Review: Unveiling the effect of beta-nerve growth factor on the reproductive function in llamas and cows.

The actions of the beta-nerve growth factor (ß-NGF) on the neuroendocrine and reproductive system have challenged classical views on the control of reproductive function. After endometrial absorption, ß-NGF triggers ovulation and promotes the development of functional corpora lutea in camelids. In this article, we review evidence showing that, in camelids, ß-NGF exerts its actions by acting in both the hypothalamus and the ovary. In the hypothalamus, ß-NGF may induce gonadotropin-releasing hormone (GnRH) release by interacting with neurons or glial cells expressing receptors for ß-NGF. The LH surge occurs under the influence of ovarian estradiol and requires the release of GnRH into the portal vessels to reach the pituitary gland. In the ovary, ß-NGF may be promoting the differentiation of follicular to luteal cells by modifying the steroidogenic profile of ovarian follicular cells in both camelids and ruminants. Although the mechanisms for these actions are largely undetermined, we aim to offer an update on the current understanding of the effects of ß-NGF controlling reproductive function in camelids and ruminants.

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.

Is seminal nerve growth factor-induced luteinizing hormone release in camelids mediated at the hypothalamus?

In brief: Seminal nerve growth factor induces ovulation in camelids by influencing the secretion of gonadotrophin-releasing hormone (GnRH) into the portal vessels of the pituitary gland. We show that the nerve growth factor-induced release of GnRH is not mediated directly through interaction with hypothalamic neurons. Abstract: Ovulation in camelids is triggered by seminal nerve growth factor (NGF). The mechanism of action of NGF appears to occur via the central nervous system. In this study, we tested the hypothesis that NGF acts in the hypothalamus to induce GnRH release. To determine if NGF-induced ovulation is associated with a rise in NGF concentrations in the cerebrospinal fluid (CSF), llamas were i) mated with an urethrostomized male, ii) mated with intact male, or given intrauterine iii) seminal plasma or i.v.) saline (Experiment 1). To characterize the luteinizing hormone (LH) response after central vs peripheral administration, llamas were treated with saline (negative control) or NGF either by i.v. or intracerebroventricular (ICV) administration (Experiment 2). To determine the role of kisspeptin, the effect of ICV infusion of a kisspeptin receptor antagonist on NGF-induced LH secretion and ovulation was tested in llamas (Experiment 3). In Experiment 1, a surge in circulating concentrations of LH was detected only in llamas mated with an intact male and those given intrauterine seminal plasma, but no changes in CSF concentrations of NGF were detected. In Experiment 2, peripheral administration (i.v.) of NGF induced an LH surge and ovulation, whereas no response was detected after central (ICV) administration. In Experiment 3, the kisspeptin receptor antagonist had no effect on the LH response to NGF. In conclusion, results did not support the hypothesis that NGF-induced ovulation is mediated via a trans-synaptic pathway within the hypothalamus, but rather through a releasing effect on tanycytes at the median eminence.

Oocyte Quality, In Vitro Fertilization and Embryo Development of Alpaca Oocytes Collected by Ultrasound-Guided Follicular Aspiration or from Slaughterhouse Ovaries.

The morphological quality and the in vitro developmental competence of cumulus-oocyte complexes (COCs) collected from in vivo or slaughtered alpacas was compared. COCs were recovered from ovarian follicles using: (i) manual aspiration in ovaries of alpacas (n = 15) sacrificed at a local slaughterhouse, or (ii) transrectal ultrasound-guided follicular aspiration (or ovum-pick-up, OPU) in live alpacas (n = 13) 4 days after the administration of an ovarian superstimulation protocol (200 UI eCG). COCs recovered from both groups were morphologically evaluated and graded. Grade I to III COCs were in vitro matured for 26 h and in vitro fertilized afterwards for 20 h using fresh alpaca epididymal spermatozoa. Presumptive zygotes from both groups were in vitro cultured for 7 days. The proportion of COCs recovered over the total number of follicles punctured was similar between groups, but the mean number of COCs collected from individual ovaries was greater (p < 0.05) in slaughterhouse ovaries. A significantly higher (p < 0.05) percentage of low-quality COCs (grades III and IV) and a lower (p < 0.05) percentage of grade I COCs was obtained using OPU. The number of blastocysts, regarding cleavage and COCs collected, was higher (p < 0.007 and p < 0.0002 respectively) for COCs collected by OPU; however, the total number of blastocysts per female did not differ between groups. We can conclude that the recovery rate and morphological quality of COCs was significantly higher when follicles were manually aspirated from slaughterhouse alpaca ovaries; however, a statistically higher developmental potential was observed in oocytes collected by OPU from live alpaca donors.

