Effect of the spatial-temporal specific theca cell Cyp17 overexpression on the reproductive phenotype of the novel TC17 mouse.

Background: In the ovarian follicle, the Theca Cells (TCs) have two main functions: preserving morphological integrity and, importantly, secreting steroid androgen hormones. TCs express the essential enzyme 17α-hydroxylase/17,20-desmolase (CYP17), which permits the conversion of pregnenolone and progesterone into androgens. Dysregulation of CYP17 enzyme activity due to an intrinsic ovarian defect is hypothesized to be a cause of hyperandrogenism in women.

FOXO1 mitigates the SMAD3/FOXL2 transcriptomic effect in a model of human adult granulosa cell tumor.

Background: Adult granulosa cell tumor (aGCT) is a rare type of stromal cell malignant cancer of the ovary characterized by elevated estrogen levels. aGCTs ubiquitously harbor a somatic mutation in FOXL2 gene, Cys134Trp (c.402C < G); however, the general molecular effect of this mutation and its putative pathogenic role in aGCT tumorigenesis is not completely understood.

FOXO1 Negates the Cooperative Action of FOXL2 and SMAD3 in CYP19 Expression in HGrC1 Cells by Sequestering SMAD3.

Adult granulosa cell tumor (aGCT) is a rare type of ovarian cancer characterized by estrogen excess. Interestingly, only the single somatic mutation was found across virtually all aGCTs. We previously reported that FOXL2 stimulates CYP19 transcription synergistically with SMAD3, leading to elevated estradiol synthesis in a human granulosa cell line (HGrC1).

Molecular Aspects and Clinical Relevance of GDF9 and BMP15 in Ovarian Function.

Growth and differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are oocyte-secreted factors with a leading role in the control of ovarian function in female reproduction, modulating both the cell fate of the somatic granulosa cells and the quality and developmental competence of the egg. This short review aims to consolidate the molecular aspects of GDF9 and BMP15 and their integral actions in female fertility to understand particularly their effects on oocyte quality and fetal growth. The significant consequences of mutations in the GDF9 and BMP15 genes in women with dizygotic twins as well as the clinical relevance of these oocyte factors in the pathogenesis of primary ovarian insufficiency and polycystic ovary syndrome are also addressed.

FOXL2C134W-Induced CYP19 Expression via Cooperation With SMAD3 in HGrC1 Cells.

Germline knockout studies in female mice demonstrated an essential role for forkhead box L2 (FOXL2) in early follicle development, whereas an inducible granulosa cell (GC)-specific deletion of Foxl2 in adults has shown ovary-to-testis somatic sex reprogramming. In women, over 120 different germline mutations in the FOXL2 gene have been shown to cause blepharophimosis/ptosis/epicantus inversus syndrome associated with or without primary ovarian insufficiency. By contrast, a single somatic mutation (FOXL2C134W) accounts for almost all adult-type GC tumors (aGCTs).

Activation of Endoplasmic Reticulum Stress in Granulosa Cells from Patients with Polycystic Ovary Syndrome Contributes to Ovarian Fibrosis.

Recent studies report the involvement of intra-ovarian factors, such as inflammation and oxidative stress, in the pathophysiology of polycystic ovary syndrome (PCOS), the most common endocrine disorder of reproductive age women. Endoplasmic reticulum (ER) stress is a local factor that affects various cellular events during a broad spectrum of physiological and pathological conditions. It may also be an important determinant of pro-fibrotic remodeling during tissue fibrosis.

PAI-1 in granulosa cells is suppressed directly by statin and indirectly by suppressing TGF-β and TNF-α in mononuclear cells by insulin-sensitizing drugs.

Problem: Plasminogen activator inhibitor-1 (PAI-1) is elevated in women with polycystic ovary syndrome (PCOS), but the regulation in granulosa cells (GCs) is unclear.

Method Of Study: PAI-1 expression in PCOS ovaries was investigated immunohistologically. PAI-1 expressions in HGrC1, a human GC cell line, were investigated at mRNA and activity levels.

Growth and differentiation factor 9 promotes oocyte growth at the primary but not the early secondary stage in three-dimensional follicle culture.

Purpose: Factors that differentially regulate oocyte and granulosa cell growth within the early preantral follicle and how these factors differ at each stage of follicle growth remain poorly understood. The aim of this study was to isolate and evaluate the effect of recombinant growth and differentiation factor 9 (GDF9) on oocyte and granulosa cell growth at the primary and early secondary stages of preantral follicle growth during in vitro culture.

