Kelle H. Moley

Hyperglycemia induces apoptosis in pre-implantation embryos through cell death effector pathways

Although perinatal mortality rates have improved for pregnant diabetic women because of insulin therapy and tight metabolic control, infants of diabetics still experience significantly higher rates of congenital malformations and spontaneous miscarriages compared with those of non-diabetic women. Our results here indicate that hyperglycemic conditions, either in vivo or in vitro, modulate the expression of an apoptosis regulatory gene as early as the pre-implantation blastocyst stage in the mouse. Apoptosis in the mammalian pre-implantation blastocyst is a normal process, thought to protect the early embryo by eliminating abnormal cells2. Here we demonstrate that expression of Bax, a Bcl-2-like protein, is increased at the blastocyst stage in the presence of high concentrations of glucose, and that these changes correlate morphologically with increased DNA fragmentation. Expression of Bax and caspase are necessary for this in vitro glucose-induced apoptotic event, and ceramide is involved in mediating this embryotoxic effect of glucose. We also show that these apoptotic cellular changes can be prevented in vivo by treating hyperglycemic mice with insulin before and immediately after conception. These findings emphasize the importance of tight glycemic control in diabetic women at the earliest stages after conception.

SLC2A9 Is a High-Capacity Urate Transporter in Humans

Background Serum uric acid levels in humans are influenced by diet, cellular breakdown, and renal elimination, and correlate with blood pressure, metabolic syndrome, diabetes, gout, and cardiovascular disease. Recent genome-wide association scans have found common genetic variants of SLC2A9 to be associated with increased serum urate level and gout. The SLC2A9 gene encodes a facilitative glucose transporter, and it has two splice variants that are highly expressed in the proximal nephron, a key site for urate handling in the kidney. We investigated whether SLC2A9 is a functional urate transporter that contributes to the longstanding association between urate and blood pressure in man. Methods and Findings We expressed both SLC2A9 splice variants in Xenopus laevis oocytes and found both isoforms mediate rapid urate fluxes at concentration ranges similar to physiological serum levels (200–500 μM). Because SLC2A9 is a known facilitative glucose transporter, we also tested whether glucose or fructose influenced urate transport. We found that urate is transported by SLC2A9 at rates 45- to 60-fold faster than glucose, and demonstrated that SLC2A9-mediated urate transport is facilitated by glucose and, to a lesser extent, fructose. In addition, transport is inhibited by the uricosuric benzbromarone in a dose-dependent manner (K i = 27 μM). Furthermore, we found urate uptake was at least 2-fold greater in human embryonic kidney (HEK) cells overexpressing SLC2A9 splice variants than nontransfected kidney cells. To confirm that our findings were due to SLC2A9, and not another urate transporter, we showed that urate transport was diminished by SLC2A9-targeted siRNA in a second mammalian cell line. In a cohort of men we showed that genetic variants of SLC2A9 are associated with reduced urinary urate clearance, which fits with common variation at SLC2A9 leading to increased serum urate. We found no evidence of association with hypertension (odds ratio 0.98, 95% confidence interval [CI] 0.9 to 1.05, p > 0.33) by meta-analysis of an SLC2A9 variant in six case–control studies including 11,897 participants. In a separate meta-analysis of four population studies including 11,629 participants we found no association of SLC2A9 with systolic (effect size −0.12 mm Hg, 95% CI −0.68 to 0.43, p = 0.664) or diastolic blood pressure (effect size −0.03 mm Hg, 95% CI −0.39 to 0.31, p = 0.82). Conclusions This study provides evidence that SLC2A9 splice variants act as high-capacity urate transporters and is one of the first functional characterisations of findings from genome-wide association scans. We did not find an association of the SLC2A9 gene with blood pressure in this study. Our findings suggest potential pathogenic mechanisms that could offer a new drug target for gout.

The facilitative glucose transporter GLUT3: 20 years of distinction.