Neuroanatomical basis of the nerve growth factor ovulation-induction pathway in llamas†.

The objective of the study was to characterize the anatomical framework and sites of action of the nerve growth factor (NGF)-mediated ovulation-inducing system of llamas. The expression patterns of NGF and its receptors in the hypothalamus of llamas (n = 5) were examined using single and double immunohistochemistry/immunofluorescence. We also compare the expression pattern of the P75 receptor in the hypothalamus of llama and a spontaneous ovulator species (sheep, n = 5). Both NGF receptors (TrkA and P75) were highly expressed in the medial septum and diagonal band of Broca, and populations of TrkA cells were observed in the periventricular and dorsal hypothalamus. Unexpectedly, we found NGF immunoreactive cell bodies with widespread distribution in the hypothalamus but not in areas endowed with NGF receptors. The organum vasculosum of the lamina terminalis (OVLT) and the median eminence displayed immunoreactivity for P75. Double immunofluorescence using vimentin, a marker of tanycytes, confirmed that tanycytes were immunoreactive to P75 in the median eminence and in the OVLT. Additionally, tanycytes were in close association with GnRH and kisspeptin in the arcuate nucleus and median eminence of llamas. The choroid plexus of llamas contained TrkA and NGF immunoreactivity but no P75 immunoreactivity. Results of the present study demonstrate sites of action of NGF in the llama hypothalamus, providing support for the hypothesis of a central effect of NGF in the ovulation-inducing mechanism in llamas.

Laterality of Ovulation and Presence of the Embryo Do Not Affect Uterine Horn Blood Flow During the First Month of Gestation in Llamas.

We determined if laterality of ovulation and intrauterine embryo location differentially induces changes in the mesometrial/endometrial vascularization area (MEVA) between uterine horns, during and after embryo migration, elongation and implantation in llamas. Adult, non-pregnant and non-lactating llamas (n = 30) were subjected to daily B-mode ultrasound scanning of their ovaries. Llamas with a growing follicle ≥8 mm in diameter in the left (n = 15) or right (n = 15) ovary were assigned to a single mating with an adult fertile or vasectomized male. Power-doppler ultrasonography was used to determine the MEVA in a cross section of the middle segment of both uterine horns. MEVA was determined by off-line measurements using the ImageJ software. MEVA measurements were performed before mating (day 0) and on days 5, 10, 15, 20, 25, and 30 after mating in pregnant [llamas with left- (n = 6) or right-sided (n = 6) ovulations] and non-pregnant [llamas with left- (n = 6) or right-sided (n = 6) ovulations] females. Ovulation was confirmed by the disappearance of a follicle (≥8 mm) detected previously. Pregnancy was confirmed by the presence of the embryo proper. MEVA was analyzed by one-way ANOVA for repeated measures using the MIXED Procedure in SAS. If significant (P ≤ 0.05) main effects or interactions were detected, Tukey's post-hoc test for multiple comparisons was used. Ovulation rate did not differ (P = 0.4) between females mated to an intact or vasectomized male and between right- or left-sided ovulations. Three females mated to the intact and 3 to the vasectomized male did not ovulate and were excluded of the study. First observation of fluid inside the gestational sac and of embryo proper, were made exclusively in the left uterine horn, on day 15.8 ± 3.8 and 22 ± 2.7, and 16.7± 2.6 and 27.5 ± 2.8 for pregnant llamas ovulating in the right and left ovary, respectively. Although the MEVA of both uterine horns was affected by time (P < 0.05), it was not affected by physiological status (pregnant vs. non-pregnant; P = 0.9) or laterality of ovulation (P = 0.4). Contrary to expectations, regardless of the laterality of ovulation, in pregnant llamas the left horn did not display a greater MEVA before or after embryo arrival, a trend that was observed during the first 30 days of gestation.

Effect of mating on mRNA and protein expression of beta nerve growth factor and its receptor, TrKA, in the oviduct of llama (Lama glama).

