Tissue sources of protein kinase activities in human seminal fluid: studies of normal, oligozoospermic, and vasectomized men*

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1 FRTILITY AND STRILITY Copyright 1983 The American Fertility Society Vol. 4, No. I, J\1Jy 1983 Printed in U.8A. Tissue sources of protein kinase activities in human seminal fluid: studies of normal, oligozoospermic, and vasectomized men* Michael J. Wilson, Ph.D.H Keith W. Kaye, M.D. Veterans Administration Medical Center and University of Minnesota, Minneapolis, Minnesota Protein kinase activities in seminal fluids of normo-, hypo-, and oligozoospermic and vasectomized men were measured using lysine-rich histones and partially de phosphorylated phosvitin as acceptor substrates. There was a significant relationship of histone kinase but not phosvitin kinase activities with the number of spermatozoa originally present in the semen. Histone kinase and phosvitin kinase activities were diminished 88% and 62%, respectively, in vasectomy seminal fluid. The sex accessory gland sources of seminal fluid protein kinase activities not associated with spermatozoa were examined in split ejaculates of vasectomized men. Histone kinase activity was greater in the first fractions, suggesting that the prostate is its predominant contributor; whereas the distribution of phosvitin kinase activity did not indicate any preferential accessory gland source of this enzyme. Fertil Steril4:15, 1983 Since the demonstration of cyclic adenosine 3':5' monophosphate (camp) involvement in the regulation of spermatozoan motility, l studies of protein phosphorylation reactions in semen have focused on the camp-dependent protein kinase of spermatozoa.2-6 There are, however, protein kinases also present in seminal plasma. These enzymes are not stimulated by camp,7, 8 although Received August 23, 1982; revised and accepted March 2, *Supported in part by the General Medical Research Funds of the Veterans Administration and the Minnesota Medical Foundation grant CRF ttoxicology Research Laboratory, Veterans Administration Medical Center, and Department of Laboratory Medicine and Pathology, University of Minnesota. treprint requests: Dr. Michael J. Wilson, Toxicology Research Laboratory, Veterans Administration Medical Center, Minneapolis, Minnesota Department of Surgery Service, Veterans Administration Medical Center, and Urologic Surgery, University of Minnesota. one kinase would appear to be the free catalytic subunit of a camp-dependent enzyme since it is inhibited by protein kinase inhibitor protein. 8 In this study, we examine the tissue sources that contribute protein kinase activities to seminal plasma. MATRIALS MATRIALS AND MTHODS Lysine-rich histones were obtained from Worthington Biochemical, Freehold, NJ, and 32Pi from ICN, Irvine, CA. All other chemicals were of the highest grade available. Preparation of 32p_ labeled adenosine triphosphate (ATP) and partially dephosphorylated phosvitin was carried out as previously described. 9 MTHODS Fresh semen samples from men attending the Fertility Clinics, University of Minnesota Hospi- Vol. 4, No.1, July 1983 Wilson and Kaye Tissue origins of protein kinases in seminal fluid 15

2 1 Table 1. Protein Phosphokinase Activities in Human Seminal Fluid-Relationship of Protein Kinase Activities Toward Phosvitin and Lysine-Rich Histones to the Relative Number of Sperm in the jaculate Sperm no. n jaculate Volume Protein Nanomoles 32p per milligram protein per hour Nanomoles 32p per milliliter per hour Phosvitin Liriine-rich Phosvitin Lysine-rich. stones histones orrnozoosperrnia (;;. 5 X 1Q6/ml) IIypozoospermia (2-5 x 1Q6/ml) Oligozoospermia «2 x 1Q6/ml) Azoospermia (vasectornized) 1 a± Standard deviation. bn = 8. cp <.2 versus normozoospermic seminal fluids ml mg/ml 3. ±.8a 58.3 ± ± ± ± ± ± b± ±.7 121b± ± ± ± ± ± ±.9 3.2c± ± c± 88.8 ± ±.5 5 ± ± 24 tal, or attending for postvasectomy examination, St. Louis Park Medical Center, were used in this study. Split ejaculates of 5 to 6 fractions lo were collected from five vasectomized men in the Urology Clinic at the University of Minnesota Hospital. These donors were asked to collect each orgasmic contraction into individual tubes; any additional contractions beyond the sixth were collected into the sixth tube. The number of spermatozoa of the non vasectomy semen samples was determined using a hemocytometer; and the percentage of viability, motility, and abnormal forms of spermatozoa were determined by microscopic examination and the eosin-nigrosin staining method. 