Contributions of white adipose tissue to energy requirements for female reproduction.

Body composition impacts female fertility and there are established relationships between adipose tissue and the reproductive system. Maintaining functional adipose tissue is vital for meeting the energetic demands during the reproductive process, from ovulation to delivery and lactation. White adipose tissue (WAT) shows plastic responses to daily physiology and secretes diverse adipokines that affect the hypothalamic-pituitary-ovarian axis, but many other interorgan interactions remain to be determined. This review summarizes the current state of research on the dialogue between WAT and the female reproductive system, focusing on the impact of this crosstalk on ovarian and endometrial factors essential for fecundity.

Global SUMOylation in mouse oocytes maintains oocyte identity and regulates chromatin remodeling and transcriptional silencing at the end of folliculogenesis.

Meiotically competent oocytes in mammals undergo cyclic development during folliculogenesis. Oocytes within ovarian follicles are transcriptionally active, producing and storing transcripts required for oocyte growth, somatic cell communication and early embryogenesis. Transcription ceases as oocytes transition from growth to maturation and does not resume until zygotic genome activation. Although SUMOylation, a post-translational modification, plays multifaceted roles in transcriptional regulation, its involvement during oocyte development remains poorly understood. In this study, we generated an oocyte-specific knockout of Ube2i, encoding the SUMO E2 enzyme UBE2I, using Zp3-cre+ to determine how loss of oocyte SUMOylation during folliculogenesis affects oocyte development. Ube2i Zp3-cre+ female knockout mice were sterile, with oocyte defects in meiotic competence, spindle architecture and chromosome alignment, and a premature arrest in metaphase I. Additionally, fully grown Ube2i Zp3-cre+ oocytes exhibited sustained transcriptional activity but downregulated maternal effect genes and prematurely activated genes and retrotransposons typically associated with zygotic genome activation. These findings demonstrate that UBE2I is required for the acquisition of key hallmarks of oocyte development during folliculogenesis, and highlight UBE2I as a previously unreported orchestrator of transcriptional regulation in mouse oocytes.

Generation of a novel Stra8-driven Cre recombinase strain for use in pre-meiotic germ cells in mice†.

The development of oocytes occurs over a broad time frame, starting at the earliest stages of embryogenesis and continuing into adulthood. Conditional knockout technologies such as the Cre/loxP recombination system are useful for analyzing oocyte development at specific stages, but not every time frame has appropriate Cre drivers, for instance, during oocyte meiotic initiation through early prophase I in the embryo. Here, we generated a novel knockin mouse line that produces a bicistronic transcript from the endogenous Stra8 locus that includes a "self-cleaving" 2A peptide upstream of cre. This allows for high efficiency cleavage and production of both proteins individually and results in expression of cre in both male and female gonads at the biologically relevant stage. Fluorescent reporter analysis confirms that this line recapitulates endogenous Stra8 expression in both sexes and does not affect fertility of heterozygous nor homozygous mice. This line, named Stra8P2Acre, adds to the repertoire of germ-cell specific cre driver lines and, importantly, allows for deletion of target genes during key embryonic oocyte developmental stages, including early events in meiosis. Summary Sentence Generation of a novel cre recombinase knockin to the Stra8 locus allows production of Stra8 and cre without affecting fertility.

Sumoylation regulates functional properties of the oocyte transcription factors SOHLH1 and NOBOX.

SOHLH1 and NOBOX are oocyte-expressed transcription factors with critical roles in ovary development and fertility. In mice, Sohlh1 and Nobox are essential for fertility through their regulation of the oocyte transcriptional network and cross-talk to somatic cells. Sumoylation is a posttranslational modification that regulates transcription factor function, and we previously showed that mouse oocytes deficient for sumoylation had an altered transcriptional landscape that included significant changes in NOBOX target genes. Here, we show that mouse SOHLH1 is modified by SUMO2/3 at lysine 345 and mutation of this residue alters SOHLH1 nuclear to cytoplasmic localization. In NOBOX, we identify a non-consensus SUMO site, K97, that eliminates NOBOX mono-SUMO2/3 conjugation, while a point mutation at K125 had no effect on NOBOX sumoylation. However, NOBOXK97R/K125R double mutants showed loss of mono-SUMO2/3 and altered higher molecular weight modifications, suggesting cooperation between these lysine's. NOBOXK97R and NOBOXK97R/K125R differentially regulated NOBOX promoter targets, with increased activity on the Gdf9 promoter, but no effect on the Pou5f1 promoter. These data implicate sumoylation as a novel regulatory mechanism for SOHLH1 and NOBOX, which may prove useful in refining their roles during oogenesis as well as their function during reprogramming to generate de novo germ cells.

