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Kelle H Moley

Publications and source records attributed to Kelle H Moley.

At least 19 recordsLinked to original sources
PubMed2007

A randomized, prospective study comparing laser-assisted hatching and assisted hatching using acidified medium.

OBJECTIVE: To compare the clinical outcomes of patients whose transferred embryos underwent either laser-assisted hatching or hatching with acidified medium. DESIGN: Randomized, prospective, double-blinded study. SETTING: University-based IVF center. PATIENT(S): Clinical outcomes following IVF were compared in 159 patient cycles who randomly had all transferred embryos undergo laser-assisted hatching (Laser; n = 57) or hatching with acidified medium (Acid; n = 54). Patients whose embryos had zonae thickness <13 mum were not hatched (Thin; n = 8) and patients with > or =4 embryos at the 7- to 8-cell stage or higher on Day 3 were transferred on Day 5 (Day 5; n = 40). MAIN OUTCOME MEASURE(S): Implantation rates, clinical pregnancy rates, ongoing pregnancy rates, multiple pregnancy rates. RESULT(S): No significant differences were noted in clinical, ongoing, or multiple pregnancy rates between the four groups. The implantation rate for Day 5 transfers was significantly greater than both the Laser and Acid treatment groups. CONCLUSION: The use of a 1.48-micron infrared diode laser beam provides a safe and rapid method for performing assisted hatching and did not have a negative impact on patient care when compared to outcomes achieved using acidified medium.

Adult↗
PubMed2006

AMPK regulation of mouse oocyte meiotic resumption in vitro.

We have previously shown that the adenosine analog 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR), an activator of AMP-activated protein kinase (AMPK), stimulates an increase in AMPK activity and induces meiotic resumption in mouse oocytes [Downs, S.M., Hudson, E.R., Hardie, D.G., 2002. A potential role for AMP-activated protein kinase in meiotic induction in mouse oocytes. Dev. Biol, 245, 200-212]. The present study was carried out to better define a causative role for AMPK in oocyte meiotic maturation. When microinjected with a constitutively active AMPK, about 20% of mouse oocytes maintained in meiotic arrest with dibutyryl cAMP (dbcAMP) were stimulated to undergo germinal vesicle breakdown (GVB), while there was no effect of catalytically dead kinase. Western blot analysis revealed that germinal vesicle (GV)-stage oocytes cultured in dbcAMP-containing medium plus AICAR possessed elevated levels of active AMPK, and this was confirmed by AMPK assays using a peptide substrate of AMPK to directly measure AMPK activity. AICAR-induced meiotic resumption and AMPK activation were blocked by compound C or adenine 9-beta-d-arabinofuranoside (araA, a precursor of araATP), both inhibitors of AMPK. Compound C failed to suppress adenosine uptake and phosphorylation, indicating that it did not block AICAR action by preventing its metabolism to the AMP analog, ZMP. 2'-deoxycoformycin (DCF), a potent adenosine deaminase inhibitor, reversed the inhibitory effect of adenosine on oocyte maturation by modulating intracellular AMP levels and activating AMPK. Rosiglitazone, an anti-diabetic agent, stimulated AMPK activation in oocytes and triggered meiotic resumption. In spontaneously maturing oocytes, GVB was preceded by AMPK activation and blocked by compound C. Collectively, these results support the proposition that active AMPK within mouse oocytes provides a potent meiosis-inducing signal in vitro.

AMP-Activated Protein Kinases↗
PubMed2006

Glucose utilization and the PI3-K pathway: mechanisms for cell survival in preimplantation embryos.

The maintenance of optimal glucose utilization during the preimplantation period is critical for embryo survival. A decrease in glucose transport during preimplantation development has been linked to the early steps of programmed cell death in these embryos. Decreased glucose transport is not thought to be simply a consequence of cell death, rather it is thought to be a trigger that can initiate the apoptotic cascade. Extensive apoptosis during the preimplantation period may manifest later in pregnancy as a malformation-- or miscarriage, if cell loss is excessive. Phosphatidylinositol 3-kinase (PI3-K) is a known regulator of a number of physiologic responses including cellular proliferation, growth, and survival as well as glucose metabolism. Studies performed in other cell systems have demonstrated that the PI3-K pathway plays a critical role in maintaining glucose transport and metabolism. This review will present the current evidence that suggests that PI3-K is vital for preimplantation embryo survival and development. In addition, data demonstrating that PI3-K activity is important for glucose metabolism during this early developmental period will be discussed.

