OBOX regulates mouse zygotic genome activation and early development.

Zygotic genome activation (ZGA) activates the quiescent genome to enable the maternal-to-zygotic transition1,2. However, the identity of transcription factors that underlie mammalian ZGA in vivo remains elusive. Here we show that OBOX, a PRD-like homeobox domain transcription factor family (OBOX1-OBOX8)3-5, are key regulators of mouse ZGA. Mice deficient for maternally transcribed Obox1/2/5/7 and zygotically expressed Obox3/4 had a two-cell to four-cell arrest, accompanied by impaired ZGA. The Obox knockout defects could be rescued by restoring either maternal and zygotic OBOX, which suggests that maternal and zygotic OBOX redundantly support embryonic development. Chromatin-binding analysis showed that Obox knockout preferentially affected OBOX-binding targets. Mechanistically, OBOX facilitated the 'preconfiguration' of RNA polymerase II, as the polymerase relocated from the initial one-cell binding targets to ZGA gene promoters and distal enhancers. Impaired polymerase II preconfiguration in Obox mutants was accompanied by defective ZGA and chromatin accessibility transition, as well as aberrant activation of one-cell polymerase II targets. Finally, ectopic expression of OBOX activated ZGA genes and MERVL repeats in mouse embryonic stem cells. These data thus demonstrate that OBOX regulates mouse ZGA and early embryogenesis.

Developmental estrogen exposure in mice disrupts uterine epithelial cell differentiation and causes adenocarcinoma via Wnt/ß-catenin and PI3K/AKT signaling.

Tissue development entails genetically programmed differentiation of immature cell types to mature, fully differentiated cells. Exposure during development to non-mutagenic environmental factors can contribute to cancer risk, but the underlying mechanisms are not understood. We used a mouse model of endometrial adenocarcinoma that results from brief developmental exposure to an estrogenic chemical, diethylstilbestrol (DES), to determine causative factors. Single-cell RNA sequencing (scRNAseq) and spatial transcriptomics of adult control uteri revealed novel markers of uterine epithelial stem cells (EpSCs), identified distinct luminal and glandular progenitor cell (PC) populations, and defined glandular and luminal epithelium (LE) cell differentiation trajectories. Neonatal DES exposure disrupted uterine epithelial cell differentiation, resulting in a failure to generate an EpSC population or distinguishable glandular and luminal progenitors or mature cells. Instead, the DES-exposed epithelial cells were characterized by a single proliferating PC population and widespread activation of Wnt/ß-catenin signaling. The underlying endometrial stromal cells had dramatic increases in inflammatory signaling pathways and oxidative stress. Together, these changes activated phosphoinositide 3-kinase/AKT serine-threonine kinase signaling and malignant transformation of cells that were marked by phospho-AKT and the cancer-associated protein olfactomedin 4. Here, we defined a mechanistic pathway from developmental exposure to an endocrine disrupting chemical to the development of adult-onset cancer. These findings provide an explanation for how human cancers, which are often associated with abnormal activation of PI3K/AKT signaling, could result from exposure to environmental insults during development.

Unexpected nuclear hormone receptor and chromatin dynamics regulate estrous cycle dependent gene expression.

Chromatin changes in response to estrogen and progesterone are well established in cultured cells, but how they control gene expression under physiological conditions is largely unknown. To address this question, we examined in vivo estrous cycle dynamics of mouse uterus hormone receptor occupancy, chromatin accessibility and chromatin structure by combining RNA-seq, ATAC-seq, HiC-seq and ChIP-seq. Two estrous cycle stages were chosen for these analyses, diestrus (highest estrogen) and estrus (highest progesterone). Unexpectedly, rather than alternating with each other, estrogen receptor alpha (ERα) and progesterone receptor (PGR) were co-bound during diestrus and lost during estrus. Motif analysis of open chromatin followed by hypoxia inducible factor 2A (HIF2A) ChIP-seq and conditional uterine deletion of this transcription factor revealed a novel role for HIF2A in regulating diestrus gene expression patterns that were independent of either ERα or PGR binding. Proteins in complex with ERα included PGR and cohesin, only during diestrus. Combined with HiC-seq analyses, we demonstrate that complex chromatin architecture changes including enhancer switching are coordinated with ERα and PGR co-binding during diestrus and non-hormone receptor transcription factors such as HIF2A during estrus to regulate most differential gene expression across the estrous cycle.