Control of ovarian follicle development by TGFß family signaling.

The reproductive lifespan of female mammals is limited and ultimately depends on the production of a sufficient number of high quality oocytes from a pool of non-growing primordial follicles that are set aside during embryonic and perinatal development. Recent studies show multiple signaling pathways are responsible for maintaining primordial follicle arrest and regulation of activation. Identification of these pathways and their regulatory mechanisms is essential for developing novel treatments for female infertility, improving existing in vitro fertilization techniques, and more recently, restoring the function of cryopreserved ovarian tissue. This review focuses on recent developments in transforming growth factor beta (TGFß) family signaling in ovarian follicle development and its potential application to therapeutic design. Mouse models have been an essential tool for discovering genes critical for fertility, and recent advancements in human organ culture have additionally allowed for the translation of murine discoveries into human research and clinical settings.

Deletion of Gremlin-2 alters estrous cyclicity and disrupts female fertility in mice†.

Members of the differential screening-selected gene aberrative in neuroblastoma (DAN) protein family are developmentally conserved extracellular binding proteins that antagonize bone morphogenetic protein (BMP) signaling. This protein family includes the Gremlin proteins, GREM1 and GREM2, which have key functions during embryogenesis and adult physiology. While BMPs play essential roles in ovarian follicle development, the role of the DAN family in female reproductive physiology is less understood. We generated mice null for Grem2 to determine its role in female reproduction in addition to screening patients with primary ovarian insufficiency (POI) for variants in GREM2. Grem2-/- mice are viable, but female Grem2-/- mice have diminished fecundity and irregular estrous cycles. This is accompanied by significantly reduced production of ovarian anti-Müllerian hormone (AMH) from small growing follicles, leading to a significant decrease in serum AMH. Surprisingly, as AMH is a well-established marker of the ovarian reserve, morphometric analysis of ovarian follicles showed maintenance of primordial follicles in Grem2-/- mice like wild-type (WT) littermates. While Grem2 mRNA transcripts were not detected in the pituitary, Grem2 is expressed in hypothalami of WT female mice, suggesting the potential for dysfunction in multiple tissues composing the hypothalamic-pituitary-ovarian axis that contribute to the subfertility phenotype. Additionally, screening 106 women with POI identified one individual with a heterozygous variant in GREM2 that lies within the predicted BMP-GREM2 interface. In total, these data suggest that Grem2 is necessary for female fecundity by playing a novel role in regulating the HPO axis and contributing to female reproductive disease.

Ube2i deletion in adipocytes causes lipoatrophy in mice.

OBJECTIVE: White adipose tissue (WAT) expansion regulates energy balance and overall metabolic homeostasis. The absence or loss of WAT occurring through lipodystrophy and lipoatrophy contributes to the development of hepatic steatosis and insulin resistance. We previously demonstrated that sole small ubiquitin-like modifier (SUMO) E2-conjugating enzyme Ube2i represses human adipocyte differentiation. The role of Ube2i during WAT development remains unknown. METHODS: To determine how Ube2i impacts body composition and energy balance, we generated adipocyte-specific Ube2i knockout mice (Ube2ia-KO). CRISPR/Cas9 gene editing inserted loxP sites flanking exons 3 and 4 at the Ube2i locus. Subsequent genetic crosses to Adipoq-Cre transgenic mice allowed deletion of Ube2i in white and brown adipocytes. We measured multiple metabolic endpoints that describe energy balance and carbohydrate metabolism in Ube2ia-KO and littermate controls during postnatal growth. RESULTS: Surprisingly, Ube2ia-KO mice developed hyperinsulinemia and hepatic steatosis. Global energy balance defects emerged from dysfunctional WAT marked by pronounced local inflammation, loss of serum adipokines, hepatomegaly, and near absence of major adipose tissue depots. We observed progressive lipoatrophy that commences in the early adolescent period. CONCLUSIONS: Our results demonstrate that Ube2i expression in mature adipocytes allows WAT expansion during postnatal growth. Deletion of Ube2i in fat cells compromises and diminishes adipocyte function that induces WAT inflammation and ectopic lipid accumulation in the liver. Our findings reveal an indispensable role for Ube2i during white adipocyte expansion and endocrine control of energy balance.

