Role of advanced glycation end-products in obesity-related ovarian dysfunction.

Obesity affects ovarian function, one of the main regulators of female fertility. Tissue levels of the proinflammatory advanced glycation end-products (AGEs) and their receptors (RAGE) are elevated in obesity. AGEs are key contributors to perturbations in the ovarian microenvironment. On this basis, the present review focuses on clinical and experimental studies supporting the role of AGE-RAGE system as a contributor to obesity-related ovarian dysfunction. Particular emphasis has been given to changes in AGEs, RAGE and the anti-inflammatory soluble receptor (sRAGE) levels in obesity state and following dietary interventions (high-fat diet and weight loss). Ovarian sensitivity, in particular granulosa cell function and oocyte meiosis, to the pro-inflammatory AGE-RAGE system as well as the relationship of follicular fluid AGEs and sRAGE to in vitro fertilization outcome are also discussed. Overall, obesity, with its alterations in the AGE-RAGE system, can disrupt the ovarian microenvironment potentially compromising oocyte competence and fertility. This review underscores a critical need to uncover the mechanistic actions of AGE-RAGE system in obesity-related ovarian dysfunction. Clinical and basic studies focusing on elucidating the patterns of accumulation and role of the AGE-RAGE system in human ovarian follicles are key steps in understanding their contribution to the health of human oocytes and embryos.

Müllerian inhibitory substance induces growth of rat preantral ovarian follicles.

Müllerian inhibitory substance (MIS), also known as anti-Müllerian hormone, is best known as the hormone that regulates the regression of the Müllerian duct in males. In females, MIS is expressed in granulosa cells of preantral and early antral follicles. The specific MIS type II receptor is present in granulosa and theca cells of these small, growing follicles. Because the role of MIS in preantral follicle development is unknown, we have evaluated the effect of MIS on the growth, differentiation, and apoptosis of intact preantral follicles in a serum-free culture system. In this system, treatment with FSH induces an increase in both follicle diameter, cell number, and follicle cell differentiation based on increased inhibin-alpha synthesis. Of interest, treatment with MIS enhances the effect of FSH both on follicle diameter and cell number. Although treatment with activin A also enhances FSH effects on follicle growth, treatment with transforming growth factor (TGF)-ss inhibits the FSH effects on follicle growth. Based on in situ staining of fragmented DNA, MIS was found to have no effect on follicle cell apoptosis, unlike its proapoptotic action on Müllerian ducts. In contrast to MIS and activin, TGF-ss was a potent proapoptotic factor for preantral follicles in culture. Analysis of inhibin-alpha expression of cultured preantral follicles further indicated that in contrast to activin, treatment with MIS did not enhance FSH-stimulated follicle differentiation. Thus, MIS is a unique factor that promotes preantral follicle growth but not preantral follicle cell differentiation and apoptosis.

In vivo treatment with GDF-9 stimulates primordial and primary follicle progression and theca cell marker CYP17 in ovaries of immature rats.

Growth differentiation factor (GDF)-9 is a cystine knot-containing hormone of the transforming growth factor-beta superfamily produced by the oocyte. In GDF-9 null mice, follicle development is arrested at the primary stage and GDF-9 treatment in vitro enhances preantral follicle growth. Immature female rats were treated with recombinant GDF-9 for 7 or 10 days. At 10 days, treatment with GDF-9 augmented ovarian weights, concomitant with an increase in the number of primary and small preantral follicles by 30 and 60%, respectively. Furthermore, the number of primordial follicles was decreased by 29%, but the number of large preantral follicles was not affected. In contrast, treatment with FSH increased the number of small and large preantral follicles by 36 and 177% but did not influence the number of primary and primordial follicles. Immunoblot analysis showed an increase of CYP17, a theca cell marker, in the ovarian homogenate after treatment with GDF-9 but not FSH. The present results indicate that in vivo treatment with GDF-9 enhances the progression of primordial and primary follicles into small preantral follicles. Thus, GDF-9 treatment could provide an alternative approach to stimulate early follicle development in addition to the widely used FSH that acts mainly on the development of more advanced follicles.

