Domestic Animal Endocrinology - Articles in Press

Effects of unsaturated fatty acids on progesterone secretion and selected protein kinases in goat granulosa cells - Corrected Proof

S. Coyral-Castel, C. Ramé, A. Fatet, J. Dupont — 2010-01-25

Abstract: Previous studies in cattle have shown influences of dietary unsaturated fatty acid (UFA) supplementation on ovarian function. However, it is unclear whether these UFA exert direct or indirect effects on ovarian steroid production or their mechanisms of action. We have recently shown that 5′AMP-activated protein kinase (AMPK) regulates progesterone secretion through mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 (MAPK ERK1/2) in rodent granulosa cells. Here, we investigated the effects of 3 UFAs, oleic acid (OA), linoleic acid (LA), and α-linolenic acid (ALA) on progesterone secretion in goat granulosa cells. Finally, we examined the effects of UFAs on MAPK ERK1/2 and AMPK phosphorylation in these granulosa cells. Oleic acid and LA (10μM each), but not ALA (100μM), increased progesterone secretion (P<0.05) in the presence or absence of insulin-like growth factor (IGF)-1 (10-8 M) or FSH (5×10−8M). The different AMPK subunits, except for γ3, are present in the goat ovary. Treatment with metformin (10mM), an activator of AMPK, increased AMPK phosphorylation (P<0.05) and reduced progesterone secretion by 50% (P<0.05) in the basal state and in response to IGF-1 or FSH in goat granulosa cells. Oleic acid and LA had no effect on AMPK phosphorylation, whereas they rapidly increased MAPK ERK1/2 phosphorylation (P<0.05). Finally, U0126, a MAPK ERK1/2 inhibitor, decreased OA- and LA-induced progesterone secretion (P<0.05), suggesting that these UFAs could stimulate progesterone secretion partly through MAPK ERK1/2 in the absence of IGF-1 and FSH in goat granulosa cells. The involvement of AMPK in this process remains to be demonstrated. Taken together, some fatty acids could improve ovarian steroidogenesis through the MAPK ERK1/2 signaling pathway and, consequently, have beneficial effects on goat fertility.

Plasma ghrelin and oxyntomodulin concentrations in lactating dairy cows receiving abomasal soybean oil, corn starch, and casein infusions - Corrected Proof

A.E. Relling, S.C. Loerch, C.K. Reynolds — 2010-01-25

Abstract: The effects of increased postruminal supply of casein, corn starch, and soybean oil on plasma concentrations of the gastrointestinal hormones ghrelin and oxyntomodulin (OXM) were investigated. Four mid-lactation Holstein cows were used in a 4×4 Latin square. Treatments were continuous abomasal infusions (23h/d) for 7 d of water, soybean oil (500g/d), corn starch (1100g/d), or casein (800g/d). Jugular vein plasma was obtained every 30min for 7h on days 1 and 7. Soybean oil and casein infusion decreased preprandial plasma ghrelin concentration by approximately 20% on both d (time-by-treatment P<0.10); however, dry matter intake (DMI) was depressed only after 7 d of oil infusion. Infusion of soybean oil, corn starch, or casein did not change the plasma OXM concentration (P>0.20). The present data indicate that plasma ghrelin concentration is depressed immediately before feeding by the postruminal infusion of soybean oil and casein, but it is not affected during the postprandial period. Plasma ghrelin concentration was not altered (P>0.20), pre- or postfeeding, by increased postruminal supply of corn starch. In addition, plasma OXM concentration did not respond (P>0.20) to postruminal nutrient infusion. In conclusion, a decrease in DMI when fat is infused could be partially explained by the decrease in prefeeding plasma ghrelin concentration, but a decrease in prefeeding plasma ghrelin concentration is not always associated with a decrease in DMI, as observed for the infusion of casein. Plasma OXM concentration was not affected by postruminal infusion of macronutrients.

