The effect of maternal high-fat/high-sugar diet on offspring oocytes and early embryo development.

Observational human data and several lines of animal experimental data indicate that maternal obesity impairs offspring health. Here, we comprehensively tested the model that maternal obesity causes defects in the next three generations of oocytes and embryos. We exposed female F0 mice to a high-fat/high-sugar (HF/HS) diet for 6 weeks before conception until weaning. Sires, F1 offspring and all subsequent generations were fed control chow diet. Oocytes from F1, F2 and F3 offspring of obese mothers had lower mitochondrial mass and less ATP and citrate than oocytes from offspring of control mothers. F0 blastocysts from HF/HS-exposed mice, but not F1 and F2 blastocysts, had lower mitochondrial mass and membrane potential, less citrate and ATP and smaller total cell number than F0 blastocysts from control mothers. Finally, supplementation of IVF media with the anti-oxidant mito-esculetin partially prevented the oocyte mitochondrial effects caused by maternal HF/HS diet. Our results support the idea that maternal obesity impairs offspring oocyte quality and suggest that antioxidant supplementation should be tested as a means to improve IVF outcomes for obese women.

Obesity-induced oocyte mitochondrial defects are partially prevented and rescued by supplementation with co-enzyme Q10 in a mouse model.

STUDY QUESTION: Does supplementation with co-enzyme Q10 (CoQ10) improve the oocyte mitochondrial abnormalities associated with obesity in mice? SUMMARY ANSWER: In an obese mouse model, CoQ10 improves the mitochondrial function of oocytes. WHAT IS KNOWN ALREADY: Obesity impairs oocyte quality. Oocytes from mice fed a high-fat/high-sugar (HF/HS) diet have abnormalities in mitochondrial distribution and function and in meiotic progression. STUDY DESIGN, SIZE, DURATION: Mice were randomly assigned to a normal, chow diet or an isocaloric HF/HS diet for 12 weeks. After 6 weeks on the diet, half of the mice receiving a normal diet and half of the mice receiving a HF/HS diet were randomly assigned to receive CoQ10 supplementation injections for the remaining 6 weeks. PARTICIPANTS/MATERIALS, SETTING, METHODS: Dietary intervention was initiated on C57Bl6 female mice at 4 weeks of age, CoQ10 versus vehicle injections were assigned at 10 weeks, and assays were conducted at 16 weeks of age. Mice were super-ovulated, and oocytes were collected and stained to assess mitochondrial distribution, quantify reactive oxygen species (ROS), assess meiotic spindle formation, and measure metabolites. In vitro fertilization was performed, and blastocyst embryos were transferred into control mice. Oocyte number, fertilization rate, blastulation rate and implantation rate were compared between the four cohorts. Bivariate statistics were performed appropriately. MAIN RESULTS AND THE ROLE OF CHANCE: HF/HS mice weighed significantly more than normal diet mice (29 versus 22 g, P< 0.001). CoQ10 supplementation did not influence weight. Levels of ATP, citrate, and phosphocreatine were lower and ROS levels were higher in HF/HS mice than in controls (P< 0.001). CoQ10 supplementation significantly increased the levels of metabolites and decreased ROS levels in oocytes from normal diet mice but not in oocytes from HF/HS mice. However, CoQ10 completely prevented the mitochondrial distribution abnormalities observed in the HF/HS mice. Overall, CoQ10 supplementation significantly increased the percentage of normal spindle and chromosome alignment (92.3 versus 80.2%, P= 0.039). In the sub-analysis by diet, the difference did not reach statistical significance. When undergoing IVF, there were no statistically significant differences in the number of mature oocytes, the fertilization rate, blastocyst formation rates, implantation rates, resorption rates or litter size between HF/HS mice receiving CoQ10 or vehicle injections. LIMITATIONS, REASONS FOR CAUTION: Experiments were limited to one species and strain of mice. The majority of experiments were performed after ovulation induction, which may not represent natural cycle fertility. WIDER IMPLICATIONS OF THE FINDINGS: Improvement in oocyte mitochondrial distribution and function of normal, chow-fed mice and HF/HS-fed mice demonstrates the importance of CoQ10 and the efficiency of the mitochondrial respiratory chain in oocyte competence. Clinical studies are now needed to evaluate the therapeutic potential of CoQ10 in women's reproductive health. STUDY FUNDING/COMPETING INTERESTS: C.E.B. received support from the National Research Training Program in Reproductive Medicine sponsored by the National Institute of Health (T32 HD040135-13) and the Scientific Advisory Board of Vivere Health. K.H.M received support from the American Diabetes Association and the National Institute of Health (R01 HD083895). There are no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: This study is not a clinical trial.

