The vaginal epithelium undergoes remarkable changes during menopause, presumably due to the physiologic estrogen reduction. Surprisingly few studies however, have quantified the histologic changes associated with estrogen supplementation. The current study was undertaken to investigate basic histologic differences between pre- and postmenopausal women and compare those with postmenopausal women using exogenous estrogen thus providing insight into both endocrinologic and age-related changes in vaginal epithelium. Analysis of histologic specimens discarded following vaginal surgery. Specimens of vaginal epithelium were obtained after prolapse surgery from premenopausal women (PRE, n = 8), postmenopausal women (POST, n = 8) and postmenopausal women using vaginal estrogen cream (EST, n = 8). After formalin fixation tissues were embedded in paraffin and sectioned and stained with Hematoxylin/Eosin or Periodic Acid Schiff. The slides were analyzed for epithelial thickness, number of epithelial layers and glycogen content. Measurements were done by 2 blinded assessors. Intraclass correlation coefficients (SPSSS 19) were used to assess interobserver agreement. Means were compared using ANOVA. The intraclass correlation coefficient between the two blinded observers for measurements of epithelial thickness in the was .921 (P < .001). The correlation between observers of the number of epithelial layers counted was not significant. Mean epithelial thickness was 167μm, 332μm and 298μm in POST, PRE and EST, respectively (P <0.05). The reduction in glycogen in the POST samples was not seen in the EST samples. Measurement of epithelial thickness is a more objective method of assessment of vaginal epithelial thickness than counting epithelial layers. Postmenopausal samples have the thinnest epithelia and the lowest glycogen content. In postmenopausal women the use of vaginal estrogen cream is associated with restoration of vaginal epithelial thickness and glycogen content.

Members of the transforming growth factor beta type (TGFbeta) superfamily and their receptors are expressed in the testis, and are believed to play important paracrine and autocrine roles during testicular development and spermatogenesis. The Smad proteins are downstream mediators for the family of TGFbeta growth factors. Smad2 and Smad3 are associated with both TGFbeta and activin signaling. However, very little is known about the expression and regulation of the Smad signaling proteins in the testis. In the present study, we have determined that Smad2 and Smad3 proteins are expressed in the postnatal testes of rats from 5 days to 60 days of age. Expression levels for both proteins are higher in young rats than in sexually mature rats. Smad2 and Smad3 messenger RNA levels parallel protein expression. Smad2 and Smad3 proteins are mainly localized in the cytoplasm of meiotic germ cells, Sertoli cells, and Leydig cells. Smad3 protein is localized to the nucleus of preleptotene to zygotene primary spermatocytes in young rats. Both proteins are expressed throughout all stages of the cycle of seminiferous tubules but are expressed at their lowest levels at stages VII-VIII in the seminiferous epithelium of adult rats. The presence of these downstream mediators in these cell types supports a role for TGFbeta and activin during spermatogenesis. The difference between the expression of Smad2 and Smad3 suggests that they may have different functions within the testis.

The response of pituitary gonadotropes to gonadotropin-releasing hormone (GnRH) correlates directly with the concentration of GnRH receptors (GnRHR) on the cell surface, which is mediated in part at the level of gene expression. Several factors are known to affect expression of the mouse GnRHR (mGnRHR) gene, including GnRH and activin. We have previously shown that activin augments GnRH-mediated transcriptional activation of mGnRHR gene, and that region -387/-308 appears to be necessary to mediate this effect. This region contains two overlapping cis-regulatory elements of interest: GnRHR activating sequence (GRAS) and a putative SMAD-binding element (SBE). This study investigates the role of these elements and their cognate transcription factors in transactivation of the mGnRHR gene. Transfection studies confirm the presence of GnRH- and activin-response elements within -387/-308 of mGnRHR gene promoter. Competition electrophoretic mobility shift assay experiments using -335/-312 as probe and alphaT3-1 nuclear extract or SMAD, Jun, and Fos proteins demonstrate direct binding of AP-1 (Fos/Jun) protein complexes to -327/-322 and SMAD proteins to -329/-328. Further transfection studies using mutant constructs of these cis-regulatory elements confirm that both are functionally important. These data define a novel cis-regulatory element comprised of an overlapping SBE and newly characterized non-consensus AP-1 binding sequence that integrates the stimulatory transcriptional effects of both GnRH and activin on the mGnRHR gene.

