Regulation of SeaUrchin Sperm Cyclic AMP-Dependent Protein Kinasesby an EggAssociatedFactor'

Transcription

1 BIOLOGY OF REPRODUCTION 22, 526â 532 (198) Regulation of SeaUrchin Sperm Cyclic AMP-Dependent Protein Kinasesby an EggAssociatedFactor' DAVID L. GARBERS, D. JANETTE TUBB and GREGORY S. KOPF Departments ofpharmacology and Physiology, and The Howard Hughes Medical Institute Laboratory at Vanderbilt University, Nashville, Tennessee ABSTRACT Experiments were designed to determine whether the elevation of sea urchin sperm cyclic AMP (camp) concentrations results in an activation of sperm camp-dependent protein kinases. The protein kinase activity ratio (enyme activity in the absence of added camp/enyme activity in the presence of 1.8 pm camp) varied between.1 â.2 under basal conditions, but increased to â œ -â.3 in cells treated with 1.5 mm theophylline. A fucose-sulfate rich factor (FS-1) purified from the jelly coat of sea urchin eggs markedly increased sperm camp concentrations and increased the protein kinase activity ratio to values >.8. After addition of FS-1 to the spermatooa, the protein kinase activity ratio was increased to maximal values within 15 sec, but declined to near basal values within 5 mm. The sperm camp concentrations followed a similar time course in response to FS-1. In the absence of added Ca2, FS-1 failed to elevate either sperm camp concentrations or the protein kinase activity ratios. Ca2@transport antagonists such as verapamil and D-6, when added to spermatooa in the presence of Ca2, blocked both FS-1-induced elevations of camp concentrations and the activation of the camp-dependent protein kinase. These data demonstrate that the elevation of sperm camp concentrations result in an activation of the sperm camp-dependent protein kinase. Since D-6 and verapamil block the elevation of camp and the activation of the camp-dependent protein kinase in response to FS-1, it appears that Ca2@may function as the mediator of the effects of FS-1 on sperm camp concentrations. INTRODUCTION A factor(s) associated with the jelly coat layer of sea urchin eggs is known to cause marked elevations of sea urchin sperm camp concentrations (Garbers and Hardman, 1975, 1976) and to activate adenylate cyclase when added to intact, but not broken, sperm cell preparations (Watkins et a!., 1978). Both the activation of adenylate cyclase and the ele vation of camp concentrations require the presence of extracellular Ca2@ (Watkins et a!., 1978; Tubb et a!., 1978). The mechanism by which elevated sperm camp concentrations might bring about changes in sperm function is not known at this time, but it has been sug gested that elevated camp concentrations result in the activation of camp-dependent protein kinases, with resultant phosphorylation of specific, functional sperm proteins (Hoskins Accepted December 11, Received September 18, â Supported by NIH grants HD-125 and HD and Casillas, 197, 1975). Experiments de signed to test such a hypothesis have met with limited success, however, due to limited incor poration of added radiolabeled phosphate into cellular ATP (Babcock et al., 1975). Huacuja ct al. (1977), however, have reported a camp dependent phosphorylation of specific mem brane components of human spermatooa and Brandt et a!. (1979) have recently observed a camp-mediated increase in the phosphorylation of bovine sperm proteins they believe to be associated with motility. To date, however, there have been no reports demonstrating that sperm camp-dependent protein kinase activity is modulated in vivo by a hormone or other agent. In these studies a purified, fucose-rich factor (FS-1) obtained from the jelly coat of sea urchin eggs is shown to activate sea urchin sperm camp-dependent protein kinases. Both the elevation of camp concentrations and the activation of the sperm camp-dependent protein kinases were shown to be Ca2+@ dependent events that could be blocked by the Ca2@ transport antagonists, D-6 and verapamil. 526