Copulation produces different stimuli in the female reproductive tract in camelids, which lead to ovulation. Expression of ß-nerve growth factor (ß-NGF) and its specific receptor, tropomyosin receptor kinase A (TrKA), was studied comparing the oviductal microenvironment of mated and nonmated llamas. ß-NGF and TrKA were expressed in the llama ampulla, isthmus, and utero-tubal-junction (UTJ), and they were mainly colocalized in the apical region of the oviductal mucosa. A TrKA immunosignal was also found in muscle cells and blood vessels, with the highest mark in UTJ muscle cells of copulated females. Both ß-NGF and TrKA transcripts were expressed in the three oviductal segments. Relative TrKA abundance did not differ between mated and nonmated females, but relative ß-NGF abundance was higher in the UTJ of copulated females (p < .05). ß-NGF might not be secreted into the oviductal fluid (OF) since the protein was not found in the OF of mated or nonmated females. Therefore, it can be concluded that the llama oviduct expresses the ß-NGF/TrKA system and that an increase in ß-NGF gene expression in the UTJ 24 h after copulation along with an increase in TrKA protein expression may indicate an important role in the gamete transport and fertilization process in llamas.

Ovulation mechanism in South American Camelids: The active role of ß-NGF as the chemical signal eliciting ovulation in llamas and alpacas.

The ovulation-inducing effect of seminal plasma was first suggested in Bactrian camels over 30 years ago, initiating a long search to identify the 'ovulation-inducing factor' (OIF) present in camelids semen. During the last decade, primarily in llamas and alpacas, this molecule has been intensively studied characterizing its biological and chemical properties and ultimately identifying it as ß-Nerve Growth Factor (ß-NGF). The high concentration of OIF/ß-NGF in seminal plasma of llamas and alpacas, and the striking effects of seminal fluid on ovarian function strongly support the notion of an endocrine mode of action. Also, have challenged the dogma of mating induced ovulation in camelid species, questioning the classical definition of reflex ovulators, which at the light of new evidence should be revised and updated. On the other hand, the presence of OIF/ß-NGF and its ovulatory effect in camelids confirm the notion that seminal plasma is not only a transport and survival medium for sperm but also, a signaling agent targeting female tissues after insemination, generating relevant physiological and reproductive consequences. The presence of this molecule, conserved among induced as well as spontaneous ovulating species, clearly suggests that the potential impacts of this reproductive feature extend beyond the camelid species and may have broad implications in mammalian fertility. The aim of the present review is to provide a brief summary of all research efforts undertaken to isolate and identify the ovulation inducing factor present in the seminal plasma of camelids. Also to give an update of the current understanding of the mechanism of action of seminal ß-NGF, at central and ovarian level; finally suggesting possible brain targets for this molecule.

New Insights Into the Role of ß-NGF/TrKA System in the Endometrium of Alpacas During Early Pregnancy.

One striking reproductive feature in South American camelids is that more than 90% of gestations are established in the left uterine horn (LUH). This phenomenon could be related to a differential vascular irrigation of the LUH. An increase of vascularization in llama endometrium was observed after systemic administration of Beta Nerve Growth Factor (ß-NGF), a neurotrophin present in the uterus and placenta of various mammals that is involved in pregnancy development. We hypothesized that the ß-NGF signaling pathway is related to embryo implantation in the LUH in camelids. The aim of this study was to characterize the spatial expression of ß-NGF and its high-affinity receptor, TrKA, between LUH and right uterine horn (RUH) of non-pregnant (NP) and early pregnant alpacas (15 and 30 days of gestation, 15 and 30P, respectively). In addition, ß-NGF, TrKA, and Vascular Endothelium Growth Factor A (VEGFA) temporal gene expression patterns and counting of blood vessels were evaluated among groups. The ß-NGF and TrKA were localized in the luminal, glandular, and vascular epithelium of the alpaca uterus and in the embryonic membranes of the 30-days-old conceptus. ß-NGF and TrKA immunosignal were stronger in 15P females than that of NP and 30P. In addition, TrKA signal was higher in the LUH luminal epithelium of NP and 15P alpacas than that of NP-RUH and 15P-RUH. ß-NGF mRNA relative abundance was higher in the 30P-RUH than that of NP-RUH; whereas TrKA mRNA abundance only differed between 15P-RUH and NP-LUH. VEGFA mRNA relative abundance was higher in NP females compared to the LUH of 15P and 30P alpacas, and lower to their right counterparts. The number of vessels per field was higher in 15P than that of 30P. A positive correlation was observed between the number of vessels per field and ß-NGF immunosignal in 15P-LUH. In contrast, the area occupied by vessels was higher in 30P alpacas than of NP and 15P females. The changes of ß-NGF/TrKA expression pattern in the peri-implantation endometria between LUH and RUH and their localization in the extraembryonic membranes support the implication of the neurotrophin during implantation and pregnancy development in South American Camelids.

