New insights into the assembly of the periaxonemal structures in mammalian spermatozoa

Disruption of Ube2b in the mouse has revealed that the regular and symmetric organization of the fibrous sheath of the sperm flagella is dependent on expression of the ubiquitin-conjugating enzyme UBE2B. These data could cast light on how a component of the ubiquitin-proteasome pathway participates in the assembly of flagellar periaxonemal structures. Data in the literature support the notion of involvement of ubiquitin-proteasome pathways in the assembly of cytoskeletal components in somatic cells. This review attempts to integrate recent knowledge regarding flagellar components that could be related to proteasome components and, therefore, could be targets of UBE2B in the spermatid. An attempt is made to characterize the human flagellar anomalies of infertile patients, which are the closest to those of Ube2b-deficient mice. These new insights regarding the assembly of mammalian sperm flagella provide a basis for studying the ontogenesis of flagellar accessory structures and suggest leads for medical and genetic investigations.     

Protamine-DNA association in mammalian spermatozoa

We have previously identified two subsets of basic nuclear proteins of mouse sperm: the protamines and a group of less basic proteins and, with the aid of a polyvalent antiserum, we have demonstrated their differential extractibility by NaCl in reducing solution (Rodman et al., J cell sci 53 (1982) 227) [9]. By affinity purification with isolated mouse sperm protamines we have obtained a protamine-specific fraction of that antiserum and a fraction that contains antibodies to the subset of less basic proteins. With those immunochemical probes we have shown the following The antigenic sites recognized by the protamine-specific antibodies are accessible, intranuclearly, only after the DNA has been removed by DNase I. The antibodies and DNA compete for binding sites on the protamines. DNA removal and consequent availability of the antigenic sites of the protamine molecules to the antibodies are possible only after displacement of the less basic proteins and chromatin decondensation have been induced. Immunoreactivity by the less basic proteins takes place without intervention of DNase. Those data indicate that the protamines are DNA-bound but that the less basic proteins are not or, alternatively, their putative DNA-binding sites do not coincide with their immuno-reactive sites. Those data also suggest that a function of the subset of less basic proteins may be to provide a shield for the protamine-DNA complex. The mouse protamine-affinity-bound antibodies are highly cross-reactive with protamines of other mammalian sperm suggesting that, despite considerable molecular diversity among mammalian protamines, the DNA-binding sites are conserved.     

Proteasomal activity in mammalian spermatozoa

The proteasome is a multicatalytic cellular complex, which possess three different enzymatic activities, trypsin-like, chymotrypsin-like, and peptidylglutamyl peptidase. Its function is to remove abnormal or aged proteins. Recently, it has been suggested the participation of the sperm proteasome during mammalian fertilization. In this study, we present evidence that indicates that sperm extracts from several mammalian species, including hamster, mice, rats, bovine, rabbits, and humans all possess proteasome activity. We characterized the three specific activities of the proteasome using specific synthetic substrates and specific proteasome inhibitors. The results indicates that the highest specific activity detected was in mouse sperm toward the trypsin substrates and it was 1,114% of the activity of human sperm toward the chymotrypsin substrate Suc-Leu-Leu-Val-Tyr-AMC (SLLVY-AMC, which was considered as 100%). In all cases, the lowest activity was toward substrates for the peptidylglutamyl peptidase hydrolyzing activity, and it was lowest for rabbit sperm (1.7% of the activity of human sperm toward the chymotrypsin substrate SLLVY-AMC). In addition, specific proteasome inhibitors were able to block all proteasome activities almost 100%, with the exception of clasto-Lactacystin beta-lactone upon rat sperm. All sperm extracts tested evidenced bands of about 29-32 kDa by Western blots using a monoclonal antibody against proteasome subunits alpha 1, 2, 3, 5, 6, and 7. In conclusion, sperm from several mammals possess enzymatic activities that correspond to the proteasome. The proteasome from the different species hold similar but distinctive enzymatic characteristics.     

Cohesive properties of mammalian epididymal spermatozoa

Changing spermatozoan associations were observed in the epididymides of several mammals. These associations ranged from closely interwoven cylindrical bodies, found in the proximal part of the epididymis, to disorganized masses of spermatozoa, found in the distal part of the duct. It is suggested that changes in the cohesive properties of epididymal spermatozoa resulted in the formation and fragmentation of cylindrical bodies. These bodies, differeing in pattern and complexity according to the species, were found in all investigated mammals, including man. Cohesiveness appeared first in the upper part of the epididymidis, where it was confined to the spermatozoan tails. In general, there was a diminution of cohesive forces as the spermatozoa passed down the epididymal duct; consequently, the cylindrical bodies turned into disorganized masses of spermatozoa. There are indications that changes in the cohesive properties of spermatozoa may represent one aspect of spermatozoan maturation.     

