Ovulation-inducing Factor In Seminal Plasma Research Articles

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.

188 EFFECT OF RABBIT SEMINAL PLASMA IN OVULATING RESPONSE

The presence of an ovulation-inducing factor (OIF) in the seminal plasma (SP) of several species with spontaneous and induced ovulation, included the rabbit, has been documented. The biochemical identity of OIF in SP remains unknown, but it seems that OIF is a protein (Ratto et al. 2011 Reprod. Biol. Endocrinol. 9, 24). The aim of this study was to determine if the protein present in the rabbit SP could induce ovulation in a dose manner and provoke changes in plasma hormone concentrations [LH and progesterone (P4)]. Semen was collected from 12 male rabbits using an artificial vagina, pooled, centrifugated at 3000g for 30 min twice and analysed by Bradford method to determine protein concentration that was 7 mg protein mL–1 of SP. After storage at –80°C, the SP was lyophilized for use at different concentrations. Twenty-four females were synchronized with an i.m. injection of 25 IU of equine chorionic gonadotropin and randomly assigned to 4 groups (n = 6). Forty-eight hours later (day 0) they were given a single i.m. dose of 1) 1 mL of saline solution (SS; negative control), 2) 20 µg of gonadorelin (GnRH; positive control), 3) 1 mL of lyophilized SP diluted in SS containing 7 mg of protein (SP7), 4) 1 mL of lyophilized SP diluted in SS containing 14 mg of protein (SP14). Blood samples for LH measurement were taken every 30 min from 30 min before injection to 2 h after treatment. Blood samples for P4 measurement were taken every 2 days from Day 0 to Day 6. Hormone determinations were made by enzyme immunoassay. Ovulation rate (OR), number of corpora lutea (CL), follicles higher than 1 mm, and total number of hemorrhagic follicles were determined after euthanasia on Day 7. Statistical analysis was performed by ANOVA. The OR was significantly higher (P < 0.0001) in GnRH than in SS, SP7, and SP14 groups (OR: 100, 0, 0, and 8.3%, respectively). Total number of CL counted in does that ovulated in GnRH and SP14 groups was not different (13.7 ± 0.8 and 9 ± 0.0 CL, respectively; P < 0.0001). No statistical differences were observed between groups on the number of follicles higher than 1 mm (GnRH: 17 ± 2.4; SS: 15 ± 1.6; SP7: 11.7 ± 2.6; SP14: 14.8 ± 0.9) and anovulatory hemorrhagic follicles (GnRH: 2.3 ± 0.9; SS: 0.2 ± 0.2; PS: 1.7 ± 0.8; PS 14: 1.7 ± 1.5). Treatment was followed by a surge in plasma LH concentration beginning 30 min after treatment to 120 min in GnRH group ranging ~75 ng mL–1, whereas in the other groups it remained at basal levels (around 20 ng mL–1; P < 0.0001). Plasma P4 concentrations were significantly increased from Day 2 to 6 (4.7 ± 0.7 to 22.3 ± 3.7 ng mL–1; P < 0.0001) only in rabbits treated with GnRH. Plasma P4 concentrations did not vary throughout the experimental period in all OIF-treated rabbits. The present study failed to demonstrate the effect of 3 different dosages of OIF of the rabbit SP on induction of ovulation. More studies are necessary to elucidate if rabbit SP could induce ovulation in rabbit females. We acknowledge CM and MEC for funding.

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.

Evidence for the conservation of biological activity of ovulation-inducing factor in seminal plasma

An ovulation-inducing factor (OIF) in the seminal plasma of llamas and alpacas (induced ovulators) and cattle (spontaneous ovulators) suggests that OIF is a conserved constituent of seminal plasma among mammals. In this study, three experiments were designed to determine the biological effects of OIF in different species. In experiment 1, superstimulated prepubertal female CD-1 mice (n=36 per group) were given a single 0.1 ml i.p. dose of 1) phosphate-buffered saline (PBS), 2) 5 μg gonadotropin-releasing hormone (GNRH), 3) 5 IU hCG, or 4) llama seminal plasma. The proportion of mice that ovulated was similar among groups treated with GNRH, hCG, or seminal plasma, and all were higher than the saline-treated group (P<0.001). In experiment 2, female llamas (n=8 or 9 per group) were intramuscularly treated with 1) 2 ml PBS, 2) 1 ml diluted llama seminal plasma, 3) 3 ml equine seminal plasma, or 4) 3 ml porcine seminal plasma. Experiment 3 was the same as experiment 2 except that the dose of equine and porcine seminal plasma was increased to 8 and 10 ml respectively. All llamas that were treated with llama seminal plasma ovulated and none that were treated with saline ovulated (P<0.0001). The proportion of llamas that ovulated in response to equine and porcine seminal plasma was intermediate. We conclude that the mechanism for the biological response to OIF is present in prepubertal CD-1 mice and that OIF is present in equine and porcine seminal plasma.

