Llama Seminal Plasma Research Articles

Development and Biodistribution of a Nerve Growth Factor Radioactive Conjugate for PET Imaging.

The purpose of these studies was to develop a nerve growth factor (NGF) radiometal-chelator conjugate to determine the biodistribution and brain uptake of NGF by positron emission tomography/computerized tomography (PET-CT). Purified NGF from llama seminal plasma was conjugated with FITC, and the chelator NOTA or DFO. NGF conjugates were evaluated for bioactivity. NOTA- and DFO-conjugated NGF were radiolabeled with gallium-68 or zirconium-89 ([68Ga]GaCl3, half-life = 68min; [89Zr]Zr(oxalate)4, half-life = 3.3days). [89Zr]Zr-NGF was evaluated for biodistribution (0.5, 1, or 24h), PET imaging (60min), and brain autoradiography in mice. Cell-based in vitro assays confirmed that the NGF conjugates maintained NGF receptor-binding and biological activity. Zirconium-89 and gallium-68 radiolabeling showed a high efficiency; however, only[89Zr]Zr-NGF was stable in vitro. Biodistribution studies showed that, as with most small proteins < 70kDa, [89Zr]Zr-NGF uptake was predominantly in the kidney and was cleared rapidly with almost complete elimination of NGF at 24h. Dynamic PET imaging from 0-60min showed a similar pattern to ex vivo biodistribution with some transient liver uptake. Interestingly, although absolute brain uptake was very low, at 24h after treatment, cerebral cortex uptake was higher than any other brain area examined and blood. We conclude that conjugation of DFO to NGF through a thiourea linkage allows effective radiolabeling with zirconium-89 while maintaining NGF bioactivity. Following intravenous administration, the radiolabeled NGF targets non-neuronal tissues (e.g., kidney, liver), and although absolute brain uptake was very low, the brain uptake that was observed was restricted to the cortex.

Characterization of proteases in the seminal plasma and spermatozoa of llama

Camelids' semen has peculiar characteristics that differentiate it from other species, including the highly viscous aspect of seminal plasma that greatly difficult sperm manipulation and the development of techniques such as cryopreservation, artificial insemination, and/or in vitro fertilization. The presence of proteases in the seminal plasma is responsible for semen liquefaction, and sperm functionality to achieve fertilization. The enzymatic and molecular composition of the semen of llama remains unknown. Therefore, the goal of the study was to characterize the protease activity and composition of the seminal plasma and sperm of llama semen. The proteolytic activity was performed using gelatine zymography and the composition by mass-spectrometry.Metallo-proteases were the major source of gelatinolytic activity in seminal plasma, while serine-peptidases were the main enzymes of sperm cells. Matrix Metalloproteinase 2 (MMP2) was the most prominent metallo-protease of llama seminal plasma characterized under the exposure of different inhibitors (EDTA and benzamidine) and by a specific immunodetection. Moreover, the prostate and epididymis were identified as potential sites of its synthesis and secretion.Outstandingly, this metalloproteinase was undetectable in llama sperm. Regarding, the molecular composition of semen by mass-spectrometry, 4 metallo-, 9 serine-, 8 threonine-, and 1 aspartic-peptidases were identified alongside 15 regulators in the sperm cell; where 24 were directly or indirectly interacting. Whereas 6 metallo-, 12 serine-, 3 cysteine-, and 1 aspartic-peptidases were identified, besides 7 inhibitors and 5 regulators in llama seminal plasma where 30 of them were directly or indirectly interconnected. This is the first study describing a partial degradome of llama seminal plasma and spermatozoa suggesting significant differences especially the absence of MMP2 in spermatozoa in contrast to data observed in other species. The characterization of proteases in llama semen will provide a better understanding of the molecular mechanisms involved in the in vivo or in vitro fertilization process in this species.

β-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 (β-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 β-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 β-NGF, β-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 β-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 3β-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. β-NGF also enhanced progesterone synthesis, which was prevented by the prior application of the MAPK inhibitor U0126. Finally, western blot analysis confirmed that β-NGF activates the ERK1/2 signaling pathway. In conclusion, our results indicate that β-NGF exerts direct luteotropic effects on llama ovarian tissue via the ERK 1/2 pathway.

