Richard G. Lea

DNA methylation, insulin resistance, and blood pressure in offspring determined by maternal periconceptional B vitamin and methionine status

A complex combination of adult health-related disorders can originate from developmental events that occur in utero. The periconceptional period may also be programmable. We report on the effects of restricting the supply of specific B vitamins (i.e., B12 and folate) and methionine, within normal physiological ranges, from the periconceptional diet of mature female sheep. We hypothesized this would lead to epigenetic modifications to DNA methylation in the preovulatory oocyte and/or preimplantation embryo, with long-term health implications for offspring. DNA methylation is a key epigenetic contributor to maintenance of gene silencing that relies on a dietary supply of methyl groups. We observed no effects on pregnancy establishment or birth weight, but this modest early dietary intervention led to adult offspring that were both heavier and fatter, elicited altered immune responses to antigenic challenge, were insulin-resistant, and had elevated blood pressure–effects that were most obvious in males. The altered methylation status of 4% of 1,400 CpG islands examined by restriction landmark genome scanning in the fetal liver revealed compelling evidence of a widespread epigenetic mechanism associated with this nutritionally programmed effect. Intriguingly, more than half of the affected loci were specific to males. The data provide the first evidence that clinically relevant reductions in specific dietary inputs to the methionine/folate cycles during the periconceptional period can lead to widespread epigenetic alterations to DNA methylation in offspring, and modify adult health-related phenotypes.

In utero exposure to low doses of environmental pollutants disrupts fetal ovarian development in sheep

Epidemiological studies of the impact of environmental chemicals on reproductive health demonstrate consequences of exposure but establishing causative links requires animal models using ‘real life’ in utero exposures. We aimed to determine whether prolonged, low-dose, exposure of pregnant sheep to a mixture of environmental chemicals affects fetal ovarian development. Exposure of treated ewes (n = 7) to pollutants was maximized by surface application of processed sewage sludge to pasture. Control ewes (n = 10) were reared on pasture treated with inorganic fertilizer. Ovaries and blood were collected from fetuses (n = 15 control and n = 8 treated) on Day 110 of gestation for investigation of fetal endocrinology, ovarian follicle/oocyte numbers and ovarian proteome. Treated fetuses were 14% lighter than controls but fetal ovary weights were unchanged. Prolactin (48% lower) was the only measured hormone significantly affected by treatment. Treatment reduced numbers of growth differentiation factor (GDF9) and induced myeloid leukaemia cell differentiation protein (MCL1) positive oocytes by 25–26% and increased pro-apoptotic BAX by 65% and 42% of protein spots in the treated ovarian proteome were differently expressed compared with controls. Nineteen spots were identified and included proteins involved in gene expression/transcription, protein synthesis, phosphorylation and receptor activity. Fetal exposure to environmental chemicals, via the mother, significantly perturbs fetal ovarian development. If such effects are replicated in humans, premature menopause could be an outcome.

Dietary omega-3 and -6 polyunsaturated fatty acids affect the composition and development of sheep granulosa cells, oocytes and embryos

The evidence that omega-3 (n-3) and -6 (n-6) polyunsaturated fatty acids (PUFAs) have differential effects on ovarian function, oocytes and embryo quality is inconsistent. We report on the effects of n-3 versus n-6 PUFA-enriched diets fed to 36 ewes over a 6-week period, prior to ovarian stimulation and follicular aspiration, on ovarian steroidogenic parameters and embryo quality. Follicle number and size were unaltered by diet, but follicular-fluid progesterone concentrations were greater in n-3 PUFA-fed ewes than in n-6 PUFA-fed ewes. The percentage of saturated FAs (mostly stearic acid) was greater in oocytes than in either granulosa cells or plasma, indicating selective uptake and/or de novo synthesis of saturated FAs at the expense of PUFAs by oocytes. High-density lipoproteins (HDLs) fractionated from sera of these ewes increased granulosa cell proliferation and steroidogenesis relative to the FA-free BSA control during culture, but there was no differential effect of n-3 and n-6 PUFAs on either oestradiol or progesterone production. HDL was ineffective in delivering FAs to embryos during culture, although n-6 PUFA HDL reduced embryo development. All blastocysts, irrespective of the treatment, contained high levels of unsaturated FAs, in particular linoleic acid. Transcripts for HDL and low-density lipoprotein (LDL) receptors (SCARB1 and LDLR) and stearoyl-CoA desaturase (SCD) are reported in sheep embryos. HDL reduced the expression of transcripts for LDLR and SCD relative to the BSA control. The data support a differential effect of n-3 and n-6 PUFAs on ovarian steroidogenesis and pre-implantation development, the latter in the absence of a net uptake of FAs.