Insights into the mechanism by which kisspeptin stimulates a preovulatory LH surge and ovulation in seasonally acyclic ewes: Potential role of estradiol - Corrected Proof

2010-01-25

Abstract: We have previously demonstrated that a constant intravenous infusion of kisspeptin (Kp) for 48h in anestrous ewes induces a preovulatory luteinizing hormone (LH) surge followed by ovulation in approximately 75% of animals. The mechanisms underlying this effect are unknown. In this study, we investigated whether Kp-induced preovulatory LH surges in anestrous ewes were the result of the general activation of the whole gonadotropic axis or of the direct activation of central GnRH neurons required for the GnRH/LH surge. In the first experiment, a constant iv infusion of ovine kisspeptin 10 (Kp; 15.2 nmol/h) was given to 11 seasonally acyclic ewes over 43h. Blood samples were taken every 10min for 15h, starting 5h before the infusion, and then hourly until the end of the infusion. We found that the infusion of Kp induced a well-synchronized LH surge (around 22h after the start of the Kp infusion) in 82% of the animals. In all ewes with an LH surge, there was an immediate but transient increase in the plasma concentrations of LH, follicle-stimulating hormone (FSH), and growth hormone (GH) at the start of the Kp infusion. Mean (± SEM) concentrations for the 5-h periods preceding and following the start of the Kp infusion were, respectively, 0.33 ± 0.09 vs 2.83 ± 0.49 ng/mL (P = 0.004) for LH, 0.43 ± 0.05 vs 0.55 ± 0.03 ng/mL (P = 0.015) for FSH, and 9.34 ± 1.01 vs 11.51 ± 0.92 ng/mL (P = 0.004) for GH. In the first experiment, surges of LH were observed only in ewes that also had a sustained rise in plasma concentrations of estradiol (E2) in response to Kp. Therefore, a second experiment was undertaken to determine the minimum duration of Kp infusion necessary to induce such a pronounced and prolonged increase in plasma E2 concentration. Kisspeptin (15.2 nmol/h) was infused for 6, 12, or 24h in seasonally acyclic ewes (N = 8), and blood samples were collected hourly for 28h (beginning 5h before the start of infusion), then every 2h for the following 22h. Kisspeptin infused for 24h induced LH surges in 75% of animals, and this percentage decreased with the duration of the infusion (12h = 50%; 6h = 12.5%). The plasma concentration of E2 was greater in ewes with an LH surge compared to those without LH surges; mean (± SEM) concentrations for the 5-h period following the Kp infusion were, respectively, 2.23 ± 0.16 vs 1.27 ± 0.13 pg/mL (P < 0.001). Collectively, our results strongly suggest that the systemic delivery of Kp induced LH surges by activating E2-positive feedback on gonadotropin secretion in acyclic ewes.

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

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.

Platelet-derived growth factor acts via both the Rho-kinase and p38 signaling enzymes to stimulate contraction in an in vitro model of equine wound healing - Corrected Proof

E.J. Watts, M.T. Rose — 2009-12-28

Abstract: Horses are more prone to complications in the wound healing process than other species, and problems such as chronic inflammation, delayed epithelialization, poor wound contraction, and exuberant granulation tissue are commonly seen, particularly in wounds on the distal limbs. In comparison, wounds of the oral mucosa heal rapidly in a scarless fashion with a high degree of wound contraction. The effect of platelet-derived growth factor BB (PDGF), insulin-like growth factor (IGF)-1, and transforming growth factor β1 (TGFβ1) on the contraction of a fibroblast-populated collagen matrix (FPCM) as a model of equine wound contraction was investigated using equine oral fibroblasts. The fibroblasts were embedded into floating FPCM and treated with PDGF, IGF-1, and TGFβ1. The surface areas of the FPCM were determined daily for 5 d. Platelet-derived growth factor significantly stimulated the contraction of the FPCM at an optimal concentration of 10 ng/mL (P=0.025). Insulin-like growth factor-1 and TGFβ1 did not significantly affect the contraction of the FPCM relative to the control. To elucidate the mechanisms by which PDGF stimulated contraction of FPCM, the Rho-kinase and p38 cell signaling pathways were blocked, resulting in a significant inhibition (P<0.001) of PDGF-stimulated contraction. Platelet-derived growth factor BB is a potent stimulator of fibroblast migration, and hence the FPCM contraction generated here is probably a result of its effects on cell migration. The results of the present experiment suggest that this effect is stimulated via both the Rho-kinase and p38 signaling pathways in equine oral fibroblasts.

