Publications by authors named "Carmen J Williams"

76 Publications

p53 convergently activates Dux/DUX4 in embryonic stem cells and in facioscapulohumeral muscular dystrophy cell models.

Nat Genet 2021 Jul 15. Epub 2021 Jul 15.

Howard Hughes Medical Institute, Department of Oncological Sciences and Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, UT, USA.

In mammalian embryos, proper zygotic genome activation (ZGA) underlies totipotent development. Double homeobox (DUX)-family factors participate in ZGA, and mouse Dux is required for forming cultured two-cell (2C)-like cells. Remarkably, in mouse embryonic stem cells, Dux is activated by the tumor suppressor p53, and Dux expression promotes differentiation into expanded-fate cell types. Long-read sequencing and assembly of the mouse Dux locus reveals its complex chromatin regulation including putative positive and negative feedback loops. We show that the p53-DUX/DUX4 regulatory axis is conserved in humans. Furthermore, we demonstrate that cells derived from patients with facioscapulohumeral muscular dystrophy (FSHD) activate human DUX4 during p53 signaling via a p53-binding site in a primate-specific subtelomeric long terminal repeat (LTR)10C element. In summary, our work shows that p53 activation convergently evolved to couple p53 to Dux/DUX4 activation in embryonic stem cells, embryos and cells from patients with FSHD, potentially uniting the developmental and disease regulation of DUX-family factors and identifying evidence-based therapeutic opportunities for FSHD.
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http://dx.doi.org/10.1038/s41588-021-00893-0DOI Listing
July 2021

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

Mol Hum Reprod 2020 11;26(11):797-800

Reproductive Medicine Group, National Institute of Environmental Health Sciences, Durham, NC, USA.

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.
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http://dx.doi.org/10.1093/molehr/gaaa066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7648930PMC
November 2020

Modulators of calcium signalling at fertilization.

Open Biol 2020 07 15;10(7):200118. Epub 2020 Jul 15.

Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.

Calcium (Ca) signals initiate egg activation across the animal kingdom and in at least some plants. These signals are crucial for the success of development and, in the case of mammals, health of the offspring. The mechanisms associated with fertilization that trigger these signals and the molecules that regulate their characteristic patterns vary widely. With few exceptions, a major contributor to fertilization-induced elevation in cytoplasmic Ca is release from endoplasmic reticulum stores through the IP3 receptor. In some cases, Ca influx from the extracellular space and/or release from alternative intracellular stores contribute to the rise in cytoplasmic Ca. Following the Ca rise, the reuptake of Ca into intracellular stores or efflux of Ca out of the egg drive the return of cytoplasmic Ca back to baseline levels. The molecular mediators of these Ca fluxes in different organisms include Ca release channels, uptake channels, exchangers and pumps. The functions of these mediators are regulated by their particular activating mechanisms but also by alterations in their expression and spatial organization. We discuss here the molecular basis for modulation of Ca signalling at fertilization, highlighting differences across several animal phyla, and we mention key areas where questions remain.
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http://dx.doi.org/10.1098/rsob.200118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7574550PMC
July 2020

Developmentally Programmed Tankyrase Activity Upregulates β-Catenin and Licenses Progression of Embryonic Genome Activation.

Dev Cell 2020 06 21;53(5):545-560.e7. Epub 2020 May 21.

Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA. Electronic address:

Embryonic genome activation (EGA) is orchestrated by an intrinsic developmental program initiated during oocyte maturation with translation of stored maternal mRNAs. Here, we show that tankyrase, a poly(ADP-ribosyl) polymerase that regulates β-catenin levels, undergoes programmed translation during oocyte maturation and serves an essential role in mouse EGA. Newly translated TNKS triggers proteasomal degradation of axin, reducing targeted destruction of β-catenin and promoting β-catenin-mediated transcription of target genes, including Myc. MYC mediates ribosomal RNA transcription in 2-cell embryos, supporting global protein synthesis. Suppression of tankyrase activity using knockdown or chemical inhibition causes loss of nuclear β-catenin and global reductions in transcription and histone H3 acetylation. Chromatin and transcriptional profiling indicate that development arrests prior to the mid-2-cell stage, mediated in part by reductions in β-catenin and MYC. These findings indicate that post-transcriptional regulation of tankyrase serves as a ligand-independent developmental mechanism for post-translational β-catenin activation and is required to complete EGA.
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http://dx.doi.org/10.1016/j.devcel.2020.04.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7335218PMC
June 2020

Uterine Patterning, Endometrial Gland Development, and Implantation Failure in Mice Exposed Neonatally to Genistein.

