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Carmen J Williams

Publications and source records attributed to Carmen J Williams.

At least 19 recordsLinked to original sources
PubMed2006

Expression of epithelial membrane protein-2 is associated with endometrial adenocarcinoma of unfavorable outcome.

BACKGROUND: Epithelial membrane protein 2 (EMP2) is an estrus-regulated tetraspan protein that is required for endometrial competence in blastocyst implantation. EMP2 controls surface levels of several classes of integrin and other cell-interaction molecules, and their trafficking to glycolipid-enriched lipid raft domains is important in receptor signaling. These features suggest that EMP2 may contribute to neoplastic traits of endometrial cancer. The objective of this study was to determine the prevalence of EMP2 expression in endometrial neoplasms and its clinical significance. METHODS: EMP2 immunophenotype, histologic diagnosis, grade, the presence of lymphovascular invasion, disease stage, and clinical follow-up were determined for 99 endometrial cancers. RESULTS: Significant EMP2 expression (EMP2 positive) was observed in 12 of 99 cancers (9 endometrioid [6 International Federation of Gynecology and Obstetrics Grade 3], 1 serous, 1 mixed endometrioid and serous, and 1 mixed endometrioid and clear cell), and weak EMP2 expression was observed in 11 cancers. EMP2-positive tumors were more likely than others to be myometrium invasive, high stage, and recurrent, persistent, or fatal. The overall median survival for patients with EMP2-positive tumor was only 23 months, whereas the medial survival was not reached for patients with EMP2-weak and EMP2-negative tumors. The median disease-free interval was only 11 months for patients with EMP2-positive tumors and was not reached for patients with EMP2-weak and EMP2-negative tumors. A multivariate analysis of disease-free survival demonstrated independent, negative prognostic significance for EMP2 expression, high stage, and high-risk histologic subtypes. CONCLUSIONS: EMP2 expression is a feature of some prognostically unfavorable endometrial cancers. Its utility for clinical decision making and its biologic role in endometrial cancer deserves further study in a larger series of patients.

Adenocarcinoma↗
PubMed2006

Calmodulin-dependent protein kinase II triggers mouse egg activation and embryo development in the absence of Ca2+ oscillations.

Fertilization in mammalian eggs is accompanied by oscillatory changes in intracellular Ca(2+) concentration, which are critical for initiating and completing egg activation events and the developmental program. Ca(2+)/Camodulin-dependent protein kinase II (CaMKII) is a multifunctional enzyme that is postulated to be the downstream transducer of the Ca(2+) signal in many cell types. We tested the hypothesis that CaMKII is the major integrator of Ca(2+)-induced egg activation events and embryo development by microinjecting a cRNA that encodes a constitutively active (Ca(2+)-independent) mutant form of CaMKII (CA-CaMKII) into mouse eggs. Expression of this cRNA, which does not increase intracellular Ca(2+), induced a sustained rise in CaMKII activity and triggered egg activation events, including cell cycle resumption, and degradation and recruitment of maternal mRNAs; cortical granule exocytosis, however, did not occur normally. Furthermore, when mouse eggs were injected with sperm devoid of Ca(2+)-releasing activity and activated with either CA-CaMKII cRNA or by SrCl(2), similar rates and incidence of development to the blastocyst stage were observed. These results strongly suggest that CaMKII is a major integrator of the Ca(2+) changes that occur following fertilization.

Animals↗
PubMed2006

Knockdown of the tetraspan protein epithelial membrane protein-2 inhibits implantation in the mouse.

The establishment of pregnancy requires a successful molecular interaction between the trophectoderm cells of the blastocyst stage embryo and the endometrial cells of the uterus. These interactions are complex and require synchronous development and coordinated endocrine, paracrine, and autocrine communication. In this study, we demonstrate that the tetraspan protein epithelial membrane protein-2 (EMP2) is involved in these molecular interactions during implantation. EMP2, which is highly expressed in the uterus, translocates from an intracellular location to the apical surface of the endometrial epithelium during the window of implantation and is expressed in decidualized stromal cells. We developed plasmid constructs that utilized either ribozyme-mediated or short hairpin RNA-mediated mechanisms to target endometrial EMP2 mRNA for destruction. These constructs were transfected into the mouse uterus on day 1 of pregnancy using the technique of in vivo reproductive tract gene transfer. Reduction in EMP2 expression by either method resulted in a significant decrease in the number of implantation sites in the treated uterine horns as compared to control horns. These studies indicate a previously unknown function of tetraspan proteins in implantation and could provide a molecular framework for the development of therapeutic modalities for both contraception and fertility.