PMCA1 depletion in mouse eggs amplifies calcium signaling and impacts offspring growth†.

Egg activation in mammals is triggered by oscillations in egg intracellular calcium (Ca2+) level. Ca2+ oscillation patterns can be modified in vitro by changing the ionic composition of culture media or in vivo by conditions affecting mitochondrial function, such as obesity and inflammation. In mice, disruption of Ca2+ oscillations in vitro impacts embryo development and offspring growth. Here we tested the hypothesis that, even without in vitro manipulation, abnormal Ca2+ signaling following fertilization impacts offspring growth. Plasma membrane Ca2+ ATPases (PMCA) extrude cytosolic Ca2+ to restore Ca2+ homeostasis. To disrupt Ca2+ signaling in vivo, we conditionally deleted PMCA1 (cKO) in oocytes. As anticipated, in vitro fertilized cKO eggs had increased Ca2+ exposure relative to controls. To assess the impact on offspring growth, cKO females were mated to wild type males to generate pups that had high Ca2+ exposure at fertilization. Because these offspring would be heterozygous, we also tested the impact of global PMCA1 heterozygosity on offspring growth. Control heterozygous pups that had normal Ca2+ at fertilization were generated by mating wild type females to heterozygous males; these control offspring weighed significantly less than their wild type siblings. However, heterozygous offspring from cKO eggs (and high Ca2+ exposure) were larger than heterozygous controls at 12 week-of-age and males had altered body composition. Our results show that global PMCA1 haploinsufficiency impacts growth and support that abnormal Ca2+ signaling after fertilization in vivo has a long-term impact on offspring weight. These findings are relevant for environmental and medical conditions affecting Ca2+ handling and for design of culture conditions and procedures for domestic animal and human assisted reproduction.

Methionine metabolism is essential for SIRT1-regulated mouse embryonic stem cell maintenance and embryonic development.

Methionine metabolism is critical for epigenetic maintenance, redox homeostasis, and animal development. However, the regulation of methionine metabolism remains unclear. Here, we provide evidence that SIRT1, the most conserved mammalian NAD+-dependent protein deacetylase, is critically involved in modulating methionine metabolism, thereby impacting maintenance of mouse embryonic stem cells (mESCs) and subsequent embryogenesis. We demonstrate that SIRT1-deficient mESCs are hypersensitive to methionine restriction/depletion-induced differentiation and apoptosis, primarily due to a reduced conversion of methionine to S-adenosylmethionine. This reduction markedly decreases methylation levels of histones, resulting in dramatic alterations in gene expression profiles. Mechanistically, we discover that the enzyme converting methionine to S-adenosylmethionine in mESCs, methionine adenosyltransferase 2a (MAT2a), is under control of Myc and SIRT1. Consistently, SIRT1 KO embryos display reduced Mat2a expression and histone methylation and are sensitive to maternal methionine restriction-induced lethality, whereas maternal methionine supplementation increases the survival of SIRT1 KO newborn mice. Our findings uncover a novel regulatory mechanism for methionine metabolism and highlight the importance of methionine metabolism in SIRT1-mediated mESC maintenance and embryonic development.

TRPM7 and CaV3.2 channels mediate Ca2+ influx required for egg activation at fertilization.

The success of mammalian development following fertilization depends on a series of transient increases in egg cytoplasmic Ca2+, referred to as Ca2+ oscillations. Maintenance of these oscillations requires Ca2+ influx across the plasma membrane, which is mediated in part by T-type, CaV3.2 channels. Here we show using genetic mouse models that TRPM7 channels are required to support this Ca2+ influx. Eggs lacking both TRPM7 and CaV3.2 stop oscillating prematurely, indicating that together they are responsible for the majority of Ca2+ influx immediately following fertilization. Fertilized eggs lacking both channels also frequently display delayed resumption of Ca2+ oscillations, which appears to require sperm-egg fusion. TRPM7 and CaV3.2 channels almost completely account for Ca2+ influx observed following store depletion, a process previously attributed to canonical store-operated Ca2+ entry mediated by STIM/ORAI interactions. TRPM7 serves as a membrane sensor of extracellular Mg2+ and Ca2+ concentrations and mediates the effects of these ions on Ca2+ oscillation frequency. When bred to wild-type males, female mice carrying eggs lacking TRPM7 and CaV3.2 are subfertile, and their offspring have increased variance in postnatal weight. These in vivo findings confirm previous observations linking in vitro experimental alterations in Ca2+ oscillatory patterns with developmental potential and offspring growth. The identification of TRPM7 and CaV3.2 as key mediators of Ca2+ influx following fertilization provides a mechanistic basis for the rational design of culture media that optimize developmental potential in research animals, domestic animals, and humans.