Environ Health Perspect 2020 03 18;128(3):37001. Epub 2020 Mar 18.

Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina, USA.

Background: Embryo implantation relies on precise hormonal regulation, associated gene expression changes, and appropriate female reproductive tract tissue architecture. Female mice exposed neonatally to the phytoestrogen genistein (GEN) at doses similar to those in infants consuming soy-based infant formulas are infertile due in part to uterine implantation defects.

Objectives: Our goal was to determine the mechanisms by which neonatal GEN exposure causes implantation defects.

Methods: Female mice were exposed to GEN on postnatal days (PND)1-5 and uterine tissues collected on PND5, PND22-26, and during pregnancy. Analysis of tissue weights, morphology, and gene expression was performed using standard histology, confocal imaging with three-dimensional analysis, real-time reverse transcription polymerase chain reaction (real-time RT-PCR), and microarrays. The response of ovariectomized adults to (E2) and artificial decidualization were measured. Leukemia inhibitory factor (LIF) injections were given intraperitoneally and implantation sites visualized. Gene expression patterns were compared with curated data sets to identify upstream regulators.

Results: GEN-exposed mice exhibited reduced uterine weight gain in response to E2 treatment or artificial decidualization compared with controls; however, expression of select hormone responsive genes remained similar between the two groups. Uteri from pregnant GEN-exposed mice were posteriorized and had reduced glandular epithelium. Implantation failure was not rescued by LIF administration. Microarray analysis of GEN-exposed uteri during early pregnancy revealed significant overlap with several conditional uterine knockout mouse models, including , , and . These models exhibit reduced endometrial glands, features of posteriorization and implantation failure. Expression of , , and , as well as genes important for neonatal uterine differentiation (, , and ), were severely disrupted on PND5 in GEN-exposed mice.

Discussion: Our findings suggest that neonatal GEN exposure in mice disrupts expression of genes important for uterine development, causing posteriorization and diminished gland function during pregnancy that contribute to implantation failure. These findings could have implications for women who consumed soy-based formulas as infants. https://doi.org/10.1289/EHP6336.
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http://dx.doi.org/10.1289/EHP6336DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7138129PMC
March 2020

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

Mol Reprod Dev 2020 02 15;87(2):284-292. Epub 2020 Jan 15.

Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina.

Calcium (Ca ) 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 Ca from the endoplasmic reticulum (ER), initiating an oscillatory pattern of Ca transients that is modulated by egg factors including Ca influx channels, Ca transporters, and phosphoinositide-regulating enzymes. Here we compared characteristics of Ca oscillations following in vitro fertilization (IVF) and ER Ca 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 Ca transient, frequency of oscillations, and ER Ca stores. With male strain held constant, the differences in Ca oscillation patterns observed result from variation in egg factors across different mouse strains. Our results support the importance of egg-intrinsic properties in determining Ca oscillation patterns and have important implications for the interpretation and comparison of studies on Ca dynamics at fertilization.
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http://dx.doi.org/10.1002/mrd.23316DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7044019PMC
February 2020

Oviductal Retention of Embryos in Female Mice Lacking Estrogen Receptor α in the Isthmus and the Uterus.

Endocrinology 2020 02;161(2)

School of Molecular Biosciences, Center for Reproductive Biology, College of Veterinary Medicine, Washington State University, Pullman, Washington, US.