11 Semen samples were kept at room temperature for 3 to 6 minutes and then were centrifuged at 9 x g for 1 minutes at 2 C to 24 C for sedimentation of most spermatozoa «5% remained in the supernatant) and other cell types present. These samples were stored an additional hour at 4 C before the supernatant was centrifuged for 3 minutes at 2, x gat 4 C. The supernatant of the second centrifugation was stored frozen at - 2 C and was referred to as the seminal fluid. It has been shown previously that there is no change in protein kinase acti vi ties of seminal fluid prepared from semen samples stored up to 4 hours at either room temperature or 4 C. 6, 8 NZYM ASSAYS Partially dephosphorylated phosvitin and lysine-rich histones were used as model acidic and basic phosphoprotein substrates. The measurement of protein kinase-mediated transfer of 32p from -y_32p_atp into these proteins was carried out as detailed previously.8 The standard reaction medium (.5 ml final volume) for assaying activity toward lysine-rich histones consisted of 3 mm Tris-HCI (final reaction ph 8. at 37 C), 1 mm dithiothreitol, 6 mm MgCI2,.5 mm (-y_32p)atp (specific radioactivity - 9 dpm/nmol), and 6 mg/ml lysine-rich histones. Measurements of protein kinase activity toward partially dephosphorylated phosvitin as substrate employed a standard reaction medium consisting of 3 mm Tris-HCI (ph 7.4 at 37 C), 1 mm dithiothreitol, 16 mm NaCI, 6 mm MgCI2, 3 mm (-y_32p)atp (specific radioactivity - 15 dpmlnmol), and 6 mg/ml phosvitin. Reactions employing these protein substrates were initiated by addition of seminal fluid diluted with.25 M sucrose (5 to 15 j-lg protein in reactions with lysine-rich histones as substrate and 15 to 4 j-lg protein in reactions with phosvitin) and were incubated for 2 minutes at 37 C in a shaking water bath. The reactions were terminated by addition of.5 ml cold 3% (wt/vol) trichloroacetic acid containing 2 mm Pi and 3% sodium pyrophosphate and the precipitates were washed and prepared for liquid scintillation counting.8 The prostatic acid phosphatase activities of split ejaculate samples (.1 to.5 j-lg protein/assay) were determined using sodium thymolphthalein monophosphate as substrate. 12 OTHR MTHODS The protein content of seminal fluid was assayed by the method of Lowry et ai.,13 using bovine serum albumin for standard. 16 Wilson and Kaye Tissue origins of protein kinases in seminal fluid Fertility and Sterility

3 ... Q). ::3 Z... Q) a. (f) DPV., LRH ' Protein Kinase Activity (nmoles 32P/mg Protein/hr) Figure 1 The distribution relationship of seminal fluid phosvitin kinase (DPV) and histone kinase (LRH) activities and relative sperm number. STATISTICAL ANALYSIS The relationship of protein kinase activities of seminal fluid with various parameters of sperm in semen were evaluated by the Pearson correlation coefficient and one-way analysis of variance of grouped data. RSULTS Semen samples were classified as to the number of sperm per milliliter. Normal levels of sperm were considered to be ;;:. 5 million sperm/ ml; hypozoospermia, between 2 and 5 million sperm/ml; oligozoospermia, < 2 million sperm! ml; and azoospermia (postvasectomy), absence of spermatozoa. The protein kinase activity toward lysine-rich histones decreased progressively in relation to the number of sperm originally present in the ejaculate (Table 1). That is, in hypozoospermic individuals, seminal fluid protein kinase activities decreased 32%; the decrease was 64% in oligozoospermic men and 88% in azoospermic vasectomized men. There was a high degree of correlation (Fig. 1) of seminal fluid histone kinase activities with sperm number in the semen (r =.68, P <.1, n = 26). This relationship was also significant (P <.2, F = 4.74,,:l = 29.7) by analysis of variance in the grouped data of normo-, hypo-, and oligozoospermic samples (Table 1). There was no relationship of phosvitin kinase activity with number of spermatozoa in the normo-, hypo-, and oligozoospermic seminal fluids (Table 1) by analysis of variance of these grouped data (P >.5, F =.56,,:l = 5.27). There was no difference in protein concentrations among the seminal fluids of the various groups (Table 1), and there was no correlation of either protein kinase activity with ejaculate volume or the percentage of motile, viable, or abnormal sperm in the semen. The sex accessory glands appeared to be an additional source of