DNA methylation and transcriptome aberrations mediated by ERα in mouse seminal vesicles following developmental DES exposure.

Early transient developmental exposure to an endocrine active compound, diethylstilbestrol (DES), a synthetic estrogen, causes late-stage effects in the reproductive tract of adult mice. Estrogen receptor alpha (ERα) plays a role in mediating these developmental effects. However, the developmental mechanism is not well known in male tissues. Here, we present genome-wide transcriptome and DNA methylation profiling of the seminal vesicles (SVs) during normal development and after DES exposure. ERα mediates aberrations of the mRNA transcriptome in SVs of adult mice following neonatal DES exposure. This developmental exposure impacts differential diseases between male (SVs) and female (uterus) tissues when mice reach adulthood due to most DES-altered genes that appear to be tissue specific during mouse development. Certain estrogen-responsive gene changes in SVs are cell-type specific. DNA methylation dynamically changes during development in the SVs of wild-type (WT) and ERα-knockout (αERKO) mice, which increases both the loss and gain of differentially methylated regions (DMRs). There are more gains of DMRs in αERKO compared with WT. Interestingly, the methylation changes between the two genotypes are in different genomic loci. Additionally, the expression levels of a subset of DES-altered genes are associated with their DNA methylation status following developmental DES exposure. Taken together, these findings provide an important basis for understanding the molecular and cellular mechanism of endocrine-disrupting chemicals (EDCs), such as DES, during development in the male mouse tissues. This unique evidence contributes to our understanding of developmental actions of EDCs in human health.

Unscrambling the oocyte and the egg: clarifying terminology of the female gamete in mammals.

Most reproductive biologists who study female gametes will agree with the 16th century anatomist William Harvey's doctrine: 'Ex Ovo Omnia'. This phrase, which literally translates to 'everything from the egg', recognizes the centrality of the egg in animal development. Eggs are most impressive cells, capable of supporting development of an entirely new organism following fertilization or parthenogenetic activation. Not so uniformly embraced in the field of reproductive biology is the nomenclature used to refer to the female germ cell. What is an oocyte? What is an egg? Are these terms the same, different, interchangeable? Here we provide functional definitions of the oocyte and egg, and how they can be used in the context of mammalian gamete biology and beyond.

Mouse strain-dependent egg factors regulate calcium signals at fertilization.

Calcium (Ca2+ ) signals triggered at fertilization initiate resumption of the cell cycle and initial steps of embryonic development. In mammals, the sperm factor phospholipase Cζ triggers the release of Ca2+ from the endoplasmic reticulum (ER), initiating an oscillatory pattern of Ca2+ transients that is modulated by egg factors including Ca2+ influx channels, Ca2+ transporters, and phosphoinositide-regulating enzymes. Here we compared characteristics of Ca2+ oscillations following in vitro fertilization (IVF) and ER Ca2+ stores among nine common laboratory mouse strains: CF1, C57BL6, SJL, CD1, DBA, FVB, 129X1, BALBc, 129S1, and the F1 hybrid B6129SF1. Sperm from B6SJLF1/J males was used for all IVF experiments. There were significant differences among the strains with respect to duration and maximum amplitude of the first Ca2+ transient, frequency of oscillations, and ER Ca2+ stores. With male strain held constant, the differences in Ca2+ oscillation patterns observed result from variation in egg factors across different mouse strains. Our results support the importance of egg-intrinsic properties in determining Ca2+ oscillation patterns and have important implications for the interpretation and comparison of studies on Ca2+ dynamics at fertilization.