Animals↗
PubMed2006

Knockdown of the cAMP-dependent protein kinase (PKA) Type Ialpha regulatory subunit in mouse oocytes disrupts meiotic arrest and results in meiotic spindle defects.

In mammalian oocytes, cyclic AMP-dependent protein kinase (PKA) is responsible for maintaining meiotic arrest. We examined the role of the predominant regulatory subunit, RIalpha in regulating PKA activity during mouse oocyte maturation by knocking down the protein levels using an RNA interference approach. In oocytes in which RIalpha protein was reduced to non-detectable levels, compensatory decreases were also observed in the RIIalpha and catalytic (Calpha) subunit levels. These oocytes resumed meiosis, despite culture under conditions that maintain elevated intracellular cAMP levels, suggesting that the remaining Calpha was not sufficient to maintain meiotic arrest. The resulting eggs, however, displayed meiotic spindle abnormalities and abnormal cleavage planes leading to extrusion of large polar bodies. These results demonstrate that RIalpha is required for regulating PKA activity in maturing oocytes and that compensatory upregulation of RII does not occur. Furthermore, we implicate PKA as a modulator of spindle morphology and function during meiosis.

Animals↗
PubMed2005

Epithelial membrane protein-2 regulates surface expression of alphavbeta3 integrin in the endometrium.

The four-transmembrane protein epithelial membrane protein-2 (EMP2) was recently identified as an endometrial protein necessary for blastocyst implantation, but the mechanism of this role is uncertain. In other cell types, EMP2 controls delivery of certain classes of proteins to the cell surface, including various integrin isoforms (a class of receptors implicated in endometrial-blastocyst interaction). Since alphavbeta3 integrin is an important endometrial molecule involved in blastocyst interaction, we evaluated the role of EMP2 in modulating integrin expression in the HEC1A endometrial cell line and endometrial epithelium in vivo. Elevation of EMP2 expression in HEC1A cells selectively increased the expression of alphavbeta3 integrin on the plasma membrane and was functional as judged by increased cell binding to an alphavbeta3 ligand, fibronectin. Conversely, reduction in EMP2 expression using an EMP2 specific ribozyme decreased the cell alphavbeta3 surface expression. The influence of EMP2 on alphavbeta3 integrin was also observed in vivo as reduction of EMP2 using ribozymes or short hairpin RNA diminished alphavbeta3 integrin expression in glandular and luminal uterine epithelium. Colocalization and coimmunoprecipitation studies suggested that EMP2 and alphavbeta3 integrin predominantly exist in a physically associated state. This study demonstrates for the first time the influence of EMP2 on alphavbeta3 surface expression and suggests that surface trafficking of integrin alphavbeta3 by EMP2 during the window of implantation may be a mechanism for its requirement in endometrial-blastocyst interaction.

Animals↗
PubMed2005

PAR-3 defines a central subdomain of the cortical actin cap in mouse eggs.

The evolutionarily conserved partitioning defective (PAR) protein PAR-3 is pivotal for establishing and maintaining cell polarity. During mammalian oocyte maturation, the radially symmetric oocyte is transformed into a highly polarized metaphase II (MII)-arrested egg. We therefore examined several aspects of PAR-3 expression during oocyte maturation. We cloned two novel PAR-3 transcripts from an oocyte library that likely encode proteins of Mr = 73 K and 133 K that are phosphorylated during maturation. PAR-3, which is found throughout the GV-intact oocyte, becomes asymmetrically localized during meiosis. Following germinal vesicle breakdown, PAR-3 surrounds the condensing chromosomes and associates with the meiotic spindles. Prior to emission of the first and second polar bodies, PAR-3 is located within a central subdomain of the polarized actin cap, which overlies the spindle. This cortical PAR-3 localization depends on intact microfilaments. These results suggest a role for PAR-3 in establishing asymmetry in the egg and in defining the future site of polar body emission.

Actin Cytoskeleton↗
PubMed2005

Phosphorylated MARCKS: a novel centrosome component that also defines a peripheral subdomain of the cortical actin cap in mouse eggs.

