Diet-induced obesity impairs endometrial stromal cell decidualization: a potential role for impaired autophagy.

STUDY QUESTION: What effect does diet-induced obesity have on endometrial stromal cell (ESC) decidualization? SUMMARY ANSWER: Diet-induced obesity impairs ESC decidualization. WHAT IS KNOWN ALREADY: Decidualization is important for successful implantation and subsequent health of the pregnancy. Compared with normal-weight women, obese women have lower pregnancy rates (both spontaneous and by assisted reproductive technology), higher rates of early pregnancy loss and poorer oocyte quality. STUDY DESIGN, SIZE, DURATION: Beginning at 6 weeks of age, female C57Bl/6J mice were fed either a high-fat/high-sugar diet (HF/HS; 58% Fat Energy/Sucrose) or a diet of standard mouse chow (CON; 13% Fat) for 12 weeks. At this point, metabolic parameters were measured. Some of the mice (n = 9 HF/HS and 9 CON) were mated with reproductively competent males, and implantation sites were assessed. Other mice (n = 11 HF/HS and 10 CON) were mated with vasectomized males, and artificial decidualization was induced. For in vitro human studies of primary ESCs, endometrial tissue was obtained via biopsy from normo-ovulatory patients without history of infertility (obese = BMI > 30 kg/m(2), n = 11 and lean = BMI < 25 kg/m(2), n = 7) and from patients consented for hysterectomies for a benign indication (n = 4). In vitro studies were also performed with immortalized human ESCs. ESCs were decidualized in culture for nine 9 days in the presence or absence of palmitic acid (PA), and the degree of decidualization was assessed by measuring expression of decidualization markers. PARTICIPANTS/MATERIALS, SETTING, METHODS: The sizes of implantation sites and fetuses were analyzed in mice mated with reproductively competent males. In mice mated with vasectomized males, decidualization was induced, and uterine tissues were analyzed via hematoxylin and eosin staining, quantitative RT-PCR (RT-qPCR), and western blots. Human ESCs were cultured in vitro and induced to decidualize by treatment with cAMP and medroxyprogesterone. The level of expression of decidualization markers was assessed by RT-qPCR (mRNA) and western blotting (protein). ATP content of ESCs was measured, and levels of autophagy were assessed by western blotting of the autophagy regulators acetyl coa carboxylase (ACC) and ULK1 (Ser 317). Autophagic flux was measured by western blot of the marker LC3b-II. MAIN RESULTS AND THE ROLE OF CHANCE: Mice exposed to an HF/HS diet became obese and metabolically impaired. HF/HS-exposed mice mated to reproductively competent males had smaller implantation sites in early pregnancy (P <0.001) and larger fetuses at term (P <0.05) than CON-exposed mice. In the artificial decidualization experiments, mice exposed to the HF/HS diet developed 50% smaller deciduomas than mice exposed to CON diet (P< 0.001). Human ESCs cultured in the presence of PA had markedly decreased mRNA expression of the decidualization markers, decidual prolactin (PRL) (P< 0.0001) and insulin-like growth factor binding protein 1 (IGFBP1) (P< 0.0001). Expression of PRL and IGFBP1 by mRNA were also significantly lower in early follicular phase ESCs of obese women than in those of normal-weight women (P< 0.05). Protein expression of phosphorylated ACC and phosphorylated ULK1, both activated forms, were lower in deciduomas of HF/HS mice than in those of control mice (P < 0.01). In immortalized human ESCs, LC3b-II levels were higher in decidualized cells than in controls, indicating increased autophagy. PA treatment abrogated this increase. LIMITATIONS, REASONS FOR CAUTION: Many aspects of obesity and metabolic impairment could contribute to the decidualization defects observed in the HF/HS-exposed mice. Although our findings suggest that both autophagy and decidualization are impaired by exposure to PA, the underlying mechanisms should be elucidated. Finally, our human patient sample size was small. WIDER IMPLICATIONS OF THE FINDINGS: Although many factors contribute to poor reproductive outcome and early pregnancy loss in obese women, our study suggests the importance of decidualization defects. Such defects may contribute to compromised endometrial receptivity and poor implantation. If defects in autophagy contribute to impaired decidualization, therapeutics could be developed to improve this process and thus improve implantation and pregnancy outcomes in obese women. STUDY FUNDING/COMPETING INTERESTS: Grants include NIH 5T32HD040135-12 (J.S.R.), R01 HD065435 (K.H.M.), NIH T32 HD049305 (J.L.S.) and ACOG Research Grant (M.B.S.). The authors report no conflicts of interest.