Animals↗
PubMed2005

Mouse glucose transporter 9 splice variants are expressed in adult liver and kidney and are up-regulated in diabetes.

A novel glucose transporter (GLUT), mouse GLUT9 (mGLUT9), was recently cloned from mouse 7-d embryonic cDNA. Several splice variants of mGLUT9 were described, two of which were cloned (mGLUT9a and mGLUT9a Delta 209-316). This study describes the cloning and characterization of another splice variant, mGLUT9b. Cloned from adult liver, mGLUT9b is identical to mGLUT9a except at the amino terminus. Based on analysis of the genomic structure, the different amino termini result from alternative transcriptional/translational start sites. Expression and localization of these two mGLUT9 splice variants were examined in control and diabetic adult mouse tissues and in cell lines. RT-PCR analysis demonstrated expression of mGLUT9a in several tissues whereas mGLUT9b was observed primarily in liver and kidney. Using a mGLUT9-specific antibody, Western blot analysis of total membrane fractions from liver and kidney detected a single, wide band, migrating at approximately 55 kDa. This band shifted to a lower molecular mass when deglycosylated with peptide-N-glycosidase F. Both forms were present in liver and kidney. Immunohistochemical localization demonstrated basolateral distribution of mGLUT9 in liver hepatocytes and the expression of mGLUT9 in specific tubules in the outer cortex of the kidney. To investigate the alternative amino termini, mGLUT9a and mGLUT9b were overexpressed in kidney epithelium cell lines. Subcellular fractions localized both forms to the plasma membrane. Immunofluorescent staining of polarized Madin Darby canine kidney cells overexpressing mGLUT9 depicted a basolateral distribution for both splice variants. Finally, mGLUT9 protein expression was significantly increased in the kidney and liver from streptozotocin-induced diabetic mice compared with nondiabetic animals.

Age Factors↗
PubMed2005

Phosphatidylinositol 3-kinase activity is critical for glucose metabolism and embryo survival in murine blastocysts.

The phosphatidylinositol 3-kinase (PI3K) signal transduction pathway is a well known mediator of cell growth, proliferation, and survival signals. Whereas the expression and function of this pathway has been documented during mammalian development, evidence demonstrating the physiologic importance of this pathway in murine preimplantation embryos is beginning to emerge. This study demonstrates that inhibition of the PI3K pathway leads to the induction of apoptosis in both murine blastocysts and trophoblast stem cells. The apoptosis induced in both model systems correlates with a decrease in the expression of the glucose transporter GLUT1 at the plasma membrane. In addition, blastocysts cultured in the presence of the PI3K inhibitor LY-294002 display a decrease in both 2-deoxyglucose uptake and hexokinase activity as compared with control blastocysts. To determine the impact of PI3K inhibition on pregnancy outcome, embryo transfer experiments were performed. Blastocysts cultured in the presence of LY-294002 demonstrate a dramatic increase in fetal resorptions as compared with control embryos. Finally, we demonstrate that impairment of glucose metabolism via iodoacetate, a glyceraldehyde-3-phosphate dehydrogenase inhibitor, is sufficient to induce apoptosis in both blastocysts and trophoblast stem cells. Moreover, blastocysts treated with iodoacetate result in poor pregnancy outcome as determined by embryo transfer experiments. Taken together these data demonstrate the critical importance of the PI3K pathway in preimplantation embryo survival and pregnancy outcome and further emphasize the importance of glucose utilization and metabolism in cell survival pathways.

Animals↗
PubMed2005

The PI3K/Akt pathway is present and functional in the preimplantation mouse embryo.