MARCKS (myristoylated alanine-rich C-kinase substrate) is a major substrate for protein kinase C (PKC), a kinase that has multiple functions during oocyte maturation and egg activation, for example, spindle function and cytoskeleton reorganization. We examined temporal and spatial changes in p-MARCKS localization during maturation of mouse oocytes and found that p-MARCKS is a novel centrosome component based its co-localization with pericentrin and gamma-tubulin within microtubule organizing centers (MTOCs). Like pericentrin, p-MARCKS staining at the MI spindle poles was asymmetric. Based on this asymmetry, we found that one end of the spindle was preferentially extruded with the first polar body. At MII, however, the spindle poles had symmetrical p-MARCKS staining. p-MARCKS also was enriched in the periphery of the actin cap overlying the MI or MII spindle to form a ring-shaped subdomain. Because phosphorylation of MARCKS modulates its actin crosslinking function, this localization suggests p-MARCKS functions as part of the contractile apparatus during polar body emission. Our finding that an activator of conventional and novel PKC isoforms did not increase the amount of p-MARCKS suggested that an atypical isoform was responsible for MARCKS phosphorylation. Consistent with this idea, immunostaining revealed that the staining patterns of p-MARCKS and the active form of the atypical PKC zeta/lambda isoform(s) were very similar. These results show that p-MARCKS is a novel centrosome component and also defines a previously unrecognized subdomain of the actin cap overlying the spindle.

Actins↗
PubMed2005

Role of MAP kinase and myosin light chain kinase in chromosome-induced development of mouse egg polarity.

During maturation, the mouse oocyte is transformed into a highly polarized egg, characterized by an actin cap and cortical granule-free domain (CGFD) overlying the meiotic spindle that is in close proximity to the cortex. The presence of spindle/chromosomes or microinjected sperm chromatin in the cortical region initiates this cortical reorganization, but the pathway is unknown. We report that cortical reorganization induced by microinjected sperm chromatin is blocked by inhibitors of microfilament assembly or disassembly. Active mitogen-activated protein kinase (MAPK), which becomes enriched in the region of sperm chromatin, is required for cortical reorganization, because microinjected sperm chromatin fails to induce cortical reorganization in Mos-/- eggs, which lack MAPK activity. Last, myosin light chain kinase (MLCK), which can be directly phosphorylated and activated by MAPK, appears involved, because the MLCK inhibitors ML-7 and Peptide 18 prevent sperm chromatin-induced cortical reorganization. These results provide new insights into how cortical reorganization occurs independently of extracellular signals to generate egg polarity.

Actins↗
PubMed2005

Differential RNA expression and polyribosome loading of alternative transcripts of the Akap4 gene in murine spermatids.

An X chromosome-linked gene, Akap4, is expressed only during spermiogenesis and encodes the major fibrous sheath protein of the mouse sperm flagellum. All sperm contain the AKAP4 protein even though only X chromosome-bearing spermatids express the gene, indicating that the Akap4 mRNA and/or protein must be shared among the conjoined spermatids via the intercellular bridges. There are two mouse Akap4 cDNA clones, Akap82 and Fsc1, which represent mRNAs that arise by alternative processing of a single gene. Although Akap82 and Fsc1 encode identical mature proteins, they differ in their 5' UTRs. We hypothesized that the expression pattern of these two mRNAs might be relevant to the issue of mRNA and/or protein transport into adjacent spermatids. Expression of both transcripts began in round spermatids, but the amount of the Akap82 transcript in condensing spermatids increased twofold relative to Fsc1. Significantly, only the Akap82 transcript was found on polyribosomes and translated in spermatids. These results indicate that the Akap82 transcript and/or its protein must be shared among the conjoined X and Y chromosome-bearing spermatids. Although Fsc1 was not polysomal, both the Akap82 and Fsc1 transcripts were deadenylated during spermiogenesis, suggesting that deadenylation is not always correlated with loading of mRNAs onto polyribosomes in germ cells. The distinct 5' UTR sequences in Akap82 and Fsc1 did not differ in their ability to regulate translation of reporter constructs either in vivo or in vitro. Antisense RNA transcripts complementary to both the Akap82 and Fsc1 mRNAs were present, suggesting that translatability may be regulated by these RNAs.

