Maternal obesity in sheep impairs foetal hepatic mitochondrial respiratory chain capacity.

BACKGROUND: Changes in the nutritional environment in utero induced by maternal obesity (MO) lead to foetal metabolic dysfunction predisposing offspring to later-life metabolic diseases. Since mitochondria play a crucial role in hepatic metabolism and function, we hypothesized that MO prior to conception and throughout pregnancy programmes foetal sheep liver mitochondrial phenotype. MATERIAL AND METHODS: Ewes ate an obesogenic diet (150% requirements; MO), or 100% requirements (CTR), from 60 days prior to conception. Foetal livers were removed at 0.9 gestation. We measured foetal liver mitochondrial DNA copy number, activity of superoxide dismutase, cathepsins B and D and selected protein content, total phospholipids and cardiolipin and activity of mitochondrial respiratory chain complexes. RESULTS: A significant decrease in activities of mitochondrial complexes I, II-III and IV, but not aconitase, was observed in MO. In the antioxidant machinery, there was a significant increase in activity of total superoxide dismutase (SOD) and SOD2 in MO. However, no differences were found regarding autophagy-related protein content (p62, beclin-I, LC3-I, LC3-II and Lamp2A) and cathepsin B and D activities. A 21.5% decrease in total mitochondrial phospholipid was observed in MO. CONCLUSIONS: The data indicate that MO impairs foetal hepatic mitochondrial oxidative capacity and affects total mitochondrial phospholipid content. In addition, MO affects the regulation of foetal liver redox pathways, indicating metabolic adaptations to the higher foetal lipid environment. Consequences of in utero programming of foetal hepatic metabolism may persist and compromise mitochondrial bioenergetics in later life, and increase susceptibility to metabolic diseases.

A heretical view: rather than a solely placental protective function, placental 11ß hydroxysteroid dehydrogenase 2 also provides substrate for fetal peripheral cortisol synthesis in obese pregnant ewes.

Exposure to glucocorticoid levels higher than appropriate for current developmental stages induces offspring metabolic dysfunction. Overfed/obese (OB) ewes and their fetuses display elevated blood cortisol, while fetal Adrenocorticotropic hormone (ACTH) remains unchanged. We hypothesized that OB pregnancies would show increased placental 11ß hydroxysteroid dehydrogenase 2 (11ß-HSD2) that converts maternal cortisol to fetal cortisone as it crosses the placenta and increased 11ß-HSD system components responsible for peripheral tissue cortisol production, providing a mechanism for ACTH-independent increase in circulating fetal cortisol. Control ewes ate 100% National Research Council recommendations (CON) and OB ewes ate 150% CON diet from 60 days before conception until necropsy at day 135 gestation. At necropsy, maternal jugular and umbilical venous blood, fetal liver, perirenal fat, and cotyledonary tissues were harvested. Maternal plasma cortisol and fetal cortisol and cortisone were measured. Fetal liver, perirenal fat, cotyledonary 11ß-HSD1, hexose-6-phosphate dehydrogenase (H6PD), and 11ß-HSD2 protein abundance were determined by Western blot. Maternal plasma cortisol, fetal plasma cortisol, and cortisone were higher in OB vs. CON (p < 0.01). 11ß-HSD2 protein was greater (p < 0.05) in OB cotyledonary tissue than CON. 11ß-HSD1 abundance increased (p < 0.05) in OB vs. CON fetal liver and perirenal fat. Fetal H6PD, an 11ß-HSD1 cofactor, also increased (p < 0.05) in OB vs. CON perirenal fat and tended to be elevated in OB liver (p < 0.10). Our data provide evidence for increased 11ß-HSD system components responsible for peripheral tissue cortisol production in fetal liver and adipose tissue, thereby providing a mechanism for an ACTH-independent increase in circulating fetal cortisol in OB fetuses.

A Review of Maternal Nutrition during Pregnancy and Impact on the Offspring through Development: Evidence from Animal Models of Over- and Undernutrition.