Estrogen receptor α (ESR1; encoded by Esr1) is a crucial nuclear transcription factor for female reproduction and is expressed throughout the female reproductive tract. To assess the function of ESR1 in reproductive tissues without confounding effects from a potential developmental defect arising from global deletion of ESR1, we generated a mouse model in which Esr1 was specifically ablated during postnatal development. To accomplish this, a progesterone receptor Cre line (PgrCre) was bred with Esr1f/f mice to create conditional knockout of Esr1 in reproductive tissues (called PgrCreEsr1KO mice) beginning around 6 days after birth. In the PgrCreEsr1KO oviduct, ESR1 was most efficiently ablated in the isthmic region. We found that at 3.5 days post coitus (dpc), embryos were retrieved from the uterus in control littermates while all embryos were retained in the PgrCreEsr1KO oviduct. Additionally, serum progesterone (P4) levels were significantly lower in PgrCreEsr1KO compared to controls at 3.5 dpc. This finding suggests that expression of ESR1 in the isthmus and normal P4 levels allow for successful embryo transport from the oviduct to the uterus. Therefore, alterations in oviductal isthmus ESR1 signaling and circulating P4 levels could be related to female infertility conditions such as tubal pregnancy.
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http://dx.doi.org/10.1210/endocr/bqz033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7295936PMC
February 2020

Endocrine Disruption and Reproductive Pathology.

Toxicol Pathol 2019 12;47(8):1049-1071

Experimental Pathology Laboratories, Inc, Sterling, VA, USA.

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.
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http://dx.doi.org/10.1177/0192623319879903DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8008741PMC
December 2019

SIX1 Regulates Aberrant Endometrial Epithelial Cell Differentiation and Cancer Latency Following Developmental Estrogenic Chemical Exposure.

Mol Cancer Res 2019 12 9;17(12):2369-2382. Epub 2019 Oct 9.

Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina.

Early-life exposure to estrogenic chemicals can increase cancer risk, likely by disrupting normal patterns of cellular differentiation. Female mice exposed neonatally to the synthetic estrogen diethylstilbestrol (DES) develop metaplastic and neoplastic uterine changes as adults. Abnormal endometrial glands express the oncofetal protein sine oculis homeobox 1 (SIX1) and contain cells with basal [cytokeratin (CK)14/18] and poorly differentiated features (CK14/18), strongly associating SIX1 with aberrant differentiation and cancer. Here, we tested whether SIX1 expression is necessary for abnormal endometrial differentiation and DES-induced carcinogenesis by using to generate conditional knockout mice lacking uterine ( ). Interestingly, corn oil (CO) vehicle-treated mice develop focal endometrial glandular dysplasia and features of carcinoma as compared with CO wild-type ( ) mice. Furthermore, mice neonatally exposed to DES had a 42% higher incidence of endometrial cancer relative to DES mice. Although DES mice had >10-fold fewer CK14/18 basal cells within the uterine horns as compared with DES mice, the appearance of CK14/18 cells remained a feature of neoplastic lesions. These findings suggest that SIX1 is required for normal endometrial epithelial differentiation, CK14/18 cells act as a cancer progenitor population, and SIX1 delays DES-induced endometrial carcinogenesis by promoting basal differentiation of CK14/18 cells. In human endometrial biopsies, 35% of malignancies showed CK14/18 expression, which positively correlated with tumor stage and grade and was not present in normal endometrium. IMPLICATIONS: Aberrant epithelial differentiation is a key feature in both the DES mouse model of endometrial cancer and human endometrial cancer. The association of CK14/18 cells with human endometrial cancer provides a novel cancer biomarker and could lead to new therapeutic strategies.
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http://dx.doi.org/10.1158/1541-7786.MCR-19-0475DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6891210PMC
December 2019

Epithelial membrane protein 2 governs transepithelial migration of neutrophils into the airspace.

J Clin Invest 2020 01;130(1):157-170

Immunity, Inflammation and Disease Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, USA.

Whether respiratory epithelial cells regulate the final transit of extravasated neutrophils into the inflamed airspace or are a passive barrier is poorly understood. Alveolar epithelial type 1 (AT1) cells, best known for solute transport and gas exchange, have few established immune roles. Epithelial membrane protein 2 (EMP2), a tetraspan protein that promotes recruitment of integrins to lipid rafts, is highly expressed in AT1 cells but has no known function in lung biology. Here, we show that Emp2-/- mice exhibit reduced neutrophil influx into the airspace after a wide range of inhaled exposures. During bacterial pneumonia, Emp2-/- mice had attenuated neutrophilic lung injury and improved survival. Bone marrow chimeras, intravital neutrophil labeling, and in vitro assays suggested that defective transepithelial migration of neutrophils into the alveolar lumen occurs in Emp2-/- lungs. Emp2-/- AT1 cells had dysregulated surface display of multiple adhesion molecules, associated with reduced raft abundance. Epithelial raft abundance was dependent upon putative cholesterol-binding motifs in EMP2, whereas EMP2 supported adhesion molecule display and neutrophil transmigration through suppression of caveolins. Taken together, we propose that EMP2-dependent membrane organization ensures proper display on AT1 cells of a suite of proteins required to instruct paracellular neutrophil traffic into the alveolus.
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http://dx.doi.org/10.1172/JCI127144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6934223PMC
January 2020