Silver-staining patterns of mammalian epididymal spermatozoa

Epididymal spermatozoa from 12 species of mammals were stained using silver nitrate and examined with the light microscope. Silver nitrate differentiates many of the gross morphological features of spermatozoa, including the acrosome, subacrosomal region, perforatorium, postacrosomal sheath, neck, dense outer fibers of the core of the midpiece, annulus, principal piece, and end piece. Silver-staining patterns of spermatozoa reveal both species-specific and strain-specific differences, particularly of the sperm head. The biochemical basis of silver staining may be due in part to the presence of sulfhydryl- and disulfide-rich proteins; however, it cannot be explained entirely by the presence of these moieties. The detail obtained using silver nitrate staining, coupled with the ease and rapidity of the procedure, should be useful to workers in many areas of biological and medical research.     

Selective fluorescence of eosinophilic structures in grasshopper and mammalian testis stained with haematoxylin-eosin

After staining with Mayer's haematoxylin and eosin Y, paraffin sections of grasshopper and mouse testis were analysed by both transmitted light and fluorescence microscopy. Under violet-blue (436 nm) light excitation, a bright green emission was observed in all eosinophilic structures. Meiotic spindles (fibres and poles), mitochondrial aggregates, centriolar adjuncts in grasshopper spermatids, the basal lamina, flagellar bundles and remaining cytoplasmic droplets in the lumen of seminiferous tubules showed the most striking fluorescence induced by eosin Y. No emission was found in these structures after haemalum staining. Fluorescent microtubular components also revealed a positive immunoperoxidase reaction for -tubulin. All fixation and embedding procedures (Bouin, Zenker, formaldehyde alone or followed by dichromate or glutaraldehyde, freeze-substitution) were suitable for observation by fluorescence microscopy. Acetylation, deamination, and prolonged washing of stained sections with water, salt solution or ethanol strongly reduced eosin Y fluorescence, while it slightly increased after methylation. These results show that routine haematoxylin-eosin stained tissue sections can be routinely analysed by fluorescence microscopy. The emission of eosin Y allows easy and precise recognition of eosinophilic structures, which are poorly visible under bright field illumination.     

Selective solubilization of chlorosome proteins in Chloroflexus aurantiacus

Proteins were solubilized selectively from chlorosomes of Chloroflexus aurantiacus by electrophoretic gel filtration according to Griebenow et al. Whereas the 11 kDa and 18 kDa proteins were extracted almost completely, the remaining modified chlorosomes contained high amounts of pigment and c-protein. It was concluded that the c-protein in contradiction to the publication by Griebenow et al. is indeed localized in the interior of Chloroflexus chlorosomes.     

DNA loop domains in mammalian spermatozoa

The highly condensed and tightly packaged DNA of hamster spermatozoa was found to be organized into topologically constrained DNA loop domains attached at their bases to a nuclear matrix. The loop domains of the sperm nuclei differed from somatic cell loop domains from the same animal in two aspects. Sperm loop domains were 60% smaller than somatic cell loop domains, with an average DNA length of 46±7 kb in sperm as compared with 16±11 kb in brain. Secondly, unlike virtually all somatic cell DNA known which is negatively supercoiled, sperm DNA was devoid of detectable supercoiling. The presence of the loop domain structure in the highly condensed DNA of mammalian spermatozoa suggests that this motif is a fundamental aspect of eukaryotic DNA organization.     

Voltage-dependent anion channel in mammalian spermatozoa

Voltage-dependent anion channel (VDAC) is a multi-functional channel protein in the mitochondrial outer membrane of all eukaryotes. It is involved in extensive physiological and pathophysiological processes. However there is only scant information about VDAC in mammalian reproduction, fertility and development in the past. It is now recognized through recent studies that VDAC is present in male mammalian germinal tissues and cells, and plays crucial roles in spermatogenesis, sperm maturation and fertilization. This review will discuss the presence, localization and function of VDAC in mammalian spermatozoa.     

Cryogenic preservation of fish and mammalian spermatozoa

Various combinations of sucrose, reduced glutathione and potassium bicarbonate were tested for the cryogenic preservation of salmon spermatozoa. When a fast freezing procedure was followed, the extender that gave the best results was composed of 1 part of dimethyl sulphoxide (DMSO), as a protective agent, and 7 parts of a medium containing 125 mM-sucrose, 6.50 mM-reduced glutathione and 100 mM-potassium bicarbonate. Salmon and cod spermatozoa were kept frozen in this extender for 1 year. Freezing resulted in a reduction in the number of motile spermatozoa but had no significant effect on the degree of progression of motile spermatozoa or on their fertilizing capacity. When glycerol replaced DMSO in the extender and a slow freezing procedure was followed, similar results were obtained for the spermatozoa of bull or man; although the number of motile spermatozoa was reduced, there was no effect on the progressive motility score.