Glucose metabolism is vital to most mammalian cells, and the passage of glucose across cell membranes is facilitated by a family of integral membrane transporter proteins, the GLUTs. There are currently 14 members of the SLC2 family of GLUTs, several of which have been the focus of this series of reviews. The subject of the present review is GLUT3, which, as implied by its name, was the third glucose transporter to be cloned (Kayano T, Fukumoto H, Eddy RL, Fan YS, Byers MG, Shows TB, Bell GI. J Biol Chem 263: 15245-15248, 1988) and was originally designated as the neuronal GLUT. The overriding question that drove the early work on GLUT3 was why would neurons need a separate glucose transporter isoform? What is it about GLUT3 that specifically suits the needs of the highly metabolic and oxidative neuron with its high glucose demand? More recently, GLUT3 has been studied in other cell types with quite specific requirements for glucose, including sperm, preimplantation embryos, circulating white blood cells, and an array of carcinoma cell lines. The last are sufficiently varied and numerous to warrant a review of their own and will not be discussed here. However, for each of these cases, the same questions apply. Thus, the objective of this review is to discuss the properties and tissue and cellular localization of GLUT3 as well as the features of expression, function, and regulation that distinguish it from the rest of its family and make it uniquely suited as the mediator of glucose delivery to these specific cells.

Zika virus infection damages the testes in mice

Infection of pregnant women with Zika virus (ZIKV) can cause congenital malformations including microcephaly, which has focused global attention on this emerging pathogen. In addition to transmission by mosquitoes, ZIKV can be detected in the seminal fluid of affected males for extended periods of time and transmitted sexually. Here, using a mouse-adapted African ZIKV strain (Dakar 41519), we evaluated the consequences of infection in the male reproductive tract of mice. We observed persistence of ZIKV, but not the closely related dengue virus (DENV), in the testis and epididymis of male mice, and this was associated with tissue injury that caused diminished testosterone and inhibin B levels and oligospermia. ZIKV preferentially infected spermatogonia, primary spermatocytes and Sertoli cells in the testis, resulting in cell death and destruction of the seminiferous tubules. Less damage was caused by a contemporary Asian ZIKV strain (H/PF/2013), in part because this virus replicates less efficiently in mice. The extent to which these observations in mice translate to humans remains unclear, but longitudinal studies of sperm function and viability in ZIKV-infected humans seem warranted.

Diet-Induced Obesity Model: Abnormal Oocytes and Persistent Growth Abnormalities in the Offspring

Associations between maternal obesity and adverse fetal outcomes are well documented, but the mechanisms involved are largely unknown. Most previous work has focused on postconceptional events, however, our laboratory has shown pre- and periconceptional aberrations in maternal glucose metabolism have adverse effects on oocytes and embryos that carry on to the fetus. To demonstrate effects of maternal obesity in the pre- and periconceptional periods, we compared reproductive tissues from diet-induced obese female mice to those of control mice. Ovaries were either stained for follicular apoptosis or dissected and evaluated for oocyte size and meiotic maturation. Mice were also mated and followed for reproductive outcomes including preimplantation embryonic IGF-I receptor (IGF-IR) immunostaining, midgestation fetal growth, and midgestational placental IGF receptor 2 (Igf2r) mRNA. Delivered pups were followed for growth and development of markers of metabolic syndrome. Compared with controls, obese mice had significantly more apoptotic ovarian follicles, smaller and fewer mature oocytes, decreased embryonic IGF-IR staining, smaller fetuses, increased placental Igf2r mRNA, and smaller pups. All weaned pups were fed a regular diet. At 13 wk pups delivered from obese mice were significantly larger, and these pups demonstrated glucose intolerance and increased cholesterol and body fat suggesting early development of a metabolic-type syndrome. Together, our findings suggest maternal obesity has adverse effects as early as the oocyte and preimplantation embryo stage and that these effects may contribute to lasting morbidity in offspring, underscoring the importance of optimal maternal weight and nutrition before conception.