The PI3K/Akt signal transduction pathway is a well-known mediator of growth promoting and cell survival signals. While the expression and function of this pathway have been documented during early and late stages of the reproductive process, currently, there is no evidence demonstrating either the presence or function of the PI3K/Akt pathway in murine preimplantation embryos. We found, using confocal immunofluorescent microscopy and Western blot analysis, that the p 85 and p110 subunits of PI3K and Akt are expressed from the 1-cell through the blastocyst stage of murine preimplantation embryo development. These proteins were localized predominantly at the cell surface from the 1-cell through the morula stage. At a blastocyst stage, both PI3K and Akt exhibited an apical staining pattern in the trophectoderm cells. Interestingly, phosphorylated Akt was detected throughout murine preimplantation development, and its presence at the plasma membrane is a reflection of its activation status. Inhibition of Akt activity had significant effects on the normal physiology of the blastocyst. Specifically, inhibition of this pathway resulted in a reduction in insulin-stimulated glucose uptake. In addition, inhibiting Akt activity resulted in a significant delay in blastocyst hatching, a developmental step facilitating implantation. Finally, we established the presence of this pathway in trophoblast stem (TS) cells, a potentially useful in vitro model to study this signaling cascade. Taken together, these data are the first to demonstrate the presence and function of the PI3K/Akt pathway in mammalian preimplantation embryos.

Androstadienes↗
PubMed2005

Factors associated with preterm delivery in mothers of children with Beckwith-Wiedemann syndrome: a case cohort study from the BWS registry.

Pregnancy that results in a child with Beckwith-Wiedemann syndrome (BWS) is associated with preterm delivery. Based on previous case series, we hypothesized that preterm delivery of a child with BWS was due to known risk factors for preterm delivery such as polyhydramnios and gestational hypertension. A case cohort study using the BWS Registry at Washington University School of Medicine was undertaken. Cases were pregnancies that resulted in the birth of a child with BWS, controls were pregnancies resulting in the birth of siblings without BWS. Univariate analyses of maternal complications and logistic regression to predict preterm delivery were used. Children with BWS (n = 304) were delivered preterm at a significantly higher rate than their siblings (n = 269) odds ratio 19.1 (95% CI 9.1-40.2). Polyhydramnios, gestational hypertension, and vaginal bleeding also occurred at high rates in the BWS group with odds ratios of 31.6 (95% CI 12.6-79.1), 2.4 (95% CI 1.4-4.1), and 3.9 (95% CI 2.3-6.4), respectively. In a multivariate logistic regression model within the BWS group, polyhydramnios, vaginal bleeding, and gestational hypertension were significant predictors of preterm delivery, odds ratios of 2.9 (95% CI 1.6-5.4), 2.6 (95% CI 1.3-5.0), and 5.3 (2.3-12.0), respectively. However, a significant proportion, 6.5% (18 of 277), of patients in the BWS group delivered preterm without known risk factors. Preterm delivery of a child with BWS is associated with an increased frequency of polyhydramnios, gestational hypertension, and vaginal bleeding in the mother. However, preterm delivery also occurs in the absence of these risk factors.

Adolescent↗
PubMed2005

Maternal diabetes adversely affects preovulatory oocyte maturation, development, and granulosa cell apoptosis.

Maternal diabetes adversely affects preimplantation embryo development and pregnancy outcomes. The objective of this study was to determine whether diabetes has an impact at an earlier stage of development, the preovulatory oocyte. Models of both acute and chronic insulin-dependent diabetes were used. Acute hyperglycemia was induced by a single streptozotocin injection. Akita mice, which harbor an autosomal dominant mutation causing them to be chronically hypoinsulinemic and hyperglycemic, were used. In both models, preovulatory oocytes were markedly smaller when compared with control animals. A significantly greater number of control oocytes had progressed to meiotic maturation before diabetic oocytes. Both models were found to have smaller, less developed ovarian follicles with a greater number of apoptotic foci by histological evaluation as well as by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling staining. Immunohistochemistry displayed a greater amount of TNF-related apoptosis-inducing ligand (TRAIL) and KILLER, a key murine ligand and receptor involved in the extrinsic pathway, expressed in cumulus cells from hyperglycemic mice compared with controls, suggesting that this apoptotic pathway may be up-regulated under diabetic stress. Elevated KILLER expression was also confirmed through Western blotting. Connexin-43 expression was found to be lower by immunohistochemistry and Western blot analysis in the diabetic samples. Both models of maternal hyperglycemia and hypoinsulinemia may have a detrimental effect on oocyte maturation and development as detailed by the smaller sizes of oocytes and developing ovarian follicles, the lowered percentage reaching germinal vesicle breakdown, and the greater amount of apoptosis. In addition, there may be dysfunctional or decreased communication in diabetic oocytes, as demonstrated by lower expression of connexin-43.

