CASK interacts with PMCA4b and JAM-A on the mouse sperm flagellum to regulate Ca2+ homeostasis and motility.

Deletion of the highly conserved gene for the major Ca(2+) efflux pump, Plasma membrane calcium/calmodulin-dependent ATPase 4b (Pmca4b), in the mouse leads to loss of progressive and hyperactivated sperm motility and infertility. Here we first demonstrate that compared to wild-type (WT), Junctional adhesion molecule-A (Jam-A) null sperm, previously shown to have motility defects and an abnormal mitochondrial phenotype reminiscent of that seen in Pmca4b nulls, exhibit reduced (P < 0.001) ATP levels, significantly (P < 0.001) greater cytosolic Ca(2+) concentration ([Ca(2+) ](c)) and ∼10-fold higher mitochondrial sequestration, indicating Ca(2+) overload. Investigating the mechanism involved, we used co-immunoprecipitation studies to show that CASK (Ca(2+) /calmodulin-dependent serine kinase), identified for the first time on the sperm flagellum where it co-localizes with both PMCA4b and JAM-A on the proximal principal piece, acts as a common interacting partner of both. Importantly, CASK binds alternatively and non-synergistically with each of these molecules via its single PDZ (PDS-95/Dlg/ZO-1) domain to either inhibit or promote efflux. In the absence of CASK-JAM-A interaction in Jam-A null sperm, CASK-PMCA4b interaction is increased, resulting in inhibition of PMCA4b's enzymatic activity, consequent Ca(2+) accumulation, and a ∼6-fold over-expression of constitutively ATP-utilizing CASK, compared to WT. Thus, CASK negatively regulates PMCA4b by directly binding to it and JAM-A positively regulates it indirectly through CASK. The decreased motility is likely due to the collateral net deficit in ATP observed in nulls. Our data indicate that Ca(2+) homeostasis in sperm is maintained by the relative ratios of CASK-PMCA4b and CASK-JAM-A interactions.

Acidic hyaluronidase activity is present in mouse sperm and is reduced in the absence of SPAM1: evidence for a role for hyaluronidase 3 in mouse and human sperm.

The molecular mechanisms underlying sperm penetration of the physical barriers surrounding the oocyte have not been completely delineated. Although neutral-active or "reproductive" hyaluronidases (hyases), exemplified by Sperm Adhesion Molecule 1 (SPAM1), are thought to be responsible for hyaluronan digestion in the egg vestments and for sperm-zona binding, their roles in mouse sperm have been recently questioned. Here we report that acidic "somatic" Hyaluronidase 3 (HYAL3), a homolog of SPAM1 with 74.6% structural similarity, exists in two isoforms in human ( approximately 47 and approximately 55 kDa) and mouse ( approximately 44 and approximately 47 kDa) sperm, where it resides on the plasma membrane over the head and midpiece. Mouse isoforms are differentially distributed in the soluble (SAP), membrane (MBP), and acrosome-reacted (AR) fraction where they are most abundant. Comparisons of zymography of Hyal3 null and wild-type (WT) AR and MBP fractions show significant HYAL3 activity at pH 3 and 4, and less at pH 7. At pH 4, a second acid-active hyase band at approximately 57 kDa is present in the AR fraction. HYAL3 activity was confirmed using immunoprecipitated HYAL3 and spectrophotometry. In total proteins, hyase activity was higher at pH 6 than at 4, where Spam1 nulls had significantly (P < 0.01) diminished activity implicating an acidic optima for murine SPAM1. Although fully fertile, Hyal3 null sperm showed delayed cumulus penetration and reduced acrosomal exocytosis. HYAL3 is expressed in epididymal tissue/fluid, from where it is acquired by caudal mouse sperm in vitro. Our results reveal concerted activity of both neutral- and acid-active hyaluronidases in sperm.

Clusterin facilitates exchange of glycosyl phosphatidylinositol-linked SPAM1 between reproductive luminal fluids and mouse and human sperm membranes.

Glycosyl phosphatidylinositol (GPI)-linked proteins, which are involved in post-testicular maturation of sperm and have a role in fertilization, are acquired on the sperm surface from both vesicular and membrane-free soluble fractions of epididymal luminal fluid (LF) and uterine LF. Herein, we investigate the mechanism of uptake of these proteins from the soluble fraction of LFs using sperm adhesion molecule 1 (SPAM1) as a model. Ultracentrifugation and native Western blot analysis of the soluble fraction revealed that SPAM1 is present in low-molecular-weight (monomeric) and high-molecular-weight (oligomeric) complexes. The latter are incapable of transferring SPAM1 and may serve to produce monomers. Monomers are stabilized by hydrophobic interactions with clusterin (CLU), a lipid carrier that is abundantly expressed in LFs. We show that CLU is involved in the transfer of SPAM1 monomers, whose delivery was decreased by anti-CLU antibody under normal and apolipoprotein-enhanced conditions. Coimmunoprecipitation revealed an intimate association of CLU with SPAM1. Both plasma and recombinant CLU had a dose-related effect on transfer efficiency: high concentrations reduced and low concentrations enhanced delivery of SPAM1 to human and mouse sperm membranes, reflecting physiological states in the epididymal tract. We propose a lipid exchange model (akin to the lipid-poor model for cholesterol efflux) for the delivery of GPI-linked proteins to sperm membranes via CLU. Our investigation defines specific conditions for membrane-free GPI-linked protein transfer in vitro and could lead to technology for improving fertility or treating sperm pathology by the addition of relevant GPI-linked proteins critical for successful fertilization in humans and domestic animals.

Anatase titanium dioxide nanoparticles in mice: evidence for induced structural and functional sperm defects after short-, but not long-, term exposure.

Titanium dioxide (TiO 2 ) nanoparticles (TNPs) are widely used commercially and exist in a variety of products. To determine if anatase TNPs (ATNPs) in doses smaller than previously used reach the scrotum after entry in the body at a distant location and induce sperm defects, 100% ATNP (2.5 or 5 mg kg-1 body weight) was administered intraperitoneally to adult males for three consecutive days, followed by sacrifice 1, 2, 3, or 5 weeks later (long-) or 24, 48 or 120 h (short-term exposure). Transmission electron microscopy revealed the presence of ANTP in scrotal adipose tissues collected 120 h postinjection when cytokine evaluation showed an inflammatory response in epididymal tissues and fluid. At 120 h and up to 3 weeks postinjection, testicular histology revealed enlarged interstitial spaces. Significantly increased numbers of terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling-positive (apoptotic) germ (P = 0.002) and interstitial space cells (P = 0.04) were detected in treated males. Caudal epididymal sperm from the short-term, but not a long-term, arm showed significantly (P < 0.001) increased frequencies of flagellar abnormalities, excess residual cytoplasm (ERC), and unreacted acrosomes in treated versus controls (dose-response relationship). A novel correlation between ERC and unreacted acrosomes was uncovered. At 120 h, there were significant decreases in hyperactivated motility (P < 0.001) and mitochondrial membrane potential (P < 0.05), and increased reactive oxygen species levels (P < 0.00001) in treated versus control sperm. These results indicate that at 4-8 days postinjection, ANTP induce structural and functional sperm defects associated with infertility, and DNA damage via oxidative stress. Sperm defects were transient as they were not detected 10 days to 5 weeks postinjection.