Expression and Pattern of SP22 Immunolocalization in Stallion Semen Collected in Two Seasons

 

N. Wrench, C. R. F. Pinto¹, G. R. Klinefelter² C. E. Farin

 

¹Department of Population Health and Pathobiology, College of Veterinary Medicine, North Carolina State University

²US Environmental Protection Agency, National Health and Environmental Effects Research Laboratory, Research Triangle Park, NC

 

Abstract

Sperm membrane protein 22 (SP22) is a protein located over the equatorial region of the head of rat spermatozoa.  Concentrations of SP22 have been correlated with fertility of rat spermatozoa from the cauda epididymis (Klinefelter et al 2002; J Androl 23:48).  Here we report the semen characteristics and pattern of SP22 immunolocalization for stallions of known fertility during the summer and fall seasons.  Semen was collected from 6 fertile stallions during the summer (June) and fall (September) of 2005.  Samples were assessed for motility, concentration, morphology, viability, membrane and acrosome integrity.  For SP22 immunocytochemistry, samples were stained using a sheep anti-rat recombinant SP22 primary antibody and FITC-conjugated secondary antibody.  At least 200 stained spermatozoa were counted using a fluorescent microscope which were categorized into one of two patterns: Pattern 1, overlying the equatorial region (ER) only; Pattern 2, overlying the acrosomal and equatorial region (AER), neck (N) and tail (T).  Data were analyzed using ANOVA.  The majority of semen parameters did not differ between summer and fall collections: morphology (86±3%, 81±3%), primary abnormalities (12±2%, 15±2%), secondary abnormalities (2±1%, 4±1%), progressive motility (66±2%, 70±2%), acrosome integrity (96±1%, 95±1%), viability (83±3%, 80±3%) and total spermatozoa (x10⁹; 19±3, 12±3) for summer and fall collections, respectively.  However, the proportion of spermatozoa having intact plasma membranes was significantly (p<0.05) greater for spermatozoa collected in the summer (79±3%) compared to the fall (69±3%).  The percentage of spermatozoa staining for SP22 tended to differ (p = 0.07) between summer (35.2 ± 4.6%) and fall (50.7 ± 4.9%).  In both summer and fall, the proportion of spermatozoa demonstrating Pattern 1 SP22 staining (ER only; 85.3 ± 3.2% and 82.6 ± 2.9%, respectively) was significantly (p<0.05) greater than those demonstrating Pattern 2 staining (AER, N and T; 14.7 ± 3.2 % and 17.5 ± 2.9%, respectively).  Thus, regardless of season the predominant localization of SP22 was equatorial, comparable to the localization found for the rat.   There was a tendency (p = 0.07) for total SP22 staining of spermatozoa to be greater in fall compared to summer.  Spermatozoa collected in fall represent cells that developed during the peak breeding season whereas those collected in summer were formed during the spring transition into the breeding season.  Therefore, spermatozoa collected in fall may represent cells produced during periods of peak testis function as reflected by an increased accumulation of SP22 in their membranes.  Because SP22 is known to be associated with fertility in other species, it is possible that this protein could serve as a marker of fertility for stallion spermatozoa.