2 SPERM PROTEIN KINASE 527 Materials MATERIALS AND METHODS All common chemicals were of highest reagent grade and were obtained from Sigma Chemical Co. or Fisher Scientific Co. Amersham Corp. supplied the [3H1-cAMP and [7-'2PJ-ATP. The [y-32p1-atp was synthesied by the method of Walseth and Johnson (1979). Histone Il-A (calf thymus), ATP and camp were from Sigma Chemical Co. Theophylline was from Merck Laboratory Chemicals and Dowex 5 (AG 5W-X8, 1â 2 mesh) was from Bio-Rad Labora tories. Verapamil and D-6 were purchased from Knoll, Ag (West Germany) and were stored in a solution containing 3 parts ethanol: 1 part dimethyl formamide, at â 2 C. Activated coconut charcoal (5â 2 mesh) and Whatmann 3 mm filter papers were obtained from Fisher Scientific Co. Strongylo centrotus purpuratus sea urchins were obtained from Pacific Bio-Marine, Venice, CA. Methods Preparation of spermatoo Sperm were collected and washed in normal sea water (ph 7.9) as described previously (Garbers and Hardman, 1976) and then were resuspended to a final concentration of between 9â 125 mg (wet weight)/ml. Cells were stored at â 2 Cuntil assay. Preincubation of spermatooa prior to assay of camp-dependent protein kinase activity. Washed sperm suspension (1 ml) was added to 2 p1 sea water or to 2 p1 of a solution containing various test substances and incubated for various periods of time at 16 C as indicated in the legends to the figures. These incubations were terminated by the addition of 5 ml buffer containing 5 mm NaCl and 1 mm potassium phosphate (ph 6.5). The cell suspensions were homogenied with one 1 sec burst using an Ultraturrax homogenier and then immediately froen on dry ice and centrifuged at 26, X g in a Beckman JA-17 rotor for 15 mm at  C.The resultant superna tant fractions were assayed for protein kinase activity as described below. Protein kinase assay. The protein kinase activity was estimated in the absence or presence of 1.8 pm camp in an assay mixture containing 9. mm MgC12, 9 mg/ml histone la,.18 mm ATP, 36 mm NaF, 91 pm 3-isobutyl-1-methylxanthine, 1â 2 X 16 dpm b'-32p1-atp, 5 mm (N-[2-acetamidoj -iminodiacetic acid) (ADA) buffer at ph 6.8 and 2 p1 of the sperm supernatant fluid in a final reaction volume of 11 p1. Assay mixtures were incubated at 3 Cfor 5 mm; reactions were then terminated and protein kinase activity determined as described by Corbin et al. (1973). The rate of histone phosphorylation was constant as a function of time for at least 1 mm at 3 C.The data are presented as the protein kinase activity ratio, which is determined by comparing enyme activity in the absence of added camp to the activity in the presence of 1.8 pm camp. Determination of sperm camp concentrations. When sperm camp concentrations were determined, the cells were incubated basically as described for the protein kinase preincubation. Sperm suspension (5 pl) was added to 1 p1 sea water containing various agents and incubated at 16 C for various periods of time as indicated in the legends to the figures. Incu bations were terminated by the addition of 1 ml.5 N perchloric acid, containing tracer amounts of [3HJ camp for estimation of sperm cyclic nucleotide recoveries. Time-ero cyclic nucleotide concentrations were estimated by adding spermatooa directly to sea water containing the perchloric acid. The acidified cell suspensions were then froen and thawed 5 times prior to purification of camp on Dowex 5 columns (.7 X 15. cm) as described by Schult et al. (197). Column fractions containing camp were lyophilied and then dissolved in.5â 1. ml de ionied water. Cyclic AMP was assayed by the method of Gilman (197), as modified by Brostrom and Kon (197) and also by the modified (Harper and Brooker, 1975) radioimmunoassay method of Steiner et al. (1969). Purification of FS-1. The activity of the factor was followed throughout all purification steps by its ability to elevate sperm camp concentrations. Basic ally, the purification of FS-1 was as follows: Egg factors were obtained after S. purpuratus eggs were allowed to stand (2% suspensions v/v) in sea water at ph 5. for 2 mm at  C. The supernatant fluid containing the egg factors was obtained by mild centrifugation of the eggs (2 X g; 2 mm;  C)and the fluid was then applied to charcoal columns (2.6 X 3 cm) preequilibrated with distilled water. FS-1 was not adsorbed to this column and was recovered in the effluent. A small molecular weight, sperm respiratory stimulating factor, however, was