Evidence that P36, a human sperm acrosomal antigen involved in the fertilization process is triosephosphate isomerase

Abstract

P36 is one of the immunodominant sperm antigens identified by antibodies eluted from the spermatozoa of infertile men. In a previous study, we isolated and characterized this auto-antigen as a glycoprotein with several isoforms. Specific rabbit antibodies were produced to investigate sperm topography and the role of P36 in the fertilization process and we showed that P36 is present on the equatorial segment of acrosome-reacted spermatozoa and is involved in sperm-binding and the penetration of zona-free hamster oocytes. In the present study, we demonstrated, by means of immunofluorescence and electron microscopy, that P36 is present all over the acrosomal membranes of non-reacted spermatozoa. We also investigated the role of P36 in the acrosome reaction and sperm binding to the zona pellucida (ZP). The exposure of capacitated spermatozoa to rabbit anti-P36 antibodies had no effect on primary fixation to the ZP, but inhibited secondary binding to the ZP and the Ca2+ ionophore-induced acrosome reaction. These results suggest that P36, an acrosomal antigen, is involved in several steps of the fertilization process. On two-dimensional Western blots, human anti-sperm antibodies (ASA) and rabbit anti-P36 antibodies recognized five to six isoforms of P36, all 36/37 kDa in size, with a pI between 5.1 and 5.7. Two major spots were identified as human triosephosphate isomerase (TPI) by MALDI-TOF mass spectrometry. Anti-TPI antibodies were shown to react with the isoforms recognized by human and rabbit anti-P36 antibodies. We also demonstrated the presence of TPI in human sperm heads. Further studies are underway to establish whether there is a sperm-specific isoform of TPI and its role in sperm function.

Identification and validation of mouse sperm proteins correlated with epididymal maturation

“…A testis-specific mRNA for TPI1 has been isolated from the rat testis [68]. Immunofluorescence experiments demonstrated that TPI is localized in the human sperm head [69]; however, further analysis should be performed to confirm the proper localization of the TPI protein using permeabilized sperm. In this report, we demonstrate the presence of three isoforms of TPI1 protein in epididymal mouse sperm.…”

Sequential Reactions of Surface- Tethered Glycolytic Enzymes

“…Other germ cell-specific glycolytic isozymes have been described that differ either in sequence, activity, or other characteristics for aldolase (Gillis and Tamblyn, 1984), triose-phosphate isomerase (Auer et al, 2004), glyceraldehyde 3-phosphate dehydrogenase (GAPD-S) (Welch et al, 1992), phosphoglycerate kinase (Boer et al, 1987), enolase (Edwards and Grootegoed, 1983), and pyruvate kinase (Feiden et al, 2007), as well as for the extra-glycolytic but related enzyme lactate dehydrogenase (LDH-C4) (Zhong and Kleene, 1999) (Zinkham, 1968). …”

Spatio-Developmental Distribution of the Prion-Like Protein Doppel in Mammalian Testis: A Comparative Analysis Focusing on Its Presence in the Acrosome of Spermatids1

“…Mouse IgG was purchased from Sigma. The pAb against P36/TPI, which stains the acrosomal membrane, was a gift from J. Auer [14]. Rabbit polyclonal antibodies against RAB6 (a Golgi vesicle marker) and against RAB5 (an endocytic vesicle marker) were obtained from Santa Cruz Biotechnology.…”