Preliminary Evaluation of Seminal Plasma Proteins and Immunoreactivity of Nerve Growth Factor as Indicative of an Ovulation Inducing Factor in Odontocetes

In the seminal plasma of terrestrial mammalian species known as induced (e.g., camels) and spontaneous (e.g., cattle) ovulators, an ovulation-inducing factor (OIF) with a protein structure similar to beta-nerve growth factor (β-NGF) has been identified. Detection of an OIF/NGF in the seminal plasma of cetaceans would have both basic and applied implications in reproductive biology and conservation management programs. A preliminary evaluation was conducted to characterize the distribution and abundance of seminal plasma proteins in aquarium-based belugas and a Pacific white-sided and bottlenose dolphin. Initially, SDS-PAGE was used with 50 μg of total protein for separation; thereafter, Western immunoblot was used with anti-NGF. In addition to odontocete seminal plasma, a purified fraction of llama seminal plasma (100 ng protein) and an extract of mouse brain (20 μg total protein) were included as positive controls for NGF. Within the two belugas, visual inspection of the protein bands indicated similar distribution and intensity. However, among the belugas and Pacific white-sided and bottlenose dolphins there was more diversity than similarity in the distribution and abundance of seminal plasma proteins. While immunoreactivity of NGF was distinctly evident in the llama and mouse positive controls, there was no visual reactivity in any of the odontocete samples. These preliminary results provide novel information indicating more homogeneity within and heterogeneity among seminal plasma proteins of ondentocetes. Although NGF was not immunologically detected, future studies are required to address the apparent limitations of immuno-detection of NGF, especially if the post-translational form of β-NGF is in low abundance in the seminal plasma of belugas and Pacific white-sided and bottlenose dolphins.

123 Nerve growth factor-induced ovulation in llamas: Evidence of hypothalamic refractoriness to nerve growth factor during the declining phase of the luteinising hormone surge

Nerve growth factor (NGF) in semen is responsible for triggering ovulation after copulation in camelids. Interaction of NGF with its cognate receptors results in a preovulatory luteinising hormone (LH) surge that leads to ovulation, but the pharmacokinetics of NGF and the mechanism by which it mediates LH release are unknown. In an effort to elucidate the site and mechanism of action involved, the objective of this study was to determine whether the decline in the LH surge occurs as a result of pituitary depletion (i.e. diminished response to gonadotrophin-releasing hormone (GnRH)) or as an obtunded response at the level of the hypothalamus (diminished GnRH). Adult nonpregnant, nonlactating llamas (n=18) were synchronized by the administration of an ovulatory dose of a GnRH analogue (100μg IM; Fertiline, Vetoquinol). At 10 to 14 days later, the ovaries were examined to confirm the presence of a dominant follicle measuring ≥ 7mm, and a jugular catheter was put in place. The following day, a pretreatment blood sample was taken, and llamas were treated intravenously with 1mg of purified NGF from llama seminal plasma. Blood samples were taken every 30min for 7h from the time of NGF treatment. After the last blood sample was taken, llamas were treated with NGF (n=6; 1mg IV), GnRH (n=5; 100μg IV), or saline (Sal; IV; n=6), and blood samples were taken every 30min for another 7h. The ovaries were examined 48h after initial NGF treatment, via transrectal ultrasonography, to detect ovulation. Plasma was harvested and stored for analysis of LH concentration by radioimmunoassay. Data were compared using ANOVA for repeated measures, and single-point data were analysed using paired t-tests. As expected, most llamas ovulated in response to the initial NGF treatment (5 out of 5 in NGF-GnRH; 5 of 6 in NGF-NGF; 5 of 6 in NGF-Sal). Compared with pretreatment values, all llamas showed a 4-fold increase in plasma LH concentrations within 2h of the initial NGF treatment (P&amp;lt;0.05). Plasma LH concentrations peaked at 3h after initial NGF treatment and began to decline 4.5 to 5h after treatment (P&amp;lt;0.05). Plasma LH concentrations continued to decline following the second dose of NGF or Sal, whereas a transient elevation of LH was detected in llamas treated with GnRH (P&amp;lt;0.05). The LH concentration returned to basal levels (pretreatment) 8, 12, and 13h after NGF treatment in llamas treated with NGF-Sal, NGF-GnRH, and NGF-NGF, respectively. We conclude that the lack of LH response to the second dose of NGF is not because of pituitary depletion but rather due to diminished GnRH. The latter may be attributed to either a downregulation of NGF receptors within the hypothalamus or to temporary depletion of GnRH at the nerve terminals within the median eminence. Research was supported by the Natural Sciences and Engineering Research Council of Canada.

The effect of seminal plasma \u03b2-NGF on follicular fluid hormone concentration and gene expression of steroidogenic enzymes in llama granulosa cells

BackgroundNerve growth factor (β-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 β-Nerve Growth Factor (β-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.MethodsExperiment 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 β-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 β-NGF treatment.ResultsExperiment I: There was a significant increase in the progesterone/estradiol ratio in mated llamas or treated with GnRH or purified β-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 β-NGF with respect to the control group. All treatments except β-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 β-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 β-NGF (10 and 20 h). Experiment II: Only β-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 β-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 β-NGF treatment.ConclusionsSystemic administration of purified β-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 β-NGF-treated llamas suggest local effects of seminal components on the preovulatory follicle.