retained by this column (Kopf et al., 1979). Three liters of 95% ethanol were added to each liter of effluent obtained from the charcoal column; this mixture was constantly sitrred at â 2 Cand then centrifuged at 35 X g for 15 mm. The pellet containing the large molecular weight factor (FS-1) was dissolved in, and dialyed extensively against, distilled water. After dialysis and lyophiliation of the dissolved pellet, the dried powder was dissolved in a solution containing.25 M NaCI,.25 M sucrose and 1 mm Tris (ph 7.9). The solution (8 ml) containing FS-1 (1 mg/ml) was then layered over a solution (13 ml) containing 5% sucrose,.25 M NaC1 and 1 mm Tris (ph 7.9) and centrifuged at 16, X g for 16 h at  Cusing a Beckman 5.2 Ti rotor. FS-1 was pelleted by this procedure and ap peared as a clear gel at the bottom of the tube. A material containing sialic acid, similar to the one described recently by SeGall and Lennar (1979), did not centrifuge into the pellet. The pellet was dissolved in.5 M NaCl, 1 mm Tris (ph 7.9) and then chro matographed on a Sephadex G-2 column (2.6 X 3 cm). The gel filtration step separated residual traces of a sperm respiratory-stimulating factor from FS-1. Purified FS-1 migrated as a single, alcian blue-staining band upon electrophoresis on cellulose acetate. The factor contained principally fucose and sulfate and some associated protein (12%). Although SeGall and Lennar (1979) have recently suggested that the fucose-sulfate present in egg jelly exists as a polymer without covalently linked protein, they also reported from â 12% protein associated with the polysac charide. The quantity of FS-1 added in subsequent experiments was monitored by measuring fucose content, as described by Dische and Shettles (198).

3 528 GARBERS ET AL. Calcium dependent effects of PS-i. The effects of Ca2 on the factor-induced changes in sperm cyclic AMP concentrations and protein kinase activity were examined using cells washed and stored in sea water (ph 7.9). Procedures for the determination of protein kinase activity and camp concentrations were as previously described except that varying amounts of added to the preincubation and incubation mixtures. RESULTS Cyclic AMP-Dependent Protein Kinase Activity in Sea Urchin Sperm Homogenates Spermatooa were routinely homogenied in a buffer containing 1 mm potassium phos phate, 1 mm EDTA,.5 M NaCI and.5 mm 3-isobutyl-1-methylxanthine, at ph 6.5. In some instances, a hypotonic buffer containing 25 mm triethanolamine and 1 mm dithio threitol at ph 7.6 was used for homogeniation. Basal protein kinase activity in the high salt buffer averaged 19.7 ± 1.2 (n = 23) pmol 32p incorporated into histone/rnin/mg wet weight cells extracted. The activity was in creased to 168 ±5.2 (n = 22) pmol 32P incor porated/min/mg wet weight cells extracted in the presence of 1.8 pm camp. In hypotonic buffer, basal protein kinase activity averaged 21.3 ±2.1 (n = 11) and was increased to 166 ±.3 (n = 12) pmol 32P incorporated into histone/ mm/mg wet weight cells by the addition of camp. Thus, the protein kinase activity ratio (â camp/+ camp) in control cells averaged between.12â >- I > I U) â A n- I I I I CELL DITION (mqwetwt/mi Hamo@.Bufter) FIG. 1. Effects of dilution on the FS-1 activation of the camp-dependent protein kinase of sea urchin spermatoo Intact cells were incubated in the ab sence or presence of the factor for 1 mm and the cells were then diluted as shown on the abscissa and homog enied in either a hypotonic buffer or a high salt buffer according to the methods outlined in Materials and Methods. Open circles (), no factor, high salt medium; open triangles (@ ), factor, high salt medium; closed boxes (.), factor, hypotonic medium. The values represent the means from 2 individual experi ments. of FS-1 did not alter the total protein kinase activity, since in the presence of maxima! amounts of FS-1 the protein kinase approached an activity of'\'168 pmol 32P incorporated into histone/min/mg wet weight cells. Conditions Required to Stabilie the Equilibrium Between the Protein Kinase Subunits In initial experiments, the buffer containing the.5 M NaC1 was compared to the hypotonic buffer for effects on protein kinase activity