Interspecific Variation in One-Carbon Metabolism within the Ovarian Follicle, Oocyte, and Preimplantation Embryo: Consequences for Epigenetic Programming of DNA Methylation.

One-carbon (1C) metabolism provides methyl groups for the synthesis and/or methylation of purines and pyrimidines, biogenic amines, proteins, and phospholipids. Our understanding of how 1C pathways operate, however, pertains mostly to the (rat) liver. Here we report that transcripts for all bar two genes (i.e., BHMT, MAT1A) encoding enzymes in the linked methionine-folate cycles are expressed in all cell types within the ovarian follicle, oocyte, and blastocyst in the cow, sheep, and pig; as well as in rat granulosa cells (GCs) and human KGN cells (a granulosa-like tumor cell line). Betaine-homocysteine methyltransferase (BHMT) protein was absent in bovine theca and GCs, as was activity of this enzyme in GCs. Mathematical modeling predicted that absence of this enzyme would lead to more volatile S-adenosylmethionine-mediated transmethylation in response to 1C substrate (e.g., methionine) or cofactor provision. We tested the sensitivity of bovine GCs to reduced methionine (from 50 to 10 µM) and observed a diminished flux of 1C units through the methionine cycle. We then used reduced-representation bisulfite sequencing to demonstrate that this reduction in methionine during bovine embryo culture leads to genome-wide alterations to DNA methylation in >1600 genes, including a cohort of imprinted genes linked to an abnormal fetal-overgrowth phenotype. Bovine ovarian and embryonic cells are acutely sensitive to methionine, but further experimentation is required to determine the significance of interspecific variation in BHMT expression.

Impact of di-ethylhexylphthalate exposure on metabolic programming in P19 ECC-derived cardiomyocytes.

Di(2-ethylhexyl)phthalate (DEHP) is the most common plasticizer in plastic devices of everyday use. It is a ubiquitous environmental contaminant and primarily known to impair male gonadal development and fertility. Studies concerning the long-term effects of prenatal DEHP exposure on certain diseases [The Developmental Origins of Health and Disease paradigm (DOHaD) hypothesis] are scarce although it is proven that DEHP crosses the placenta. Rising environmental pollution during the last centuries coincides with an increasing prevalence of cardiovascular and metabolic diseases. We have investigated the effects of an early embryonic DEHP exposure at different developmental stages on cardiomyogenesis. We used an in-vitro model, the murine P19 embryonic carcinoma cell line (P19 ECC), mimicking early embryonic stages up to differentiated beating cardiomyocytes. P19 ECC were exposed to DEHP (5, 50, 100 µg ml(-1)) at the undifferentiated stage for 5 days and subsequently differentiated to beating cardiomyocytes. We analyzed the expression of metabolic (Pparg1, Fabp4 and Glut4), cardiac (Myh6, Gja1) and methylation (Dnmt1, Dnmt3a) marker genes by quantitative real-time PCR (qRT-PCR), beating rate and the differentiation velocity of the cells. The methylation status of Pparg1, Ppara and Glut4 was investigated by pyrosequencing. DEHP significantly altered the expression of all investigated genes. The beating rate and differentiation velocity were accelerated. Exposure to DEHP led to small but statistically significant increases in methylation of specific CpGs within Ppara and Pparg1, which otherwise were generally hypomethylated, but methylation of Glut4 was unaltered. Early DEHP exposure of P19 ECC alters the expression of genes associated with cellular metabolism and the functional features of cardiomyocytes.

Enhanced progesterone support during stimulated cycles of transvaginal follicular aspiration improves bovine in vitro embryo production.

