The effect of seminal plasma ß-NGF on follicular fluid hormone concentration and gene expression of steroidogenic enzymes in llama granulosa cells.

BACKGROUND: Nerve growth factor (ß-NGF) from llama seminal plasma has been described as a potent ovulatory and luteotrophic molecule after intramuscular or intrauterine infusion in llamas and alpacas. We tested the hypothesis that systemic administration of purified ß-Nerve Growth Factor (ß-NGF) during the preovulatory stage will up-regulate steroidogenic enzymes and Vascular Endothelial Growth Factor (VEGF) gene expression in granulosa cells inducing a change in the progesterone/estradiol ratio in the follicular fluid in llamas. METHODS: Experiment I: Female llamas (n = 64) were randomly assigned to receive an intramuscular administration of: a) 50 µg gonadorelin acetate (GnRH, Ovalyse, Pfizer Chile SA, Santiago, Chile, n = 16), b) 1.0 mg of purified llama ß-NGF (n = 16), or c) 1 ml phosphate buffered saline (PBS, negative control group, n = 16). An additional group of llamas (n = 16) were mated with a fertile male. Follicular fluid and granulosa cells were collected from the preovulatory follicle at 10 or 20 h after treatment (Time 0 = administration of treatment, n = 8/treatment/time point) to determine progesterone/estradiol concentration and steroidogenic enzymes and VEGF gene expression at both time points. Experiment II: Granulosa cells were collected from preovulatory follicles from llamas (n = 24) using ultrasound-guided transvaginal follicle aspiration for in vitro culture to determine mRNA relative expression of Steroidogenic Acute Regulatory Protein (StAR) and VEGF at 10 or 20 h (n = 4 replicates) and progesterone secretion at 48 h (n = 4 replicates) after LH or ß-NGF treatment. RESULTS: Experiment I: There was a significant increase in the progesterone/estradiol ratio in mated llamas or treated with GnRH or purified ß-NGF. There was a significant downregulation in the mRNA expression of Aromatase (CYP19A1/P450 Arom) for both time points in llamas mated or treated with GnRH or llama purified ß-NGF with respect to the control group. All treatments except ß-NGF (20 h) significantly up regulated the mRNA expression of 3-beta-hydroxysteroid dehydrogenase (HSD3B) whereas the expression of StAR and Side-Chain cleavage enzyme (CYP11A1/P450scc) where significantly up regulated only by mating (20 h), or ß-NGF at 10 or 20 h after treatment. VEGF was up regulated only in those llamas submitted to mating (10 h) or treated with purified ß-NGF (10 and 20 h). Experiment II: Only ß-NGF treatment induced an increase of mRNA abundance of StAR from llama granulosa cells at 20 h of in vitro culture. There was a significant increase on mRNA abundance of VEGF at 10 and 20 h of in vitro culture from granulosa cells treated with ß-NGF whereas LH treatment increases VEGF mRNA abundance only at 20 h of in vitro culture. In addition, there was a significant increase on progesterone secretion from llama granulosa cells 48 h after LH or ß-NGF treatment. CONCLUSIONS: Systemic administration of purified ß-NGF from llama seminal fluid induced a rapid shift from estradiol to progesterone production in the preovulatory follicle. Differences in gene expression patterns of steroidogenic enzymes between GnRH and mated or ß-NGF-treated llamas suggest local effects of seminal components on the preovulatory follicle.

Prediction efficiency by near-infrared spectroscopy of immunoglobulin G in liquid and dried bovine colostrum samples.

The objective of this study was to compare the prediction efficiency of IgG concentration in bovine colostrum by NIRS, using liquid and dried (Dry-Extract Spectroscopy for Infrared Reflectance, DESIR) samples by transflectance and reflectance modes, respectively. Colostrum samples (157), obtained from 2 commercial Holstein dairy farms, were collected within the first hour after calving and kept at -20 °C until analysis. After thawing and homogenisation, a subsample of 500 mg of liquid colostrum was placed in an aluminium mirror transflectance cell (0·1 mm path length), in duplicate, to collect the spectrum. A glass fiber filter disc was infused with another subsample of 500 mg of colostrum, in duplicate, and dried in a forced-air oven at 60 °C for 20 min. The samples were placed in cells for dry samples to collect the spectra. The spectra in the VIS-NIR region (400-2500 nm) were obtained with a NIRSystems 6500 monochromator. Mathematical treatments, scatter correction treatments and number of cross-validation groups were tested to obtain prediction equations for both techniques. Reference analysis for IgG content was performed by radial immunodiffusion. The DESIR technique showed a higher variation in the spectral regions associated with water absorption bands, compared with liquid samples. The best equation for transflectance method (liquid samples) obtained a higher coefficient of determination for calibration (0·95 vs. 0·94, respectively) and cross validation (0·94 vs. 0·91, respectively), and a lower error of cross validation (9·03 vs. 11·5, respectively) than the best equation for reflectance method (DESIR samples). In final, both methods showed excellent capacity for quantitative analysis, with residual predictive deviations above 3. It is concluded that, regarding accuracy of prediction and time for obtaining results of IgG from bovine colostrum, NIRS analysis of liquid samples (transflectance) is recommended over dried samples (DESIR technique by reflectance).