after treatment of spermatooa with FS-1. As first reported by Corbin et a!. (1973) the presence of high sodium chloride concen trations appeared to prevent the reassociation of the regulatory and catalytic subunits of the protein kinase (Fig. 1). When cells were diluted to various degrees in the high salt homogeni ation medium, there was no change in the protein kinase activity ratio in either the absence (open circles) or presence (open triangles) of FS-1. The hypotonic buffer, however, did not appear to prevent this re association (Fig. 1; closed boxes). The addition Concentration-Response Relationship Between FS-1 and Protein Kinase Activity In the initial experiments, the amount of FS-1 added (expressed as pg added fucose) was varied to compare the effects of FS-1 on camp concentrations and the protein kinase activity ratios. Two different preparations of FS-1 resulted in the composite concentration response curves shown in Fig. 2. Half-maximal stimulation of the protein kinase activity ratio occurred at â \@.5pg added fucose in the absence of theophylline and at â \â.8 pg added fucose in the presence of theophylline (Fig. 2). The camp concentrations of these sperm samples were also determined and were found to be closely correlated with the protein kinase activity ratios (Fig. 3). Thus, an approximately

4 SPERM PROTEIN KINASE > U w U) FUCOSEAODED(LIg) FIG. 2. Concentration-response relationship be tween the purified FS-1 and sperm protein kinase activity ratios. Various concentrations of factor (expressed in terms of pg fucose) were added to sper matooa in the absence (o) or presence (t@)of 1.5 mm theophylline and were incubated for 1 mm prior to homogeniation. Protein kinase was assayed as described in Materials and Methods. The data presented represent the means of 2 individual experi ments. 8-fold elevation of camp concentrations resulted in an increase in protein kinase activity from 3). â œ -â 1% to 8% of maxima! activity (Fig. Time Dependent Changes in Sperm camp Concentrations and camp-dependent Protein Kinase Activity The addition of a maximal concentration of FS-1 (5 pg fucose/ml) caused a near maximal activation of the camp-dependent protein kinase within 15 sec (Fig. ). The protein kinaseactivityratioremained elevatedforâ \i3 mm and then declined rapidly, reaching a basal activity ratio between 5â 1 mm. The cyclic AMP concentrations also reached a sharp peak at â œ 1mm and then declined precipitously (Fig. ). Although the camp concentrations decreased at a faster rate than the protein kinase activity ratios, they remained at elevated levels until 5â 1mm after the beginning of the CYCLICAMP (nmol/g wet weightsperm) FIG. 3. Correlation between sperm camp concen trations and protein kinase activity ratios. Both parameters were modulated by the addition of pun fied FS-1 in the same experiments described in the legend to Fig. 2. Protein kinase activity ratios (Fig. 2) were plotted against camp concentrations for a given factor concentration (expressed as pg added fucose/ ml). incubation. Theophylline, alone, also caused a slightincreasein both the protein kinase activity ratios and camp concentrations. Ca2 Requirement The effects of FS-1 (5 pg added fucose/mi) on camp elevations and the camp-dependent protein kinase were studied in the presence or absence of Ca2@. In the absence of added Ca2@ neither the sperm camp concentrations nor the protein kinase activity ratios were increased when cells were incubated with a maximal concentration of the factor (Fig. 5). Calcium, at concentrations of. mm or greater restored the camp elevations and protein kinase activity ratio responses to the added factor. The calcium dependence of these two events was also studied using two putative antagonists of Ca2@ transport, D-6 and verapamil. These compounds have been demonstrated to reduce the transmembrane Ca2 conductivity in cardiac muscle (Kohlhardt et a!., 1972) and have recently been shown to block 5Ca2@-uptake and the acrosome reaction of sea urchin sper

