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Granulosa cells (GCs) must respond appropriately to follicle-stimulating hormone (FSH) for proper follicle maturation. FSH activates protein kinase A (PKA) leading to phosphorylation of the cyclic AMP response element binding protein-1 (CREB1). We identified a unique A-kinase anchoring protein (AKAP13) containing a Rho guanine nucleotide exchange factor (RhoGEF) region that was induced in GCs during folliculogenesis. AKAPs are known to coordinate signaling cascades, and we sought to evaluate the role of AKAP13 in GCs in response to FSH. Aromatase reporter activity was increased in COV434 human GCs overexpressing AKAP13. Addition of FSH, or the PKA activator forskolin, significantly enhanced this activity by 1.5- to 2.5-fold, respectively (p < 0.001). Treatment with the PKA inhibitor H89 significantly reduced AKAP13-dependent activation of an aromatase reporter (p = 0.0067). AKAP13 physically interacted with CREB1 in co-immunoprecipitation experiments and increased the phosphorylation of CREB1. CREB1 phosphorylation increased after FSH treatment in a time-specific manner, and this effect was reduced by siRNA directed against AKAP13 (p = 0.05). CREB1 activation increased by 18.5-fold with co-expression of AKAP13 in the presence of FSH (p < 0.001). Aromatase reporter activity was reduced by inhibitors of the RhoGEF region, C3 transferase and A13, and greatly enhanced by the RhoGEF activator, A02. In primary murine and COV43 GCs, siRNA knockdown of Akap13/AKAP13 decreased aromatase and luteinizing hormone receptor transcripts in cells treated with FSH, compared with controls. Collectively, these findings suggest that AKAP13 may function as a scaffolding protein in FSH signal transduction via an interaction with CREB, resulting in phosphorylation of CREB.

Geophagy has been observed in nearly every long‐term study of folivorous primates. Yet despite frequent observations of this behavior, conclusive explanations for soil consumption remain ambiguous. This study tests the most frequently proposed hypotheses for geophagy using data collected on the geophagic behavior of the Milne‐Edwards’ sifaka (Propithecus edwardsi) living in two forests with varying levels of disturbance within Ranomafana National Park, Madagascar. Field data were collected from December 2002 to November 2003, during which time soil samples were collected for mineral analyses from 102 sites selected for geophagy and 42 control sites at which no geophagy was recorded. Results indicate that control soils differ significantly between the two study sites and that this difference is primarily attributable to varying levels of habitat disturbance. The frequency of soil consumption also does not vary significantly by sex or between logged and unlogged habitats. Soil consumption significantly correlated with fruit/seed consumption overall, but to a lesser degree in logged compared with unlogged sites. Clay minerals found in soils likely prevent absorption of dietary toxins in the gut and/or may mediate gut pH. This provides strong evidence for the protection hypothesis for geophagy, which may be especially important in areas where logging, or other forms of habitat disturbance, has been experienced. Abstract in French is available with online material.

Background/Aims Recent evidence suggests that there are numerous benefits to scheduling postpartum visits as early as 3 weeks post-delivery. However, findings are not conclusive due to methodological limitations. This report discusses the unique aspects of a randomized controlled trial's (RCT) design, intervention, and strategies to maintain participant retention. Methods This study was a four-year, prospective, open-label RCT conducted at the Virginia Commonwealth University Medical Center. Women who recently delivered a healthy, full-term baby vaginally, were randomized to receive a 3–4 or 6–8 weeks postpartum appointment and were followed for 18 months. Results A total of 364 women participated in this study. A large proportion of women were retained in the study as demonstrated by the high completion rates at the 18-month follow-up interview (Total sample: 87.6%; 3–4 weeks group: 88.0%; 6–8 weeks group: 87.3%). Similarly, high adherence to the protocol-directed postpartum visit schedule was reported in the overall study sample (79.7%), as well as in the 3–4 (70.5%) and 6–8 (90.0%) week postpartum groups. Conclusion The study design offered unique features which ensured excellent participant completion and adherence rates, despite the presence of hard-to-track women who typically do not return for their postpartum visits.