Animals↗
PubMed2005

Association between Beckwith-Wiedemann syndrome and assisted reproductive technology: a case series of 19 patients.

OBJECTIVE: An association between assisted reproductive technique (ART) and specific imprinting mutations, such as Beckwith-Wiedemann syndrome (BWS), has recently been documented. Based on experiments in farm animals that demonstrated an association between alterations in culture media during ART and large offspring syndrome, we hypothesized that the culture media could be implicated as a common factor among the children with BWS conceived after ART. DESIGN: Retrospective case series. SETTING: Registry from Academic Medical Center. PATIENT(S): Nineteen children born after ART were identified within the registry. MAIN OUTCOME MEASURE(S): Demographics of patients, type of ART, culture media, IVF parameters. RESULT(S): Twelve of the 19 medical records from the reproductive endocrine centers were successfully obtained. Ten of 12 mothers of children with BWS had IVF, but no single, consistent culture media was used in this group. Half of the patients underwent IVF with intracytoplasmic sperm injection (ICSI; n = 5), whereas the other half had routine IVF. One child was conceived through clomiphene citrate (CC) stimulation and artificial insemination, whereas another patient conceived through gonadotropin stimulation with intrauterine insemination (IUI). The gonadotropin dosage and quantity of embryos transferred also varied significantly. The only consistent finding was that all 12 women received some type of ovarian stimulation medication. CONCLUSION(S): Large epidemiologic studies are needed to further study the association between BWS and ART.

Adolescent↗
PubMed2005

GLUT8 contains a [DE]XXXL[LI] sorting motif and localizes to a late endosomal/lysosomal compartment.

Glucose transporter 8 (GLUT8) contains a cytoplasmic N-terminal dileucine motif and localizes to a thus far unidentified intracellular compartment. Translocation of GLUT8 to the plasma membrane (PM) was found in insulin-treated mouse blastocysts. Using overexpression of GLUT8 in adipocytes and neuronal cells however, insulin treatment or depolarization of the cells did not lead to GLUT8 PM translocation in other studies. In addition, other experiments showing dynamin-dependent endocytosis of GLUT8 suggested that GLUT8 recycles between an endosomal compartment and the PM. To reveal the functional/physiological role of GLUT8, we studied its subcellular localization in 3T3L1, HEK293 and CHO cells. We show that GLUT8 does not co-localize with GLUT4 and does not redistribute to the PM after treatment with insulin, ionophores or okadaic acid in these cell lines. Once endocytosed, GLUT8 does not recycle to the PM. GLUT8 localizes to late endosomes and lysosomes. An interspecies GLUT8 - sequence alignment revealed the presence of a highly conserved late endosomal/lysosomal-targeting motif ([DE]XXXL[LI]). Changing the glutamate to arginine as found in GLUT4 (RRXXXLL) alters GLUT8 endocytosis and retains the transporter at the PM. Furthermore, sorting GLUT8 to late endosomes/lysosomes does not require prior presence of GLUT8 at the PM followed by its endocytosis. In summary, GLUT8 does not reside in a recycling vesicle pool and is distinct from GLUT4. From our data, we postulate a role for GLUT8 in transport of hexoses across intracellular membranes, for example in specific compartments of GLUT8 expression such as the acrosome of mature spermatozoa or secretory granules in neurons. Furthermore, a role for GLUT8 in hexose transport across the lysosomal membrane, a transport mechanism that has long been suggested but unexplained, is discussed.

Amino Acid Sequence↗
PubMed2005

Gametes and embryo epigenetic reprogramming affect developmental outcome: implication for assisted reproductive technologies.