Dizygotic twinning is not linked to variation at the alpha-inhibin locus on human chromosome 2.

Natural multiple pregnancy in women leading to dizygotic (DZ) twins is familial and varies across racial groups, suggesting a genetic predisposition. Mothers of DZ twins have a higher incidence of spontaneous multiple ovulation and elevated FSH concentrations. FSH release is controlled by feedback of inhibin peptides from the ovary, and immunization against inhibin alpha-subunit results in an increased ovulation rate in animals. The inhibin alpha-subunit is therefore a candidate gene for mutations that may increase the frequency of DZ twinning. Restriction digests of a PCR product from exon 1 with the enzyme SpeI detects a C/T polymorphism at bp 128 with two alleles of 447 and 323/124 bp. The polymorphism was typed in 1,125 individuals from 326 pedigrees with 717 mothers of spontaneous DZ twins. The alpha-inhibin locus mapped within 3 centimorgans of D2S164, and linkage with DZ twinning was excluded [decimal log odds ratio (LOD) score, -2.81 at theta = 0]. There was complete exclusion of linkage (LOD, less than -2) of a gene conferring relative risk 1.8 (lambdas, >1.8) across the chromosome, except at the p-terminus region and a small peak (maximum LOD score, 0.6) in the region of D2S151-D2S326. Analysis using either recessive or dominant models excluded linkage with DZ twinning in this population (LOD score, less than -2.5) across chromosome 2. We conclude that dizygotic twinning is not linked to variation in the alpha-inhibin locus. The results also suggest that mutations in other candidates on chromosome 2, including the receptor for FSH and the betaB-inhibin subunit (INHBB) cannot be major contributors to risk for DZ twinning.

Hormonal regulation of early follicle development in the rat ovary.

Although earlier studies focused on the hormonal regulation of antral and preovulatory follicles, recent studies indicate the importance of the hormonal control mechanism for preantral follicles. The endocrine hormone FSH is not only a survival factor for early antral follicles but also a potent growth and differentiation factor for preantral follicles. In addition, KGF secreted by theca cells and c-kit ligand secreted by granulosa cells play paracrine roles in the regulation of preantral follicle growth and development. Furthermore oocyte-derived GDF-9 promotes the growth and differentiation of early follicles by acting on somatic cells in the follicle. It is likely that the genetic makeup of an oocyte could determine the secretion of oocyte hormones which would, in turn, regulate the growth and differentiation of the surrounding somatic cells of that follicle. A better understanding of the hormonal mechanisms underlying early follicle development could provide a refined culture system for the in vitro maturation of fertilizable oocytes and future design of fertility and contraceptive agents.

The regulation of apoptosis in preantral ovarian follicles.

Less than 1% of ovarian follicles ever mature to ovulation. The remainder undergo atretic degeneration via apoptosis during development. Though the regulation of antral and preovulatory survival has been studied for many years, very little is known about the regulation of survival and development of preantral follicles. This review discusses recent findings regarding preantral follicle development with emphasis on the regulation of preantral follicle apoptosis.

Initial and cyclic recruitment of ovarian follicles.

Mammalian ovaries consist of follicles as basic functional units. The total number of ovarian follicles is determined early in life, and the depletion of this pool leads to reproductive senescence. Each follicle develops to either ovulate or, more likely, to undergo degeneration. The dynamics of ovarian follicle development have interested endocrinologists and developmental biologists for many years. With the advent of assisted reproductive techniques in humans, the possibility of regulating follicle development in vivo and in vitro has gained clinical relevance. In this review, we focus upon key branching points during the development of ovarian follicles as well as factors involved in determining the eventual destiny of individual follicles. We discuss inconsistencies in the literature regarding the definitions of follicle recruitment and selection and propose to name the two major steps of follicle development as initial and cyclic recruitment, respectively. Because some of these disparities have arisen due to differences in the animal systems studied, we also compare the development of the ovarian follicles of both humans and rats. We also review the status of knowledge of several puzzling clinical issues that may provide important clues toward unlocking the mechanisms of follicle development.

Keratinocyte growth factor promotes the survival, growth, and differentiation of preantral ovarian follicles.