The in vitro production (IVP) of cattle embryos requires that germinal-vesicle stage oocytes undergo a period of maturation in vitro prior to fertilization and culture to the blastocyst stage. Success of IVP in taurine cattle is enhanced following ovarian stimulation prior to oocyte retrieval (OPU), particularly if preceded by a short period of FSH withdrawal ('coasting'). However, evidence regarding the importance of progesterone (P4) support during OPU-IVP is equivocal. The current study, therefore, determined the effects of increased peripheral P4 concentrations during FSH-stimulated ('coasted') cycles of OPU. Progesterone support was provided by either an active corpus luteum (CL) and/or one of two intravaginal P4 releasing devices (i.e., CIDR® [1.38 g P4] or PRID® Delta [1.55 g P4]). Expt. 1 established an initial estrus prior to OPU, allowing CL formation (single luteal phase) spanning the first two of five cycles of OPU; the remaining three cycles were supported by either a CIDR® or PRID® Delta. Expt. 2 commenced with two cycles of dominant follicle removal (including prostaglandin F2α) undertaken seven days apart prior to six cycles of OPU. The absence of a CL meant that these cycles were supported only by a CIDR® or PRID® Delta. As each experiment involved several sequential cycles of OPU, the cumulative effects of device use on vaginal discharges were also assessed. Each experiment involved 10 sexually mature Holstein heifers. In the absence of a CL, peak plasma P4 concentrations were greater (P = 0.002) for the PRID® Delta (4.3 ± 0.22) than for the CIDR® (2.9 ± 0.22). In Expt. 1 there was an interaction (P < 0.05) between CL presence at OPU and P4 device on Day 8 blastocyst yields, indicating an effect of P4 device only when the CL was absent. The percentage hatching/hatched blastocysts of matured oocytes for the CIDR® and PRID® Delta was 44.3 ± 5.04 and 41.0 ± 5.40 in the presence, and 17.1 ± 3.48 and 42.2 ± 3.76 in the absence, of a CL (P = 0.018). Combined analyses of data from Expt. 1 and 2, when no CL was present, confirmed that Day 8 blastocyst yields were greater (P = 0.022) for the PRID® Delta than the CIDR®. Vaginal discharge scores were higher (P < 0.001) for the PRID® Delta than the CIDR® in Expt. 1 but not in Expt 2; however scores were low, did not increase with repeated use, and thus were deemed of no clinical or welfare concern. In conclusion, enhanced P4 support during FSH-stimulated cycles of OPU-IVP can improve in vitro embryo development.

Developmental, cytogenetic and epigenetic consequences of removing complex proteins and adding melatonin during in vitro maturation of bovine oocytes.

Background: In vitro maturation (IVM) of germinal vesicle intact oocytes prior to in vitro fertilization (IVF) is practiced widely in animals. In human assisted reproduction it is generally reserved for fertility preservation or where ovarian stimulation is contraindicated. Standard practice incorporates complex proteins (CP), in the form of serum and/or albumin, into IVM media to mimic the ovarian follicle environment. However, the undefined nature of CP, together with batch variation and ethical concerns regarding their origin, necessitate the development of more defined formulations. A known component of follicular fluid, melatonin, has multifaceted roles including that of a metabolic regulator and antioxidant. In certain circumstances it can enhance oocyte maturation. At this stage in development, the germinal-vesicle intact oocyte is prone to aneuploidy and epigenetic dysregulation. Objectives: To determine the developmental, cytogenetic and epigenetic consequences of removing CP and including melatonin during bovine IVM. Materials and methods: The study comprised a 2 x 2 factorial arrangement comparing (i) the inclusion or exclusion of CP, and (ii) the addition (100 nM) or omission of melatonin, during IVM. Cumulus-oocyte complexes (COCs) were retrieved from stimulated cycles. Following IVM and IVF, putative zygotes were cultured to Day 8 in standard media. RNAseq was performed on isolated cumulus cells, cytogenetic analyses (SNP-based algorithms) on isolated trophectoderm cells, and DNA methylation analysis (reduced representation bisulfite sequencing) on isolated cells of the inner-cell mass. Results: Removal of CP during IVM led to modest reductions in blastocyst development, whilst added melatonin was beneficial in the presence but detrimental in the absence of CP. The composition of IVM media did not affect the nature or incidence of chromosomal abnormalities but cumulus-cell transcript expression indicated altered metabolism (primarily lipid) in COCs. These effects preceded the establishment of distinct metabolic and epigenetic signatures several days later in expanded and hatching blastocysts. Conclusions: These findings highlight the importance of lipid, particularly sterol, metabolism by the COC during IVM. They lay the foundation for future studies that seek to develop chemically defined systems of IVM for the generation of transferrable embryos that are both cytogenetically and epigenetically normal.