Loss of the E2 SUMO-conjugating enzyme Ube2i in oocytes during ovarian folliculogenesis causes infertility in mice.

The number and quality of oocytes within the ovarian reserve largely determines fertility and reproductive lifespan in mammals. An oocyte-specific transcription factor cascade controls oocyte development, and some of these transcription factors, such as newborn ovary homeobox gene (NOBOX), are candidate genes for primary ovarian insufficiency in women. Transcription factors are frequently modified by the post-translational modification SUMOylation, but it is not known whether SUMOylation is required for function of the oocyte-specific transcription factors or if SUMOylation is required in oocytes during their development within the ovarian follicle. To test this, the sole E2 SUMO-conjugating enzyme, Ube2i, was ablated in mouse oocytes beginning in primordial follicles. Loss of oocyte Ube2i resulted in female infertility with major defects in stability of the primordial follicle pool, ovarian folliculogenesis, ovulation and meiosis. Transcriptomic profiling of ovaries suggests that loss of oocyte Ube2i caused defects in both oocyte- and granulosa cell-expressed genes, including NOBOX and some of its known target genes. Together, these studies show that SUMOylation is required in the mammalian oocyte during folliculogenesis for both oocyte development and communication with ovarian somatic cells.

RUNX3 Promotes the Tumorigenic Phenotype in KGN, a Human Granulosa Cell Tumor-Derived Cell Line.

Granulosa cell tumors of the ovary (GCT) are the predominant type of ovarian sex cord/stromal tumor. Although prognosis is generally favorable, the outcome for advanced and recurrent GCT is poor. A better understanding of the molecular pathogenesis of GCT is critical to developing effective therapeutic strategies. Here we have examined the potential role of the runt-related transcription factor RUNX3. There are only two GCT cell lines available. While RUNX3 is silenced in the GCT cell line KGN cells, it is highly expressed in another GCT cell line, COV434 cells. Re-expression of RUNX3 promotes proliferation, anchorage-independent growth, and motility in KGN cells in vitro and tumor formation in mice in vivo. Furthermore, expression of a dominant negative form of RUNX3 decreases proliferation of COV434 cells. To address a potential mechanism of action, we examined expression of cyclin D2 and the CDK inhibitor p27Kip1, two cell cycle regulators known to be critical determinants of GCT cell proliferation. We found that RUNX3 upregulates the expression of cyclin D2 at the mRNA and protein level, and decreases the level of the p27Kip1 protein, but not p27Kip1 mRNA. In conclusion, we demonstrate that RUNX proteins are expressed in GCT cell lines and human GCT specimens, albeit at variable levels, and RUNX3 may play an oncogenic role in a subset of GCTs.

The TGF-ß Family in the Reproductive Tract.

The transforming growth factor ß (TGF-ß) family has a profound impact on the reproductive function of various organisms. In this review, we discuss how highly conserved members of the TGF-ß family influence the reproductive function across several species. We briefly discuss how TGF-ß-related proteins balance germ-cell proliferation and differentiation as well as dauer entry and exit in Caenorhabditis elegans. In Drosophila melanogaster, TGF-ß-related proteins maintain germ stem-cell identity and eggshell patterning. We then provide an in-depth analysis of landmark studies performed using transgenic mouse models and discuss how these data have uncovered basic developmental aspects of male and female reproductive development. In particular, we discuss the roles of the various TGF-ß family ligands and receptors in primordial germ-cell development, sexual differentiation, and gonadal cell development. We also discuss how mutant mouse studies showed the contribution of TGF-ß family signaling to embryonic and postnatal testis and ovarian development. We conclude the review by describing data obtained from human studies, which highlight the importance of the TGF-ß family in normal female reproductive function during pregnancy and in various gynecologic pathologies.

