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.

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.

Creatine enhances the duration of sperm capacitation: a novel factor for improving in vitro fertilization with small numbers of sperm.

STUDY QUESTION: Why are many sperm required for successful fertilization of oocytes in vitro, even though fertilization occurs in vivo when only a few sperm reach the oocyte? SUMMARY ANSWER: Creatine produced in the ovary promotes efficient fertilization in vivo; however, in vitro, creatine is not contained in the in vitro fertilization (IVF) medium. WHAT IS KNOWN ALREADY: The IVF medium enables capacitation of sperm. However, the IVF medium does not fully mimic the in vivo environment during fertilization. Consequently, fertilization in vitro is more inefficient than in the oviduct. STUDY DESIGN, SIZE, DURATION: Follicular and oviductal fluids were collected and then analyzed for creatine and glucose levels. To determine the physiological functions of creatine, the creatine antagonist 3-guanidinopropionic acid (GPA) was injected into hormonally primed mice. Using conventional IVF protocols, sperm were pre-incubated in IVF medium with creatine and then co-cultured with 10 ovulated cumulus-oocyte complexes (1-1000 per oocyte) in 50 µl medium droplets. PARTICIPANTS/MATERIALS, SETTING, METHODS: Glucose and creatine levels were measured using commercial enzymatic assay kits. The effect of creatine in vivo was assessed by mating experiments using mice treated with or without GPA just before ovulation. To assess the functions of sperm incubated in IVF medium containing creatine, we analyzed (1) the motility of sperm using computer-assisted sperm assay, (2) the capacitation level of sperm by western blot analyses, and (3) the condition of sperm acrosomes by peanut agglutinin lectin-FITC staining. MAIN RESULTS AND THE ROLE OF CHANCE: Oviductal creatine levels were significantly increased following ovulation. Injecting mice with GPA just before ovulation significantly reduced the number of fertilized oocytes. The addition of creatine to IVF medium enhanced sperm capacitation by increasing ATP levels. Successful fertilization was achieved with as few as five sperm/oocyte in the creatine group, and the number of fertilized oocytes was significantly higher than in the control without creatine (P < 0.01). LIMITATIONS, REASONS FOR CAUTION: In the present study, a pharmacological approach, creatine antagonist (GPA) treatment, but not a knockout mouse model, was used to understand the role of creatine in vivo. The role of creatine in fertilization processes can only be shown in a mouse model. WIDER IMPLICATIONS OF THE FINDINGS: A modified IVF technique using creatine-containing medium was developed and shown to markedly improve fertilization with small numbers of sperm. This approach has the potential to be highly beneficial for human assisted reproductive technologies, especially for patients with a limited number of good quality sperm. STUDY FUNDING/COMPETING INTEREST(S): This work was supported in part by JSPS KAKENHI Grant numbers JP24688028, JP16H05017 (to M.S.), and JP15J05331 (to T.U.), the Japan Agency for Medical Research and Development (AMED) (16gk0110015h0001 to M.S.), and National Institutes of Health (NIH-HD-076980 to J.S.R). The authors have nothing to disclose.

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.

CBP-CITED4 is required for luteinizing hormone-triggered target gene expression during ovulation.

Pituitary-secreted luteinizing hormone (LH) induces ovulation by activating an extracellular regulated kinase 1/2 (ERK1/2) cascade. However, little is known regarding the ERK1/2 downstream effectors that are involved in regulating rapid, transient expression of LH-target gene in ovulatory follicles. By comparing the gene expression profiles of LH-stimulated wild type with ERK1/2-deleted ovarian granulosa cells (GCs), we identified Cited4 as a previously unknown LH target gene during ovulation. LH induced Cited4 expression in pre-ovulatory follicles in an ERK1/2-dependent manner. CITED4 formed an endogenous protein complex and docked on the promoters of LH and ERK1/2 target genes. Both CITED4 expression and CBP acetyltransferase activity leading to histone acetylation were indispensable for LH-induced ovulation-related events. LH induced dynamic histone acetylation changes in pre-ovulatory GCs, including the acetylation of histone H2B (Lys5) and H3 (Lys9). This was essential for the rapid responses and dramatic increases of LH target gene expressions by the ordered activation of ERK1/2 and CITED4-CBP. In addition, histone deacetylases (HDACs) antagonized CITED4-CBP to turn off expression of these genes after exposure to LH. Thus, we determined that CITED4 was a novel LH and ERK1/2 target for triggering ovulation. These results support the proposition that LH induces rapid, significant gene expression in pre-ovulatory follicles by modulating histone acetylation status.

