A low-protein diet during pregnancy alters glucose metabolism and insulin secretion

In pancreatic islets, glucose metabolism is a key process for insulin secretion, and pregnancy requires an increase in insulin secretion to compensate for the typical insulin resistance at the end of this period. Because a low-protein diet decreases insulin secretion, this type of diet could impair glucose homeostasis, leading to gestational diabetes. In pancreatic islets, we investigated GLUT2, glucokinase and hexokinase expression patterns as well as glucose uptake, utilization and oxidation rates. Adult control non-pregnant (CNP) and control pregnant (CP) rats were fed a normal protein diet (17%), whereas low-protein non-pregnant (LPNP) and low-protein pregnant (LPP) rats were fed a low-protein diet (6%) from days 1 to 15 of pregnancy. The insulin secretion in 2.8 mmol l(-1) of glucose was higher in islets from LPP rats than that in islets from CP, CNP and LPNP rats. Maximal insulin release was obtained at 8.3 and 16.7 mmol l(-1) of glucose in LPP and CP groups, respectively. The glucose dose-response curve from LPNP group was shifted to the right in relation to the CNP group. In the CP group, the concentration-response curve to glucose was shifted to the left compared with the CNP group. The LPP groups exhibited an "inverted U-shape" dose-response curve. The alterations in the GLUT2, glucokinase and hexokinase expression patterns neither impaired glucose metabolism nor correlated with glucose islet sensitivity, suggesting that β-cell sensitivity to glucose requires secondary events other than the observed metabolic/molecular events.     

Use of sucrose during removal of cryoprotectants after thawing eight-cell mouse embryos

Eight-cell mouse embryos were frozen in 0.5-ml plastic straws in modified Dulbecco's phosphate buffered saline (PBS) plus 5% steer serum plus either 1.32 M dimethyl sulfoxide (DMSO) or 1.32 M glycerol. Upon thawing, embryos were diluted 1:4 with 0.0, 0.2, 0.6, or 1.0 M sucrose solutions within the straws. Thawing was either in air at ambient temperature or in 8 degrees C or 38 degrees C water. After 48 h of culture, more embryos frozen in DMSO and thawed in 8 degrees C and 37 degrees C water developed to blastocysts (87 and 93%, respectively) than embryos thawed in air (75%; P < 0.05). No significant differences in development were noted among the three thawing regimens when embryos were frozen with glycerol. There was no significant effect of concentration of sucrose during dilution on development of embryos postthaw. With glycerol as the cryoprotectant, damage to zonae pellucidae increased as thawing rates increased, whereas the opposite was observed with DMSO as the cryoprotectant (P < 0.05).     

Assisted reproduction technologies alter steroid delivery to the mouse fetus during pregnancy

Assisted reproduction technologies (ART) include in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI), and are common treatments for infertility. Although generally successful, ART warrant further investigations due to emerging perinatal issues, especially low birth weight. Herein we extend our previous work demonstrating higher steroid clearance in murine ART placentas by examining steroid biosynthesis and the directional flow of steroids in the maternal-placental-fetal units. The activities of the major steroidogenic enzymes 3β-hydroxysteroid dehydrogenase (3β-HSD) and cytochrome P450 17-αhydroxylase (CYP17) were assessed in maternal liver and ovaries and fetal livers as were levels of cholesterol, progesterone, estrone (E1), and estradiol (E2) in the maternal, placental and fetal units. No structural abnormalities were found in placentas from ART. Although ART increased 3β-HSD activity in maternal livers, there were no other changes in 3β-HSD- or CYP17-mediated steroidogenesis. Cholesterol levels were significantly lower in maternal livers of ICSI pregnancies and in placentas from both IVF and ICSI pregnancies but not altered in the fetal livers. Progesterone levels were higher in maternal and fetal livers in IVF and ICSI, respectively, but were significantly lowered in ICSI placentas, compared to normal fertilization. For estrogenic hormones, no differences in E1 or E2 levels were observed in maternal livers but ICSI significantly increased both E1 and E2 levels in placentas while both IVF and ICSI significantly lowered E1 but raised E2 levels in fetal livers. In summary, while steroid production was normal, steroid diffusion/flow from mother to fetus was altered in murine pregnancies conceived by ART. This appears to occur, at least in part; through placental mechanisms. Impaired cholesterol and steroid transfer may affect correct regulation of fetal growth and development.     