Adverse effects of obesity and/or high-fat diet on oocyte quality and metabolism are not reversible with resumption of regular diet in mice.

Obesity adversely affects reproduction and results in oocyte defects in both mice and humans. In the present study we used a mouse model to examine whether the adverse effects of an obesogenic diet on oocyte metabolism and morphology can be reversed by return to a control diet. The intervention group consisted of C57BL6/J mice placed on a high-fat diet (HFD; 35.8% fat and 20.2% protein by nutritional content) for 6 weeks and then switched to an isocaloric control diet (CD; 13% fat and 25% protein) for 8 weeks (HFD/CD mice). The control group consisted of age-matched C57BL6/J mice maintained on CD for 14 weeks (CD/CD mice). Although metabolic parameters (weight, glucose tolerance and cholesterol levels) of HFD/CD mice returned to normal after this 'diet reversal' period, several oocyte defects were not reversible. These HFD/CD oocytes demonstrated significantly higher percentages of abnormal meiotic spindles, lower mitochondrial membrane potential and lower ATP and citrate levels, and higher percentages of abnormal lipid accumulation and mitochondrial distribution compared with CD/CD mice. These results suggest that the negative effects of an obesogenic diet on oocyte quality are not reversible, despite reversal of metabolic parameters. These data may provide better insight when counselling obese women regarding reproductive options and success.

Modeling the effect of cigarette smoke on hexose utilization in spermatocytes.

We set out to determine whether the addition of an aryl hydrocarbon receptor (AHR) antagonist has an effect on glucose/fructose utilization in the spermatocyte when exposed to cigarette smoke condensate (CSC). We exposed male germ cells to 5 and 40 µg/mL of CSC ± 10 µmol/L of AHR antagonist at various time points. Immunoblot expression of specific glucose/fructose transporters was compared to control. Radiolabeled uptake of 2-deoxyglucose (2-DG) and fructose was also performed. Spermatocytes utilized fructose nearly 50-fold more than 2-DG. Uptake of 2-DG decreased after CSC + AHR antagonist exposure. Glucose transporters (GLUTs) 9a and 12 declined after CSC + AHR antagonist exposure. Synergy between CSC and the AHR antagonist in spermatocytes may disrupt the metabolic profile in vitro. Toxic exposures alter energy homeostasis in early stages of male germ cell development, which could contribute to later effects explaining decreases in sperm motility in smokers.

The fatty acid beta-oxidation pathway is important for decidualization of endometrial stromal cells in both humans and mice.

Embryo implantation and development requires the endometrial stromal cells (ESCs) to undergo decidualization. This differentiation process requires glucose utilization, and blockade of the pentose phosphate pathway inhibits decidualization of ESCs both in vitro and in vivo. Glucose and fatty acids are energy substrates for many cell types, and fatty acid beta-oxidation is critical for embryo implantation. Here, we investigated whether beta-oxidation is required for decidualization of ESCs. As assessed by marker gene expression, decidualization of human primary ESCs was blocked by reducing activity of carnitine calmitoyltransferase I, the rate-limiting enzyme in beta-oxidation, either by short hairpin RNA-mediated silencing or by treatment with the inhibitor etomoxir. Ranolazine (RAN), a partial beta-oxidation inhibitor, blocked early decidualization of a human ESC line. However, decidualization resumed after several days, most likely due to a compensatory up-regulation of GLUT1 expression and an increase in glucose metabolism. Simultaneous inhibition of the beta-oxidation pathway with RAN and the pentose phosphate pathway with glucosamine (GlcN) impaired in vitro decidualization of human ESCs more strongly than inhibition of either pathway alone. These findings were confirmed in murine ESCs in vitro, and exposure to RAN plus GlcN inhibited decidualization in vivo in a deciduoma model. Finally, intrauterine implantation of time-release RAN and GlcN pellets reduced pup number. Importantly, pup number returned to normal after the end of the pellet-active period. This work indicates that both fatty acids and glucose metabolism pathways are important for ESC decidualization, and suggests novel pathways to target for the design of future nonhormonal contraceptives.