A subset of patients with recurrent spontaneous abortion is deficient in transforming growth factor β-2-producing suppressor cells in uterine tissue near the placental attachment site

PROBLEM: To determine if patients with unexplained recurrent miscarriage have a deficiency of decidual immunosuppressor cells that produce transforming growth factor β type 2, as has been found in mice with abortion due to rejection and/or trophoblast failure.

Ontogeny of the expression of leptin and its receptor in the murine fetus and placenta.

Leptin is a 167-amino acid protein that is secreted from adipose cells and expressed in placental tissues. It is important nutritionally in the regulation of energy balance, but also has other functions such as a role in reproduction. To investigate the function of the leptin system in fetal development we examined, primarily by in-situ hybridization and immunohistochemistry, the expression (both mRNA and protein) of leptin and its receptor (including the signalling splice variant) in tissues from 11.5, 13.5, 16.5 and 18.5 d postcoitus murine fetuses and associated placentas. We detected leptin mRNA (at low levels) and protein predominantly in the cytotrophoblasts of the labyrinth part of the placenta, an area of nutrient exchange between the developing fetus and the placenta, and in the trophoblast giant cells situated in the junctional zone at the maternal interface. In addition, leptin was strongly expressed in the fetal cartilage-bone and at a lower level in the hair follicles, heart, and liver of the murine fetus at differing stages of development. The leptin receptor, including the signalling splice variant, was also identified in specific fetal tissues. The physiological importance of expression of both leptin and the leptin receptor (OB-R and OB-Rb) in the placenta remains to be determined. In addition, the high levels of expression of leptin and its receptor in discrete areas of the murine fetus suggest that leptin has a critical role in fetal development.

Immunoendocrine aspects of endometrial function and implantation

Effective ovarian and uterine function relies on a complex interplay between the endocrine and immune systems. It is generally accepted that in reproductive tissues, oestradiol and progesterone have pro- and anti-inflammatory activities respectively and, in this regard, the paracrine effects of the sex steroids on the ovary are similar to the endocrine effects on the uterus. Ovarian leukocyte recruitment and cytokine release are central to follicle development, ovulation and corpus luteum function. At the uterine level, the cyclical changes in sex steroids regulate the number and distribution of endometrial and decidual immune cells as well as other immune signalling and surveillance factors. The uterine mucosa is unique, in that it must tolerate sperm and the allogeneic blastocyst in a way that does not compromise uterine immune surveillance against bacteria, yeast and viruses. Crosstalk between the sex steroids and immune mediators (systemic and local) are central to these functions, and this article will review these mechanisms and their importance for successful reproductive function and pregnancy success.

Immunohistochemical evidence for an endocrine/paracrine role for ghrelin in the reproductive tissues of sheep

BackgroundThe gut hormone, ghrelin, is involved in the neuroendocrine and metabolic responses to hunger. In monogastric species, circulating ghrelin levels show clear meal-related and body weight-related changes. The pattern of secretion and its role in ruminant species is less clear. Ghrelin acts via growth hormone secretagogue receptors (GHSR-1a) to alter food intake, fat utilization, and cellular proliferation. There is also evidence that ghrelin is involved in reproductive function. In the present study we used immunohistochemistry to investigate the presence of ghrelin and GHSR-1a in sheep reproductive tissues. In addition, we examined whether ghrelin and GHSR-1a protein expression is developmentally regulated in the adult and fetal ovine testis, and whether there is an association with markers of cellular proliferation, i.e. stem cell factor (SCF) and proliferating cell nuclear antigen (PCNA).MethodsAntibodies raised against ghrelin and its functional receptor, GHSR-type 1a, were used in standard immunohistochemical protocols on various reproductive tissues collected from adult and fetal sheep. GHSR-1a mRNA presence was also confirmed by in situ hybridisation. SCF and PCNA immunoexpression was investigated in fetal testicular samples. Adult and fetal testicular immunostaining for ghrelin, GHSR-1a, SCF and PCNA was analysed using computer-aided image analysis. Image analysis data were subjected to one-way ANOVA, with differences in immunostaining between time-points determined by Fishers least significant difference.ResultsIn adult sheep tissue, ghrelin and GHSR-1a immunostaining was detected in the stomach (abomasum), anterior pituitary gland, testis, ovary, and hypothalamic and hindbrain regions of the brain. In the adult testis, there was a significant effect of season (photoperiod) on the level of immunostaining for ghrelin (p < 0.01) and GHSR-1a (p < 0.05). In the fetal sheep testis, there was a significant effect of gestational age on the level of immunostaining for ghrelin (p < 0.001), GHSR-1a (p < 0.05), SCF (p < 0.05) and PCNA (p < 0.01).ConclusionEvidence is presented for the presence of ghrelin and its receptor in various reproductive tissues of the adult and fetal sheep. In addition, the data indicate that testicular expression of ghrelin and its receptor is physiologically regulated in the adult and developmentally regulated in the fetus. Therefore, the ghrelin ligand/receptor system may have a role (endocrine and/or paracrine) in the development (cellular proliferation) and function of the reproductive axis of the sheep.