Juvenile Granulosa Cell Tumor of the Ovary: A Clinicopathologic Study.

STUDY OBJECTIVE: To report on the clinical characteristics and outcome of pediatric patients with juvenile granulosa cell tumor (JGCT) of the ovary. DESIGN, SETTING, PARTICIPANTS, INTERVENTIONS, AND MAIN OUTCOME MEASURES: Patients with histopathologically confirmed ovarian JGCT diagnosed between 1990 and 2016 were identified. Data on the clinical presentation, surgical management, oncologic management, laboratory investigation, follow-up, and outcome were collected. Tumors were staged according to the International Federation of Gynecology and Obstetrics criteria. RESULTS: Eight patients were diagnosed with ovarian JGCT during the study period. The median age at presentation was 3 years (range, 0.7-14 years). Precocious puberty was the presenting symptom in all five prepubertal children; abdominal distension due to mass effect was the presenting symptom in three children older than 9 years of age. In patients who had preoperative serologic testing, estradiol (n = 3) and inhibin (n = 3) levels were elevated. Five patients had stage I disease, and three had stage III. All stage I patients underwent salpingo-oophorectomy as the only treatment. Stage III patients received adjuvant chemotherapy. After a median follow-up of 6.2 years, six patients (75%) were alive without evidence of disease. One stage I patient with germline p53 mutation and phosphatase and tensin homolog mutation, died because of subsequent liposarcoma. One patient with stage IIIB disease developed recurrence detected according to an elevated inhibin serum level, and died due to progressive disease despite receiving multiple chemotherapy regimens. CONCLUSION: Juvenile granulosa cell tumor has a favorable prognosis in patients with stage I disease after surgical resection alone. Adjuvant chemotherapy might be indicated in patients with higher-stage tumors.

SMAD Signaling Is Required for Structural Integrity of the Female Reproductive Tract and Uterine Function During Early Pregnancy in Mice.

Pregnancy is a complex physiological process tightly controlled by the interplay among hormones, morphogens, transcription factors, and signaling pathways. Although recent studies using genetically engineered mouse models have revealed that ligands and receptors of transforming growth factor beta (TGFbeta) and bone morphogenetic protein (BMP) signaling pathways are essential for multiple reproductive events during pregnancy, the functional role of SMAD transcription factors, which serve as the canonical signaling platform for the TGFbeta/BMP pathways, in the oviduct and uterus is undefined. Here, we used a mouse model containing triple conditional deletion of the BMP receptor signaling Smads (Smad1 and Smad5) and Smad4, the central mediator of both TGFbeta and BMP signaling, to investigate the role of the SMADs in reproductive tract structure and function in cells from the Amhr2 lineage. Unlike the respective single- or double-knockouts, female Smad1(flox/flox) Smad5(flox/flox) Smad4(flox/flox) Amhr2(cre/+)conditional knockout (i.e., Smad1/5/4-Amhr2-cre KO) mice are sterile. We discovered that Smad1/5/4-Amhr2-cre KO females have malformed oviducts that subsequently develop oviductal diverticuli. These oviducts showed dysregulation of multiple genes essential for oviduct and smooth muscle development. In addition, uteri from Smad1/5/4-Amhr2-cre KO females exhibit multiple defects in stroma, epithelium, and smooth muscle layers and fail to assemble a closed uterine lumen upon embryo implantation, with defective uterine decidualization that led to pregnancy loss at early to mid-gestation. Taken together, our study uncovers a new role for the SMAD transcription factors in maintaining the structural and functional integrity of oviduct and uterus, required for establishment and maintenance of pregnancy.

Regulation of germ cell function by SUMOylation.