DHEA-mediated inhibition of the pentose phosphate pathway alters oocyte lipid metabolism in mice.

Women with polycystic ovary syndrome (PCOS) and hyperandrogenism have altered hormone levels and suffer from ovarian dysfunction leading to subfertility. We have attempted to generate a model of hyperandrogenism by feeding mice chow supplemented with dehydroepiandrosterone (DHEA), an androgen precursor that is often elevated in women with PCOS. Treated mice had polycystic ovaries, low ovulation rates, disrupted estrous cycles, and altered hormone levels. Because DHEA is an inhibitor of glucose-6-phosphate dehydrogenase, the rate-limiting enzyme in the pentose phosphate pathway, we tested the hypothesis that oocytes from DHEA-exposed mice would have metabolic disruptions. Citrate levels, glucose-6-phosphate dehydrogenase activity, and lipid content in denuded oocytes from these mice were significantly lower than controls, suggesting abnormal tricarboxylic acid and pentose phosphate pathway metabolism. The lipid and citrate effects were reversible by supplementation with nicotinic acid, a precursor for reduced nicotinamide adenine dinucleotide phosphate. These findings suggest that elevations in systemic DHEA can have a negative impact on oocyte metabolism and may contribute to poor pregnancy outcomes in women with hyperandrogenism and PCOS.

Glucosamine inhibits decidualization of human endometrial stromal cells and decreases litter sizes in mice.

Embryo implantation in the uterus depends on decidualization of the endometrial stromal cells (ESCs), and glucose utilization via the pentose phosphate pathway is critical in this process. We hypothesized that the amino sugar glucosamine may block the pentose phosphate pathway via inhibition of the rate-limiting enzyme glucose-6-phosphate dehydrogenase in ESCs and therefore impair decidualization and embryo implantation, thus preventing pregnancy. Both human primary and immortalized ESCs were decidualized in vitro in the presence of 0, 2.5, or 5 mM glucosamine for 9 days. Viability assays demonstrated that glucosamine was well tolerated by human ESCs. Exposure of human ESCs to glucosamine resulted in significant decreases in the activity and expression of glucose-6-phosphate dehydrogenase and in the mRNA expression of the decidual markers prolactin, somatostatin, interleukin-15, and left-right determination factor 2. In mouse ESCs, expression of the decidual marker Prp decreased upon addition of glucosamine. In comparison with control mice, glucosamine-treated mice showed weak artificial deciduoma formation along the stimulated uterine horn. In a complementary in vivo experiment, a 60-day-release glucosamine (15, 150, or 1500 µg) or placebo pellet was implanted in a single uterine horn of mice. Mice with a glucosamine pellet delivered fewer live pups per litter than those with a control pellet, and pup number returned to normal after the end of the pellet-active period. In conclusion, glucosamine is a nonhormonal inhibitor of decidualization of both human and mouse ESCs and of pregnancy in mice. Our data indicate the potential for development of glucosamine as a novel, reversible, nonhormonal contraceptive.

Slc2a8 deficiency in mice results in reproductive and growth impairments.