Adiponectin and adiponectin receptors in the mouse preimplantation embryo and uterus.

BACKGROUND: Adiponectin (Adipoq), a protein secreted by adipocytes in inverse proportion to the adipose mass present, modulates energy homeostasis and increases insulin sensitivity. Tissue Adipoq signaling decreases in settings of maternal diabetes, polycystic ovary syndrome (PCOS) and endometriosis, conditions which are associated with reproductive difficulty. Our objective was to define the expression and hormonal regulation of Adipoq and its receptors in the mouse preimplantation embryo and uterus. METHODS AND RESULTS: By real-time quantitative PCR, mRNA transcripts for Adipoq, AdipoR1, AdipoR2, Ppara, Ppard, FATP1 (SLC27A1) and acyl CoA oxidase (Acox1) were identified in mouse 2-cell and 8-cell embryos, while blastocyst stage embryos and trophoblast stem (TS) cells expressed mRNA for all genes except Adipoq. Protein expression of Adipoq, AdipoR1, AdipoR2, the insulin sensitive transporters GLUT8 (Slc2A8), GLUT12 (Slc2A12) and p-PRKAA1 was identified by immunofluorescence staining in all stages of preimplantation embryos including the blastocyst. In situ hybridization demonstrated the presence of Adipoq, AdipoR1 and AdipoR2 mRNA in the mouse decidual cells of the implantation site and in artificially decidualized cells, and the expression of these proteins was confirmed by western blotting. Flow cytometry confirmed cell surface expression of AdipoR1 and AdipoR2 in TS cells and decidual cells. CONCLUSIONS: These results suggest for the first time that Adipoq signaling may play an important role in preimplantation embryo development and uterine receptivity by autocrine and paracrine methods in the mouse. Implantation failures and pregnancy loss, specifically those experienced in women with maternal metabolic conditions such as diabetes, obesity and PCOS, may be the result of aberrant Adipoq and AdipoR1 and AdipoR2 expression and suboptimal decidualization in the uterus.

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 cells. 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.

Hyperglycemia and apoptosis: mechanisms for congenital malformations and pregnancy loss in diabetic women.

Congenital malformations are the leading cause of perinatal death among infants of diabetic women. Abnormal fuel metabolism and hyperglycemia have been shown to disturb embryogenesis during the earliest pre- and postimplantation stages in mice. This review presents a new model to explain, in part, adverse pregnancy outcomes associated with diabetes. In this model, by altering gene expression in developing tissues, raised glucose concentrations led to premature programmed cell death in key progenitor cells of the mouse blastocyst or in emerging organ structures in the mouse postimplantation embryo, resulting in abnormal morphogenesis or miscarriage. Although recent studies are still somewhat speculative and have currently only been explored in the mouse, this paradigm is supported by examples in other cell systems, which include human-derived cell lines, thereby suggesting that these findings are also applicable to human pregnancy.

Cigarette smoke-induced cell death of a spermatocyte cell line can be prevented by inactivating the Aryl hydrocarbon receptor.