Oogenesis and spermatogenesis are tightly regulated complex processes that are critical for fertility. Germ cells undergo meiosis to generate haploid cells necessary for reproduction. Errors in meiosis, including the generation of chromosomal abnormalities, can result in reproductive defects and infertility. Meiotic proteins are regulated by post-translational modifications including SUMOylation, the covalent attachment of small ubiquitin-like modifier (SUMO) proteins. Here, we review the role of SUMO proteins in controlling germ cell development and maturation based on recent findings from mouse models. Several studies have characterized the localization of SUMO proteins in male and female germ cells. However, a deeper understanding of how SUMOylation regulates proteins with essential roles in oogenesis and spermatogenesis will provide useful insight into the underlying mechanisms of germ cell development and fertility.

Uterine activin receptor-like kinase 5 is crucial for blastocyst implantation and placental development.

Members of the transforming growth factor ß (TGF-ß) superfamily are key regulators in most developmental and physiological processes. However, the in vivo roles of TGF-ß signaling in female reproduction remain uncertain. Activin receptor-like kinase 5 (ALK5) is the major type 1 receptor for the TGF-ß subfamily. Absence of ALK5 leads to early embryonic lethality because of severe defects in vascular development. In this study, we conditionally ablated uterine ALK5 using progesterone receptor-cre mice to define the physiological roles of ALK5 in female reproduction. Despite normal ovarian functions and artificial decidualization in conditional knockout (cKO) mice, absence of uterine ALK5 resulted in substantially reduced female reproduction due to abnormalities observed at different stages of pregnancy, including implantation defects, disorganization of trophoblast cells, fewer uterine natural killer (uNK) cells, and impairment of spiral artery remodeling. In our microarray analysis, genes encoding proteins involved in cytokine-cytokine receptor interactions and NK cell-mediated cytotoxicity were down-regulated in cKO decidua compared with control decidua. Flow cytometry confirmed a 10-fold decrease in uNK cells in cKO versus control decidua. According to these data, we hypothesize that TGF-ß acts on decidual cells via ALK5 to induce expression of other growth factors and cytokines, which are key regulators in luminal epithelium proliferation, trophoblast development, and uNK maturation during pregnancy. Our findings not only generate a mouse model to study TGF-ß signaling in female reproduction but also shed light on the pathogenesis of many pregnancy complications in human, such as recurrent spontaneous abortion, preeclampsia, and intrauterine growth restriction.

FOXO1/3 and PTEN Depletion in Granulosa Cells Promotes Ovarian Granulosa Cell Tumor Development.

The forkhead box (FOX), FOXO1 and FOXO3, transcription factors regulate multiple functions in mammalian cells. Selective inactivation of the Foxo1 and Foxo3 genes in murine ovarian granulosa cells severely impairs follicular development and apoptosis causing infertility, and as shown here, granulosa cell tumor (GCT) formation. Coordinate depletion of the tumor suppressor Pten gene in the Foxo1/3 strain enhanced the penetrance and onset of GCT formation. Immunostaining and Western blot analyses confirmed FOXO1 and phosphatase and tensin homolog (PTEN) depletion, maintenance of globin transcription factor (GATA) 4 and nuclear localization of FOXL2 and phosphorylated small mothers against decapentaplegic (SMAD) 2/3 in the tumor cells, recapitulating results we observed in human adult GCTs. Microarray and quantitative PCR analyses of mouse GCTs further confirmed expression of specific genes (Foxl2, Gata4, and Wnt4) controlling granulosa cell fate specification and proliferation, whereas others (Emx2, Nr0b1, Rspo1, and Wt1) were suppressed. Key genes (Amh, Bmp2, and Fshr) controlling follicle growth, apoptosis, and differentiation were also suppressed. Inhbb and Grem1 were selectively elevated, whereas reduction of Inha provided additional evidence that activin signaling and small mothers against decapentaplegic (SMAD) 2/3 phosphorylation impact GCT formation. Unexpectedly, markers of Sertoli/epithelial cells (SRY [sex determining region Y]-box 9/keratin 8) and alternatively activated macrophages (chitinase 3-like 3) were elevated in discrete subpopulations within the mouse GCTs, indicating that Foxo1/3/Pten depletion not only leads to GCTs but also to altered granulosa cell fate decisions and immune responses. Thus, analyses of the Foxo1/3/Pten mouse GCTs and human adult GCTs provide strong evidence that impaired functions of the FOXO1/3/PTEN pathways lead to dramatic changes in the molecular program within granulosa cells, chronic activin signaling in the presence of FOXL2 and GATA4, and tumor formation.

