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 β-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 β-NGF as an important mediator of seminal plasma-induced ovulation in llamas because ovulation does not occur if β-NGF levels do not increase in the bloodstream, a change that occurs promptly after copulation with an intact male or IUI of seminal plasma.
BackgroundThe 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.MethodsUsing 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.ResultsOvulation 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.ConclusionCetrorelix (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.
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 β-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 β-NGF in the llama hypothalamus. Finally, we described the presence of β-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.
Background Nerve 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. 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 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. Results Experiment 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 ...
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