Cigarette smoke exposure causes germ cell death during spermatogenesis. Our earlier studies demonstrated that cigarette smoke condensate (CSC) causes spermatocyte cell death in vivo and growth arrest of the mouse spermatocyte cell line (GC-2spd(ts)) in vitro via the aryl hydrocarbon receptor (AHR). We hypothesize here that inactivation of AHR could prevent the CSC-induced cell death in spermatocytes. We demonstrate that CSC exposure generates oxidative stress, which differentially regulates mitochondrial apoptosis in GC-2spd(ts) and wild type (WT) and AHR knockout (AHR-KO) mouse embryonic fibroblasts (MEFs). SiRNA-mediated silencing of Ahr augments the extent of CSC-mediated cellular damage while complementing the AHR-knockout condition. Pharmacological inhibition using the AHR-antagonist (CH223191) modulates the CSC-altered expression of apoptotic proteins and significantly abrogates DNA fragmentation though the cleavage of PARP appears AHR independent. Pretreatment with CH223191 at concentrations above 50 µM significantly prevents the CSC-induced activation of caspase-3/7 and externalization of phosphatidylserine in the plasma membrane. However, MAPK inhibitors alone or together with CH223191 could not prevent the membrane damage upon CSC addition and the caspase-3/7 activation and membrane damage in AHR-deficient MEF indicates the interplay of multiple cell signaling and cytoprotective ability of AHR. Thus the data obtained on one hand signifies the protective role of AHR in maintaining normal cellular homeostasis and the other, could be a potential prophylactic therapeutic target to promote cell survival and growth under cigarette smoke exposed environment by receptor antagonism via CH223191-like mechanism. Antagonist-mediated inactivation of the aryl hydrocarbon receptor blocks downstream events leading to cigarette smoke-induced cell death of a spermatocyte cell line.

Mcl-1 is a key regulator of the ovarian reserve.

A majority of ovarian follicles are lost to natural death, but the disruption of factors involved in maintenance of the oocyte pool results in a further untimely follicular depletion known as premature ovarian failure. The anti-apoptotic B-cell lymphoma 2 (Bcl-2) family member myeloid cell leukemia-1 (MCL-1) has a pro-survival role in various cell types; however, its contribution to oocyte survival is unconfirmed. We present a phenotypic characterization of oocytes deficient in Mcl-1, and establish its role in maintenance of the primordial follicle (PMF) pool, growing oocyte survival and oocyte quality. Mcl-1 depletion resulted in the premature exhaustion of the ovarian reserve, characterized by early PMF loss because of activation of apoptosis. The increasingly diminished surviving cohort of growing oocytes displayed elevated markers of autophagy and mitochondrial dysfunction. Mcl-1-deficient ovulated oocytes demonstrated an increased susceptibility to cellular fragmentation with activation of the apoptotic cascade. Concomitant deletion of the pro-apoptotic Bcl-2 member Bcl-2-associated X protein (Bax) rescued the PMF phenotype and ovulated oocyte death, but did not prevent the mitochondrial dysfunction associated with Mcl-1 deficiency and could not rescue long-term breeding performance. We thus recognize MCL-1 as the essential survival factor required for conservation of the postnatal PMF pool, growing follicle survival and effective oocyte mitochondrial function.

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.

Does intrauterine insemination offer an advantage to cervical cap insemination in a donor insemination program?

OBJECTIVE: To compare pregnancy outcome after IUI versus cervical cap insemination in a donor insemination program. DESIGN: A randomized prospective clinical trial in which patients were alternately inseminated with cryopreserved human semen using either IUI or cervical cap insemination methods. SETTING: The donor insemination program at Washington University School of Medicine. PATIENTS: Forty-two women with either isolated male factor or male factor plus corrected ovulatory dysfunction using clomiphene citrate underwent 141 cycles of donor insemination. MAIN OUTCOME MEASURES: Clinical pregnancy rates (PRs) defined as a viable intrauterine gestation > 12 weeks or delivered were compared between groups using the chi 2 test. RESULTS: Clinical PRs were significantly higher in the IUI group (16.4%) compared with the cervical cap insemination group (5.9%). The spontaneous abortion rates were similar between the IUI (1.4%) and cervical cap insemination groups (4.4%). CONCLUSIONS: These findings suggest an advantage to IUI over cervical cap insemination in a donor insemination program.

Ovarian follicular growth, ovulation and atresia. Endocrine, paracrine and autocrine regulation.

