Combining Biomarker Calibration Data to Reduce Measurement Error.

Biomarker assay measurement often consists of a two-stage process where laboratory equipment yields a relative measure which is subsequently transformed to the unit of interest using a calibration curve. The calibration curve establishes the relation between the measured relative units and sample biomarker concentrations using stepped samples of known biomarker concentrations. Samples from epidemiologic studies are often measured in multiple batches or plates, each with independent calibration experiments. Collapsing calibration information across batches before statistical analysis has been shown to reduce measurement error and improves estimation. Additionally, collapsing in practice can also create an additional layer of quality control (QC) and optimization in a part of the laboratory measurement process that is often highly automated. Principled recalibration is demonstrated via. a three-step process of identifying batches where recalibration might be beneficial, forming a collapsed calibration curve and recalibrating identified batches, and using QC data to assess the appropriateness of recalibration. Here, we use inhibin B measured in biospecimens from the BioCycle study using 50 enzyme-linked immunosorbent assay (ELISA) batches (3875 samples) to motivate and display the benefits of collapsing calibration experiments, such as detecting and overcoming faulty calibration experiments, and thus improving assay coefficients of variation from reducing unwanted measurement error variability. Differences in the analysis of inhibin B by testosterone quartile are also demonstrated before and after recalibration. These simple and practical procedures are minor adjustments implemented by study personnel without altering laboratory protocols which could have positive estimation and cost-saving implications especially for population-based studies.

Endocrine disrupting chemicals in seminal plasma and couple fecundity.

Growing evidence supports the importance of men's exposure to non-persistent endocrine disruptors (EDCs) and couple fecundability, as measured by time-to-pregnancy (TTP). This evolving literature contrasts with the largely equivocal findings reported for women's exposures and fecundity. While most evidence relies upon urinary concentrations, quantification of EDCs in seminal plasma may be more informative about potential toxicity arising within the testes. We analyzed 5 chemical classes of non-persistent EDCs in seminal plasma for 339 male partners of couples who were recruited prior to conception and who were followed daily until pregnant or after one year of trying. Benzophenones, bisphenols, parabens, and phthalate metabolites and phthalate diesters were measured using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) except for phthalate diesters, which were analyzed using gas chromatography-mass spectrometry. Cox regression with discrete-time was used to estimate fecundability odds ratios (FORs) and 95% confidence intervals (CIs) for each chemical to estimate the probability of pregnancy. While most EDCs were detected in seminal plasma, concentrations were lower than urinary concentrations previously analyzed for the cohort. None of the EDCs were significantly associated with fecundability even after covariate adjustment, though benzophenones consistently yielded FORs <1.0 (ranging from 0.72 to 0.91) in couple-adjusted models suggestive of diminished fecundity (longer TTP). The findings underscore that a range of EDCs can be quantified in seminal plasma, but the lower concentrations may require a large cohort for assessing couple fecundability, as well as the need to consider other fecundity outcomes such as semen quality.

Preconception seminal plasma concentrations of endocrine disrupting chemicals in relation to semen quality parameters among male partners planning for pregnancy.

BACKGROUND: Some non-persistent endocrine disruptors (EDCs) are adversely associated with semen quality and few studies have measured those EDCs in seminal plasma. OBJECTIVE: To find an association between EDCs in seminal plasma and semen quality parameters. METHODS: Five chemical classes of non-persistent EDCs were quantified in seminal plasma from 339 male partners who participated in a prospective pregnancy study. Bisphenols, benzophenone UV-filters, antimicrobials and phthalate diesters and their monoester metabolites were measured using high performance liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry. Semen samples underwent next day analysis using a standardized protocol for the quantification of 35 endpoints. Linear mixed-effects models of EDCs that were log transformed and rescaled by their standard deviations or dichotomized at the 75th percentile for each exposure and outcomes with covariate adjustment were performed. EDCs in seminal plasma were also assessed relative to clinical reference values of semen quality endpoints using logistic regression or generalized estimating equations. RESULTS: The most consistent findings supporting adverse associations between seminal EDCs and semen quality were observed for some phthalate metabolites. For example, seminal plasma mono-ethyl, mono-n-butyl, mono-2-isobutyl and mono-benzyl phthalate concentrations were associated with decreased odds of having semen volume above clinical reference values (mEP: aOR=0.46; 95%CI= 0.32, 0.66; mBP: aOR=0.40; 95%CI= 0.28, 0.57; miBP: aOR=0.39; 95%CI= 0.27, 0.56), and mBzP: aOR= 0.34; 95%CI= 0.24, 0.49). CONCLUSIONS: Environmentally relevant concentrations of specific phthalates in seminal plasma were associated with diminished semen volume, sperm motility, viability, and morphological alterations in sperm heads such that semen volume and sperm viability fall below reference values.