Protein metabolism in the mouse during pregnancy and lactation.

Protein synthesis was measured in vivo in the whole body and in a number of individual tissues in mice at various stages of pregnancy and lactation. The absolute rate of protein synthesis in the whole body increased from 640 mg/day in virgin mice to 1590 mg/day by day 18 of pregnancy, and to 2100 mg/day by day 15 of lactation. Large proportions of these increments were contributed by the rapidly growing foetuses and placentae in the pregnant animals and by protein synthesis in the mammary glands during lactation. In addition, a substantial stimulation of growth and protein synthesis was also observed in the liver and the gastrointestinal tract. Gastrocnemius muscle showed no changes in protein metabolism, indicating that in the well-fed mouse this tissue is not required to play a role as a protein reserve during pregnancy and lactation.     

Synthesis and distribution of carbohydrate chains in cleavage-stage mouse embryos carrying the t12 lethal mutation

Carbohydrate chains on the t12 mutant embryos were analyzed. No abnormalities of the synthesis of the carbohydrate chains were observed in the t12 homozygotes until the late morula stage, when radiolabeled carbohydrate chains were analyzed by Sephadex G-50 column chromatography. Furthermore, polarization of the Con A receptor occurred normally at the eight-cell stage. Therefore, the possible defects of the carbohydrate chains on the t12 embryos were suggested to be neither gross abnormality of the synthesis nor abnormal localization on the surface, but rather minor structural changes on a specific molecule.     

Maternal diet alters exencephaly frequency in SELH/Bc strain mouse embryos

BACKGROUND: The SELH/Bc mouse inbred strain, with a high frequency of nonsyndromic, genetically-multifactorial exencephaly, is a model for human cranial neural tube defects (NTDs). Maternal diet affects risk of human NTDs. METHODS: Exencephaly frequencies in SELH/Bc embryos were compared in 8 studies in which dams were fed alternative commercial Purina diets (5015 and 5001) or semisynthetic diets, and in several studies in which maternal diet was supplemented with a specific nutrient, either in drinking water or food before and during pregnancy, or by intraperitoneal injection on E7 and/or E8. RESULTS: The exencephaly frequency in SELH/Bc embryos was 2- to 8-fold higher when the dams were fed Purina 5015 (averaging 23% exencephaly) or a semisynthetic diet modeled on Purina 5015 (averaging 28%) or NIH-31 standard diet (23%), compared with Purina 5001 (averaging 7%). The exencephaly frequency remained high (41%) on a semisynthetic diet modeled on Purina 5001. The exencephaly frequency was not reduced significantly by maternal supplementation with folic acid, nor with each of zinc, methionine, niacin, brewers' yeast, riboflavin, vitamin B12, or inositol. Nor was it reduced by maternal diets with supplemental methyl donors and cofactors or with reduced fat. CONCLUSIONS: The frequency of exencephaly in SELH/Bc embryos is strongly influenced by a specific unidentified aspect of the commercial ration Purina 5001 that prevents 55-85% of exencephaly in SELH/Bc embryos, when directly compared with an alternative commercial ration Purina 5015 or its semisynthetic mimic. This strong maternal diet effect on NTD frequency may point to novel nutritional approaches to prevention of human NTDs.     

Endothelial-derived nitric oxide and angiotensinogen: blood pressure and metabolism during mouse pregnancy

The regulation of blood pressure during pregnancy involves several biological pathways. Candidate genes implicated in hypertensive diseases during pregnancy include those of the renin-angiotensin system and nitric oxide synthase (NOS). We evaluated blood pressure and metabolic characteristics during pregnancy in mutant mice. These included mice with a null mutation in the endothelial NOS (eNOS) gene (Nos3(-/-)), four copies of the angiotensinogen gene (Agt(2/2)), and mutations in both genes [four copies of Agt and heterozygous deficient for eNOS (Agt(2/2)Nos3(+/-)), four copies of Agt and homozygous deficient for eNOS (Agt(2/2)Nos3(-/-))]. Blood pressure measurements of nulliparous females from mutant strains were compared with two common laboratory strains C57Bl6/J and SV129 throughout their first pregnancy. Serum and urine analysis for the evaluation of renal and liver physiology were measured in the prepregnant state and during the third trimester of pregnancy. Throughout pregnancy blood pressures in all mutant strains were higher compared with controls. Agt(2/2)Nos3(-/-) showed the highest blood pressures and C57Bl6/J the lowest. Control mice, but not mutant mice, showed a second trimester decline in blood pressure. No immediate differences were noted regarding behavioral characteristics, renal or liver function parameters. Mice deficient for eNOS, mice with overexpression of Agt, and mice with mutations in both genes demonstrated higher blood pressure throughout pregnancy. There was no evidence of renal dysfunction, liver dysfunction, or hemolysis among any of the strains studied. We conclude that Nos3 and Agt are important genes in the regulation of blood pressure during pregnancy.     