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

Biol Reprod 2019 08;101(2):306-317

Department of Physiological Sciences, University of Florida, Gainesville, Florida, USA.

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 17β-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.
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http://dx.doi.org/10.1093/biolre/ioz097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7302517PMC
August 2019

TRPM7 and Ca3.2 channels mediate Ca influx required for egg activation at fertilization.

Proc Natl Acad Sci U S A 2018 10 15;115(44):E10370-E10378. Epub 2018 Oct 15.

Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709;

The success of mammalian development following fertilization depends on a series of transient increases in egg cytoplasmic Ca, referred to as Ca oscillations. Maintenance of these oscillations requires Ca influx across the plasma membrane, which is mediated in part by T-type, Ca3.2 channels. Here we show using genetic mouse models that TRPM7 channels are required to support this Ca influx. Eggs lacking both TRPM7 and Ca3.2 stop oscillating prematurely, indicating that together they are responsible for the majority of Ca influx immediately following fertilization. Fertilized eggs lacking both channels also frequently display delayed resumption of Ca oscillations, which appears to require sperm-egg fusion. TRPM7 and Ca3.2 channels almost completely account for Ca influx observed following store depletion, a process previously attributed to canonical store-operated Ca entry mediated by STIM/ORAI interactions. TRPM7 serves as a membrane sensor of extracellular Mg and Ca concentrations and mediates the effects of these ions on Ca oscillation frequency. When bred to wild-type males, female mice carrying eggs lacking TRPM7 and Ca3.2 are subfertile, and their offspring have increased variance in postnatal weight. These in vivo findings confirm previous observations linking in vitro experimental alterations in Ca oscillatory patterns with developmental potential and offspring growth. The identification of TRPM7 and Ca3.2 as key mediators of Ca 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.
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http://dx.doi.org/10.1073/pnas.1810422115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6217414PMC
October 2018

Prenatal Bisphenol A Exposure Alters Epithelial Cell Composition in the Rhesus Macaque Fetal Oviduct.

Toxicol Sci 2019 02;167(2):450-457

California National Primate Research Center, University of California.

Bisphenol A (BPA) is an endocrine disrupting compound that is a pervasive environmental contaminant. Although it has been reported to affect the development of a variety of fetal reproductive tissues, data on the effect of fetal BPA exposure on oviducts were extremely limited and were only available in mice. To determine if there are adverse effects of gestational BPA exposure on fetal oviduct, we exposed pregnant rhesus macaques with female fetuses to oral or nonoral BPA during the last trimester of gestation (day 100 to term). After the treatment, fetal oviducts were collected for morphology evaluation. BPA exposure altered the percentages of different cell types (ciliated, nonciliated, and secretory) in the fetal oviduct and resulted in a significant high ciliated cell population in the BPA-exposed fetal oviduct. The distribution of ciliated cells on the epithelium in the BPA-exposed fetal oviduct was also altered. Gestational BPA exposure reduced the expression of mucosubstance and uteroglobin in secretory cells in the fetal oviduct. A comparison of the outcome of the fetal oviduct studies with similar outcomes previously reported in the lung from the same fetuses demonstrates that BPA exhibits opposite effects in these two organs. In conclusion, the BPA-associated alterations in the fetal oviduct could potentially affect the oviduct morphology and function later in life with a negative impact on fertility. The mechanisms of action of the differential response in the oviduct and the lung to BPA exposure require further investigation.
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http://dx.doi.org/10.1093/toxsci/kfy251DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6358242PMC
February 2019

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

Toxicol Pathol 2018 07 12;46(5):574-596. Epub 2018 Jun 12.

2 Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina, USA.

