No abstract

Different aspects of matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) have been used as discovery tools to obtain global and time-correlated information on the local proteomic composition of the sexually mature mouse epididymis from both qualitative and semiquantitative points of view. Tissue sections and laser captured microdissected cells and secretory products were analyzed by MALDI-MS and from the recovered protein profiles, over 400 different proteins were monitored. Over 50 of these, some of which have been identified, displayed regionalized behavior from caput to cauda within the epididymis. Combining the information obtained from high-resolution imaging mass spectrometry and laser captured microdissection experiments, numerous proteins were localized within the epididymis at the cellular level. Furthermore, from the signal intensities observed in the different protein profiles organized in space, semiquantitative information for each protein was obtained.

A monoclonal antibody, designated TES101, was raised by immunizing BALB/c mice with an allogenic mouse testicular homogenate followed by immunohistochemical selection as the initial screening method. By searching the expressed sequence tag (EST) database with the N-terminal amino acid sequence of TES101 reactive protein, we found that the predicted amino acid sequence encoded by a mouse testicular EST clone matched the TES101 protein sequence. Sequence analysis of the clone revealed no homologous molecule in the DNA/protein database. Based on data obtained from N-terminal amino acid analysis of the TES101 protein, the derived amino acid sequence contained a signal peptide region of 25 amino acids and a mature protein region of 225 amino acids, which translated into a protein with a molecular weight of 24 093. Northern blot analysis showed that mRNA of the TES101 protein was found in testis but not in any other mouse tissues examined. Western blot analysis revealed that TES101 reacted with a 38-kDa band on SDS-PAGE under nonreducing conditions, and this reactivity was abrogated under reducing conditions. Immunoelectron microscopic studies demonstrated that the molecule was predominantly located on the plasma membrane of spermatocytes and spermatids but not in Sertoli cells or interstitial cells, including Leydig cells. Thus, the TES101 protein is a novel molecule present primarily on the surface of developing male germ cells. TES101 protein may play a role in the processes underlying male germ cell formation.

In previous reports from this laboratory, we identified the presence of a novel alpha-D-mannosidase on the surface of rat, mouse, hamster, and human spermatozoa [J Cell Biol 1989; 109:1257-1267 and Biol Reprod 1990; 42:843-858]. Since it has been suggested that mannosyl residues on the egg zona pellucida may be important for sperm-egg binding, studies were undertaken to examine the potential role of the sperm alpha-D-mannosidase during fertilization. Incubation of mouse spermatozoa in the presence of increasing concentrations of the inhibitory sugars, alpha-methyl mannoside, alpha-methyl glucoside, D-mannose, or D-mannitol, resulted in a dose-dependent decrease in the number of spermatozoa bound per egg without a deleterious effect on sperm motility or on the sperm acrosome, and a dose-dependent inhibition of the sperm mannosidase activity. Galactose, however had no effect on sperm-egg binding or on sperm mannosidase activity. Two nucleotide sugars (UDP-GlcNAc and UDP-gal) were also tested and shown to reduce sperm-egg binding but with only a minimal effect on sperm mannosidase activity. In additional studies, spermatozoa incubated in the presence of a mannose-containing oligosaccharide exhibited a dramatic reduction in sperm-egg binding that correlated with a similar inhibition of sperm mannosidase activity. The oligosaccharide substrate did not affect sperm motility or the sperm acrosome. These studies suggest that the sperm alpha-D-mannosidase may play an important role during fertilization.

Abstract. During the course of a study of glycoprotein processing mannosidases in the rat epididymis, we have made an intriguing discovery regarding the presence of a novel ot-o-mannosidase on the rat sperm plasma membranes. Unlike the sperm acrosomal "acid" mannosidase which has a pH optimum of 4.4, the newly discovered Ot-D-mannosidase has a pH optimum of 6.2, and 6.5 when assayed in sperm plasma membranes and intact spermatozoa, respectively. In addition, the two enzymes show different substrate specificity. The acrosomal Ot-D-mannosidase is active mainly towards synthetic substrate, p-nitrophenyl a-Dmannopyranoside, whereas the sperm plasma membrane O~-D-mannosidase shows activity mainly towards mannose-containing oligosaccharides. Evidence is presented which suggest that the sperm plasma membrane Ot-D-mannosidase is different from several processing mannosidases previously characterized from the rat liver.The newly discovered a-D-mannosidase appears to be an intrinsic plasma membrane component, since washing of the purified membranes with buffered 0.4 M NaCI did not release the enzyme in soluble form. The enzyme requires nonionic detergent (Triton X-100) for complete solubilization. The enzyme is activated by Co 2+ and Mn 2÷. However, Cu 2÷ and Zn 2÷ are potent inhibitors of the sperm plasma membrane Ot-D-mannosidase. At a concentration of 0.1 mM, these divalent cations caused nearly complete inactivation of the sperm enzyme. In addition methyl-c~-D-mannoside, methyl-ot-D-glucoside, mannose, 2-deoxy-D-glucose, and D-mannosamine are inhibitors of the sperm surface a-D-mannosidase. The physiological role of the newly discovered enzyme is not yet known. Several published reports in three species, including the rat, suggest that the sperm surface c~-o-mannosidase may have a role in binding to mannose-containing saccharides presumably present on the zona pellucida.I X is generally accepted that one step in the fertilization process requires interaction between complementary molecules present on the surface of the spermatozoon and the zona pellucida. The chemical nature of these complementary recognition sites is poorly understood, although there is growing evidence that carbohydrate moieties on surface membrane glycoconjugates are involved in these interactions (1, 34).In mammals, several sperm proteins have been suggested to bind to zona pellucida (28). Some of these macromolecules have enzymatic activity and are thought to form a stable enzyme-substrate complex by binding to the oligosaccharide units present on the zona pellucida glycoproteins. In mice, galactosyltransferase present on the head region of the spermatozoa mediates sperm-egg binding by interacting with its substrate on the zona pellucida (23). There is also evidence that trypsin-like protease present on spermatozoa of mouse (4, 31) initiates sperm-egg binding. Mouse spermatozoa also contain sialyltransferase (13) and fucosyltransferase (30). The latter enzyme has been suggested to be involved in some aspect of sperm-egg recognition (Apter, E M., ...