Effects of omega-3 and -6 polyunsaturated fatty acids on ovine follicular cell steroidogenesis, embryo development and molecular markers of fatty acid metabolism.

We previously reported increased follicular fluid progesterone (P(4)) concentrations in ewes fed an n-3 compared to an n-6 polyunsaturated fatty acid (PUFA)-enriched diet, but detected no differential effect of n-3 and n-6 PUFA-enriched high-density lipoproteins (HDL) on granulosa cell (GC) steroidogenesis in vitro. Moreover, net n-6 PUFA-enriched HDL reduced early embryo development, but in the absence of a net uptake of FA. Consequently, we hypothesised that a) effects of n-3 PUFA on ovarian steroidogenesis are mediated by theca rather than GCs and b) during embryo culture lipids are acquired solely from the albumin fraction of serum, so that albumin-delivered n-3 and n-6 PUFA exert a greater differential effect on embryo development than either low-density lipoprotein (LDL)- or HDL-delivered PUFA. Data confirmed that n-3 PUFA increases P(4) production solely in theca cells and that this is associated with an increase in STAR transcript expression. Furthermore, LDL- and HDL-delivered n-3 PUFA are equally efficacious in this regard during the first 96 h of culture, but thereafter only HDL-delivered n-3 PUFA induces this effect in partially luteinised theca cells. We also demonstrate that albumin is the sole serum fraction that leads to a net uptake of FA during embryo culture. PUFA-enriched serum and albumin increased the yield of morphologically poorer quality blastocysts with increased transcript expression for the antioxidant enzyme SOD1. Important differential effects of n-3 and n-6 PUFA on ovarian steroidogenesis acting solely on theca cells are identified, but differential effects of PUFA on embryo development are less apparent.

Tight junction protein ZO-2 expression and relative function of ZO-1 and ZO-2 during mouse blastocyst formation.

Apicolateral tight junctions (TJs) between epithelial cells are multiprotein complexes regulating membrane polarity and paracellular transport and also contribute to signalling pathways affecting cell proliferation and gene expression. ZO-2 and other ZO family members form a sub-membranous scaffold for binding TJ constituents. We investigated ZO-2 contribution to TJ biogenesis and function during trophectoderm epithelium differentiation in mouse preimplantation embryos. Our data indicate that ZO-2 is expressed from maternal and embryonic genomes with maternal ZO-2 protein associated with nuclei in zygotes and particularly early cleavage stages. Embryonic ZO-2 assembled at outer blastomere apicolateral junctional sites from the late 16-cell stage. Junctional ZO-2 first co-localised with E-cadherin in a transient complex comprising adherens junction and TJ constituents before segregating to TJs after their separation from the blastocyst stage (32-cell onwards). ZO-2 siRNA microinjection into zygotes or 2-cell embryos resulted in specific knockdown of ZO-2 mRNA and protein within blastocysts. Embryos lacking ZO-2 protein at trophectoderm TJs exhibited delayed blastocoel cavity formation but underwent normal cell proliferation and outgrowth morphogenesis. Quantitative analysis of trophectoderm TJs in ZO-2-deficient embryos revealed increased assembly of ZO-1 but not occludin, indicating ZO protein redundancy as a compensatory mechanism contributing to the mild phenotype observed. In contrast, ZO-1 knockdown, or combined ZO-1 and ZO-2 knockdown, generated a more severe inhibition of blastocoel formation indicating distinct roles for ZO proteins in blastocyst morphogenesis.

One-Carbon Metabolism: Linking Nutritional Biochemistry to Epigenetic Programming of Long-Term Development.