The present study examined how a gene related to functioning of the dopaminergic system, catechol-O-methyltransferase (COMT), and estradiol were related to brain functioning in healthy postmenopausal women. Participants were 118 healthy, cognitively normal postmenopausal women between the ages of 50-60 years. All women provided a blood sample for COMT and estradiol analyses and underwent a magnetic resonance imaging scan. Working memory performance and related brain activation were measured with BOLD functional magnetic resonance imaging during the N-back task. Results were examined across each COMT genotype and a median split was performed on the circulating estradiol levels to create high and low estradiol groups for each genotype. COMT genotype and estradiol level were hypothesized to be proxy measures for brain dopamine levels with the Met/Met and high estradiol group having the most dopamine and Val/Val and low estradiol group having the least dopamine. The functional magnetic resonance imaging results showed that the N-back task activated the expected bilateral frontal and bilateral parietal working memory network. However, no main effects of COMT genotype or estradiol group were found. There was COMT-estradiol interaction found in a small area of decreased activation in the right precentral gyrus (Brodmann Area 6) that was related to the increasing hypothesized dopamine level. Specifically, women with a Met/Met genotype in the high estradiol group had the least activation in this frontal lobe working memory region. Women with a Val/Val genotype in the low estradiol group had greater activation in this region relative to the other groups. Performance on the N-back task did not show any group differences. These data indicate that after menopause COMT genotype and potentially the menopause-related changes to the dopaminergic system are not related to cognition. Future studies should examine how the relationship between COMT, estradiol, and cognition around the menopause transition as there appear to be differences in this relationship for premenopausal and postmenopausal women.

Background. Delays in postpartum contraceptive use may increase risk for unintended or rapid repeat pregnancies. The postpartum care visit (PPCV) is a good opportunity for women to discuss family planning options with their health care providers. This study examined the association between PPCV attendance and modern contraceptive use using data from a managed care organization. Methods. Claims and demographic and administrative data came from a nonprofit managed care organization in Virginia (2008–2012). Information on the most recent delivery for mothers with singleton births was analyzed (N = 24,619). Routine PPCV (yes, no) and modern contraceptive use were both dichotomized. Descriptive analyses provided percentages, frequencies, and means. Multiple logistic regression was conducted and ORs and 95% CIs were calculated. Results. More than half of the women did not attend their PPCV (50.8%) and 86.9% had no modern contraceptive use. After controlling for the effects of confounders, women with PPCV were 50% more likely to use modern contraceptive methods than women with no PPCV (OR = 1.50, 95% CI = 1.31, 1.72). Conclusions. These findings highlight the importance of PPCV in improving modern contraceptive use and guide health care policy in the effort of reducing unintended pregnancy rates.

The postpartum care visit (PPCV) plays an important role in ensuring the well-being of mother and infant. This study sought to assess correlates of PPCV attendance among women who are at high risk of nonattendance.This study used deidentified medical claims data from Virginia Premier-a nonprofit Managed Care Organization that provides health insurance for Medicaid beneficiaries. The association between various correlates and PPCV attendance was examined using multiple logistic regression analyses.Of the 25,692 women in the study, more than half (50.5%) did not attend a postpartum visit. Racial/ethnic minorities and women receiving the majority of their care at hospitals, Health Departments, or Federally Qualified Health Centers were more likely to attend their postpartum visit. Women who smoked and those who did not attend prenatal care had reduced odds of postpartum visit attendance. Age, education, and delivery method were not found to be significantly associated with PPCV attendance.Our results highlight factors associated with attendance of PPCVs in low income populations. The continued disparity in postpartum care utilization compels additional efforts to improve access to health services across socioeconomic and demographic boundaries.

Preantral follicle development has become an increasingly recognized area of study in the last two decades. Factors that regulate the growth survival and differentiation of these small, yet complex structures have been identified. The field of fertility preservation and a need for increased numbers of mature oocytes for stem cell research revealed how little we knew of how follicles got from the primordial stage to the antral stage with a healthy and competent oocyte inside. This work discusses the role of gonadotropins in regulating preantral follicles and also the role of the TGF-β family members and their associated Smad signaling molecules in preantral follicle development. Preantral follicle development is a necessary step to fertility in females and further understanding of this process is essential for progress in both infertility care and the enlarging field of in vitro folliculogenesis.