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.
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http://dx.doi.org/10.1177/0192623318779326DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6027618PMC
July 2018

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

Proc Natl Acad Sci U S A 2018 05 16;115(18):E4189-E4198. Epub 2018 Apr 16.

Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709;

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.
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http://dx.doi.org/10.1073/pnas.1719010115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5939078PMC
May 2018

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

Nucleic Acids Res 2018 06;46(11):5487-5503

Reproductive & Developmental Biology Laboratory, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC 27709, USA.

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.
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http://dx.doi.org/10.1093/nar/gky260DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6009594PMC
June 2018

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

Biol Reprod 2018 04;98(4):449-464

Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA.

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.
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http://dx.doi.org/10.1093/biolre/ioy004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6279059PMC
April 2018

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

EMBO J 2017 11 11;36(21):3175-3193. Epub 2017 Oct 11.

Signal Transduction Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA

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 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.
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http://dx.doi.org/10.15252/embj.201796708DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5666621PMC
November 2017

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

J Pathol 2017 06 3;242(2):246-259. Epub 2017 May 3.

Department of Pathology and Laboratory Medicine, Geffen School of Medicine at UCLA, Los Angeles, CA, 90095, USA.

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.
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http://dx.doi.org/10.1002/path.4893DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5444952PMC
June 2017

Store-operated Ca entry is not required for fertilization-induced Ca signaling in mouse eggs.

Cell Calcium 2017 07 11;65:63-72. Epub 2017 Feb 11.

Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA. Electronic address:

Repetitive oscillations in cytoplasmic Ca due to periodic Ca release from the endoplasmic reticulum (ER) drive mammalian embryo development following fertilization. Influx of extracellular Ca to support the refilling of ER stores is required for sustained Ca oscillations, but the mechanisms underlying this Ca influx are controversial. Although store-operated Ca entry (SOCE) is an appealing candidate mechanism, several groups have arrived at contradictory conclusions regarding the importance of SOCE in oocytes and eggs. To definitively address this question, Ca influx was assessed in oocytes and eggs lacking the major components of SOCE, the ER Ca sensor STIM proteins, and the plasma membrane Ca channel ORAI1. We generated oocyte-specific conditional knockout (cKO) mice for Stim1 and Stim2, and also generated Stim1/2 double cKO mice. Females lacking one or both STIM proteins were fertile and their ovulated eggs displayed normal patterns of Ca oscillations following fertilization. In addition, no impairment was observed in ER Ca stores or Ca influx following store depletion. Similar studies were performed on eggs from mice globally lacking ORAI1; no abnormalities were observed. Furthermore, spontaneous Ca influx was normal in oocytes from Stim1/2 cKO and ORAI1-null mice. Finally, we tested if TRPM7-like channels could support spontaneous Ca influx, and found that it was largely prevented by NS8593, a TRPM7-specific inhibitor. Fertilization-induced Ca oscillations were also impaired by NS8593. Combined, these data robustly show that SOCE is not required to support appropriate Ca signaling in mouse oocytes and eggs, and that TRPM7-like channels may contribute to Ca influx that was previously attributed to SOCE.
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http://dx.doi.org/10.1016/j.ceca.2017.02.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5461193PMC
July 2017

CNOT3-Dependent mRNA Deadenylation Safeguards the Pluripotent State.

Stem Cell Reports 2016 11 13;7(5):897-910. Epub 2016 Oct 13.

Epigenetics and Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA. Electronic address:

Poly(A) tail length and mRNA deadenylation play important roles in gene regulation. However, how they regulate embryonic development and pluripotent cell fate is not fully understood. Here we present evidence that CNOT3-dependent mRNA deadenylation governs the pluripotent state. We show that CNOT3, a component of the Ccr4-Not deadenylase complex, is required for mouse epiblast maintenance. It is highly expressed in blastocysts and its deletion leads to peri-implantation lethality. The epiblast cells in Cnot3 deletion embryos are quickly lost during diapause and fail to outgrow in culture. Mechanistically, CNOT3 C terminus is required for its interaction with the complex and its function in embryonic stem cells (ESCs). Furthermore, Cnot3 deletion results in increases in the poly(A) tail lengths, half-lives, and steady-state levels of differentiation gene mRNAs. The half-lives of CNOT3 target mRNAs are shorter in ESCs and become longer during normal differentiation. Together, we propose that CNOT3 maintains the pluripotent state by promoting differentiation gene mRNA deadenylation and degradation, and we identify poly(A) tail-length regulation as a post-transcriptional mechanism that controls pluripotency.
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http://dx.doi.org/10.1016/j.stemcr.2016.09.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5106518PMC
November 2016

Soy Formula and Epigenetic Modifications: Analysis of Vaginal Epithelial Cells from Infant Girls in the IFED Study.

