Identification and purification of a sperm surface protein with a potential role in sperm-egg membrane fusion.

Abstract

Abstract. Sperm-egg plasma membrane fusion during fertilization was studied using guinea pig gametes and mAbs to sperm surface antigens. The rnAb, PH-30, strongly inhibited sperm-egg fusion in a concentration-dependent fashion. When zona-free eggs were inseminated with acrosome-reacted sperm preincubated in saturating (140 lxg/ml) PH-30 mAb, the percent of eggs showing fusion was reduced 75 %. The average number of sperm fused per egg was also reduced by 75 %. In contrast a control mAb, PH-I, preincubated with sperm at 400 gg/ml, caused no inhibition. The PH-30 and PH-1 mAbs apparently recognize the same antigen but bind to two different determinants. Both mAbs immunoprecipitated the same two ~5I-labeled polypeptides with Mr 60,000 (60 kD) and Mr 44,000 (44 kD). Boiling a detergent extract of sperm severely reduced the binding of PH-30 but had essentially no effect on the binding of PH-1, indicating that the two mAbs recognize different epitopes.Immunoelectron microscopy revealed that PH-30 mAb binding was restricted to the sperm posterior head surface and was absent from the equatorial region. The PH-30 and PH-1 mAbs did not bind to sperm from the testis, the caput, or the corpus epididymis. PH-30 mAb binding was first detectable on sperm from the proximal cauda epididymis, i.e., sperm at the developmental stage where fertilization competence appears.After purification by mAb affinity chromatography, the PH-30 protein retained antigenic activity, binding both the PH-30 and PH-1 mAbs. The purified protein showed two polypeptide bands of 60 and 44 kD on reducing SDS PAGE. The two polypeptides migrated further (to ,,o49 kD and ,033 kD) on nonreducing SDS PAGE, showing that they do not contain interchain disulfide bonds, but probably have intrachain disulfides. 44 kD appears not to be a proteolytic fragment of 60 kD because V8 protease digestion patterns did not reveal related peptide patterns from the 44-and 60-kD bands. In the absence of detergent, the purified protein precipitates, suggesting that either 60 or 44 kD could be an integral membrane polypeptide.

Expression and role of mannose receptor/terminal high-mannose type oligosaccharide on osteoclast precursors during osteoclast formation

“…In viral infections such as influenza virus (Anders et al 1990) or HIV (Lifson et al 1986, Matthews et al 1987 infection, the involvement of terminal highmannose type oligosaccharide has been reported. Cell-cell fusion reactions such as sperm-egg fusion (Primakoff et al 1987, Blobel et al 1990, myoblast fusion (Kaufman et al 1985, Rosenberg et al 1985, Menko & Boettiger 1987, Rosen et al 1992, monocyte fusion (Most et al 1990), or preosteoclast fusion (Kurachi et al 1993) are also protein mediated. From these observations (Kurachi et al 1994), we have already reported that mannose residues are expressed on the outer membranes of monocytes during osteoclast differentiation using pradimicin derivatives, which are antiviral and antifungal both in vitro and in vivo, and recognize and bind specific sugars such as mannose residues (Oki et al 1988(Oki et al , 1990.…”

“…Terminal high-mannose type oligosaccharide on the cell surface mediates cell-cell fusion reactions such as sperm-egg fusion (Primakoff et al 1987, Blobel et al 1990 and myoblast fusion (Kaufman et al 1985, Rosenberg et al 1985, Menko & Boettiger 1987, Rosen et al 1992. The involvement of terminal high-mannose type oligosaccharide has also been reported in the events of viral infections such as the influenza virus (Anders et al 1990) or human immunodeficiency virus (HIV) (Lifson et al 1986, Matthews et al 1987.…”

Chronological gene expression of ADAMs during testicular development: Prespermatogonia (gonocytes) express fertilin β (ADAM2)

“…The designation disintegrin refers to its property of disrupting integrin binding to classic ligands, including fibronectin and laminin. The ␣ and ␤ subunits of the heterodimeric sperm protein fertilin (also designated ADAM-1 and ADAM-2, respectively; see http:// www.people.Virginia.edu/ϳjw7g/ Table_of_the_ADAMs.html for a table of ADAM gene and protein nomenclature) were the first members of the ADAM family to be recognized as spermatogenic cell-specific and implicated in sperm-egg membrane fusion (Primakoff et al, 1987). In addition to fertilin ␣ and fertilin ␤, the gene expression of several members of the ADAM family has been detected in somatic and spermatogenic cell components of prepubertal and adult rat testis and other organs (Frayne et al, 1997).…”

In VivoAnalysis of Membrane Fusion

“…Currently it remains unknown what the fusogen is that mediates this process. Early experiments implicated a family of proteins known as the fertilins (Primakoff et al, 1987). The fertilins a and b are the founding members of the ADAM (A Disintegrin and Metalloprotease) family.…”