TGFß signaling promotes juvenile granulosa cell tumorigenesis by suppressing apoptosis.

Molecular changes that give rise to granulosa cell tumors of the ovary are not well understood. Previously, we showed that deletion in granulosa cells of the bone morphogenetic protein receptor-signaling transcription factors, Smad1 and Smad5, causes development of metastatic granulosa cell tumors that phenocopy the juvenile form of granulosa cell tumors (JGCTs) in humans. The TGFß-SMAD2/3 pathway is active in JGCTs, but its role is unknown. We tested the in vivo contribution of TGFß-SMAD signaling to JGCT development by genetically deleting the common Smad4 from Smad1/5 double knockout mice. Smad1/5/4 triple knockout mice were sterile and had significantly increased survival and delayed tumor development compared to those for the Smad1/5 double knockout mice. The few tumors that did develop were smaller, showed no evidence of metastasis, and had increased apoptosis. In the human JGCT cell line COV434, TGFß1 increased viability by inhibiting apoptosis through a TGFß type I receptor-dependent repression of caspase activity and inhibition of poly(ADP-ribose) polymerase cleavage. These data support a tumor-promoting function of TGFß in JGCTs through its ability to repress apoptosis.

Soy promotes juvenile granulosa cell tumor development in mice and in the human granulosa cell tumor-derived COV434 cell line.

Soy attracts attention for its health benefits, such as lowering cholesterol or preventing breast and colon cancer. Soybeans contain isoflavones, which act as phytoestrogens. Even though isoflavones have beneficial health effects, a role for isoflavones in the initiation and progression of diseases including cancer is becoming increasingly recognized. While data from rodent studies suggest that neonatal exposure to genistein (the predominant isoflavone in soy) disrupts normal reproductive function, its role in ovarian cancers, particularly granulosa cell tumors (GCT), is largely unknown. Our study aimed to define the contribution of a soy diet in GCT development using a genetically modified mouse model for juvenile GCTs (JGCT; Smad1 Smad5 conditional double knockout mice) as well as a human JGCT cell line (COV434). While dietary soy cannot initiate JGCT development in mice, we show that it has dramatic effects on GCT growth and tumor progression compared to a soy-free diet. Loss of Smad1 and Smad5 alters estrogen receptor alpha (Esr1) expression in granulosa cells, perhaps sensitizing the cells to the effects of genistein. In addition, we found that genistein modulates estrogen receptor expression in the human JGCT cell line and positively promotes cell growth in part by suppressing caspase-dependent apoptosis. Combined, our work suggests that dietary soy consumption has deleterious effects on GCT development.

Activin-like kinase 2 functions in peri-implantation uterine signaling in mice and humans.

Implantation of a blastocyst in the uterus is a multistep process tightly controlled by an intricate regulatory network of interconnected ovarian, uterine, and embryonic factors. Bone morphogenetic protein (BMP) ligands and receptors are expressed in the uterus of pregnant mice, and BMP2 has been shown to be a key regulator of implantation. In this study, we investigated the roles of the BMP type 1 receptor, activin-like kinase 2 (ALK2), during mouse pregnancy by producing mice carrying a conditional ablation of Alk2 in the uterus (Alk2 cKO mice). In the absence of ALK2, embryos demonstrate delayed invasion into the uterine epithelium and stroma, and upon implantation, stromal cells fail to undergo uterine decidualization, resulting in sterility. Mechanistically, microarray analysis revealed that CCAAT/enhancer-binding protein ß (Cebpb) expression is suppressed during decidualization in Alk2 cKO females. These findings and the similar phenotypes of Cebpb cKO and Alk2 cKO mice lead to the hypothesis that BMPs act upstream of CEBPB in the stroma to regulate decidualization. To test this hypothesis, we knocked down ALK2 in human uterine stromal cells (hESC) and discovered that ablation of ALK2 alters hESC decidualization and suppresses CEBPB mRNA and protein levels. Chromatin immunoprecipitation (ChIP) analysis of decidualizing hESC confirmed that BMP signaling proteins, SMAD1/5, directly regulate expression of CEBPB by binding a distinct regulatory sequence in the 3' UTR of this gene; CEBPB, in turn, regulates the expression of progesterone receptor (PGR). Our work clarifies the conserved mechanisms through which BMPs regulate peri-implantation in rodents and primates and, for the first time, uncovers a linear pathway of BMP signaling through ALK2 to regulate CEBPB and, subsequently, PGR during decidualization.