SLC2A8, also known as GLUT8, is a facilitative glucose transporter expressed in the testis, brain, liver, heart, uterus, ovary, and fat. In this study we examined the effect of Slc2a8 deficiency on mouse gamete, preimplantation embryo, and implantation phenotype, as well as postnatal growth and physiology. For this model, the transcriptional start site and exons 1-4 were targeted and a lack of protein expression was confirmed by Western immunoblot. Oocytes obtained from Slc2a8(-/-) mice demonstrated abnormal metabolism and ATP production. In addition, deletion of Slc2a8 resulted in impaired decidualization, a critical step in the differentiation of endometrial stromal cells (ESCs), necessary for implantation. This indicates a role for SLC2A8 in decidualization, which is supported by Slc2a8 mRNA expression in both mouse and human ESCs, which increases dramatically in response to hormonal changes occurring during the process of implantation. Ovarian transplantation studies confirm that lack of SLC2A8 affects both the embryo and the implantation processes. This phenotype leads to decreased litter size, and smaller pups at weaning that continue to display an abnormally small growth phenotype into adulthood. The Slc2a8 null mice display decreased body fat by magnetic resonance imaging, and, interestingly, they are resistant to a diet high in fat and carbohydrates.

An intercellular pathway for glucose transport into mouse oocytes.

Glucose is an essential nutrient for mammalian cells. Emerging evidence suggests that glucose within the oocyte regulates meiotic maturation. However, it remains controversial as to whether, and if so how, glucose enters oocytes within cumulus-oocyte complexes (COCs). We used a fluorescent glucose derivative (6-NBDG) to trace glucose transport within live mouse COCs and employed inhibitors of glucose transporters (GLUTs) and gap junction proteins to examine their distinct roles in glucose uptake by cumulus cells and the oocyte. We showed that fluorescent glucose enters both cumulus-enclosed and denuded oocytes. Treating COCs with GLUT inhibitors leads to simultaneous decreases in glucose uptake in cumulus cells and the surrounded oocyte but no effect on denuded oocytes. Pharmacological blockade of of gap junctions between the oocyte and cumulus cells significantly inhibited fluorescent glucose transport to oocytes. Moreover, we find that both in vivo hyperglycemic environment and in vitro high-glucose culture increase free glucose levels in oocytes via gap junctional channels. These findings reveal an intercellular pathway for glucose transport into oocytes: glucose is taken up by cumulus cells via the GLUT system and then transferred into the oocyte through gap junctions. This intercellular pathway may partly mediate the effects of high-glucose condition on oocyte quality.

Insulin-stimulated glucose uptake occurs in specialized cells within the cumulus oocyte complex.

The oocyte exists within the mammalian follicle surrounded by somatic cumulus cells. These cumulus cells metabolize the majority of the glucose within the cumulus oocyte complex and provide energy substrates and intermediates such as pyruvate to the oocyte. The insulin receptor is present in cumulus cells and oocytes; however, it is unknown whether insulin-stimulated glucose uptake occurs in either cell type. Insulin-stimulated glucose uptake is thought to be unique to adipocytes, skeletal and cardiac muscle, and the blastocyst. Here, we show for the first time that many of the components required for insulin signaling are present in both cumulus cells and oocytes. We performed a set of experiments on mouse cumulus cells and oocytes and human cumulus cells using the nonmetabolizable glucose analog 2-deoxy-d-glucose to measure basal and insulin-stimulated glucose uptake. We show that insulin-stimulated glucose uptake occurs in both compact and expanded cumulus cells of mice, as well as in human cumulus cells. Oocytes, however, do not display insulin-stimulated glucose uptake. Insulin-stimulated glucose uptake in cumulus cells is mediated through phosphatidylinositol 3-kinase signaling as shown by inhibition of insulin-stimulated glucose uptake and Akt phosphorylation with the specific phosphatidylinositol 3-kinase inhibitor, LY294002. To test the effect of systemic in vivo insulin resistance on insulin sensitivity in the cumulus cell, cumulus cells from high fat-fed, insulin-resistant mice and women with polycystic ovary syndrome were examined. Both sets of cells displayed blunted insulin-stimulated glucose uptake. Our studies identify another tissue that, through a classical insulin-signaling pathway, demonstrates insulin-stimulated glucose uptake. Moreover, these findings suggest insulin resistance occurs in these cells under conditions of systemic insulin resistance.

Live imaging reveals the link between decreased glucose uptake in ovarian cumulus cells and impaired oocyte quality in female diabetic mice.