FZD1 regulates cumulus expansion genes and is required for normal female fertility in mice.

WNT4 is required for normal ovarian follicle development and female fertility in mice, but how its signal is transduced remains unknown. Fzd1 encodes a WNT receptor whose expression is markedly induced in both mural granulosa cells and cumulus cells during the preovulatory period, in a manner similar to Wnt4. To study the physiological roles of FZD1 in ovarian physiology and to determine whether it serves as receptor for WNT4, Fzd1-null mice were created by gene targeting. Whereas rare Fzd1(-/-) females were sterile because of uterine fibrosis and ovarian tubulostromal hyperplasia, most were subfertile, producing ≈1 fewer pup per litter on average relative to controls. Unlike WNT4-deficient mice, ovaries from Fzd1(-/-) mice had normal weights, numbers of follicles, steroid hormone production, and WNT4 target gene expression levels. Microarray analyses of granulosa cells from periovulatory follicles revealed few genes whose expression was altered in Fzd1(-/-) mice. However, gene expression analyses of cumulus-oocyte complexes (COCs) revealed a blunted response of both oocyte (Zp3, Dppa3, Nlrp5, and Bmp15) and cumulus (Btc, Ptgs2, Sema3a, Ptx3, Il6, Nts, Alcam, and Cspg2) genes to the ovulatory signal, whereas the expression of these genes was not altered in WNT4-deficient COCs from Wnt4(tm1.1Boer/tm1.1Boer);Tg (CYP19A1-cre)1Jri mice. Despite altered gene expression, cumulus expansion appeared normal in Fzd1(-/-) COCs both in vitro and in vivo. Together, these results indicate that Fzd1 is required for normal female fertility and may act in part to regulate oocyte maturation and cumulus cell function, but it is unlikely to function as the sole ovarian WNT4 receptor.

Partially redundant functions of Adamts1 and Adamts4 in the perinatal development of the renal medulla.

Adamts4 encodes a widely-expressed proteinase thought to be involved in processes ranging from cartilage metabolism to ovarian follicle development. To study its physiological roles, Adamts4-null mice were created by gene targeting. Unexpectedly, these were found to be phenotypically normal, suggesting that other gene(s) may compensate for its loss. Adamts4(-/-) mice were, therefore, crossed with a strain lacking Adamts1, whose pattern of expression and substrate specificity overlap that of Adamts4. Most (>95%) Adamts1(-/-) ;Adamts4(-/-) mice died within 72 hr after birth with a marked thinning of the renal medulla. The renal defect was not observed in embryonic Adamts1(-/-) ;Adamts4(-/-) kidneys, but became apparent around birth. The few (<5%) Adamts1(-/-) ;Adamts4(-/-) animals to reach adulthood had the same renal phenotype seen in newborns. This study is thus the first to report Adamts4 expression and function in the mammalian kidney, and to demonstrate that Adamts1 and Adamts4 play redundant and essential roles in perinatal kidney development.

Toll-like receptors (TLR) 2 and 4 on human sperm recognize bacterial endotoxins and mediate apoptosis.

BACKGROUND: Bacterial infections of the genital tract are one of the most serious causes of infertility in males. In some human patients with poor semen quality, leukocytospermia has been observed. Because leukocytes express the bacterial-lipopolysaccharide (LPS) responsive Toll-like receptor (TLR) signaling cascade and secrete tumor necrosis factor-α, secreted cytokines comprise one, but probably not the only, class of factors that can impact sperm motility. METHODS AND RESULTS: In this study, we documented that bacterial endotoxins, LPS and peptidoglycan, can be detected in human semen. Furthermore, the addition of endotoxins in the absence of leukocytes directly and significantly reduced the motility and increased the apoptotic rate of both human and mouse sperm and suppressed fertilization by mouse sperm both in vivo and in vitro. The well-known LPS receptor, TLR4, and peptidoglycan receptor, TLR2, were expressed in human and mouse sperm. In Tlr2/4(-/-) double-mutant mice, the negative effects of endotoxins on sperm functions were blocked, suggesting that the bacterial endotoxins mediated activation of TLR-dependent pathways in sperm leading to apoptosis. CONCLUSIONS: Sperm can recognize bacterial endotoxins by TLRs present in their membranes. The activated TLRs reduce sperm motility, induce sperm apoptosis and significantly impair the potential for fertilization.