Paracrine and autocrine growth factors can affect many different aspects of ovarian follicle development. Many members of the transforming growth factor beta (TGFbeta) family of growth factors and their receptors are expressed in developing follicles. However, the presence and function of the family of the TGFbeta signaling molecules known as Smads have not been evaluated during follicle development. We have demonstrated that two Smad family members that function as mediators for both activin and TGFbeta are expressed in granulosa cells of preantral follicles but not in large antral follicles. Smad2 expression, but not Smad3 expression, returns in luteal cells. Both Smad2 and Smad3 are translocated to the nucleus of granulosa cells in response to treatment with either TGFbeta or activin. However, Smad2 is more responsive to activin stimulation, and Smad3 is more responsive to TGFbeta stimulation. Stage-specific expression and differing ligand sensitivity of signaling molecules may work together to allow different effects of TGFbeta family ligands using the same signaling pathways over the course of follicular development.

Objective: The TGFβ family of growth factors has long been thought to play a role in ovarian follicle development, however until recently the mechanisms of action of these growth factors have been unknown. A group of proteins that functions as intracellular mediators of TGFβ family signaling has recently been identified and named Smad. Though there are 9 Smads so far, two of the receptor-specific smads: Smad2 and Smad3 are both associated with both TGFβ and activin signaling. Receptor complexes that have bound ligand can activate receptor-specific Smads by phosphorylation. Phosphorylated Smads translocate to the nucleus where they modulate transcription. In this study we evaluated the capacity of TGFβ and activin to induce nuclear translocation of Smad2 and Smad3 in cultured granulosa cells.Design: Intracellular localization of Smad2 and Smad3 proteins was performed on cells treated with either TGFβ or Activin.Materials/Methods: Granulosa cells obtained from 25-day-old rat ovaries were cultured on glass coverslips for 24 hours in medium containing 10% serum. The medium was then changed to serum-free medium containing FSH (10 ng/ml). After 24 hours of treatment with FSH, groups of cells were treated with either Activin (20 ng/ml) or TGFβ (1 ng/ml) for 30 minutes. Cells were washed with PBS and fixed in 4% paraformadehyde prior to immunofluorescence staining with antibodies to either Smad2 or Smad3. Slides were visualized using a fluorescence microscope fitted with a 510 nm filter. 100 cells from each group were counted based on a semi-random grid with at least one field from each of the four quadrants. Virtually all of the cultured cells exhibited some level of staining for both proteins. Negative control slides did not have cellular staining. For each counted cell, the observer (who was blinded to the treatment group) determined visually whether the nuclear staining was greater than the cytoplasmic staining. This was defined as a nuclear stained cell. The counts were reported as a percent of all cells with staining based on this definition. The experiment was repeated with different cell isolations. Statistical significance between mean values was determined by analysis of variance followed by post-hoc testing and was accepted at 0.05 level.Results: Both Smad2 and Smad3 proteins are primarily expressed in cytoplasm of cultured granulosa cells in the absence of growth factors. In FSH-treated cells, Smad2 is localized to the nucleus in 14 ± 1.2% of cells and Smad3 is localized to the nucleus in 22 ± 1.7%. When FSH-treated cells are then exposed 30 minutes to either TGFβ (1 ng/ml) or activin A (20 ng/ml), nuclear staining for both Smad2 and Smad3 is markedly increased. TGFβ treatment results in 30 ± 4.4% and 50 ± 2.3% nuclear staining for Smad2 and Smad3, respectively (p < 0.05). But, activin treatment increases the level of nuclear staining for Smad2 to 53 ± 4.6% and Smad3 to 36 ± 4.1% (P < 0.05).% Nuclear localization. Tabled 1FSH onlyFSH + TGFβFSH + activinSmad2143053Smad3225036 Open table in a new tab Conclusions: In granulosa cells derived from large antral follicles, both Smad2 and Smad3 are activated and undergo nuclear transport in response to TGFβ and activin treatment. However, Smad2 is more responsive to stimulation by activin and Smad3 is more responsive to stimulation by TGFβ.Supported By: The Ortho-MacNeil/ASRM Grant in Reproductive Medicine. Objective: The TGFβ family of growth factors has long been thought to play a role in ovarian follicle development, however until recently the mechanisms of action of these growth factors have been unknown. A group of proteins that functions as intracellular mediators of TGFβ family signaling has recently been identified and named Smad. Though there are 9 Smads so far, two of the receptor-specific smads: Smad2 and Smad3 are both associated with both TGFβ and activin signaling. Receptor complexes that have bound ligand can activate receptor-specific Smads by phosphorylation. Phosphorylated Smads translocate to the nucleus where they modulate transcription. In this study we evaluated the capacity of TGFβ and activin to induce nuclear translocation of Smad2 and Smad3 in cultured granulosa cells. Design: Intracellular localization of Smad2 and Smad3 proteins was performed on cells treated with either TGFβ or Activin. Materials/Methods: Granulosa cells obtained from 25-day-old rat ovaries were cultured on glass coverslips for 24 hours in medium containing 10% serum. The medium was then changed to serum-free medium containing FSH (10 ng/ml). After 24 hours of treatment with FSH, groups of cells were treated with either Activin (20 ng/ml) or TGFβ (1 ng/ml) for 30 minutes. Cells were washed with PBS and fixed in 4% paraformadehyde prior to immunofluorescence staining with antibodies to either Smad2 or Smad3. Slides were visualized using a fluorescence microscope fitted with a 510 nm filter. 100 cells from each group were counted based on a semi-random grid with at least one field from each of the four quadrants. Virtually all of the cultured cells exhibited some level of staining for both proteins. Negative control slides did not have cellular staining. For each counted cell, the observer (who was blinded to the treatment group) determined visually whether the nuclear staining was greater than the cytoplasmic staining. This was defined as a nuclear stained cell. The counts were reported as a percent of all cells with staining based on this definition. The experiment was repeated with different cell isolations. Statistical significance between mean values was determined by analysis of variance followed by post-hoc testing and was accepted at 0.05 level. Results: Both Smad2 and Smad3 proteins are primarily expressed in cytoplasm of cultured granulosa cells in the absence of growth factors. In FSH-treated cells, Smad2 is localized to the nucleus in 14 ± 1.2% of cells and Smad3 is localized to the nucleus in 22 ± 1.7%. When FSH-treated cells are then exposed 30 minutes to either TGFβ (1 ng/ml) or activin A (20 ng/ml), nuclear staining for both Smad2 and Smad3 is markedly increased. TGFβ treatment results in 30 ± 4.4% and 50 ± 2.3% nuclear staining for Smad2 and Smad3, respectively (p < 0.05). But, activin treatment increases the level of nuclear staining for Smad2 to 53 ± 4.6% and Smad3 to 36 ± 4.1% (P < 0.05). % Nuclear localization. Tabled 1FSH onlyFSH + TGFβFSH + activinSmad2143053Smad3225036 Open table in a new tab Conclusions: In granulosa cells derived from large antral follicles, both Smad2 and Smad3 are activated and undergo nuclear transport in response to TGFβ and activin treatment. However, Smad2 is more responsive to stimulation by activin and Smad3 is more responsive to stimulation by TGFβ. Supported By: The Ortho-MacNeil/ASRM Grant in Reproductive Medicine.