Introduction: α7 nicotinic acetylcholine receptors (nAChRs) play an important role in vagus nerve-based cholinergic anti-inflammatory effects. This study was designed to assess the role of α7 nAChRs in dextran sodium sulfate (DSS)-induced colitis in male and female mouse. We first compared disease activity and pathogenesis of colitis in α7 knockout and wild-type mice. We then evaluated the effect of several α7 direct and indirect agonists on the severity of disease in the DSS-induced colitis. Methods: Male and female adult mice were administered 2.5% DSS solution freely in the drinking water for 7 consecutive days and the colitis severity (disease activity index) was evaluated as well as colon length, colon histology, and levels of tumor necrosis factor-alpha colonic levels. Results: Male, but not female, α7 knockout mice displayed a significantly increased colitis severity and higher tumor necrosis factor-alpha levels as compared with their littermate wild-type mice. Moreover, pretreatment with selective α7 ligands PHA-543613, choline, and PNU-120596 decreased colitis severity in male but not female mice. The anti-colitis effects of these α7 compounds dissipated when administered at higher doses. Conclusions: Our results suggest the presence of a α7-dependent anti-colitis endogenous tone in male mice. Finally, our results show for the first time that female mice are less sensitive to the anti-colitis activity of α7 agonists. Ovarian hormones may play a key role in the sex difference effect of α7 nAChRs modulation of colitis in the mouse. Implications: Our collective results suggest that targeting α7 nAChRs could represent a viable therapeutic approach for intestinal inflammation diseases such as ulcerative colitis with the consideration of sex differences.

From alleles to ecosystems and landscapes, anthropogenic activity continues to affect the environment, with particularly adverse effects on biodiversity hotspots such as Madagascar. Selective logging has been proposed as a “win-win” conservation strategy, yet its effects on different components of biodiversity are still not fully understood. Here we examine biotic factors (i.e., dietary differences) that may be driving differences in biogeochemical stocks between disturbed and undisturbed forests. We present the stable nitrogen (δ15N) and carbon (δ13C) isotope composition of hair from the lemur Propithecus edwardsi and of whole bodies of its obligate ectoparasite, the louse-fly Allobosca crassipes, from sites in Ranomafana National Park (RNP) that are comparable except for the history of logging and subsequent forest regeneration. P. edwardsi and A. crassipes from the disturbed (i.e., heavily selectively logged) site are lower in 15N and 13C relative to P. edwardsi and A. crassipes from sites that were minimally selectively logged or not commercially logged at all. There is a ∼3‰ decrease in 15N between disturbed and undisturbed sites that corresponds to a difference of nearly a full trophic level. Flowers from Bakerella clavata, a staple food source for P. edwardsi in disturbed habitats and a fallback food for P. edwardsi in primary forests, were also analyzed isotopically. B. clavata is δ15N-depleted in both disturbed and undisturbed sites. Data from longitudinal behavioral surveys of P. edwardsi in RNP and other forests in eastern Madagascar point to significant differences in consumption patterns of B. clavata, with P. edwardsi in disturbed forests consuming almost twice as much of this plant. Depletion of 15N in animal tissues is a complex issue, but likely the result of the interaction of physiological and ecological factors. Anthropogenic disturbance in RNP from selective logging has had both biotic and biogeochemical effects that are observable trophically.

Follicle-stimulating hormone (FSH) is the major regulator of folliculogenesis, but other factors modulate its action, including members of the transforming growth factor (TGF) beta family. The intersection of signal transduction pathways that integrate the follicular response to FSH remains to be elucidated. Herein, we investigated the role of Smad3, a critical molecule mediating the intracellular TGFbeta family proteins, in follicle development and the expression of FSH receptors. We found that gonadotropin stimulation could not induce normal ovulation in Smad3-deficient mice. Moreover, FSH could not stimulate early follicle growth in Smad3-deficient mice in in vivo or in vitro systems. Cultured granulosa cells from Smad3-deficient animals had reduced cell division rates following FSH treatment compared with granulosa cells derived from the ovaries of wild-type (WT) mice. Whole ovaries and isolated granulosa cells from Smad3-deficient animals had lower basal expression of FSH receptor (Fshr), aromatase (Cyp19a1), and cyclin D2 (Ccnd2) mRNA compared with WT mice. Follicle-stimulating hormone treatment of granulosa cells from WT ovaries upregulated Fshr, Cyp19a1, and Ccnd2 expression. However, FSH did not increase these mRNAs in Smad3-deficient granulosa cells. When Smad3 was introduced into Smad3-deficient granulosa cells with adenovirus vectors, FSH responsiveness was restored, and FSH was able to upregulate Fshr expression. Furthermore, SMAD3 interacts with a palindromic SMAD binding element in the Fshr promoter, and TGFB can activate promoter constructs containing this element. Collectively, these observations establish an essential role for Smad3 in regulating the response of ovarian follicles to FSH.