WNT4 is required for normal ovarian follicle development and female fertility.

To study the role of WNT4 in the postnatal ovary, a mouse strain bearing a floxed Wnt4 allele was created and mated to the Amhr2(tm3(cre)Bhr) strain to target deletion of Wnt4 to granulosa cells. Wnt4(flox/-);Amhr2(tm3(cre)Bhr/+) mice had reduced ovary weights and produced smaller litters (P<0.05). Serial follicle counting demonstrated that Wnt4(flox/-);Amhr2(tm3(cre)Bhr/+) mice were born with a normal ovarian reserve and maintained normal numbers of small follicles until puberty but had only 25.2% of the normal number of healthy antral follicles. Some Wnt4(flox/-);Amhr2(tm3(cre)Bhr/+) mice had no antral follicles or corpora lutea and underwent premature follicle depletion. RT-PCR analyses of Wnt4(flox/-);Amhr2(tm3(cre)Bhr/+) granulosa cells and cultured granulosa cells that overexpress WNT4 demonstrated that WNT4 regulates the expression of Star, Cyp11a1, and Cyp19, steroidogenic genes previously identified as downstream targets of the WNT signaling effector CTNNB1. Decreased serum progesterone levels were found in immature, gonadotropin-treated Wnt4(flox/-);Amhr2(tm3(cre)Bhr/+) mice (P<0.05). WNT4- and CTNNB1-overexpressing cultured granulosa cells were analyzed by microarray for alterations in gene expression, which showed that WNT4 regulates additional genes involved in late follicle development via the WNT/CTNNB1 signaling pathway. Together, these data indicate that WNT4 is required for normal antral follicle development and may act by regulating granulosa cell functions including steroidogenesis.

Large-scale DNA demethylation occurs in proliferating ovarian granulosa cells during mouse follicular development.

During ovarian follicular development, granulosa cells proliferate and progressively differentiate to support oocyte maturation and ovulation. To determine the underlying links between proliferation and differentiation in granulosa cells, we determined changes in 1) the expression of genes regulating DNA methylation and 2) DNA methylation patterns, histone acetylation levels and genomic DNA structure. In response to equine chorionic gonadotropin (eCG), granulosa cell proliferation increased, DNA methyltransferase (DNMT1) significantly decreased and Tet methylcytosine dioxygenase 2 (TET2) significantly increased in S-phase granulosa cells. Comprehensive MeDIP-seq analyses documented that eCG treatment decreased methylation of promoter regions in approximately 40% of the genes in granulosa cells. The expression of specific demethylated genes was significantly increased in association with specific histone modifications and changes in DNA structure. These epigenetic processes were suppressed by a cell cycle inhibitor. Based on these results, we propose that the timing of sequential epigenetic events is essential for progressive, stepwise changes in granulosa cell differentiation.

Decidual PTEN expression is required for trophoblast invasion in the mouse.

Trophoblast invasion likely depends on complex cross talk between the fetal and maternal tissues and may involve the modulation of phosphatidylinositol 3-kinase (PI3K)/AKT signaling activity in maternal decidual cells. In this report, we studied implantation in Pten(tm1Hwu/tm1Hwu);Amhr2(tm3(cre)Bhr/+) mice, which lack the PI3K signaling antagonist gene Pten in myometrial and stromal/decidual cells. Primiparous Pten(tm1Hwu/tm1Hwu);Amhr2(tm3(cre)Bhr/+) mice were found to be subfertile because of increased fetal mortality at e11.5. Histopathological analyses revealed a failure of decidual regression in these mice, accompanied by reduced or absent invasion of fetal trophoblast glycogen cells and giant cells, abnormal development of the placental labyrinth, and frequent apparent intrauterine fetal growth restriction. Unexpectedly, the loss of phosphate and tensin homolog deleted on chromosome 10 (PTEN) expression in Pten(tm1Hwu/tm1Hwu);Amhr2(tm3(cre)Bhr/+) decidual cells was not accompanied by a detectable increase in AKT phosphorylation or altered expression or activation of PI3K/AKT downstream effectors such as mammalian target of rapamycin or glycogen synthase kinase-3ß. Terminal deoxynucleotidyl transferase-mediated nick end labeling and bromodeoxyuridine incorporation analyses attributed to the lack of decidual regression mainly to decreased apoptosis in Pten(tm1Hwu/tm1Hwu);Amhr2(tm3(cre)Bhr/+) decidual cells, rather than to increased proliferation. Remodeling of the maternal vasculature was delayed in Pten(tm1Hwu/tm1Hwu);Amhr2(tm3(cre)Bhr/+) uteri at e11.5, as evidenced by persistence of vascular smooth muscle and decreased infiltration of uterine natural killer cells. In addition, thickening of the myometrium and disorganization of the muscle fibers were observed before and throughout gestation. Almost all Pten(tm1Hwu/tm1Hwu);Amhr2(tm3(cre)Bhr/+) mice failed to carry a second litter to term, apparently attributable to endometrial hyperplasia and uterine infections. Together, these data demonstrate novel roles of PTEN in the mammalian uterus and its requirement for proper trophoblast invasion and decidual regression.

