Domestic Animal Endocrinology

Editorial Board

2010-04-01

VEGF modulates the effects of gonadotropins in granulosa cells

L.K. Doyle, C.A. Walker, F.X. Donadeu — 2009-10-08

Abstract: Follicle selection is associated with an increase in the expression of vascular endothelial growth factor (VEGF) and its receptors in granulosa cells, however, the roles of VEGF in regulating the function of these or other non-endothelial cells in the ovary have not been explored in detail. The current study used bovine cell cultures to investigate potential roles of VEGF in the regulation of granulosa cell function during follicle development. Granulosa cells were obtained from morphologically healthy follicles 4 to 8mm or 9 to 14mm in diameter (corresponding to diameters before and after the establishment of dominance, respectively, during a bovine follicular wave) and exposed to a range of VEGF concentrations (1 to 100ng/mL) encompassing concentrations found naturally in bovine dominant follicles. A concentration of VEGF of 1ng/mL induced significant proliferation of granulosa cells from 4- to 8-mm follicles (P=0.024) and increased the proliferative response of these cells to follicle-stimulating hormone (FSH; P=0.045); whereas higher doses of VEGF had no effect on proliferation (P=0.9). Treatment with VEGF induced an overall increase in mean extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation (P=0.02). In contrast, VEGF, alone or in combination with FSH, had no effect on expression of the steroidogenic enzyme, CYP11A1, by cells from 4- to 8-mm follicles (P=0.9). Granulosa cells from 9- to 14-mm follicles responded to 1ng/mL VEGF with an increase in expression of the ovulation-associated gene, PTGS2 (P=0.003) but higher VEGF doses had no effect (P=0.9). The PTGS2 response to 1ng/mL VEGF was similar to that induced by treatment with luteinizing hormone (LH). Interestingly, the stimulatory effects of LH on ERK1/2 phosphorylation (P=0.003) and PTGS2 expression (P<0.01) in granulosa cells from 9- to 14-mm follicles were abolished (P=0.2) by specific chemical inhibition of VEGF receptor 2 (VEGFR2). These results suggest novel and important roles of VEGF and its receptor, VEGFR2, in mediating and/or enhancing the effects of gonadotropins in granulosa cells.

Presence of anti-insulin natural autoantibodies in healthy cats and its interference with immunoassay for serum insulin concentrations

2009-10-22

Abstract: A substance interfering with the enzyme-linked immunosorbent assay (ELISA) for feline insulin concentration was investigated in healthy cats. An insulin-binding substance isolated from feline serum showed 2 bands at 25 and 50kDa in SDS-PAGE, suggesting the presence of immunoglobulin G (IgG). Insulin-binding IgG from healthy cats indeed reduced insulin immunoreactivity in the ELISA for determining insulin concentration. The insulin-binding IgG was polyclonal/polyreactive and showed certain specificity, high affinity, and high binding capacity, which was evaluated by liquid-phase radioimmunoassay with Scatchard plot analysis. Epitope analysis revealed that the insulin-binding IgG showed significant binding at residues A1-5 and B20-30 of the insulin molecule. Removal of the antibodies from serum enabled the determination of serum insulin concentrations by ELISA. Our data indicated that serum from healthy cats contained substantial amounts of natural autoantibodies combined with insulin, and that the antibodies interfered with the heterologous immunoassay for serum insulin concentration.

Maternal social stress during late pregnancy affects hypothalamic-pituitary-adrenal function and brain neurotransmitter systems in pig offspring

W. Otten, E. Kanitz, D. Couret, I. Veissier, A. Prunier, E. Merlot — 2009-11-02

Abstract: Maternal stress in pregnant sows may induce long-lasting alterations in the behavior, physiology, and immunity of their offspring. The aim of the present study was to investigate the consequences of repeated social stress during late gestation on determinants of the hypothalamic–pituitary–adrenal axis and on hippocampal neurotransmitter profiles in pig offspring. All pregnant gilts were housed in pairs. Each Stress gilt was mixed with an unfamiliar gilt twice a week between days 77 and 105 of gestation (n=18). Control gilts were housed in stable pairs over the same period (n=18). Plasma cortisol and corticosteroid binding globulin (CBG) were measured in 1 male and 1 female per litter in a basal situation on postnatal days (PND) 4, 26, and 60 and in a stressful situation at PND 28 (2 d after weaning) and 62 (2 d after relocation to a new building). Prenatal stress had no effect on plasma cortisol, but it decreased CBG at PND 26. Brain and adrenals were collected from 1 female per litter after weaning or relocation at PND 28 and PND 62. Adrenals were additionally collected at PND 4. Glucocorticoid receptor binding in the hippocampus and hypothalamus was not affected by prenatal treatment. However, prenatal stress increased the expression of 11β-hydroxysteroid dehydrogenase type 1 mRNA in the hippocampus after weaning (P<0.05) and after relocation (P=0.08). In addition, prenatally stressed piglets showed an increased 5-hydroxyindole-3-acetic acid to 5-hydroxytryptamine ratio in the hippocampus after weaning and increased hippocampal c-fos mRNA expression and noradrenaline concentration after relocation (P<0.05). Prenatal stress also increased the relative adrenal weight at PND 4 and the cell density in the cortex and the medulla at PND 28, whereas no difference was found for activities of catecholamine-synthesising enzymes in the medulla. Overall, our data indicate that repeated social stress during pregnancy has long-lasting consequences on hypothalamic–pituitary–adrenal axis and hippocampal neurotransmitter activity in the offspring of pigs.

