Search

In previous studies a series of Mabs against boar capacitated sperm have been produced. One of these Mabs--4B12--was found to recognize a surface membrane-associated protein located in the acrosome portion of the spermatozoa that became accessible to antibody after capacitation. In biological experiments it was shown that Mab 4B12 significantly inhibited boar sperm-porcine ZP binding. In attempts to investigate the mechanisms by which Mab 4B12 affected sperm-ZP binding, the role of the cognate protein on some functional parameters such as sperm motility and ability of the capacitated spermatozoa to undergo AR was studied. Experimental models of premature AR and AR physiologically induced with ZP were applied to study the effect of Mab 4B12 on boar sperm AR using PSA staining to estimate the acrosome-reacted state of spermatozoa. Sperm motility characteristics were determined by the time-exposure photokinesigraphic method. The results obtained in the present study, together with previously established inhibition of sperm-ZP binding by Mab 4B12, documented the participation of the 4B12 protein in primary sperm-ZP binding. The protein is not connected with sperm motility and secondary sperm-ZP binding.

Certain morphological changes such as rearrangement of cytoskeletal proteins of the mammalian spermatozoa are detectable during the AR. The type of changes differs according to the studied sperm species and follows the course of AR. Relocation of cytoskeletal structures was previously observed especially in the case of actin-, alpha-, gamma-tubulin- and spectrin-containing structures. To prove these findings we used specific inhibitors of cytoskeletal proteins (eg. colcemide, cytochalasine B, nocodazole and vinblastine). It has been shown that the AR is influenced by cytoskeletal inhibitors, but the obtained results also document that cytoskeletal proteins actin, tubulin and spectrin play a significant role in the course of AR in vitro. Our results of confocal and electron microscopy also demonstrate visible changes of actin-, tubulin- and spectrin-containing structures after the AR. Our data indicate that specific cytoskeletal inhibitors influence the AR and they prove the role of cytoskeletal proteins in this process.

A new monoclonal antibody designated Hs-14 was generated after immunization of BALB/c mice with the acid extract of human sperm. In indirect immunofluorescence Hs-14 mAb binds to the acrosome of permeabilized sperm cells and consequently recognizes some intra-acrosomal protein. Western blotting analysis revealed that under non-reducing conditions the Hs-14 mAb detects a protein with a molecular mass of 220 kDa. Under reducing conditions the Hs-14 recognizes several peptide bands within the range from 55 kDa to 110 kDa. Beside human sperm the antibody positively reacts also with sperm of some other mammalian species. Using Hs-14 mAb it is possible to evaluate the acrosomal integrity of spermatozoa and to reveal sperm pathology.

Several cytoskeletal proteins (alpha-tubulin, beta-tubulin, actin, spectrin, tropomyosin, vimentin and cytokeratin) were studied in human and boar spermatozoa. Their localization was observed by means of specific antibodies using indirect immunofluorescence technique. Immunocytochemical results were confirmed by the Western blot technique. Cytoskeletal proteins were examined in ejaculated spermatozoa before and after acrosome reaction induced by the ionophore A23187. The immunofluorescence assay revealed that localization of the studied cytoskeletal proteins in human and boar spermatozoa differ remarkably. In human spermatozoa, the localization of actin and spectrin changed after acrosome reaction; on the other hand, in boar spermatozoa, the changes of localization concerned alpha-tubulin, beta-tubulin, actin and spectrin. The type of changes differs in the studied species and follows probably the course of acrosome reaction. The results suggest that cytoskeleton participates in the process of acrosome reaction of mammalian spermatozoa.

Monoclonal antibody ACR.4 recognizing specifically the 28-kDa intra-acrosomal protein was prepared by immunization of mice with acetic acid extract of boar spermatozoa, but cross-reacted also with bull intra-acrosomal protein. This monoclonal antibody was used for immunostaining analysis of bull spermatozoa before and during capacitation and ionophore-induced acrosome reaction. Immunostaining analysis showed changes of 28-kDa protein in the acrosome during capacitation and loss of this protein after induced acrosome reaction by ionophore A23187. Therefore, this monoclonal antibody can be used in the bull spermatozoa as an immunological test for detection of the acrosome state after manipulation with spermatozoa or after freezing/thawing. This test could be useful (apart from morphology and motility) for the selection of suitable spermatozoa for insemination or in vitro fertilization.