Progesterone receptor-induced gene expression in primary mouse granulosa cell cultures.

The progesterone receptor (PGR) is induced by luteinizing hormone (LH) in granulosa cells of preovulatory follicles, and the PGR-A isoform is essential for ovulation based on the phenotypes of Pgr isoform-specific knockout mice. Although several genes regulated by PGR-A in vivo have been identified, whether these genes are primary targets of PGR-A or if their expression also depends on other signaling molecules that are induced by the LH surge has not been resolved. Therefore, to identify genes that are either induced or repressed by PGR in the absence of LH-mediated signaling cascades, we infected primary cultures of mouse granulosa cells with either PGR-A or PGR-B adenoviral vectors without or with R-5020 as a PGR ligand. Total RNA was extracted from infected cells at 16 h and analyzed by Affymetrix Mouse 430 2.0 microarrays. PGR-A in the presence or absence of ligand significantly induced approximately 50 genes 2-fold or more (local pooled error test at P

New insights into ovarian function.

Infertility adversely affects many couples worldwide. Conversely, the exponential increase in world population threatens our planet and its resources. Therefore, a greater understanding of the fundamental cellular and molecular events that control the size of the primordial follicle pool and follicular development is of utmost importance to develop improved in vitro fertilization as well as to design novel approaches to regulate fertility. In this review we attempt to highlight some new advances in basic research of the mammalian ovary that have occurred in recent years focusing primarily on mouse models that have contributed to our understanding of ovarian follicle formation, development, and ovulation. We hope that these new insights into ovarian function will trigger more research and translation to clinically relevant problems.

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.

Immune-like mechanisms in ovulation.

Ovulation is the unique biological process by which a mature oocyte (egg) and surrounding somatic cells, the cumulus cell-oocyte complex (COC), are released from the surface of the ovary into the oviduct for transport and fertilization. Ovulation is similar to an inflammatory response: the follicles become hyperemic, produce prostaglandins and synthesize a hyaluronan-rich extracellular matrix. However, this view of ovulation might be too restrictive and needs to be broadened to encompass the innate immune cell surveillance-response system. This hypothesis is being proposed because ovarian granulosa cells and cumulus cells express and respond to innate immune cell-related surveillance proteins (Toll-like receptors 2 and 4) and cytokines, such as interleukin 6 (IL-6), during ovulation.

Targeted disruption of Pten in ovarian granulosa cells enhances ovulation and extends the life span of luteal cells.

FSH activates the phosphatidylinositol-3 kinase (PI3K)/acute transforming retrovirus thymoma protein kinase pathway and thereby enhances granulosa cell differentiation in culture. To identify the physiological role of the PI3K pathway in vivo we disrupted the PI3K suppressor, Pten, in developing ovarian follicles. To selectively disrupt Pten expression in granulosa cells, Ptenfl/fl mice were mated with transgenic mice expressing cAMP response element recombinase driven by Cyp19 promoter (Cyp19-Cre). The resultant Pten mutant mice were fertile, ovulated more oocytes, and produced moderately more pups than control mice. These physiological differences in the Pten mutant mice were associated with hyperactivation of the PI3K/acute transforming retrovirus thymoma protein kinase pathway, decreased susceptibility to apoptosis, and increased proliferation of mutant granulosa cells. Strikingly, corpora lutea of the Pten mutant mice persisted longer than those of control mice. Although the follicular and luteal cell steroidogenesis in Ptenfl/fl;Cyp19-Cre mice was similar to controls, viable nonsteroidogenic luteal cells escaped structural luteolysis. These findings provide the novel evidence that Pten impacts the survival/life span of granulosa/luteal cells and that its loss not only results in the facilitated ovulation but also in the persistence of nonsteroidogenic luteal structures in the adult mouse ovary.

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.

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.