Alcohol exposure alters DNA methylation profiles in mouse embryos at early neurulation

Alcohol exposure during development can cause variable neurofacial deficit and growth retardation known as fetal alcohol spectrum disorders (FASD). The mechanism underlying FASD is not fully understood. However, alcohol, which is known to affect methyl donor metabolism, may induce aberrant epigenetic changes contributing to FASD. Using a tightly controlled whole-embryo culture, we investigated the effect of alcohol exposure (88mM) at early embryonic neurulation on genome-wide DNA methylation and gene expression in the C57BL/6 mouse. The DNA methylation landscape around promoter CpG islands at early mouse development was analyzed using MeDIP (methylated DNA immunoprecipitation) coupled with microarray (MeDIP-chip). At early neurulation, genes associated with high CpG promoters (HCP) had a lower ratio of methylation but a greater ratio of expression. Alcohol-induced alterations in DNA methylation were observed, particularly in genes on chromosomes 7, 10, and X; remarkably, a >10 fold increase in the number of genes with increased methylation on chromosomes 10 and X was observed in alcohol-exposed embryos with a neural tube defect phenotype compared to embryos without a neural tube defect. Significant changes in methylation were seen in imprinted genes, genes known to play roles in cell cycle, growth, apoptosis, cancer, and in a large number of genes associated with olfaction. Altered methylation was associated with significant (p     

Epoxyeicosatrienoic Acid inhibition alters renal hemodynamics during pregnancy

In this study we examined the expression of cytochrome P450 (CYP) 2C and CYP2J isoforms in renal proximal tubules and microvessels isolated from rats at different stages of pregnancy. We also selectively inhibited epoxyeicosatrienoic acid (EET) production by the administration of N-methanesulfonyl-6-(2-proparyloxyphenyl)hexanamide (MSPPOH 20 mg/kg/day iv) to rats during Days 14-17 of gestation and to age-matched virgin rats and determined the consequent effects on renal function. Western blot analysis showed that CYP2C11, CYP2C23, and CYP2J2 expression was significantly increased in the renal microvessels of pregnant rats on Day 12 of gestation. In the proximal tubules, CYP2C23 expression was significantly increased throughout pregnancy, while the expression of CYP2C11 was increased in early and late pregnancy and the expression of CYP2J2 was increased in middle and late pregnancy. MSPPOH treatment significantly increased pregnant rats' mean arterial pressure, renal vascular resistance, and sodium balance but significantly decreased renal blood flow, glomerular filtration rate, and urinary sodium excretion, as well as fetal pups' body weight and length. In contrast, MSPPOH treatment had no effect on renal hemodynamics or urinary sodium excretion in age-matched virgin rats. In pregnant rats, MSPPOH treatment also caused selective inhibition of renal cortical EET production and significantly decreased the expression of CYP2C11, CYP2C23, and CYP2J2 in the renal cortex, renal microvessels, and proximal tubules. These results suggest that upregulation of renal vascular and tubular EETs contributes to the control of blood pressure and renal function during pregnancy.     

