Epididymal SPAM1 Is a Marker for Sperm Maturation in the Mouse1

Abstract

Sperm adhesion molecule 1 (SPAM1), is a glycosyl phoshatidylinositol-linked sperm membrane protein that is dually expressed in testis and epididymis. Epididymal SPAM1 is secreted in all three regions of the epididymis in all mammalian species studied, including humans. It shares the same molecular mass and neutral hyaluronidase activity as the testicular and sperm isoforms that are responsible for the penetration of the cumulus during fertilization. Using wild-type (W/T) sperm and those from mice homozygous for either a null (Spam1-/-) or mutant Spam1 allele, which results in decreased mRNA and protein, we demonstrate that sperm binding of epididymal SPAM1 occurs in vitro after exposure to W/T sperm-free epididymal luminal fluid (ELF). Binding or adsorption that occurred after incubation at room temperature or 32 degrees C was detected immunocytochemically and confirmed quantitatively using flow cytometry. The localization of SPAM1 on the plasma membrane of Spam1-null sperm mimicked that seen in the W/T. The remarkable increase in binding on W/T caudal sperm indicates that they are not fully saturated with SPAM1 during storage, and suggests that uptake of epididymal SPAM1 in vivo augments testicular SPAM1. Spam1-null sperm exposed to W/T ELF for 45-60 min during in vitro capacitation to allow epididymal SPAM1 binding showed a highly significant (P < 0.001) increase in cumulus penetration after 6-7 h compared to those incubated in ELF from null males. Similarly, the number of cumulus-free oocytes was also highly significantly greater (P < 0.001) than that for sperm capacitated in W/T SPAM1-antibody-inhibited ELF. Because epididymal SPAM1 uptake significantly increases cumulus penetration, we conclude that it is a marker of sperm maturation.

Murine SPAM1 is secreted by the estrous uterus and oviduct in a form that can bind to sperm during capacitation: acquisition enhances hyaluronic acid-binding ability and cumulus dispersal efficiency

“…This would be similar to epididymal SPAM1 that has been shown to be transferred to the sperm plasma membrane in vitro (Chen et al 2006) and has a GPI anchor on the vesicular component of the ELF (Zhang & Martin-DeLeon 2003a). Our study therefore suggests that SPAM1, like other GPIlinked proteins, may be involved in the post-testicular remodeling of the sperm surface that occurs not only during their maturation and storage in the cauda, but also during capacitation (Kirchoff et al 1997).…”

“…The eggs were monitored every 30 min for 5 h, and the percentage of eggs in each stage of cumulus dispersal was recorded and graphed against time (in 30 min increments). The stages of cumulus dispersal were assessed similar to Chen et al (2006) and Miller et al (2007) using the following criteria: stage 1 eggs are surrounded by tightly packed cumulus cells such that they occlude the view of the oocytes. Stage 2 eggs retain most of their cumulus mass, however, enough cumulus cells are dispersed so that the oocytes are partially visible.…”

“…Recently, our laboratory has shown that caudal mouse sperm can acquire SPAM1 in vitro from the epididymal luminal fluid (ELF) and that this uptake increases sperm fertilizing competence (Chen et al 2006). We have also demonstrated that, similar to its expression in the male reproductive tract, SPAM1 is expressed in the glandular and surface epithelia of the female tract (Zhang & Martin-DeLeon 2003b).…”

The contribution of proteomics to understanding epididymal maturation of mammalian spermatozoa

“…However, among the epididymal fluid proteins, other components less soluble are GPI anchor proteins, i.e., CD52 [Kirchhoff 1996], the prion protein [Ecroyd et al 2005;Gatti et al 2002], SPAM1 [Chen et al 2006], the serine protease PRSS21 [Netzel-Arnett et al 2009], or proteins with highly hydrophobic properties [Gatti et al 1999]. Such poorly soluble proteins can be in part explained by the presence of particulate components, either in the form of membrane vesicles named epididymosomes (for 'epididymal exosomes') or of 'micelle-like structures' [Frenette et al 2002;Gatti et al 2005] in the fluid.…”

Testicular descent, sperm maturation and capacitation. Lessons from our most distant relatives, the monotremes

“…Of particular interest are the proteins that are secreted by the epididymis and interact with spermatozoa to modify their function. A number have been identified in eutherian mammals, including SPAM1 (formerly PH20; Chen et al 2006), SED1 (Ensslin and Shur 2003), α-d-mannosidase (Jin et al 1999), CRISP1 (Eberspaecher et al 1995), CD52 (Kirchhoff et al 1993), ASF (Thomas et al 1984), HE1 (Kirchhoff et al 1996) and p26H (Coutu et al 1996). However, whilst knockout mice for SPAM1 (Baba et al 2002) and SED1 (Ensslin and Shur 2003) have reduced fertility compared with wild-type, none of the knockouts is infertile.…”