Widespread enhancer activation via ERα mediates estrogen response in vivo during uterine development.

Little is known regarding how steroid hormone exposures impact the epigenetic landscape in a living organism. Here, we took a global approach to understanding how exposure to the estrogenic chemical, diethylstilbestrol (DES), affects the neonatal mouse uterine epigenome. Integration of RNA- and ChIP-sequencing data demonstrated that ∼80% of DES-altered genes had higher H3K4me1/H3K27ac signal in close proximity. Active enhancers, of which ∼3% were super-enhancers, had a high density of estrogen receptor alpha (ERα) binding sites and were correlated with alterations in nearby gene expression. Conditional uterine deletion of ERα, but not the pioneer transcription factors FOXA2 or FOXO1, prevented the majority of DES-mediated changes in gene expression and H3K27ac signal at target enhancers. An ERα dependent super-enhancer was located at the Padi gene locus and a topological connection to the Padi1 TSS was documented using 3C-PCR. Chromosome looping at this site was independent of ERα and DES exposure, indicating that the interaction is established prior to ligand signaling. However, enrichment of H3K27ac and transcriptional activation at this locus was both DES and ERα-dependent. These data suggest that DES alters uterine development and consequently adult reproductive function by modifying the enhancer landscape at ERα binding sites near estrogen-regulated genes.

The histone methyltransferase EZH2 is required for normal uterine development and function in mice†.

Enhancer of zeste homolog 2 (EZH2) is a rate-limiting catalytic subunit of a histone methyltransferase, polycomb repressive complex, which silences gene activity through the repressive histone mark H3K27me3. EZH2 is critical for epigenetic effects of early estrogen treatment, and may be involved in uterine development and pathologies. We investigated EZH2 expression, regulation, and its role in uterine development/function. Uterine epithelial EZH2 expression was associated with proliferation and was high neonatally then declined by weaning. Pre-weaning uterine EZH2 expression was comparable in wild-type and estrogen receptor 1 knockout mice, showing neonatal EZH2 expression is ESR1 independent. Epithelial EZH2 was upregulated by 17ß-estradiol (E2) and inhibited by progesterone in adult uteri from ovariectomized mice. To investigate the uterine role of EZH2, we developed a EZH2 conditional knockout (Ezh2cKO) mouse using a cre recombinase driven by the progesterone receptor (Pgr) promoter that produced Ezh2cKO mice lacking EZH2 in Pgr-expressing tissues (e.g. uterus, mammary glands). In Ezh2cKO uteri, EZH2 was deleted neonatally. These uteri had reduced H3K27me3, were larger than WT, and showed adult cystic endometrial hyperplasia. Ovary-independent uterine epithelial proliferation and increased numbers of highly proliferative uterine glands were seen in adult Ezh2cKO mice. Female Ezh2cKO mice were initially subfertile, and then became infertile by 9 months. Mammary gland development in Ezh2cKO mice was inhibited. In summary, uterine EZH2 expression is developmentally and hormonally regulated, and its loss causes aberrant uterine epithelial proliferation, uterine hypertrophy, and cystic endometrial hyperplasia, indicating a critical role in uterine development and function.

Endocrine Disruption and Reproductive Pathology.

During the past 20 years, investigations involving endocrine active substances (EAS) and reproductive toxicity have dominated the landscape of ecotoxicological research. This has occurred in concert with heightened awareness in the scientific community, general public, and governmental entities of the potential consequences of chemical perturbation in humans and wildlife. The exponential growth of experimentation in this field is fueled by our expanding knowledge into the complex nature of endocrine systems and the intricacy of their interactions with xenobiotic agents. Complicating factors include the ever-increasing number of novel receptors and alternate mechanistic pathways that have come to light, effects of chemical mixtures in the environment versus those of single EAS laboratory exposures, the challenge of differentiating endocrine disruption from direct cytotoxicity, and the potential for transgenerational effects. Although initially concerned with EAS effects chiefly in the thyroid glands and reproductive organs, it is now recognized that anthropomorphic substances may also adversely affect the nervous and immune systems via hormonal mechanisms and play substantial roles in metabolic diseases, such as type 2 diabetes and obesity.