Evaluation of the effect of mating, intrauterine deposition of raw seminal plasma or seminal plasma purified β-NGF on endometrial vascularization in llamas

The aim of this study was to evaluate the endometrial vascularization area (EVA) of both uterine horns in llamas subjected to different intrauterine treatments resembling physiological conditions after a single mating. Llamas with a growing follicle (≥8 mm) were randomly assigned to: a) single mating with a fertile male (mating; positive control; n = 6); b) intramuscular administration of 50 μg of gonadorelin acetate plus an intrauterine infusion of 4 ml of PBS (GnRH; negative control; n = 4); c) intrauterine infusion of 4 ml of raw llama seminal plasma (SP; n = 4) or d) intrauterine infusion of 10 mg of β-NGF purified from llama semen diluted in 4 ml of PBS (spβ-NGF; n = 6). Females in GnRH, SP and NGF group received 50% of treatment volume into each horn by guiding an insemination pipet through the cervix. Ovaries were examined by ultrasonography every 12 h until Day2 (Day 0 = Day of treatment) to determine ovulation. Power-Doppler ultrasonography evaluation of EVA in a cross-section of the middle segment of each horn was conducted at 1 h before and 1, 3, 6, 12 and 24 h (intensive evaluation) and 2, 4, 6 and 8 days (long-term evaluation) after treatment administration. Serial EVA data was analyzed as a 2-by-2 factorial design for repeated measures using the MIXED procedure. The analysis included main effects of treatment (mating, SP, spβ-NGF or GnRH), uterine horn (left vs right), time, and their interactions. According to the 2 by 2 analysis there was no effect of uterine horn on EVA during the first 24 h and from Day 2 to Day 8 after treatment; therefore, data were grouped based on treatment type regardless of uterine horn for both periods of observation. Thus, EVA was affected by time (P < 0.04) and treatment by time interaction (P < 0.02) and tended (P = 0.07) to be influenced by type of treatment during the intensive evaluation period. Females on mating and spβ-NGF group showed a significant increase in EVA at 3 and 12 h after treatment compared to GnRH and SP groups. However, no effect of treatment, time or their interaction was observed during the long-term evaluation period. In spite of the limited number of animals used in this study, our results allow us to concluded that natural mating and intrauterine deposition of 10 mg of spβ-NGF induce a symmetrical increase in endometrial vascularization of both uterine horns during the first 24 h post treatment administration in llamas; however, this effect did not persist beyond that period.

13 EFFECTS OF NERVE GROWTH FACTOR-β, PURIFIED FROM BULL SEMINAL PLASMA, ON CORPUS LUTEUM FUNCTION AND CONCEPTUS DEVELOPMENT IN COWS

Nerve growth factor-β (NGF) has been identified in the seminal plasma of livestock and is required for the induction of ovulation in camelids. Until recently, it was thought to play a negligible role in species with spontaneous ovulation. On the contrary, recent studies have shown that systemic administration of NGF, purified from llama seminal plasma, was associated with larger corpus luteum (CL) diameter and higher concentrations of progesterone (P) following ovulation. The objective of the current project was to determine if systemic administration of NGF, purified from bovine seminal plasma, would improve CL formation and enhance embryonic development. Our hypothesis was that systemic administration of NGF at the time of artificial insemination in cows would lead to increased CL volume, increased P secretion, and improved expression of markers of conceptus development and maternal recognition of pregnancy. Seminal plasma was harvested from semen collected by electroejaculation in Angus cross bulls. Purification of NGF was performed using a combination of anion- and cation-exchange chromatography and gradient elution. Beef cows were randomly assigned to CONT (n = 30) or NGF (n = 30) groups and synchronized using a 7-day Co-Synch + CIDR program. At time of insemination (day 0), NGF cows received 296 µg of purified NGF, reconstituted in 12 mL of PBS, and CONT cows received 12 mL of PBS intramuscularly. Blood samples were collected from the coccygeal vein of each cow at days 0, 3, 7, 10, 14, 19, 21, 28, 31, 38, 45, and 66. Ultrasound was performed at each time point for determination of ovarian structures (day 0), corpus luteum volume (all time points), and fetus detection (day 28). Statistical analysis was performed using analysis of variance with repeated-measures in R (R version 3.2.2; https://www.r-project.org/). At day 28, 17/30 (57%) CONT cows and 21/30 (70%) NGF cows were diagnosed as pregnant (P = 0.15). At day 0, cows that later became pregnant had a larger follicular diameter (1.46 cm) than those diagnosed as open (1.16 cm; P &lt; 0.01). Follicular diameter at day 0 did not differ significantly between CONT and NGF groups for either open (P = 0.35) or pregnant (P = 0.90) cows. CL volume in open cows was affected by day (P &lt; 0.001) with no treatment (P = 0.84) or treatment by day (P = 0.42) interaction. CL volume in pregnant cows was affected by both day (P &lt; 0.001) and treatment (P &lt; 0.001), with CONT cows having a higher CL volume than NGF cows. Though NGF appeared to have an inhibitory effect on CL volume in pregnant cows, results are still pending for quantification of P, insulin-like growth factor 1, pregnancy-specific protein B, and interferon-stimulated genes, which will more accurately assess the effects that NGF may have on conceptus development. The results of this study will help us to better understand the role of the seminal plasma protein, NGF, at ovulation and determine if it can be utilised to enhance insemination programs in cattle.

