Masatoshi Matsuzaki

Maternal and Fetal Growth, Body Composition, Endocrinology, and Metabolic Status in Undernourished Adolescent Sheep

Abstract The influence of relative maternal undernutrition on growth, endocrinology, and metabolic status in the adolescent ewe and her fetus were investigated at Days 90 and 130 of gestation. Singleton pregnancies to a single sire were established, and thereafter ewes were offered an optimal control (C; n = 14) or low (L [0.7 × C]; n = 21) dietary intake. Seven ewes receiving the L intake were switched to the C intake on Day 90 of gestation (L-C). At Day 90, live weight and adiposity score were reduced (P < 0.001) in L versus C dams. Plasma insulin and IGF1 concentrations were decreased (P < 0.02), whereas glucose concentrations were preserved in L relative to C intake dams. Fetal and placental mass was independent of maternal nutrition at this stage. By Day 130 of gestation, when compared to C and L-C dams, maternal adiposity was further depleted in L intake dams; concentrations of insulin, IGF1, and glucose were reduced; and nonesterified fatty acids increased. At Day 130, placental mass remained independent of maternal nutrition, but body weight was reduced (P < 0.01) in L compared with C fetuses (3555 g vs. 4273 g). Body weight was intermediate (3836 g) in L-C fetuses. Plasma glucose (P < 0.03), insulin (P < 0.07), and total liver glycogen content (P < 0.04) were attenuated in L fetuses. Fetal carcass analyses revealed absolute reductions (P < 0.05) in dry matter, crude protein, and fat, and a relative (g/kg) increase in carcass ash (P < 0.01) in L compared with C fetuses. Thus, limiting maternal intake during adolescent pregnancy gradually depleted maternal body reserves, impaired fetal nutrient supply, and slowed fetal soft tissue growth.

Serial Measurement of Uterine Blood Flow From Mid to Late Gestation in Growth Restricted Pregnancies Induced by Overnourishing Adolescent Sheep Dams

Uterine blood flow (UtBF) is a major regulator of transplacental fetal nutrient supply. The aim was to serially measure uterine blood flow from mid to late pregnancy in a paradigm of relatively late onset placental and fetal growth restriction. Singleton bearing adolescent dams was fed high (H) or control (C) nutrient intakes to induce putatively compromised or normal pregnancies, respectively. A perivascular flow probe was attached to the uterine artery of the gravid horn on Day 83 of gestation and UtBF was then recorded continuously for 2h, three times weekly until approximately Day 135, when pregnancies were either terminated or ewes allowed to deliver at term (approximately Day 145). Pregnancy outcome was determined at term in contemporaneous ewes without UtBF assessment. Placental and fetal weights were lower (P<0.001) in H compared with C intake groups and were independent of flow probe surgery and monitoring. Uterine blood flow was lower in H compared with C groups at the first assessment (Day 88, P<0.001) and was positively correlated with adjusted fetal weight at term, irrespective of treatment group (P<0.01). UtBF increased throughout the second half of gestation in both groups. Linear regression analysis of UtBF against day of gestation revealed that the slope was equivalent (5.5 vs. 5.3ml/min per day) and the mean intercept lower (212 vs. 370ml/min, P<0.001) in H compared with C groups, respectively. This study demonstrates the feasibility of serially measuring UtBF within the same individual sheep for a protracted period during the second half of gestation. UtBF was already lower at mid gestation in putatively growth restricted compared with control pregnancies, ahead of any reduction in placental and fetal weight, but increased similarly during the second half of gestation in both groups. These data are commensurate with the reported decrease in placental angiogenic growth factor expression at mid gestation, and, indicate that attenuated UtBF is an early defect in this adolescent paradigm.

Overnourishing pregnant adolescent ewes preserves perirenal fat deposition in their growth-restricted fetuses.