Urinary Phytoestrogen Concentrations Are Not Associated with Incident Endometriosis in Premenopausal Women.

BACKGROUND: Phytoestrogens have been associated with subtle hormonal changes, but their effects on endometriosis are largely unknown. OBJECTIVE: We assessed the association between urinary concentrations of phytoestrogens and incident endometriosis. METHODS: We included an operative sample of 495 premenopausal women aged 18-44 y undergoing laparoscopies and laparotomies at 14 clinical sites between 2007 and 2009 and a general population sample of 131 women from the same geographic area who were matched on age and menstruation status. Endometriosis in the surgical sample was assessed by surgical visualization (clinical gold standard), whereas disease in the general population sample was assessed with the use of a pelvic MRI. Urine concentrations of genistein, daidzen, O-desmethylangolensin, equol, enterodiol, and enterolactone were measured at baseline. Poisson regression with robust error variance was used to estimate the risk of an endometriosis diagnosis for each sample after adjusting for age and body mass index (in kg/m2). Separate models were run for each phytoestrogen. RESULTS: Overall geometric mean urine concentrations of phytoestrogens were as follows: genistein [88 nmol/L (95% CI: 72, 108 nmol/L)], daidzein [194 nmol/L (95% CI: 160, 236 nmol/L)], O-desmethylangolensin [4 nmol/L (95% CI: 3, 6 nmol/L)], equol [4 nmol/L (95% CI: 4, 6 nmol/L)], enterodiol [29 nmol/L (95% CI: 22, 38 nmol/L)], and enterolactone [355 nmol/L (95% CI: 395, 544 nmol/L)]. Geometric mean concentrations of phytoestrogens did not significantly differ by endometriosis status in either sample. Adjusted RRs for endometriosis ranged from 0.87 to 1.09 for the 6 phytoestrogens measured, with all CIs including a value ≥1. Phytoestrogens were not associated with the severity of endometriosis when restricting the analysis to women with moderate-to-severe disease per the revised American Society for Reproductive Medicine criteria. Furthermore, no associations were observed between self-reported high soy intake and endometriosis. CONCLUSIONS: Despite endometriosis being an estrogen-dependent disease, we found no evidence that urinary phytoestrogens were associated with a higher risk of an endometriosis diagnosis in either a sample of premenopausal women or in a surgical sample.

Membrane-type 6 matrix metalloproteinase regulates the activation-induced downmodulation of CD16 in human primary NK cells.

CD16 (FcγRIIIa), the low-affinity receptor for IgG, expressed by the majority of human NK cells, is a potent activating receptor that facilitates Ab-dependent cell-mediated cytotoxicity (ADCC). ADCC dysfunction has been linked to cancer progression and poor prognosis for chronic infections, such as HIV; thus, understanding how CD16 expression is regulated by NK cells has clinical relevance. Importantly, CD16 cell-surface expression is downmodulated following NK cell activation and, in particular, exposure to stimulatory cytokines (IL-2 or IL-15), likely owing to the action of matrix metalloproteinases (MMPs). In this article, we identify membrane-type 6 (MT6) MMP (also known as MMP25) as a proteinase responsible for CD16 downmodulation. IL-2-induced upregulation of MT6/MMP25 cell-surface expression correlates with CD16 downmodulation. MT6/MMP25, sequestered in intracellular compartments in unstimulated NK cells, translocates to the cell surface after stimulation; moreover, it polarizes to the effector-target cell interface of the CD16-mediated immunological synapse. siRNA-mediated disruption of MT6/MMP25 expression enhances the ADCC capacity of NK cells, emphasizing the important functional role of MT6/MMP25 in the regulation of ADCC activity. Thus, this study uncovers a previously unknown role of MT6/MMP25 in human NK cells, and suggests that inhibition of MT6/MMP25 activity could improve ADCC efficacy of therapeutically administered NK cells that require IL-2 for culture and expansion.