5' Untranslated Regions↗
PubMed2005

The "soluble" adenylyl cyclase in sperm mediates multiple signaling events required for fertilization.

Mammalian fertilization is dependent upon a series of bicarbonate-induced, cAMP-dependent processes sperm undergo as they "capacitate," i.e., acquire the ability to fertilize eggs. Male mice lacking the bicarbonate- and calcium-responsive soluble adenylyl cyclase (sAC), the predominant source of cAMP in male germ cells, are infertile, as the sperm are immotile. Membrane-permeable cAMP analogs are reported to rescue the motility defect, but we now show that these "rescued" null sperm were not hyperactive, displayed flagellar angulation, and remained unable to fertilize eggs in vitro. These deficits uncover a requirement for sAC during spermatogenesis and/or epididymal maturation and reveal limitations inherent in studying sAC function using knockout mice. To circumvent this restriction, we identified a specific sAC inhibitor that allowed temporal control over sAC activity. This inhibitor revealed that capacitation is defined by separable events: induction of protein tyrosine phosphorylation and motility are sAC dependent while acrosomal exocytosis is not dependent on sAC.

Acrosome↗
PubMed2005

Genomic imprinting and assisted reproductive technology: connections and potential risks.

Assisted reproductive technology (ART) has become the standard of care for the treatment of many types of infertility. As a result, the numbers of children born after ART worldwide has escalated dramatically, and attention has turned to the potential risks of these procedures to the health and development of the children. In addition to the well-established risks of multiple gestations, recent reports have suggested a link between ART and rare disorders of imprinting including Beckwith-Wiedemann syndrome and Angelman syndrome. Here we review molecular mechanisms of genomic imprinting, consider how ART procedures could influence imprinting of gametes and embryos, and review the reports connecting imprinting disorders to ART. We highlight the importance of long-term follow-up of children born after ART, and emphasize the need for an improved understanding of the mechanisms of imprinting at the molecular level so that methods to prevent disruption of this critical epigenetic process can be developed.

Angelman Syndrome↗
PubMed2004

Transgenic RNA interference reveals role for mouse sperm phospholipase Czeta in triggering Ca2+ oscillations during fertilization.

A sperm-specific phospholipase (PL) C, termed PLCzeta, is proposed to be the soluble sperm factor that induces Ca(2+) oscillations in mammalian eggs and, thus, initiates egg activation in vivo. We report that sperm from transgenic mice expressing short hairpin RNAs targeting PLCzeta mRNA have reduced amounts of PLCzeta protein. Sperm derived from these transgenic mice trigger patterns of Ca(2+) oscillations following fertilization in vitro that terminate prematurely. Consistent with the perturbation in patterns of Ca(2+) oscillations is the finding that mating of transgenic founder males to females results in lower rates of egg activation and no transgenic offspring. These data strongly suggest that PLCzeta is the physiological trigger of Ca(2+) oscillations required for activation of development.

Animals↗
PubMed2004

Calreticulin on the mouse egg surface mediates transmembrane signaling linked to cell cycle resumption.

Calreticulin, a protein best known as an endoplasmic reticulum chaperone, also is found on the extracellular plasma membrane surface of many cell types where it serves as a mediator of adhesion and as a regulator of the immune response. In this report, we demonstrate that calreticulin is present on the extracellular surface of the mouse egg plasma membrane and is increased in the perivitelline space after egg activation. The extracellular calreticulin appears to be secreted by vesicles in the egg cortex that are distinct from cortical granules. An anticalreticulin antibody binds to extracellular calreticulin on live eggs and inhibits sperm-egg binding but not fusion. In addition, engagement of cell surface calreticulin by incubation of mouse eggs in the presence of anticalreticulin antibodies results in alterations in the localization of cortical actin and the resumption of meiosis as indicated by alterations in chromatin configuration, decreases in cdc2/cyclin B1 and MAP kinase activities, and pronuclear formation. These events occur in the absence of any observable alterations in intercellular calcium. These data demonstrate that calreticulin functionally interacts with the egg cytoskeleton and can mediate transmembrane signaling linked to cell cycle resumption. These studies suggest a role for calreticulin as a lectin that may be involved in signal transduction events during or after sperm-egg interactions at fertilization.

Actins↗
PubMed2004

Requirements for glucose beyond sperm capacitation during in vitro fertilization in the mouse.