LH release and ovulatory response after intramuscular, intravenous, and intrauterine administration of β-nerve growth factor of seminal plasma origin in female llamas

The objective of the study was to compare the pituitary and ovarian responses after intramuscular, intravenous, or intrauterine administration of β-nerve growth factor (β-NGF) of seminal plasma origin (SP-NGF) in llamas. In experiment 1, mature female llamas with a growing follicle of 7 mm or greater were assigned randomly to four groups (n = 7/group) and given 2 mg of purified SP-NGF in a volume of 2 mL by (1) intramuscular administration, (2) intravenous administration, and (3) intrauterine infusion, or (4) intrauterine infusion of 2 mL of PBS (negative control). Because ovulations were not detected after intrauterine infusion in experiment 1, a second experiment was done to determine if a higher dose of SP-NGF given by intrauterine infusion, similar to a natural dose during copulation, will elicit an ovulatory response. In experiment 2, llamas with a growing follicle of 7 mm or greater were assigned randomly to three groups (n = 6/per group) given an intrauterine infusion of (1) 4 mL of raw seminal plasma, (2) 4 mL of PBS containing 20 mg of purified llama SP-NGF, or 3) 4 mL of PBS (negative control). In both experiments, the ovaries were examined daily by transrectal ultrasonography using a B-mode scanner and power Doppler mode to detect ovulation and to monitor CL growth, regression, and vascularization. Blood samples were collected to determine plasma LH and progesterone concentrations. In experiment 1, only llamas treated by intramuscular or intravenous administration of SP-NGF ovulated (7 of 7 and 6 of 7, respectively). Plasma LH concentration did not differ between the intramuscular and intravenous SP-NGF–treated groups, nor did CL diameter, CL vascularization, or plasma progesterone concentration profiles. In experiment 2, the ovulation rate was 100% for llamas treated by intrauterine infusion of raw seminal plasma or llama SP-NFG, whereas no ovulations were detected in females treated with PBS. Plasma LH concentrations did not differ between groups that ovulated, nor did CL diameter, CL vascularization, or plasma progesterone concentration profiles. We conclude that β-NGF from llama seminal plasma origin elicits a preovulatory LH surge, followed by ovulation and the development of a functional CL, regardless of the route of administration. However, the dose required to elicit pituitary and ovarian responses is higher when administered by intrauterine infusion than by intramuscular or intravenous routes.

Bioactivity of ovulation inducing factor (or nerve growth factor) in bovine seminal plasma and its effects on ovarian function in cattle

To understand the role of ovulation-inducing factor (or nerve growth factor) (OIF [NGF]) in bovine seminal plasma, we (1) used an in vivo llama bioassay to test the hypothesis that bovine seminal plasma induces ovulation and CL development in llamas similar to that of llama seminal plasma when the dose of seminal plasma is adjusted to ovulation-inducing factor content (experiment 1) and (2) determined the effect of bovine seminal plasma on the interval to ovulation and luteal development in heifers (experiment 2). Within species, seminal plasma was pooled (n = 160 bulls, n = 4 llamas), and the volume of seminal plasma used for treatment was adjusted to a total dose of 250 μg of ovulation-inducing factor. In experiment 1, mature female llamas were assigned randomly to four groups and treated intramuscularly with either 10 mL of PBS (negative control, n = 5), 50-μg GnRH (positive control, n = 5), 6-mL of llama seminal plasma (n = 6), or 12 mL of bull seminal plasma (n = 6). Ovulation and CL development were monitored by transrectal ultrasonography. In experiment 2, beef heifers were given a luteolytic dose of prostaglandin followed by 25-mg porcine LH (pLH) 12 hours later to induce ovulation. Heifers were assigned randomly to three groups and given 12 mL bovine seminal plasma intramuscularly 12 hours after pLH treatment (n = 10), within 4 hours after ovulation (n = 9), or no treatment (control, n = 10). Ovulation was monitored by ultrasonography every 4 hours, and the CL development was monitored daily until the next ovulation. In experiment 1, ovulation was detected in 0/5, 4/5, 4/6, 4/6 llamas in the PBS, GnRH, llama seminal plasma, and bovine seminal plasma groups, respectively (P < 0.05). Luteal development was not different among groups. In experiment 2, the interval to ovulation was more synchronous (range: 4 vs. 22 hours; P < 0.0001) in heifers treated with seminal plasma before ovulation compared with the other groups. Luteal development was not different among groups; however, plasma progesterone concentrations tended to be greater in the postovulation treatment group compared with other groups. In summary, results confirmed the presence of bioactive ovulation-inducing factor in bull seminal plasma and supported the hypothesis that bovine and llama seminal plasma have similar ovulatory effects, using a llama bioassay. Treatment with bovine seminal plasma resulted in greater synchrony of ovulation in heifers pretreated with pLH. Plasma progesterone concentration tended to be higher in heifers given bovine seminal plasma within 4 hours after ovulation, suggesting that bovine ovulation-inducing factor is luteotrophic.

