Walter T. Dixon

AMPK activation increases uncoupling protein‐3 expression and mitochondrial enzyme activities in rat muscle without fibre type transitions

The present study examined the effect of chronic activation of 5′‐AMP‐activated protein kinase (AMPK) on the metabolic profile, including uncoupling protein‐3 (UCP‐3) and myosin heavy chain (MHC)‐based fibre phenotype of rodent fast‐twitch tibialis anterior muscle. Sprague‐Dawley rats were given daily injections of 5‐aminoimidazole‐4‐carboxamide‐1‐β‐D‐ribofuranoside (AICAR), a known activator of AMPK, or vehicle (control) for 28 days. After AICAR treatment, UCP‐3 expression at the mRNA level was elevated 1.6 ± 0.1‐fold (P < 0.006) and corresponded to a 3.3 ± 0.2‐fold increase in UCP‐3 protein content (P < 0.0001). In addition, the activities of the mitochondrial reference enzymes citrate synthase (EC 4.1.3.7) and 3‐hydroxyacyl‐CoA‐dehydrogenase (EC 1.1.1.35), which are known to increase in proportion to mitochondrial volume density, were elevated 1.6‐fold (P < 0.006), while the activity of lactate dehydrogenase (EC 1.1.1.27) was reduced to 80 % of control (P < 0.02). No differences were detected after AICAR treatment in the activities of the glycolytic reference enzymes glyceraldehydephosphate dehydrogenase (EC 1.2.1.12) or phosphofructokinase (EC 2.7.1.11), nor were MHC‐based fibre‐type transitions observed, using immunohistochemical or electrophoretic analytical methods. These changes could not be attributed to variations in inter‐organ signalling by metabolic substrates or insulin. We conclude that an AMPK‐dependent pathway of signal transduction does mimic some of the metabolic changes associated with chronic exercise training, but does not affect expression of the MHC‐based structural phenotype. Thus, the metabolic and MHC‐based fibre types do not appear to be regulated in a co‐ordinated way, but may be independently modified by different signalling pathways.

Nutritional restriction in lactating primiparous sows selectively affects female embryo survival and overall litter development

This study explored the possibility of sex-specific effects on embryonic survival in primiparous sows subjected to restricted feed intake during the last week of lactation and bred after weaning (Restrict; n = 16), compared with control sows fed close to ad libitum feed intakes (Control; n = 17). Restrict sows were in a substantial negative net energy balance at weaning, and lost 13% of estimated protein and 17% of fat mass during lactation, yet the weaning-to-oestrous interval and ovulation rate were not different between treatments. However, embryonic survival at Day 30 of gestation was lower (P < 0.05) in Restrict than Control sows, and selectively reduced the proportion of female embryos surviving (P < 0.01). A decrease in weight and crown-rump length of surviving female (P < 0.05) and male (P < 0.05) embryos was seen in Restrict litters. The mechanisms mediating this sex-specific effect on embryonic loss in feed-restricted sows are unclear. The data presented here indicate that feed-restriction during the last week of lactation in primiparous sows causes a selective decrease in survival of female embryos and limits the growth of all surviving embryos.

Biomarkers of in vivo fertility in sperm and seminal plasma of fertile stallions

The global proteome of sperm and seminal plasma of fertile stallions was investigated to determine whether associations with relative in vivo fertility exist. Seven stallions at stud in a commercial breeding station were collected throughout the breeding season and bred to a total of 164 mares to determine conception rates. On three occasions during the breeding season, raw semen was obtained from a regular collection for proteomic analysis using two-dimensional electrophoresis and also assessed for routine semen quality end points. First cycle conception rate was negatively related to ejaculate volume (r = -0.43, P = 0.05) and total IGF1 content (ng) per ejaculate (r = -0.58, P = 0.006), whereas overall pregnancy rate was positively related to sperm concentration (r = 0.56, P = 0.01). The abundance of three proteins known to be involved in carbohydrate metabolism in sperm was positively related to fertility. Furthermore, the abundance of four seminal plasma proteins were identified as being negatively related to fertility; these were identified as kallikrein-1E2 (KLK2), clusterin, and seminal plasma proteins 1 (SP1) and 2 (SP2). Abundance of cysteine-rich secretory protein 3 (CRISP3) was positively related to first cycle conception rate (r = 0.495, P = 0.027) and may provide a good marker of fertility. Based on stepwise regression analysis, clusterin and SP1 in seminal plasma together with sperm citrate synthase were predictive of fertility (r = 0.77, P < 0.0001). This study identified proteins within sperm and seminal plasma that could serve as biomarkers of semen quality and fertility in stallions.