There is concern about the health of children who are conceived with the use assisted reproductive technologies (ART). In addition to reports of low birth weight and chromosomal anomalies, there is evidence that ART may be associated with increased epigenetic disorders in the infants who are conceived using these procedures. Epigenetic reprogramming is critical during gametogenesis and at preimplantation stage and involves DNA methylation, imprinting, RNA silencing, covalent modifications of histones, and remodeling by other chromatin-associated complexes. Epigenetic regulation is involved in early embryo development, fetal growth, and birth weight. Disturbances in epigenetic reprogramming may lead to developmental problems and early mortality. Recent reports suggest the increased incidence of imprinting disorders such as Beckwith-Wiedemann syndrome, Angelman syndrome, and retinoblastoma in children who are conceived with the use of ART. These may result from an accumulation of epigenetic alterations during embryo culture and/or by altered embryonic developmental timing. Further research is urgently needed to determine whether a causal relationship between ART and epigenetic disorders exists. Until then, cautious review of both short-term and long-term ART outcomes at a national level is recommended.

Embryo, Mammalian↗
PubMed2004

TRAIL and KILLER are expressed and induce apoptosis in the murine preimplantation embryo.

TRAIL (tumor necrosis factor [TNF]-related apoptosis-inducing ligand) and KILLER are a death-inducing ligand and receptor pair that belong to the TNF and TNF-receptor superfamilies, respectively. To date, only one apoptosis-inducing TRAIL receptor (murine KILLER [MK]) has been identified in mice, and it is a homologue of human Death Receptor 5. Whereas the expression of other death receptors, such as Fas and TNF receptor 1 have been documented in mammalian preimplantation embryos, no evidence currently demonstrates either the presence or the function of TRAIL and its corresponding death receptor, MK. Using reverse transcription-polymerase chain reaction and confocal immunofluorescent microscopy, we found that both TRAIL and MK are expressed from the 1-cell through the blastocyst stage of murine preimplantation embryo development. These proteins are localized mainly at the cell surface from the 1-cell through the morula stage. At the blastocyst stage, both TRAIL and MK exhibit an apical staining pattern in the trophectoderm cells. Finally, using the TUNEL assay, we demonstrated that MK induces apoptosis in blastocysts sensitized to TRAIL via actinomycin D. Taken together, these data are the first to demonstrate the presence and function of TRAIL and MK, a death-inducing ligand and its receptor, in mammalian preimplantation embryos.

Animals↗
PubMed2004

Identification and characterization of human glucose transporter-like protein-9 (GLUT9): alternative splicing alters trafficking.

The recently cloned human GLUT9 gene, which maps to chromosome 4p15.3-p16, consists of 12 exons coding for a 540-amino acid protein. Based on a sequence entry (NCBI accession number BC018897) and screening of expressed sequence tags, we have cloned an alternative splice variant of GLUT9 from human kidney cDNA. The RNA of this splice variant consists of 13 exons and codes for a putative protein of 512 amino acids (GLUT9DeltaN). The predicted proteins differ only in their N terminus, suggesting a different subcellular localization and possible physiological role. Screening human tissue RNA by reverse transcription-PCR showed that GLUT9 is expressed mainly in kidney, liver, placenta, and leukocytes, whereas GLUT9DeltaN was detected only in kidney and placenta. The GLUT9 protein localized by immunohistochemistry to human kidney proximal tubules, and subcellular fractionation of human kidney revealed the GLUT9 protein in plasma membranes and high density microsomal membranes. Treatment of kidney membrane proteins with peptide N-glycosidase F showed that GLUT9 and GLUT9DeltaN are expressed in vivo. Localization of GLUT9 and GLUT9DeltaN in three kidney-derived cell lines revealed a plasma membrane distribution for GLUT9 in COS-7 and HEK293 cells, whereas GLUT9DeltaN showed a perinuclear pattern and plasma membrane staining in COS-7 and HEK293 cells, respectively. In polarized Madin-Darby canine kidney cells, GLUT9 trafficked to the basolateral membrane, whereas GLUT9DeltaN localized to the apical membrane. Using heterologous expression of GLUT9 in Xenopus oocytes, GLUT9 appears to be a functional isoform with low affinity for deoxyglucose. Deoxyglucose transport mediated by GLUT9 was not inhibited by cytochalasin B. GLUT9 did not bind cytochalasin B as shown by a cytochalasin B binding assay, indicating a similar behavior of GLUT9 compared with GLUT5.

Alternative Splicing↗
PubMed2004

Septate uterus with cervical duplication and longitudinal vaginal septum: a report of five new cases.

