Multiple protein kinase activities required for activation of sperm flagellar motility.

A specific peptide inhibitor of the cyclic AMP (cAMP)-dependent protein kinase (PKI-peptide) is a very effective inhibitor of the cAMP-dependent activation of motility of Ciona spermatozoa, when PKI-peptide is present at the beginning of incubation of demembranated spermatozoa with cAMP and ATP. Under conditions where approximately 120 sec is required for full activation of motility, the window of sensitivity to the PKI-peptide lasts for only 25-30 sec. Examination of sperm pellet proteins labeled with 32P ATP during activation reveals a major 25 kDa phosphoprotein and 2 minor phosphoproteins whose phosphorylation is highly sensitive to to inhibition by the PKI-peptide and essentially complete during this early phase. These sperm proteins appear to be immediate substrates for cAMP-dependent protein kinase, and phosphorylation of one or more of these appears to be requires, but not sufficient, for activation of motility. The phosphorylation of other proteins is reduced or eliminated when PKI-peptide is present at the beginning of incubation, but is unaffected by later addition of PKI-peptide. Some of these substrates appear to be likely candidates for axonemal proteins that must be phosphorylated during the later stages of incubation in order to complete the activation process. This selection is based upon a high degree of inhibition by inclusion of PKI-peptide or other inhibitors at the start of the incubation process, on near-completion of their phosphorylation by the end of the 2 min incubation period required for the activation of motility, and evidence that these proteins are phosphorylated during in vivo activation of motility. Although these observations suggest the presence of a second kinase activity that is upregulated by the initial activation of the cAMP-dependent protein kinase, assays using exogenous substrates have not yet been able to identify such a kinase activity.

Polyamines in ejaculated ram spermatozoa and their relationship with sperm motility.

Intracellular polyamine levels in ejaculated spermatozoa and seminal fluid from rams were determined by fluorescent spectroscopy of their dansyl derivatives. Relationships between the sperm polyamine content and sperm motility of six mature and eight pubescent rams were studied. Samples were collected from both groups once a month from August through October. Mature rams had a greater percentage of motile sperm cells than lambs (94% versus 73% in September and 92% versus 78% in October); higher spermidine content (36 versus 9 pmol/10(8) cells in September and 162 versus 55 pmol/10(8) cells in October); higher spermine content (984 versus 205 pmol/10(8) cells in September and 1,229 versus 414 pmol/10(8) cells in October); and higher total sperm polyamine content (1,021 versus 216 pmol/10(8) cells in September and 2,258 versus 973 pmol/10(8) cells in October). In the lambs, spermidine content increased (55 versus 9 pmol/10(8) cells); spermine content increased (414 versus 205 pmol/10(8) cells); and total sperm polyamine content increased (973 versus 215 pmol/10(8) cells) in October compared to September. Ejaculates with sperm motility higher than 85% had greater spermine (848 versus 234 pmol/10(8) cells in September and 1064 versus 449 pmol/10(8) cells in October), and total sperm polyamine content (882 versus 244 pmol/10(8) cells in September and 2,015 versus 1,008 pmol/10(8) cells in October) than ejaculates with less than 450 pmol total sperm polyamines/10(8) cells was 68% +/- 6% compared to 90% +/- 4% in cells with greater than 450 pmol (average for all ejaculates) total sperm polyamines/10(8) cells. These data suggest a positive relationship between sperm polyamine constant and sperm motility.(ABSTRACT TRUNCATED AT 250 WORDS)

Isotypes of protein kinase C in bovine sperm.

Protein kinase C is present in bovine epididymal sperm. The enzyme was partially purified by gel filtration on Sephacryl S-300. The Ca2+/phosphatidylserine-dependent histone phosphotransferase activity elutes from the gel filtration column in a manner corresponding to a Mr approximately 80 kDa. The activity peak also corresponds with [3H]phorbol 12,13-dibutyrate binding activity. Immunoblot analysis of the partially purified enzyme with isozyme-specific monoclonal antibodies revealed the presence of alpha-, beta-, and gamma-subspecies of protein kinase C. Indirect immunofluorescence showed that the antibodies against alpha-, beta-, and gamma-subspecies produced prominent staining of the postacrosomal region of the sperm head. In addition, beta-subspecies antibodies produced minor staining of the midpiece and gamma-subspecies antibodies produced a minor staining of the acrosomal region.

Casein kinase I in bovine sperm: purification and characterization.