Maternal diabetes has been demonstrated to adversely affect preimplantation embryo development and pregnancy outcomes. Emerging data suggest that these effects are associated with compromised oocyte quality. However, direct evidence of a pathway by which maternal diabetes exerts its effects on the oocyte is still lacking. Cumulus cells are metabolically coupled to oocytes, and bidirectional communication between them is essential for the development and functions of both compartments. The primary focus of this work was to evaluate the connection between glucose uptake in cumulus cells and oocyte quality in diabetic mice. This experiment has been difficult, because cumulus cells need to be separated from oocytes and labeled with isotope in the process of measuring glucose uptake. Here, we report a method for live imaging glucose transport in single cumulus-oocyte complexes using a fluorescent glucose analog (6-(N-(7-nitrobenz-2-oxa-1,3-diazol- 4-yl)amino)-6-deoxyglucose). By tracking the ATP content and spindle/chromosome status in individual oocytes surrounded by cumulus cells with differing glucose uptake activity, we reveal that compromised oocyte quality in diabetic mice is linked to decreased glucose uptake in cumulus cells.

Preimplantation exposure of mouse embryos to palmitic acid results in fetal growth restriction followed by catch-up growth in the offspring.

Free fatty acids (FFAs) are energy substrates for many cell types, but in excess, some FFAs can accumulate in nonadipose cells, inducing apoptosis. Also known as lipotoxicity, this phenomenon may play a role in the development of obesity-related disease. Obesity is common among reproductive age women and is associated with adverse pregnancy and fetal outcomes; however, little is known about the effects of excess FFAs on embryos and subsequent fetal development. To address this knowledge gap, murine blastocysts were cultured in excess palmitic acid (PA), the most abundant saturated FFA in human serum, and ovarian follicular fluid. Targets susceptible to aberrations in maternal physiology, including embryonic IGF1 receptor (IGF1R) expression, glutamic pyruvate transaminase (GPT2) activity, and nuclei count, were measured. PA-exposed blastocysts demonstrated altered IGF1R expression, increased GPT2 activity, and decreased nuclei count. Trophoblast stem cells derived from preimplantation embryos were also cultured in PA. Cells exposed to increasing doses of PA demonstrated increased apoptosis and decreased proliferation. To demonstrate long-term effects of brief PA exposure, blastocysts cultured for 30 h in PA were transferred into foster mice, and pregnancies followed through Embryonic Day (ED)14.5 or delivery. Fetuses resulting from PA-exposed blastocysts were smaller than controls at ED14.5. Delivered pups were also smaller but demonstrated catch-up growth and ultimately surpassed control pups in weight. Altogether, our data suggest brief PA exposure results in altered embryonic metabolism and growth, with lasting adverse effects on offspring, providing further insight into the pathophysiology of maternal obesity.

A differential autophagic response to hyperglycemia in the developing murine embryo.

Autophagy is critical to the process of development because mouse models have shown that lack of autophagy leads to developmental arrest during the pre-implantation stage of embryogenesis. The process of autophagy is regulated through signaling pathways, which respond to the cellular environment. Therefore, any alteration in the environment may lead to the dysregulation of the autophagic process potentially resulting in cell death. Using both in vitro and in vivo models to study autophagy in the pre-implantation murine embryo, we observed that the cells respond to environmental stressors (i.e. hyperglycemic environment) by increasing activation of autophagy in a differential pattern within the embryo. This upregulation is accompanied by an increase in apoptosis, which appears to plateau at high concentrations of glucose. The activation of the autophagic pathway was further confirmed by an increase in GAPDH activity in both in vivo and in vitro hyperglycemic models, which has been linked to autophagy through the activation of the Atg12 gene. Furthermore, this increase in autophagy in response to a hyperglycemic environment was observed as early as the oocyte stage. In conclusion, in this study, we provided evidence for a differential response of elevated activation of autophagy in embryos and oocytes exposed to a hyperglycemic environment.

Decreased IRS signaling impairs beta-cell cycle progression and survival in transgenic mice overexpressing S6K in beta-cells.

