Biochemical isolation and purification of ovulation-inducing factor (OIF) in seminal plasma of llamas

BACKGROUND The objective of the present study was to isolate and purify the protein fraction(s) of llama seminal plasma responsible for the ovulation-inducing effect of the ejaculate. METHODS Semen collected from male llamas by artificial vagina was centrifuged and the seminal plasma was harvested and stored frozen. Seminal plasma was thawed and loaded onto a Type 1 macro-prep ceramic hydroxylapatite column and elution was carried out using a lineal gradient with 350 mM sodium phosphate. Three protein fractions were identified clearly (Fractions A, B, and C), where a prominent protein band with a mass of 14 kDa was identified in Fraction C. Fraction C was loaded into a sephacryl gel filtration column for further purification using fast protein liquid chromatography (FPLC). Isocratic elution resulted in 2 distinct protein fractions (Fractions C1 and C2). An in vivo bioassay (n=10 to 11 llamas per group) was used to determine the ovarian effect of each fraction involving treatment with saline (negative control), whole seminal plasma (positive control), or seminal plasma Fractions A, B or C2. Ultrasonography was done to detect ovulation and CL formation, and blood samples were taken to measure plasma progesterone and LH concentrations. RESULTS Ovulation and CL formation was detected in 0/10, 10/11, 0/10, 2/11, and 10/11 llamas treated with saline, whole seminal plasma, Fractions A, B and C2 respectively (P<0.001). A surge in circulating concentrations of LH was detected within 2 hours only in llamas treated with either whole seminal plasma or Fraction C2. Plasma progesterone concentration and CL diameter profiles were greatest (P<0.05) in llamas treated with Fraction C2. CONCLUSION Ovulation-inducing factor was isolated from llama seminal plasma as a 14 kDa protein molecule that elicits a preovulatory LH surge followed by ovulation and CL formation in llamas, suggesting an endocrine effect at the level of the hypothalamus (release of GnRH) or the pituitary (gonadotrophs).