultures on Matrigel. The results of the present study suggest that culture on Matrigel promotes the differentiation of human granulosa cells and provides a useful tool which may improve the efficiency of in vitro fertilization.

Long-term growth and steroidogenic potential of human granulosa-lutein cells immortalized with SV40 large T antigen

Studies of human ovarian granulosa cells have been limited by the small numbers and short life span in culture of cells currently obtainable from clinical material. Using SV40 large T antigen, we have reproducibly immortalized freshly explanted human granulosa cells obtained through an In Vitro Fertilization Program. Of 69 independently isolated clones, 17 grew progressively into lines. In the presence of 1 mM 8-Br-cyclic AMP and 50 ng/ml pregnenolone, 15 of these 17 lines secreted progesterone. Seven lines derived from two patients were analyzed in detail. The lines differed in their responsiveness to cyclic AMP and pregnenolone, and in the timing of their steroidogenic responses. In response to cyclic AMP plus pregnenolone, progesterone secretion increased up to 20-fold. Three clones tested all responded to forskolin and cholera toxin with up to 7-fold increases in progesterone secretion. One line responded inconsistently to 1 IU/ml hCG but not to FSH. The steroidogenic responses to cyclic AMP were accompanied by morphologic cell rounding. The immortalized cell lines underwent 40–60 population doublings, thus, providing a theoretical yield of up to 10 18 cells per line. These results show that a high proportion of SV40 immortalized lines is steroidogenic. These lines provide a new experimental model for studies of cell lines that appear representative of different states of differentiation of human granulosa cells.

Mediation of gonadotrophin-stimulated growth and differentiation of human granulosa cells by adenosine-3',5'-monophosphate: one molecule, two messages.

To determine how the second messenger adenosine-3',5'-monophosphate (cyclic AMP) is able to mediate divergent actions of FSH and LH on granulosa cell growth and differentiation in human ovaries. Human granulosa cells were cultured for 96 hours in serum-free medium 199 containing increasing doses of either FSH, LH or dibutyryl cyclic AMP. Extra and intra-cellular cyclic AMP levels were determined by radioimmunoassay. Tritiated thymidine uptake and cell number were measured as indices of cell growth, and spent medium was assayed for steroids (oestradiol and progesterone) to reflect differentiation. 'Mature' granulosa cells were aspirated from preovulatory follicles in the ovaries of clomiphene-stimulated patients undergoing laparoscopic sterilization; 'luteinized' granulosa cells were aspirated from periovulatory follicles in the ovaries of gonadotrophin-stimulated in-vitro fertilization patients. LH consistently inhibited, whereas FSH maintained or stimulated, basal granulosa cell numbers. Steroidogenesis was dose-dependently increased by both gonadotrophins, with LH having the significantly greater effect over the entire dose-response range (1-100 micrograms/l). LH also induced significantly more cyclic AMP production than FSH, both intra and extra-cellularly, providing a basis for differential post-receptor signalling via a common second messenger. Addition of dibutyryl cyclic AMP, at low concentrations (10-250 mumol/l) to the cultured cells in the absence of gonadotrophins mimicked FSH effects with stimulation/maintenance of cell numbers and moderate steroidogenesis. High concentrations of dibutyryl cyclic AMP (500-1000 mumol/l) caused a significant inhibition of cell numbers together with maximal steroidogenesis, simulating LH action. These results suggest that granulosa cell maturation in the follicular phase of the menstrual cycle (controlled by FSH) is associated with a low cyclic AMP tone that favours cell growth and expression of aromatase activity in the developing preovulatory follicle. During the early luteal phase (dominated by LH), the intracellular cycle AMP tone increases to allow maximal progesterone production and inhibition of cell growth in the corpus luteum. Thereby one second messenger can mediate divergent gonadotrophic effects on granulosa cell growth and differentiation in the human ovary.

Synergistic Effect of Human Chorionic Gonadotropin and Extracellular Matrix on in Vitro Differentiation of Human Granulosa Cells: Progesterone Production and Gap Junction Formation*

The effects of extracellular matrix (ECM) in the form of a native basement membrane and of hCG on progesterone production, cellular morphology, and gap junction formation in cultured human granulosa cells (GCs) were examined. GCs were harvested from human preovulatory follicles in the course of oocyte retrieval for in vitro fertilization. Cells grown on ECM secreted by bovine corneal endothelial cells (BCE) produced significantly higher amounts of progesterone (up to 3-fold) than cells on uncoated dishes. Culturing of cells on BCE-ECM in the presence of hCG resulted in a further increase in progesterone production; in the first 2 days an additive effect was noted, and on days 3, 4, and 5 a synergistic effect became evident (1.2- to 1.8-fold above the sum of the individual treatments). LH/hCG receptor content on day 3 of culture was 4-fold higher in GCs grown on BCE-ECM compared to that in uncoated dishes. A similar elevation was found when cells were grown on ECM secreted by PFHR-9 mouse endodermal cells. Cells grown on both types of ECM maintained their epithelioid shape but, in addition, formed cytoplasmic processes interconnected with neighboring cells by gap junctions. Morphometric analysis revealed that the area of cell membrane occupied by gap junctions increased by 3- to 4-fold on ECM compared to that in cells grown on plastic. Enlargement of endoplasmic reticulum was evident on ECM along with clustering of numerous mitochondria, lipid droplets, and lysosomes characteristic of highly luteinized cells. Cells grown in the presence of hCG on ECM showed morphological modulation similar to that of cells cultured on ECM alone. These observations suggest that growing of human GCs on a ECM similar to a naturally produced basement membrane enhances morphological and biochemical differentiation, and that ECM and gonadotropin affect synergistically the extent of luteinization in these cells.

Aromatase activity in human granulosa cells during follicular development and the modulation by follicle-stimulating hormone and insulin

Granulosa cells were isolated from follicles that ranged in size from 0.4 to 2.0 cm in diameter, obtained from patients during the follicular phase of the menstrual cycle. Aromatase activity, assessed by the release of tritiated water from [1β-3H]testosterone, was undetectable in follicles with a diameter of less than 1.0 cm; thereafter, there was a direct correlation between aromatase activity and follicular diameter. When cultured in the presence of 200 ng of National Institutes of Health follicle-stimulating hormone-15 per milliliter, aromatase activity was stimulated in cells isolated from all sizes of follicles from 0.4 to 1.5 cm. Insulin (500 ng/ml) further augmented follicle-stimulating hormone (FSH)-induced aromatase activity, as evidenced both by an increase in 17β-estradiol production and by the release of tritiated water from [1β-3H]testosterone by granulosa cells. This study further delineates the role of FSH in estrogen production during human follicular development and suggests that insulin or insulin-like growth factors may play a role in modifying the FSH-dependent cellular differentiation of human granulosa cells.