The effect of repeated administrations of llama ovulation-inducing factor (OIF/NGF) during the peri-ovulatory period on corpus luteum development and function in llamas

The objective of the study was to test the hypothesis that repeated administrations of OIF/NGF during the peri-ovulatory period (pre-ovulatory, ovulatory, early post-ovulatory), will enhance the luteotrophic effect in llamas. Female llamas were examined daily by transrectal ultrasonography in B- and Doppler-mode using a scanner equipped with a 7.5-MHz linear-array transducer to monitor ovarian follicle and luteal dynamics. When a growing follicle ≥7mm was detected, llamas were assigned randomly to one of the three groups and given 1mg of purified OIF/NGF im (intramuscular) (a) pre-ovulation (single dose; n=12), (b) pre-ovulation and at the time of ovulation (2 doses, n=10), or (c) pre-ovulation, at the time of ovulation, and 24h after ovulation (3 doses, n=10). The pre-ovulatory follicle diameter at the time of treatment, ovulation rate and the first day of CL detection did not differ (P=0.3) among groups. However, maximum CL diameter was greatest (P=0.003) in llamas in the 2-dose group, and smallest in the 3-dose group. Accordingly, the 2 dose-group had the largest day-to-day profile for CL diameter (P<0.01), area of CL vascularization (<0.01), and plasma progesterone concentration (P=0.01) compared to the other groups. Interestingly, the luteal response to 3-doses of OIF/NGF during the peri-ovulatory period was not different from a single dose. In conclusion, OIF/NGF isolated from llama seminal plasma is luteotrophic and the effect on CL size and function is affected by the number and timing of treatments during the peri-ovulatory period.

Ovulation-inducing factor (OIF/NGF) from seminal plasma origin enhances Corpus Luteum function in llamas regardless the preovulatory follicle diameter

Ovulation-inducing factor (OIF) is a protein present in llama seminal plasma that has recently been identified as β-Nerve Growth Factor (NGF) and it induces not only a high rate of ovulation but also appears to have luteotrophic properties in this species. A 2-by-2 experimental design was used to determine the effect of treatments (OIF/NGF vs GnRH) and categories of preovulatory follicle diameter (7–10 vs >10mm) on ovulation rate, CL diameter and function in llamas. Llamas (n=32 llamas per group) were randomly assigned to receive an intramuscular dose of: (a) 1mg purified OIF/NGF in the presence of a follicle of 7–10mm in diameter; (b) 50μg of GnRH in the presence of a follicle of 7–10mm in diameter; (c) 1mg purified OIF/NGF in the presence of a follicle >10mm in diameter; (d) 50μg of GnRH in the presence of a follicle >10mm in diameter. Llamas were examined by ultrasonography every 12h from treatment to Day 2 (Day 0=treatment) to detect ovulation, and again on Day 8 to determine CL diameter. Ovulation rates did not differ among groups. There was an effect of preovulatory follicle size on Corpus Luteum diameter at Day 8 (P<0.001), however plasma progesterone concentration (n=15/per group) was higher (P<0.05) in the OIF/NGF – than that of the GnRH – treated group by the same day. We conclude that OIF/NGF treatment enhances CL function regardless preovulatory follicle size at the time of treatment.