Expression of leukemia inhibitory factor and leukemia inhibitory factor receptor in the canine pituitary gland and corticotrope adenomas - Corrected Proof

J.M. Hanson, J.A. Mol, B.P. Meij — 2009-12-28

Abstract: Leukemia inhibitory factor (LIF) is a pleiotropic cytokine of the IL-6 family that activates the hypothalamic-pituitary-adrenal axis and promotes corticotrope cell differentiation during development. The aim of this study was to investigate the expression of LIF and its receptor (LIFR) in the canine pituitary gland and in corticotrope adenomas, and to perform a mutation analysis of LIFR. Using immunohistochemistry, immunofluorescence, and quantitative expression analysis, LIF and LIFR expression were studied in pituitary glands of control dogs and in specimens of corticotrope adenoma tissue collected through hypophysectomy in dogs with pituitary-dependent hypercortisolism (PDH, Cushing's disease). Using sequence analysis, cDNA was screened for mutations in the LIFR. In the control pituitary tissues and corticotrope adenomas, there was a low magnitude of LIF expression. The LIFR, however, was highly expressed and co-localized with ACTH1-24 expression. Cytoplasmatic immunoreactivity of LIFR was preserved in corticotrope adenomas and adjacent nontumorous cells of pars intermedia. No mutation was found on mutation analysis of the complete LIFR cDNA. Surprisingly, nuclear to perinuclear immunoreactivity for LIFR was present in nontumorous pituitary cells of the pars distalis in 10 of 12 tissue specimens from PDH dogs. These data show that LIFR is highly co-expressed with adrenocorticotropic hormone (ACTH) and α-melanocyte-stimulating hormone (α-MSH) in the canine pituitary gland and in corticotrope adenomas. Nuclear immunoreactivity for LIFR in nontumorous cells of the pars distalis may indicate the presence of a corticotrope adenoma.

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 - Corrected Proof

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.

Endometrial and conceptus expression of HoxA10, transforming growth factor β1, leukemia inhibitory factor, and prostaglandin H synthase-2 in early pregnant pigs with gonadotropin-induced estrus - Corrected Proof

A. Blitek, M.M. Kaczmarek, J. Kiewisz, A.J. Ziecik — 2009-12-21

Abstract: This study was conducted to evaluate the effect of estrus induction with gonadotropins on endometrial and conceptus expression of HoxA10, transforming growth factor (TGF) β1, leukemia inhibitory factor (LIF), and prostaglandin H synthase-2 (PGHS-2) during early pregnancy in pigs. Twenty-four prepubertal gilts received 750 IU of pregnant mare serum gonadotropin (PMSG) and 500 IU of human chorionic gonadotropin (hCG) 72h later. Gilts in the control group (n=23) were observed daily for estrus behavior. Endometrial tissue samples, conceptuses, blood serum, and uterine luminal flushings (ULFs) were collected on days 10, 11, 12, and 15 after insemination. There was no effect of estrus induction on estradiol content in ULFs, or on ovulation and fertilization rates in studied gilts. However, the content of progesterone in the blood serum was greater in naturally ovulated gilts in comparison to gonadotropin-treated animals on day 12 of pregnancy (P<0.05). HoxA10 expression was up-regulated in the endometrium of pregnant gilts, with natural ovulation on days 12 (P<0.05) and 15 (P<0.001) in comparison to days 10 and 11. When compared to control gilts, administration of PMSG/hCG resulted in decreased expression of endometrial HoxA10, TGFβ, LIF, and PGHS-2 on day 12 of pregnancy (P<0.05). Conceptus expression of studied factors was not affected by gonadotropin treatment. Overall, these results suggest improper endometrial preparation for implantation in prepubertal gilts induced to ovulate with PMSG/hCG.