In the paper, “Inhibin Biosynthesis and Activity Are Limited by a Prodomain-Derived Peptide,” Walton et al (1) purport that processing of inhibin-α can result in a 43-amino acid pro-α peptide from an alternate cleavage site. This propeptide is projected to have an amphipathic α-helical structure that can wrap around the open hand or butterfly-shaped mature inhibin protein, thereby hindering inhibin association with β-glycan. The function of this proinhibin α-peptide is investigated as an inhibitor of mature inhibin action. The inhibins and activins are produced as prepro-proteins in which nonactive domains that are important to protein folding and dimerization of the molecules are cleaved, releasing the final active products. The β-subunits for inhibin and activin have only one classical cleavage site, but the α-inhibin subunit has another cleavage site between the pro-α-peptide and the N-peptide (Figure 1A). Uncleaved or incompletely cleaved inhibins have been described for many years as high-molecular-weight or big inhibins, with varying demonstrations of bioactivity (2,–4). A free α-subunit in association with the C portion, without the N portion, has also been described and is produced at high levels by a variety of normal and abnormal tissues (5, 6), but this is the first evaluation of a functional small prodomain cleavage peptide for inhibin. Figure 1. A, Inhibin precursor with the cleavage sites. Note the two cleavage sites on the α-subunit and the single cleavage site on the β-subunit. B, Inhibin A/B binding to β-glycan. C, Pro-α-peptide intercalating between inhibin ... The authors also noted some time ago that three hydrophobic amino acids in the prodomain of the inhibin α-subunit were critical to proper folding and secretion of mature inhibins (7). In that work they used mutagenesis evidence to propose a model of interaction of the N-terminal prodomain with a region near the C terminus of the proprotein to hold it in a coiled conformation that would allow the covalent bonds with the similarly held β-proprotein subunits and promote further processing. In that work they also proposed that the entire cleaved prodomain could remain associated with the active inhibin in a noncovalent way, such as the fact that the latency-associated peptide remains associated with TGFβ (8). In this work they have focused on the role of the small 43-amino acid propeptide. Not only do they demonstrate the necessity of cleavage of this promolecule from the N and C regions of inhibin for production of mature peptide to occur, but they also provide strong evidence for a role of this unassuming peptide in modulating the bioactivity of the mature inhibins. Yet it does not seem to interfere with activin activity. Thus, inhibin protein may come along with its own tiny inhibitor after processing, with the little associated prodomain peptide intercalating itself between the interface with inhibin A or B and the β-glycan receptor (Figure 1, B and C). Yet a function of β-glycan is to inhibit the activation of the activin receptor signaling cascade (9, 10). So the inhibitor is packaged with its own inhibitor, like a functional mini-me, providing direct modulation of inhibin function. This has profound implications biologically if this relationship is shown to occur in vivo. Activin action is already known to be tightly regulated on many levels, both directly and indirectly. From the expression of excess inhibin α-subunits (favoring inhibin production over activin), to follistatin or α-macroglobin binding, to production of inhibitory smads that can directly modulate the activin signaling cascade; the action of activin is tightly regulated in tissue-specific ways, and its effects are profoundly concentration and tissue specific (11, 12). Entry into this already complex system regulating activin, the action of a small amphipathic molecule that could be delivered to a specific site to block inhibin and thereby increase activin action and possibly result in increased unbound β-glycan may have great usefulness clinically and as a research tool across several overlapping fields of endocrinology.

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.