OBJECTIVE: To determine the effect of treatment with keratinocyte growth factor (KGF) on the survival of cells in cultured preantral follicles and on the growth and differentiation of preantral follicles. DESIGN: Preantral follicles (140-150 microm) were dissected mechanically from the ovaries of 14-day-old rats and cultured for 24 hours with and without KGF. Genomic DNA was extracted, labeled with [32P]-dideoxyadenosine triphosphate, and fractionated through agarose gels. For growth studies, the follicles were cultured individually in 96-well dishes. After 72 hours, the follicles were collected and their protein or DNA content was evaluated and their inhibin-alpha content was determined. RESULT(S): Keratinocyte growth factor suppressed apoptosis in cultured preantral follicles by 60%. Treatment with KGF or FSH increased follicle diameter by 8% and 16%, respectively, and combined treatment with KGF and FSH increased follicle diameter by 26%. Western blot analysis demonstrated increased expression of inhibin-alpha content after treatment with KGF (2-fold), treatment with FSH (4-fold), and combined treatment with FSH and KGF (12-fold), demonstrating the effect of KGF on preantral follicle differentiation. CONCLUSION(S): Treatment with KGF promotes the survival, growth, and differentiation of cultured preantral follicles. Keratinocyte growth factor produced by theca cells may play a role in the progression of early follicle development.

Recombinant growth differentiation factor-9 (GDF-9) enhances growth and differentiation of cultured early ovarian follicles.

Transgenic mice with deletion of the GDF-9 (growth differentiation factor-9) gene are characterized by the arrest of ovarian follicle development at the primary stage. Based on the hypothesis that GDF-9 is important for early follicle development, we isolated rat GDF-9 complementary DNA (cDNA) and generated recombinant GDF-9 protein to study its physiological role. Using bacteria-derived GDF-9-glutathione S-transferase (GST) fusion protein, specific antibodies to the mature form of GDF-9 was generated. Immunohistochemical staining of ovarian sections indicated the localization of GDF-9 protein in the oocyte of primary, secondary and preantral follicles, whereas immunoblotting demonstrated the secretion of GDF-9 by mammalian cells transfected with GDF-9 cDNAs. Recombinant GDF-9 was shown to be an N-glycosylated protein capable of stimulating early follicle development. Growth of preantral follicles isolated from immature rats was enhanced by treatment with either GDF-9 or FSH whereas the combined treatment showed an additive effect. In addition, treatment with GDF-9, like forskolin, also stimulated inhibin-alpha content in explants of neonatal ovaries. In contrast, the stimulatory effects of GDF-9 were not mimicked by amino-terminal tagged GDF-9 that was apparently not bioactive. Thus, the present study demonstrates the important role of GDF-9 in early follicle growth and differentiation. The availability of recombinant bioactive GDF-9 allows future studies on the physiological role of GDF-9 in ovarian development in vivo.

Restricted expression of WT1 messenger ribonucleic acid in immature ovarian follicles: uniformity in mammalian and avian species and maintenance during reproductive senescence.

WT1 is a zinc finger protein with transcriptional repressor activity on several growth factor and growth factor receptor genes. In the ovary, a potential role for WT1 in the suppression of the development of immature follicles has been demonstrated. Here, gel retardation assays further showed that recombinant WT1 protein interacted with consensus DNA sequences in the inhibin-alpha gene promoter. We investigated the pattern of WT1 expression in a wide variety of species and also over the reproductive life span in rats. In chicken ovaries, Northern blot analysis revealed the presence of WT1 transcript in small healthy white follicles (1-5 mm in diameter) and its absence in small yellow (6-12 mm in diameter) or larger follicles (F1-F5). In pig and monkey ovaries, WT1 expression was limited to granulosa cells of preantral follicles, as shown by in situ hybridization analysis. In rats, Northern blot analyses demonstrated the presence of WT1 transcript in the ovaries of young (3-mo-old) and middle-aged (9-mo-old) rats on the proestrous day, with a decrease in old (12-mo-old) rats in persistent estrus. In situ hybridization analysis further suggested that the decrease in WT1 expression in aging ovaries was associated with fewer immature follicles. Thus, WT1 expression is restricted to immature follicles in diverse avian and mammalian species and over the reproductive life span in rats. These data demonstrated that WT1 is a marker for immature follicles and suggested a potential role of this transcriptional repressor in the slow growth of early follicles.

