Two-dimensional polyacrylamide gel electrophoresis of equine seminal plasma proteins and their correlation with fertility

摘要

The objectives of this study were to identify proteins found in stallion seminal plasma, using 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) in conjunction with Western blotting, and to determine if any of these proteins were correlatedwith stallion fertility, using regression analysis. Fertility was quantified by assigning a breeding score to each stallion, calculated by dividing the number of conceptions by the number of breedings for 4 successive breeding seasons (1992-1995). Ejaculates from 6 stallions of known fertility were collected with a Missouri-style artificial vagina. Immediately after collection, the semen sample was filtered and the gel fraction removed. The resultant spermatozoa-rich fraction was centrifuged at 10 000X g and the seminal plasma aspirated from the pelleted sperm cells. Two-dimensional PAGE of the seminal plasma was performed under denaturing conditions; 14 proteins were common to all stallions in the research population. Four of these proteins (SP-1, SP-2, SP-3, and SP-4) were found to be significantly (P<0.05) correlated with the breeding score assigned to each stallion. Regression analyses of protein optical densities with breeding score were performed; SP-1 (72 kDa, isoelectric point (pI) =5.6) waspositively correlated with fertility (P<0.05, r2 = 0.706); SP-2 (75 kDa, pI = 6.0), SP-3 (18 kDa, pI = 4.3) and SP-4 (16 kDa, pI = 6.5) were negatively correlated (P<0.05, r2 = 0.762, 0.730 and 0.775 respectively) with fertility. Western blotting of SP-1 was performed; the protein had antigenic homology with a cattle 55 kDa fertility-associated seminal plasma protein identified in a previous study [see Killian, J. G.; Chapman, D. A.; Rogowski L. A., Biology of Reproduction (1993) 49, 1202-1207]. It issuggested that the 2 proteins have a similar physiological role and common biological properties.