Similarities in offspring phenotype due to maternal under- or over-nutrition during gestation have been observed in studies conducted at University of Wyoming. In these studies, ewes were either nutrient-restricted (NR) from early to mid-gestation, or fed an obesogenic diet (MO) from preconception through term. Offspring necropsies occurred at mid-gestation, late-gestation, and after parturition. At mid gestation, body weights of NR fetuses were ~30% lighter than controls, whereas MO fetuses were ~30% heavier than those of controls. At birth, lambs born to NR, MO, and control ewes exhibited similar weights. This was a consequence of accelerated fetal growth rates in NR ewes, and reduced fetal growth rates in MO ewes in late gestation, when compared to their respective controls. These fetal growth patterns resulted in remarkably similar effects of increased susceptibility to obesity, cardiovascular disease, and glucose intolerance in offspring programmed mostly during fetal stages of development. These data provide evidence that maternal under- and over-nutrition similarly induce the development of the same cadre of physical and metabolic problems in postnatal life.

Rapid Communication: Reduced maternal nutrition during early- to mid-gestation elevates newborn lamb plasma cortisol concentrations and eliminates the neonatal leptin surge.

Human epidemiological and animal studies show that maternal nutrient reduction (MNR) and maternal overnutrition/obesity (MO) alter fetal growth and development, predisposing offspring (F1) to endocrine and appetite dysregulation. Compared to F1 of control-fed ewes, F1 of MO ewes display hypercortisolemia at birth and fail to exhibit the neonatal leptin surge implicated in lifelong appetite regulation. Here, we determined if MNR also elevates newborn lamb plasma cortisol and eliminates the neonatal leptin surge. Starting 30 d prior to conception, nulliparous control (CON, n = 6) ewes ate 100% NRC recommendations through parturition. Nutrient-reduced (NR, n = 6) ewes ate a CON diet through day 27 of gestation. From gestational days 28 to 78, NR ewes ate 50% of the CON diet before realimentation to 100% NRC recommendations. Jugular blood was collected daily from lambs from birth (day 0) through postnatal day 10, to determine plasma cortisol and leptin. Newborn NR plasma cortisol concentrations were increased (P < 0.0001) vs. CON and were similar to concentrations in MO lambs. Plasma leptin concentrations were similar between groups through postnatal day 7. The leptin surge, seen in CON lambs on postnatal days 8 to 10 was not present in NR lambs. These data show that, similar to MO lambs, early pregnancy MNR elevates newborn lamb plasma cortisol and eliminates the neonatal leptin surge. In the light of the similar elevation of neonatal cortisol in MNR and MO lambs, we conclude that cortisol plays a central role in regulating the neonatal lamb leptin surge.

Maternal obesity in the ewe increases cardiac ventricular expression of glucocorticoid receptors, proinflammatory cytokines and fibrosis in adult male offspring.

Obesity during human pregnancy predisposes offspring to obesity and cardiovascular disease in postnatal life. In a sheep model of maternal overnutrition/obesity we have previously reported myocardial inflammation and fibrosis, as well as cardiac dysfunction in late term fetuses, in association with chronically elevated blood cortisol. Significant research has suggested a link between elevated glucocorticoid exposure in utero and hypertension and cardiovascular disease postnatally. Here we examined the effects of maternal obesity on myocardial inflammation and fibrosis of their adult offspring. Adult male offspring from control (CON) mothers fed 100% of National Research Council (NRC) recommendations (n = 6) and male offspring from obese mothers (MO) fed 150% NRC (n = 6), were put on a 12-week ad libitum feeding challenge then necropsied. At necropsy, plasma cortisol and left and right ventricular thickness were markedly increased (P<0.05) in adult male MO offspring. Myocardial collagen content and collagen-crosslinking were greater (P<0.05) in MO offspring compared to CON offspring in association with increased mRNA and protein expression of glucocorticoid receptors (GR). No group difference was found in myocardial mineralocorticoids receptor (MR) protein expression. Further, mRNA expression for the proinflammatory cytokines: cluster of differentiation (CD)-68, transforming growth factor (TGF)-ß1, and tumor necrosis factor (TNF)-α were increased (P < 0.05), and protein expression of CD-68, TGF-ß1, and TNF-α tended to increase (P<0.10) in MO vs. CON offspring. These data provide evidence for MO-induced programming of elevated plasma cortisol and myocardial inflammation and fibrosis in adult offspring potentially through increased GR.

Maternal obesity programs reduced leptin signaling in the pituitary and altered GH/IGF1 axis function leading to increased adiposity in adult sheep offspring.

