Evaluation of the expression of sperm proteins in normozoospermic and asthenozoospermic men using monoclonal antibodies

Recent studies have shown that infertility affects estimated 15% of all couples. Male infertility is the primary or contributory cause in 60% of these cases. Consequently, the application of assisted reproduction is increasing. These methods could benefit from an extended evaluation of sperm quality. For this reason, we analyzed sperm proteins from 30 men with normal spermiograms and 30 men with asthenozoospermia. Ejaculates of both groups were tested by flow cytometry （FCM） and fluorescence with a set of well-characterized anti-human sperm Hs-monoclonal antibodies （MoAbs）, which were generated in our laboratory. No statistically significant differences were found between normospermics and asthenospermics in the expression of the sperm surface protein clusterin, evaluated with Hs-3 MoAb, and semenogelin, evaluated with Hs-9 MoAb. However, FCM revealed quantitative differences in the acrosomal proteins between normozoospermic and asthenozoospermic men, namely, in glyceraldehyde-3-phosphate dehydrogenase, evaluated with Hs-8 MoAb, valosin-containing protein, evaluated with Hs-14 MoAb, and ATP synthase （cAMP-dependent protein kinase II, PRKAR2A）, evaluated with MoAb Hs-36. Asthenozoospermic men displayed a highly reduced expression of intra-acrosomal proteins, with a likely decrease in sperm quality, and thus a negative impact on successful reproduction. Asthenozoospermia seems to be a complex disorder involving intra-acrosomal proteins.

The antibody against a nuclear autoantigenic sperm protein can result in reproductive failure

To study whether the antibody against the testis form of the nuclear autoantigenic sperm protein(tNASP)could result in reproductive failure,we successfully cloned and expressed a 339-bp cDNA fragment of mouse tNASP(mtNASP).Using mouse as a model,recombinant mtNASP(rmtNASP)and a synthetic peptide,human tNASP393-408(htNASP393-408),were investigated for their antifertility effect.Active immunization with rmtNASP or the synthesized peptide raised high antibody titers in the immunized mice.Sperm-egg binding and fusion assay were carried out in 8-10-week-old BALB/c mice.Sperm-egg binding and in vitro fertilization of mouse oocytes were inhibited by co-incubation of zona-free mouse oocytes with capacitated mouse spermatozoa in the presence of varying concentrations of the antisera against rmtNASP.There was a significant antifertility effect in animals immunized with rmtNASP or the synthesized peptide.The effect on fertility in the mice immunized with the synthesized peptide was reversible.Our data indicate that active immunization with rmtNASP antigen may induce a strong antibody response that causes an inhibition of fertility.

Analysis of Sperm Membrane Protein Relevant to Antisperm Antibody by Two-Dimensional Gel Electrophoresis and Western Blotting

Objective To identify the sperm membrane proteins that are associated with antisperm antibody Methods Using antisperm antibody positive serum through unidimensional polyacrylamide gel electrophoresis and 2-dimensional gel electrophoresis followed by Western blot analysis to determine the molecular weights (MW) and isoelectric points (pI) of sperm membrane proteins that are associated with antisperm antibody. Results Eight kinds of MW with more than ten sperm membrane proteins can be recognized by antisperm antibody positive serum, of which the MWs and pI were 23 kD, 31 kD, 32 kD, 34 kD, 41 kD, 51 kD, 60 kD, 78 kD and 5.3, 5.5,5.7, 5.0, 5.3, 5.8, 6.0, 5.5～6.2, 4.6,5.1,5.5～5.8 respectively. The identification ratios of the sperm membrane proteins on 78 kD (60.7%), 60 kD (71.4%), 51 kD (14.9%) and 23 kD (14.29%) were higher. Conclusion The sperm membrane proteins with MW of 78 kD, 60 kD, 51 kD and 23 kD were associated with antisperm antibody and immunological infertility. Two- dimensional gel electrophoresis and Western blotting can precisely identify the sperm membrane proteins that are associated with antisperm antibody.