Regulator of G-protein signaling 2 (RGS2) suppresses premature calcium release in mouse eggs.

During oocyte maturation, capacity and sensitivity of Ca(2+) signaling machinery increases dramatically, preparing the metaphase II (MII)-arrested egg for fertilization. Upon sperm-egg fusion, Ca(2+) release from IP3-sensitive endoplasmic reticulum stores results in cytoplasmic Ca(2+) oscillations that drive egg activation and initiate early embryo development. Premature Ca(2+) release can cause parthenogenetic activation prior to fertilization; thus, preventing inappropriate Ca(2+) signaling is crucial for ensuring robust MII arrest. Here, we show that regulator of G-protein signaling 2 (RGS2) suppresses Ca(2+) release in MII eggs. Rgs2 mRNA was recruited for translation during oocyte maturation, resulting in ∼ 20-fold more RGS2 protein in MII eggs than in fully grown immature oocytes. Rgs2-siRNA-injected oocytes matured to MII; however, they had increased sensitivity to low pH and acetylcholine (ACh), which caused inappropriate Ca(2+) release and premature egg activation. When matured in vitro, RGS2-depleted eggs underwent spontaneous Ca(2+) increases that were sufficient to cause premature zona pellucida conversion. Rgs2(-/-) females had reduced litter sizes, and their eggs had increased sensitivity to low pH and ACh. Rgs2(-/-) eggs also underwent premature zona pellucida conversion in vivo. These findings indicate that RGS2 functions as a brake to suppress premature Ca(2+) release in eggs that are poised on the brink of development.

Mediator complex component MED13 regulates zygotic genome activation and is required for postimplantation development in the mouse.

Understanding factors that regulate zygotic genome activation (ZGA) is critical for determining how cells are reprogrammed to become totipotent or pluripotent. There is limited information regarding how this process occurs physiologically in early mammalian embryos. Here, we identify a mediator complex subunit, MED13, as translated during mouse oocyte maturation and transcribed early from the zygotic genome. Knockdown and conditional knockout approaches demonstrate that MED13 is essential for ZGA in the mouse, in part by regulating expression of the embryo-specific chromatin remodeling complex, esBAF. The role of MED13 in ZGA is mediated in part by interactions with E2F transcription factors. In addition to MED13, its paralog, MED13L, is required for successful preimplantation embryo development. MED13L partially compensates for loss of MED13 function in preimplantation knockout embryos, but postimplantation development is not rescued by MED13L. Our data demonstrate an essential role for MED13 in supporting chromatin reprogramming and directed transcription of essential genes during ZGA.

Differentiation Patterns of Uterine Carcinomas and Precursor Lesions Induced by Neonatal Estrogen Exposure in Mice.

Developmental exposure to estrogenic chemicals is an established risk factor for cancer of the female reproductive tract. This increase in risk has been associated with disruption of normal patterns of cellular differentiation during critical stages of morphogenesis. The goal of this study was to document uterine epithelial phenotypes over time following neonatal treatment with the synthetic estrogen diethylstilbestrol (DES) or the soy phytoestrogen genistein (GEN) in female CD-1 mice. Both DES and GEN induced three distinct populations of abnormal endometrial epithelial cells: luminal (SIX1+/P63-/CK14-/CK18+), basal (SIX1+/P63+/CK14+/CK18-), and mixed/bipotential (SIX1+/P63-/CK14+/CK18+), which were all established by early adulthood. In older animals, DES and GEN resulted in uterine carcinomas with mixed glandular, basal, and squamous cell elements. All carcinomas were composed largely of the three abnormal cell types. These findings identify novel epithelial differentiation patterns in the uterus and support the idea that disruption of cellular programming in early development can influence cancer risk later in life.

Epithelial membrane protein 2 (EMP2) deficiency alters placental angiogenesis, mimicking features of human placental insufficiency.