Methods: Primary stage follicles (diameters of 50-89 μm) and early secondary stage follicles (diameters of 90-120 μm) were isolated from immature mice, and individual, intact follicles were cultured in vitro in the presence and absence of recombinant GDF9.

A Novel Letrozole Model Recapitulates Both the Reproductive and Metabolic Phenotypes of Polycystic Ovary Syndrome in Female Mice.

Polycystic ovary syndrome (PCOS) pathophysiology is poorly understood, due partly to lack of PCOS animal models fully recapitulating this complex disorder. Recently, a PCOS rat model using letrozole (LET), a nonsteroidal aromatase inhibitor, mimicked multiple PCOS phenotypes, including metabolic features absent in other models. Given the advantages of using genetic and transgenic mouse models, we investigated whether LET produces a similar PCOS phenotype in mice.

Decreased inhibin B responses following recombinant human chorionic gonadotropin administration in normal women and women with polycystic ovary syndrome.

Objective: To determine whether granulosa cells contribute to excess androgen production, by assessing inhibin B (Inh B) responses to hCG in women with polycystic ovary syndrome (PCOS) and in normal women.

Design: Prospective study.

Setting: Academic medical center.

Paracrine regulation of theca androgen production by granulosa cells in the ovary.

Objective: To test whether and to what extent inhibin mediates Cyp17 messenger RNA (mRNA) expression in theca cells (TCs) in response to FSH stimulation of granulosa cells (GCs).

Design: Ex vivo and in vitro experimental study.

Setting: University.

GRK-6 mediates FSH action synergistically enhanced by estrogen and the oocyte in rat granulosa cells.

Estrogen is known to play a pivotal role in granulosa cell responses to follicle-stimulating hormone (FSH) that is critical for the establishment of dominant follicles and subsequent ovulation in mammals. Thus, elucidating the cellular and molecular mechanisms that regulate FSH activity is important to understand female fertility. We previously discovered that the oocyte is required for estrogen to exert its positive effects on FSH activity in rat granulosa cells.

Granulosa cell tumor mutant FOXL2C134W suppresses GDF-9 and activin A-induced follistatin transcription in primary granulosa cells.

A single somatic FOXL2 mutation (FOXL2(C134W)) was identified in almost all granulosa cell tumor (GCT) patients. In the pituitary, FOXL2 and Smad3 coordinately regulate activin stimulation of follistatin transcription. We explored whether a similar regulation occurs in the ovary, and whether FOXL2(C134W) has altered activity.

Essential but differential role of FOXL2wt and FOXL2C134W in GDF-9 stimulation of follistatin transcription in co-operation with Smad3 in the human granulosa cell line COV434.

The FOXL2(C134W) mutation has been identified in virtually all adult granulosa cell tumors (GCTs). Here we show that the exogenous FOXL2 expression is necessary for GDF-9 stimulation of follistatin transcription in the human GCT cell line, COV434 that lacks endogenous FOXL2 expression. Interestingly, in the presence of Smad3 co-expression, FOXL2(C134W) negated GDF-9 stimulation of follistatin transcription.

Identification and characterization of canine growth differentiation factor-9 and its splicing variant.

Growth differentiation factor-9 (GDF-9), a member of the transforming growth factor-β (TGF-β) superfamily, is expressed exclusively in the oocyte within the ovary and plays essential roles in the ovarian function in mammals. However, a possible involvement of GDF-9 in canine ovarian physiology that has a unique ovulation process among mammals has not been studied. Interestingly, we have isolated two types of cDNA clones generated by an alternative splicing from a canine ovarian total RNA.

Integral role of GDF-9 and BMP-15 in ovarian function.

The oocyte plays an important role in regulating and promoting follicle growth, and thereby its own development, by the production of oocyte growth factors that predominantly act on supporting granulosa cells via paracrine signaling. Genetic studies in mice demonstrated critical roles of two key oocyte-derived growth factors belonging to the transforming growth factor-β (TGF-β) superfamily, growth and differentiation factor-9 (GDF-9) and bone morphogenetic protein-15 (BMP-15), in ovarian function. The identification of Bmp15 and Gdf9 gene mutations as the causal mechanism underlying the highly prolific or infertile nature of several sheep strains in a dosage-sensitive manner also highlighted the crucial role these two genes play in ovarian function.

Impaired production of BMP-15 and GDF-9 mature proteins derived from proproteins WITH mutations in the proregion.

Mutations in the bone morphogenetic protein-15 (BMP-15) and growth and differentiation factor-9 (GDF-9) genes have been identified in women with primary ovarian insufficiency (POI) and mothers of dizygotic twins. Here, we show that biological activities of the conditioned media from human embryonic kidney 293F cells transfected with two representative BMP-15 and GDF-9 mutants identified in the affected women have significantly reduced biological activities compared with the corresponding wild-type. Moreover, this difference is due to decreased production of the mature proteins, attributed most likely to impaired posttranslational processing of the proprotein.

