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.     

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.     

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.     

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.     

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.     

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.     

Selective solubilization of high-molecular-mass neurofilament subunit during nerve regeneration

A reduction in neurofilament (NF) protein synthesis and changes in their phosphorylation state are observed during nerve regeneration. To investigate how such metabolic changes are involved in the reorganization of the axonal cytoskeleton, we studied the injury-induced changes in the solubility and axonal transport of NF proteins as well as their phosphorylation states in the rat sciatic nerve. In the control nerve, 15-25% of high-molecular-mass NF subunit (NF-H) was recovered in the 1% Triton-soluble fraction when fractionated in the presence of phosphatase inhibitors. After a complete loss of NF proteins distal to the injury site (70-75 mm from the spinal cord) 1 week after injury, NF-H detected in the regenerating sprouts at 2 weeks or later exhibited higher solubility (>50%) and lower C-terminal phosphorylation level than NF-H in the control nerve. Solubility increase was also apparent with L-[35S]methionine-labeled NF-H that was in transit in the proximal axon at the time of injury. The low-molecular-mass subunit remained in the insoluble fraction in both the normal and the regenerating nerves, indicating that selective solubilization of NF-H rather than total filament disassembly occurs during regeneration.     

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.     

Detergent solubilization of mammalian cardiac and hepatic beta-adrenergic receptors.

The solubilization of canine cardiac and hepatic β-adrenergic receptors was characterized with 19 different ionic and nonionic surfactants and surfactant combinations. The effects of alterations in the hydrophobic and hydrophilic moieties of the nonpolar detergents were examined in relation to their efficacy in solubilizing these receptor molecules. Within this group of surfactants the more effective agents contained an average of 9–10 polyoxyethylene units per molecule. The best degree of β-receptor solubilization for both heart and liver was obtained with 1% Brij 96 or a combination of 1% digitonin with 0.25% Triton X-100. Hepatic but not cardiac β-receptors were solubilized by polyoxyethylene ether W-1 or by Triton X-100. Solubilization time courses indicated that the maximum degree of receptor solubilization occurred within 5 min at 0–4 °C. Solubilization temperatures were varied from 0 to 37 °C. Temperatures up to 30 °C increased numbers of cardiac receptors solubilized by 30% over those obtained at 0 °C. The same temperature changes had no significant effects on liver β-receptor solubilization. Increasing the solubilization temperature to 37 °C decreased the detectable number of β-receptors by 91 (liver) and 72% (heart). β-Receptors solubilized in the absence of receptor ligand were extremely labile with a half-life on the order of 90 min at 4 °C for both heart and liver. Occupation of the receptors by [¹²⁵I]-iodohydroxybenzylpindolol prior to solubilization conferred a certain degree of stability on the receptors.     

Apparent densities of spermatozoa of various mammalian species

The apparent densities of sperm cells in several mammalian species were determined by means of Percoll density-gradient centrifugation. Semen samples were obtained from (1) ejaculates from the bull, human, and rabbit, and (2) from the cauda epididymis of the goat, golden hamster, house musk shrew, mastomys, and mouse. The profiles of sperm distribution showed a single peak after the centrifugation in the first group and goat, whereas two separate main peaks in the second group (except goat). This disparity in sperm distribution profiles may be due mainly to differences in the degree of maturity of the sperm. Highly motile and mature sperm were obtained at higher densities, whereas immotile or immature sperm were found at the apparent densities. Thus, in mammals, the profiles of sperm distribution in the Percoll density gradient are classified into two types, those with a single peak and those with two separate peaks. The apparent density of sperm cells may be of importance in sperm physiology.