The nerve of ovulation-inducing factor in semen.

A component in seminal fluid elicits an ovulatory response and has been discovered in every species examined thus far. The existence of an ovulation-inducing factor (OIF) in seminal plasma has broad implications and evokes questions about identity, tissue sources, mechanism of action, role among species, and clinical relevance in infertility. Most of these questions remain unanswered. The goal of this study was to determine the identity of OIF in support of the hypothesis that it is a single distinct and widely conserved entity. Seminal plasma from llamas and bulls was used as representative of induced and spontaneous ovulators, respectively. A fraction isolated from llama seminal plasma by column chromatography was identified as OIF by eliciting luteinizing hormone (LH) release and ovulation in llamas. MALDI-TOF revealed a molecular mass of 13,221 Da, and 12-23 aa sequences of OIF had homology with human, porcine, bovine, and murine sequences of ß nerve growth factor (ß-NGF). X-ray diffraction data were used to solve the full sequence and structure of OIF as ß-NGF. Neurite development and up-regulation of trkA in phaeochromocytoma (PC(12)) cells in vitro confirmed NGF-like properties of OIF. Western blot analysis of llama and bull seminal plasma confirmed immunorecognition of OIF using polyclonal mouse anti-NGF, and administration of ß-NGF from mouse submandibular glands induced ovulation in llamas. We conclude that OIF in seminal plasma is ß-NGF and that it is highly conserved. An endocrine route of action of NGF elucidates a previously unknown pathway for the direct influence of the male on the hypothalamo-pituitary-gonadal axis of the inseminated female.

Administration of Beta-Nerve Growth Factor during the Preovulatory Stage Improves Endocrine and Luteal Function in Dairy Heifers.

The neurotrophin beta-nerve growth factor (NGF), which is present in the semen of different mammals, elicits potent ovulatory and luteotrophic actions in llamas following systemic administration. Here, we determine if purified NGF given intramuscularly (IM) during the preovulatory stage affects the corpus luteum (CL), hormone production, endometrial gene expression, and pregnancy rate of dairy heifers. Holstein-Friesian heifers were estrus-synchronized using estradiol benzoate (EB) plus an intravaginal progesterone (P4) device (DIB). After eight days, the device was removed and cloprostenol was given IM; the next day (day 9), heifers received EB IM plus one of the following: (i) 1 mg of NGF (NGF D9 group), (ii) 1 mg of NGF 32 h after EB (NGF D10 group), or (iii) phosphate buffer saline (control group). To measure pregnancy rates, heifers were treated similarly, then artificially inseminated with sexed semen 48-52 h after DIB removal, then an ultrasound was conducted 30 days after insemination. The females given NGF along with EB (NGF D9) showed significantly higher luteinizing hormone (LH) concentrations, larger CL vascular areas, and higher plasma P4 concentrations than the NGF D10 and control animals. Downregulation of the P4 receptor (PGR), and upregulation of both lipoprotein lipase (LPL) and Solute Carrier Family 6 member 14 (SLC6A14) endometrial genes, were detected in NGF D9 heifers. Furthermore, these heifers had a 10% higher pregnancy rate than the control group. We conclude that the higher P4 output, in response to the early NGF administration, led to the enhanced gene expression of transcripts related to uterine receptivity that may result in enhanced pregnancy rates.

Cetrorelix suppresses the preovulatory LH surge and ovulation induced by ovulation-inducing factor (OIF) present in llama seminal plasma.