OBJECTIVE: To describe a unique congenital müllerian anomaly. DESIGN: Case report. SETTING: A university-based reproductive endocrine center. PATIENT(S): Five reproductive-age, nulligravida patients who underwent clinical, radiologic, and surgical work-up. INTERVENTION(S): Retrospective review of prior medical records and studies. MAIN OUTCOME MEASURE(S): Definition of abnormal pelvic anatomy. RESULT(S): Five patients from a university-based, reproductive endocrine center were found to have cervical duplication with a longitudinal vaginal septum, uterine septum, and a normal fundus. The patients most often presented initially to their primary obstetrician-gynecologists with symptomatic complaints secondary to their vaginal septums. Diagnoses were obtained with physical examinations, ultrasound imaging, hysterosalpingograms, magnetic resonance imaging, and surgical evaluation. CONCLUSION(S): These findings call into question the classic hypothesis of unidirectional (caudal to cranial) müllerian development and supports an alternative embryologic hypothesis of Müller et al., which states that fusion and resorption begins at the isthmus and proceeds simultaneously in both the cranial and caudad directions. The high number of cases reported here might be due to the increased accessibility and accuracy of such imaging modalities as magnetic resonance imaging. These patients will be followed longitudinally so that it can be determined whether this anomaly affects fertility and so that the optimal treatment plans can be developed.

Adult↗
PubMed2003

GLUT9 is differentially expressed and targeted in the preimplantation embryo.

During preimplantation development in the mouse, it is crucial that glucose metabolism not be compromised. Any decrease in glucose uptake at this stage in development can compromise the developing embryo. We have cloned another member of the glucose transporter family, GLUT9, which is expressed embryonically. Three different isoforms were identified. We have shown that two of the mouse GLUT9 isoforms transport glucose at a rate significantly greater than controls. Expression analysis of the preimplantation blastocyst identifies only the presence of the shorter GLUT9 isoform, RT-PCR and Western immunoblot confirmed this finding. A differential pattern of expression was seen with GLUT9 present at the plasma membrane in one- and two-cell zygotes and in an intracellular compartment in trophectoderm cells at a blastocyst stage. Although blocking GLUT9 expression during preimplantation development had no effect on glucose transport or apoptosis, transfer of these embryos into pseudopregnant mice resulted in increased pregnancy loss, suggesting that GLUT9 is critical for early preimplantation development.

Amino Acid Sequence↗
PubMed2003

Syntaxin 4 expression affects glucose transporter 8 translocation and embryo survival.

Target-soluble N-ethylmaleimide-sensitive factor attachment protein receptors (t-SNAREs) are receptors that facilitate vesicle and target membrane fusion. Syntaxin 4 is the t-SNARE critical for insulin-stimulated glucose transporter 4 (GLUT4)-plasma membrane fusion in adipocytes. GLUT8 is a novel IGF-I/insulin-regulated glucose transporter expressed in the mouse blastocyst. Similar to GLUT4, GLUT8 translocates to the plasma membrane to increase glucose uptake at a stage in development when glucose serves as the main substrate. Any decrease in GLUT8 cell surface expression results in increased apoptosis and pregnancy loss. Previous studies have also shown that disruption of the syntaxin 4 (Stx4a) gene results in early embryonic lethality before embryonic d 7.5. We have now demonstrated that syntaxin 4 protein is localized predominantly to the apical plasma membrane of the murine blastocyst. Stx4a inheritance, as detected by protein expression, occurs with the expected Mendelian frequency up to embryonic d 4.5. In parallel, 22% of the blastocysts from Stx4a+/- matings had no significant insulin-stimulated translocation of GLUT8 whereas 77% displayed either partial or complete translocation to the apical plasma membrane. This difference in GLUT8 translocation directly correlated with one-third of blastocysts from Stx4a+/- mating having reduced rates of insulin-stimulated glucose uptake and 67% with wild-type rates. These data demonstrate that the lack of syntaxin 4 expression results in abnormal movement of GLUT8 in response to insulin, decreased insulin-stimulated glucose uptake, and increased apoptosis. Thus, syntaxin 4 functions as the necessary t-SNARE protein responsible for correct fusion of the GLUT8-containing vesicle with the plasma membrane in the mouse blastocyst.

Animals↗