Roehler9s Coryphodon Catastrophe Quarry (Wasatch Formation, lower Eocene) in the Washakie Basin, Wyoming is a paucispecific assemblage containing a minimum of 10 individuals from a single population of cf. Coryphodon anthracoideus . The RCCQ Coryphodon assemblage provides a relatively rare opportunity to assess intraspecific variation in a genus that has historically been difficult to classify due to variation from specimen to specimen and species to species. Variation in metric features of the dentition (i.e., length, width, and molar area) in the RCCQ Coryphodon is within the range observed in comparable sexually dimorphic extant species. The RCCQ Coryphodon assemblage is also compared to another paucispecific, single population assemblage of Coryphodon molestus from New Mexico. Intraspecific metric variation characterizing these two assemblages is also comparable. In both assemblages, overall intraspecific metric variation is most pronounced in the third molar, suggesting sexual dimorphism as the source of variation.

Paracrine and autocrine growth factors can affect many different aspects of ovarian follicle development. Many members of the transforming growth factor b (TGFb) family of growth factors and their receptors are expressed in developing follicles. However, the presence and function of the family of the TGFb 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 TGFb 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 TGFb or activin. However, Smad2 is more responsive to activin stimulation, and Smad3 is more responsive to TGFb stimulation. Stage-specific expression and differing ligand sensitivity of signaling molecules may work together to allow different effects of TGFb family ligands using the same signaling pathways over the course of follicular development. activin, corpus luteum, follicular development, granulosa cells, growth factors

Fibromyalgia (FMS) is a chronic pain syndrome with a complex but poorly understood pathogenesis affecting approximately 10 million adults in the United States. The lack of a clear etiology of FMS has limited the effective diagnosis and treatment of this debilitating condition. The objective of this secondary data analysis was to examine plasma cytokine levels in women with FMS using the Bio-Plex Human Cytokine 17-plex Assay. Post hoc analysis of plasma cytokine levels was performed to evaluate patterns that were not specifieda priori. Upon examination, patients with FMS exhibited a marked reduction inTH2cytokines such as IL-4, IL-5, and IL-13. The finding of this pattern of altered cytokine milieu not only supports the role of inflammation in FMS but also may lead to more definitive diagnostic tools for clinicians treating FMS. TheTH2suppression provides strong evidence of immune dysregulation in patients with FMS.

Mullerian inhibitory substance (MIS), also known as anti-Mullerian hormone, is best known as the hormone that regulates the regression of the Mullerian 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-α 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)-β inhibits the FSH effects on fol...

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.

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.

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 μm) 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 [ 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-α 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-α 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.

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.

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-a 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 middleaged (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.

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 in...

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.

OBJECTIVE: Our purpose was to determine the effects of transforming growth factor-β 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-β 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- β 1 on the enzymatic activity of P450c17 and 3 β HSD, the expression of immunodetectable P450c17 protein, and the expression of messenger ribonucleic acid for P450scc, P450c17, and 3 β HSD were determined. RESULTS: Basal steroid secretion, steroidogenic enzyme activity, enzyme protein, and messenger ribonucleic acid expression were not affected by transforming growth factor-β 1 alone. Forskolin treatment significantly stimulated steroid production and the enzymatic activity of P450c17 and 3 β HSD up to 10-fold above basal levels. However, transforming growth factor- β 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 β HSD activity. Forskolin-stimulated immunodetectable P450c17 α protein was markedly inhibited by transforming growth factor- β 1 . In addition, transforming growth factor- β 1 markedly inhibited the forskolin-stimulation of P450c17 messenger ribonucleic acid, while not significantly altering P450scc or 3 β HSD messenger ribonucleic acid expression. CONCLUSION: Forskolin stimulated human ovarian thecal-like tumor cell steroidogenesis, P450c17 and 3βHSD activity, immunodetectable P450c17, and messenger ribonucleic acid content for P450scc, P450c17, and 3βHSD. Transforming growth factor-β 1 inhibited forskolin stimulation of androstenedione production through the inhibition of P450c17 expression. (AM J OBSTET GYNECOL 1996;174:1109-17.)

