Targeted deletion of NR2F2 and VCAM1 in theca cells impacts ovarian follicular development: insights into polycystic ovary syndrome?†.

Defining features of polycystic ovary syndrome (PCOS) include elevated expression of steroidogenic genes, theca cell androgen biosynthesis, and peripheral levels of androgens. In previous studies, we identified vascular cell adhesion molecule 1 (VCAM1) as a selective androgen target gene in specific NR2F2/SF1 (+/+) theca cells. By deleting NR2F2 and VCAM1 selectively in CYP17A1 theca cells in mice, we documented that NR2F2 and VCAM1 impact distinct and sometimes opposing theca cell functions that alter ovarian follicular development in vivo: including major changes in ovarian morphology, steroidogenesis, gene expression profiles, immunolocalization images (NR5A1, CYP11A1, NOTCH1, CYP17A1, INSL3, VCAM1, NR2F2) as well as granulosa cell functions. We propose that theca cells impact follicle integrity by regulating androgen production and action, as well as granulosa cell differentiation/luteinization in response to androgens and gonadotropins that may underlie PCOS.

Polyploid giant cancer cells and ovarian cancer: new insights into mitotic regulators and polyploidy†.

Current first-line treatment of patients with high-grade serous ovarian cancer (HGSOC) involves the use of cytotoxic drugs that frequently lead to recurrent tumors exhibiting increased resistance to the drugs and poor patient survival. Strong evidence is accumulating to show that HGSOC tumors and cell lines contain a subset of cells called polyploidy giant cancer cells (PGCCs) that act as stem-like, self-renewing cells. These PGCCs appear to play a key role in tumor progression by generating drug-resistant progeny produced, in part, as a consequence of utilizing a modified form of mitosis known as endoreplication. Thus, developing drugs to target PGCCs and endoreplication may be an important approach for reducing the appearance of drug-resistant progeny. In the review, we discuss newly identified regulatory factors that impact mitosis and which may be altered or repurposed during endoreplication in PGCCs. We also review recent papers showing that a single PGCC can give rise to tumors in vivo and spheroids in culture. To illustrate some of the specific features of PGCCs and factors that may impact their function and endoreplication compared to mitosis, we have included immunofluorescent images co-localizing p53 and specific mitotic regulatory, phosphoproteins in xenografts derived from commonly used HGSOC cell lines.

Differences in gynecologic tumor development in Amhr2-Cre mice with KRASG12D or KRASG12V mutations.

How different KRAS variants impact tumor initiation and progression in vivo has not been thoroughly examined. We hypothesize that the ability of either KRASG12D or KRASG12V mutations to initiate tumor formation is context dependent. Amhr2-Cre mice express Cre recombinase in tissues that develop into the fallopian tubes, uterus, and ovaries. We used these mice to conditionally express either the KRASG12V/+ or KRASG12D/+ mutation. Mice with the genotype Amhr2-Cre Pten(fl/fl) KrasG12D/+(G12D mice) had abnormal follicle structures and developed low-grade serous ovarian carcinomas with 100% penetrance within 18 weeks. In contrast, mice with the genotype Amhr2-Cre Pten(fl/fl) KrasG12V/+ (G12V mice) had normal follicle structures, and about 90% of them developed uterine tumors with diverse histological features resembling those of leiomyoma and leiomyosarcoma. Granulosa cell tumors also developed in G12V mice. Differences in cell-signaling pathways in the uterine tissues of G12D and G12V mice were identified using RNA sequencing and reverse-phase protein array analyses. We found that CTNNB1, IL1A, IL1B, TNF, TGFB1, APP, and IL6 had the higher activity in G12V mice than in G12D mice. These mouse models will be useful for studying the differences in signaling pathways driven by KrasG12V/+ or KrasG12D/+ mutations to aid development of targeted therapies for specific KRAS mutant variants. Our leiomyoma model driven by the KrasG12V/+ mutation will also be useful in deciphering the malignant progression from leiomyoma to leiomyosarcoma.

S100a4-Cre-mediated deletion of Patched1 causes hypogonadotropic hypogonadism: role of pituitary hematopoietic cells in endocrine regulation.