BACKGROUND: The purpose of the study was to determine if the effect of llama OIF on LH secretion is mediated by stimulation of the hypothalamus or pituitary gland. METHODS: Using a 2-by-2 factorial design to examine the effects of OIF vs GnRH with or without a GnRH antagonist, llamas with a growing ovarian follicle greater than or equal to 8 mm were assigned randomly to four groups (n = 7 per group) and a) pre-treated with 1.5 mg of GnRH antagonist (cetrorelix acetate) followed by 1 mg of purified llama OIF, b) pre-treated with 1.5 mg of cetrorelix followed by 50 micrograms of GnRH, c) pre-treated with a placebo (2 ml of saline) followed by 1 mg of purified llama OIF or d) pre-treated with a placebo (2 ml of saline) followed by 50 micrograms of GnRH. Pre-treatment with cetrorelix or saline was given as a single slow intravenous dose 2 hours before intramuscular administration of either GnRH or OIF. Blood samples for LH measurement were taken every 15 minutes from 1.5 hours before to 8 hours after treatment. The ovaries were examined by ultrasonography to detect ovulation and CL formation. Blood samples for progesterone measurement were taken every-other-day from Day 0 (day of treatment) to Day 16. RESULTS: Ovulation rate was not different (P = 0.89) between placebo+GnRH (86%) and placebo+OIF groups (100%); however, no ovulations were detected in llamas pre-treated with cetrorelix. Plasma LH concentrations surged (P < 0.01) after treatment in both placebo+OIF and placebo+GnRH groups, but not in the cetrorelix groups. Maximum plasma LH concentrations and CL diameter profiles did not differ between the placebo-treated groups, but plasma progesterone concentrations were higher (P < 0.05), on days 6, 8 and 12 after treatment, in the OIF- vs GnRH-treated group. CONCLUSION: Cetrorelix (GnRH antagonist) inhibited the preovulatory LH surge induced by OIF in llamas suggesting that LH secretion is modulated by a direct or indirect effect of OIF on GnRH neurons in the hypothalamus.

Bovine Fetal Mesenchymal Stem Cells Obtained From Omental Adipose Tissue and Placenta Are More Resistant to Cryoprotectant Exposure Than Those From Bone Marrow.

Recent studies have shown promise for the development of cellular therapies with mesenchymal stem cells (MSCs) in livestock species, specifically bovines, and cryopreservation is highly relevant for the advancement of these applications. The use of permeable and/or non-permeable cryoprotectant solutions is necessary to reduce cell damage during freezing and thawing, but these same compounds can also cause negative effects on MSCs and their therapeutic properties. Another important factor to consider is the tissue source of MSCs, since it is now known that MSCs from different tissues of the same individual do not behave the same way, so optimizing the type and concentration of cryoprotectants for each cell type is essential to achieve a large and healthy population of MSCs after cryopreservation. Furthermore, sources of MSCs that could provide great quantities, non-invasively and without ethical concerns, such as placental tissue, have great potential for the development of regenerative medicine in livestock species, and have not been thoroughly evaluated. The objective of this study was to compare the viability of bovine fetal MSCs extracted from bone marrow (BM), adipose tissue (AT), and placenta (PT), following their exposure (15 and 30 min) to several solutions of permeable (dimethyl sulfoxide and ethylene glycol) and non-permeable (trehalose) cryoprotectants. Viability assays were performed with Trypan Blue to assess post-exposure plasma membrane integrity. The apoptotic potential was estimated analyzing the mRNA abundance of BAX and BCL-2 genes using quantitative rt-PCR. Based on the results of the study, BM-MSC exhibited significantly lower viability compared to AT-MSC and PT-MSC, at both 15 and 30 min of exposure to cryoprotectant solutions. Nevertheless, viability did not differ among treatments for any of the cell types or timepoints studied. BCL-2 expression was higher in BM-MSC compared to AT-MSC, however, BAX/BCL-2 ratio did not differ. In conclusion, AT-MSC and PT-MSC were more resistant that BM-MSC, which showed higher sensitivity to experimental conditions, regardless of the exposure times, and cryoprotectant solutions used in the study.

ß-NGF Stimulates Steroidogenic Enzyme and VEGFA Gene Expression, and Progesterone Secretion via ERK 1/2 Pathway in Primary Culture of Llama Granulosa Cells.