Overnourishing the adolescent sheep promotes rapid maternal growth at the expense of the gravid uterus. The growth of the placenta is impaired and results in the premature delivery of low-birthweight lambs. The present study details fetal adipose tissue development in these growth-restricted pregnancies. Singleton pregnancies were established by embryo transfer and, thereafter, adolescent ewes were offered a high (H; n = 12) or moderate (M; n = 14) level of a complete diet until necropsy on Day 131 of gestation. Fetal weight was lower (P < 0.001) in H compared with M groups. High maternal intake preserved brain and perirenal fat weight (P < 0.003), whereas relative weights of the heart, lungs, spleen and liver were unaltered. High nutrient intake resulted in significantly elevated maternal plasma concentrations of insulin, leptin, prolactin and glucose, no significant changes in fetal insulin, leptin or non-esterified fatty acids and attenuated fetal prolactin concentrations. Irrespective of nutritional intake, maternal plasma leptin, prolactin and glucose concentrations were negatively correlated with fetal weight and were positively correlated with fetal perirenal fat proportion (all P < 0.01). The mRNA expression for leptin, prolactin receptor and uncoupling protein (UCP) 1 in fetal perirenal fat was equivalent between groups, but, irrespective of maternal nutrition, UCP1 mRNA levels were negatively correlated with fetal weight (P < 0.01). Thus, overnourishing pregnant adolescent sheep preserves fat deposition in their growth-restricted fetuses, which may have implications for neonatal thermogenesis and for programming of postnatal adiposity.

Localization of interleukin-18 and its receptor in somatotrophs of the bovine anterior pituitary gland

A pro-inflammatory cytokine, interleukin 18 (IL-18), induces intracellular expression of IL-1 and the release of IL-6. IL-1 and IL-6 has been detected in anterior pituitary cells, suggesting that IL-18 is produced in anterior pituitary cells and may serve to aid immuno-endocrine regulation. In the present study, we addressed this hypothesis by investigating the intracellular localization of IL-18 and its receptor in bovine anterior pituitary gland. IL-18 mRNA and its protein were detected in the anterior pituitary gland by RT-PCR and Western blotting. In situ hybridization showed that IL-18 mRNA was expressed in the anterior pituitary cells. Immunohistochemistry of IL-18 and specific hormones revealed the presence of IL-18 in somatotrophs. Furthermore, the expression of GH mRNA in IL-18 immunoreactive cells was confirmed by immuno-laser microdissection. These results first demonstrated that somatotrophs produced IL-18. Subsequently, the distribution of the IL-18 receptor alpha (IL-18Rα) was investigated in order to understand IL-18 signaling among the anterior pituitary cells. Bovine IL-18Rα cDNA was partially sequenced and detected in the anterior pituitary gland by RT-PCR. Immunohistochemistry of IL-18Rα, IL-18 and GH showed that IL-18Rα was co-localized in IL-18 immunoreactive cells or somatotrophs. These data suggest that IL-18 acts on somatotrophs as an immuno-endocrine mediator through the autocrine pathway.

Placental Growth, Angiogenic Gene Expression, and Vascular Development in Undernourished Adolescent Sheep

Abstract Limiting maternal nutrient intake during ovine adolescent pregnancy progressively depleted maternal body reserves, impaired fetal nutrient supply, and slowed fetal soft tissue growth. The present study examined placental growth, angiogenic gene expression, and vascular development in this undernourished adolescent model at Days 90 and 130 of gestation. Singleton pregnancies were established, and ewes were offered an optimal control (C; n = 14) or low (L [0.7 × C]; n = 21) dietary intake. Seven ewes receiving L intakes were switched to C intakes on Day 90 of gestation (L-C). Fetal body weight (P < 0.01) and glucose concentrations (P < 0.03) were reduced in L versus C pregnancies by Day 130, whereas L-C group values were intermediate. Placental cellular proliferation, gross morphology, and mass were independent of maternal nutrition at both Day 90 and 130. In contrast, capillary area density in the maternal caruncular portion of the placentome was reduced by 20% (P < 0.001) at both stages of gestation in L compared with C groups. Caruncular capillary area density was equivalent in the L and L-C groups at Day 130. Placental mRNA expression of five key angiogenic ligands or receptors increased (P < 0.001) between Days 90 and 130 of gestation. VEGFA mRNA expression was higher (P < 0.04) in L compared with C and L-C pregnancies at Day 130, but otherwise gene expression of the remaining angiogenic factors and receptors analyzed was unaffected by maternal intake. Undernourishing the pregnant adolescent dam restricts fetal growth independently of changes in placental mass. Alterations in maternal placental vascular development may, however, play a role in mediating the previously reported reduction in maternal and hence fetal nutrient supply.

