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H. Hyatt and R. B. L. Gwatkin

Summary. The acrosomal matrix of hamster spermatozoa was enriched and characterized. Acrosomal matrices were released from spermatozoa with shaking in a pH 5·2 buffer containing Triton X-100 and protease inhibitors, and enriched on a glass-bead column. Phase-contrast microscopy indicated that 70–80% of the acrosomal matrices were released from the spermatozoa and only minor contamination from sperm heads was detected. Transmission electron microscopy confirmed the low level of contamination in the preparation and revealed a bilaminar structure similar but not identical to that of guinea-pig acrosomal matrix. One- and two-dimensional SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed the acrosomal matrix to be a complex structure enriched for several polypeptides. Proteinase activity was demonstrated by gelatin–SDS-PAGE. The major activity corresponded to bands of relative molecular masses (M r) of 56 000, 51 000 and 48 000 with two minor bands of M r 30 000 and 28 000. The lectin Pisum sativum agglutinin (PSA) bound to the anterior head of spermatozoa and isolated acrosomal matrix as judged by fluorescence microscopy using FITC–PSA. Western blots of spermatozoa and acrosomal matrices followed by overlay with biotinylated PSA indicated that there are at least two PSA-binding glycoproteins of M r 60 000 and 72 000.

Keywords: acrosome; acrosomal matrix; hamster spermatozoa; acrosin; proacrosin

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M A Hyatt, E A Butt, H Budge, T Stephenson and M E Symonds

Maternal cold exposure of pregnant sheep promotes fetal growth, whereas nutrient restriction (NR) can reverse this effect. The present study was designed to establish whether cold exposure induced by winter shearing of the mother at 70 days gestation (term=147 days), with or without NR (induced by a 50% reduction in maternal food intake from 110 days gestation), has specific effects on mRNA abundance of hepatic genes related to growth and liver energy metabolism that could regulate postnatal body and liver growth. Measurements of hepatic gene expression for the GH secretagog receptor-1a (GHSR-1A), peroxisome proliferator-activated receptor (PPAR)α, phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase activity together with glycogen content were made in the livers of offspring at 1 and 30 days of age. Maternal NR reduced liver mass at day 1, whereas offspring of cold-exposed mothers had larger livers at day 30 irrespective of maternal diet. Cold exposure resulted in the up-regulation of GHSR-1A mRNA abundance and reduced glucose-6-phosphatase activity at 1, but not 30 days of age, whereas IGF-II mRNA was decreased at 1 and 30 days. PPARα mRNA abundance was enhanced, while PEPCK was reduced in 30-day old offspring of cold-exposed mothers. NR caused reductions in IGF-I mRNA and, at 1-day postnatal age, down-regulated GHR, while, at 30 days, reduced GHSR-1A gene expression and hepatic glycogen content. In conclusion, we have shown that maternal cold exposure and NR have different effects on the hepatic GH–IGF and metabolic axis that may contribute to changes in liver growth over the first month of life.

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D P Yakubu, A Mostyn, M A Hyatt, L O Kurlak, H Budge, T Stephenson and M E Symonds

This study investigated the developmental and nutritional programming of two important mitochondrial proteins, namely voltage-dependent anion channel (VDAC) and cytochrome c, in the sheep kidney, liver and lung. The effect of maternal nutrient restriction between early and mid-gestation (i.e. 28- to 80-day gestation, the period of maximal placental growth) on the abundance of these proteins was also examined in fetal and juvenile offspring. Fetuses were sampled at 80 and 140 days of gestation (term ~147 days), and postnatal animals at 1 and 30 days and 6 months of age. The abundance of VDAC peaked at 140 days of gestation in the lung, compared with 1 day after birth in the kidney and liver, whereas cytochrome c abundance was greatest at 140 days of gestation in the liver, 1 day after birth in the kidney and 6 months of age in lungs. This differential ontogeny in mitochondrial protein abundance between tissues was accompanied with very different tissue-specific responses to changes in maternal food intake. In the liver, maternal nutrient restriction only increased mitochondrial protein abundance at 80 days of gestation, compared with no effect in the kidney. In contrast, in the lung mitochondrial protein, abundance was raised near to term, whereas VDAC abundance was decreased by 6 months of age. These findings demonstrate the tissue-specific nature of mitochondrial protein development that reflects differences in functional adaptation after birth. The divergence in mitochondrial response between tissues to maternal nutrient restriction early in pregnancy further reflects these differential ontogenies.