Luteotrophic effect of ovulation-inducing factor/nerve growth factor present in the seminal plasma of llamas

The hypothesis that ovulation-inducing factor/nerve growth factor (OIF/NGF) isolated from llama seminal plasma exerts a luteotrophic effect was tested by examining changes in circulating concentrations of LH and progesterone, and the vascular perfusion of the ovulatory follicle and developing CL. Female llamas with a growing follicle of 8 mm or greater in diameter were assigned randomly to one of three groups (n = 10 llamas per group) and given a single intramuscular dose of PBS (1 mL), GnRH (50 μg), or purified OIF/NGF (1.0 mg). Cineloops of ultrasonographic images of the ovary containing the dominant follicle were recorded in brightness and power Doppler modalities. Llamas were examined every 4 hours from the day of treatment (Day 0) until ovulation, and every other day thereafter to Day 16. Still frames were extracted from cineloops for computer-assisted analysis of the vascular area of the preovulatory follicle from treatment to ovulation and of the growing and regressing phases of subsequent CL development. Blood samples were collected for the measurement of plasma LH and progesterone concentrations. The diameter of the dominant follicle at the time of treatment did not differ among groups (P = 0.48). No ovulations were detected in the PBS group but were detected in all llamas given GnRH or OIF/NGF (0/10, 10/10, and 10/10, respectively; P < 0.0001). No difference was detected between the GnRH and OIF/NGF groups in the interval from treatment to ovulation (32.0 ± 1.9 and 30.4 ± 5.7 hours, respectively; P = 0.41) or in maximum CL diameter (13.1 ± 0.4 and 13.5 ± 0.3 mm, respectively; P = 0.44). The preovulatory follicle of llamas treated with OIF/NGF had a greater vascular area at 4 hours after treatment than that of the GnRH group (P < 0.001). Similarly, the luteal tissue of llamas treated with purified OIF/NGF had a greater vascular area than that of the GnRH group on Day 6 after treatment (P < 0.001). The preovulatory surge in plasma LH concentration began, and peaked 1 to 2 hours later in the OIF/NGF group than in the GnRH group (P < 0.05). Plasma progesterone concentration was higher on Day 6 in the OIF/NGF group than in the GnRH group (P < 0.001). Results support the hypothesis that OIF/NGF exerts a luteotrophic effect by altering the secretion pattern of LH and enhancing tissue vascularization during the periovulatory period and early stages of CL development.

Llama oviductal sperm reservoirs: involvement of bulbourethral glands

The aim of this study was to elucidate the role of llama seminal plasma in the formation of oviductal sperm reservoirs. Female llamas with follicles in the mature phase were mated with a bulbourethral glands-removed male. Females mated with nonbulbourethral glands-removed males were used as control. Oviducts were obtained by surgery 24 h after mating. The uterotubal junction and isthmus were examined by scanning electron microscopy, and mucopolysaccharides were identified by Alcian blue staining. To know the proteins probably involved in sperm reservoir formation, SDS-PAGE of seminal plasma (8% and 18% resolving gel) was made. Spermatozoa only adhered to the oviductal mucosa surface of uterotubal junction of females mated with nonbulbourethral glands-removed males confirming that seminal plasma and, in particular, bulbourethral secretions are related with the oviductal sperm reservoir formation. Histological sections showed sperm in the lumen, immersed in substance, positive for acid mucopolysaccharides. Alcian blue staining of seminal plasma proteins SDS-PAGE showed a band of high molecular weight containing mucopolysaccharides, only present in nonbulbourethral glands-removed males. Bulbourethral glands would secrete at least eight different proteins that most likely participate in the process of sperm storage in the oviduct.

182 EFFECT OF OVULATION-INDUCING FACTOR ON DEVELOPMENT AND VASCULAR PERFUSION OF THE OVULATORY FOLLICLE AND CORPUS LUTEUM IN LLAMAS

The aim of the study was to determine if purified ovulation-inducing factor (OIF) from llama seminal plasma evokes changes in tissue vascular area of the preovulatory follicle and CL. Mature non-lactating, non-pregnant, female llamas (n = 20) were monitored by transrectal ultrasonography using a 7.5-MHz linear-array transducer (MyLab 5, Canadian Veterinary Imaging) to determine ovarian follicular status. Llamas with a growing follicle (for 3 consecutive days) ≥8 mm were assigned randomly to 2 groups (n = 10/group) and given an i.m. dose of (1) 50 µg of gonadorelin acetate or (2) 1 mg of purified OIF from llama seminal plasma. Llamas were examined daily by transrectal ultrasonography using B-mode and power Doppler mode from Day 0 (day of treatment) to Day 16 to detect ovulation and CL development, and to calculate the area of blood-flow signal in the preovulatory follicle at 12 h after treatment and in the CL on Days 2, 4, 6, 8, 10, 12, 14, and 16. Power Doppler images were recorded, edited, and analysed using the ImageJ software (National Institutes of Health, Bethesda, MD, USA). Data were compared between groups by Student t-test, chi-square, and ANOVA for repeated measures. No differences were detected between GnRH and OIF groups, respectively, in the diameter of the preovulatory follicle at the time of treatment (9.7 ± 0.3 v. 9.9 ± 0.4 mm), ovulation rate (10/10 v. 10/10), interval from treatment to ovulation (32.0 ± 0.6 v. 30.4 ± 1.8 h), maximum CL diameter (13.05 ± 0.4 v. 13.5 ± 0.3 mm), or the day on which the CL reached the maximum diameter (8.7 ± 0.3 v. 8.2 ± 0.2). The preovulatory follicle from llamas treated with purified OIF had a greater (P ≤ 0.0001) blood-flow signal area after treatment than that of the GnRH group. Similarly, the luteal tissue of llamas treated with purified OIF had a greater (P ≤ 0.001) blood-flow signal area than that of the GnRH group on Days 4, 6, 12, and 16 after treatment. We conclude that OIF purified from llama seminal plasma increases the vascular perfusion of the preovulatory follicle and the subsequent CL, consistent with the hypothesis that OIF is luteotrophic. Research funded by Chilean National Science and Technology Research Council (Fondecyt Regular 1120518), the Natural Sciences and Engineering Research Council of Canada, and the Alpaca Research Foundation.