One-carbon (1C) metabolism comprises a series of interlinking metabolic pathways that include the methionine and folate cycles that are central to cellular function, providing 1C units (methyl groups) for the synthesis of DNA, polyamines, amino acids, creatine, and phospholipids. S-adenosylmethionine is a potent aminopropyl and methyl donor within these cycles and serves as the principal substrate for methylation of DNA, associated proteins, and RNA. We propose that 1C metabolism functions as a key biochemical conduit between parental environment and epigenetic regulation of early development and that interindividual and ethnic variability in epigenetic-gene regulation arises because of genetic variants within 1C genes, associated epigenetic regulators, and differentially methylated target DNA sequences. We present evidence to support these propositions, drawing upon studies undertaken in humans and animals. We conclude that future studies should assess the epigenetic effects of cumulative (multigenerational) dietary imbalances contemporaneously in both parents, as this better represents the human experience.

Society for Reproductive Biology Founders' Lecture 2003. The making of an embryo: short-term goals and long-term implications.

During early development, the eutherian mammalian embryo forms a blastocyst comprising an outer trophectoderm epithelium and enclosed inner cell mass (ICM). The short-term goal of blastocyst morphogenesis, including epithelial differentiation and segregation of the ICM, is mainly regulated autonomously and comprises a combination of temporally controlled gene expression, cell polarisation, differentiative cell divisions and cell-cell interactions. This aspect of blastocyst biogenesis is reviewed, focusing, in particular, on the maturation and role of cell adhesion systems. Early embryos are also sensitive to their environment, which can affect their developmental potential in diverse ways and may lead to long-term consequences relating to fetal or postnatal growth and physiology. Some current concepts of embryo-environment interactions, which may impact on future health, are also reviewed.

Adaptive responses by mouse early embryos to maternal diet protect fetal growth but predispose to adult onset disease.

Poor maternal nutrition during pregnancy can alter postnatal phenotype and increase susceptibility to adult cardiovascular and metabolic diseases. However, underlying mechanisms are largely unknown. Here, we show that maternal low protein diet (LPD), fed exclusively during mouse preimplantation development, leads to offspring with increased weight from birth, sustained hypertension, and abnormal anxiety-related behavior, especially in females. These adverse outcomes were interrelated with increased perinatal weight being predictive of later adult overweight and hypertension. Embryo transfer experiments revealed that the increase in perinatal weight was induced within blastocysts responding to preimplantation LPD, independent of subsequent maternal environment during later pregnancy. We further identified the embryo-derived visceral yolk sac endoderm (VYSE) as one mediator of this response. VYSE contributes to fetal growth through endocytosis of maternal proteins, mainly via the multiligand megalin (LRP2) receptor and supply of liberated amino acids. Thus, LPD maintained throughout gestation stimulated VYSE nutrient transport capacity and megalin expression in late pregnancy, with enhanced megalin expression evident even when LPD was limited to the preimplantation period. Our results demonstrate that in a nutrient-restricted environment, the preimplantation embryo activates physiological mechanisms of developmental plasticity to stablize conceptus growth and enhance postnatal fitness. However, activation of such responses may also lead to adult excess growth and cardiovascular and behavioral diseases.

Mouse embryo culture induces changes in postnatal phenotype including raised systolic blood pressure.

A key factor in the use of assisted reproductive technologies (ART) for diverse species is the safety of procedures for long-term health. By using a mouse model, we have investigated the effect of in vitro culture and embryo transfer (ET) of superovulated embryos on postnatal growth and physiological activity compared with that of embryos developing in vivo. Embryo culture from two-cell to blastocyst stages in T6 medium either with or without a protein source reduced blastocyst trophectoderm and inner cell mass cell number compared with that of embryos developing in vivo. Embryo culture and ET had minimal effects on postnatal growth when compared with in vivo development with an equivalent litter size. However, embryo culture, and to a lesser extent ET, led to an enhanced systolic blood pressure at 21 weeks compared with in vivo development independent of litter size, maternal origin, or body weight. Moreover, activity of enzymatic regulators of cardiovascular and metabolic physiology, namely, serum angiotensin-converting enzyme and the gluconeogenesis controller, hepatic phosphoenolpyruvate carboxykinase, were significantly elevated in response to embryo culture and/or ET in female offspring at 27 weeks, independent of maternal factors and postnatal growth. These animal data indicate that postnatal physiological criteria important in cardiovascular and metabolic health may be more sensitive to routine ART procedures than growth.