protein kinase activities in seminal fluid since their activities, although greatly reduced, were not entirely absent in the semen of vasectomized men. The tissue sources of the protein kinase activities, which were not associated with spermatozoa in semen, were examined in split ejaculates collected from vasectomized men. The prostate gland appears to be a major contributor to this second source of histone kinase activities since its specific activities were higher in the first fractions of the split ejaculate (Fig. 2). However, the seminal vesicles or another male accessory organ may also be the origin of some of the histone kinase activities since these enzymic activities did not decline to the same extent in fractions 3, 4, and 5 as did prostatic acid phosphatase. The latter activity was a marker of the relative prostate secretory contribution to each fraction. The distribution pattern of phosvitin kinase activities does not indicate any preferential accessory gland source of this enzyme. DISCUSSION The presence of histone kinase activity in seminal fluid correlates with the number of spermatozoa originally present in the ejaculate. Since there is no leakage of enzyme activity from spermatozoa during storage, it is presumed that these enzymes were originally associated with spermatozoa. Thus, the kinase may have been released from spermatozoa during a maturational phase in development or in an activation step during initiation of motility prior to or simultaneous with ejaculation. Motility of caudal epididymal sper- Vol. 4, No.1, July 1983 Wilson and Kaye Tissue origins of protein kinases in seminal fluid 17

4 3 A ';;. III.c Cl. 1.5 ct "A!.; 2 :i 1. V>.c Cl. I N..c " V> u of <5.c l- 'iii V>.3- " :l.. 14 B T += ;; Q V> 8 b.c 1 Cl..; 8 6 '; V> <5 Cl..c I 6 N.c U 4. V>.;:: <5 4.c iii I- 2., -' " I 2 < Fraction Number Figure 2 The phosvitin kinase, histone kinase, and prostatic acid phosphatase activities in sequential fractions of a split ejaculate of a vasectomized man. This distribution of protein kinases is representative of that observed in split ejaculates collected from five vasectomized men. The specific activities are expressed per milligram of protein (A) and per milliliter of fluid (8). matozoa is stimulated by exogenous camp,!,14 and endogenous levels of the cyclic nucleotide are increased upon activating motility of bovine caudal spermatozoa by dilution.15 Majumder6 has shown camp to activate a protein kinase on the outer surface of epididymal sperm with the simultaneous release of this enzyme activity into the surrounding medium. This is consistent with our observation tpat the majority of the histone kinase activity in human seminal fluid is apparently the free catalytic subunit of a camp-dependent protein kinase since it is strongly inhibited by the camp-dependent protein kinase inhibitor protein.8, 16 There is little phosphorylation of endogenous proteins when spermatozoa are incubated with ),_32p_ATP,6 and.5 :l.. only a few proteins are phosphorylated when disrupted spermatozoa are studied. These include membrane proteins,4 tubulin,17 and a "motility protein.,,18 The significance of protein kinases released from spermatozoa into the surrounding milieu is not understood. It may represent an enzyme needed for a particular function external to the spermatozoon, or it may be an enzyme that has completed a function and is discarded. Although the majority of histone kinase activity in seminal fluid appears to be derived from spermatozoa, the prostate gland (to a lesser extent, the seminal vesicles or another accessory organ) appears to be an important contributor of non-spermatozoa-associated histone kinase in seminal fluid, as judged by the distribution of these activities in split ejaculates of vasectomized men. On the other hand, phosvitin kinase activities do not appear to be contributed preferentially by anyone accessory gland. The presence of protein kinases in human prostate secretion has been substantiated by demonstration of these activities in fluid expressed by prostate massage19 and in secretion of rat ventral prostate.16 The differential distribution of histone kinase and phosvitin kinase activities in semen possessing different numbers of spermatozoa and in split ejaculates of vasectomized men further substantiates the separate identity of these enzymes.8 Protein kinases have generally been found to be involved in regulatory mechanisms of a wide variety of cellular processes,2 including