Richard G. Lea

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., B(12) 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.

Transforming growth factor beta (TGF beta) is a multifunctional protein which has been suggested to play a central role in the pathogenesis of chronic inflammation and fibrosis. Nasal polyposis is a condition affecting the upper airways characterized by the presence of chronic inflammation and varying degrees of fibrosis. To examine the potential role of TGF beta in the pathogenesis of this condition, we investigated gene expression and cytokine production in nasal polyp tissues as well as in the normal nasal mucosa. By Northern blot analysis using a porcine TGF beta 1 cDNA probe, we detected TGF beta 1-specific mRNA in nasal polyp tissues, as well as in the tissue from a patient with allergic rhinitis, but not in the normal nasal mucosa. By the combination of tissue section staining with chromotrope 2R with in situ hybridization using the same TGF beta 1 probe, we found that approximately 50% of the eosinophils infiltrating the polyp tissue express the TGF beta 1 gene. In addition, immunohistochemical localization of TGF beta 1 was detected associated with extracellular matrix as well as in cells in the stroma. These results suggest that in nasal polyposis where eosinophils are the most prevalent inflammatory cell, TGF beta 1 synthesized by these cells may contribute to the structural abnormalities such as stromal fibrosis and basement membrane thickening which characterize this disease.

Leptin expression in third trimester placenta (p) and leptin concentrations in umbilical cord blood (cb) were investigated in normal pregnancies [n = 10 (p), 31 (cb)] and abnormal pregnancies complicated with (i) maternal insulin-dependent diabetes [IDDM: n = 3 (p), 13 (cb)], (ii) gestational diabetes [GD: n = 2 (p), 10 (cb)] and (iii) fetal growth retardation [FGR: n = 5 (p), 5 (cb)]. By in-situ hybridization and immunohistochemistry, placental leptin mRNA and protein were co-localized to the syncytiotrophoblast and villous vascular endothelial cells. Leptin receptor was immunolocalized to the syncytiotrophoblast. Relative to controls, the FGR group was characterized by low concentrations of placental and cord blood leptin. In a twin pregnancy, the normal-sized infant exhibited more placental and cord blood leptin than its growth-retarded twin. In contrast, both diabetic groups exhibited high concentrations of placental leptin mRNA and protein. The IDDM group exhibited the highest concentrations of leptin in cord blood. No change was observed in the expression of the leptin receptor in either the growth-retarded or diabetic pregnancies. In conclusion, the localization of placental leptin suggests that it may be released into both maternal and fetal blood. Furthermore, in fetal growth-retarded and diabetic pregnancies, the changes in leptin expression in the placenta and in leptin concentrations in umbilical cord blood appear to be related.

Maternal iron deficiency during pregnancy induces anaemia in the developing fetus; however, the severity tends to be less than in the mother. The mechanism underlying this resistance has not been determined. We have measured placental expression of proteins involved in iron transfer in pregnant rats given diets with decreasing levels of iron and examined the effect of iron deficiency on iron transfer across BeWo cell layers, a model for placental iron transfer. Transferrin receptor expression was increased at both mRNA and protein levels. Similarly, expression of the iron-responsive element (IRE)-regulated form of the divalent metal transporter 1 (DMT1) was also increased. In contrast, the non-IRE regulated isoform showed no change in mRNA levels. Protein levels of DMT1 increased significantly. Iron efflux is thought to be mediated by the metal transporter protein, IREG1/ferroportin1/MTP1, and oxidation of Fe(II) to Fe(III) prior to incorporation into fetal transferrin is carried out by the placental copper oxidase. Expression of IREG1 was not altered by iron deficiency, whereas copper oxidase activity was increased. In BeWo cells made iron deficient by treatment with desferrioxamine ('deferioxamine'), iron accumulation from iron-transferrin increased, in parallel with increased expression of the transferrin receptor. At the same time, iron efflux also increased, showing a higher flux of iron from the apical to the basolateral side. The data show that expression of placental proteins of iron transport are up-regulated in maternal iron deficiency, resulting in an increased efficiency of iron flux and a consequent minimization of the severity of fetal anaemia.

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.