This chapter describes a finely tuned series of events that results in ovarian morphological changes including follicular growth, dominant follicle selection, oocyte development, ovulation, and corpus luteum formation. These changes are under the obligate control of the pituitary gonadotropins FSH and LH. The signaling mechanisms whereby the gonadotropins stimulate these changes in the ovary are now well described. The gonadotropin membrane receptors have been cloned and the cellular events proceeding from receptor binding to phosphorylation of protein gene regulators, such as CREB, have been elucidated. A whole series of intraovarian paracrine and autocrine regulators have been described as having the capability of "fine tuning" the effects of gonadotropins (see Tables I and II), and they are likely involved in dominant follicle selection and the demise of the corpus luteum. The roles of the individual paracrine regulators are, as of yet, not well known, but the IGF system offers an attractive paradigm for these intraovarian factors. Lastly, we have described the explosion of data on intracellular and intranuclear regulators. The challenge for us will be to understand the physiological significance of the seemingly unlimited number of regulatory possibilities. Much has been learned concerning the regulation of the morphological changes seen in the ovary, but much more remains to be elucidated.

Pelvic inflammatory disease. Correlation of severity with CA-125 levels.

OBJECTIVE: To determine whether elevated levels of cancer antigen 125 (CA-125) correlate with the severity and clinical outcome of pelvic inflammatory disease. STUDY DESIGN: CA-125 levels were measured prospectively in 36 women with pelvic inflammatory disease using a commercial immunoassay. Initial inclusion criteria were abdominal tenderness, cervical motion tenderness and adnexal tenderness. Patients then were categorized into three groups based on the severity of the illness. Group 1 included patients with (1) temperature > or = 38.0 but < 38.5 degrees C or (2) WBC count > or = 10 but < 15 x 10(3) cells/microL. Group 2 included patients with (1) temperature > or = 38.5 degrees C or (2) WBC count > or = 15 x 10(3) cells/microL. Group 3 included patients who fulfilled the same criteria as group 2 but who also had an adnexal mass. Differences in CA-125 levels were compared statistically using the unpaired Student t test. RESULTS: All patients with the diagnosis of pelvic inflammatory disease had CA-125 levels > 30 U/mL, with a range of 48-656. Moreover, the levels were significantly different among the three groups (group 1 vs. 2, P = .002; 1 vs. 3, P = .001; 2 vs. 3, P = .001), and a correlation between severity of disease and CA-125 levels was found. All patients with a presumed pelvic abscess had CA-125 levels in the range for neoplastic processes. CONCLUSION: Elevated CA-125 levels correlate with the severity of pelvic inflammatory disease and outcome. Pelvic inflammatory disease must be considered in the differential diagnosis of elevated CA-125 levels and pelvic masses.

Expression of the GLUT1 and GLUT9 facilitative glucose transporters in embryonic chondroblasts and mature chondrocytes in ovine articular cartilage.

Glucose transport across the chondrocyte membrane is essential for chondrogenesis and the development of the skeletal system. We have previously used RT-PCR to show that fully developed human articular chondrocytes express transcripts for the GLUT1 and GLUT9 glucose transporters. In this study we report on the expression and immunohistochemical localization of the GLUT1 and GLUT9 proteins in embryonic and mature ovine cartilage. We also provide Western blot evidence for GLUT1 and GLUT9 expression in mature ovine chondrocytes. Ovine embryos (developmental stages E32 to E36 and E42 to E45) were obtained from pregnant ewes humanely killed by injection with sodium pentobarbitone. Embryos were fixed and processed for immunohistochemistry. Polyclonal antibodies to GLUT1 and GLUT9 revealed that both transporters are expressed in developing chondrocytes in ovine embryos and in the superficial, middle and deep layers of ovine cartilage from mature animals. GLUT1 expression was observed in erythrocytes and organs including heart, liver, and kidney. GLUT9 was also found in heart, kidney and liver. Western blotting confirmed the presence of the GLUT1 protein which migrated between the 50 and 64 kDa markers and two specific GLUT9 bands migrating under the 50 and 60 kDa markers, respectively. The presence of GLUT1 and GLUT9 in developing joints of ovine embryos suggests that these proteins may be important in glucose delivery to developing chondroblasts. Expression of these GLUT isoforms may be an important bioenergetic adaptation for chondrocytes in the extracellular matrix of developing cartilage.