Hormones produced by the anterior pituitary gland regulate an array of important physiological functions, but pituitary hormone disorders are not fully understood. Herein we report that genetically-engineered mice with deletion of the hedgehog signaling receptor Patched1 by S100a4 promoter-driven Cre recombinase (S100a4-Cre;Ptch1fl/fl mutants) exhibit adult-onset hypogonadotropic hypogonadism and multiple pituitary hormone disorders. During the transition from puberty to adult, S100a4-Cre;Ptch1fl/fl mice of both sexes develop hypogonadism coupled with reduced gonadotropin levels. Their pituitary glands also display severe structural and functional abnormalities, as revealed by transmission electron microscopy and expression of key genes regulating pituitary endocrine functions. S100a4-Cre activity in the anterior pituitary gland is restricted to CD45+ cells of hematopoietic origin, including folliculo-stellate cells and other immune cell types, causing sex-specific changes in the expression of genes regulating the local microenvironment of the anterior pituitary. These findings provide in vivo evidence for the importance of pituitary hematopoietic cells in regulating fertility and endocrine function, in particular during sexual maturation and likely through sexually dimorphic mechanisms. These findings support a previously unrecognized role of hematopoietic cells in causing hypogonadotropic hypogonadism and provide inroads into the molecular and cellular basis for pituitary hormone disorders in humans.

VCAM1 Is Induced in Ovarian Theca and Stromal Cells in a Mouse Model of Androgen Excess.

Ovarian theca androgen production is regulated by the pituitary LH and intrafollicular factors. Enhanced androgen biosynthesis by theca cells contributes to polycystic ovary syndrome (PCOS) in women, but the ovarian consequences of elevated androgens are not completely understood. Our study documents the molecular events that are altered in the theca and stromal cells of mice exposed to high androgen levels, using the nonaromatizable androgen DHT. Changes in ovarian morphology and function were observed not only in follicles, but also in the stromal compartment. Genome-wide microarray analyses revealed marked changes in the ovarian transcriptome of DHT-treated females within 1 week. Particularly striking was the increased expression of vascular cell adhesion molecule 1 (Vcam1) specifically in the NR2F2/COUPTF-II lineage theca cells, not granulosa cells, of growing follicles and throughout the stroma of the androgen-treated mice. This response was mediated by androgen receptors (ARs) present in theca and stromal cells. Human theca-derived cultures expressed both ARs and NR2F2 that were nuclear. VCAM1 mRNA and protein were higher in PCOS-derived theca cells compared with control theca and reduced markedly by the AR antagonist flutamide. In the DHT-treated mice, VCAM1 was transiently induced by equine chorionic gonadotropin, when androgen and estrogen biosynthesis peak in preovulatory follicles, and was potently suppressed by a superovulatory dose of human chorionic gonadotropin. High levels of VCAM1 in the theca and interstitial cells of DHT-treated mice and in adult Leydig cells indicate that there may be novel functions for VCAM1 in reproductive tissues, including the gonads.

WOMEN IN REPRODUCTIVE SCIENCE: Discovering science and the ovary: a career of joy.

My career has been about discovering science and learning the joys of the discovery process itself. It has been a challenging but rewarding process filled with many exciting moments and wonderful colleagues and students. Although I went to college to become a French major, I ultimately stumbled into research while pursuing a Masters Degree in teaching. Thus, my research career began in graduate school where I was studying NAD kinase in the ovary as a possible regulator of steroidogenesis, a big issue in the late 1960s. After a short excursion of teaching in North Dakota, I became a postdoctoral fellow at the University of Michigan, where radio-immuno assays and radio receptor assays had just come on the scene and were transforming endocrinology from laborious bioassays to quantitative science and of course these assays related to the ovary. From there I went to Baylor College of Medicine, a mecca of molecular biology, cloning genes and generating mouse models. It has been a fascinating and joyous journey.

Zinc Oxide nanoparticles induce oxidative and proteotoxic stress in ovarian cancer cells and trigger apoptosis Independent of p53-mutation status.