Ghrelin and Leptin Did Not Improve Meiotic Maturation of Porcine Oocytes Cultured In Vitro

To improve culture system for in vitro maturation (IVM) of porcine oocytes, ghrelin, leptin or growth hormone (GH), at concentration of 0, 0.5, 5, 50 and 500 ng/ml were added to the porcine follicular fluid (pFF)-supplemented medium NCSU23, and their effects on the maturation and cytoskeletal distribution of the oocytes with or without cumulus cells were compared. In the cumulus-denuded oocytes, no significant changes were noted in the maturation rate by different hormone treatments due to a marked decline in the controls. Maturation of the cumulus intact oocytes was moderately interfered by ghrelin (0.5-50 ng/ml, p < 0.01), but not significantly affected by leptin and GH. Distribution density of the cytoplasmic microtubules was decreased significantly by addition of ghrelin (by approximately 30% in 50-500 ng/ml, p < 0.01), whereas no remarkable effect was noted by leptin supplementation. High concentration (500 ng/ml) of ghrelin or leptin decreased significantly the cytoplasmic microfilaments in density (by 43% and 38%, p < 0.01, respectively). GH did not affect cytoskeletal distribution. The results suggest, in the culture system using pFF-supplemented medium that (i) ghrelin may have some inhibitory effect on the organization of microtubules and microfilaments, probably being a factor in lowered maturation rate and (ii) the addition of higher concentration of leptin may decrease microfilaments in density with no effect on meiotic maturation of the porcine oocytes.

Lactogenic hormones alter cellular and extracellular microRNA expression in bovine mammary epithelial cell culture

BackgroundBovine milk contains not only a variety of nutritional ingredients but also microRNAs (miRNAs) that are thought to be secreted by the bovine mammary epithelial cells (BMECs). The objective of this study was to elucidate the production of milk-related miRNAs in BMECs under the influence of lactogenic hormones.ResultsAccording to a microarray result of milk exosomal miRNAs prior to cellular analyses, a total of 257 miRNAs were detected in a Holstein cow milk. Of these, 18 major miRNAs of interest in the milk were selected for an expression analysis in BMEC culture that was treated with or without dexamethasone, insulin, and prolactin (DIP) to induce a lactogenic differentiation. Quantitative polymerase chain reaction (qPCR) results showed that the expressions of miR-21–5p (P = 0.005), miR-26a (P = 0.016), and miR-320a (P = 0.011) were lower in the DIP-treated cells than in the untreated cells. In contrast, the expression of miR-339a (P = 0.017) in the cell culture medium were lower in the DIP-treated culture than in the untreated culture. Intriguingly, the miR-148a expression in cell culture medium was elevated by DIP treatment of BMEC culture (P = 0.018). The medium-to-cell expression ratios of miR-103 (P = 0.025), miR-148a (P < 0.001), and miR-223 (P = 0.013) were elevated in the DIP-treated BMECs, suggesting that the lactogenic differentiation-induced secretion of these three miRNAs in BMECs. A bioinformatic analysis showed that the miRNAs down-regulated in the BMECs were associated with the suppression of genes related to transcriptional regulation, protein phosphorylation, and tube development.ConclusionThe results suggest that the miRNAs changed by lactogenic hormones are associated with milk protein synthesis, and mammary gland development and maturation. The elevated miR-148a level in DIP-treated BMECs may be associated with its increase in milk during the lactation period of cows.

