Characterization of the major polypeptides of equine seminal plasma by two-dimensional polyacrylamide gel electrophoresis

The functions of the various proteins in equine seminal plasma (ESP) are poorly understood, including what, if any, effect these proteins have on fertility. In light of the encouraging reports from the bovine literature, we separated the polypeptides in ESP using 2D-PAGE technology to determine if there is a characteristic profile for the polypeptides and, if so, to present a nomenclature for future comparisons of the identified spots. Ejaculates were obtained from 14 stallions, and protein concentration was standardized to 50 mu g/10 mu l. Isoelectric points (pI) and molecular weights (MW) of ESP polypeptides were determined by measuring spot mobility following 2D-PAGE (15% polyacrylamide) and Coomassie Blue staining. The ''a'' constellation consisted of 2 distinct rows (30 kDa; 28 kDa), ranging from pI 5.9 to pI 6.5, with no ejaculate containing all 8 polypeptides. The ''b'' constellation also consisted of 2 distinct rows (22 kDa; 19 kDa), ranging from pI 4.2 to pI 5.2. Three ''b'' polypeptides (b1, b2, b4) were present in all 14 ejaculates; polypeptide c1 was present in 10 ejaculates (21 kDa; pI 6.0); and polypeptide dl was present in all 14 ejaculates (18.2 kDa; pI 6.9). The less frequently observed polypeptides were e1 (16.3 kDa; pI 7.3), e2 (14.6 kDa; pI 7.1), f1 (14.6 kDa; pI 7.6), and f2 (12.4 kDa; pI 7.8). One polypeptide (''g'' band) had a MW 27.9 kDa and appeared as a distinct band in 7 ejaculates, ranging from pI 7.0 to pI 8.0. In 6 other ejaculates the ''g'' polypeptide appeared as a faintly stained spot at the basic end of the pi range. Polypeptides b4 and d1 were prominent in all 14 ejaculates, and may correspond to the recently identified stallion seminal plasma proteins, HSP-1 and HSP-2. Other prominent polypeptides were f2 and a7. The relative protein composition of polypeptide d1 exceeded that of b4 in 10 ejaculates, and they were equal in another. The 3 ejaculates in which b4 content exceeded that of d1 also had the most widespread distribution of the polypeptides. To our knowledge this is the first report describing the 2D-PAGE profile of the polypeptides in stallion seminal plasma.