Urinary concentrations of phthalates in couples planning pregnancy and its association with 8-hydroxy-2'-deoxyguanosine, a biomarker of oxidative stress: longitudinal investigation of fertility and the environment study.

Oxidative stress has been recognized as one of the most important contributors to infertility in both males and females. Exposure to many environmental chemicals, such as phthalates, has been shown to induce oxidative stress. In a longitudinal study designed to assess exposure to environmental chemicals and fecundity in couples who were planning pregnancy, 894 urine samples were collected from 469 couples from Michigan and Texas during 2005-2009. The concentrations of 14 phthalate metabolites and a marker of oxidative stress, 8-hydroxy-2'-deoxyguanosine (8-OHdG), were determined in these samples. Concentrations, profiles, and estimated daily intakes (DIs) of phthalates were positively associated with 8-OHdG. The median concentrations of monomethyl phthalate (mMP), monoethyl phthalate (mEP), mono(3-carboxypropyl) phthalate (mCPP), mono-n-butyl phthalate (mBP), mono(2-isobutyl) phthalate (miBP), monobenzyl phthalate (mBzP), Σ5mEHP (sum of five metabolites of di(2-ethylhexyl) phthalate (DEHP)) and Σ14phthalates (sum of 14 urinary phthalate metabolites) were 0.48, 85.2, 4.50, 7.66, 4.36, 3.80, 54.8, and 249 µg/g creatinine, respectively. The estimated DI values for DEHP in 39 individuals were above the U.S. Environmental Protection Agency's (EPA) reference dose (RfD) of 20 µg/kg-bw/day. The mean and median concentrations of 8-OHdG were 6.02 and 3.13 µg/g creatinine, respectively, which were significantly higher in females than in males. Statistically significant associations were found between 8-OHdG and urinary concentrations of mEP, and Σ5mEHP for females. Similarly, a significant association was found between 8-OHdG and DIs estimated for select phthalates. Our results suggested that phthalate exposure increases oxidative stress, which can be a mechanism for the diminished fertility observed in couples who were highly exposed to select phthalates.

Toso, a functional IgM receptor, is regulated by IL-2 in T and NK cells.

We find that the cell surface receptor Toso is dramatically downregulated by in vitro stimulation of human T and NK cells with IL-2 in a STAT5-dependent manner. The fact that IL-2 is known to prime NK and T cells for Fas/TNF-mediated activation-induced cell death (AICD) fits nicely with the original and recent descriptions of Toso as an inhibitor of Fas/TNF-induced apoptosis. In support of this possibility, effector memory T cells express markedly lower levels of Toso than those of naive T cells, indicating that activation in vivo correlates with the downregulation of Toso. Moreover, in vitro activation of memory T cells through TCR dramatically downregulates Toso expression compared with that of naive CD4 T cells. However, overexpression of Toso in human NK cells and Jurkat T cells does not inhibit Fas-mediated apoptosis, and, in agreement with other recent reports, Toso clearly functions as an IgM receptor. Unlike CD16, Toso expression by NK cells does not convey cytotoxic potential, but its ligation does trigger intracellular signaling in NK cells. In summary, our data indicate that Toso is a functional IgM receptor that is capable of activating signaling molecules, is regulated by IL-2, and is not inherently an antiapoptotic molecule.