DEFT, a novel death effector domain-containing molecule predominantly expressed in testicular germ cells.

Apoptosis is a physiological process by which multicellular organisms eliminate unwanted cells. Death factors such as Fas ligand induce apoptosis by triggering a series of intracellular protein-protein interactions mediated by defined motifs found in the signaling molecules. One of these motifs is the death effector domain (DED), a stretch of about 80 amino acids that is shared by adaptors, regulators, and executors of the death factor pathway. We have identified the human and rat complementary DNAs encoding a novel protein termed DEFT (Death EFfector domain-containing Testicular molecule). The N-terminus of DEFT shows a high degree of homology to the DEDs found in FADD (an adaptor molecule) as well as procaspase-8/FLICE and procaspase-10/Mch4 (executors of the death program). Northern blot hybridization experiments have shown that the DEFT messenger RNA (mRNA) is expressed in a variety of human and rat tissues, with particularly abundant expression in the testis. In situ hybridization analysis further indicated the expression of DEFT mRNA in meiotic male germ cells. In a model of germ cell apoptosis induction, an increase in testis DEFT mRNA was found in immature rats after 2 days of treatment with a GnRH antagonist. Unlike FADD and procaspase-8/FLICE, overexpression of DEFT did not induce apoptosis in Chinese hamster ovary cells. Although cotransfection studies indicated that DEFT is incapable of modulating apoptosis effected by FADD and procaspase-8/FLICE, interactions between DEFT and uncharacterized DED-containing molecules in the testis remain to be studied in the future. In conclusion, we have identified a novel DED-containing protein with high expression in testis germ cells. This protein may be important in the regulation of death factor-induced apoptosis in the testis and other tissues.

Cell death and survival during ovarian follicle development.

Mammalian germ cells arise in the yolk sac endoderm at the caudal aspect of the embryo and migrate to the mesodermally-derived gonadal ridge early in development. After the oogonia reach the gonadal ridge, the process of meiosis begins which coincides with the first major wave of apoptosis of female germ cells (Coucouvanis et al., 1993). Subsequently, oocytes progress to the dictyate stage of prophase I where they remain arrested until ovulation.

Follicle-stimulating hormone enhances the development of preantral follicles in juvenile rats.

The stimulatory effects of gonadotropins on antral and preovulatory follicles are well known, but conflicting results have been reported regarding the gonadotropin responsiveness and dependency of preantral follicles. Taking advantage of the relatively uniform development of the first wave of follicles in the postnatal rat ovary, we evaluated the role of endogenous and exogenous gonadotropins on preantral follicle development. Reduction of the high levels of gonadotropins present in juvenile rats by either hypophysectomy (at Day 15) or GnRH antagonist treatment (starting from Day 11 of age) resulted in decreased ovarian weight at Day 19 of age that was associated with a reduced number of developing follicles and increased atresia of remaining follicles. In contrast, treatment with FSHctp (a long-acting FSH agonist) in intact (Days 5-19 of age), hypophysectomized (Days 15-19), or GnRH antagonist-treated (Days 11-19) animals resulted in increased ovarian weight and follicle development as determined histologically and by inhibin-alpha expression. A dose-dependent stimulatory effect of hCG on ovarian weight was seen when animals were cotreated with FSHctp and the GnRH antagonist. At low doses of hCG, augmentation of antral follicle formation occurred, whereas higher doses of hCG led to morphological signs of luteinization. These findings demonstrate the important role of endogenous gonadotropins in preantral follicle development and indicate that preantral follicles are highly responsive to exogenous gonadotropins.

The effect of transforming growth factor-beta on steroidogenesis and expression of key steroidogenic enzymes with a human ovarian thecal-like tumor cell model.