5 53 GARBERS ET AL > TIME (mm) FIG.. The time course of purified FS-1 (5 pg fucose/ml)-induced elevation of sperm camp concen trations and activation of the camp-dependent protein kinase. Parallel experiments measuring camp concen trations and protein kinase activity were carried out as described in Materials and Methods., â, no A, factor; open figures, camp concentrations; closed figures, protein kinase activity ratios. The data presented represent the means of 3 individual expeni ments. matooa (Schackmann et al., 1978). D-6 or verapamil (1 pm) almost completely blocked the ability of FS-1 to elevate sperm camp concentrations (not shown) or to activate the camp-dependent protein kinase in the presence of 9.6 mm Ca2@(Fig. 6). DISCUSSION This report describes the first correlation between the in vivo activity of camp-dependent protein kinase and the intracellular camp concentrations of spermatoo The enyme activity ratios in spermatooa were increased to values of >.8 by increasing intracellular camp concentrations <8-fold. The increase in enyme activity did not appear to be an artifact occurring as a secondary consequence of cell homogeniation procedures. Such possi bilities were ruled out by the dilution experi ments (Fig. 1). FS-1 appeared to be very potent with respect to its ability to increase the sperm protein kinase activity ratios. If one assumes a Ct?@mM FIG. 5. Ca2 requirements for the FS-1 (5 pg fucose/mi)-induced sperm camp elevation and camp dependent protein kinase activation. Parallel experi ments measuring camp concentrations and enyme activity were run using cells washed and suspended in Ca2@-free sea water. Incubations were for 1 mm. Incubation mixtures contained various concentrations of CaC12 to give the final Ca2@concentration indicated on the absciss Assays were run as described in Materials and Methods. a, â, no factor; A, A, factor; open figures, camp concentrations; closed figures, protein kinase activity ratios. The values represent the means of 2 separate experiments. minimal molecular weight of 5 X 16 (this probably represents the minimal molecular weight, since the factor migrates at the void volume of A-Sm columns and continues to pellet after centrifugation on 6% cesium chloride) for the factor and a composition of % fucose (by weight), it can be calculated that 17 and 3 molecules (factor)/sperm cell can bring about the observed half-maximal activation of the enyme in the absence and presence of theophylline, respectively. The physiological consequences of the sperm camp-dependent protein kinase activation are not yet clear. Since the enyme has been found to be present in substantial quantities in all spermatooa studied to date (Hoskins et a!., 1972; Garbers et al., 1973; Lee and Iverson, 1976), it would appear that the protein may have important regulatory function(s). Whereas the mammalian spermatooa may contain multiple forms of protein kinase (Hoskins et a!., 1972; Garbers et a!., 1973), sea urchin sper

6 SPERM PROTEIN KINASE D6O@VERAPAMIL 2 FIG. 6. Effects of D-6 and verapamil on the FS-1 (5 pg fucose/ml)-induced elevation of the sperm camp-dependent protein kinase activity ratios. D-6 and verapamil were added to sperm cells suspended in sea water containing a maximal concentration of factor. Incubations were for 1 mm. The drug controls were ethanol:dimethylformamide (3: 1) in place of D-6 or verapamil. Assays were as described in Materials and Methods and the final calcium concen tration was 9.6 mm. (.), solvent + factor (ratio =.86) and (.), solvent alone (ratio.25); (), D-6; (tx), verapamil. The values presented represent the means from 2 separate experiments. matooa may contain only one form (Lee and Iverson, 1976). It is of interest to note that the activation of the protein kinase by FS-1 is Ca2@ dependent and is, therefore, similar to the absolute Ca2 requirement for the induction of the sperm acrosome reaction (Dan, 195). The activation of the protein kinase could, therefore, be related to this morphological process. Phosphorylation of specific cytosolic or membrane proteins by the enyme could be required for, or be associated with, the induc tion of the acrosome reaction. Alternatively, or in conjunction with effects on the acrosome reaction, protein phosphorylation could be associated with alterations in the sperm motil ity state (Brandt et al., 1979). REFERENCES (pm) Babcock, D. F., First, N. L. and Lardy, H. A. (1975). Transport mechanism for succinateand phos phate localied in the plasma membrane of bovine spermatoo J. Biol. Chem. 25, 688â 695. Brandt, H., Brehmer, L. and Hoskins, D. D. (1979). Evidence for the existence of sperm camp dependent phosphorylated motility proteins. Fed. Proc.38,315. Brostrom, C.. and Kon, D. (197). An improved protein binding assay for cyclic AMP. Anal. Biochem. 