Characterization of cAMP-dependent protein kinase and its endogenous substrate proteins in ram testicular,cauda epididymal,and ejaculated spermatozoa

Mammalian spermatozoa have been shown to possess cAMP-dependent protein kinase (A-PK) and endogenous substrate proteins for this enzyme. A study of the kinase system was undertaken to determine changes that may be associated with sperm maturation by comparing immature testicular with mature cauda epididymal and ejaculated spermatozoa. Absolute activity levels of A-PK, stimulated over a concentration range of 10^?9 to 10^?5 M, was significantly greater in testicular than ejaculated spermatozoa. At an optimal cAMP concentration (10^?6M), testicular spermatozoa had significantly greater amounts of cAMP-dependent protein kinase activity than did cauda or ejaculated spermatozoa. Electrophoretic analysis and autoradiography of NP-40-soluble protein extracts revealed the presence of two substrate proteins (M_r = 62,000 and 44,000) in all three types of spermatozoa. In addition, a phosphoprotein (M_r = 20,000) was detected in mature cauda and ejaculated but not immature testicular spermatozoa. The phosphorylation of these substrate proteins was both dose and time dependent. Examination of cyclic AMP phosphodiesterase activity revealed significantly higher levels in testicular than ejaculated spermatozoa. These results indicate marked alterations in cAMP-modulated protein phosphorylation and dephosphorylation systems in ram spermatozoa during epididymal maturation.     

The zymogen form of acrosin in testicular,epididymal, and ejaculated spermatozoa from ram

The sperm-specific proteinase acrosin (EC 3.4.21.10) is found in spermatozoa as a zymogen. We have looked for different forms of this zymogen in testicular, epididymal, and ejaculated spermatozoa from ram and have compared total sperm extracts made immediately after cell disruption with extracts made later from isolated sperm heads. We have concluded that the autoactivatable zymogen form, known generally as proacrosin, is the only form of acrosin within intact mature ram spermatozoa; no other zymogen form was detected, although lower levels of proacrosin were found in some samples of testicular spermatozoa. From studies of the activation process, it appears that ram proacrosin is truly autoactivatable; no evidence could be found for the involvement of any auxiliary enzyme. Estimations of the molecular weight of proacrosin using gel chromatography (60,000) and SDS-polyacrylamide gel electrophoresis (51,300) indicated that the zymogen is monomeric. Comparison with the molecular weight of ram acrosin (44,000 or 40,000, using the two respective methods) indicated that a single acrosin molecule is derived from each zymogen molecule. The sperm acrosin inhibitor (molecular weight 11,000 or 8,000) was present in testicular spermatozoa as well as in ejaculated spermatozoa; there was no evidence that it was produced as a result of zymogen activation.     

Effects of pH on the reactivation of human spermatozoa demembranated with Triton X-100

The aim of this work was to study the role of different parameters involved in the motility of human spermatozoa. Human spermatozoa were totally demembranated with 0.05% Triton X-100, and the demembranation was checked using electron microscopy. We have shown that, with a concentration of ATP-Mg lower than 2 mM, a pH effect was observed with a dose-dependent motility reactivation at pH 7.1, with 14% +/- 2.0% motile cells at 1 mM ATP-Mg and a straight line velocity (VSL) of 12.0 +/- 1.4 microns/sec. However, at pH 7.8, more than 65% of the spermatozoa were reactivated with as low as 0.02 mM ATP-Mg and 77.8% +/- 2.5% of them were motile at 1 mM ATP-Mg and had a VSL of 23.4 +/- 3.9 microns/sec. The depletion of free calcium by the addition of 0.5 mM EGTA in the reactivation medium (RM) improved the percentage of motile cells and the VSL most markedly at low ATP-Mg and low pH. If no MgSO4 was added in RM, cells were not motile at pH 7.8, but 30-40% reactivated at pH 7.1. If 5 mM Ca2+ was added to the RM, up to 88% of the cells became reactivated at both pHs, but the beat frequencies were very low, suggesting different mechanisms of reactivation when Mg2+ or when Ca2+ is present in the RM.     

Effect of triton X-100 on ultrastructure,reactivation, and motility characteristics of ram spermatozoa

For optimal extraction and reactivation of ram sperm, glutamate, dithiothreitol, magnesium, and cyclic AMP were required in a medium of pH 7.9. On extraction with 0.01 % Triton X-100, ram sperm were only partially demembranated, and extensive areas of plasma membrane remained intact especially in the midpiece region. Treatment with 0.1% Triton X-100 removed all plasma membranes and extracted the mitochondrial membrane and matrix. In the absence of ATP, 16.6% ± 0.4 of the partially demembranated sperm were motile, but sperm extracted with 0.1% Triton X-100 were completely immotile. On adding ATP partially demembranated sperm reactivated better (81.6% ± 2.8) than sperm completely demembranated in 0.1 % Triton X-100 (39.5% ± 4.6). The release of intracellular LDH rose linearly with increasing concentrations of the detergent from 0.01 to 0.05%, at which it plateaued. There was a significant increase in beat frequency and forward velocity of partially demembranated sperm when treated with ATP. Partially demembranated sperm had intact mitochondria that presumably were still able to produce ATP, although the spermatozoan movement was stimulated by exogenous ATP.     