A spline polynomial model to describe serum IGF-I concentration from birth to slaughter in calves: effects of weaning age, pre-weaning concentrate feeding and breed

M. Blanco, I. Casasús, D. Villalba — 2009-12-21

Abstract: The statistical analysis of hormones sampled throughout the production cycle is complicated because factors such as age and weight at the measuring date interfere. Spline curves constructed from pieces of low-degree, random-effects polynomials could be used for a more accurate analysis of data. Concentration of insulin-like growth factor-1 (IGF-1), weight gain, and concentrate intake of Parda de Montaña (PM) (n=27) and Pirenaica calves (n=14) were modeled with a spline model according to age at weaning, pre-weaning concentrate feeding, and breed. At birth, calves were randomly assigned to early weaning (EW) at 90d or traditional weaning (TW) at 150d. During lactation, half of PM calves received concentrates (S), whereas the remainder received no concentrates (NS). After weaning, calves received concentrates on an ad libitum basis until they reached a weight of 450kg. The spline model had better likelihood than a polynomial of 6 degrees or a split-plot model. Serum IGF-1 concentration was greatly affected by age at weaning and pre-weaning concentrate feeding, but not by breed. In NS calves, IGF-1 concentration was greater in EW than in TW calves from 120 to 300d, irrespective of breed. During lactation, S calves had greater IGF-1 concentration than NS calves. After weaning, EWNS calves reached the IGF-1 concentration of EWS calves after 4mo on concentrates, whereas TWNS calves attained IGF-1 concentration of TWS calves after only 2mo, because of their increased concentrate intake relative to TWS calves. Concentration of IGF-1 was positively correlated with the immediate weight gains and intake, but it was not a good predictor of performance in the long term.

Adipose tissue depots of Holstein cows are immune responsive: Inflammatory gene expression in vitro

M. Mukesh, M. Bionaz, D.E. Graugnard, J.K. Drackley, J.J. Loor — 2009-11-16

Abstract: The transcriptional response of adipose tissue depots with respect to their immune responsiveness in dairy cows remains largely unknown. Thus, we examined mRNA expression and responsiveness of subcutaneous (SUB) and mesenteric (MES) adipose tissue from nonpregnant dairy cows to a short-term (2h), in vitro lipopolysaccharide (LPS) challenge (20μg/mL in physiological saline). Abundance of mRNA for tumor necrosis factor-α (TNFA), interleukin-6 (IL6), serum amyloid A3 (SAA3), toll-like receptor 4 (TLR4), monocyte chemoattractant protein-1 (CCL2), and RANTES/chemokine C-C motif ligand 5 (CCL5) were analyzed using quantitative polymerase chain reaction (PCR) from tissue samples collected at slaughter from 5 nonpregnant/nonlactating Holstein cows. Prior to LPS challenge, SAA3 mRNA abundance was greater in MES than SUB tissue. Regardless of depot site, LPS led to greater mRNA abundance of TNFA and IL6 and was more pronounced for IL6 in MES. We also observed a marked increased in expression of CCL2, CCL5, TLR4, IL6, and TNFA in both MES and SUB during the 2-h incubation with saline alone (ie, the control). Because mRNA expression of the apoptotic markers B-cell CLL/lymphoma 2 (BCL2) and tumor protein p53 (TP53) did not differ during the 2-h incubation, it is less likely that the response to saline was a result of increased rate of cell death during incubation. Analysis using semiquantitative PCR of the 16s rRNA gene in cDNA from tissue explants revealed the presence of bacteria likely arising from contamination during sample collection. Furthermore, surfactant medium from about 50% of explant cultures had viable aerobic bacteria without differences between treatments or tissue samples. Thus, the presence of bacteria could partly explain the large increase in inflammatory-related genes after 2-h incubation with saline. The higher SAA3 expression in MES suggests that this acute-phase protein has a role in lipid metabolism and/or transport during an immune challenge. Overall, results provided evidence that adipose depots of dairy cows are capable of synthesizing chemokines and are immune responsive when exposed to inflammatory conditions that can arise from a pathogenic insult or during and soon after parturition.