Studies in rodents highlight a role for leptin in stimulation of pituitary growth hormone (GH) secretion, with an impact on body composition regulation. We have reported that maternal obesity (MO) during ovine pregnancy results in hyperphagia, glucose-insulin dysregulation, increased adiposity, hypercortisolemia and hyperleptinemia in mature offspring subjected to a bout of ad libitum feeding. We hypothesized that MO reduces leptin signaling in the pituitary and down regulates the GH/IGF1 axis and increases circulating cortisol leading to increased adiposity in their adult offspring. Male lambs born to MO (n = 6) or control (CON, n = 6) ewes were fed only to requirements until placed on a 12 week ad libitum feeding trial at maturity. The pituitary, hypothalamic arcuate nucleus, and liver were collected at necropsy and mRNA and protein expression determined. Plasma cortisol concentrations were increased (P<0.05) in MO vs. CON offspring at the end of the feeding trial. Further, serum concentrations of IGF1 decreased (P<0.01) and GH tended to decrease (P<0.08) in MO vs. CON offspring. Pituitary mRNA and leptin receptor protein expression were decreased in MO vs. CON offspring in association with decreased GH mRNA expression, and decreased IGF1 mRNA and protein expression in liver. Liver 11ß-hydroxysteroid dehydrogenase 1 (11ßHSD1) expression was increased (P<0.01) and its cofactor hexose-6-phosphate dehydrogenase tended to increase (P<0.06) in MO vs. CON offspring. 11ßHSD2 expression remained unchanged. These data indicate that MO induced an increase in liver conversion of cortisone to cortisol in adult offspring and support a role for leptin signaling in the pituitary in mediating offspring adiposity.

Increased conception rates in beef cattle inseminated with nanopurified bull semen.

Aberrant sperm phenotypes coincide with the expression of unique sperm surface determinants that can be probed by objective, biomarker-based semen analysis and targeted as ligands for semen purification. This study evaluated a nanoparticle-based magnetic purification method that removes defective spermatozoa (∼30% of sample) from bull semen and improves sperm sample viability and fertilizing ability in vitro and in vivo. Two types of nanoparticles were developed: a particle coated with antibody against ubiquitin, which is present on the surface of defective spermatozoa, and a particle coated with the lectin peanut agglutinin, which binds to glycans exposed by acrosomal damage. In a 2 yr artificial insemination field trial with 798 cows, a conception rate of 64.5% ± 3.7% was achieved with a 10 × 10(6) sperm dose of peanut agglutinin-nanopurified spermatozoa, comparable to a control nonpurified full dose of 20 × 10(6) spermatozoa per dose (63.3% ± 3.2%) and significantly higher than a 10 × 10(6) sperm dose of nonpurified control semen (53.7% ± 3.2%; P < 0.05). A total of 466 healthy calves were delivered, and no negative side effects were observed in the inseminated animals or offspring. Because the method is inexpensive and can be fully integrated in current protocols for semen cryopreservation, it is feasible for use in the artificial insemination industry to improve fertility with reduced sperm dosage inseminations. Spermatology will benefit from nanopurification methodology by gaining new tools for the identification of candidate biomarkers of sperm quality such as binder of sperm protein 5 (BSP5), described in the present study.

Intravaginal lactic Acid bacteria modulated local and systemic immune responses and lowered the incidence of uterine infections in periparturient dairy cows.

The objective of this investigation was to evaluate whether intravaginal infusion of a lactic acid bacteria (LAB) cocktail around parturition could influence the immune response, incidence rate of uterine infections, and the overall health status of periparturient dairy cows. One hundred pregnant Holstein dairy cows were assigned to 1 of the 3 experimental groups as follows: 1) one dose of LAB on wk -2 and -1, and one dose of carrier (sterile skim milk) on wk +1 relative to the expected day of parturition (TRT1); 2) one dose of LAB on wk -2, -1, and +1 (TRT2), and 3) one dose of carrier on wk -2, -1, and +1 (CTR). The LAB were a lyophilized culture mixture composed of Lactobacillus sakei FUA3089, Pediococcus acidilactici FUA3138, and Pediococcus acidilactici FUA3140 with a cell count of 108-109 cfu/dose. Blood samples and vaginal mucus were collected once a week from wk -2 to +3 and analyzed for content of serum total immunoglobulin G (IgG), lipopolysaccharide-binding protein (LBP), serum amyloid A (SAA), haptoglobin (Hp), tumor necrosis factor (TNF), interleukin (IL)-1, IL-6, and vaginal mucus secretory IgA (sIgA). Clinical observations including rectal temperature, vaginal discharges, retained placenta, displaced abomasum, and laminitis were monitored from wk -2 to +8 relative to calving. Results showed that intravaginal LAB lowered the incidence of metritis and total uterine infections. Intravaginal LAB also were associated with lower concentrations of systemic LBP, an overall tendency for lower SAA, and greater vaginal mucus sIgA. No differences were observed for serum concentrations of Hp, TNF, IL-1, IL-6 and total IgG among the treatment groups. Administration with LAB had no effect on the incidence rates of other transition cow diseases. Overall intravaginal LAB lowered uterine infections and improved local and systemic immune responses in the treated transition dairy cows.