OBJECTIVE: Our purpose was to determine the effects of transforming growth factor-beta on steroidogenesis and regulation of steroidogenic enzyme expression by use of a human ovarian thecal-like tumor cell culture system. STUDY DESIGN: Human ovarian thecal-like tumor cells were treated in serum-free medium in the presence or absence of forskolin and transforming growth factor-beta 1. The accumulation of progesterone and androstenedione in the culture medium was evaluated by radioimmunoassay. The effects of forskolin with or without transforming growth factor-beta 1 on the enzymatic activity of P450c17 and 3 beta HSD, the expression of immunodetectable P450c17 protein, and the expression of messenger ribonucleic acid for P450scc, P450c17, and 3 beta HSD were determined. RESULTS: Basal steroid secretion, steroidogenic enzyme activity, enzyme protein, and messenger ribonucleic acid expression were not affected by transforming growth factor-beta 1 alone. Forskolin treatment significantly stimulated steroid production and the enzymatic activity of P450c17 and 3 beta HSD up to 10-fold above basal levels. However, transforming growth factor-beta 1 inhibited forskolin-stimulated androstenedione production to near basal levels and increased progesterone 1.4- to 2-fold while suppressing P450c17 enzyme activity to near basal levels, but it did not affect 3 beta HSD activity. Forskolin-stimulated immunodetectable P450c17 alpha protein was markedly inhibited by transforming growth factor-beta 1. In addition, transforming growth factor-beta 1 markedly inhibited the forskolin-stimulation of P450c17 messenger ribonucleic acid, while not significantly altering P450scc or 3 beta HSD messenger ribonucleic acid expression. CONCLUSION: Forskolin stimulated human ovarian thecal-like tumor cell steroidogenesis, P450c17 and 3 beta HSD activity, immunodetectable P450c17, and messenger ribonucleic acid content for P450scc, P450c17, and 3 beta HSD. Transforming growth factor-beta 1 inhibited forskolin stimulation of androstenedione production through the inhibition of P450c17 expression.

Human ovarian tumor cells: a potential model for thecal cell steroidogenesis.

Ovarian thecal cell production of C19 steroids (i.e. dehydroepiandrosterone, androstenedione, and testosterone) is necessary to provide substrate for granulosa cell biosynthesis of estrogen; however, excessive production of C19 steroids can lead to disorders associated with androgen excess. Because of difficulties in obtaining adequate numbers of thecal cells, the biomolecular regulation of C19 steroid production and expression of steroidogenic enzymes is not well defined. We have overcome this obstacle by developing a highly dependable and unique human ovarian thecal-like tumor (HOTT) cell culture model system from an ovarian tumor found to produce excessive amounts of C19 steroids. Aliquots of freshly dispersed tumor cells were frozen for future use. Once placed in monolayer culture, HOTT cells proliferated and could be maintained for extended periods. Acutely, cultured HOTT cells increased progesterone and cAMP production in response to 2 h of forskolin treatment. These cells were, however, unresponsive to treatment with LH. Steroid hormone production continued in cells that were maintained in culture for up to 2 months. Analysis of the steroids produced by HOTT cells was accomplished using RIA and high performance liquid chromatography. Under basal conditions, HOTT cells produced mainly 17 alpha-hydroxyprogesterone and progesterone. Treatment with forskolin or dibutyryl cAMP (dbcAMP) increased the production of progesterone and 17 alpha-hydroxyprogesterone as well as C19 steroids. Treatment of monolayer cultures of HOTT cells with forskolin (0.01 to 20 mumol/L) or dbcAMP (0.01 to 1 mmol/L) for 48 h increased the production of androstenedione (8- to 15-fold) and progesterone (2- to 5-fold). In HOTT cells chronically treated with forskolin or dbcAMP (up to 72 h), progesterone production was observed to plateau, although the amount of androstenedione continued to increase. The enzymatic activities of both 3 beta-hydroxysteroid dehydrogenase (6-fold), and 17 alpha-hydroxylase P450 (P450c17; 9-fold) were also increased by activation of the protein kinase A messenger pathway. Treatment of HOTT cells with forskolin caused a time-dependent induction of the messenger RNAs for cholesterol side-chain cleavage P450, 3 beta-hydroxysteroid dehydrogenase, and P450c17. No changes in steroidogenic enzyme expression were observed following treatment with LH. In conclusion, these data demonstrate that certain ovarian tumor cells may serve well as appropriate models to study the molecular mechanisms regulating human ovarian thecal cell C19 steroidogenesis and the expression of steroid-metabolizing enzymes.