The beta-nerve growth factor (ß-NGF) from llama seminal plasma exerts ovulatory and luteotrophic effects following intramuscular or intrauterine infusion in llamas and alpacas. In this study, we investigate the in vitro effect of llama ß-NGF on the expression of genes involved in angiogenesis and progesterone synthesis as well as progesterone release in preovulatory llama granulosa cells; we also determine whether these changes are mediated via the ERK1/2 signaling pathway. From adult female llamas, we collected granulosa cells from preovulatory follicles by transvaginal ultrasound-guided follicle aspiration; these cells were pooled and incubated. After 80% confluence, the cultured granulosa cells were treated with ß-NGF, ß-NGF plus the MAPK inhibitor U0126, or luteinizing hormone, and the abundance of angiogenic and steroidogenic enzyme mRNA transcripts were quantified after 10 and 20 h by RT-qPCR. We also quantified the progesterone concentration in the media after 48 h by radioimmunoassay. We found that application of ß-NGF increases the abundance of mRNA transcripts of the vascular endothelial growth factor (VEGFA) and the steroidogenic enzymes cytochrome P450 side-chain cleavage (P450scc/CYP11A1), steroidogenic acute regulatory protein (STAR), and 3ß-hydroxysteroid dehydrogenase (HSD3B1) at 10 and 20 h of treatment. Application of the MAPK inhibitor U0126 resulted in downregulation of the genes encoding these enzymes. ß-NGF also enhanced progesterone synthesis, which was prevented by the prior application of the MAPK inhibitor U0126. Finally, western blot analysis confirmed that ß-NGF activates the ERK1/2 signaling pathway. In conclusion, our results indicate that ß-NGF exerts direct luteotropic effects on llama ovarian tissue via the ERK 1/2 pathway.

Novel Brn3a cis-acting sequences mediate transcription of human trkA in neurons.

TrkA, the receptor tropomyosin-related kinase for nerve growth factor, is critical not only for the correct spatial and temporal development of sensory neurons during embryogenesis but also for the survival of sensory neurons, the differentiation and apoptosis of neuronal tumors and suppression of latent herpes simplex virus genomes. While the regulation of the expression of trkA is a complex process, the transcription factor Brn3a is known to play an important role as an enhancer of trkA transcription during development in the mouse. Despite considerable information on the regulation of trkA during embryogenesis, the mechanisms by which the expression of trkA is regulated in differentiated neurons, or the factors that influence its expression in tumor cells, have not been identified. We initiated studies to determine whether Brn3a/trkA promoter interactions may be important in a model of differentiated neurons and in medulloblastoma cells. We constructed a plasmid that contains 1043 base pairs of genomic sequences that extend to 30 nucleotides upstream of trkA coding region. In contrast to previous data, a short 190 bp region that lies proximal to the trkA initiation codon was sufficient for Brn3a responsiveness in Vero cells. This region was also sufficient for Brn3a trans-activation in nerve growth factor-differentiated PC12 cells. At least two portions of the 190 bp fragment bind to Brn3a with an affinity high enough to be detected in electromobility shift assays. In addition, Brn3a increased levels of endogenous trkA transcripts in PC12 cells and initiated trkA expression in medulloblastoma cells, which normally do not express trkA.

Zhangfei, a novel regulator of the human nerve growth factor receptor, trkA.

The replication of herpes simplex virus (HSV) in epithelial cells, and during reactivation from latency in sensory neurons, depends on a ubiquitous cellular protein called host cell factor (HCF). The HSV transactivator, VP16, which initiates the viral replicative cycle, binds HCF as do some other cellular proteins. Of these, the neuronal transcription factor Zhangfei suppresses the ability of VP16 to initiate the replicative cycle. It also suppresses Luman, another cellular transcription factor that binds HCF. Interactions of nerve growth factor (NGF) and its receptor tropomyosin-related kinase (trkA) appear to be critical for maintaining HSV latency. Because the neuronal transcription factor Brn3a, which regulates trkA expression, has a motif for binding HCF, we investigated if Zhangfei had an effect on its activity. We found that Brn3a required HCF for activating the trkA promoter and Zhangfei suppressed its activity in non-neuronal cells. However, in neuron-like NGF-differentiated PC12 cells, both Brn3a and Zhangfei activated the trkA promoter and induced the expression of endogenous trkA. In addition, capsaicin, a stressor, which activates HSV in in vitro models of latency, decreased levels of Zhangfei and trkA transcripts in NGF-differentiated PC12 cells.

Cloning and nucleotide sequence of the equine and elk pituitary pre-prolactin cDNA.