Ovarian cancer continues to be the most lethal among gynecological malignancies and the major cause for cancer-associated mortality among women. Limitations of current ovarian cancer therapeutics is highlighted by the high frequency of drug-resistant recurrent tumors and the extremely poor 5-year survival rates. Zinc oxide nanoparticles (ZnO-NPs) have shown promise in various biomedical applications including utility as anti-cancer agents. Here, we describe the synthesis and characterization of physical properties of ZnO-NPs of increasing particle size (15 nm - 55 nm) and evaluate their benefits as an ovarian cancer therapeutic using established human ovarian cancer cell lines. Our results demonstrate that the ZnO-NPs induce acute oxidative and proteotoxic stress in ovarian cancer cells leading to their death via apoptosis. The cytotoxic effect of the ZnO-NPs was found to increase slightly with a decrease in nanoparticle size. While ZnO-NPs caused depletion of both wild-type and gain-of-function (GOF) mutant p53 protein in ovarian cancer cells, their ability to induce apoptosis was found to be independent of the p53-mutation status in these cells. Taken together, these results highlight the potential of ZnO-NPs to serve as an anti-cancer therapeutic agent for treating ovarian cancers independent of the p53 mutants of the cancer cells.

Endocrine, Paracrine, and Autocrine Signaling Pathways That Regulate Ovulation.

The central role of luteinizing hormone (LH) and its receptor (LHCGR) in triggering ovulation has been recognized for decades. Because the LHCGR is present in the mural (outermost) granulosa cell layer of preovulatory follicles (POFs), the LH-initiated signal has to be transmitted to another somatic cell type (cumulus granulosa cells) and the oocyte to release a fertilizable oocyte. Recent studies have shown that activation of the LHCGR initiates vectorial transfer of information among the two somatic cell types and the oocyte and the molecules and signaling pathways involved are now better understood. This review summarizes the newer developments on the complex signaling pathways that regulate ovulation.

USP15-dependent lysosomal pathway controls p53-R175H turnover in ovarian cancer cells.

Gain-of-function p53 mutants such as p53-R175H form stable aggregates that accumulate in cells and play important roles in cancer progression. Selective degradation of gain-of-function p53 mutants has emerged as a highly attractive therapeutic strategy to target cancer cells harboring specific p53 mutations. We identified a small molecule called MCB-613 to cause rapid ubiquitination, nuclear export, and degradation of p53-R175H through a lysosome-mediated pathway, leading to catastrophic cancer cell death. In contrast to its effect on the p53-R175H mutant, MCB-613 causes slight stabilization of p53-WT and has weaker effects on other p53 gain-of-function mutants. Using state-of-the-art genetic and chemical approaches, we identified the deubiquitinase USP15 as the mediator of MCB-613's effect on p53-R175H, and established USP15 as a selective upstream regulator of p53-R175H in ovarian cancer cells. These results confirm that distinct pathways regulate the turnover of p53-WT and the different p53 mutants and open new opportunities to selectively target them.

The Cell Type-Specific Expression of Lhcgr in Mouse Ovarian Cells: Evidence for a DNA-Demethylation-Dependent Mechanism.

The luteinizing hormone receptor (LHCGR) is expressed at low levels in mural granulosa cells and cumulus cells of antral follicles and is induced dramatically in granulosa cells but not in cumulus cells by follicle-stimulating hormone (FSH). Therefore, we hypothesized that FSH not only activates transcription factors controlling Lhcgr expression but also alters other events to permit and enhance Lhcgr expression in granulosa cells but not in cumulus cells. In granulosa cells, the level of DNA methylation in the Lhcgr promoter region was significantly decreased by equine chorionic gonadotropin (eCG) in vivo. However, in cumulus cells, hypermethylation of the Lhcgr promoter remained after eCG stimulation. eCG induced estrogen production from testosterone (T) and retinoic acid (RA) synthesis in granulosa cells. When either T or RA in the presence or absence of FSH was added to granulosa cell cultures, the combined treatment with FSH and RA induced demethylation of Lhcgr-promoter region and Lhcgr expression. FSH-dependent RA synthesis was negatively regulated by coculture of granulosa cells with denuded oocytes, suggesting that oocyte-secreted factors downregulate RA production in cumulus cells where Lhcgr expression was not induced. Strikingly, treatment of cultured cumulus-oocyte complexes with a SMAD inhibitor, SB431542, significantly induced RA production, demethylation of Lhcgr-promoter region, and Lhcgr expression in cumulus cells. These results indicate the demethylation of the Lhcgr-promoter region is mediated, at least in part, by RA synthesis and is a key mechanism regulating the cell type-specific differentiation during follicular development.