Complex regulation of human NKG2D-DAP10 cell surface expression: opposing roles of the γc cytokines and TGF-ß1.

Natural killer (NK) cells help protect the host against viral infections and tumors. NKG2D is a vital activating receptor, also expressed on subsets of T cells, whose ligands are up-regulated by cells in stress. Ligation of NKG2D leads to phosphorylation of the associated DAP10 adaptor protein, thereby activating immune cells. Understanding how the expression of NKG2D-DAP10 is regulated has implications for immunotherapy. We show that IL-2 and TGF-ß1 oppositely regulate NKG2D-DAP10 expression by NK cells. IL-2 stimulation increases NKG2D surface expression despite a decrease in NKG2D mRNA levels. Stimulation with IL-2 results in a small increase of DAP10 mRNA and a large up-regulation of DAP10 protein synthesis, indicating that IL-2-mediated effects are mostly posttranscriptional. Newly synthesized DAP10 undergoes glycosylation that is required for DAP10 association with NKG2D and stabilization of NKG2D expression. TGF-ß1 has an opposite and dominant effect to IL-2. TGF-ß1 treatment decreases DAP10, as its presence inhibits the association of RNA polymerase II with the DAP10 promoter, but not NKG2D mRNA levels. This leads to the down-regulation of DAP10 expression and, as a consequence, NKG2D protein as well. Finally, we show that other γ(c) cytokines act similarly to IL-2 in up-regulating DAP10 expression and NKG2D-DAP10 surface expression.

"What was the vibe you were going for?" SAM Behaviors and Socialization During the COVID-19 Pandemic, a Qualitative Approach.

Introduction: Marijuana use is at historic highs amongst college-aged adults, who are more likely to engage in simultaneous alcohol and marijuana use (SAM) than other age cohorts. For college students, the COVID-19 pandemic is a unique transitory phenomenon that led to isolation, as well as changes in socialization, academic environments, and substance use. This exploratory qualitative study aims to understand SAM socialization and motivation behaviors among college students. Methods: Semi-structured qualitative interviews (N=32) were conducted across the United States from January 2021-April 2021via Zoom. Interviews were then transcribed, then a thematic analysis was conducted in Atlas.ti. Results: The sample was primarily college juniors (mage=21). Since the pandemic, half of the participants increased SAM, whereas the other half decreased SAM. SAM was reported in different categories including primarily with friends, but, much less with partners and with roommates. More than half of the sample indicated that they used SAM alone. Motivations to engage in SAM included relaxing, socializing, offsetting stressors specific to the COVID-19 pandemic, and relieving general stress, anxiety and boredom. Conclusion: The COVID-19 pandemic impacted college students' substance use in interesting ways. Understanding the behaviors of SAM in the context of the COVID-19 pandemic is crucial due to the legalization of marijuana in many states. This understanding has significant implications for prevention strategies and potential policy interventions. Our study yielded findings regarding the impact of socialization on SAM. We discovered that not only does socialization affect SAM, but the specific contexts and motivations behind these behaviors also play a crucial role, which adds to our developing understanding of SAM behavior.

Pregnancy Loss and Iodine Status: The LIFE Prospective Cohort Study.

Iodine deficiency in pregnancy is a common problem in the United States and parts of Europe, but whether iodine deficiency is associated with increased pregnancy loss has not been well studied. The LIFE study provided an excellent opportunity to examine the relationship between iodine status and pregnancy loss because women were monitored prospectively to ensure excellent ascertainment of conceptions. The LIFE study, a population-based prospective cohort study, monitored 501 women who had discontinued contraception within two months to become pregnant; 329 became pregnant, had urinary iodine concentrations measured on samples collected at enrollment, and were followed up to determine pregnancy outcomes. Of the 329, 196 had live births (59.5%), 92 (28.0%) had losses, and 41 (12.5%) withdrew or were lost to follow up. Urinary iodine concentrations were in the deficiency range in 59.6% of the participants. The risk of loss, however, was not elevated in the mildly deficient group (hazard ratio 0.69, 95% confidence interval 0.34, 1.38), the moderately deficient group (hazard ratio 0.81, 95% confidence interval 0.43, 1.51), or the severely deficient group (hazard ratio 0.69, 95% confidence interval 0.32, 1.50). Iodine deficiency, even when moderate to severe, was not associated with increased rates of pregnancy loss. This study provides some reassurance that iodine deficiency at levels seen in many developed countries does not increase the risk of pregnancy loss.

