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.

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.

Radiation-induced ovarian follicle loss occurs without overt stromal changes.

Radiation damage due to total body irradiation (TBI) or targeted abdominal radiation can deplete ovarian follicles and accelerate reproductive aging. We characterized a mouse model of low-dose TBI to investigate how radiation affects the follicular and stromal compartments of the ovary. A single TBI dose of either 0.1 Gy or 1 Gy (Cesium-137 γ) was delivered to reproductively adult CD1 female mice, and sham-treated mice served as controls. Mice were euthanized either 2 weeks or 5 weeks post exposure, and ovarian tissue was harvested. To assess the ovarian reserve, we classified and counted the number of morphologically normal follicles in ovarian histologic sections for all experimental cohorts using an objective method based on immunohistochemistry for an oocyte-specific protein (MSY2). 0.1 Gy did not affect that total number of ovarian follicles, whereas 1 Gy resulted in a dramatic loss. At two weeks, there was a significant reduction in all preantral follicles, but early antral and antral follicles were still present. By five weeks, there was complete depletion of all follicle classes. We examined stromal quality using histologic stains to visualize ovarian architecture and fibrosis and by immunohistochemistry and quantitative microscopy to assess cell proliferation, cell death and vasculature. There were no differences in the ovarian stroma across cohorts with respect to these markers, indicating that this compartment is more radio-resistant relative to the germ cells. These findings have implications for reproductive health and the field of fertility preservation because the radiation doses we examined mimic scatter doses experienced in typical therapeutic regimens.

C57BL/6 Substrains Exhibit Different Responses to Acute Carbon Tetrachloride Exposure: Implications for Work Involving Transgenic Mice.

Biological differences exist between strains of laboratory mice, and it is becoming increasingly evident that there are differences between substrains. In the C57BL/6 mouse, the primary substrains are called 6J and 6N. Previous studies have demonstrated that 6J and 6N mice differ in response to many experimental models of human disease. The aim of our study was to determine if differences exist between 6J and 6N mice in terms of their response to acute carbon tetrachloride (CCl4) exposure. Mice were given CCl4 once and were euthanized 12 to 96 h later. Relative to 6J mice, we found that 6N mice had increased liver injury but more rapid repair. This was because of the increased speed with which necrotic hepatocytes were removed in 6N mice and was directly related to increased recruitment of macrophages to the liver. In parallel, enhanced liver regeneration was observed in 6N relative to 6J mice. Hepatic stellate cell activation occurred earlier in 6N mice, but there was no difference in matrix metabolism between substrains. Taken together, these data demonstrate specific and significant differences in how the C57BL/6 substrains respond to acute CCl4, which has important implications for all mouse studies utilizing this model.

Reproductive age-associated fibrosis in the stroma of the mammalian ovary.

Under normal physiological conditions, tissue remodeling in response to injury leads to tissue regeneration without permanent damage. However, if homeostasis between synthesis and degradation of extracellular matrix (ECM) components is altered, fibrosis - or the excess accumulation of ECM - can disrupt tissue architecture and function. Several organs, including the heart, lung and kidney, exhibit age-associated fibrosis. Here we investigated whether fibrosis underlies aging in the ovary - an organ that ages chronologically before other organs. We used Picrosirius Red (PSR), a connective tissue stain specific for collagen I and III fibers, to evaluate ovarian fibrosis. Using bright-field, epifluorescence, confocal and polarized light microscopy, we validated the specific staining of highly ordered PSR-stained fibers in the ovary. We next examined ovarian PSR staining in two mouse strains (CD1 and CB6F1) across an aging continuum and found that PSR staining was minimal in ovaries from reproductively young adult animals, increased in distinct foci in animals of mid-to-advanced reproductive age, and was prominent throughout the stroma of the oldest animals. Consistent with fibrosis, there was a reproductive age-associated increase in ovarian hydroxyproline content. We also observed a unique population of multinucleated macrophage giant cells, which are associated with chronic inflammation, within the ovarian stroma exclusively in reproductively old mice. In fact, several genes central to inflammation had significantly higher levels of expression in ovaries from reproductively old mice relative to young mice. These results establish fibrosis as an early hallmark of the aging ovarian stroma, and this altered microenvironment may contribute to the age-associated decline in gamete quality.