AKAP13 Enhances CREB1 Activation by FSH in Granulosa Cells.

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.

Sex Differences and Drug Dose Influence the Role of the α7 Nicotinic Acetylcholine Receptor in the Mouse Dextran Sodium Sulfate-Induced Colitis Model.

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.

Regulators of ovarian preantral follicle development.

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-ß 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.

Activation of the LH receptor up regulates the type 2 adiponectin receptor in human granulosa cells.

PURPOSE: Adiponectin is a predominantly adipocyte-derived hormone which influences insulin sensitivity and energy homeostasis through at least two receptors, AdipoR1 and AdipoR2. In animal models, adiponectin may regulate ovarian steroidogenesis, folliculogenesis, and ovulation. The receptors AdipoR1 and AdipoR2 are present in the human ovary, but their regulation is unknown. In these studies, we determined the effects of LH receptor activation on the expression and function of the two adiponectin receptors in human granulosa cells. METHODS: Granulosa cells were obtained at the time of oocyte retrieval in women undergoing in vitro fertilization (IVF). Cells were isolated and cultured for 48 h in DMEM/F12 medium with 5 % FBS and 50 ug/ml gentamicin. Medium was changed to low serum for 12 h and cells were treated with hCG (100 ng/ml), forskolin (30 µMol/L), or FSH (1 IU/ml) for 24 h for mRNA experiments. mRNA was isolated and RT PCR was performed using Taqman assays and quantification with the delta delta CT method. For immunocytochemistry, cells were grown on chamber slides and treated with hCG for 1 to 24 h and fixed with acetone. ICC was performed with polyclonal rabbit primary antibodies followed by alexa fluor goat anti-rabbit antibody and imaging with a fluorescence microscope and Zeiss software analysis. 3ß-hydroxysteroid dehydrogenase (3ßHSD) enzyme activity was determined by measuring the progesterone produced when cells were provided with an excess of 22-hydroxy-cholesterol as substrate following an incubation with hCG (1 IU/ml) and/or adiponectin (10 ng/ml). Progesterone content in the media was determined by ELISA. RESULTS: Messenger RNA for the two Adiponectin receptors is differentially regulated by activation of LHR with hCG treatment. AdipoR2 was increased nearly 4-fold (p < 0.05), whereas AdipoR1 expression was not changed by hCG treatment. Treatment with either FSH or forskolin (an activator of cAMP) had similar effects. Basal AdipoR2 protein was fairly low in granulosa cells in culture however treatment of cells with hCG resulted in a discernible increase in immunodetectable cytoplasmic protein as early as 6 h after treatment and was maintained for at least 24 h. The number of cells positive for AdipoR2 at 6 h increased from a basal of 20 % to almost 60 % (p < 0.05). Adiponectin treatment of hCG-primed cells resulted in increased 3ßHSD activity by approximately 60 % over hCG alone and more than 3-fold over basal levels. CONCLUSIONS: AdipoR2 is regulated by the LH receptor function via a cAMP dependant mechanism. Increased expression of adipoR2 prior to and following ovulation may contribute to enhanced 3ßHSD activity and increased progesterone secretion by the corpus luteum of the ovary. Dysregulation of adiponectin that may occur with PCOS may impair normal progesterone production.

Smad7 is a transforming growth factor-beta-inducible mediator of apoptosis in granulosa cells.

OBJECTIVE: To determine the functional role of Smad7 in granulosa cells. DESIGN: Granulosa cell culture and molecular biological techniques were used to investigate regulation and function of Smad7. SETTING: Research laboratory. ANIMAL(S): C57bl/j hybrid mouse. INTERVENTION(S): Primary mouse granulosa cells were isolated and grown in culture for all messenger RNA expression experiments. Smad7 promoter constructs were evaluated with a luciferase reporter system in SIGC cells to determine sites activating Smad7 expression. MAIN OUTCOME MEASURE(S): Overexpression (Smad7 complementary DNA) and downregulation (Smad7 small interfering RNA) of Smad7 in primary mouse granulosa cells were used to evaluate the functional role of Smad7 in granulosa cells. RESULT(S): Smad7 expression was upregulated by treatment with transforming growth factor-ß (TGF-ß) but not activin or activation of the cyclic adenosine monophosphate pathway. The promoter of Smad7 was activated by TGF-ß. Truncation of the promoter or mutation of the Smad response element at -141 eliminated TGF-ß activation of the promoter. Smad3 was not specifically required for TGF-ß-stimulated expression of Smad7, though activation of the TGFBR1 receptor was. When Smad7 was overexpressed in granulosa cells, apoptosis was markedly increased. When Smad7 expression was reduced with small interfering RNA, then the TGF-ß-induced apoptosis was blocked. CONCLUSION(S): Smad7 mediates apoptosis induced by TGF-ß in mouse granulosa cells, suggesting that dysregulation of Smad7 could impair folliculogenesis.

Cervical cancer and risk for delivery of small-for-gestational age neonates.

OBJECTIVE: To determine if cervical intraepithelial neoplasia grade 3 (CIN-3) and cervical cancer are associated with adverse obstetrical outcomes. METHODS: 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 value <0.05. RESULTS: 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). CONCLUSIONS: Cervical cancer is a risk factor for delivery of an SGA neonate in a subsequent pregnancy.

Dynamic oxygen enhances oocyte maturation in long-term follicle culture.

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.

Smad3 is required for normal follicular follicle-stimulating hormone responsiveness in the mouse.

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.

Calcium alginate microencapsulation of ovarian follicles impacts FSH delivery and follicle morphology.

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.

Developmental and stage-specific expression of Smad2 and Smad3 in rat testis.

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.

Direct binding of AP-1 (Fos/Jun) proteins to a SMAD binding element facilitates both gonadotropin-releasing hormone (GnRH)- and activin-mediated transcriptional activation of the mouse GnRH receptor gene.

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.

Stage-specific expression of Smad2 and Smad3 during folliculogenesis.

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.