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.

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.

Objectives: The TGFβ family of growth factors is an important group of regulators of early follicle development, however there is little information about the downstream regulation of their function in the ovary. Recently, a group of signaling molecules for the TGFβ family have been identified and named SMADs. In these studies we have explored the regulation of expression of Smad2 and Smad3 in whole ovaries and granulosa cells. Design: PMSG and FSH treatments were used to determine the gonadotropin effects on ovarian expression of SMAD proteins in vivo and in vitro. Materials and Methods: The expression pattern of Smad2 and Smad3 protein was determined using immunohistochemical analysis of ovaries from immature and cycling rats. Western analysis was performed on lysates from ovaries obtained from control and PMSG-treated immature animals. Cultured granulosa cells were obtained from immature rats and plated at near confluence in serum containing medium. The next morning the medium was changed to serum-free. After 24 hours in serum-free medium, medium was replaced with a basal medium with or without FSH and/or TGFβ over a 48 hour time course. Western analysis for Smad3 was performed on the resulting cell lysates. Results: Immunohistochemical analysis reveals that Smad2 and Smad3 protein are both expressed in granulosa cells of growing follicles in immature and cycling rats. However immunostaining for both Smad2 and Smad3 is less intense in the granulosa cells of large antral follicles. Western analysis of whole ovary lysates demonstrates that treatment of immature rats with PMSG for 48 hours increases Smad3 protein content relative to control untreated rats. FSH treatment of granulosa cells in monolayer culture increases Smad3 protein content as early as 6 hours after addition of treatments and increased expression relative to controls continues for at least 48 hours. Concomitant treatment with TGFβ suppresses the FSH-induced increase to near baseline levels. Conclusions: FSH treatment regulates the expression of Smad3, a key mediator of activin and TGFβ signaling. This introduces another level of regulation to the complex interplay of gonadotropins and these key growth factors. This work was supported by the ASRM/Ortho MacNeil Grant in Reproductive Medicine.

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.

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.

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.

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.

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 mu m) were dissected mechanically from the ovaries of Id,-day-old rats and cultured for 24 hours with and without KGF. Genomic DNA was extracted, labeled with [(32)P]-dideoxy adenosine 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-a 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 16%. Western blot analysis demonstrated increased expression of inhibin-a! 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.

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.