Activin and inhibin are structurally related dimeric glycoproteins belonging to the transforming growth factor-β superfamily of proteins which are synthesized and secreted by the granulosa cells of the ovary. Although initially characterized by their ability to influence FSH secretion from pituitary cells, paracrine regulatory roles of these factors on neighboring ovarian theca interna have been suggested. While inhibin has been shown to increase and activin to decrease the production of androgens, the mechanisms of action are not well defined, partly due to difficulties in obtaining adequate numbers of thecal cells from individual patients or animal models. Using a unique human ovarian thecal-like tumor (HOTT) cell culture model system we investigated the biochemical and molecular mechanisms controlling C19 steroidogenesis and the effects of activin and inhibin on the activity and expression of key ovarian thecal steroidogenic enzymes, cholesterol side-chain cleavage cytochrome P450 (P450scc), 3β-hydroxysteroid dehydrogenase (3βHSD) and 17α-hydroxylase/17,20 lyase cytochrome P450 (P450c17). Steroid production, level of steroidogenic enzyme mRNA expression, and enzyme activity following treatment with forskolin, inhibin-A and activin-A were examined. Basal steroid production, enzyme activities, and steroidogenic enzyme mRNA levels were not markedly different following treatment with activin (25 ng/ml) or inhibin (25 ng/ml) alone. Forskolin (10 μm) markedly increased production of both androstenedione (fivefold) and progesterone (threefold) as well as the activity of 3βHSD (sevenfold), and P450c17 (sevenfold) over basal. Forskolin stimulated the expression of mRNA for P450scc (fourfold), 3βHSD (threefold), and P450c17 (eightfold) over basal. Androstenedione accumulation was decreased by 60% in the forskolin plus activin group compared with forskolin alone, while progesterone production was maintained. This was attributed to a reduction of P450c17 mRNA (45% of forskolin alone) and activity (45% of forskolin alone). In contrast, co-treatment with forskolin and inhibin increased androstenedione production by 40% while decreasing progesterone by 40% compared with forskolin alone. Concomitantly, this was associated with a higher P450c17 mRNA expression (1·5-fold) and activity (two-fold) but with minimal effects on the mRNA for 3βHSD and P450scc. HOTT cell responses to activin (0·05–50 ng/ml) and inhibin (0·05–50 ng/ml) in the presence of forskolin demonstrated dose-dependent effects on the steroid accumulation, enzymatic activity and mRNA expression of P450c17. Additionally, the differences seen on mRNA expression of steroidogenic enzymes in response to these factors were time-dependent. In summary, forskolin stimulated C19 steroid production from HOTT cells by increasing the expression of all steroidogenic enzymes examined. Inhibin and activin exerted differential effects on the expression of these enzymes which resulted in alterations in the steroid profile toward production of C19 steroids in the case of inhibin and away from C19 steroids in the case of activin. The influence of these important intraovarian factors on the expression of P450c17, a pivotal enzyme in thecal cell production of C19 steroids, could impact greatly on the follicular milieu of a normal developing follicle as well as in pathophysiological disorders such as polycystic ovarian syndrome. Journal of Endocrinology (1996) 148, 213–221

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, unresponsiv...

OBJECTIVE: We determined the relative effects of insulin and FSH on progesterone accumulation as well as activity, protein content, and mRNA expression of 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) in human luteinized granulosa cells. METHODS: Luteinized granulosa cells obtained from women undergoing in vitro fertilization were plated and grown to near confluence and treated with FSH, insulin, or a combination of insulin and FSH. Progesterone production as well as enzyme activity, protein content, and mRNA expression for 3 beta HSD were evaluated. RESULTS: Progesterone production was not affected by insulin alone but increased threefold in the presence of FSH (50 ng/microL) alone. The presence of FSH plus insulin (100 nmol/L) caused a significant increase in progesterone accumulation greater than that of FSH alone. The already high basal levels of 3 beta HSD activity were unaffected by insulin alone but increased 1.7-fold in the presence of FSH. The combination of FSH (50 ng/mL) and insulin (100 nmol/L) increased activity 1.3-fold over FSH alone (P < .02). Insulin (greater than 100 nmol/L) alone increased 3 beta HSD protein content as measured by Western analysis 1.8-2-fold over basal levels, whereas FSH alone increased protein content 2.8-fold, and was further augmented by the addition of insulin in a dose-related fashion up to 3.5-fold over basal levels. Insulin increased 3 beta HSD mRNA twofold over basal levels; FSH alone increased mRNA expression of 3 beta HSD 3.2-fold. In the presence of insulin plus FSH, 3 beta HSD mRNA expression increased 7.6-fold over basal levels. For comparison, insulin also stimulated cytochrome P450 aromatase activity, P450 aromatase protein, and mRNA but to a greater degree than that seen for 3 beta HSD. CONCLUSION: Insulin is a regulator of both 3 beta HSD and aromatase expression in human granulosa cells. Elevated insulin levels could therefore affect steroid production in human granulosa cells and presumably alter the menstrual cycle and fertility.