In both the mouse and the human, it is a point of controversy whether glucose is necessary for in vitro fertilization. Some of this controversy has resulted from a failure to distinguish between requirements for glucose during sperm capacitation versus requirements during the multistage process of fertilization. Using the mouse as a model, we performed a series of experiments designed to identify specific processes that might require glucose. We observed a positive correlation between increasing glucose concentrations during capacitation and fertilization, and increasing fertilization of zona pellucida (ZP)-intact eggs. These data supported a requirement for glucose in the fertilization medium even when sperm were first capacitated in the presence of 5.5 mM glucose. This glucose requirement was observed for both ZP-intact and ZP-free eggs. During ZP-free in vitro fertilization, some binding and fusion between the plasma membrane of the sperm and egg occurred in the absence of glucose and at concentrations less than 1 mM, suggesting that this substrate is not absolutely required. However, glucose concentrations of 1 mM or higher greatly facilitated both binding and fusion under these conditions. These subtle distinctions suggest that during ZP-free in vitro fertilization, 1 mM glucose represents a threshold level that facilitates binding and fusion. Taken as a whole, the data suggest requirements for glucose during both capacitation and fertilization under normal physiologic conditions.

Animals↗
PubMed2004

Long-term effects of culture of preimplantation mouse embryos on behavior.

Many procedures used in assisted reproductive technologies (ART) to treat human infertility entail culture of preimplantation embryos. Moreover, there is an increasing trend to culture embryos for longer periods of time before uterine transfer to identify the "best" embryos for transfer and to minimize multiple pregnancies. Embryo culture, however, can perturb embryo metabolism and gene expression, and the long-term consequences of culture are unknown. We have explored the behavioral consequences of embryo culture by using a 129S6/SvEvTac/C57BL/6J F(1) mouse model and find that adults derived from cultured embryos exhibit specific behavioral alterations in the elevated zero maze and Morris water maze tasks.

Animals↗
PubMed2003

AKAP7gamma is a nuclear RI-binding AKAP.

Spatial regulation of protein kinase A (PKA) is accomplished by its sequestration via A-kinase anchor proteins (AKAPs). PKA activity is critical for mammalian oocyte development, suggesting that PKA must be appropriately positioned in these large cells. A screen for AKAPs in oocytes identified AKAP7gamma, an AKAP originally found in pancreas. Yeast two-hybrid analysis and co-immunoprecipitation studies showed that AKAP7gamma bound the type I PKA regulatory subunit (RI) and that the RI-binding domain overlapped the previously identified type II PKA regulatory subunit (RII) binding domain. Overexpressed AKAP7gamma localized to the nuclei of HEK 293 cells via a nuclear localization signal. In addition, endogenous AKAP7gamma protein was found in both the nucleus and cytoplasm of oocytes. This work identifies AKAP7gamma as the first nuclear AKAP to bind RI and suggests that AKAP7gamma may be responsible for positioning PKA via RI and/or RII to regulate PKA-mediated gene transcription in both somatic cells and oocytes.

A Kinase Anchor Proteins↗
PubMed2003

Chromatin-mediated cortical granule redistribution is responsible for the formation of the cortical granule-free domain in mouse eggs.

A cortical granule-free domain (CGFD) overlies the metaphase chromatin in fully mature mouse eggs. Although a chromatin-induced localized release of cortical granules (CG) during maturation is thought to be a major contributing factor to its formation, there are indications that CG redistribution may also be involved in generating the CGFD. We performed experiments to determine the relative contributions of CG exocytosis and redistribution in generating the CGFD. We found that the CGFD-inducing activity was not specific to female germ cell chromatin and was heat stable but sensitive to DNase and protease treatment. Surprisingly, chelation of egg intracellular Ca(2+) levels did not prevent CGFD formation in response to microinjection of exogenous chromatin, suggesting that development of the CGFD was not a result of CG exocytosis. This finding was confirmed by the lack of CG exudate on the plasma membrane surface of the injected eggs and the absence of conversion of ZP2 to ZP2(f) during formation of the new CGFD. Moreover, clamping intracellular Ca(2+) did not prevent the formation of the CGFD during oocyte maturation, but did inhibit the maturation-associated release of CGs between metaphase I and II. Results of these experiments suggest that CG redistribution is the dominant factor in formation of the CGFD.

Animals↗