Localization and gene expression of glucose transporters in bovine mammary gland

Glucose uptake in the mammary gland is a rate-limiting step in milk synthesis. To study glucose transporters in the bovine mammary gland, the erythrocyte-type glucose transporter (GLUT1) and the insulin-responsive glucose transporter (GLUT4) proteins were assessed by Western blotting and immunohistochemical staining, using polyclonal antibodies against the C-terminal peptide of GLUT1 and GLUT4. Our results demonstrated that the bovine mammary gland expressed a relatively high level of GLUT1 protein, whereas GLUT4 protein was not detected in the mammary gland of either lactating or dry cows. The absence of GLUT4 may indicate that glucose transport is not regulated by insulin in the lactating and dry bovine mammary gland. The anti-GLUT1 antibody strongly stained the single layer of epithelial cells of mammary alveoli. The expression of GLUT1 mRNA was similar in the mammary gland of late lactation and non-lactating cows. However, a smaller molecular weight species (38 kDa) of GLUT1 protein was detected in the mammary gland of non-lactating cows where its abundance in crude membrane preparation was 80% higher than in lactating animals. There were no significant differences in GLUT1 mRNA in bovine mammary gland at 118 d and 181 d postpartum, however, GLUT1 protein expression tended to be greater at 118 d postpartum.

Seminal Plasma Proteins as Potential Markers of Relative Fertility in Boars

This study investigated whether specific proteins from distinct seminal plasma fractions of boars could be related to in vivo fertility. Nine boars with acceptable sperm motility and morphology for use in artificial insemination demonstrated major differences in total number born and pregnancy rate when low sperm doses (1.5 billion sperm) were used to breed a minimum of 50 gilts per boar. The 2 lowest-fertility and 2 highest-fertility boars were chosen for evaluation of specific seminal plasma proteins. On 4 occasions, semen was collected and separated into 3 fractions based on sperm concentration (Sperm-Peak, Sperm-Rich, and Sperm-Free), and the fractions were analyzed for total protein concentration and abundance of major seminal plasma glycoprotein (PSP-I), AWN-1, and osteopontin protein using Western blotting techniques. The concentrations of these seminal plasma proteins were lower in the Sperm-Peak fractions compared with the Sperm-Free fractions (P < .05). Seminal plasma from the pooled Sperm-Rich fraction used for artificial insemination was also subjected to two-dimensional gel electrophoresis to investigate novel protein markers related to in vivo fertility. Total piglets born (r = -0.76, P = .01) and sperm motility at day 7 (r = -0.74, P = .037) were again negatively correlated with a 22-kDa protein identified by mass spectrometry as PSP-I. However, fertility index and farrowing rate tended to be positively correlated (P < .10) with a 25-kDa protein, identified as glutathione peroxidase (GPX5), an antioxidant enzyme that may protect sperm membranes from oxidative damage. These candidate proteins merit further investigation as markers of fertility in boars.

THE EPIDERMAL MELANIN UNIT IN THE PATHOPHYSIOLOGY OF MALIGNANT MELANOMA

The epidermal melanin unit (EMU) denotes the symbiotic relationship between a melanocyte and a pool of associated keratinocytes. We propose to show that alterations in the biology of the EMU are the main determinant of the different patterns of intraepidermal growth of melanocytes in lentigo maligna melanoma (LMM) and superficial spreading melanoma (SSM). They also appear to affect the biosynthesis of melanin and melanosomes during malignant transformation. Findings in histochemical studies with monoclonal antibodies generated against melanosomal proteins to produce different stains of melanocytes of normal skin, dysplastic melanocytic nevi (DMN), common melanocytic nevi (CMN), LMM, and SSM have led to the suggestion that the altered melanosome synthesis is a main phenotype in the pathophysiology in neoplastic transformation of melanocytes. Altered melanin synthesis may also affect the carcinogenesis in malignant melanoma: pheomelanin is increased in malignant melanoma and DMN, but not in normal skin and CMN. Pheomelanin and its precursors could aid the malignant transformation of melanocytes through the generation of mutagenic ultraviolet photoproducts in familial DMN syndrome.