From Follicular Development and Ovulation to Ovarian Cancers: An Unexpected Journey.

Follicular development and ovulation are complex development processes that are regulated by multiple, interacting pathways and cell types. The oocyte, cumulus cells, granulosa cells, and theca cells communicate to impact follicular development and ovulation. Many hormones and cytokines control intracellular regulatory networks and transcription factors, some of which are cell type specific. Molecular biology approaches and mutant mouse models have contributed immensely to our knowledge of what genes and signaling cascades impact each stage of follicular development and ovulation, and how the alteration of gene expression profiles and the activation of specific signaling pathways can impact ovarian cancer development in ovarian surface epithelial cells as well as granulosa cells. This chapter explores how pathways controlling normal follicle development and ovulation can be diverted to abnormal development.

The Ovarian Cycle.

The "ovarian cycle" is an exquisite and dynamic endocrine system that includes ovarian events, hypothalamic-pituitary interactions, uterine endometrial and myometrial changes during implantation and pregnancy, cervical alterations in structure, and breast development. The ovarian cycle and the steroid hormones produced by the ovary also impact epithelial cancer development in the ovary, uterus, cervix, and breast. This chapter provides a personal view of recent developments that occur in this complex endocrine environment.

WNT-Mediated Regulation of FOXO1 Constitutes a Critical Axis Maintaining Pubertal Mammary Stem Cell Homeostasis.

Puberty is characterized by dynamic tissue remodeling in the mammary gland involving ductal elongation, resolution into the mature epithelial bilayer, and lumen formation. To decipher the cellular mechanisms underlying these processes, we studied the fate of putative stem cells, termed cap cells, present in terminal end buds of pubertal mice. Employing a p63CreERT2-based lineage-tracing strategy, we identified a unipotent fate for proliferative cap cells that only generated cells with basal features. Furthermore, we observed that dislocated "cap-in-body" cells underwent apoptosis, which aided lumen formation during ductal development. Basal lineage-specific profiling and genetic loss-of-function experiments revealed a critical role for FOXO transcription factors in mediating these proliferative versus apoptotic fates. Importantly, these studies revealed a mode of WNT signaling-mediated FOXO1 inhibition, potentially mediated through AKT. Together, these data suggest that the WNT pathway confers proliferative and survival advantages on cap cells via regulation of FOXO1 localization.

The acceleration of reproductive aging in Nrg1flox/flox ;Cyp19-Cre female mice.

Irregular menstrual cycles, reduced responses to exogenous hormonal treatments, and altered endocrine profiles (high FSH/high LH/low AMH) are observed in women with increasing age before menopause. In this study, because the granulosa cell-specific Nrg1 knockout mice (gcNrg1KO) presented ovarian and endocrine phenotypes similar to older women, we sought to understand the mechanisms of ovarian aging and to develop a new strategy for improving fertility in older women prior to menopause. In the ovary of 6-month-old gcNrg1KO mice, follicular development was blocked in bilayer secondary follicles and heterogeneous cells accumulated in ovarian stroma. The heterogeneous cells in ovarian stroma were distinguished as two different types: (i) the LH receptor-positive endocrine cells and (ii) actin-rich fibrotic cells expressing collagen. Both the endocrine and fibrotic cells disappeared following long-term treatment with a GnRH antagonist, indicating that the high levels of serum LH induced the survival of both cell types and the abnormal endocrine profile to reduce fertility. Moreover, follicular development to the antral stages was observed with reduced LH and the disappearance of the abnormal stromal cells. Mice treated with the GnRH antagonist regained normal, recurrent estrous cycles and continuously delivered pups for at least for 3 months. We conclude that endocrine and matrix alternations occur within the ovarian stroma with increasing age and that abolishing these alternations resets the cyclical release of LH. Thus, GnRH antagonist treatments might provide a new, noninvasive strategy for improving fertility in a subset of aging women before menopause.

Enhanced Inflammatory Transcriptome in the Granulosa Cells of Women With Polycystic Ovarian Syndrome.