The role of retinal and extra-retinal photostimulation in reproductive activity in broiler breeder hens - Corrected Proof

N. Mobarkey, N. Avital, R. Heiblum, I. Rozenboim — 2009-12-21

Abstract: Photostimulation of retinal photoreceptors, which are sensitive to green light, appears to inhibit reproductive activity in birds, whereas photostimulation of extra-retinal photoreceptors, which are sensitive to red light, accelerates it. The objective of this study was to determine the effect of either retinal or extra-retinal photostimulation on reproductive activities of broiler breeder hens. At 23 wk of age, Cobb hens (N=135) were divided into 9 rooms with individual cages (n=15). At 24 wk of age, 3 rooms were photostimulated (14L:10D) with white light (Control, n=45). Six rooms had 2 parallel lighting systems, red (660nm) and green (560nm), which were both on during 6 out of 14h of the light period. Then, in 3 of these rooms, the green light was turned off and hens were exposed to a total of 14h of red light (Red, n=45), and in the other 3, the red light was turned off and green lighting continued for a total of 14h (Green, n=45). The Green group had reduced egg production; reduced plasma concentrations of ovarian steroids; reduced luteinizing hormone (LH)-β, vasoactive intestinal peptide (VIP), and prolactin mRNA expression; and greater retinal green opsin mRNA expression (P ≤ 0.05). The Red group had greater egg production; greater gonadotropin-releasing hormone-I (GnRH-I) and red opsin gene expression in the hypothalamus; and lesser green opsin gene expression in the retina (P ≤ 0.05). We suggest that selective photostimulation of extra-retinal photostimulation as opposed to retinal photostimulation is a key factor in the determination of successful reproduction of broiler breeder hens.

Expression of Ki-67, PCNA, and p27kip1 in canine pituitary corticotroph adenomas - Corrected Proof

S.J. van Rijn, G.C.M. Grinwis, L.C. Penning, B.P. Meij — 2009-12-21

Abstract: Pituitary-dependent hypercortisolism (PDH), which is caused by adrenocorticotropic hormone (ACTH)-secreting pituitary adenomas, is a common endocrinopathy in dogs. Dogs with non-enlarged pituitaries harboring a microadenoma have a better prognosis than those with enlarged pituitaries. The aim of this study was to investigate the expression of the proliferation markers Ki-67 and proliferating cell nuclear antigen (PCNA) and the cell-cycle inhibitor p27kip1 in corticotroph adenomas in enlarged and non-enlarged pituitaries. The expression of Ki-67, PCNA, and p27kip1 was analyzed by immunohistochemical staining of 17 pituitary adenoma samples harvested during pituitary surgery in dogs with PDH. The labeling index was calculated by counting the number of immunopositive cells per 1,000 cells. The mean (± standard deviation) labeling index for Ki-67 was 8.4%±14.2% for the group with enlarged pituitaries, and 8.8%±5.5% for the group with non-enlarged pituitaries; that for PCNA was 35.5%±12.2% and 37.0%±15.5%; and that for p27kip1 was 29.3%±22.6% and 42.5%±27.9%, respectively. No significant differences in Ki-67, PCNA, and p27kip1 labeling indices were found between enlarged and non-enlarged pituitaries. However, a trend toward significance was observed when comparing the expression of p27kip1 in enlarged pituitaries versus normal pituitary tissue. It is concluded that Ki-67 and PCNA are not useful as proliferative markers for studying the pathobiology of pituitary corticotroph adenomas in dogs.

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

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 - Corrected Proof

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.

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

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.

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

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.

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

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.

VEGF modulates the effects of gonadotropins in granulosa cells - Corrected Proof

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.