DASH, the data and specimen hub of the National Institute of Child Health and Human Development.

The benefits of data sharing are well-established and an increasing number of policies require that data be shared upon publication of the main study findings. As data sharing becomes the new norm, there is a heightened need for additional resources to drive efficient data reuse. This article describes the development and implementation of the Data and Specimen Hub (DASH) by the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) to promote data sharing from NICHD-funded studies and enable researchers to comply with NIH data sharing policies. DASH's flexible architecture is designed to archive diverse data types and formats from NICHD's broad scientific portfolio in a manner that promotes FAIR data sharing principles. Performance of DASH over two years since launch is promising: the number of available studies and data requests are growing; three manuscripts have been published from data reanalysis, all within two years of access. Critical success factors included NICHD leadership commitment, stakeholder engagement and close coordination between the governance body and technical team.

Switching of NR5A proteins associated with the inhibin alpha-subunit gene promoter after activation of the gene in granulosa cells.

The inhibin alpha-subunit gene is transcriptionally activated by FSH in ovarian granulosa cells during follicular growth. We have investigated the roles of the NR5A family nuclear receptors steroidogenic factor 1 (SF-1) and liver receptor homolog 1 (LRH-1) in transcriptional activation of the inhibin alpha-subunit gene. Transfection assays using an inhibin alpha-subunit promoter reporter in GRMO2 granulosa cells show that LRH-1 and SF-1 act similarly to increase promoter activity, and that the activity of both transcription factors is augmented by the coactivators cAMP response element-binding protein-binding protein and steroid receptor coactivator 1. However, chromatin immunoprecipitation experiments illustrate differential dynamic association of LRH-1 and SF-1 with the alpha-subunit inhibin promoter in both primary cells and the GRMO2 granulosa cell line such that hormonal stimulation of transcription results in an apparent replacement of SF-1 with LRH-1. Transcriptional stimulation of the inhibin alpha-subunit gene is dependent on MAPK kinase activity, as is the dynamic association/disassociation of SF-1 and LRH-1 with the promoter. Inhibition of the phosphatidylinositol 3-kinase signaling pathway influences promoter occupancy and transcriptional activation by SF-1 but not LRH-1, suggesting a possible mechanistic basis for the distinct functions of these NR5A proteins in inhibin alpha-subunit gene regulation.

Sequence-specific deoxyribonucleic acid (DNA) recognition by steroidogenic factor 1: a helix at the carboxy terminus of the DNA binding domain is necessary for complex stability.

Steroidogenic factor 1 (SF1) is a member of the NR5A subfamily of nuclear hormone receptors and is considered a master regulator of reproduction because it regulates a number of genes encoding reproductive hormones and enzymes involved in steroid hormone biosynthesis. Like other NR5A members, SF1 harbors a highly conserved approximately 30-residue segment called the FTZ-F1 box C-terminal to the core DNA binding domain (DBD) common to all nuclear receptors and binds to 9-bp DNA sequences as a monomer. Here we describe the solution structure of the SF1 DBD in complex with an atypical sequence in the proximal promoter region of the inhibin-alpha gene that encodes a subunit of a reproductive hormone. SF1 forms a specific complex with the DNA through a bipartite motif binding to the major and minor grooves through the core DBD and the N-terminal segment of the FTZ-F1 box, respectively, in a manner previously described for two other monomeric receptors, nerve growth factor-induced-B and estrogen-related receptor 2. However, unlike these receptors, SF1 harbors a helix in the C-terminal segment of the FTZ-F1 box that interacts with both the core DBD and DNA and serves as an important determinant of stability of the complex. We propose that the FTZ-F1 helix along with the core DBD serves as a platform for interactions with coactivators and other DNA-bound factors in the vicinity.

