Search

Bovine sperm protease, 66 kDa (BSp66) is a serine protease previously characterized in bovine spermatozoa. Like other proteases, it may be present in sperm from other mammalian species different from bovine, playing a role in the fertilization process. In this study, we looked for BSp66 in hamster spermatozoa using heterologous antibodies against bovine BSp66. An immunoreactive protein was detected by Western blotting in mature and immature sperm. The detected protein had two isoforms similar to the ones reported in bovine sperm. Furthermore, indirect immune detection by fluorescence and electron microscopy assays, showed BSp66 signal at the acrosomal region similar to bovine sperm. As it was determined in bovine sperm, the acrosomal reaction displays the antigen within the acrosomal content. When live hamster sperm was incubated with polyclonal antibody against bovine BSp66 a decrease in the number of sperm bound to zona pellucida in homologous IVF and an impairment of head-head agglutination, were observed. These results suggest that a protease homologous to bovine BSp66 is present in golden hamster spermatozoa, with a conserved molecular mass and cellular location. Moreover, hamster BSp66 is probably involved in zona pellucida recognition.

Little is known about the evolution of vertebrate spermatozoa. In most eutherian taxa a high degree of uniformity in sperm shapes and dimensions among species was observed. The aim of this work is to trace a possible evolutionary change in sperm morphology and morphometry in dasypodids. The main difference between the spermatozoa of the studied armadillos is the shape of the sperm heads. We have classified the spermatozoa into 4 different groups according with their head shapes. Sperm from group 1 (Dasypus) are considered ancestral and are clearly separated from the others. The remaining sperm types are derivative ones; those from group 2 (Tolypeutes) are farther from those of groups 3 (Priodontes and Cabassous) and 4 (Chaetopractus, Zaedyus and Euphractus) which would have recently differentiated from each other. The sperm shape and size are not constant across taxa in armadillos; an important evolutive differentiation was established on the sperm morphology and morphometry between the different genera in Dasypodidae.

We studied in the rat epididymis the presence of membrane-bounded vesicles in the stereociliar areas of the epithelial cells. The intimate contact between principal cell stereocilia and luminal spermatozoa was also explored. The epididymidis of adult male albino rats were fixed with Mollenhauer's fixative via the thoracic aorta; they were removed and the caput and the cauda were separated and fixed for 4 additional hours at 4 degrees C. After fixation, the samples were processed with routine techniques for transmission and scanning electron microscopy. The study showed membrane-bounded vesicles in the lumen of the caput and cauda epididymidis. They are present between stereocilia, in the most peripheral regions of the epididymal lumen, and in a stereocilia-free zone in the apical plasma membrane of the principal cells. The smaller vesicles are located near the apical surface of the latter, and the larger ones are located near the tips of the stereocilia. Their contents are electron lucent in some images and electron dense in others. In several thin sections some of the vesicles are observed to have a stalk. This suggests that the possible mode of production may be an exocytotic process. Some membrane-bounded vesicles were found to be in contact with the head or the tail of maturating spermatozoa. Moreover, an intimate contact was found to exist in the epididymidis between the plasma membranes of the spermatozoa and the stereocilia. These observations seem to suggest two possible mechanisms for sperm-epididymal cell relations: 1) release of a secretion product via the membrane-bounded vesicles and 2) direct contact between stereocilia and spermatozoa.

In order to increase the value of the zona-free hamster oocyte penetration test, a comparatively simple and fast method using the fluorochrome Hoechst 33342 was developed. Human spermatozoa were washed and incubated 1 hr medium BWW for capacitation. Hamster oocytes were stripped of cumulus oophorus and zona pellucida with hyaluronidase and trypsin, washed and used immediately. Thirty oocytes were placed in a drop of BWW containing 3,5.10(6)/ml of human spermatozoa under mineral oil. The sperm-oocyte preparation was incubated for 3 hr at 37 degrees C, during the last 15 min of incubation, the fluorochrome Hoechst 33342 (H) was added and incubation was allowed to proceed until the incubation time was over. Observations showed that the female pronucleus, eccentrically placed, gives a bright green-bluish fluorescence whereas chromatin of sperm heads shows different stages of decondensation and also a bright fluorescence. This inexpensive method has given consistent results in a large number of cases and provides an additional new approach to the "penetration test" as a proof of the capacity to form a "male pronucleus".

Morphological changes in sperm are one aspect of a maturation process during epididymal transit in mammals. The literature mentions only, for different strains of rats, a remodeling and decrease in size of the acrosome. In the present work, the sperm were obtained from caput, corpus, and cauda epididymis of the albino rat. Samples were processed for scanning electron microscopy, with routine techniques, and for light microscopy and video microscopy. It appeared, with these techniques, that the acrosomal curvature and the whole head surface area of the rat sperm decrease during the epididymal transit. To measure these changes, a geometrical method was designed, and surface measurements were made using a computer program. It was found that the caput sperm head has the greatest surface area and a sharper acrosome bend than the cauda sperm. In an attempt to explain the above-mentioned changes, the suggestion is offered that some compactation of the nucleus and acrosomal material could be related to the decrease of the surface area.