58, 59â 68. Corbin, J. D., Soderling, T. R. and Park, C. R. (1973). Regulation of adenosine 3', 5'-monophosphate dependent protein kinase. I. Preliminary charac teriation of the adipose tissue enyme in crude extracts. J. Biol. Chem. 28, 1813â Dan, J. C. (195). Studies on the acrosome. III. Effect of calcium deficiency. Biol. Bull. 17, 335â 37. Dische, Z., Shettles, L. B. and Osnos, M. (199). New specific color reactions of hexoses and spectro photometric micro methods for their determi nation. Arch. Biochem. Biophys. 22, 169â 18. Garbers, D. L. and Hardman, J. G. (1975). Factors released from sea urchin eggs affect cyclic nucleo tide metabolism in sperm. Nature 257, 677 Garbers, D. L. and Hardman, J. G. (1976). Effects of egg factors on cyclic nucleotide metabolism in sea urchin sperm. J. Cycl. Nuc. Res. 2, 59â 7. Garbers, D. L., First, N. L. and Lardy, H. A. (1973). Properties of adenosine 3', 5'-monophosphate dependent protein kinases isolated from bovine epididymal spermatoo J. Biol. Chem. 28, 875â 879. Gilman, A. G. (197). A protein binding assay for adenosine 3', 5'-cyclic monophosphate. Proc. Nat. Acad. Sci. USA 67, 35â 312. Harper, J. F. and Brooker, G. (1975). Femtomole sensitive radioimmunoassay for cyclic AMP and cyclic GMP after 2'O acetylation by acetic anhydride in aqueous solution. J. Cycl. Nuc. Res. 1, 27 â 218. Hoskins, D. D. and Casillas, E. R. (197). Hormones, second-messengers, and the mammalian sper matooan. In: Advances in Sex Hormone Re search. Vol. 1. (R. L. Singhal and J. A. Thomas, eds.). University Park Press, Baltimore. pp. 283â 32. Hoskins, D. D. and Casillas, E. R. (1975). Function of cyclic nucleotides in mammalian spermato o In: Handbook of Physiology. (R.. Greep and E. B. Astwood, eds.). American Physiology Society, Washington, D.C. pp. 53â 6. Hoskins, D. D., Casillas, E. R. and Stephens, D. T. (1972). Cyclic AMP-dependent protein kinases of bovine epididymal spermatoo Biochem. Biophys. Res. Commun. 8, 1331â Huacuja, L., Delgado, N. M., Merchant, H., Pancardo, R. M. and Rosado, A. (1977). Cyclic AMP induced incorporation of 33Pi into human sper matooa membrane components. Biol. Reprod. 17, 89â 96. Kohlhardt, M., Bauer, B., Krause, H. and Fleckenstein, A. (1972). Differentiation of the transmembrane Na and Ca channels in mammalian cardiac fibers by the use of specific inhibitors. Pflugers Arch. 335, 39â 322. Kopf, G. S., Tubb, D. J. and Garbers, D. L. (1979). Activation of sperm respiration by a low molec

7 532 GARBERS ET AL. ular weight egg factor and by 8-Bromoguanosine 3', 5'-monophosphate. J. Biol. Chem. 25, 855â 856. Lee, M.Y.W. and Iverson, R. M. (1976). An adenosine 3', 5'-monophosphate dependent protein kinase from sea urchin spermatoo Biochem. Biophys. Acta 29, 123â 136. Schackmann, R. W., Eddy, E. M. and Shapiro, B. M. (1978). The acrosome reaction and activation of Strongylocentrotus purpuratus sperm: Ion re quirements and movements. Dcv. Biol. 71, 33â 8. SeGall, G. K. andlennar, W. J. (1979). Chemical characteriation of the component of the jelly coat from sea urchin eggs responsible for induc tion of the acrosome reaction. Dcv. Biol. 71, 33â 8. Steiner, A. L., Kipnis, D. M., Utiger, R. and Parker, C. (1969). Radioimmunoassay for the measure ment of adenosine 3', 5'-cyclic phosphate. Proc. Nat. Acad. Sci. USA 6,67â 73. Tubb, D. J., Kopf, G. S. and Garbers, D. L. (1978). The elevation of sperm adenosine 3', 5'-mono phosphate concentrations by factors released from eggs requires calcium. Biol. Reprod. 18, 181â 185. Walseth, T. and Johnson, R. A. (1979). The enymatic preparation of [cs-32p1 nucleoside triphosphates, cyclic [32P1 AMP and cyclic [32P] GMP. Bio chem. Biophys. Acta 562, 11â 31. Watkins, H. D., Kopf, G. S. and Garbers, D. L. (1978). Activation of sperm adenylate cyclase by factors associated with eggs. Biol. Reprod. 19, 89â 89.