Environ Health Perspect 2017 03 19;125(3):447-452. Epub 2016 Aug 19.

Epigenetics and Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina, USA.

Background: Early-life exposure to estrogenic compounds affects the development of the reproductive system in rodent models and humans. Soy products, which contain phytoestrogens such as genistein, are one source of exposure in infants fed soy formula, and they result in high serum concentrations.

Objectives: Our goal was to determine whether soy exposure is associated with differential DNA methylation in vaginal cells from soy-fed infant girls.

Methods: Using the Illumina HumanMethylation450 BeadChip, we evaluated epigenome-wide DNA methylation in vaginal cells from four soy formula-fed and six cow formula-fed girls from the Infant Feeding and Early Development (IFED) study. Using pyrosequencing we followed up the two most differentially methylated sites in 214 vaginal cell samples serially collected between birth and 9 months of age from 50 girls (28 soy formula-fed and 22 cow formula-fed). With a mouse model, we examined the effect of neonatal exposure to genistein on gene specific mRNA levels in vaginal tissue.

Results: The epigenome-wide scan suggested differences in methylation between soy formula-fed and cow formula-fed infants at three CpGs in the gene proline rich 5 like () ( < 10). Pyrosequencing of the two feeding groups found that methylation levels progressively diverged with age, with pointwise differences becoming statistically significant after 126 days. Genistein-exposed mice showed a 50% decrease in vaginal mRNA levels compared to controls.

Conclusions: Girls fed soy formula have altered DNA methylation in vaginal cell DNA which may be associated with decreased expression of an estrogen-responsive gene. Citation: Harlid S, Adgent M, Jefferson WN, Panduri V, Umbach DM, Xu Z, Stallings VA, Williams CJ, Rogan WJ, Taylor JA. 2017. Soy formula and epigenetic modifications: analysis of vaginal epithelial cells from infant girls in the IFED study. Environ Health Perspect 125:447-452; http://dx.doi.org/10.1289/EHP428.
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http://dx.doi.org/10.1289/EHP428DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5332195PMC
March 2017

Environmental factors, epigenetics, and developmental origin of reproductive disorders.

Reprod Toxicol 2017 03 12;68:85-104. Epub 2016 Jul 12.

Reproductive Medicine Group, Reproductive & Developmental Biology Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC, United States. Electronic address:

Sex-specific differentiation, development, and function of the reproductive system are largely dependent on steroid hormones. For this reason, developmental exposure to estrogenic and anti-androgenic endocrine disrupting chemicals (EDCs) is associated with reproductive dysfunction in adulthood. Human data in support of "Developmental Origins of Health and Disease" (DOHaD) comes from multigenerational studies on offspring of diethylstilbestrol-exposed mothers/grandmothers. Animal data indicate that ovarian reserve, female cycling, adult uterine abnormalities, sperm quality, prostate disease, and mating behavior are susceptible to DOHaD effects induced by EDCs such as bisphenol A, genistein, diethylstilbestrol, p,p'-dichlorodiphenyl-dichloroethylene, phthalates, and polyaromatic hydrocarbons. Mechanisms underlying these EDC effects include direct mimicry of sex steroids or morphogens and interference with epigenomic sculpting during cell and tissue differentiation. Exposure to EDCs is associated with abnormal DNA methylation and other epigenetic modifications, as well as altered expression of genes important for development and function of reproductive tissues. Here we review the literature exploring the connections between developmental exposure to EDCs and adult reproductive dysfunction, and the mechanisms underlying these effects.
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http://dx.doi.org/10.1016/j.reprotox.2016.07.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5233640PMC
March 2017

LUTs of blastocyst nuclei cell for quantification.

Mol Reprod Dev 2016 07;83(7):575

Reproductive Medicine Group, Reproductive & Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709.