Effects of NGF Addition on Llama (Lama glama) Sperm Traits After Cooling.

To provide new insights into the mechanisms through which seminal plasma proteins can protect sperm from damage caused during refrigeration, we evaluate the possibility that ß-NGF can contribute to the improvement of sperm quality after cooling. First, ß-NGF was detected in refrigerated sperm and compared with unrefrigerated sperm by western blotting of the proteins adsorbed by sperm, showing that native ß-NGF is still present even 24 h after cooling only as an active form. Then, the effect of exogenous ß-NGF on sperm quality after cooling was evaluated. A total of 12 ejaculates from male llamas (three ejaculates per male), were obtained by electro-ejaculation, diluted 4:1 with buffer Hepes-balanced salt solution and centrifuged at 800 × g for 8 min to remove the seminal plasma. Sperm were suspended in Tris-citrate-fructose-egg yolk diluent for a final concentration of 30 ×106/ml and cooled at 5°C for 24 h. After refrigeration, the extended sperm were equilibrated for 5 min at 37°C and divided into the following subgroups: sperm samples without treatment (control) and sperm samples supplemented with exogenous human ß-NGF (10, 100, and 500 ng/ml). At 5, 30, and 60 min of incubation sperm were evaluated for sperm viability (using eosin/nigrosin stain), sperm motility and vigor (observed under light microscopy), and mitochondrial activity (using the JC-1 fluorescent marker). Vigor data were analyzed with the nonparametric Kruskal-Wallis test. The rest of the variables were analyzed with a mixed models approach. Mean comparisons were performed using Fisher's LSD test with a confidence level of 95%. A principal components analysis was performed to analyze the relationships between variables. Treatment of 24 h cooled sperm with 10 or 100 ng/ml of human ß-NGF increased the percentage of total motility and vigor (p < 0.05). Besides, an incubation time of 60 min would be adequate to improve sperm quality, since all variables are positively related. The significant improvement observed in the motility and vigor of post-refrigerated sperm suggests that supplementation with exogenous ß-NGF may be profitable for the improvement of cooled llama sperm.

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.

New insights of the role of ß-NGF in the ovulation mechanism of induced ovulating species.

The type of stimuli triggering GnRH secretion has been used to classify mammalian species into two categories: spontaneous or induced ovulators. In the former, ovarian steroids produced by a mature follicle elicit the release of GnRH from the hypothalamus, but in the latter, GnRH secretion requires coital stimulation. However, the mechanism responsible for eliciting the preovulatory LH surge in induced ovulators is still not well understood and seems to vary among species. The main goal of this review is to offer new information regarding the mechanism that regulates coitus-induced ovulation. Analysis of several studies documenting the discovery of ß-NGF in seminal plasma and its role in the control of ovulation in the llama and rabbit will be described. We also propose a working hypothesis regarding the sites of action of ß-NGF in the llama hypothalamus. Finally, we described the presence of ß-NGF in the semen of species categorized as spontaneous ovulators, mainly cattle, and its potential role in ovarian function. The discovery of this seminal molecule and its ovulatory effect in induced ovulators challenges previous concepts about the neuroendocrinology of reflex ovulation and has provided a new opportunity to examine the mechanism(s) involved in the cascade of events leading to ovulation. The presence of the factor in the semen of induced as well as spontaneous ovulators highlights the importance of understanding its signaling pathways and mechanism of action and may have broad implications in mammalian fertility.

Evaluation of the effect of mating, intrauterine deposition of raw seminal plasma or seminal plasma purified ß-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 ß-NGF purified from llama semen diluted in 4 ml of PBS (spß-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, spß-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 spß-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 spß-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.

Source and localization of ovulation-inducing factor/nerve growth factor in male reproductive tissues among mammalian species.