The fertilizing capacity of ram testicular spermatozoa,freshly collected and after storage in cauda epididymal fluid

Epididymal fluid may contain substances which promote development of the fertilizing capacity of testicular spermatozoa under in vitro conditions, provided that the spermatozoa are exposed to such substances for long periods of time. In an attempt to resolve this question, the fertilizing capacity of testicular spermatozoa was assessed before and after storage in cauda epididymal fluid and comparisons made with ejaculated spermatozoa from the same rams. Of the 13 eggs examined from the group of ewes inseminated with ejaculated spermatozoa 61.5% were found to be in the 2-to 8-cell stage. No fertilized eggs were recovered from ewes impregnated with freshly collected testicular spermatozoa. Nor were any cleaved eggs obtained from the group of ewes inseminated with testicular spermatozoa stored in cauda epididymal fluid at 4°C for 7 to 11 days. We suggest there-fore, that in order to develop maximal fertilizing capacity, mammalian spermatozoa must be exposed to specific concentrated testicular and epididymal secretions in a sequential order and within strict time limits.     

Lipid composition and metabolism in testicular and ejaculated ram spermatozoa

1. Spermatozoa collected directly from the testis of the conscious ram contain 25% more phospholipid than ejaculated spermatozoa. The concentration of lecithin, phosphatidylethanolamine and ethanolamine plasmalogen was greater in testicular spermatozoa; little difference was observed in choline plasmalogen. Both types of spermatozoa had significant amounts of cardiolipin and alkyl ether phospholipid. 2. The fatty acids in the phospholipid extracted from testicular spermatozoa have a very high content of palmitic acid. The phospholipids of ejaculated spermatozoa contained less palmitic acid, but more myristic acid. 3. Ejaculated spermatozoa contained less acyl ester and cholesterol. It is suggested that lipids are a source of substrate for spermatozoa during their passage through the epididymis. 4. Testicular spermatozoa when incubated with [U-¹⁴C]glucose incorporated more radioactivity into the glycerol part of the phospholipid and neutral lipid fractions than did ejaculated cells. The distribution of radioactivity in the individual phospholipids and neutral lipids was similar for both cell types. No radioactivity was detected in choline plasmalogen, which accounted for approx. 40% of the total phospholipid. 5. Testicular spermatozoa incorporated more radioactivity from glucose into formate than into acetate, whereas a higher proportion of radioactivity was found in acetate in ejaculated cells. 6. The implications of these lipid changes in the process of spermatozoal maturation are discussed.     

Microtubules in mouse embryo fibroblasts extracted with Triton X-100

Treatment of mouse embryo fibroblasts with 1% Triton X-100 at 37 degrees C in the presence of 4M glycerol and 1 mM EGTA results in the extraction of about 80% cellular proteins. Indirect immunofluorescent staining with monospecific antibodies against tubulin showed that extracted cultures contained a well developed system of cytoplasmic microtubules, indistinguishable from a system of control non-extracted cells. Microtubules in extracted cells were sensitive to Ca2+ ions, and to cold or prolonged incubation in a glycerol-free buffer. Sodium dodecylsulphate-polyacrilamide gel electrophoresis revealed proteins co-electrophoresed with tubulin and actin in Triton-treated cultures. Electron microscopy demonstrated the presence of both microtubules and microfilament bundles in the extracted cells, but complete dissolution of plasma and intracellular membranes.     

Surface transformation of ram spermatozoa in uterine, oviduct and cauda epididymal fluids in vitro

Genital tract fluids were collected continuously from conscious ewes through catheters inserted surgically into the uterus and oviducts. Cauda epididymal spermatozoa and fluid were obtained through catheters inserted into the transected vas deferens. The washed spermatozoa were labelled using the surface-specific chloroglycoluril-Na125I procedure. High-resolution electrophoretic analysis of sperm plasma membrane preparations revealed a partial loss of a major surface component (i.e. Mr 97,000) during incubation in uterine and oviduct fluids. This specific loss resulted in a shift in radioactivity distribution toward an Mr 24,000 component which had been previously identified as a sialoglycoprotein. No significant changes in the distribution of radiolabelled surface components were detectable when the spermatozoa were incubated in synthetic medium. Incubation of unlabelled spermatozoa in 125I-labelled uterine fluid showed that adsorption of exogenous fluid components was highly selective; an Mr 16,000 polypeptide was greatly enriched on the sperm surface although it was only a minor component in the incubation fluid. Adsorption of labelled oviduct fluid components was also selective and involved predominantly high molecular weight components (i.e. Mr 140,000, 95,000, 78,000, 53,000). When spermatozoa were incubated in labelled cauda epididymal fluid after exposure to unlabelled uterine and oviduct fluids, several fluid components were incorporated by the plasma membrane, indicating that surface renovation of 'capacitated' spermatozoa may be a more general process rather than a specific event. These results suggest that capacitation of ram spermatozoa involves loss of specific surface proteins as well as selective adsorption of exogenous fluid components and point to a polypeptide in uterine fluid as an active constituent.     