Differential Proteomic Analysis of Carbon Ion Radiation in Sheep Sperm

This study is first to investigate proteomic changes in sheep sperm induced by carbon ion radiation using two-dimensional electrophoresis （2-DE） analysis in the project of breeding a new variety of sheep. Differential expression proteins were detected using the PDQuest 8.0 software after staining with Coomassie blue. Valid spots were then analyzed through liquid chromatography tandem mass spectrometry （LC-MS/MS）. Among the 480 total protein spots displayed in 2-D gels, 6 specific protein spots were observed in sperm gels. A search against protein sequences in the National Center for Biotechnology Information databases （NCBI） indicated that differentially expressed proteins correspond to two proteins, identified to be enolase and transcription factor AP-2-alpha （TFAP-2c0. The two proteins were up-regulated in the irradiated sperm. To the best of our knowledge, this study is the first to identify proteomic changes induced by carbon ion radiation in sheep sperm. The analysis of differential expression protein may be useful in identifying new breeding markers in sheep reproduction and in clarifying the mechanisms involved in irradiation or space breeding.

Expression of Human Sperm Mem brane Protein in theRecom binantSalm onella Typhim urium Vaccine

A 550 bp cDNA fragment of HSD I coding for an extracellular domain of human sperm membrane protein (hSMP 1) was ligated with an Adapter containing the universal stop codon, and the ligated fragment cDNA was then cloned into the MAS of pUC19. The desired plasmid with correct open reading frame was obtained, and was cut with EcoR I.The insert was purified and then cloned into the two asd + Salmonella expression vectors (the low copy number plasmid pYA292 and the high copy number plasmid pYA3137). The recombinant plasmid containing the insert with the correct orientation was selected by restriction enzyme digestion analysis. The recombinant plasmids were transferred into the non pathogenic Salmonella typhimurium χ4550, which was deletion of the Δcya, Δcrp and Δasd genes. Western blot analysis of the whole cell lysate of the two recombinants of S. typhimurium showed a predominant protein band at 21 KD, which reacted with the anti hSMP 1 antiserum. The result indicated that two recombinants of S. typhimurium containing the 550 bp cDNA of HSD I were constructed and the characteristics of their growth in vitro were determined. They may be used as new potential mucosal immunization antifertility.

Transformation:how do nematode sperm become activated and crawl?

Nematode sperm undergo a drastic physiological change during spermiogenesis(sperm activation).Unlike mammalian flagellated sperm,nematode sperm are amoeboid cells and their motility is driven by the dynamics of a cytoskeleton composed of major sperm protein(MSP)rather than actin found in other crawling cells.This review focuses on sperm from Caenorhabditis elegans and Ascaris suum to address the roles of external and internal factors that trigger sperm activation and power sperm motility.Nematode sperm can be activated in vitro by several factors,including Pronase and ionophores,and in vivo through the TRY-5 and SPE-8 pathways.Moreover,protease and protease inhibitors are crucial regulators of sperm maturation.MSP-based sperm motility involves a coupled process of protrusion and retraction,both of which have been reconstituted in vitro.Sperm motility is mediated by phosphorylation signals,as illustrated by identification of several key components(MPOP,MFPs and MPAK)in Ascaris and the characterization of GSP-3/4 in C.elegans.

Cytosolic Ca^(2+)as a multifunctional modulator is required for spermiogenesis in Ascaris suum

The dynamic polar polymers actin fi laments and microtu-bules are usually employed to provide the structural ba-sis for establishing cell polarity in most eukaryotic cells.Radially round and immotile spermatids from nematodes contain almost no actin or tubulin,but still have the abil-ity to break symmetry to extend a pseudopod and initiate the acquisition of motility powered by the dynamics of cytoskeleton composed of major sperm protein(MSP)during spermiogenesis(sperm activation).However,the signal transduction mechanism of nematode sperm activation and motility acquisition remains poorly under-stood.Here we show that Ca^(2+) oscillations induced by the Ca^(2+) release from intracellular Ca^(2+) store through inositol(1,4,5)-trisphosphate receptor are required for Ascaris suum sperm activation.The chelation of cytosolic Ca^(2+) suppresses the generation of a functional pseudopod,and this suppression can be relieved by introducing ex-ogenous Ca^(2+) into sperm cells.Ca^(2+) promotes MSP-based sperm motility by increasing mitochondrial membrane potential and thus the energy supply required for MSP cytoskeleton assembly.On the other hand,Ca^(2+) promotes MSP disassembly by activating Ca^(2+)/calmodulin-depend-ent serine/threonine protein phosphatase calcineurin.In addition,Ca^(2+)/camodulin activity is required for the fusion of sperm-specifi c membranous organelle with the plasma membrane,a regulated exocytosis required for sperm mo-tility.Thus,Ca^(2+)plays multifunctional roles during sperm activation in Ascaris suum.