This chapter provides insight into the fertility management of cancer survivors with compromised or absent ovarian function and without cryopreserved material. It begins with the evaluation of the cancer survivor for pregnancy. A flow-chart of cancer survivor screening is shown. Standard ovarian stimulation for assisted reproductive technology (ART) results in high levels of circulating estrogen, potentially 10 times the peak levels of spontaneous cycles. The mechanics of oocyte donation involves a number of steps: the recruitment of suitable donors, informed consent of donors and recipients, matching of donors to recipients, ovarian stimulation of the donor and the retrieval of her oocytes. Third-party reproduction is one of the most ethically complex aspects of reproductive health care. The chapter addresses, among other things, issues that occur when preservation of fertility has not been possible or has not been the best choice for a given individual.

To determine if cervical intraepithelial neoplasia grade 3 (CIN-3) and cervical cancer are associated with adverse obstetrical outcomes.Women with diagnoses of CIN-3 and cervical cancer were first identified from the University of Pittsburgh Medical Center (UPMC) Network Cancer Registry by using respective ICD-3 codes. Identified records were then linked to the Magee Obstetrical Maternal and Infant (MOMI) database to identify women who subsequently delivered pregnancies at Magee-Womens Hospital. Women with cervical disease were compared with women without known disease to determine the impact of cervical disease on various maternal and neonatal outcomes. The latter group consisted of those women who delivered singleton pregnancies at our institution, as determined by the MOMI database, but who did not have any matching records in the UPMC Cancer Registry. Statistical significance was defined by a p value0.05.We identified CIN-3 (n = 52) and cervical cancer patients (n = 83) who later had documented pregnancies delivered at Magee-Womens Hospital between 1989 and 2006. Women with cervical cancer and CIN-3 were at greater risk to deliver small-for-gestational age (SGA) neonates compared with women without known cervical disease (RR 1.54, 95% confidence interval [CI] 1.0-2.46). A secondary analysis of risk factors for SGA neonates demonstrated a significant association with cervical cancer (p = 0.04). After accounting for variables known to be risk factors for SGA, cervical cancer was associated with a 1.9-fold increased risk of a SGA delivery (OR 1.9, 95% CI 1.1-3.4).Cervical cancer is a risk factor for delivery of an SGA neonate in a subsequent pregnancy.

Traditionally, follicles have been grown in standard incubators with atmospheric oxygen concentration. However, preantral follicles exist in the avascular cortex of the ovary. This study examines the effectiveness of an oxygen delivery protocol that more closely mimics the in vivo environment of the ovary on oocyte viability, maturation, parthenogenetic activation, and fertilization from in vitro cultured rat preantral follicles. Of 54 oocytes cultured in the dynamic oxygen environment, 35 were viable while only 22 of 50 oocytes cultured within an ambient oxygen concentration remained viable (p < 0.05). Germinal vesicle breakdown was observed in 56% of oocytes from the dynamic oxygen group compared to 30% of oocytes from the ambient oxygen group (p < 0.05). Parthenogenetic activation was observed in a significant number of oocytes from the dynamic oxygen group, while none of the oocytes from the ambient oxygen group activated (p < 0.05). However, the proportions of oocytes from the dynamic oxygen group that remained viable underwent germinal vesicle breakdown, and activated were still significantly less than those from the in vivo control group (p < 0.05). Fertilization of the oocytes from the dynamic oxygen group was confirmed through a successful trial of intracytoplasmic sperm injection.