Localization of leptin and leptin receptor in the bovine adenohypophysis.

The present study was carried out to detail the cellular localization of leptin (Lep) and the leptin receptor (LepR) in the bovine adenohypophysis. Lep immunoreactivity (Lep-ir) was found in about 30% of adenohypophysial cells in the gland. Immunochemistry of Lep and specific hormones using serial sections revealed that Lep-ir was present in 60.4% of somatotrophs, 15.9% of gonadotrophs, 6.5% of mammotrophs, 6.5% of thyrotrophs and 2.4% of corticotrophs. Both the common short isoform (OBRa) and the long isoform (OBRb) of LepR mRNA were expressed in the bovine adenohypophysis. LepR immunoreactivity (LepR-ir) was found in only 2.8% of the adenohypophysial cells and over 50% of LepR-ir cells were gonadotrophs, in which most of the cells were distributed in the zona tuberalis. The findings on Lep and LepR in the adenohypophysial cells indicate that Lep may regulate gonadotroph function through autocrine/paracrine pathway in the bovine adenohypophysis.

Both Microtubules and Microfilaments Mutually Control the Distribution of Mitochondria in Two-Cell Embryos of Golden Hamsters

ABSTRACT The roles of microtubules and microfilaments on distribution of mitochondria were evaluated by using fluorescent staining in 2-cell embryos of golden hamsters with or without cytoskeletal assembly inhibitors. In 2-cell embryos without treatment (control), most mitochondria were accumulated at the perinuclear region, while some mitochondria were noted at the cell cortex. Microtubules were found around the nuclei, correlating with distribution of the mitochondria. In contrast, microfilaments were stained intensely beneath the cell membrane and especially at the cell-to-cell contact region. In most (82%) of embryos treated with nocodazole (an inhibitor of microtubule polymerization), mitochondria had extended into the subcortical (intermediate) region with varying degree, where they were aggregated in patches. After a treatment of cytochalasin D (an inhibitor of actin polymerization), distributional density of mitochondria decreased at the cell cortex, suggesting that mitochondria moved back around the nucleus. After a treatment of both inhibitors, the distribution pattern of mitochondria was almost similar to that observed after cytochalasin D treatment. Our results suggest that the translocation of mitochondria to the perinuclear region is mediated by microtubules, while the movement of mitochondria to the cell cortex is regulated by microfilaments. Microtubules and microfilaments may function as bidirectional anchors of mitochondria to the perinuclear region and to the peripheral region, respectively.

Effects of feeding level of milk replacer on body growth, plasma metabolite and insulin concentrations, and visceral organ growth of suckling calves

The objective was to evaluate effects of feeding level of milk replacer on body growth, plasma metabolite and insulin concentrations, and allometric growth of visceral organs in suckling calves. Holstein bull calves (n = 8; 3-4 days of age) were fed either a low amount (average 0.63 kgDM/day, LM) or high amount (average 1.15 kgDM/day, HM) of high protein milk replacer until they were slaughtered at 6 weeks of age. Body weight (BW) at 4, 5, and 6 weeks of age, feed intake, average daily gain, and feed efficiency were higher in the HM than LM calves. The HM group had higher plasma glucose at 3 and 4 weeks of age and insulin levels after the age of 4 weeks compared with LM calves whereas no effect was detected on plasma nonesterified fatty acid or urea nitrogen concentrations. The HM calves had greater empty body weight (EBW), viscera-free BW and most of the organs dissected than LM calves. Relative weights (% of EBW) of liver, spleen, kidneys, and internal fat were higher, whereas head and large intestine was lower in HM than LM calves. The results suggest that increased milk feeding levels would accelerate the growth of the body and specific organs.