Changes in cortisol release and heart rate variability in sport horses during long-distance road transport

2009-12-04

Abstract: It is widely accepted that transport is stressful for horses, but only a few studies are available involving horses that are transported regularly and are accustomed to transport. We determined salivary cortisol immunoreactivity (IR), fecal cortisol metabolites, beat-to-beat (RR) interval, and heart rate variability (HRV) in transport-experienced horses (N=7) in response to a 2-d outbound road transport over 1370km and 2-d return transport 8 d later. Salivary cortisol IR was low until 60min before transport but had increased (P<0.05) 30min before loading. Transport caused a further marked increase (P<0.001), but the response tended to decrease with each day of transport. Concentrations of fecal cortisol metabolites increased on the second day of both outbound and return transports and reached a maximum the following day (P<0.001). During the first 90min on Day 1 of outbound transport, mean RR interval decreased (P<0.001). Standard deviations of RR interval (SDRR) decreased transiently (P<0.01). The root mean square of successive RR differences (RMSSD) decreased at the beginning of the outbound and return transports (P<0.01), reflecting reduced parasympathetic tone. On the first day of both outbound and return transports, a transient rise in geometric HRV variable standard deviation 2 (SD2) occurred (P<0.01), indicating increased sympathetic activity. In conclusion, transport of experienced horses leads to increased cortisol release and changes in heart rate and HRV, which is indicative of stress. The degree of these changes tended to be most pronounced on the first day of both outbound and return transport.

Effects of 1,25-dihydroxyvitamin D3 on calcium and phosphorus homeostasis in sheep fed diets either adequate or restricted in calcium content

M.R. Wilkens, N. Mrochen, G. Breves, B. Schröder — 2009-12-04

Abstract: It was the aim of the present study to collect basic data on calcium (Ca) and phosphorus (P) homoeostasis in sheep. Two series of experiments were carried out to investigate the effects of 1,25-dihydroxyvitammin D3 (calcitriol) in supraphysiological dosage in combination with varying alimentary Ca supply. In the first series, blood samples were collected over 72h to determine the concentrations of total Ca (Ca), ionized Ca (Ca2+), inorganic phosphate (Pi), and the bone resorption marker CrossLaps (CL). In the second series, measurements were carried out over 12h. In addition, urine samples were collected to calculate the fractional excretions (FE) of Ca and Pi. Changes in plasma macromineral concentrations (P<0.01) as well as in CL (P<0.001) and endogenous calcitriol (P<0.05) were observed in the alimentary Ca-restricted animals, indicating that the reduction of daily Ca intake challenged the animals’ macromineral homeostatic mechanisms. However, the Ca-restricted diet had an effect on neither FE of Ca nor on FE of Pi. The treatment resulted in peak serum calcitriol concentrations between 1,900 and 2,500pg/mL, and supraphysiological concentrations were maintained for the next 48h. Irrespective of dietary Ca, calcitriol had hypercalcemic and hyperphosphatemic effects. An increase in CL was revealed only in the Ca-restricted, calcitriol-treated sheep (P<0.01), reflecting a remarkable enhancement of Ca mobilization from the bone by calcitriol exclusively in this group. From these data, it can be concluded that the sheep can be a suitable animal model for studying catabolic effects of Ca deficiency and calcitriol on bone metabolism.

MicroRNA-25 functions in regulation of pigmentation by targeting the transcription factor MITF in alpaca (Lama pacos) skin melanocytes

2009-12-28

Abstract: Although the influence of endocrine factors is well established, the molecular and cellular mechanisms controlling coat color are not completely understood. A major mechanism for post-transcriptional regulation of gene expression is through the action of microRNAs (miRNAs), which anneal to the 3’ untranslated region of mRNAs in a sequence-specific fashion and either block translation or promote transcript degradation. In this study, we investigated the expression of miRNAs in the skin of alpacas with brown vs white coat color using a microarray screen; identified potential mRNA targets for identified miRNAs among coat color genes; and subsequently determined the ability of a specific, differentially expressed miRNA (miR-25) to suppress expression of micropthalmia-associated transcription factor (MITF), a predicted miR-25 target gene that regulates genes linked to coat color. Expression of 10 different miRNA species in the skin of alpacas with brown vs white coat color was identified from microarray screens. Of the 10 alpaca skin miRNAs identified, predicted binding sites in the 3′ untranslated region of RNAs encoding for known genes linked to coat color were primarily for miR-25, but sites were also identified for miR-129 and miR-377. Potential miR-25 binding sites were present in transcripts encoding for 11 coat color genes, including MITF. An inverse relationship between transcript abundance for MITF and miR-25 was observed in skin samples collected from alpacas with white vs brown coat color. Furthermore, overexpression of miR-25 in cultured melanocytes reduced MITF mRNA and protein abundance and corresponding mRNA abundance for the MITF-regulated enzymes tyrosinase and tyrosinase-related protein 1. Results support a novel functional role for miRNA-25 in the regulation of gene expression linked to coat color.