Maternal low protein diet restricted to the preimplantation period induces a gender-specific change on hepatic gene expression in rat fetuses.

It has been shown previously that maternal low protein diet (LPD) throughout rat gestation altered hepatic gene expression and enzyme activities in offspring. Here, we investigate the effect of maternal LPD (9% casein vs. 18% control) exclusively during the preimplantation period (switched diet group) or provided throughout gestation on hepatic gene expression in day 20 fetuses. Using quantitative competitive PCR, we found that switched diet induced a two-fold increase (P = 0.008) in hepatic gene expression of phosphoenolpyruvate carboxykinase (PEPCK, a rate limiting enzyme for gluconeogenesis) in male fetuses and a 17% increase (P = 0.005) in 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1, acts primarily as a reductase to produce active glucocorticoid) in female liver compared with control fetuses. Maternal LPD administered throughout gestation increased 11beta-HSD1 expression in male fetal liver by 27% (P = 0.042) compared with controls. However, maternal LPD fed for either period did not affect fetal hepatic insulin receptor (IR), glucocorticoid receptor (GR), glycogen synthase (GS) nor placental glucose transporter 1 (Glut1) and 3 (Glut3) transcript levels. The alteration in fetal hepatic gene expression could not be attributed specifically to known regulators including insulin or glucose concentrations in fetal blood nor alteration in cAMP in fetal liver, although a combination of these regulatory factors may be responsible. Fetal hepatic glycogen level was unaffected by maternal diet. The present findings show that the long term potential of the preimplantation embryo is sensitive to maternal LPD such that basal levels of hepatic gene expression in day 20 fetuses are altered in a gender-specific manner.

Imprinted gene expression in the rat embryo-fetal axis is altered in response to periconceptional maternal low protein diet.

In our previous study, we have shown that maternal low protein diet (LPD, 9% casein vs 18% casein control) fed exclusively during the rat preimplantation period (0-4.25 day postcoitum) induced low birth weight, altered postnatal growth and hypertension in a gender-specific manner. In this study, we investigated the effect of maternal LPD restricted only to the preimplantation period (switched diet) or provided throughout gestation on fetal growth and imprinted gene expression in blastocyst and fetal stages of development. Male, but not female, blastocysts collected from LPD dams displayed a significant reduction (30%) in H19 mRNA level. A significant reduction in H19 (9.4%) and Igf2 (10.9%) mRNA was also observed in male, but not in female, fetal liver at day 20 postcoitum in response to maternal LPD restricted to the preimplantation period. No effect on the blastocyst expression of Igf2R was observed in relation to maternal diet. The reduction in H19 mRNA expression did not correlate with an observed alteration in DNA methylation at the H19 differentially methylated region in fetal liver. In contrast, maternal LPD throughout 20 days of gestation did not affect male or female H19 and Igf2 imprinted gene expression in fetal liver. Neither LPD nor switched diet treatments affected H19 and Igf2 imprinted gene expression in day 20 placenta. Our findings demonstrate that one contributor to the alteration in postnatal growth induced by periconceptional maternal LPD may derive from a gender-specific programming of imprinted gene expression originating within the preimplantation embryo itself.

The embryo and its future.

The preimplantation mammalian embryo from different species appears sensitive to the environment in which it develops, either in vitro or in vivo, for example, in response to culture conditions or maternal diet. This sensitivity may lead to long-term alterations in the characteristics of fetal and/or postnatal growth and phenotype, which have implications for clinical health and biotechnological applications. We review the breadth of environmental influences that may affect early embryos and their responses to such conditions along epigenetic, metabolic, cellular, and physiological directions. In addition, we evaluate how embryo environmental responses may influence developmental potential and phenotype during later gestation. We conclude that a complex of different mechanisms may operate to associate early embryo environment with future health.