OBJECTIVE: The purpose of this study was to evaluate the role of the S6K arm of mammalian target of rapamycin complex 1 (mTORC1) signaling in regulation of ß-cell mass and function. Additionally, we aimed to delineate the importance of in vivo S6K activation in the regulation of insulin signaling and the extent to which alteration of insulin receptor substrate (IRS) signaling modulates ß-cell mass and function. RESEARCH DESIGN AND METHODS: The current experiments describe the phenotype of transgenic mice overexpressing a constitutively active form of S6K under the control of the rat insulin promoter. RESULTS: Activation of S6K signaling in these mice improved insulin secretion in the absence of changes in ß-cell mass. The lack of ß-cell mass expansion resulted from decreased G(1)-S progression and increased apoptosis. This phenotype was associated with increased p16 and p27 and decreased Cdk2 levels. The changes in cell cycle were accompanied by diminished survival signals because of impaired IRS/Akt signaling. CONCLUSIONS: This work defines the importance of S6K in regulation of ß-cell cycle, cell size, function, and survival. These experiments also demonstrate that in vivo downregulation of IRS signaling by TORC1/S6K induces ß-cell insulin resistance, and that this mechanism could explain some of the abnormalities that ultimately result in ß-cell failure and diabetes in conditions of nutrient overload.

Mitochondrial dysfunction and apoptosis in cumulus cells of type I diabetic mice.

Impaired oocyte quality has been demonstrated in diabetic mice; however, the potential pathways by which maternal diabetes exerts its effects on the oocyte are poorly understood. Cumulus cells are in direct contact with the oocyte via gap junctions and provide essential nutrients to support oocyte development. In this study, we investigated the effects of maternal diabetes on the mitochondrial status in cumulus cells. We found an increased frequency of fragmented mitochondria, a decreased transmembrane potential and an aggregated distribution of mitochondria in cumulus cells from diabetic mice. Furthermore, while mitochondrial biogenesis in cumulus cells was induced by maternal diabetes, their metabolic function was disrupted as evidenced by lower ATP and citrate levels. Moreover, we present evidence suggesting that the mitochondrial impairments induced by maternal diabetes, at least in part, lead to cumulus cell apoptosis through the release of cytochrome c. Together the deleterious effects on cumulus cells may disrupt trophic and signaling interactions with the oocyte, contributing to oocyte incompetence and thus poor pregnancy outcomes in diabetic females.

Facilitative glucose transporter 9 expression affects glucose sensing in pancreatic beta-cells.

Facilitative glucose transporters (GLUTs) including GLUT9, accelerate the facilitative diffusion of glucose across the plasma membrane. Studies in GLUT2-deficient mice suggested the existence of another GLUT in the mammalian beta-cell responsible for glucose sensing. The objective of this study was to determine the expression and function of GLUT9 in murine and human beta-cells. mRNA and protein expression levels were determined for both isoforms of GLUT9 in murine and human isolated islets as well as insulinoma cell lines (MIN6). Immunohistochemistry and subcellular localization were performed to localize the protein within the cell. Small interfering RNA knockdown of GLUT9 was used to determine the effect of this transporter, in the presence of GLUT2, on cell metabolism and insulin secretion in MIN6 and INS cells. In this report we demonstrate that GLUT9a and GLUT9b are expressed in pancreatic islets and that this expression localizes to insulin-containing beta-cells. Subcellular localization studies indicate that mGLUT9b is found associated with the plasma membrane as well as in the high-density microsome fraction and low-density microsome fraction, whereas mGLUT9a appears to be located only in the high-density microsome and low-density microsome under basal conditions. Functionally GLUT9 appears to participate in the regulation of glucose-stimulated insulin secretion in addition to GLUT2. small interfering RNA knockdown of GLUT9 results in reduced cellular ATP levels that correlate with reductions in glucose-stimulated insulin secretion in MIN6 and INS cells. These studies confirm the expression of GLUT9a and GLUT9b in murine and human beta-cells and suggest that GLUT9 may participate in glucose-sensing in beta-cells.

cAMP pulsing of denuded mouse oocytes increases meiotic resumption via activation of AMP-activated protein kinase.