A highly purified preparation of sperm casein kinase I was obtained by sequential chromatography with phosphocellulose, gel filtration on sephacryl S-300, Affi-gel blue and DEAE-Cellulose. The chromatographic behavior and properties of the enzyme suggest that the sperm enzyme is similar to casein kinase I from other tissues. Antibodies against calf thymus casein kinase I cross-react with the sperm enzyme. A special feature of the sperm enzyme is that the activity is stimulated by spermine.

Purification and characterization of polyamine-stimulated protein kinase (casein kinase II) from bovine spermatozoa.

Casein kinase II from bovine epididymal spermatozoa was purified to apparent homogeneity by repeated chromatography with phosphocellulose and gel filtration with sephacryl S-200. The purified enzyme exhibited a molecular mass of 130 kDa by gel filtration and displayed three polypeptide bands with molecular masses of 26, 33, and 36 kDa by SDS-polyacrylamide gel electrophoresis. Antibodies raised against calf thymus casein kinase II cross reacted with the three sperm polypeptides. Incubation of the holoenzyme with either [gamma-32P]ATP or [gamma-32P]GTP resulted in the phosphorylation of the 26-kDa subunit. The enzymatic activity with casein as substrate was strongly inhibited by nanomolar heparin and greatly stimulated by micromolar spermine. With casein as substrate, the specific activity of the pure enzyme (0.5 mumol/min/mg protein) was comparable to that of casein kinase II from other sources. Endogenous substrates of the kinase were demonstrated by incubating sperm cytosolic extracts with [gamma-32P]GTP, under conditions that limit the expression of other protein kinases, and analyzing the products by SDS-PAGE and autoradiography. Similar results were obtained when sperm extracts, suitably diluted to minimize endogenous casein kinase II, were incubated with [gamma-32P]GTP and aliquots of pure sperm casein kinase II. Low concentrations (50 microM) spermine strongly enhanced the phosphorylation of 92- and 106-kDa cytosolic proteins. Our results clearly show that casein kinase II is present in spermatozoa and that it shares many of the properties of the enzyme from other sources. Further, they indicate that the enzyme plays a role in mediating the phosphorylation state of sperm proteins.

Casein kinase II activity and polyamine-stimulated protein phosphorylation of cytosolic and plasma membrane proteins in bovine sperm.

A highly purified preparation of sperm cytosolic protein kinase was obtained by repeated chromatography with phosphocellulose. The preferred substrate of the enzyme was casein and the activity was not stimulated by added Ca2+, calmodulin, or cAMP. With casein as substrate, both ATP and GTP served as phosphate donors and the activity was inhibited by low micromolar heparin and stimulated by low millimolar spermine and spermidine. These properties are characteristic of casein kinase II from other cells. Endogenous protein substrates of the enzyme in sperm cytosolic fractions and in plasma membranes were demonstrated by incubating the preparations with [gamma-32P]GTP, under conditions unfavorable to other protein kinases, and analyzing the products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Spermine greatly enhanced the phosphorylation of three (55, 92, and 106 kDa) proteins in both cytosolic and plasma membrane preparations. Our results indicate that polyamines play a role in modulating the phosphorylation state of proteins in sperm and may further regulate sperm function through this mechanism.

Calmodulin-stimulated cyclic nucleotide phosphodiesterases in plasma membranes of bovine epididymal spermatozoa.

Cyclic nucleotide phosphodiesterase in the plasma membranes of bovine epididymal spermatozoa was stimulated by added Ca2+ and calmodulin. The rate of hydrolysis and responsiveness toward calmodulin was greater for cAMP than for cGMP. The kinetic analysis of the activity revealed two forms of phosphodiesterase with apparent Km values of 7.5 and 95 microM for cAMP. Calmodulin stimulated both of the activities by increasing the Vmax without affecting the Km's. The activity response with respect to Ca2+ concentration appears to be biphasic in both the absence and presence of added calmodulin. Trifluoperazine inhibited the Ca2+- and calmodulin-sensitive enzyme activity in a dose-dependent manner. The calmodulin-stimulated phosphodiesterase activity in the sperm plasma membranes can be solubilized and absorbed to a Calmodulin-Sepharose affinity column in the presence of Ca2+.

Inhibition of human lens aldose reductase by flavonoids, sulindac and indomethacin.