Background We have previously shown that suspension culture prevents follicle flattening and maintains three-dimensional follicle architecture better than culture on flat plates. However, many of the follicles cultured in suspension do eventually rupture, as basement membrane integrity is lost and the three-dimensional structure of the follicle is altered. Therefore, the objective of this study is to support three-dimensional follicle architecture during in vitro growth of ovarian follicles through encapsulation in calcium alginate, while maintaining responsiveness to FSH stimulation. Methods Preantral follicles (150 – 160 micrometers in diameter) were isolated from the ovaries of juvenile rats and grown in culture tubes or encapsulated in calcium alginate and grown in culture tubes. Previous studies revealed that follicles maintained structural integrity but did not grow as well when encapsulated in calcium alginate. In these studies, we evaluated the effect of calcium alginate on FSH-stimulated follicle growth, survival, and morphology in suspension culture. Follicles were grown under 5 culture conditions: 1) not encapsulated; with FSH in the medium, 2) encapsulated in the absence of FSH, grown in medium without FSH, 3) encapsulated with calcium alginate containing FSH but grown in medium without FSH, 4) encapsulated without FSH but grown in medium containing FSH and 5) encapsulated with calcium alginate containing FSH and in medium containing FSH. To assess growth rates, follicles were cultured for 72 hours and analyzed for follicle size increase and DNA content. Survival analysis for encapsulated and unencapsulated follicles was performed by constructing a Kaplan Meier survival curve of daily observations of intact follicle survival. Three-dimensional architecture was assessed histologically and by analysis of the pattern of connexin 43 expression in the cultured follicles. Results In the absence of FSH, follicle diameter increased by only 6.4%. When FSH was included in the alginate bead alone or the media alone, the follicle diameter increased by 13.5% and 19.9% respectively. This was greater than follicles cultured in the absence of FSH (p < 0.05), but less than that of the FSH-treated unencapsulated follicles (p < 0.05). However, when follicles were cultured with FSH included in both the media and the bead, a 32.6% increase in follicle diameter was observed, statistically no different than the growth rate of the unencapsulated follicles grown with FSH. Conclusion Microencapsulation supports three-dimensional follicle growth, but may limit access to hormones in the medium resulting in altered development compared to unencapsulated follicles. Inclusion of FSH in the alginate bead restores the follicle growth response to FSH, while also providing a scaffold of support for three-dimensional growth. The application of tissue engineering principles to the problems of follicle culture in vitro may provide advances applicable to fertility preservation in women and endangered species.

Smad 3 is a signaling intermediate for the transforming growth factor beta (TGFβ) family; however, little is known about the role this protein plays in the regulation of the ovarian surface epithelium (OSE). Using a transgenic mouse model, we found that in the absence of Smad 3 there was a distinct morphological alteration of OSE cells. Wild-type (WT) OSE was flat with thin cells, while Smad 3-deficient (Smad 3 −/−) OSE was thick with plump cuboidal cells. WT OSE had less immunostaining for proliferating cell nuclear antigen (PCNA) and estrogen receptor alpha (ERα) than Smad 3 −/− OSE. However, there were no differences in the number of apoptotic cells or Bax and Bcl-2 levels between WT and Smad 3 −/− OSE. Although WT mice had higher levels of serum estradiol than Smad 3 −/− mice, WT and Smad 3 −/− mice had similar levels of progesterone. These data suggest that Smad 3 regulates OSE morphological appearance and proliferation in the absence of high serum estradiol levels or alterations in progesterone levels. Anat Rec Part A 273A:681–686, 2003. © 2003 Wiley-Liss, Inc.

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.

As a richly fossiliferous bone bed with over 350 bones from a single taxon, Roehler’s Coryphodon Catastrophe Quarry (RCCQ) in the Washakie Basin of Wyoming is an intriguing paleontological phenomenon. Coryphodon was a large bodied, subdigitigrade, graviportal herbivorous browser that first appeared in North America in the latest Paleocene (Clarkforkian Land Mammal Age) and was ubiquitous in early Eocene terrestrial faunas (Lucas, 1984). It is suggested that RCCQ is a mass death accumulation, but that evidence for such an event is obscured by the overprint of at least one additional stage which intervened between the time of death and the time of final deposition. This paper draws on faunal, taphonomic, and sedimentologic evidence to reconstruct the sequence of events leading to formation of the RCCQ bone bed, and to ascertain the extent and degree of bias caused by successive modifications.