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Use of salt-stored zonae pellucidae as a highly sensitive assay of immunological infertility
THERIOGENOLOGY
USE OF SALT-STOREDZONAE PELLUCIDAEAS A HIGHLYSENSITIVEASSAYOF IMMUNOLOGICAL INFERTILITY R.A. Fayrer-Hosken, C.E. McBride, and B.G. Brackett Department of Physiology and Pharmacology College of Veterinary Medicine The University of Georgia, Athens, GA., 30602. U.S.A. The rabbit salt-stored zonae (SSZI assay allows precise assessment of sperm attachment and penetration of zonae pellucidae (Fayrer-Hosken and Brackett, 1987, Gam Res 17[31:191-2011. Study of sperm-SSZ interaction has led to a biochemical explanation of rabbit sperm-egg interaction, and heterologous gamete interaction (Fayrer-Hosken et al, Biol Reprod 36(11:53#191; these mechanisms apparently hinge on sperm-specific glycosyltransferases. The objective of this research was to develop a highly sensitive assay to detect antibodies that would cause infertility. Polyclonal antibody serum to rabbit plasma and outer acrosomal membrane complex (POAC) proteins and inner acrosomal membrane (IAM) proteins in Freund’s adjuvant was produced in female rabbits (POAC-R. IAM-RI and female guinea pigs (IAM-GPI. Control serum was produced by treating a female rabbit and guinea pig with Freund’s adjuvant alone. Perturbation of the kinetics of normal sperm binding to and penetration of SSZ by serial dilutions (1 :l to 1:25601 of serum was used to assay the presence of antisperm antibodies. This was compared to a standard Ouchterlony test using the POAC and IAM antigens with the same serial serum dilutions. Spermatozoa1 binding and penetration were examined with Nomarski optics at 400 times magnification. Spermatozoa remaining on the zona surface or in the zona matrix after three aspirations with a fine bore pipette were recorded as bound spermatozoa. Spermatozoa in the same focal plane as the shrunken vitellus or whose motion was restricted by the inner surface of the zona pellucida were classified as being in the perivitelline space. Maximum sensitivity (greatest responsive dilution) of Ouchterlony tests to POAC and IAM antigens gave values of 1: 1 IAM-R and 1:20 IAM-GP. while POAC-R produced no response, compared to control serum (rabbit and guinea pig). Sperm-SSZ interaction assayed with the antibodies showed significant (p
246
JANUARY
1988 VOL. 29 NO. 1
USE OF SALT-STOREDZONAE PELLUCIDAEAS A HIGHLYSENSITIVEASSAYOF IMMUNOLOGICAL INFERTILITY R.A. Fayrer-Hosken, C.E. McBride, and B.G. Brackett Department of Physiology and Pharmacology College of Veterinary Medicine The University of Georgia, Athens, GA., 30602. U.S.A. The rabbit salt-stored zonae (SSZI assay allows precise assessment of sperm attachment and penetration of zonae pellucidae (Fayrer-Hosken and Brackett, 1987, Gam Res 17[31:191-2011. Study of sperm-SSZ interaction has led to a biochemical explanation of rabbit sperm-egg interaction, and heterologous gamete interaction (Fayrer-Hosken et al, Biol Reprod 36(11:53#191; these mechanisms apparently hinge on sperm-specific glycosyltransferases. The objective of this research was to develop a highly sensitive assay to detect antibodies that would cause infertility. Polyclonal antibody serum to rabbit plasma and outer acrosomal membrane complex (POAC) proteins and inner acrosomal membrane (IAM) proteins in Freund’s adjuvant was produced in female rabbits (POAC-R. IAM-RI and female guinea pigs (IAM-GPI. Control serum was produced by treating a female rabbit and guinea pig with Freund’s adjuvant alone. Perturbation of the kinetics of normal sperm binding to and penetration of SSZ by serial dilutions (1 :l to 1:25601 of serum was used to assay the presence of antisperm antibodies. This was compared to a standard Ouchterlony test using the POAC and IAM antigens with the same serial serum dilutions. Spermatozoa1 binding and penetration were examined with Nomarski optics at 400 times magnification. Spermatozoa remaining on the zona surface or in the zona matrix after three aspirations with a fine bore pipette were recorded as bound spermatozoa. Spermatozoa in the same focal plane as the shrunken vitellus or whose motion was restricted by the inner surface of the zona pellucida were classified as being in the perivitelline space. Maximum sensitivity (greatest responsive dilution) of Ouchterlony tests to POAC and IAM antigens gave values of 1: 1 IAM-R and 1:20 IAM-GP. while POAC-R produced no response, compared to control serum (rabbit and guinea pig). Sperm-SSZ interaction assayed with the antibodies showed significant (p
246
JANUARY
1988 VOL. 29 NO. 1