The effect of insulin and insulin-like growth factors on the expression of steroidogenic enzymes in a human ovarian thecal-like tumor cell model.

OBJECTIVE: To determine the effects of insulin and insulin-like growth factors (IGF-I and IGF-II) on steroidogenesis and steroidogenic enzyme expression in a human ovarian thecal-like tumor cell culture model system. DESIGN: Human ovarian thecal-like tumor cells treated with forskolin and insulin IGF-I or IGF-II were evaluated for media accumulation of P and androstenedione (A) as well as 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) and cytochrome P450 17 alpha-hydroxylase (P450c17) enzyme activity. Northern analysis of cytochrome P450 side chain cleavage (P450scc), P450c17, and 3 beta HSD messenger RNA (mRNA) also was performed. RESULTS: Basal hormone secretion, enzyme activity, and mRNA levels were not affected by treatment with insulin or the IGFs. Forskolin treatment stimulated steroid production, enzyme activity, and mRNA content. Forskolin-stimulated P secretion was augmented 30% by treatment with insulin and IGFs, whereas 3 beta HSD activity was augmented twofold to threefold. Forskolin stimulated A and P450c17 activity were enhanced by treatment with insulin and the IGFs. In forskolin-treated cells. P450c17 and P450scc mRNA levels were not affected by insulin (100 nM) or IGF (10 nM) treatment; however, 3 beta HSD mRNA levels were augmented by treatment with insulin and IGFs. CONCLUSIONS: We observed that forskolin-stimulated human ovarian thecal-like tumor cell steroidogenesis, P450c17, and 3 beta HSD activity, as well as mRNA content for P450scc, 3 beta HSD, and P450c17. Insulin and the IGFs augmented forskolin-stimulated production of P and the expression of 3 beta HSD, with little effect on A production, P450scc, or P450c17 expression.

Regulation of human adrenal carcinoma cell (NCI-H295) production of C19 steroids.

The regulation of biosynthesis of the adrenal C19 steroids (the so-called adrenal androgens) remains unclear. Understanding adrenal production of C19 steroids is important when the benefits of these steroids are considered on processes and diseases associated with aging. In vitro studies defining the mechanisms that regulate the production of human adrenal C19 steroids have been limited because of the difficulties in obtaining adrenal tissue. A cell line that retains differentiated adrenal functions would greatly facilitate research in this area. Herein, we describe the use of the human adrenocortical tumor H295 cell line as a model to evaluate mechanisms controlling C19 and C21 steroid production. The cells were characterized with regard to ACTH, forskolin, and dibutyryl cAMP (dbcAMP) responsiveness, as measured by increased cAMP production, synthesis of steroids, and induction of 17 alpha-hydroxylase cytochrome P450 (P450c17). Forskolin and dbcAMP, which were more effective than ACTH, enhanced the production of cortisol, dehydroepiandrosterone (DHEA), DHEA sulfate (DHEAS), and androstenedione over a 48-h treatment period. Comparison of the relative amounts of measured steroid secreted under forskolin treatment indicated that the primary product was cortisol (70%), followed by androstenedione (14%), DHEA (9%), and DHEAS (7%). Cortisol was also demonstrated to be the major steroid product by examination of UV-detectable steroids after high performance liquid chromatographic separation. The increases in steroid production caused by ACTH, forskolin, and dbcAMP occurred in a concentration- and time-dependent manner. A key enzyme in the production of C19 steroids is P450c17. ACTH, forskolin, and dbcAMP increased the activity of 17 alpha-hydroxylase by approximately 2.5-, 10-, and 10-fold, respectively. These effects on enzyme activity occurred in a concentration-dependent manner and coincided with increased levels of P450c17 mRNA. In summary, H295 cells should provide a much-needed model to study mechanisms controlling the secretion of glucocorticoids and C19 steroids, because steroid production in these cells is hormonally controlled and associated with the induction of P450c17.