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 β nerve growth factor (β-NGF). X-ray diffraction data were used to solve the full sequence and structure of OIF as β-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 β-NGF from mouse submandibular glands induced ovulation in llamas. We conclude that OIF in seminal plasma is β-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.

Ovulation-inducing factor (OIF) induces LH secretion from pituitary cells

A substance in the seminal plasma of llamas and alpacas has been discovered that induces ovulation and growth of the corpus luteum (CL) in the female of the same species. The ovarian effects of the ovulation-inducing factor (OIF) are associated with a surge release of LH into circulation. We hypothesize that OIF stimulates LH release from gonadotroph cells in the anterior pituitary gland. Four experiments were done to determine if purified OIF isolated from llama seminal plasma stimulates LH secretion in pituitary cells using tissue from an induced ovulator (llama) and spontaneous ovulator (cattle). Anterior pituitary cells were cultured in vitro for two days, and on the third day, wells were incubated for 2h with media containing no treatment (control), GnRH or OIF. Concentrations of LH in the culture medium were measured using radioimmunoassay and compared among groups by analysis of variance. In all experiments, GnRH and OIF treatments induced more LH secretion than untreated controls (P<0.05). A dose-related effect was evident in the llama pituitary cell cultures in that mean LH concentrations were greater (P<0.05) in wells treated with a higher dose of OIF (5.41±0.28ng/mL) compared to wells treated with a lower dose (2.70±0.50ng/mL), both of which were higher (P<0.05) than in wells with no treatment (0.87±0.18ng/mL). Although OIF stimulated LH release in bovine cell cultures, a dose-related effect was not detected. We conclude that OIF stimulates LH secretion from pituitary gonadotrophs in vitro.

EFECTO DE FRACCIONES DEL PLASMA SEMINAL, SEGÚN SU PESO MOLECULAR, SOBRE LA INDUCCIÓN DE LA OVULACIÓN EN LLAMAS (Lama glama).

The present study was conducted to evaluate the effect of different fractions of llama seminal plasma, based on the molecular weight (MW), on the ovulation. Fifty four female adult llamas without calf at foot and in good reproductive conditions were used. The animals were bearing a &gt;7 mm dominant follicle determined by ultrasound diagnosis during a three consecutive days. Females were randomly distributed in six groups: A, fraction of seminal plasma with MW &gt;30 kDa; B, fraction of seminal plasma with MW &gt;10 and &lt;30 kDa; C, fraction of seminal plasma with MW &gt;5 and &lt;10 kDa ; D, fraction of seminal plasma with MW &lt;5 kDa; E, complete plasma seminal; and F, PBS. Animals were treated with a 1.5 ml solution of the corresponding fraction or placebo via i.m. Ovarian ultrasound was performed at day 2 and 9 post-treatment to determine the ovulation rate and the size of the corpora lutea. Ovulation rate was 100% in groups A and E, 11.1% in group B and 0% in the other groups. No statistical differences were observed between groups on the size of the corpus luteum. The results indicated that the plasma seminal fraction with molecular weight &gt;30 kDa induce ovulation in llamas.