Increased androgen, anti-Müllerian hormone, and sporadic anovulation in healthy, eumenorrheic women: a mild PCOS-like phenotype?

CONTEXT: Hyperandrogenism is a hallmark of polycystic ovary syndrome (PCOS) in women with irregular menses, yet the relationship between androgens and ovarian dysfunction remains poorly understood in eumenorrheic women. OBJECTIVE: The objective of the study was to evaluate whether sporadic anovulation was associated with higher T and anti-müllerian hormone (AMH; marker of ovarian follicle count) concentrations in eumenorrheic women. DESIGN: This was a prospective cohort study from 2005 to 2007. SETTING: The study was conducted at the University of Buffalo in western New York state. PARTICIPANTS: A total of 259 eumenorrheic women without a self-reported history of infertility, PCOS, or other endocrine disorder participated in the study. MAIN OUTCOME MEASURES: Total T and AMH were measured five to eight times per cycle for one (n = 9) or two (n = 250) cycles per woman (n = 509 cycles) with timing of menstrual cycle phase assisted by fertility monitors. Anovulatory cycles were defined biochemically by progesterone and LH concentrations. Repeated-measures ANOVA was conducted on log-transformed data with adjustment for age. RESULTS: Compared with ovulatory cycles (n = 467), sporadic anovulatory cycles (n = 42) had marginally higher total and significantly higher free T [mean 23.7 ng/dL (95% confidence interval [CI] 21.4-26.3) vs 21.6 ng/dL (95% CI 20.9-22.3), P = .08, and 0.36 ng/dL (95% CI 0.33-0.40) vs 0.32 ng/dL (95% CI 0.31-0.33), P = .02, respectively] during menses and also throughout the luteal phase (P < .01 for all). Women with higher T had elevated AMH concentrations, increased reporting of a history of acne requiring medical treatment, but not increased hirsutism. CONCLUSIONS: Mechanisms of androgen-related ovulatory dysfunction that characterize PCOS in women with menstrual disturbances may occur across a continuum of T concentrations, including in eumenorrheic women without clinical hyperandrogenism.

Lhcgr expression in granulosa cells: roles for PKA-phosphorylated ß-catenin, TCF3, and FOXO1.

Ovarian follicles lacking FSH or FSH receptors fail to progress to a preovulatory stage, resulting in infertility. One hallmark of the preovulatory follicle is the presence of luteinizing hormone/choriogonadotropin receptors (LHCGR) on granulosa cells (GCs). However, the mechanisms by which FSH induces Lhcgr gene expression are poorly understood. Our results show that protein kinase A (PKA) and phosphoinositide 3-kinase (PI3K)/AKT pathways are required for FSH to activate both the murine Lhcgr-luciferase reporter and expression of Lhcgr mRNA in rat GCs. Based on results showing that an adenovirus (Ad) expressing a steroidogenic factor 1 (SF1) mutant that cannot bind ß-catenin abolished FSH-induced Lhcgr mRNA, we evaluated the role of ß-catenin in the regulation of Lhcgr gene expression. FSH promoted the PKA-dependent, PI3K-independent phosphorylation of ß-catenin on Ser552 and Ser665. FSH activated the ß-catenin/T-cell factor (TCF) artificial promoter-reporter TOPFlash via a PKA-dependent, PI3K-independent pathway, and dominant-negative (DN) TCF abolished FSH-activated Lhcgr-luciferase reporter and induction of Lhcgr mRNA. Microarray analysis of GCs treated with Ad-DN-TCF and FSH identified the Lhcgr as the most down-regulated gene. Chromatin immunoprecipitation results placed ß-catenin phosphorylated on Ser552 and Ser675 and SF1 on the Lhcgr promoter in FSH-treated GCs; TCF3 was constitutively associated with the Lhcgr promoter. Transduction with an Ad-phospho-ß-catenin mutant (Ser552/665/Asp) enhanced Lhcgr mRNA expression in FSH-treated cells greater than 3-fold. Finally, we identified a recognized PI3K/AKT target, forkhead box O1, as a negative regulator of Lhcgr mRNA expression. These results provide new understanding of the complex regulation of Lhcgr gene expression in GCs.