Epithelial membrane protein-2 (EMP2) is a tetraspan protein predicted to regulate placental development. Highly expressed in secretory endometrium and trophectoderm cells, previous studies suggest that it may regulate implantation by orchestrating the surface expression of integrins and other membrane proteins. In order to test the role of EMP2 in pregnancy, mice lacking EMP2 (Emp2-/- ) were generated. Emp2-/- females are fertile but have reduced litter sizes when carrying Emp2-/- but not Emp2+/- fetuses. Placentas of Emp2-/- fetuses exhibit dysregulation in pathways related to neoangiogenesis, coagulation, and oxidative stress, and have increased fibrin deposition and altered vasculature. Given that these findings often occur due to placental insufficiency resulting in an oxygen-poor environment, the expression of hypoxia-inducible factor-1 alpha (HIF-1α) was examined. Placentas from Emp2-/- fetuses had increased total HIF-1α expression in large part through an increase in uterine NK (uNK) cells, demonstrating a unique interplay between uNK cells and trophoblasts modulated through EMP2. To determine if these results translated to human pregnancy, placentas from normal, term deliveries or those complicated by placental insufficiency resulting in intrauterine growth restriction (IUGR) were stained for EMP2. EMP2 was significantly reduced in both villous and extravillous trophoblast populations in IUGR placentas. Experiments in vitro using human trophoblast cells lines indicate that EMP2 modulates angiogenesis by altering HIF-1α expression. Our results reveal a novel role for EMP2 in regulating trophoblast function and vascular development in mice and humans, and suggest that it may be a new biomarker for placental insufficiency. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

CaV3.2 T-type channels mediate Ca²âº entry during oocyte maturation and following fertilization.

Initiation of mouse embryonic development depends upon a series of fertilization-induced rises in intracellular Ca(2+). Complete egg activation requires influx of extracellular Ca(2+); however, the channels that mediate this influx remain unknown. Here, we tested whether the α1 subunit of the T-type channel CaV3.2, encoded by Cacna1h, mediates Ca(2+) entry into oocytes. We show that mouse eggs express a robust voltage-activated Ca(2+) current that is completely absent in Cacna1h(-/-) eggs. Cacna1h(-/-) females have reduced litter sizes, and careful analysis of Ca(2+) oscillation patterns in Cacna1h(-/-) eggs following in vitro fertilization (IVF) revealed reductions in first transient length and oscillation persistence. Total and endoplasmic reticulum (ER) Ca(2+) stores were also reduced in Cacna1h(-/-) eggs. Pharmacological inhibition of CaV3.2 in wild-type CF-1 strain eggs using mibefradil or pimozide reduced Ca(2+) store accumulation during oocyte maturation and reduced Ca(2+) oscillation persistence, frequency and number following IVF. Overall, these data show that CaV3.2 T-type channels have prev8iously unrecognized roles in supporting the meiotic-maturation-associated increase in ER Ca(2+) stores and mediating Ca(2+) influx required for the activation of development.

Calcium influx-mediated signaling is required for complete mouse egg activation.

Mammalian fertilization is accompanied by oscillations in egg cytoplasmic calcium (Ca(2+)) concentrations that are critical for completion of egg activation. These oscillations are initiated by Ca(2+) release from inositol 1,4,5-trisphosphate (IP(3))-sensitive intracellular stores. We tested the hypothesis that Ca(2+) influx across the plasma membrane was a requisite component of egg activation signaling, and not simply a Ca(2+) source for store repletion. Using intracytoplasmic sperm injection (ICSI) and standard in vitro fertilization (IVF), we found that Ca(2+) influx was not required to initiate resumption of meiosis II. However, even if multiple oscillations in intracellular Ca(2+) occurred, in the absence of Ca(2+) influx, the fertilized eggs failed to emit the second polar body, resulting in formation of three pronuclei. Additional experiments using the Ca(2+) chelator, BAPTA/AM, demonstrated that Ca(2+) influx is sufficient to support polar body emission and pronucleus formation after only a single sperm-induced Ca(2+) transient, whereas BAPTA/AM-treated ICSI or fertilized eggs cultured in Ca(2+)-free medium remained arrested in metaphase II. Inhibition of store-operated Ca(2+) entry had no effect on ICSI-induced egg activation, so Ca(2+) influx through alternative channels must participate in egg activation signaling. Ca(2+) influx appears to be upstream of CaMKIIγ activity because eggs can be parthenogenetically activated with a constitutively active form of CaMKIIγ in the absence of extracellular Ca(2+). These results suggest that Ca(2+) influx at fertilization not only maintains Ca(2+) oscillations by replenishing Ca(2+) stores, but also activates critical signaling pathways upstream of CaMKIIγ that are required for second polar body emission.