Effect of Iron Deficiency on Placental Cytokine Expression and Fetal Growth in the Pregnant Rat

Abstract Iron deficiency anemia is the most common nutritional disorder in the world. Anemia is especially serious during pregnancy, with deleterious consequences for both the mother and her developing fetus. We have developed a model to investigate the mechanisms whereby fetal growth and development are affected by maternal anemia. Weanling rats were fed a control or iron-deficient diet before and throughout pregnancy and were killed at Day 21. Dams on the deficient diet had lower hematocrits, serum iron concentrations, and liver iron levels. Similar results were recorded in the fetus, except that the degree of deficiency was markedly less, indicating compensation by the placenta. No effect was observed on maternal weight or the number and viability of fetuses. The fetuses from iron-deficient dams, however, were smaller than controls, with higher placental:fetal ratios and relatively smaller livers. Iron deficiency increased levels of tumor necrosis factor α (TNFα) only in the trophoblast giant cells of the placenta. In contrast, levels of type 1 TNFα receptor increased significantly in giant cells, labyrinth, cytotrophoblast, and fetal vessels. Leptin levels increased significantly in labyrinth and marginally (P = 0.054) in trophoblast giant cells. No change was observed in leptin receptor levels in any region of the placentas from iron-deficient dams. The data show that iron deficiency not only has direct effects on iron levels and metabolism but also on other regulators of growth and development, such as placental cytokines, and that these changes may, in part at least, explain the deleterious consequences of maternal iron deficiency during pregnancy.

Cytokines and the regulation of apoptosis in reproductive tissues : A Review

PROBLEM: To determine the role of apoptosis‐regulating genes bax and bcl‐2 in reproduction.

3 Macrophages and migratory cells in endometrium relevant to implantation

The implantation of an appropriately developed embryo into a suitably conditioned uterine lining depends on the synchronous maturation of the preimplantation embryo and uterine lining. The pre- and postimplantation embryo also requires protection from immunocompetent maternal immune effectors. Preimplantation embryo development is affected by genotype, intercellular communication and autocrine growth factors (polyamines, TGF-alpha, TGF-beta 1, PAF). Factors of maternal origin may also enhance embryo development (EGF, TGF-alpha, TGF-beta 1, IGF, polyamines). The preimplantation embryo signals its presence to the mother by release of factor(s) such as IFN-alpha-II and a PAF-like factor. PAF may induce EPF in the mother and enhances vascular permeability at the implantation site. Uterine or peritoneal leukocytosis may inhibit development via toxic effects of lymphokines/monokines (IL-2, IL-1?, IFN-gamma, TNF-alpha). Immunoprotection of the preimplantation embryo is conferred by embryo derived maternal factors (EPF, T-cell suppressor factors). The uterus is receptive during a limited period of time (implantation window) and the substrate adhesion molecules produced by uterine and embryonic trophectoderm cells are crucial for the initial stages of implantation. At implantation, trophoblast expression of MHC and non-MHC antigens is shut off and both immunocompetent maternal cells (macrophages, dendritic cells, granulocytes, IELs, immunocytes) and lymphatics become sparse at implantation sites. Peri-implantation cytokines of maternal origin, such as CSF-1, GM-CSF and IGF-1 binding protein, are probably important for trophoblast growth and development. Immuno-protection of the embryo at this stage may be mediated by embryo derived factors that inactivate macrophages and by a population of large, hormone dependent Lyt 2+ (CD8+) suppressor cells. It is possible that these CD8+ cells respond to progesterone and secrete molecules that inactivate natural effector (NK-type) cells against trophoblast. Prostaglandins (PGE2) may play a brief role in immunosuppression at the time of implantation but its role is probably more important with respect to the decidual response. Defects in the pre- and peri-implantation stages of pregnancy may lead to delayed failure in the form of clinical miscarriage.