107 DETECTION OF BIOTINYLATED-OVULATION-INDUCING FACTOR (OIF) IN CEREBROSPINAL FLUID AND ITS ABILITY TO INDUCE OVULATION

Abstract

Ovulation-inducing factor (OIF) is a protein in the seminal plasma of llamas that induces a preovulatory LH surge by acting directly or indirectly on the hypothalamic GnRH neurons (Silva et al. 2011 Reprod. Biol. Endocr. 9, 74). We hypothesize that OIF crosses the blood–brain barrier and reaches the hypothalamus via secretion into the cerebro-spinal fluid (CSF) through the choroid plexus. Two experiments were designed to determine whether biotinylation of OIF (as a tracer) alters its bioactivity in a llama model (Experiment 1) and whether it crosses the blood–brain barrier in a rabbit model (Experiment 2). In Experiment 1, llamas with a follicle ≥8 mm in diameter that had grown for 3 consecutive days were assigned randomly to 5 groups (n = 2/group) and given an IV dose of 1) 800 µg of OIF, 2) 800 µg of OIF biotinylated at the amino end; 3) 1600 µg of OIF biotinylated at the amino end, 4) 800 µg of OIF biotinylated at the carboxyl end, or 5) phosphate buffered saline (control). The ovaries were examined daily by transrectal ultrasonography on Day 3 and 8 after treatment (Day 0 = treatment) to detect ovulation and corpus luteum formation. In Experiment 2, adult female rabbits were assigned randomly to 3 groups and given an IV dose of (1) 250 µg of OIF, (2) 250 µg of OIF biotinylated at the amino end, or (3) 250 µg of OIF biotinylated at the carboxyl end. A 50-µL sample of CSF was collected from the cisterna magna under general anesthesia before (0 min) and 10, 20, 30, and 45 min after treatment. The presence of biotinylated OIF in CSF samples was determined by dot blot, using streptavidin-peroxidase and diaminobenzidine. In Experiment 1, the diameter of the follicle at the time of the treatment did not differ among groups (9.7 ± 0.2, 9.4 ± 0.0, 10.5 ± 1.0, 10.1 ± 0.2, 10.3 ± 0.4). Ovulation was detected in all llamas except one llama treated with 800 µg OIF biotinylated at the carboxyl end and both llamas given PBS. The diameter of the corpus luteum did not differ among OIF-treated groups. In Experiment 2, OIF biotinylated at both amino and carboxyl ends was detected in CSF samples at 10, 20, 30, and 45 min after IV administration. No signal was recorded before IV administration (0 min) or in samples from rabbits that were given nonbiotinylated OIF. We conclude that the biotinylation process did not affect OIF bioactivity, and OIF crosses the blood–brain barrier and reaches the CSF in rabbits.

Research supported by FONDECYT 1120518, the Natural Sciences and Engineering Research Council of Canada, and the Alpaca Research Foundation.