Role of the phosphatidylinositol-3-kinase and extracellular regulated kinase pathways in the induction of hypoxia-inducible factor (HIF)-1 activity and the HIF-1 target vascular endothelial growth factor in ovarian granulosa cells in response to follicle-stimulating hormone.

FSH stimulation of granulosa cells (GCs) results in increased hypoxia-inducible factor (HIF)-1alpha protein levels and HIF-1 activity that is necessary for up-regulation of certain FSH target genes including vascular endothelial growth factor. We report that the role of the phosphatidylinositol (PI)-3-kinase/AKT pathway in increasing HIF-1alpha protein in FSH-stimulated GCs extends beyond an increase in mammalian target of rapamycin-stimulated translation. FSH increases phosphorylation of the AKT target mouse double-minute 2 (MDM2); a phosphomimetic mutation of MDM2 is sufficient to induce HIF-1 activity. The PI3-kinase/AKT target forkhead box-containing protein O subfamily 1 (FOXO1) also effects the accumulation of HIF-1alpha as evidenced by the ability of a constitutively active FOXO1 mutant to inhibit the induction by FSH of HIF-1alpha protein and HIF-1 activity. Activation of the PI3-kinase/AKT pathway in GCs by IGF-I is sufficient to induce HIF-1alpha protein but surprisingly not HIF-1 activity. HIF-1 activity also appears to require a PD98059-sensitive protein (kinase) activity stimulated by FSH that is both distinct from mitogen-activated ERK kinase1/2 or 5 and independent of the PI3-kinase/AKT pathway. These results indicate that FSH-stimulated HIF-1 activation leading to up-regulation of targets such as vascular endothelial growth factor requires not only PI3-kinase/AKT-mediated activation of mammalian target of rapamycin as well as phosphorylation of FOXO1 and possibly MDM2 but also a protein (kinase) activity that is inhibited by the classic ERK kinase inhibitor PD98059 but not ERK1/2 or 5. Thus, regulation of HIF-1 activity in GCs by FSH under normoxic conditions is complex and requires input from multiple signaling pathways.

Follicle-stimulating hormone/cAMP regulation of aromatase gene expression requires beta-catenin.

Estrogens profoundly influence the physiology and pathology of reproductive and other tissues. Consequently, emphasis has been placed on delineating the mechanisms underlying regulation of estrogen levels. Circulating levels of estradiol in women are controlled by follicle-stimulating hormone (FSH), which regulates transcription of the aromatase gene (CYP19A1) in ovarian granulosa cells. Previous studies have focused on two downstream effectors of the FSH signal, cAMP and the orphan nuclear receptor steroidogenic factor-1 (NR5A1). In this report, we present evidence for beta-catenin (CTNNB1) as an essential transcriptional regulator of CYP19A1. FSH induction of select steroidogenic enzyme mRNAs, including Cyp19a1, is enhanced by beta-catenin. Additionally, beta-catenin is present in transcription complexes assembled on the endogenous gonad-specific CYP19A1 promoter, as evidenced by chromatin immunoprecipitation assays. Transient expression and RNAi studies demonstrate that FSH- and cAMP-dependent regulation of this promoter is sensitive to alterations in the level of beta-catenin. The stimulatory effect of beta-catenin is mediated through functional interactions with steroidogenic factor-1 that involve four acidic residues within its ligand-binding domain, mutation of which attenuates FSH/cAMP-induced Cyp19a1 mRNA accumulation. Together, these data demonstrate that beta-catenin is essential for FSH/cAMP-regulated gene expression in the ovary, identifying a central and previously unappreciated role for beta-catenin in estrogen biosynthesis, and a potential broader role in other aspects of follicular maturation.