Transducin-like enhancer of split-6 (TLE6) is a substrate of protein kinase A activity during mouse oocyte maturation.

Fully grown oocytes in the ovary are arrested at prophase of meiosis I because of high levels of intraoocyte cAMP that maintain increased levels of cAMP-dependent protein kinase (PKA) activity. Following the luteinizing hormone surge at the time of ovulation, cAMP levels drop, resulting in a reduction in PKA activity that triggers meiotic resumption. Although much is known about the molecular mechanisms of how PKA activity fluctuations initiate the oocyte's reentry into meiosis, significantly less is known about the requirement for PKA activity in the oocyte after exit from the prophase I arrest. Here we show that although PKA activity decreases in the oocyte upon meiotic resumption, it increases throughout meiotic progression from the time of germinal vesicle breakdown (GVBD) until the metaphase II (MII) arrest. Blocking this meiotic maturation-associated increase in PKA activity using the pharmacological inhibitor H89 resulted in altered kinetics of GVBD, defects in chromatin and spindle dynamics, and decreased ability of oocytes to reach MII. These effects appear to be largely PKA specific because inhibitors targeting other kinases did not have the same outcomes. To determine potential proteins that may require PKA phosphorylation during meiosis, we separated oocyte protein extracts on an SDS-PAGE gel, extracted regions of the gel that had corresponding immune reactivity towards an anti-PKA substrate antibody, and performed mass spectrometry and microsequencing. Using this approach, we identified transducin-like enhancer of split-6 (TLE6)-a maternal effect gene that is part of the subcortical maternal complex-as a putative PKA substrate. TLE6 localized to the oocyte cortex throughout meiosis in a manner that is spatially and temporally consistent with the localization of critical PKA subunits. Moreover, we demonstrated that TLE6 becomes phosphorylated in a narrow window following meiotic resumption, and H89 treatment can completely block this phosphorylation when added prior to GVBD but not after. Taken together, these results highlight the importance of oocyte-intrinsic PKA in regulating meiotic progression after the prophase I arrest and offer new insights into downstream targets of its activity.

Estrogen receptor alpha mediated repression of PRICKLE1 destabilizes REST and promotes uterine fibroid pathogenesis.

Uterine fibroids (leiomyomas), benign tumors of the myometrial smooth muscle layer, are present in over 75% of women, often causing severe pain, menorrhagia and reproductive dysfunction. The molecular pathogenesis of fibroids is poorly understood. We previously showed that the loss of REST ( RE -1 S ilencing T ranscription factor), a tumor suppressor, in fibroids leads to activation of PI3K/AKT-mTOR pathway. We report here a critical link between estrogen receptor alpha (ERα) and the loss of REST, via PRICKLE1. PRICKLE1 expression is markedly lower in leiomyomas, and the suppression of PRICKLE1 significantly down regulates REST protein levels. Conversely, overexpression of PRICKLE1 resulted in the restoration of REST in cultured primary leiomyoma smooth muscle cells (LSMCs). Crucially, mice exposed neonatally to environmental estrogens, proven risk factors for fibroids, expressed lower levels of PRICKLE1 and REST in the myometrium. Using mice that lack either endogenous estrogen ( Lhb -/- mice) or ERα ( Esr1 -/- mice), we demonstrate that Prickle1 expression in the myometrium is suppressed by estrogen through ERα. Enhancer of zeste homolog 2 (EZH2) is known to participate in the repression of specific ERα target genes. Uterine leiomyomas express increased levels of EZH2 that inversely correlate with the expression of PRICKLE1. Using chromatin immunoprecipitation, we provide evidence for association of EZH2 with the PRICKLE1 promoter and for hypermethylation of H3K27 within the regulatory region of PRICKLE1 in leiomyomas. Additionally, siRNA mediated knockdown of EZH2 leads to restoration of PRICKLE1 in LSMCs. Collectively, our results identify a novel link between estrogen exposure and PRICKLE1/REST-regulated tumorigenic pathways in leiomyomas.