The inhibition of human lens aldose reductase by flavonoids has been studied. Quercetin, the major pentahydroxyflavone, was observed to inhibit human lens aldose reductase by 50% at a concentration of 5 X 10(-6) M. The inhibitory activity of its 3-O-glucoside was similar to that of the parent aglycon. Glycosidation with L-sugar (quercitrin and guaijaverin), however, improved the inhibitory activity (the IC50 values being 1 X 10(-6) M and 2.5 X 10(-6) M respectively). The improvement in inhibitory activity with glycosidation with L-sugar was also apparent from the high inhibitory activity of myricitrin as compared to myricetin, although the improvement in this case of hexahydroxy flavone glycosidation was significantly less than in the case of penthahydroxy flavone glycosidation. The structure-activity relationship observed for human lens enzyme was similar to that reported previously for rat lens enzyme. Inhibitory activity on the whole however, was lower with human lens enzyme. Some known inhibitors of cyclo-oxygenase such as indomethacin, aspirin and sulindac also inhibited human lens aldose reductase. Thus, an inhibitor of one of the enzymes may actually inhibit both and, when administered, may exert mixed physiological effects.

Subcellular localization of adenylate cyclase of buffalo spermatozoa.

The intracellular localization of adenylate cyclase and 3',5'-cyclic nucleotide phosphodiesterase in buffalo sperm was examined. Adenylate cyclase activity is distributed in heads (8.4%), midpieces (16.6%), tails (49.5%) and 5.7% in the soluble supernatant; the total recovery being 81%. A 4-fold increase in specific activity was observed in the tail fraction relative to sonicated suspension. Further fractionation of the tail fraction into plasma membrane and microtubules by dialysis against low ionic strength buffer was followed by marker enzymes (Mg2+ -ATPase, 5'-nucleotidase and alkaline phosphatase) as well as by examination of fractions under electron microscope. The recovered adenylate cyclase (79%) was found in microtubules (45%) and plasma membrane (34%). Cyclic nucleotide phosphodiesterase in tails was distributed in tail plasma membrane (13.7%), microtubules (31.5%) and cytosol (34%) with a total recovery of 80%. Similar results were obtained when the distribution of adenylate cyclase and cyclic nucleotide phosphodiesterase was studied by treatment with Triton X-100; 40% activity of adenylate cyclase present in tails (about 20% relative to sperm sonicate) appeared in the soluble form by this method. The results are discussed in relation to control of cyclic AMP levels in buffalo sperm by adenylate cyclase and cyclic nucleotide phosphodiesterase.

Adenosine 3'5'-monophosphate phosphodiesterase of buffalo spermatozoa.

The cyclic AMP-phosphodiesterase (EC 3.1.4.17) of buffalo spermatozoa is distributed in the head, mid-piece and tail fractions and has multiple forms, 70% of which is in the bound form. The bound enzyme was not solubilized by Triton X-100, lubrol or hyamine 2389. Kinetic measurements of the soluble enzyme showed two apparent Km values for low and high cAMP concentrations, i.e. 4.5 and 100 micro M with Vmax values of 0.25 and 2.0 nmol cAMP hydrolysed min-1 mg protein-1. The bound enzyme had an apparent Km of 66.6 microM with a Vmax of 0.75 nmol cAMP hydrolysed min-1 mg protein-1. The pH for optimum enzyme activity was 7.5 and Mg2+ was essential for the activity of the soluble and bound enzymes. Methylxanthines, ATP, ADP and ppi inhibited the soluble and bound enzymes, ATP being the most potent inhibitor.

Distribution of beta-N-acetylglucosaminidase, hyaluronoglucosaminidase and acrosin in buffalo and goat spermatozoa.

The distribution of beta-N-acetylglucosaminidase, hyaluronoglucosaminidase and acrosin in buffalo and goat sperm acrosomes was studied. The three hydrolases were found to occur in soluble and bound forms. In the bound form, they were associated with the denuded sperm and were maximally solubilized at pH 3.0. The possible role of beta-N-acetylglucosaminidase in fertilization is discussed.

Acrosomal hydrolases in buffalo spermatozoa.

Buffalo sperm acrosome resembles its counterpart in other species, being rich in hydrolytic enzymes. Of the enzyme activities estimated, acid phosphatase, beta-N-acetylglucosaminidase and hyaluronidase were low compared to those of ram semen. However, the aryl sulphatase activity was high. GOT activity estimated in sperm preparation may not be of acrosomal origin.