2 EVIDENCE FOR THE PRESENCE OF OVULATION-INDUCING FACTOR IN PORCINE AND EQUINE SEMINAL PLASMA

The presence of an ovulation-inducing factor (OIF) in the seminal plasma of llamas and alpacas (reflex ovulators) and cattle (spontaneous ovulators) has been reported previously (Ratto MH et al. 2006 Theriogenology 66, 1102–1106). The presence of this protein in unrelated species supports the hypothesis that OIF is a conserved factor among species. The objectives of this study were to determine if OIF was present in equine and porcine seminal plasma, and whether the proportion of test animals (llamas) that ovulated in response to treatment with seminal plasma was related to dose. In Experiment 1, female llamas were assigned randomly to four groups (n = 8 or 9 per group) and treated intramuscularly with 1 mL llama seminal plasma (positive control), 3 mL equine seminal plasma, 3 mL porcine seminal plasma, or 2 mL saline (negative control). Ovulation and maximum corpus luteum diameter were compared using ultrasonography and confirmed with blood samples taken on Day 7 (Day 0 = day of treatment) to determine plasma progesterone concentration. The diameter of the preovulatory follicle at the time of treatment did not differ among groups. Equine seminal plasma induced ovulations in 3/8 (38%) llamas compared to 0/8 (0%) llamas treated with saline or porcine seminal plasma (P = 0.1). The proportion of females that ovulated was lower in the equine group (P &lt; 0.01) compared with those animals treated with llama seminal plasma (9/9; 100%). Of the animals that ovulated, maximum CL diameter did not differ between llama and equine seminal plasma-treated groups (mean ± SEM; 11.1 ± 1.1, 11.5 ± 1.5, respectively). Similarly, progesterone concentrations were not different among llamas treated with llama seminal plasma or equine seminal plasma (mean ± SEM; 3.1 ± 0.4, 3.7 ± 1.2, respectively). The design of Experiment 2 was the same, but the dose of equine and porcine seminal plasma was increased to 8 mL and 10 mL, respectively. The proportion of females that ovulated was less (P &lt; 0.05) in equine (2/9) and porcine (3/9) seminal plasma groups compared with the group treated with llama seminal plasma (9/9). There were no ovulations detected in llamas treated with saline (0/8). Although differences between equine, porcine, and negative control groups did not reach significance, results provide some evidence for the presence of OIF in equine and porcine seminal plasma. The effect of dose of equine and porcine seminal plasma is equivocal, suggesting that the concentration of OIF in the seminal plasma of these species may be very low and the optimal dose for inducing ovulation in test animals had not been reached. Research supported by the Natural Sciences and Engineering Council of Canada.

221 PREPUBERTAL MOUSE BIOASSAY FOR OVULATION-INDUCING FACTOR IN SEMINAL PLASMA

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. Ultrasonographic detection of ovulation and CL development is currently the only method available for testing the bioactive effects of OIF. The purpose of this study was to determine if a superstimulatory prepubertal mouse model could be developed as an in vivo bioassay for OIF. Prepubertal female CD1 mice (n = 144), 20 days of age and weighing 20–25 g, were housed at 24�C with lights on from 0500 to 1900 h and free access to food and water. An intramuscular dose of 5 IU of eCG (Novormon, Bioniche Animal Health, Belleville, ON, Canada) was given (Day 0) for ovarian superstimulation. On Day 2, mice were assigned randomly to 4 groups (n = 36 per group) and given a single 0.1 mL intraperitoneal dose of (1) 5 IU of hCG (Chorulon, Intervet Canada, Ltd., Whitby, ON, Canada), (2) 5 µg GnRH (gonadotropin-releasing hormone: Cystorelin, Merial, Ltd., Iselin, NJ, USA), (3) llama seminal plasma, or (4) phosphate-buffered saline (negative control). On Day 3, females were euthanized by an overdose of inhaled halothane. Oviducts were collected and oocytes were counted using trans-illumination stereomicroscopy. The proportion of mice that ovulated did not differ among groups treated with hCG, GnRH, and seminal plasma (31/36, 31/36, 28/36, respectively); however, the proportion of mice that ovulated in each treatment group was greater than that in the saline-treated group (9/36) (P &lt; 0.001). The number of oocytes counted (mean � SEM) was also similar among groups treated with hCG (25.8 � 2.9), GnRH (27.4 � 2.7), and seminal plasma (19.2 � 2.8), all of which were greater (P &lt; 0.01) than in the saline-treated group (6.2 � 2.1). We conclude that the superstimulated prepubertal CD1 mouse model is effective as an in vivo bioassay for OIF in seminal plasma. Whether the bioassay may be used for quantitative estimates of OIF activity will require dose-response trials using serial dilutions of seminal plasma.