CONTEXT: Polycystic ovarian syndrome (PCOS), the most common endocrine disorder of reproductive-aged women, is associated with systemic low-grade inflammation. OBJECTIVE: We propose that increased or altered intrafollicular inflammatory reactions also occur in periovulatory follicles of PCOS patients. DESIGN: Gene profiling and quantitative PCR (qPCR) analyses in granulosa-lutein cells (GCs) collected from PCOS and non-PCOS women undergoing in vitro fertilization were compared with serum and follicular fluid (FF) levels of cytokines and chemokines. SETTING: This was a university-based study. PATIENTS: Twenty-one PCOS and 45 control patients were recruited: demographic, hormone, body mass index, and pregnancy outcomes were abstracted from patient data files. INTERVENTIONS: GC cytokine/chemokine mRNAs were identified and analyzed by gene-chip microarrays/qPCR before and after culture with human chorionic gonadotropin, DHT, IL-6, or IL-8; serum/FF cytokine levels were also analyzed. MAIN OUTCOME MEASURES: Relative serum/FF cytokine levels and GC cytokine expression before and after culture were compared and related to body mass index. RESULTS: The following results were found: 1) PCOS GCs express elevated transcripts encoding cytokines, chemokines, and immune cell markers, 2) based on gene profiling and qPCR analyses, obese PCOS patients define a distinct PCOS disease subtype with the most dramatic increases in proinflammatory and immune-related factors, and 3) human chorionic gonadotropin and DHT increased cytokine production in cultured GCs, whereas cytokines augmented cytokine and vascular genes, indicating that hyperandrogenism/elevated LH and obesity in PCOS women augment intrafollicular cytokine production. CONCLUSIONS: Intrafollicular androgens and cytokines likely comprise a local regulatory loop that impacts GC expression of cytokines and chemokines and the presence of immune cells; this loop is further enhanced in the obese PCOS subtype.

Mutant p53 Promotes Epithelial Ovarian Cancer by Regulating Tumor Differentiation, Metastasis, and Responsiveness to Steroid Hormones.

Mutations in the tumor protein p53 (TP53) are the most frequently occurring genetic events in high-grade ovarian cancers, especially the prevalence of the Trp53(R172H)-mutant allele. In this study, we investigated the impact of the Trp53(R172H)-mutant allele on epithelial ovarian cancer (EOC) in vivo We used the Pten/Kras(G12D)-mutant mouse strain that develops serous EOC with 100% penetrance to introduce the mutant Trp53(R172H) allele (homolog for human Trp53(R172H)). We demonstrate that the Trp53(R172H) mutation promoted EOC but had differential effects on disease features and progression depending on the presence or absence of the wild-type (WT) TP53 allele. Heterozygous WT/Trp53(R172H) alleles facilitated invasion into the ovarian stroma, accelerated intraperitoneal metastasis, and reduced TP53 transactivation activity but retained responsiveness to nutlin-3a, an activator of WT TP53. Moreover, high levels of estrogen receptor α in these tumors enhanced the growth of both primary and metastatic tumors in response to estradiol. Ovarian tumors homozygous for Trp53(R172H) mutation were undifferentiated and highly metastatic, exhibited minimal TP53 transactivation activity, and expressed genes with potential regulatory functions in EOC development. Notably, heterozygous WT/Trp53(R172H) mice also presented mucinous cystadenocarcinomas at 12 weeks of age, recapitulating human mucinous ovarian tumors, which also exhibit heterozygous TP53 mutations (∼50%-60%) and KRAS mutations. Therefore, we present the first mouse model of mucinous tumor formation from ovarian cells and supporting evidence that mutant TP53 is a key regulator of EOC progression, differentiation, and responsiveness to steroid hormones. Cancer Res; 76(8); 2206-18. ©2016 AACR.

De Novo-Synthesized Retinoic Acid in Ovarian Antral Follicles Enhances FSH-Mediated Ovarian Follicular Cell Differentiation and Female Fertility.