Open access

M A Hyatt, D S Gardner, S Sebert, V Wilson, N Davidson, Y Nigmatullina, L L Y Chan, H Budge and M E Symonds

Maternal nutrition during the period of early organ development can modulate the offspring's ability to metabolise excess fat as young adults when exposed to an obesogenic environment. This study examined the hypothesis that exposing offspring to nutrient restriction coincident with early hepatogenesis would result in endocrine and metabolic adaptations that subsequently lead to increased ectopic lipid accumulation within the liver. Pregnant sheep were fed either 50 or 100% of total metabolisable energy requirements from 30 to 80 days gestation and 100% thereafter. At weaning, offspring were made obese, and at ∼1 year of age livers were sampled. Lipid infiltration and molecular indices of gluconeogenesis, lipid metabolism and mitochondrial function were measured. Although hepatic triglyceride accumulation was not affected by obesity per se, it was nearly doubled in obese offspring born to nutrient-restricted mothers. This adaptation was accompanied by elevated gene expression for peroxisome proliferator-activated receptor γ (PPARG) and its co-activator PGC1α, which may be indicative of changes in the rate of hepatic fatty acid oxidation. In contrast, maternal diet had no influence on the stimulatory effect of obesity on gene expression for a range of proteins involved in glucose metabolism and energy balance including glucokinase, glucocorticoid receptors and uncoupling protein 2. Similarly, although gene expressions for the insulin and IGF1 receptors were suppressed by obesity they were not influenced by the prenatal nutritional environment. In conclusion, excess hepatic lipid accumulation with juvenile obesity is promoted by suboptimal nutrition coincident with early development of the fetal liver.

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M G Gnanalingham, P Williams, V Wilson, J Bispham, M A Hyatt, A Pellicano, H Budge, T Stephenson and M E Symonds

In sheep, modest maternal nutrient restriction (NR) over the period of rapid placental growth restricts placentome growth and results in offspring in which glucocorticoid action is enhanced. Therefore, this study investigated the placental effects of early to mid-gestational NR on glucocorticoid receptor (GR), 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2), uncoupling protein-2 (UCP2), and IGF type-I receptor (IGF-IR) mRNA abundance together with cell proliferation and apoptosis as determined histologically, and the mitochondrial proteins voltage-dependent anion channel and cytochrome c that are involved in apoptosis. Placenta was sampled at 80 and 140 days gestation (dGA; term ~147 dGA). NR was imposed between 28 and 80 days gestation when control and nutrient-restricted groups consumed 150 or 60% respectively of their total metabolizable energy requirements. All mothers were then fed to requirements up to term. Total fetal placentome weights were decreased by NR at 80 dGA but were heavier at 140 dGA following 60 days of nutritional rehabilitation. GR and UCP2 mRNA abundance increased whilst 11βHSD2 mRNA decreased with gestational age. NR persistently up-regulated GR and UCP2 mRNA abundance. 11βHSD2 mRNA was reduced by NR at 80 dGA but increased near to term. IGF-IRmRNA abundance was only decreased at 80 dGA. Placental apoptosis and mitochondrial protein abundance were unaffected by NR, whereas cell proliferation was markedly reduced. In conclusion, placental UCP2 and local glucocorticoid action are affected by the gestational nutritional status and may result in the offspring showing enhanced glucocorticoid sensitivity, thereby predisposing them to disease in later life.

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S P Sébert, M A Hyatt, L L Y Chan, M Yiallourides, H P Fainberg, N Patel, D Sharkey, T Stephenson, S M Rhind, R C Bell, H Budge, D S Gardner and M E Symonds

The recent discovery of an association between body composition, energy intake and the fat mass and obesity-associated (FTO) gene represents a promising new therapeutic target in obesity prevention. In a well, pre-established large animal model, we investigated the regulation of FTO gene expression under conditions either leading to obesity or increased risk of obesity related disorders: i) a sedentary ‘Western’ lifestyle and ii) prenatal exposure to nutrient restriction. Pregnant sheep were either fed to fully meet their nutritional requirements throughout gestation or 50% of this amount from early-to-mid gestation. Following weaning, offspring were either made obese through exposure to a sedentary obesogenic environment or remained lean. A significant positive relationship between placental FTO gene expression and fetal weight was found at 110 days gestation. In both the newborn and adult offspring, the hypothalamus was the major site of FTO gene expression. Hypothalamic FTO gene expression was upregulated by obesity and was further increased by prenatal nutrient restriction. Importantly, we found a strong negative relationship between the hypothalamic FTO gene expression and food intake in lean animals only that may imply FTO as a novel controller of energy intake. In contrast, FTO gene expression in the heart was downregulated in obese offspring born to nutrient restricted mothers. In addition, FTO gene expression was unaffected by obesity or prenatal diet in insulin-dependent tissues, where it changed with age possibly reflecting adaptations in cellular energetic activity. These findings extend information gained from human epidemiology and provide new insights into the regulation of in vivo energy metabolism to prevent obesity.