Golgi apparatus casein kinase phosphorylates bioactive Ser-6 of bone morphogenetic protein 15 and growth and differentiation factor 9.

Bone morphogenetic protein-15 (BMP-15) and growth and differentiation factor-9 (GDF-9) are oocyte-secreted factors that play essential roles in human folliculogenesis and ovulation. Their bioactivity is tightly regulated through phosphorylation, likely to occur within the Golgi apparatus of the secretory pathway. Here we show that Golgi apparatus casein kinase (G-CK) catalyzes the phosphorylation of rhBMP-15 and rhGDF-9.

Follistatin gene expression by gonadotropin-releasing hormone: a role for cyclic AMP and mitogen-activated protein kinase signaling pathways in clonal gonadotroph LbetaT2 cells.

The purpose of the present study was to examine the signal transduction pathways involved in follistatin gene expression induced by GnRH in the LbetaT2 cell line. The LHbeta-subunit was predominantly increased by high frequency GnRH pulses (30 min interval); whereas low frequency pulses (120 min) increased FSHbeta. In a static culture, follistatin expression was significantly increased at 12 h (2.

Oocyte-specific overexpression of mouse bone morphogenetic protein-15 leads to accelerated folliculogenesis and an early onset of acyclicity in transgenic mice.

Whereas mutations in the bmp15 gene cause infertility in ewes and women due to defects in folliculogenesis, most defects in female mice lacking bone morphogenetic protein (BMP)-15 are confined to the ovulation process, supportive of the observation that functional mouse BMP-15 is barely detected in oocytes in vivo until after the LH surge. In addition, the mouse BMP-15 proprotein is not processed into the functional mature protein in transfected cells. However, a chimeric protein consisting of the human proregion, human cleavage site, and mouse mature region (termed hhmBMP-15) is processed and the mature protein secreted.

Increased androgen response to follicle-stimulating hormone administration in women with polycystic ovary syndrome.

Context: In women with polycystic ovary syndrome (PCOS), excess ovarian androgen production is driven by increased LH secretion. Studies conducted in animals suggest that the granulosa cell may influence LH-stimulated theca cell androgen production.

Objective: The objective of this study was to determine whether FSH enhances androgen production in women with PCOS compared with that of normal women.

Characterization of the post-translational modification of recombinant human BMP-15 mature protein.

Bone morphogenetic protein-15 (BMP-15) is an oocyte-secreted factor critical for the regulation of ovarian physiology. When recombinant human BMP-15 (rhBMP-15) produced in human embryonic kidney 293 cells was subjected to SDS-PAGE analysis, two mature protein forms corresponding to 16 kDa (P16) and 17 kDa (P17) were observed. Despite the physiological relevance and critical function of BMP-15 in female reproduction, little is known about the structure of rhBMP-15.

Phosphorylation of bone morphogenetic protein-15 and growth and differentiation factor-9 plays a critical role in determining agonistic or antagonistic functions.

Two highly homologous oocyte-secreted growth factors, bone morphogenetic protein (BMP)-15 and growth and differentiation factor (GDF)-9, are known to control folliculogenesis and ovulation through direct effects on granulosa cells in the developing follicles. Although much is known about the expression and biology of these proteins, the impact of posttranslational modifications of BMP-15 and GDF-9 is unknown. Here, we report that: 1) recombinant human (rh) BMP-15 and rhGDF-9 are phosphorylated; 2) the phosphorylation is essential for bioactivity; and 3) the dephosphorylated forms of rhBMP-15 and rhGDF-9 can abolish the bioactivity of rhBMP-15, rhGDF-9, and rhBMP-7, but not rh activin A.

Oocyte-derived BMP15 and FGFs cooperate to promote glycolysis in cumulus cells.

Mammalian oocytes are deficient in their ability to carry out glycolysis. Therefore, the products of glycolysis that are necessary for oocyte development are provided to oocytes by companion cumulus cells. Mouse oocytes secrete paracrine factors that promote glycolysis in cumulus cells.

Structural and biophysical coupling of heparin and activin binding to follistatin isoform functions.

Follistatin (FS) regulates transforming growth factor-beta superfamily ligands and is necessary for normal embryonic and ovarian follicle development. Follistatin is expressed as two splice variants (FS288 and FS315). Previous studies indicated differences in heparin binding between FS288 and FS315, potentially influencing the physiological functions and locations of these isoforms.