Diabetes and preimplantation events of embryogenesis.

From animal and human studies is it clear that mammalian embryos are vulnerable to injury during the earliest preimplantation stages of development. Exposure to some agents during this brief period has resulted not only in fetal loss but also in malformations. Thus, the potential for maternal induced embryotoxicity exists earlier than previously expected. This period is marked by a drastic change in metabolic needs at the late morula stage. Glucose becomes the predominant exogenous energy substrate and enters the blastocyst via one of three facilitative glucose transporters, GLUT1, GLUT2, and GLUT3. It has been shown that maternal diabetes adversely affects the preimplantation embryo. Recent work has revealed that hyperglycemia leads to a downregulation of the GLUTs at the blastocyst stage in the mouse. This downregulation results in decreased glucose transport into the blastocyst of diabetic mice and thus lower intraembryonic free glucose levels. The embryos are starving themselves of the key substrate necessary for survival. Maternal diabetes also causes a decrease in the number of cells in rat blastocyst and recently hyperglycemia has been shown to induce apoptosis in the mouse blastocyst via cell death effector pathways involving BAX and caspases. Significant loss of key progenitor cells from the blastocyst may predispose these diabetic embryos to later developmental deficiencies manifesting as dysmorphogenesis, fetal loss or early growth delay. The hypothesis that the hyperglycemia-induced decrease in glucose transport triggers apoptosis is presented and discussed as a novel mechanism to explain preimplantation diabetic embryopathy.

Glucose transport and apoptosis.

The transport and metabolism of glucose modify programmed cell death in a number of different cell types. This review presents three cell death paradigms that link a decrease in glucose transport to apoptosis. Although these pathways overlap, the glucose-dependent stimuli that trigger cell death differ. These paradigms include glucose deprivation-induced ATP depletion and stimulation of the mitochondrial death pathway cascade; glucose deprivation-induced oxidative stress and triggering of Bax-associated events including the JNK/MAPK signalling pathways; and finally hypoglycemia-regulated expression of HIF-1 alpha, stabilization of p53 leading to an increase in p53-associated apoptosis. Several examples of each paradigm are presented. Future studies of glucose transport-associated apoptotic events will allow better understanding of the role of cellular metabolism in programmed cell death.

Progestin replacement in the menopause: effects on the endometrium and serum lipids.

The benefits of estrogen replacement therapy (ERT) in the menopause have been well demonstrated and are of significant importance, particularly with regard to prevention of osteoporosis and reduction in cardiovascular morbidity and mortality. The addition of a progestin to ERT is advocated in patients with a uterus to minimize the risk of endometrial hyperplasia and cancer. Although progestins can have adverse effects on serum lipids, it is unclear whether or not these effects negate the cardioprotective effects of estrogen. Progestins are an important part of hormone replacement therapy (HRT) regimen in patients with an intact uterus. The minimum dose and duration should be given to offset potential adverse effects on serum lipids while affording adequate protection of the endometrium. Both continuous and sequential progestin regimens appear to be efficacious. The newer progestins may offer increased flexibility in minimizing progestin side-effects while protecting the endometrium. Other regimens, such as less than monthly progestin administration, may offer another alternative to achieve these goals. Future studies in these areas are warranted.

Manifestations of diabetes mellitus on mouse preimplantation development: effect of elevated concentration of metabolic intermediates.

The metabolic derangements of pregnancies complicated by diabetes mellitus, specifically hyperglycaemia and hyperketonaemia, are known to be teratogenic during the period of organogenesis in animals. We have shown previously that poorly controlled diabetes mellitus impairs in-vivo and in-vitro mouse preimplantation embryo growth, and that culturing embryos in elevated glucose concentrations only partially recreates this developmental delay. To extend this observation we examined the effect on mouse preimplantation embryo growth of elevated concentrations of other metabolic intermediates, which may be deranged in diabetes mellitus, namely lipids, lactate, glycerol, amino acids, and ketones. Two-cell embryos from ovulation-induced B6C3F1 mice were cultured for 72 h in the presence of added lipids (250 mg/dl), lactate (5 mM), glycerol (160 microM) or mixed amino acids (8.5% travasol, 7 mM) and showed no significant difference in growth over 72 h versus their control groups. However, growth of preimplantation embryos in acetoacetate (10 mM) or in the racemic mixture of DL-beta-hydroxybutyrate (16 and 32 mM) revealed marked retardation versus controls when assessed either by distribution of developmental stages over time (24, 48, 72 h, P < 0.001) or by the difference in the average rank of sums indicating a delay in maturation (P < 0.0001). We conclude that elevated ketone concentrations adversely affect preimplantation embryo development. These findings extend previous studies which correlate uncontrolled diabetes mellitus as well as hyperglycaemia with abnormal organogenesis, and demonstrate that exposure to metabolic derangements may also hinder reproductive performance at even earlier stages in gestation.