We report the equine (Equs equs) and elk (Cervus elaphus) pituitary pre-prolactin (PRL) cDNA cloning, and their nucleotide and deduced amino acid sequences. Pre-PRL cDNA was obtained by RNA ligation mediated-rapid amplification of cDNA ends (RLM-RACE) and polymerase chain reaction (PCR). The elk pre-PRL cDNA exhibits two polymorphisms at positions 96 and 672, which are silent since they encode for the same amino acids, proline and isoleucine, respectively. We found no polymorphisms in the equine pre-PRL cDNA. The deduced amino acid sequence of the equine pre-PRL is 99% identical to the previously reported protein sequence. Pre-PRL mRNA is <1 kb in length and is highly expressed in the anterior pituitary gland, as demonstrated by Northern hybridization analysis. In summary, we cloned and sequenced the equine and elk pre-PRL cDNAs. The deduced amino acid sequence of elk and equine pre-PRL appears to be moderately conserved among other mammalian species. The polymorphic sites found in the elk cDNA could potentially be used in parentage testing and gene mapping.

Zhangfei induces the expression of the nerve growth factor receptor, trkA, in medulloblastoma cells and causes their differentiation or apoptosis.

Interactions between nerve growth factor (NGF) and its receptor-the tropomyosin related kinase A (trkA)-regulate many neuronal functions including the correct development of sensory neurons during embryogenesis, the survival of sensory neurons and the differentiation and apoptosis of neuronal tumours. Zhangfei is a transcriptional factor that is expressed in differentiated neurons. Since we could detect Zhangfei in mature neurons but not in neuronal tumour cells, we hypothesised that ectopic expression of the protein in medulloblastoma cells may induce the differentiation of these cells. We show that in ONS-76 medulloblastoma cells, resveratrol, an inducer of apoptosis and differentiation, increased the expression of Zhangfei, trkA and Early Growth Response Gene 1 (Egr1), a gene normally activated by NGF-trkA signalling. ONS-76 cells stopped growing soon after treatment with resveratrol. While the induction of Zhangfei in resveratrol-treated cells was modest albeit consistent, the infection of actively growing medulloblastoma cells with an adenovirus vector expressing Zhangfei mimicked some of the effects of resveratrol. Ectopically expressed Zhangfei in ONS-76 cells led to the increased expression of trkA and Egr1, phosphorylation of extracellular signal-regulated kinase (Erk1), and caused ONS-76 cells to display markers of apoptosis. UW228, another medulloblastoma cell-line, was also susceptible to the suppressive effects of resveratrol and Zhangfei. In contrast, while resveratrol suppressed the growth of human diploid fibroblasts (MRC5), Zhangfei had relatively little effect on these cells.

Cadmium stimulates transcription of the cytochrome p450 side chain cleavage gene in genetically modified stable porcine granulosa cells.

We investigated the effects of cadmium (Cd2+) on transcription of the cytochrome p450 side chain cleavage (p450scc) gene and on progesterone synthesis in stable granulosa cells. We used the stable porcine granulosa cell line, JC-410, genetically modified to express a luciferase genomic construct carrying 2320 base pairs (bp) of the p450scc gene promoter (p450scc-2320-LUC). A construct containing only the luciferase gene, pOLUC, was used as a promoterless control. At 1 microM, cadmium chloride (CdCl2) increased transient expression of p450scc-2320-LUC in JC-410 cells by 2.6-fold after 24-h incubation. A similar pattern of stimulation by CdCl2 was observed in cells transiently transfected with a luciferase genomic construct carrying 100 bp of the p450scc gene promoter p450scc-100-LUC, whereas no stimulation by CdCl2 was observed in cells transfected with pOLUC. At 0.6, 1, and 2 microM, CdCl2 stimulated the activity of the p450scc-2320-LUC promoter in a dose-related fashion by 1.58-, 3.19-, and 2.67-fold, respectively, after 24-h incubation. Northern blot analysis showed that CdCl2 at 0.1, 1, 2, and 3 microM increased p450scc mRNA levels by 3.13-, 1.38-, 1.61-, and 1.57-fold, respectively, after 24-h incubation. After 48-h incubation, CdCl2 at 0.6, 1, and 2 microM further increased p450scc mRNA levels by 3.43-, 2.08-, and 2.4-fold, respectively. At 1, 2, and 3 microM, CdCl2 inhibited progesterone synthesis to 0.48-, 0.38-, and 0.29-fold, respectively. After 48-h incubation, CdCl2 at 0.1 microM stimulated progesterone synthesis by 1.6-fold. We conclude that Cd2+ has a dual action in stable porcine granulosa cells: Low concentrations activate, whereas high concentrations inhibit, expression of the p450scc gene and progesterone synthesis. The stimulatory effect of Cd2+ appears to be mediated via a cis-acting element located 100 bp upstream of the p450scc gene transcription start site.