The objectives of the study were to compare the presence and localization of ovulation-inducing factor (OIF)/nerve growth factor (NGF) in male reproductive organs and determine the abundance in ejaculates of species representative of both spontaneous and induced ovulators. We hypothesized that the protein is a widely conserved component of semen among mammals, but is most abundant in camelids. Immunohistochemical analysis was performed on tissues from the male reproductive system of llamas, rats, cattle, bison, elk, and white-tailed deer (n = 2 males/species), and the abundance of OIF/NGF in the seminal plasma of camelids (llamas and alpacas), cattle, horses, and pigs (n = 69, 53, 24, and 16 ejaculates, respectively) were quantified by radioimmunoassay. Based on immunoreactivity in both the glandular epithelium and glandular lumen, the prostate gland was the main source of seminal OIF/NGF in llamas, the vesicular gland and ampullae in bovids (cattle and bison), and the ampullae and prostate in cervids (elk and white-tailed deer). Camelid and bovine seminal plasma induced dendritic growth in the PC12 differentiation bioassay, but no effect was observed with equine or porcine seminal plasma. The concentration of OIF/NGF was 10 times higher in camelid than bovine seminal plasma (1.2 ± 0.21 vs. 0.10 ± 0.03; P < 0.05); OIF/NGF was not detected in equine or porcine ejaculates by radioimmunoassay. Based on tissue localization, abundance, and bioactivity, we conclude that OIF/NGF is a common protein within the male accessory glands among species, and its abundance in camelids, bovids, and cervids suggests an important role in the mechanisms of ovulation in both induced and spontaneous ovulators.

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 (ß-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 ß-NGF is in low abundance in the seminal plasma of belugas and Pacific white-sided and bottlenose dolphins.

Natural and controlled ovulation in South American camelids.

The four species of New World camelids and 2 species of Old World camelids derived from a common ancestor in North America. The reproductive characteristics, particularly those involving ovarian function and ovulation, are remarkably similar among the 6 living species of camelids, so much so that interspecies hybrids of nearly all possible combinations have been documented. Camelids are induced-ovulators, triggered by an ovulation-inducing factor in seminal plasma. The timing and mechanism of endocrine events leading to ovulation are discussed, as well as the discovery, identification and mode of action of the seminal factor responsible. The applied aspects of our present understanding are discussed with specific reference to controlled induction of ovulation, ovarian synchronization, and superovulation. Emphasis has been given to the literature on llamas and alpacas, with some reference to studies done in wild species of South American camelids and Old World camels.

Effect of oocyte maturation time, sperm selection method and oxygen tension on in vitro embryo development in alpacas.

We evaluated the effect of in vitro maturation time, sperm selection and oxygen tension on alpaca embryo development. In Experiment I, Cumulus Oocyte- Complexes (COCs) were obtained from abattoir ovaries and in vitro matured in TCM-199 for 24 (n = 217), 28 (215), or 32 h (223) at 38.5 °C, high humidity and 5% CO2 in air. Oocytes from 24 (n = 392), 28 (n = 456) or 32 (n = 368) h groups were in vitro fertilized with epididymal sperm and cultured in SOFaa at 38.5 °C, high humidity and 5% CO2, 5% O2 and 90% N2 for 7 days. Embryo development was evaluated on Day 2, 5 and Day 7 of in vitro culture (Day 0 = in vitro fertilization). In Experiment II, a 2 by 2-factorial design was used to determine the effect of sperm selection (Swim-up vs Percoll) and oxygen tension (20% vs 5%) during embryo culture and their interaction on embryo development. COCs were in vitro matured for 32 h at 38.5 °C and 5% CO2 in air and then in vitro inseminated with epididymal sperm processed by swim-up or Percoll. Zygotes were cultured in SOFaa + cumulus cells at 38.5 °C under 20 or 5% of O2 tension and high humidity for 7 days. A total of 235, 235, 253 and 240 oocytes were assigned to: swim-up+20 O2, swim-up+5 O2 or Percoll+20 O2, Percoll+5 O2, groups respectively. The proportion of oocytes reaching MII stage was highest after 32 h of in vitro maturation (P < 0.05). Blastocyst rate (29.1 ± 2.7%) was also highest for COCs matured for 32 h (Exp I). In Experiment II, Blastocysts rate (26.03 ± 4.7; 27.7 ± 4.3; 29.7 ± 3.8 and 27.6 ± 4.2% for swim-up+20 O2, swim-up+5 O2 or Percoll+20 O2, Percoll+5 O2, respectively) was not affected by sperm selection method (P = 0.8), oxygen tension (P = 0.9) or their interaction (P = 0.5).

