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

Abstract

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).

Figure 1

Protein fractions of llama seminal plasma. Fractions A, B and C were eluted on hydroxylapatite gravity chromatography columns using a lineal gradient of 10 to 400 mM sodium phosphate (left). Fraction C contained a major 14 kDa protein observed after denaturing on 12% SDS-PAGE (right).

Figure 2

Separation of protein Fraction C of llama seminal plasma. Separation was done using sephacryl gel filtration fast protein liquid chromatography (FPLC) and isocratic elution with phosphate buffered saline (left). Fraction C was isolated previously by hydroxylapatite gravity column chromatography. Vertical lines along the x-axis represent fractions collected and examined. The protein band at about 14 kDa on denaturing 12% SDS-PAGE (right) was the major constituent of Fraction C₂ (right).

Figure 3

Effect of protein fractions of llama seminal plasma on CL diameter and plasma progesterone concentrations in llamas. Female llamas were given whole seminal plasma (SP, positive control), Fractions A or B (isolated by hydroxylapatite column chromatography), Fraction C₂ (isolated by gel filtration chromatography), or phosphate buffered saline (PBS, negative control). ^abcd Within days, values with no common superscript are different (P < 0.05).

Figure 4

Effect of protein fractions of llama seminal plasma on circulating LH concentration in llamas. Female llamas were given whole seminal plasma (SP, positive control), Fractions A or B (isolated by hydroxylapatite column chromatography), Fraction C₂ (isolated by gel filtration chromatography), or phosphate buffered saline (PBS, negative control). *Interval during which values in SP and C₂ were higher (P < 0.05) than in other groups.