Effect of transportation temperature on the quality of cauda epididymal spermatozoa of ram

The objective of this study was to develop a protocol for ram epididymal sperm preservation that could be applied to wild ruminants for collection and preservation of spermatozoa from dead or hunted animals. Ram testicles collected from abattoirs were used to study the effect of two transportation temperatures viz. ambient temperature (AT) and refrigeration temperature (RT) on the cauda epididymal sperm quality at recovery and during preservation up to 72h at 4°C. For AT the testicles were transported in normal saline in a container (17.9-21.5°C) where as for RT the testicles were transported in an ice-chest (4.9-6°C). The results of the current study revealed that intact acrosome was significantly higher (P<0.01) and other quality parameters like sperm motility, live sperm count, sperm concentration and major sperm abnormalities were also higher (P>0.05) for RT than AT. The mean percent sperm motility for RT and AT was 81.67% and 78.33%, respectively. The corresponding figures were 92.08% and 90.46% for mean live sperm, 98.33% and 90.50% for intact acrosome, 0.50% and 0.33% for major sperm defects. The percent minor abnormality was 79.50% for RT and 77.67% for AT. The most prevalent minor defect was distal cytoplasmic droplet (70-80%). The mean sperm motility for RT and AT at 0h was 82.50% and 75.00%, respectively and the corresponding values at 72h of preservation were 60.00% and 45.83%. The mean live sperm at 0h for RT and AT were 92.92% and 88.92%, respectively and the corresponding figures at 72h were 81.50% and 73.17%. The mean intact acrosome at 0h for RT and AT was 98.58% and 90.58%, respectively and at 72h the corresponding values were 91.66% and 82.25%. The sperm motility, live sperm count and intact acrosome decreased significantly (P<0.05) from 0h to 72h of preservation for both transportation temperatures. The sperm motility, live sperm count and intact acrosome also varied significantly between the transportation temperatures. The major sperm abnormality for both RT and AT at each hour of preservation up to 72h was less than 0.5%. The study concluded that epididymides or testicles should be transported to the laboratory at RT (4.9-6°C) either in an ice-chest or portable refrigerator for their processing, evaluation and storage.     

Dimeric nature of apo-low density lipoprotein extracted with Triton X-100.

Human low density lipoprotein (LDL) was dissolved in 0.3 to 2.0% Triton X-100 at pH 7.5 and apo-LDL (B protein) was extracted from LDL to form B protein-Triton complex. Sedimentation equilibrium study of this complex in a solvent nearly isopycnic to Triton X-100 showed that the molecular weight of the protein in the complex was 570,000. The complex eluted almost at the void volume of a Sepharose 6B column, as would be expected for a complex with a total molecular weight of roughly 900,000, on the assumption that 0.52 g of Triton was bound to 1 g of protein (Helenius, A. and Simons, K. (1972) J. Biol. Chem. 247, 3656-3661). The sedimentation coefficient of the complex gave f/fmin = 2.2, indicating that the complex was either as asymmetric as a fibrinogen molecule or not compact. These results show that B protein exists in its complex with Triton X-100 as an elongated or a loosely expanded dimer based on the molecular weight of monomeric B protein of 270,000. B protein may also exist in LDL as a dimer.     

Adenosine triphosphate-induced motility and sliding of filaments in mammalian sperm extracted with Triton X-100

Bull sperm that had been extracted with 0.2% Triton X-100 could be reactivated with ATP, and their movement closely resembled the motion of intact live sperm. Their motility required the presence of ATP, magnesium, and a medium of suitable salt concentration and pH. When Triton-extracted bull sperm were digested breifly with trypsin at pH 9.0, they appeared to reatin most of their normal structure, but subsequent exposure of the digested sperm to ATP caused a disintegration by light microscopy, using dark-field illumination, combined with an electron microscope study of preparations of the disintegrated sperm, demonstrated the presence of an active sliding mechanism of filament interaction in bull spermatozoa. Human sperm subjected to the same procedures showed similar patterns of reactivation and of disintegration.     

Characteristics of motor apparatus in testicular, epididymal and ejaculated spermatozoa. A study using demembranated sperm models

Morphological alterations of cytoplasmic organelles occur in the early stage of the cell fusion reaction induced by HVJ (Sendai virus) and these alterations are reversed in the late stage of the reaction. When the cells were treated with the virus at 37 °C for 3 min, the mitochondria became condensed, the cisternae of the endoplasmic reticula and Golgi stacks became highly distended, the inner space of the nuclear envelopes became enlarged, and the arrangement of 100 Å filaments became disordered. All these morphological changes were gradually reversed during further incubation at 37 °C and had been completely reversed by the end of the cell fusion reaction. These changes occurred at the time of interaction of the cell membrane with HVJ, and seemed to represent functional responses of the living cell to perturbation of the membrane resulting from its interaction with the virus.