Somatic cell nuclear transfer is associated with altered expression of angiogenic factor systems in bovine placentomes at term

Low efficiency of somatic cell cloning by nuclear transfer has been associated with alterations of placental vascular architecture. Placental growth and function depend on the growth of blood vessels; VEGF-A and bFGF are the most important factors controlling neovascularization and vascular permeability in the placenta. We hypothesize that the VEGF-A and bFGF systems are disrupted in placentomes from cloned animals, contributing to the placental abnormalities that are common in these clones. We determined mRNA expression and protein tissue localization of VEGF-A, bFGF, and their receptors in placentomes from cloned and non-cloned bovine fetuses at term. Real-time RT-PCR revealed that VEGFR-2 mRNA was increased in cloned male-derived placentomes, while mRNA of bFGF and its receptors were decreased in placentomes of cloned females. VEGF-A system proteins were found to be located in placentomal endothelial, maternal and fetal epithelial and stromal cells; there was a variable pattern of cellular distribution of these proteins in both cloned and non-cloned animals. Alterations in the expression of VEGF-A and bFGF systems suggest that angiogenic factors are involved in abnormal placental development in cloned gestations, contributing to impaired fetal development and poor survival ratesFAPESP 02/07392-7CAPES (PROBRAL grant 272/7)\ud CAPES (PROBRAL grant D/06/33937

Effect of cysteamine during in vitro maturation of bovine oocytes on embryo development

In in vitro embryo production (IVP), oxidative modifications via increased reactive oxygen species (ROS) represent a major culture induced stress. Anti-oxidant systems such as glutathione (GSH) can attenuate deleterious effects of oxidative stress decreasing ROS thus protecting the zygote and early embryo. Previous studies suggest that addition of cysteamine to in vitro maturation (IVM) medium can increase intracellular GSH synthesis, improving pronucleus formation, cleavage rates and embryo development. The aim of the present work was to investigate the effects of cysteamine during IVM with conventional FSH stimulation or utilizing the IVM phase of the follicular system (FS), recently proposed by Ovarian Molecular Physiology Laboratory research group. The FS base medium consisted of TCM199 (with Earle's salts, bovine serum albumin, amikacin, pyruvate) supplemented with rhFSH, amphiregulin, insulin-like growth factor 1, estradiol and progesterone (Soares et al., Reproduction, Fertility and Development, 29:2217-2224, 2017). Five replicates were performed to compare four experimental groups: FSH (basic medium supplemented with rhFSH 10-1 UI/mL); FSH+C (FSH medium supplemented with cysteamine 1 mM/mL); FS and FS+C (FS medium supplemented with cysteamine 1 mM/mL). Ovaries were obtained from a slaughterhouse and COCs recovered by aspiration were submitted to IVM for 24h, followed by in vitro fertilization for 18h and in vitro culture (IVC) for seven days. Blastocyst rate was calculated in relation to total oocytes subjected to IVM and blastocyst cell numbers were assessed by Hoechst 33342 staining. Rates of expanded and hatched blastocysts were calculated in relation to total blastocysts. Data were arcsine transformed and compared with Tukey (parametric data) or Wilcoxon (non-parametric data) tests. Differences were considered significant when P 640.05. Addition of cysteamine did not alter blastocyst rate (P>0.05; FSH 24.67\ub15.37; FSH+C 33.50\ub14.95; FS 25.96\ub14.92; FS+C 22.95\ub15.56), expanded and hatched blastocysts rates (P>0.05; FSH 94.44\ub13.51; FSH+C 83.57\ub15.61; FS 85.83\ub14.86; FS+C 83.36\ub17.65), nor the total number of embryonic cells (P>0.05; FSH 118.40\ub17.41; FSH+C 123.39\ub17.36; SF 124.97\ub19.75; SF+C 124.37\ub19.63). In conclusion, addition of cysteamine to the IVM medium did not improve embryo production. Supported by FAPESP 2017/07588-4

Somatic cell nuclear transfer is associated with altered expression of angiogenic factor systems in bovine placentomes at term

Low efficiency of somatic cell cloning by nuclear transfer has been associated with alterations of placental vascular architecture. Placental growth and function depend on the growth of blood vessels; VEGF-A and bFGF are the most important factors controlling neovascularization and vascular permeability in the placenta. We hypothesize that the VEGF-A and bFGF systems are disrupted in placentomes from cloned animals, contributing to the placental abnormalities that are common in these clones. We determined mRNA expression and protein tissue localization of VEGF-A, bFGF, and their receptors in placentomes from cloned and non-cloned bovine fetuses at term. Real-time RT-PCR revealed that VEGFR-2 mRNA was increased in cloned male-derived placentomes, while mRNA of bFGF and its receptors were decreased in placentomes of cloned females. VEGF-A system proteins were found to be located in placentomal endothelial, maternal and fetal epithelial and stromal cells; there was a variable pattern of cellular distribution of these proteins in both cloned and non-cloned animals. Alterations in the expression of VEGF-A and bFGF systems suggest that angiogenic factors are involved in abnormal placental development in cloned gestations, contributing to impaired fetal development and poor survival rates

Maternal protein restriction affects gene expression and enzyme activity of intestinal disaccharidases in adult rat offspring

This study investigated the consequences of intrauterine protein restriction on the gastrointestinal tract and particularly on the gene expression and activity of intestinal disaccharidases in the adult offspring. Wistar rat dams were fed isocaloric diets containing 6% protein (restricted, n = 8) or 17% protein (control, n = 8) throughout gestation. Male offspring (n = 5-8 in each group) were evaluated at 3 or 16 weeks of age. Maternal protein restriction during pregnancy produced offspring with growth restriction from birth (5.7 ± 0.1 vs 6.3 ± 0.1 g; mean ± SE) to weaning (42.4 ± 1.3 vs 49.1 ± 1.6 g), although at 16 weeks of age their body weight was similar to control (421.7 ± 8.9 and 428.5 ± 8.5 g). Maternal protein restriction also increased lactase activity in the proximal (0.23 ± 0.02 vs 0.15 ± 0.02), medial (0.30 ± 0.06 vs 0.14 ± 0.01) and distal (0.43 ± 0.07 vs 0.07 ± 0.02 U·g-1·min-1) small intestine, and mRNA lactase abundance in the proximal intestine (7.96 ± 1.11 vs 2.38 ± 0.47 relative units) of 3-week-old offspring rats. In addition, maternal protein restriction increased sucrase activity (1.20 ± 0.02 vs 0.91 ± 0.02 U·g-1·min-1) and sucrase mRNA abundance (4.48 ± 0.51 vs 1.95 ± 0.17 relative units) in the duodenum of 16-week-old rats. In conclusion, the present study shows for the first time that intrauterine protein restriction affects gene expression of intestinal enzymes in offspring