Ovarian steroid metabolism during post-natal development in the normal mouse and in the adult hypogonadal (hpg) mouse

Ovarian steroid metabolism was investigated (i) during development in a normal inbred strain in which post-natal follicle growth has been described and (ii) in adult hypogonadal (hpg) mice which lack GnRH and have very low serum concentrations of gonadotrophins. Tissue was incubated with [3H]pregnenolone or [3H]androstenedione and metabolites separated by t.l.c. or h.p.l.c. Progesterone was the major metabolite formed at all ages while androstenedione was the major androgen. Between 7 and 21 days there was an overall increase in steroidogenic enzyme activity with a peak of 5 alpha-reductase between 21 and 29 days. The major metabolite of progesterone around puberty was 5 alpha-pregnane-3 alpha-ol-20-one. A sharp increase in 20 alpha-hydroxysteroid dehydrogenase was observed after 38 days due, presumably, to the appearance of corpora lutea. Unlike the rat, androstanediol levels were low at all ages. Oestradiol was the major oestrogen formed from androstenedione with a peak of production at 38 days. In the adult hpg mouse metabolism was similar to that of the 7-day normal mouse although 17-ketosteroid reductase and aromatase levels were very low compared to normal animals of any age, indicating that gonadotrophin stimulation is involved in the expression of activity by these enzymes.     

A possible effect of the Na+ concentration in oviductal fluid on amino acid uptake by cleavage-stage mouse embryos

Two- and four-cell mouse embryos exhibited both Na+-dependent and Na+-independent components of zwitterionic alpha-amino acid transport, which we tentatively ascribe to the A and L amino acid transport systems, respectively. Uptake of taurine was virtually all Na+-dependent and is probably via the beta system. Na+-independent L-lysine uptake by two-cell embryos may have been via system y+. The small amount of lysine transport which was Na+-dependent (30% of the total) could not be attributed to any well known transport system and may have been due to the early ontogenetic expression of a newly described transport system which predominates in preimplantation blastocysts. We conclude that the rate of Na+-dependent amino acid transport in two-cell mouse embryos could be significantly affected in situ by changes in the [Na+] which are known to occur in oviductal fluid.     

Effects of removal of necrotic blastomeres from mouse cryopreserved embryos on blastocyst formation and hatching

To evaluate whether the developmental potential of embryos that were partially damaged after freezing and thawing can be improved by removal of necrotic blastomeres. Eight-cell mouse embryos were cryopreserved using 1,2-propanediol and sucrose as cryoprotectant with slow cooling procedure. After thawing, blastocyst formation and hatching of fully intact embryos were compared between no treatment and with laser-assisted hatching. For partially intact embryos, the effects of removal of necrotic blastomeres with micromanipulation were evaluated. Laser-assisted hatching of mouse cryopreserved fully intact embryos significantly increased blastocyst hatching (63.4% versus 48.3%, P<0.05), but had little effect on blastocyst formation (72.0% versus 70.1%, P>0.05). The removal of necrotic blastomeres from partially damaged mouse cryopreserved embryos with micromanipulation significantly increased blastocyst formation (52.9% versus 32.0%, P<0.05) and blastocyst hatching (41.2% versus 22.0%, P>0.05) compared with the control group. The developmental potential of partially damaged cryopreserved embryos can be improved by removal of necrotic blastomeres with micromanipulation.     

Synthesis of hydroxylated sterols in transgenic Arabidopsis plants alters growth and steroid metabolism

To explore mechanisms in plant sterol homeostasis, we have here increased the turnover of sterols in Arabidopsis (Arabidopsis thaliana) and potato (Solanum tuberosum) plants by overexpressing four mouse cDNA encoding cholesterol hydroxylases (CHs), hydroxylating cholesterol at the C-7, C-24, C-25, or C-27 positions. Compared to the wild type, the four types of Arabidopsis transformant showed varying degrees of phenotypic alteration, the strongest one being in CH25 lines, which were dark-green dwarfs resembling brassinosteroid-related mutants. Gas chromatography-mass spectrometry analysis of extracts from wild-type Arabidopsis plants revealed trace levels of α and β forms of 7-hydroxycholesterol, 7-hydroxycampesterol, and 7-hydroxysitosterol. The expected hydroxycholesterol metabolites in CH7-, CH24-, and CH25 transformants were identified and quantified using gas chromatography-mass spectrometry. Additional hydroxysterol forms were also observed, particularly in CH25 plants. In CH24 and CH25 lines, but not in CH7 ones, the presence of hydroxysterols was correlated with a considerable alteration of the sterol profile and an increased sterol methyltransferase activity in microsomes. Moreover, CH25 lines contained clearly reduced levels of brassinosteroids, and displayed an enhanced drought tolerance. Equivalent transformations of potato plants with the CH25 construct increased hydroxysterol levels, but without the concomitant alteration of growth and sterol profiles observed in Arabidopsis. The results suggest that an increased hydroxylation of cholesterol and/or other sterols in Arabidopsis triggers compensatory processes, acting to maintain sterols at adequate levels.     