those of male accessory organs.21, 22 However, the possible physiologic significance of accessory gland protein kinases in phosphoprotein metabolism of seminal constituents has received scant attention. The identification of the endogenous phosphoprotein substrates, whether associated with spermatozoa, seminal fluid, or the female reproductive tract, and the control of their phosphorylation by these enzymes is an important step toward that goal. Acknowledgments. We gratefully acknowledge the excellent technical assistance of Roger Salem, Amy Setzer, and Renae Roelofs in performing the protein kinase assays; Dr. Khalil Ahmed for reading the manuscript; and the consultation of Dr. Ulysses S. Seal with respect to statistical analyses. The kind cooperation of Connie Warvin, Pat Norgren, and Sheila Coombs of the Department of Obstetrics and Gynecology, University of Minnesota; Marion Quinn, St. Louis Park Medical Center; and Doris iselmayer, Department of Urologic Surgery, University of Minnesota, for clinical analyses of semen samples is greatly appreciated. 18 Wilson and Kaye Tissue origins of protein kinases in seminal fluid Fertility and Sterility

5 RFRNCS 1. Garbers DL, Lust WD, First NL, Lardy HA: ffects of phosphodiesterase inhibitor and cyclic nucleotides on sperm respiration and motility. Biochemistry 1:1825, Garbers DL, First NL, Lardy HA: Properties of adenosine 3',5' -monophosphate-dependent protein kiases isolated from bovine epididymal spermatozoa. J BIOI Chem 248: 875, Lee MYW, Iverson RM: An adenosine 3':5' monophosphate-dependent protein kinase from sea urchin spermatozoa. Biochim Biophys Acta 429:123, Huacuja L, Delgado NM, Merchant H, Pancardo RM, Rasado A: Cyclic AMP induced incorporation of 33Pi into human spermatozoa membrane components. BioI Reprod 17:89, Hoskins DD, Casillas R, Stephens DT: Cyclic AMP-dependent protein kinases of bovine epididymal spermatozoa. Biochem Biophys Res Commun 48:1331, Majumder GC: Occurrence of a cyclic AMP-dependent protein kinase on the outer surface of rat epididymal spermatozoa. Biochem Biophys Res Commun 83:829, Majumder GC: Characterization of a protein kinase from human seminal plasma. Biochem Biophys Res Commun 81:1217, Wilson MJ, Steer RC, Kaye KW: Presence and characterization of two protein kinase activities in human seminal fluid. Biochim Biophys Acta 7:26, Ahmed K, Wilson MJ, Davis AT: Phosvitin phosphate content: implication for protein kinase assay. Biochim Biophys Acta 377:8, liasson R, Lindholmer C: Functions of male accessory genital organs. In Human Semen and Fertility Regulation in Men, dited by S Hafez. St. Louis, C. V. Mosby, 1976, p Amelar RD, Dubin L: Semen analysis. In Male Infertility, dited by RD Amelar, L Dubin, PC Walsh. Philadelphia, W. B. Saunders Co., 1977, p Roy A V, Brower M, Hayden J: Sodium thymolphthalein monophosphat a new acid phosphatase substrate with greater specificity for the prostatic enzyme in serum. Clin Chem 17:193, Lowry OH, Rosebrough NH, Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent. J BioI Chem 193:265, Hoskins DD, Stephens DT, Hall ML: Cyclic adenosine 3':5' -monophosphate and protein kinase levels in developing bovine spermatozoa. J Reprod Fertil 37:131, Cascieri M, Amann RP, Hammerstedt RH: Ademne nucleotide changes at initiation of bull sperm motility. J BioI Chem 251:787, Wilson MJ, Steer RC, Kaye KW: Studies of human seminal fluid: presence of protein phosphokinase activities. In Male Reproduction and Fertility, dited by A Negro-Villar. New York, Raven Press, 1983, p Tongkao D, Chulavatnatol M: Phosphorylation of microtubules of rat spermatozoa during epididymal maturation. In The Spermatozoan, dited by DW Fawcett, JM Bedford. Baltimore, Urban and Schwartzenberg, 1979, p Brandt H, Hoskins DD: A camp-dependent phosphorylated motility protein in bovine epididymal sperm. J BioI Chem 255:982, Wilson MJ, Steer RC, Kaye KW: Unpublished data 2. Rubin CS, Rosen OM: Protein phosphorylation. Annu Rev Biochem 44:831, Ahmed K, Wilson MJ, Goueli SA, Norvitch M: Testosterone effects on the prostatic nucleus. In ffects of Drugs in the Cell Nucleus, dited by H Busch, Y Daskal, S Crooke. New York, Academic Press, 1979, p Ahmed K, Wilson MJ, Goueli SA, Steer RC: Functional biochemistry. In Accessory Glands of the Male Reproductive Tract, dited by Spring-Mills, S Hafez. Ann Arbor, Ann Arbor Science Publishers, 1979, p 69 Vol. 4, No.1, July 1983 Wilson and Kaye Tissue origins of protein kinases in seminal fluid 19