Diet reduction to requirements in obese/overfed ewes from early gestation prevents glucose/insulin dysregulation and returns fetal adiposity and organ development to control levels.

Obesity at conception and excess gestational weight gain pose significant risks for adverse health consequences in human offspring. This study evaluated the effects of reducing dietary intake of obese/overfed ewes beginning in early gestation on fetal development. Sixty days prior to conception, ewes were assigned to a control diet [CON: 100% of National Research Council (NRC) recommendations], a diet inducing maternal obesity (MO: 150% of NRC recommendations), or a maternal obesity intervention diet (MOI: 150% of NRC recommendations to day 28 of gestation, then 100% NRC) until necropsy at midgestation (day 75) or late (day 135) gestation. Fetal size and weight, as well as fetal organ weights, were greater (P < 0.05) at midgestation in MO ewes than those of CON and MOI ewes. By late gestation, whereas fetal size and weight did not differ among dietary groups, cardiac ventricular weights and wall thicknesses as well as liver and perirenal fat weights remained elevated in fetuses from MO ewes compared with those from CON and MOI ewes. MO ewes and fetuses exhibited elevated (P < 0.05) plasma concentrations of triglycerides, cholesterol, insulin, glucose, and cortisol at midgestation compared with CON and MOI ewes and fetuses. In late gestation, whereas plasma triglycerides and cholesterol, insulin, and cortisol remained elevated in MO vs. CON and MOI ewes and fetuses, glucose concentrations were elevated in both MO and MOI fetuses compared with CON fetuses, which was associated with elevated placental GLUT3 expression in both groups. These data are consistent with the concept that reducing maternal diet of obese/overfed ewes to requirements from early gestation can prevent subsequent alterations in fetal growth, adiposity, and glucose/insulin dynamics.

Sperm proteasomes degrade sperm receptor on the egg zona pellucida during mammalian fertilization.

Despite decades of research, the mechanism by which the fertilizing spermatozoon penetrates the mammalian vitelline membrane, the zona pellucida (ZP) remains one of the unexplained fundamental events of human/mammalian development. Evidence has been accumulating in support of the 26S proteasome as a candidate for echinoderm, ascidian and mammalian egg coat lysin. Monitoring ZP protein degradation by sperm during fertilization is nearly impossible because those few spermatozoa that penetrate the ZP leave behind a virtually untraceable residue of degraded proteins. We have overcome this hurdle by designing an experimentally consistent in vitro system in which live boar spermatozoa are co-incubated with ZP-proteins (ZPP) solubilized from porcine oocytes. Using this assay, mimicking sperm-egg interactions, we demonstrate that the sperm-borne proteasomes can degrade the sperm receptor protein ZPC. Upon coincubation with motile spermatozoa, the solubilized ZPP, which appear to be ubiquitinated, adhered to sperm acrosomal caps and induced acrosomal exocytosis/formation of the acrosomal shroud. The degradation of the sperm receptor protein ZPC was assessed by Western blotting band-densitometry and proteomics. A nearly identical pattern of sperm receptor degradation, evident already within the first 5 min of coincubation, was observed when the spermatozoa were replaced with the isolated, enzymatically active, sperm-derived proteasomes. ZPC degradation was blocked by proteasomal inhibitors and accelerated by ubiquitin-aldehyde(UBAL), a modified ubiquitin protein that stimulates proteasomal proteolysis. Such a degradation pattern of ZPC is consistent with in vitro fertilization studies, in which proteasomal inhibitors completely blocked fertilization, and UBAL increased fertilization and polyspermy rates. Preincubation of intact zona-enclosed ova with isolated active sperm proteasomes caused digestion, abrasions and loosening of the exposed zonae, and significantly reduced the fertilization/polyspermy rates after IVF, accompanied by en-mass detachment of zona bound sperm. Thus, the sperm borne 26S proteasome is a candidate zona lysin in mammals. This new paradigm has implications for contraception and assisted reproductive technologies in humans, as well as animals.