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.

The mechanism responsible for poor reproductive outcomes in type 1 diabetic males is not well understood. In light of new evidence that the Sertoli cells of the testis secrete insulin, it is currently unclear whether diabetic subfertility is the result of deficiency of pancreatic insulin, testicular insulin, or both. In this study, the Akita mouse diabetic model, which expresses a mutant, nonfunctional form of ins2 in testes and pancreas, was used to distinguish between systemic and local effects of insulin deficiency on the process of spermatogenesis and fertility. We determined that Akita homozygous male mice are infertile and have reduced testis size and abnormal morphology. Spermatogonial germ cells are still present but are unable to mature into spermatocytes and spermatids. Exogenous insulin treatment regenerates testes and restores fertility, but this plasma insulin cannot pass through the blood-testis barrier. We conclude that insulin does not rescue fertility through direct interaction with the testis; instead, it restores function of the hypothalamic-pituitary-gonadal axis and, thus, normalizes hormone levels of luteinizing hormone and testosterone. Although we show that the Sertoli cells of the testis secrete insulin protein, this insulin does not appear to be critical for fertility.

Recurrent miscarriages affect about 1–2% of couples trying to conceive; however, mechanisms leading to this complication are largely unknown. Most studies focus on the early embryo, but proper development and implantation of the blastocyst are also dependent on optimal endometrial progression into a receptive state. One of the key steps in the uterine preparation for embryo receptivity, known as decidualization, is the differentiation of endometrial stromal cells (ESCs) into decidual cells. During this transition, the ESCs undergo a drastic change in glucose metabolism. The efficiency of glucose uptake is determined by a family of facilitative glucose transporters (GLUTs), and many have been identified in the stroma. The primary focus of this work was to quantify the absolute amount of GLUT mRNAs in this cell type before and after decidualization. We used primary ESCs isolated from murine and human uteri. We developed and validated cDNA-based calibration curves for each GLUT and used these primers to arrive at absolute mRNA copy numbers. Here, we report all the GLUT mRNAs that are present in the ESCs and their abundance under both conditions, control and decidualized. GLUT1 mRNA is the most abundant and critical transporter in ESCs of both species, because knocking down this GLUT with sort hairpin RNA leads to dramatically reduced decidualization. These findings suggest that GLUT1 mRNA expression is essential for decidualization and we are the first to determine a possible mechanism to explain how maternal conditions of abnormal glucose utilization may impair implantation at the level of the ESCs.

Facilitative glucose transport molecules (glucose transporters, GLUTs) are responsible for glucose transport across cellular membranes. Of the 14 family members, expression of nine has been reported in the murine uterus and seven in the human uterus. Some studies reveal that adequate glucose uptake and metabolism are essential for the proper differentiation of the uterine endometrium toward a receptive state capable of supporting embryo implantation. However, the mechanistic role of GLUTs in endometrial function remains poorly understood. This review aims to present the current knowledge about GLUT expression in the uterus and distribution among the different cell types within the endometrium. In addition, it analyzes the available data in the context of roles GLUTs may play in normal uterine physiology as well as the pathological conditions of infertility, endometrial cancer, and polycystic ovarian syndrome.

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.