Retinoic acid (RA) is the active form of vitamin A and is synthesized from retinol by two key enzymes, alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH). As the physiological precursor of RA, retinol impacts female reproductive functions and fertility. The expression of Adh1 and Adh5 as well as Aldh1a1 and Aldh1a7 are significantly increased in the ovaries of mice treated with equine chorionic gonadotropin/FSH. The RA receptor is expressed and localized in granulosa cells and is activated by endogenous RA as indicated by LacZ expression in granulosa cells of RA-responsive transgene-LacZ transgenic mice (RA reporter mice). Coinjection of the ADH inhibitor, 4-methylpyrazole, with equine chorionic gonadotropin significantly decreases the number and developmental competence of oocytes ovulated in response to human chorionic gonadotropin/LH as compared with controls. Injections of RA completely reverse the effects of the inhibitor of ovulation and oocyte development. When mice were fed a retinol-free, vitamin A-deficient diet that significantly reduced the serum levels of retinol, the expression of the LH receptor (Lhcgr) was significantly lower in the ovaries of the vitamin A-deficient mice, and injections of human chorionic gonadotropin failed to induce genes controlling ovulation. These results indicate that ovarian de novo biosynthesis of RA is required for the follicular expression of Lhcgr in granulosa cells and their ability to respond to the ovulatory LH surge.

Growth Arrest Specific-1 (GAS1) Is a C/EBP Target Gene That Functions in Ovulation and Corpus Luteum Formation in Mice.

Ovulation and luteinization are initiated in preovulatory follicles by the luteinizing hormone (LH) surge; however, the signaling events that mediate LH actions in these follicles remain incompletely defined. Two key transcription factors that are targets of LH surge are C/EBPalpha and C/EBPbeta, and their depletion in granulosa cells results in complete infertility. Microarray analyses of these mutant mice revealed altered expression of a number of genes, including growth arrest specific-1 (Gas1). To investigate functions of Gas1 in ovulation- and luteinization-related processes, we crossed Cyp19a1-Cre and Gas1(flox/flox) mice to conditionally delete Gas1 in granulosa and cumulus cells. While expression of Gas1 is dramatically increased in granulosa and cumulus cells around 12-16 h post-human chorionic gonadotropin (hCG) stimulation in wild-type mice, this increase is abolished in Cebpa/b double mutant and in Gas1 mutant mice. GAS1 is also dynamically expressed in stromal cells of the ovary independent of C/EBPalpha/beta. Female Gas1 mutant mice are fertile, exhibit enhanced rates of ovulation, increased fertility, and higher levels of Areg and Lhcgr mRNA in granulosa cells. The morphological appearance and vascularization of corpora lutea appeared normal in these mutant females. Interestingly, levels of mRNA for a number of genes (Cyp11a1, Star, Wnt4, Prlr, Cd52, and Sema3a) associated with luteinization are decreased in corpora lutea of Gas1 mutant mice as compared with controls at 24 h post-hCG; these differences were no longer detectable by 48 h post-hCG. The C/EBP target Gas1 is induced in granulosa cells and is associated with ovulation and luteinization.

Specific TP53 Mutants Overrepresented in Ovarian Cancer Impact CNV, TP53 Activity, Responses to Nutlin-3a, and Cell Survival.

Evolutionary Action analyses of The Cancer Gene Atlas data sets show that many specific p53 missense and gain-of-function mutations are selectively overrepresented and functional in high-grade serous ovarian cancer (HGSC). As homozygous alleles, p53 mutants are differentially associated with specific loss of heterozygosity (R273; chromosome 17); copy number variation (R175H; chromosome 9); and up-stream, cancer-related regulatory pathways. The expression of immune-related cytokines was selectively related to p53 status, showing for the first time that specific p53 mutants impact, and are related to, the immune subtype of ovarian cancer. Although the majority (31%) of HGSCs exhibit loss of heterozygosity, a significant number (24%) maintain a wild-type (WT) allele and represent another HGSC subtype that is not well defined. Using human and mouse cell lines, we show that specific p53 mutants differentially alter endogenous WT p53 activity; target gene expression; and responses to nutlin-3a, a small molecular that activates WT p53 leading to apoptosis, providing "proof of principle" that ovarian cancer cells expressing WT and mutant alleles represent a distinct ovarian cancer subtype. We also show that siRNA knock down of endogenous p53 in cells expressing homozygous mutant alleles causes apoptosis, whereas cells expressing WT p53 (or are heterozygous for WT and mutant p53 alleles) are highly resistant. Therefore, despite different gene regulatory pathways associated with specific p53 mutants, silencing mutant p53 might be a suitable, powerful, global strategy for blocking ovarian cancer growth in those tumors that rely on mutant p53 functions for survival. Knowing p53 mutational status in HGSC should permit new strategies tailored to control this disease.