Replication timing: histone genes replicate during early S phase in cleavage-stage embryos of sea urchin.

Newly synthesized DNA was separated from the bulk of the DNA by pulse-labeling with BUdR and centrifugation in an alkaline CsCl buoyant density gradient. The content of histone gene in the newly synthesized DNA was determined by DNA dot hybridization. The gene contents in DNA replicated during the early half of S phase and during the whole S phase were compared. Results showed that histone genes were replicated during the first half of the S phase in embryos in the early cleavage stage.     

Cotransfer of parthenogenetic embryos improves the pregnancy and implantation of nuclear transfer embryos in mouse

The majority of somatic cell nuclear transfer (SCNT) clones dies in the peri- or postimplantation period. Improvement of the full-term healthy pregnancy rates is a key issue for the economical viability and animal welfare profile of SCNT technology. In this study the effects of cotransfer of parthenogenetic or fertilized embryos on the pregnancy and implantation of SCNT mouse embryos have been investigated. SCNT embryos were produced by transferring cumulus cell nuclei into enucleated B6D2F1 mouse oocytes, whereas parthenogenetically activated (PA) and fertilized embryos were derived from ICR mice by artificial activation with strontium and in vivo fertilization, respectively. SCNT embryos were inferior in their developmental capacity to blastocyst compared to either PA or fertilized embryos. SCNT embryos were transferred alone (SCNT), or cotransferred with two to three PA (SCNT + PA) or fertilized (SCNT + Fert) embryos into the oviducts of an ICR recipient. Both pregnancy and implantation rates originating from clones in the SCNT + PA group were significantly higher than those of SCNT group (p < 0.05). The weight of placentas of clones derived from SCNT, SCNT + PA, or SCNT + Fert was in all cases significantly higher than that of fertilized controls (p < 0.001). Most of the clones derived from SCNT embryos cotransferred with PA or fertilized embryos survived to adulthood and were fertile and healthy according to histopathological observations. Our results demonstrate in mouse that cotransfer of PA embryos improves the pregnancy and implantation of SCNT embryos without compromising the overall health of the resulting clones.     

Anaerobic metabolism in fowl embryos during normal incubation

Lactate concentration in blood, liver, yolk, amniotic and allantoic fluid and blood pyruvate was measured in embryos in the final week of incubation. Blood lactate was low up to day 18. The blood lactate/pyruvate ratio and liver lactate increased from day 19 until hatching. From day 14 to 19, lactate concentration in amniotic fluid remained constant, it increased 2-fold in yolk and 10-fold in allantoic fluid. There was only a 48% net accumulation of lactate in the three cavities. In conclusion, fowl embryos do not turn to anaerobic metabolism until the hatching process starts on day 19.     

Mycoplasma contamination alters 2'-deoxyadenosine metabolism in deoxycoformycin-treated mouse leukemia cells

Deoxycoformycin-treated P388 and L1210 mouse leukemia cells salvage 2'-deoxyadenosine from the medium only inefficiently, because deoxyadenosine deamination is blocked and its phosphorylation is limited by feedback controls. Mycoplasma contamination at a level that had no significant effect on the growth of the cells increased the salvage of deoxyadenosine greater than 10 fold over a 90 min period of incubation at 37 degrees C, but in this case deoxyadenosine was mainly incorporated into ribonucleotides and RNA via adenine formed from deoxyadenosine by mycoplasma adenosine phosphorylase. Deoxyadenosine was an efficient substrate for this enzyme, in contrast to 2',3'-dideoxyadenosine which was not phosphorolyzed. Mycoplasma infection was confirmed by the presence of uracil phosphoribosyltransferase activity and by culture isolation. The contaminant has been identified as Mycoplasma orale. Mycoplasma infection had no effect on the deamination and phosphorylation of deoxyadenosine and adenosine, on the salvage of hypoxanthine and adenine, or on the degradation of dAMP and dATP by the cells or on their acid and alkaline phosphatase activities.