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.

Hyperglycemia-induced apoptotic cell death in the mouse blastocyst is dependent on expression of p53.

Murine preimplantation embryos exposed to hyperglycemia experience decreased glucose transport, and overexpression of the proapoptotic protein BAX, leading to increased apoptosis. These changes may account for the increased rates of miscarriages and malformations seen in women with diabetes mellitus. To test whether p53 expression is necessary for hyperglycemia-induced apoptosis, p53+/+, +/-, -/- embryos were obtained by superovulation. Two-cell embryos were cultured to a blastocyst stage in 52 mM D- or L-glucose. Apoptosis was detected using terminal dUTP nick end labeling (TUNEL) assays. In vivo studies were performed in the same manner using blastocysts recovered from streptozotocin-induced diabetic mothers. Both in vitro and in vivo studies showed that wildtype embryos had a significantly higher percentage of TUNEL-positive nuclei than p53+/- and -/- embryos. To test whether p53 is upstream of BAX, immunofluorescent confocal microscopy and immunoprecipitation/ immunoblotting were performed on blastocysts cultured in high vs. control glucose conditions. Blastocysts from p53+/+ mice exhibited increased BAX staining vs. p53+/- and -/- embryos. Next, to determine whether a decrease in glucose transport was upstream or downstream of p53, deoxyglucose transport was measured in individual blastocysts from p53+/+ and +/- diabetic vs. nondiabetic mice. Embryos from diabetic p53+/- mice exhibit a 44% decrease in glucose transport, similar to the 38% decrease seen in embryos from diabetic p53+/+ mice. Taken together, these results strongly indicate that p53 plays a role in hyperglycemia-induced apoptosis, upstream of BAX overexpression and downstream of the decrease in glucose transport experienced by the mouse preimplantation embryo.

Preimplantation exposure to high insulin-like growth factor I concentrations results in increased resorption rates in vivo.

BACKGROUND: Women with polycystic ovarian syndrome suffer increased rates of miscarriage. Elevated insulin and insulin-like growth factor I (IGF-I) concentrations have been implicated. Here, we hypothesize that the high concentrations of IGF-I result in miscarriage, represented by decreased normal pregnancy rates and increased resorption rates in a mouse model. METHODS: In-vitro studies: 2-cell embryos were cultured in either 1.3 or 130 nmol/l IGF-I; or 500 nmol/l IGF-I receptor (IGF-IR) sense and antisense oligoprobes for 72 h. Embryos were then transferred into pseudo-pregnant ICR females. In-vivo studies: IGF-I-containing slow-release pellets or mock pellets were implanted within the uterine horn in ICR female mice. For both studies, the recipient females were killed on day 14.5 and the numbers of normal implantation sites versus resorption sites were recorded. RESULTS: In-vitro studies: blastocysts cultured in low IGF-I exhibited significantly higher normal implantation rates than blastocysts cultured in high IGF-I concentrations (P < 0.01). Blastocysts cultured in IGF-IR sense oligoprobes exhibited a significantly higher normal implantation rate than blastocysts cultured in antisense oligoprobes. In-vivo studies: mice implanted with IGF-I-containing pellets exhibited significantly lower normal implantation rates as compared with mock-pellet controls (P < 0.01). CONCLUSIONS: High preimplantation IGF-I concentrations in vitro or in vivo lead to increased resorption rates in the mouse.