128 BOVINE SEMINAL PLASMA INDUCES OVULATION IN LLAMAS

Ovulation-inducing factor (OIF) is a protein present in the seminal plasma of several species, including llamas, alpacas, pigs, cattle, sheep, horses and rabbits. In an initial study (Ratto et al. 2006 Theriogenology 66, 1102–1106), bovine seminal plasma induced ovulations in 26% (5/19) of llamas compared with 0% (0/19) in the placebo group, but induced proportionately less than in llamas treated with alpaca or llama seminal plasma (100%). It is important to highlight that treatments were based on volume of seminal plasma; the actual dose of OIF was unknown. In a later study (Tanco et al. 2011 Biol. Reprod. doi:10.1095/biolreprod.111.091876), OIF from llama seminal plasma had a dose-dependent effect on ovulation rate, corpus luteum (CL) diameter and progesterone production in llamas. The present study was designed to test the hypothesis that bovine seminal plasma induces ovulation and CL development in llamas comparable with that of llama seminal plasma, based on total dose of OIF. Within species, seminal plasma was pooled from 1 to 4 ejaculates per male (n = 145 bulls, n = 4 llamas). The concentration of OIF in the pooled seminal plasma was measured by radioimmunoassay and the volume of seminal plasma used for treatment was adjusted to reach a total dose of 250 μg of OIF. Mature female llamas were assigned randomly to 4 groups and given a single intramuscular dose of 10 mL of PBS (negative control, n = 5), 50 μg of gonadotropin-releasing hormone (GnRH; positive control, n = 5), 6 mL of llama seminal plasma (n = 6), or 12 mL of bull seminal plasma (n = 6). Ovulation and CL development were monitored by transrectal ultrasonography. The incidence of ovulation was compared among groups by Fisher's exact test. Nonserial data (i.e. follicle size at treatment, maximum CL diameter, day of maximum CL diameter and first day of CL detection) were compared among groups by ANOVA. The diameter of the preovulatory follicle at treatment did not differ among groups (P = 0.10). The incidence of ovulation was 0/5, 4/5, 3/6 and 4/6 in the groups treated with PBS, GnRH, llama seminal plasma and bovine seminal plasma, respectively (P &lt; 0.05). The incidence of ovulation did not differ among llamas treated with GnRH, llama seminal plasma, or bovine seminal plasma. Among the treatments that elicited ovulation, neither the maximum CL diameter nor the day of maximum CL diameter differed (P = 0.30 and P = 0.24, respectively). In addition, no difference was detected in the day of first detection of the CL (P = 0.25). Results document the bioactivity of OIF in the bovine seminal plasma of Bos taurus. These findings further support the notion that OIF is highly conserved among mammals and that seminal plasma exerts its effect in an OIF dose-related manner. This research was supported by the Natural Sciences and Engineering Research Council of Canada.

Is an ovulation-inducing factor (OIF) present in the seminal plasma of rabbits?

The objectives of this study were (1) to determine the effect of rabbit seminal plasma on LH secretion and ovulation using the llama animal model as an in vivo ovulation bioassay and (2) to determine the effect of llama or rabbit seminal plasma on ovulation induction in the rabbit model. In Experiment 1, llamas with a growing follicle ≥8 mm in diameter were assigned randomly to one of three groups ( n = 5 per group) and given an intramuscular dose of 1 mL of: (a) llama seminal plasma, (b) rabbit seminal plasma, or (c) phosphate buffered saline (PBS; negative control). Blood samples for LH measurement were taken every 15 min from 1.5 h before to 8 h after treatment (Day 0: starting of treatment). Llamas were examined by ultrasonography every 12 h from treatment to ovulation, and then every other day until Day 16 after treatment to evaluate corpus luteum (CL) development. Blood samples for progesterone measurement were taken every other day from Day 0 to Day 16. Ovulation was detected in 4 of 5, 5 of 5, and 0 of 0 llamas treated with llama or rabbit seminal plasma and PBS, respectively ( P < 0.001). After treatment, plasma LH concentration increased and decreased ( P < 0.01) in the llama and rabbit seminal plasma group but not in the PBS-treated group. No differences were observed on CL development ( P ≥ 0.3) and progesterone secretion ( P > 0.05) between both seminal plasma treated groups. In Experiment 2, receptive female rabbits ( n = 5–7 per group) were given an intramuscular dose of: (a) 0.5, (b) 1.0 and (c) 2.0 mL of either rabbit or llama seminal plasma, (d) 0.5 mL PBS (negative control), or (e) 25 μg of gonadoreline acetate (GnRH; positive control). Does were submitted to laparotomy 24–36 h after treatment to determine the ovulatory response and the presence of antral and hemorrhagic anovulatory follicles. Ovulation sites (7.0 ± 0.6) were only detected in GnRH-treated does ( P < 0.01). There was an increase ( P < 0.01), in the total number of follicles (antral plus hemorraghic follicles) in those females treated with 1 mL of rabbit seminal plasma and there was a tendency ( P = 0.08) for more hemorrhagic anovulatory follicles in does treated with 1.0 and 2.0 mL of either rabbit or llama seminal plasma. Results document the presence of OIF in the seminal plasma of rabbits. The differential ovulatory response between species, however, requires further investigation.