GDF9 modulates the reproductive and tumor phenotype of female inha-null mice.

Intraovarian factors play important roles in coordinating germ cell and somatic cell growth in the ovary. Prior to the onset of gonadotropin stimulation and reproductive cyclicity, follicle development is dependent upon locally produced growth factors, such as the transforming growth factor beta family members inhibin, activin, and GDF9. In the absence of inhibin in prepubertal mice (Inha(-/-)), there are marked alterations in preantral follicle growth, but no evidence of ovarian tumors characteristic of adult Inha-null mice. To ascertain the contribution of GDF9 to the Inha-null phenotype, we analyzed folliculogenesis in postnatal Inha Gdf9 double knockout mice. Deletion of Gdf9 from Inha(-/-) rescues the initial growth defects found at early follicle stages in Inha(-/-) ovaries, but surprisingly enhances the onset of pretumor lesions. The normalization of growth dynamics between granulosa cells and oocytes of Inha Gdf9 double knockout mice is also accompanied by a reduction in levels of the activin/inhibin beta B subunit, Inhbb, which is upregulated in Inha(-/-) ovaries. However, at later ages, Inha Gdf9 double knockout ovaries are similar to Inha(-/-) ovaries, and show upregulation of the activin/inhibin subunits and downregulation of the growth factor, kit ligand, thus resulting in a local environment that is growth-promoting for granulosa cells but growth-inhibitory for oocytes. These data suggest a sequential mechanism of action initiated by GDF9 in the Inha knockout mouse that promotes defective folliculogenesis. These studies thus provide a novel role for GDF9 in causing reproductive defects and suppressing tumor initiation in the Inha(-/-) mouse model.

SMAD4 haploinsufficiency associates with augmented colonic inflammation in select humans and mice.

SMAD4 is a common mediator of the TGF-beta signaling pathway. One of the members of this pathway, TGF-beta 1, has an important role in controlling gut inflammation in relation to the continuous stimulation of the intestinal microbiota. SMAD4 haploinsufficiency in humans has been linked to juvenile polyposis hereditary hemorrhagic telangiectasia syndrome (JP/HHT; OMIM#17505). Hematochezia and colonic mucosal inflammation suggestive of inflammatory bowel diseases (IBD) have been reported in JP/HHT. Stimulated by recent experience with two affected pediatric patients presented here, we explored the potential role of Smad4 haploinsufficiency in a murine model of colonic inflammation. Smad4(+/-) mice were maintained on a mixed C57/129SvEv background. Chronic colitis was induced with repeated administration of dextran sulfate sodium (DSS) in drinking water. The colonic mucosal microbiota was interrogated by massively parallel pyrosequencing of the bacterial 16S rRNA gene. 66.7% of Smad4(+/-) mice were sensitive to DSS colitis compared to 14.3% of wild type (Chi-Square p=0.036). The augmented colitis was associated with microbiota separation in the Smad4(+/-) mice. Enterococcus and Enterococcus faecalis specifically was increased in abundance in the colitis-prone animals. Smad4 haploinsufficiency can associate with increased susceptibility to large bowel inflammation in mammals with variable penetrance in association with the colonic mucosal microbiota. These findings may reveal implications not only towards colonic inflammation in the setting of SMAD4 haploinsufficiency, but for colorectal cancer as well.