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.

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.

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.

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.

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.

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.

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.

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.

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.