High Fat Diet Induced Developmental Defects in the Mouse: Oocyte Meiotic Aneuploidy and Fetal Growth Retardation/Brain Defects

Background Maternal obesity is associated with poor outcomes across the reproductive spectrum including infertility, increased time to pregnancy, early pregnancy loss, fetal loss, congenital abnormalities and neonatal conditions. Furthermore, the proportion of reproductive-aged woman that are obese in the population is increasing sharply. From current studies it is not clear if the origin of the reproductive complications is attributable to problems that arise in the oocyte or the uterine environment. Methodology/Principal Findings We examined the developmental basis of the reproductive phenotypes in obese animals by employing a high fat diet mouse model of obesity. We analyzed very early embryonic and fetal phenotypes, which can be parsed into three abnormal developmental processes that occur in obese mothers. The first is oocyte meiotic aneuploidy that then leads to early embryonic loss. The second is an abnormal process distinct from meiotic aneuploidy that also leads to early embryonic loss. The third is fetal growth retardation and brain developmental abnormalities, which based on embryo transfer experiments are not due to the obese uterine environment but instead must be from a defect that arises prior to the blastocyst stage. Conclusions/Significance Our results suggest that reproductive complications in obese females are, at least in part, from oocyte maternal effects. This conclusion is consistent with IVF studies where the increased pregnancy failure rate in obese women returns to the normal rate if donor oocytes are used instead of autologous oocytes. We postulate that preconceptional weight gain adversely affects pregnancy outcomes and fetal development. In light of our findings, preconceptional counseling may be indicated as the preferable, earlier target for intervention in obese women desiring pregnancy and healthy outcomes.

Somatic Cell-Like Features of Cloned Mouse Embryos Prepared with Cultured Myoblast Nuclei

Abstract Cloning by somatic cell nuclear transfer requires silencing of the donor cell gene expression program and the initiation of the embryonic gene expression program (nuclear reprogramming). Failure to silence the donor cell program could lead to altered embryonic phenotypes. Cloned mouse embryos produced using myoblast nuclei fail to thrive in standard embryo culture media but flourish in somatic cell culture media favored by the donor myoblasts themselves, forming blastocysts at a significant rate, with robust morphologies, high total cell number, and a normal allocation of cells to the inner cell mass in most embryos. Myoblast cloned embryos continue expressing the GLUT4 glucose transporter, which is typically expressed in muscle but not in preimplantation stage embryos. Myoblast clones also exhibit precocious enrichment of GLUT1 at the cell surface. Both myoblast and cumulus cell cloned embryos exhibit enhanced rates of glucose uptake. These observations indicate that silencing of the donor cell genome during cloning either is incomplete or occurs progressively over the course of preimplantation development. As a result, cloned embryos initially exhibit many somatic cell-like characteristics. Tetraploid constructs, which possess a transplanted somatic cell genome plus the oocyte-derived chromosomes, exhibit a more embryonic-like pattern of gene expression and culture preference. We conclude that preimplantation stage cloned embryos have profoundly altered characteristics that are donor cell type specific and that exposure of cloned embryos to standard embryo culture conditions may lead to disruptions in basic homeostasis and inhibition of a range of essential processes including further nuclear reprogramming, contributing to cloned embryo demise.

High insulin-like growth factor 1 (IGF-1) and insulin concentrations trigger apoptosis in the mouse blastocyst via down-regulation of the IGF-1 receptor.

Women with polycystic ovary syndrome have significantly higher rates of pregnancy loss, as well as elevated insulin and IGF-1 levels. In this study, preimplantation embryos exposed to high concentrations of IGF-1 or insulin undergo extensive apoptosis of the ICM nuclei. Lack of BAX expression, the caspase inhibitor, zVAD, or the ceramide synthase inhibitor, fumonisin B1, prevents this event, suggesting involvement of programmed cell death effector pathways. In other systems, the IGF-1 concentration regulates IGF-1R expression and thus high concentrations lead to down-regulation of the receptor. Here, data show a decrease in IGF-1 receptor protein expression, both by confocal immunofluorescent microscopy and by Western analysis upon exposure to 130 nM IGF-1. Insulin-stimulated glucose uptake, an event regulated via the IGF-1 receptor, is decreased upon exposure to excess IGF-1, suggesting decreased function of the receptor. The data also show that, by blocking receptor signal transduction or by decreasing receptor expression, the apoptotic event can be recreated, thus strongly suggesting that the mechanism of high IGF-1 induced apoptosis is decreased downstream IGF-1 receptor signaling. This embryotoxic insult by high IGF-1 levels may be responsible for the high incidence of pregnancy loss seen in women with polycystic ovary syndrome.