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http://dx.doi.org/10.1002/mrd.22677DOI Listing
July 2016

SIX1 Oncoprotein as a Biomarker in a Model of Hormonal Carcinogenesis and in Human Endometrial Cancer.

Mol Cancer Res 2016 09 3;14(9):849-58. Epub 2016 Jun 3.

Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, North Carolina. Curriculum in Toxicology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.

Unlabelled: The oncofetal protein sine oculis-related homeobox 1 (SIX1) is a developmental transcription factor associated with carcinogenesis in several human cancer types but has not been investigated in human endometrial cancer. In a model of hormonal carcinogenesis, mice neonatally exposed to the soy phytoestrogen genistein (GEN) or the synthetic estrogen diethylstilbestrol (DES) develop endometrial cancer as adults. Previously, we demonstrated that SIX1 becomes aberrantly expressed in the uteri of these mice. Here, we used this mouse model to investigate the role of SIX1 expression in endometrial carcinoma development and used human tissue microarrays to explore the utility of SIX1 as a biomarker in human endometrial cancer. In mice neonatally exposed to GEN or DES, the Six1 transcript level increased dramatically over time in uteri at 6, 12, and 18 months of age and was associated with development of endometrial carcinoma. SIX1 protein localized within abnormal basal cells and all atypical hyperplastic and neoplastic lesions. These findings indicate that developmental estrogenic chemical exposure induces persistent endometrial SIX1 expression that is strongly associated with abnormal cell differentiation and cancer development. In human endometrial tissue specimens, SIX1 was not present in normal endometrium but was expressed in a subset of endometrial cancers in patients who were also more likely to have late-stage disease. These findings identify SIX1 as a disease biomarker in a model of hormonal carcinogenesis and suggest that SIX1 plays a role in endometrial cancer development in both mice and women.

Implications: The SIX1 oncoprotein is aberrantly expressed in the endometrium following developmental exposure to estrogenic chemicals, correlates with uterine cancer, and is a biomarker in human endometrial cancers. Mol Cancer Res; 14(9); 849-58. ©2016 AACR.
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http://dx.doi.org/10.1158/1541-7786.MCR-16-0084DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5025359PMC
September 2016

Oviductal estrogen receptor α signaling prevents protease-mediated embryo death.

Elife 2015 Dec 1;4:e10453. Epub 2015 Dec 1.

Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, United States.

Development of uterine endometrial receptivity for implantation is orchestrated by cyclic steroid hormone-mediated signals. It is unknown if these signals are necessary for oviduct function in supporting fertilization and preimplantation development. Here we show that conditional knockout (cKO) mice lacking estrogen receptor α (ERα) in oviduct and uterine epithelial cells have impaired fertilization due to a dramatic reduction in sperm migration. In addition, all successfully fertilized eggs die before the 2-cell stage due to persistence of secreted innate immune mediators including proteases. Elevated protease activity in cKO oviducts causes premature degradation of the zona pellucida and embryo lysis, and wild-type embryos transferred into cKO oviducts fail to develop normally unless rescued by concomitant transfer of protease inhibitors. Thus, suppression of oviductal protease activity mediated by estrogen-epithelial ERα signaling is required for fertilization and preimplantation embryo development. These findings have implications for human infertility and post-coital contraception.
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http://dx.doi.org/10.7554/eLife.10453DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4718728PMC
December 2015

CaV3.2 T-type channels mediate Ca²⁺ entry during oocyte maturation and following fertilization.

J Cell Sci 2015 Dec 19;128(23):4442-52. Epub 2015 Oct 19.

Reproductive and Developmental Biology Laboratory, National Institutes of Health, Research Triangle Park, NC 27709, USA

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.
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http://dx.doi.org/10.1242/jcs.180026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4712821PMC
December 2015

Lou Guillette--in memorandum.

Mol Reprod Dev 2015 Oct;82(10):Fmi-v

College of Life and Environmental Sciences, University of Exeter.

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http://dx.doi.org/10.1002/mrd.22587DOI Listing
October 2015

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

Development 2015 Aug 9;142(15):2633-40. Epub 2015 Jul 9.

Department of Cell Biology, UConn Health, Farmington, CT 06030, USA

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.
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http://dx.doi.org/10.1242/dev.121707DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4529029PMC
August 2015