cAMP plays a critical role in the control of oocyte maturation, as a high level of cAMP maintains oocyte arrest at the first meiotic prophase. Yet this study shows that pulsing meiotically arrested denuded oocytes (DO) with cAMP induces oocyte maturation through the activation of AMP-activated protein kinase (PRKA). Short-term (3 h) pulsing of meiotically arrested oocytes with forskolin, an adenyl cyclase (AC) activator, increased oocyte cAMP, led to elevated AMP, and induced oocyte meiotic resumption compared to oocytes continuously cultured in the control medium with or without forskolin. Western analysis showed that germinal vesicle (GV)-stage oocytes after forskolin pulsing contained increased levels of phospho-acetyl CoA carboxylase (pACACA), a primary substrate of PRKA. Pulsing oocytes with the phosphodiesterase (PDE)-sensitive cAMP analog, 8-bromo-cAMP (8-Br-cAMP), also increased pACACA and pPRKA levels in GV-stage oocytes and induced oocyte meiotic resumption. Moreover, the PRKA inhibitors, compound C and araA, prevented 8-Br-cAMP pulsing-induced maturation. The lack of effect on meiotic induction and PRKA activation when oocytes were pulsed with the PDE-resistant activators of cAMP-dependent protein kinase, Sp-cAMP-AM and Sp-5,6-DCI-cBIMPS, suggests that cAMP degradation is required for pulsing-induced maturation. Pulsing oocytes with the exchange protein directly activated by cAMP (Epac)-specific activator, 8-CPT-2'-O-Me-cAMP, had no stimulatory effect on oocyte maturation, suggesting Epac is not involved in the pulsing-induced maturation. Taken together, these data support the idea that a transient increase in oocyte cAMP can induce meiotic resumption via activation of PRKA.

Maternal diabetes causes mitochondrial dysfunction and meiotic defects in murine oocytes.

The adverse effects of maternal diabetes on embryo development and pregnancy outcomes have recently been shown to occur as early as the one-cell zygote stage. The hypothesis of this study was that maternally inherited mitochondria in oocytes from diabetic mice are abnormal and thus responsible in part for this latency of developmental compromise. In ovulated oocytes from diabetic mice, transmission electron microscopy revealed an alteration in mitochondrial ultrastructure, and the quantitative analysis of mitochondrial DNA copy number demonstrated an increase. The levels of ATP and tricarboxylic acid cycle metabolites in diabetic oocytes were markedly reduced compared with controls, suggesting a mitochondrial metabolic dysfunction. Abnormal distribution of mitochondria within maturing oocytes also was seen in diabetic mice. Furthermore, oocytes from diabetic mice displayed a higher frequency of spindle defects and chromosome misalignment in meiosis, resulting in increased aneuploidy rates in ovulated oocytes. Collectively, our results suggest that maternal diabetes results in oocyte defects that are transmitted to the fetus by two routes: first, meiotic spindle and chromatin defects result in nondisjunction leading to embryonic aneuploidy; second, structural and functional abnormalities of oocyte mitochondria, through maternal transmission, provide the embryo with a dysfunctional complement of mitochondria that may be propagated during embryogenesis.

Role of glucose in cloned mouse embryo development.

Cloned mouse embryos display a marked preference for glucose-containing culture medium, with enhanced development to the blastocyst stage in glucose-containing medium attributable mainly to an early beneficial effect during the first cell cycle. This early beneficial effect of glucose is not displayed by parthenogenetic, fertilized, or tetraploid nuclear transfer control embryos, indicating that it is specific to diploid clones. Precocious localization of the glucose transporter SLC2A1 to the cell surface, as well as increased expression of glucose transporters and increased uptake of glucose at the one- and two-cell stages, is also seen in cloned embryos. To examine the role of glucose in early cloned embryo development, we examined glucose metabolism and associated metabolites, as well as mitochondrial ultrastructure, distribution, and number. Clones prepared with cumulus cell nuclei displayed significantly enhanced glucose metabolism at the two-cell stage relative to parthenogenetic controls. Despite the increase in metabolism, ATP content was reduced in clones relative to parthenotes and fertilized controls. Clones at both stages displayed elevated concentrations of glycogen compared with parthenogenetic controls. There was no difference in the number of mitochondria, but clone mitochondria displayed ultrastructural alterations. Interestingly, glucose availability positively affected mitochondrial structure and localization. We conclude that cloned embryos may be severely compromised in terms of ATP-dependent processes during the first two cell cycles and that glucose may exert its early beneficial effects via positive effects on the mitochondria.