We previously identified a coregulator, repressor of estrogen receptor activity (REA), that directly interacts with estrogen receptor (ER) and represses ER transcriptional activity. Decreasing the intracellular level of REA by using small interfering RNA knockdown or antisense RNA approaches in cells in culture resulted in a significant increase in the level of up-regulation of estrogen-stimulated genes. To elucidate the functional activities of REA in vivo, we have used targeted disruption to delete the REA gene in mice. The targeting vector eliminated, by homologous recombination, the REA exon sequences encoding amino acids 12 to 201, which are required for REA repressive activity and for interaction with ER. The viability of heterozygous animals was similar to that of the wild type, whereas homozygous animals did not develop, suggesting a crucial role for REA in early development. Female, but not male, heterozygous animals had an increased body weight relative to age-matched wild-type animals beginning after puberty. REA mRNA and protein levels in uteri of heterozygous animals were half that of the wild type, and studies with heterozygous animals revealed a greater uterine weight gain and epithelial hyperproliferation in response to estradiol (E2) and a substantially greater stimulation by E2 of a number of estrogen up-regulated genes in the uterus. Even more dramatic in REA heterozygous animals was the loss of down regulation by E2 of genes in the uterus that are normally repressed by estrogen in wild-type animals. Mouse embryo fibroblasts derived from heterozygous embryos also displayed a greater transcriptional response to E2. These studies demonstrate that REA is a significant modulator of estrogen responsiveness in vivo: it normally restrains estrogen actions, moderating ER stimulation and enhancing ER repression of E2-regulated genes.The biological activity of estrogens, acting through estrogen receptors (ERs), is critically dependent on coregulator proteins (coactivators or corepressors) that are recruited to the ligand-receptor complex at various gene regulatory sites. Recent work indicates that this is a highly dynamic situation, with the equilibrium between corepressors and coactivators in a cell and the nature of the hormonal ligand determining the state of nuclear receptor activation or inhibition (4,8,11,17,21,30,32,34,35,40,43,44,47).Coactivator and corepressor proteins assemble into distinct, dynamic multiprotein complexes. In the case of coactivators, these complexes are constituted of the SRC/p160 family of proteins, CREB binding protein (CBP) and/or p300, and other factors that are recruited in a temporally ordered fashion (4, 44) and up-regulate nuclear receptor activity, at least in part, through enhanced histone acetyltransferase activity (8,30,32). ATP-dependent chromatin remodeling complexes, such as SWI/SNF, and the TRAP-DRIP-ARC (mediator) complex, which act sequentially or combinatorially, also enhance gene transcription by facilitating RNA polymerase II recruitment to promoters and ...

Embryo implantation is a highly synchronized event between an activated blastocyst and a receptive endometrium. The success of this process relies on the dynamic interplay of estrogen (E(2)) and progesterone (P(4)), however, the details of this interaction are not entirely clear. Recent data implicate E(2) and P(4) in the regulation of glucose utilization by affecting facilitative glucose transporter (GLUT) expression. In this study we examine GLUT1 expression in murine and human endometrial stromal cells (ESCs) using a primary culture system. We show that expression of GLUT1 is increased during ESC decidualization in vitro. P(4) up-regulates, whereas E(2) down-regulates, GLUT1 expression. In addition, P(4) increases and E(2) decreases glucose uptake in ESCs, suggesting that GLUT1 may be a major player in glucose utilization in these cells. Moreover, GLUT1 expression is increased in human ESCs when decidualized in vitro with P(4) and dibutyryl cAMP, suggesting a similar role for P(4) in human endometrium. In conclusion, an imbalance between P(4) and E(2) seen in patients with polycystic ovary syndrome, luteal phase defect, and recurrent pregnancy loss may have a critical impact on glucose utilization in the endometrial stroma, and, thus, may be responsible for endometrial dysfunction and failure of embryo implantation in these patient populations.

Endometrial stromal cells (ESC) must undergo a hormone-driven differentiation to form decidual cells as a requirement of proper embryo implantation. Recent studies from our laboratory have demonstrated that decidualizing cells require glucose transporter 1 expression and an increase in glucose use to complete this step. The present study focuses on the glucose-dependent molecular and metabolic pathways, which are required by ESC for decidualization. Inhibition of glycolysis had no effect on decidualization. However, blockade of the pentose phosphate pathway (PPP) with pharmacologic inhibitors 6-aminonicotinamide or dehydroepiandrosterone (DHEA), and short hairpin RNA-mediated knockdown of glucose-6-phosphate dehydrogenase, the rate-limiting step in the PPP, both led to strong decreases in decidual marker expression in vitro and decreased decidualization in vivo. Additionally, the studies demonstrate that inhibition is due, at least in part, to ribose-5-phosphate depletion, because exogenous nucleoside administration restored decidualization in these cells. The finding that PPP inhibition prevents decidualization of ESC is novel and clinically important, because DHEA is an endogenous hormone produced by the adrenal glands and elevated in a high proportion of women who have polycystic ovary syndrome, the most common endocrinopathy in reproductive age women. Together, this data suggest a mechanistic link between increased DHEA levels, use of glucose via the PPP, and pregnancy loss.