Seminal Plasma Induces Ovulation in Llamas in the Absence of a Copulatory Stimulus: Role of Nerve Growth Factor as an Ovulation-Inducing Factor.

Llamas are considered to be reflex ovulators. However, semen from these animals is reported to be rich in ovulation-inducing factor(s), one of which has been identified as nerve growth factor (NGF). These findings suggest that ovulation in llamas may be elicited by chemical signals contained in semen instead of being mediated by neural signals. The present study examines this notion. Llamas displaying a preovulatory follicle were assigned to four groups: group 1 received an intrauterine infusion (IUI) of PBS; group 2 received an IUI of seminal plasma; group 3 was mated to a male whose urethra had been surgically diverted (urethrostomized male); and group 4 was mated to an intact male. Ovulation (detected by ultrasonography) occurred only in llamas mated to an intact male or given an IUI of seminal plasma and was preceded by a surge in plasma LH levels initiated within an hour after coitus or IUI. In both ovulatory groups, circulating ß-NGF levels increased within 15 minutes after treatment, reaching values that were greater and more sustained in llamas mated with an intact male. These results demonstrate that llamas can be induced to ovulate by seminal plasma in the absence of copulation and that copulation alone cannot elicit ovulation in the absence of seminal plasma. In addition, our results implicate ß-NGF as an important mediator of seminal plasma-induced ovulation in llamas because ovulation does not occur if ß-NGF levels do not increase in the bloodstream, a change that occurs promptly after copulation with an intact male or IUI of seminal plasma.

Granulocyte-macrophage colony stimulating factor (GM-CSF) enhances cumulus cell expansion in bovine oocytes.

BACKGROUND: The objectives of the study were to characterize the expression of the α- and ß-subunits of granulocyte-macrophage colony stimulating factor (GM-CSF) receptor in bovine cumulus cells and oocytes and to determine the effect of exogenous GM-CSF on cumulus cells expansion, oocyte maturation, IGF-2 transcript expression and subsequent competence for embryonic development. METHODS: Cumulus-oocyte complexes (COC) were obtained by aspirating follicles 3- to 8-mm in diameter with an 18 G needle connected to a vacuum pump at -50 mmHg. Samples of cumulus cells and oocytes were used to detect GM- CSF receptor by immunofluorescence. A dose-response experiment was performed to estimate the effect of GM-CSF on cumulus cell expansion and nuclear/cytoplasmic maturation. Also, the effect of GM-CSF on IGF-2 expression was evaluated in oocytes and cumulus cells after in vitro maturation by Q-PCR. Finally, a batch of COC was randomly assigned to in vitro maturation media consisting of: 1) synthetic oviductal fluid (SOF, n = 212); 2) synthetic oviductal fluid supplemented with 100 ng/ml of GM-CSF (SOF + GM-CSF, n = 224) or 3) tissue culture medium (TCM 199, n = 216) and then subsequently in vitro fertilized and cultured for 9 days. RESULTS: Immunoreactivity for both α and ß GM-CSF receptors was localized in the cytoplasm of both cumulus cells and oocytes. Oocytes in vitro matured either with 10 or 100 ng/ml of GM-CSF presented a higher (P < 0.05) cumulus cells expansion than that of the control group (0 ng/ml of GM-CSF). GM-CSF did not affect the proportion of oocytes in metaphase II, cortical granules dispersion and IGF-2 expression. COC exposed to 100 ng/ml of GM-CSF during maturation did not display significant differences in terms of embryo cleavage rate (50.4% vs. 57.5%), blastocyst development at day 7 (31.9% vs. 28.7%) and at day 9 (17.4% vs. 17.9%) compared to untreated control (SOF alone, P = 0.2). CONCLUSIONS: GM-CSF enhanced cumulus cell expansion of in vitro matured bovine COC. However, GM-CSF did not increase oocyte nuclear or cytoplasmic maturation rates, IGF-2 expression or subsequent embryonic development.