Crosstalk between the AMP-activated kinase and insulin signaling pathways rescues murine blastocyst cells from insulin resistance.

Maternal insulin resistance results in poor pregnancy outcomes. In vivo and in vitro exposure of the murine blastocyst to high insulin or IGF1 results in the down-regulation of the IGF1 receptor (IGF1R). This in turn leads to decreased glucose uptake, increased apoptosis, as well as pregnancy resorption and growth restriction. Recent studies have shown that blastocyst activation of AMP-activated protein kinase (AMPK) reverses these detrimental effects; however, the mechanism was not clear. The objective of this study was to determine how AMPK activation rescues the insulin-resistant blastocyst. Using trophoblast stem (TS) cells derived from the blastocyst, insulin resistance was recreated by transfecting with siRNA to Igf1r and down-regulating expression of the protein. These cells were then exposed to AMPK activators 5-aminoimidazole-4-carboxamide riboside and phenformin, and evaluated for apoptosis, insulin-stimulated 2-deoxyglucose uptake, PI3-kinase activity, and levels of phospho-AKT, phospho-mTor, and phospho-70S6K. Surprisingly, disrupted insulin signaling led to decreased AMPK activity in TS cells. Activators reversed these effects by increasing the AMP/ATP ratio. Moreover, this treatment increased insulin-stimulated 2-deoxyglucose transport and cell survival, and led to an increase in PI3-kinase activity, as well as increased P-mTOR and p70S6K levels. This study is the first to demonstrate significant crosstalk between the AMPK and insulin signaling pathways in embryonic cells, specifically the enhanced response of PI3K/AKT/mTOR to AMPK activation. Decreased insulin signaling also resulted in decreased AMPK activation. These findings provide mechanistic targets in the AMPK signaling pathway that may be essential for improved pregnancy success in insulin-resistant states.

Maternal diabetes adversely affects AMP-activated protein kinase activity and cellular metabolism in murine oocytes.

Maternal diabetes is associated with an increased risk of miscarriages and congenital anomalies. Preovulatory oocytes in murine models also experience maturational delay and greater granulosa cell apoptosis. The objective of this study was to examine whether maternal diabetes influences preovulatory oocyte metabolism and impacts meiotic maturation. Streptozotocin-induced diabetic B6SJLF1 mice were superovulated, and oocytes were collected at 0, 2, and 6 h after human chorionic gonadotropin (hCG) injection. Individual oocyte concentrations of ATP, 5'-AMP, glycogen, and fructose-1,6-phosphate (FBP) and enzyme activities of glucose-6-phosphate dehydrogenase (G6PDH), adenylate kinase, hydroxyacyl-CoA dehydrogenase (Hadh2), and glutamic pyruvate transaminase (Gpt2) were measured. Protein levels of phosphorylated AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) were also measured. ATP levels were significantly lower in oocytes from diabetic mice, and the percent change in the AMP-to-ATP ratio was significantly higher in these oocytes. In contrast, activities of Hadh2 and Gpt2, two enzymes activated by AMPK, were significantly less in these oocytes. Additionally, glycogen and FBP levels, both endogenous inhibitors of AMPK, were elevated. Phosphorylated ACC, a downstream target of AMPK, and phosphorylated AMPK were both decreased in diabetic oocytes, thus confirming decreased AMPK activity. Finally, addition of the activator AICAR to the in vitro maturation assay restored AMPK activity and corrected the maturation defect experienced by the oocytes from diabetic mice. In conclusion, maternal diabetes adversely alters cellular metabolism leading to abnormal AMPK activity in murine oocytes. Increasing AMPK activity in these oocytes during the preovulatory phase reverses the metabolic changes and corrects delays in meiotic maturation.