Effect of satellite cell ablation on low‐frequency‐stimulated fast‐to‐slow fibre‐type transitions in rat skeletal muscle

The purpose of this study was to determine whether satellite cell ablation within rat fast‐twitch muscles exposed to chronic low‐frequency stimulation (CLFS) would limit fast‐to‐slow fibre‐type transitions. Twenty‐nine male Wistar rats were randomly assigned to one of three groups. Satellite cells of the left tibialis anterior were ablated by weekly exposure to a 25 Gy dose of γ‐irradiation during 21 days of CLFS (IRR‐Stim), whilst a second group received only 21 days of CLFS (Stim). A third group received weekly doses of γ‐irradiation (IRR). Non‐irradiated right legs served as internal controls. Continuous infusion of 5‐bromo‐2′‐deoxyuridine (BrdU) revealed that CLFS induced an 8.0‐fold increase in satellite cell proliferation over control (mean ±s.e.m.: 23.9 ± 1.7 versus 3.0 ± 0.5 mm−2, P < 0.0001) that was abolished by γ‐irradiation. M‐cadherin and myogenin staining were also elevated 7.7‐ and 3.8‐fold (P < 0.0001), respectively, in Stim compared with control, indicating increases in quiescent and terminally differentiating satellite cells; these increases were abolished by γ‐irradiation. Myonuclear content was elevated 3.3‐fold (P < 0.0001) in Stim, but remained unchanged in IRR‐Stim. Immunohistochemical analyses revealed attenuation of fast‐to‐slow fibre‐type transitions in IRR‐Stim compared with Stim. Comparable changes were observed at the protein level by SDS‐PAGE. It is concluded that although considerable adaptive potential exists within myonuclei, satellite cells play a role in facilitating fast‐to‐slow fibre‐type transitions.

Biological Markers of Boar Fertility

The semen evaluation techniques used in most commercial artificial insemination centers, which includes sperm motility and morphology measurements, provides a very conservative estimate of the relative fertility of individual boars. As well, differences in relative boar fertility are masked by the widespread use of pooled semen for commercial artificial insemination (AI) in many countries. Furthermore, the relatively high sperm numbers used in commercial AI practice usually compensate for reduced fertility, as can be seen in some boars when lower numbers of sperm are used for AI. The increased efficiency of pork production should involve enhanced use of boars with strong reproductive efficiency and the highest genetic merit for important production traits. Given that the current measures of semen quality are not always indicative of fertility and reproductive performance in boars, accurate and predictive genetic and protein markers are still needed. Recently, significant efforts have been made to identify reliable markers that allow for the identification and exclusion of sires with reduced reproductive efficiency. This paper reviews the current status of proteomic and genomic markers of fertility in boars in relation to other livestock species.

Feed Restriction and Insulin Treatment Affect Subsequent Luteal Function in the Immediate Postovulatory Period in Pigs: Progesterone Production In Vitro and Messenger Ribonucleic Acid Expression for Key Steroidogenic Enzymes

Abstract Progesterone production and release in vitro, and mRNA expression for key steroidogenic enzymes, were studied in luteal tissue recovered in the immediate postovulatory period from cyclic gilts allocated to one of three treatments: moderate feed restriction during the first (RH) or second week of the estrous cycle, with (HR+I) or without (HR) concomitant injections of long-acting insulin. Time of feed restriction affected neither progesterone production or release, nor mRNA expression for several key steroidogenic enzymes. However, luteal tissue from RH but not from HR gilts responded to LH stimulation by increasing progesterone production and release (P < 0.05). Insulin treatment increased progesterone production and release, restored luteal tissue responsiveness to LH, up-regulated steroidogenic enzyme mRNA expression, and down-regulated the tissue inhibitor of metalloproteinase-I mRNA expression in HR+I compared with HR gilts (P < 0.05). In vitro progesterone production and gene expression were affected by time of tissue collection after ovulation in RH and HR gilts but not in HR+I gilts, and were correlated with temporal changes in oviductal and peripheral plasma progesterone concentrations. Inherent differences in luteal function therefore appear to mediate latent effects of nutrition and insulin treatment on circulating progesterone concentrations in the critical postovulatory period in gilts.

Nitric oxide synthase inhibition prevents activity-induced calcineurin–NFATc1 signalling and fast-to-slow skeletal muscle fibre type conversions

•  Exercise is known to trigger skeletal muscle structural and functional adaptations. •  Control of these adaptive alterations is a complex process involving multiple signalling pathways and levels of regulation. •  The well‐characterized calcineurin–nuclear factor of activated T‐cells (NFATc1) signalling pathway is involved in the regulation of activity‐dependent alterations in skeletal muscle myosin heavy chain expression. Myosin heavy chain is a contractile protein that largely dictates a muscles speed of contraction. •  We show that a signalling molecule called nitric oxide may be regulating alterations in myosin heavy chain expression via activity‐modulated calcineurin–NFATc1 signalling. •  These findings increase our understanding of how skeletal muscle adaptive alterations are regulated.