Maternal hyperglycemia alters glucose transport and utilization in mouse preimplantation embryos

Glucose utilization was studied in preimplantation embryos from normal and diabetic mice. With use of ultramicrofluorometric enzyme assays, intraembryonic free glucose in single embryos recovered from control and streptozotocin-induced hyperglycemic mice was measured at 24, 48, 72, and 96 h after mating. Free glucose concentrations dropped significantly in diabetics at 48 and 96 h, corresponding to the two-cell and blastocyst stages (48 h: diabetic 0.23 +/- 0.09 vs. control 2.30 +/- 0.43 mmol/kg wet wt; P < 0.001; 96 h: diabetic 0.31 +/- 0.29 vs. control 5.12 +/- 0.17 mmol/kg wet wt; P < 0.001). Hexokinase activity was not significantly different in the same groups. Transport was then compared using nonradioactive 2-deoxyglucose uptake and microfluorometric enzyme assays. The 2-deoxyglucose uptake was significantly lower at both 48 and 96 h in embryos from diabetic vs. control mice (48 h diabetic, 0.037 +/- 0. 003; control, 0.091 +/- 0.021 mmol . kg wet wt-1 . 10 min-1, P < 0. 05; 96 h diabetic, 0.249 +/- 0.008; control, 0.389 +/- 0.007 mmol . kg wet wt-1 . 10 min-1, P < 0.02). When competitive quantitative reverse transcription-polymerase chain reaction was used, there was 44 and 68% reduction in the GLUT-1 mRNA at 48 h (P < 0.001) and 96 h (P < 0.05), respectively, in diabetic vs. control mice. GLUT-2 and GLUT-3 mRNA values were decreased 63 and 77%, respectively (P < 0.01, P < 0.01) at 96 h. Quantitative immunofluorescence microscopy demonstrated 49 +/- 6 and 66 +/- 4% less GLUT-1 protein at 48 and 96 h and 90 +/- 5 and 84 +/- 6% less GLUT-2 and -3 protein, respectively, at 96 h in diabetic embryos. These findings suggest that, in response to a maternal diabetic state, preimplantation mouse embryos experience a decrease in glucose utilization directly related to a decrease in glucose transport at both the mRNA and protein levels.

Associations between free fatty acids, cumulus oocyte complex morphology and ovarian function during in vitro fertilization

OBJECTIVE To determine if follicular free fatty acid (FFA) levels are associated with cumulus oocyte complex (COC) morphology. DESIGN Prospective cohort study. SETTING University in vitro fertilization (IVF) practice. PATIENT(S) A total of 102 women undergoing IVF. INTERVENTION(S) Measurement of FFAs in serum and ovarian follicular fluid. MAIN OUTCOME MEASURE(S) Total and specific follicular and serum FFA levels, correlations between follicular and serum FFAs, and associations between follicular FFA levels and markers of oocyte quality, including COC morphology. RESULT(S) Predominant follicular fluid and serum FFAs were oleic, palmitic, linoleic, and stearic acids. Correlations between follicular and serum FFA concentrations were weak (r=0.252, 0.288, 0.236, 0.309, respectively for specific FFAs; r=0.212 for total FFAs). A receiver operating characteristic curve determined total follicular FFAs≥0.232 μmol/mL distinguished women with a lower versus higher percentage of COCs with favorable morphology. Women with elevated follicular FFAs (n=31) were more likely to have COCs with poor morphology than others (n=71; OR 3.3, 95% CI1.2-9.2). This relationship held after adjusting for potential confounders, including age, body mass index, endometriosis, and amount of gonadotropin administered (β=1.2; OR 3.4, 95% CI 1.1-10.4). CONCLUSION(S) Elevated follicular FFA levels are associated with poor COC morphology. Further work is needed to determine what factors influence follicular FFA levels and if these factors impact fertility.