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Effect of relaxin and antirelaxin serum on porcine sperm motility
Animal Reproduction Science, 20 (1989) 21-29 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
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E f f e c t of R e l a x i n and A n t i r e l a x i n S e r u m on Porcine Sperm Motility H.H. JUANG, A.I. MUSAH, C. SCHWABE, and L.L. ANDERSON ~
Department of Animal Science, Iowa State University, Ames, IA 50011 "U.S.A.) and ~Department of Biochemistry, Medical University of South Carolina, Charleston, SC 29495 (U.S.A.) (Accepted 14 February 1989)
ABSTRAC2 Juang, H.H., Musah, A.I., Schwabe, C. and Anderson, L.L., 1989. Effect of relaxin and antirelaxin serum on porcine sperm motility. Anim. Reprod. Sci., 20: 21-29. Sperm motility was estimated by a transmembrane migration ratio (TMMR) method, which is based on the proportion of sperm that migrate across a membrane containing micropores with a diameter slightly greater than the width of the sperm nucleus. Relaxin (1000, 333 and 167 ng/ ml) attenuated (P < 0.01) loss of sperm motility during 120 rain (50 vs 40% ). The addition of 1000 ng/ml relaxin to semen improved sperm motility (P < 0.05) stored at 12 °C for 5 days with compared controls (43 vs 32 % ). Antirelaxin serum (R6) and normal rabbit serum ( NRS; 1 : 9000 ) were added to semen and the sperm motility was determined at 0, 10, 40, 80 and 120 rain. Antirelaxin serum abruptly inhibited sperm motility (P < 0.01) within 10 min compared with NRStreated control semen (25 vs 58% ). Caffeine and relaxin stimulated motility (P< 0.01) of frozenthawed porcine semen; however, relaxin maintained greater sperm motility (P< 0.01) than caffeine after 80 min (30 vs 16% ). These results indicate that purified porcine relaxin can maintain or increase sperm motility; in contrast, porcine antirelaxin abruptly inhibits sperm motility.
INTRODUCTION R e l a x i n is a p e p t i d e h o r m o n e o f a b o u t 6000 D f o u n d p r i m a r i l y in r e p r o d u c tive t i s s u e s d u r i n g p r e g n a n c y in s e v e r a l m a m m a l i a n species b u t also in t h e m a l e r e p r o d u c t i v e t r a c t ( A n d e r s o n , 1987). R e l a x i n h a s b e e n localized in t h e r o o s t e r testis ( S t e i n e t z et al., 1959 ); r e l a x i n i m m u n o a c t i v i t y in t h e b l o o d p l a s m a o f t h e r a m , b o a r a n d h u m a n m a l e ( B r y a n t , 1972). R e l a x i n - l i k e i m m u n o c y t o logic r e a c t i o n w a s r e p o r t e d in i n t e r s t i t i a l a n d S e r t o l i cells of t h e b o a r t e s t i s ( D u b o i s a n d D a c h e u x , 1978) a n d g e n i t a l t r a c t o f o t h e r species ( C a m e r o n et al., 1982; K e n d a l l et al., 1982; Y k i - J a r v i n e n et al., 1983). S u b s e q u e n t s t u d i e s i n d i c a t e d t h a t r e l a x i n m a i n t a i n e d m o t i l i t y o f w a s h e d or a g e d u n w a s h e d h u m a n s p e r m a t o z o a ( E s s i g et al., 1982; L e s s i n g et al., 1986). R e l a x i n w a s l a t e r s h o w n
0378-4320/89/$03.50
© 1989 Elsevier Science Publishers B.V.
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to be necessary for proper penetration of cervival mucus by human spermatozoa (Brenner et al., 1984). The addition of antirelaxin serum, but not antiinsulin serum, to washed human spermatozoa causes marked inhibition of motility (Sarosi et al., 1983ab; Lessing et al., 1984). The addition of relaxin to frozen-thawed human semen significantly increased sperm motility, although adding relaxin before freezing the semen, has no effect on sperm motility after thawing (Lessing et al., 1985). Prostaglandin E2 (PGE2) and relaxin antagonize each other's action on sperm motility (Colon et al., 1986). These results indicate that porcine relaxin has a specific stimulatory effect on the motility of human spermatozoa, though the mechanism of action is unknown. The objective of this study was to determine the effect of purified porcine relaxin and antirelaxin serum on the motility of fresh, 12 ° C stored, and frozenthawed boar sperm by using the transmembrane migration ratio (TMMR) method. MATERIALS AND METHODS
Ejaculates were collected from three fertile Yorkshire boars. The semen was filtered through cheese-cloth and assessed immediately for motility and sperm concentration.
Estimation of sperm motility The transmembrane migration ratio (TMMR) apparatus (Fig. 1 ) was modified from Hong et al. (1981, 1985). This method is based on the proportion of sperm that migrate from an inner well across a membrane containing micropores with a diameter slightly greater than the width of the sperm nucleus. The upper chambers were fabricated from a plastic sperm chamber (Durawel, Taipei,
!ii! t upperchamber .]
Rubber Ring 0.1 ml Semen Nuclepore Membrane
t
~
--Culture-Well
•
1
i
Ixm
Lower Chamber - - 1 ml PBS Buffer
8
m
I
8 ~m
Fig. 1. The apparatus for measuring the transmembrane migration ratio of porcine sperm.
23 Taiwan). The lower part of the upper chamber consisted of a sheet (13 mm diameter, 10/~m thickness) of polycarbonate membrane (8~m pore, Nuclepore, Pleasanton, CA) bonded with methylene chloride. The upper chamber was inserted into a 24-well multicell culture plate (Fisher Scientific, Pittsburg, PA) containing 1 ml phosphate buffer saline (PBS; 136 m M NaC1, 2.68 m M KC1, 4.29 ~Y/Na2HPO4, 1.47 m M KH2PO4) forming the lower chamber. An aliquot of prepared boar semen (100 zl) was pipetted into the chamber and the apparatus incubated in a water bath for 3 h at 37°C. After incubation, the upper chamber was removed, and the concentration of sperm in the lower chamber was estimated (n = 4) by hemocytometer counts. The proportion of sperm crossing the membrane into the lower chamber was highly correlated with the percentage of progressively moving sperm and the results are expressed in this form. There is a 0.96 correlation between T M M R and percent of sperm which show progressive motility as evaluated by light microscopy (Hong et al., 1981, 1985). It is thus a simple quantitative and reproducible method which requires small aliquots of semen to evaluate the effects of hotmones and drugs on sperm motility. Effect of relaxin on fresh sperm Six ejaculates were diluted in tubes with PBS (pH 7.3 ) to 1.0-1.5 × 10s sperm/ ml, and porcine relaxin (3000 IU/mg; Biillesbach and Schwabe, 1985) in PBS was added in three volumes (1 : 2, v/v) to give final concentrations of 1000, 333, 167, 83 and 0 ng/ml. Aliquots were taken at 0, 40, 80, 120 and 180 min for T M M R measurement. Effect of antirelaxin on fresh sperm Antirelaxin serum (Dual Rabbit R6; Dr. B.G. Steinetz, New York University, Tuxedo, NY) and normal rabbit serum (NRS; Antibodies Inc., Davis, CA) were diluted in PBS and added (1:2, v/v) to three boar semen samples. The final concentration was 1 : 9000. T M M R was assessed at 0, 10, 40, 80 and 120 rain. Effect of relaxin on sperm stored at 12 ° C Five ejaculates were treated as in the first experiment except that they were diluted (2:1) with Beltsville Thawing Solution (BTS) (205 m M glucose, 2.04 m M trisodium citrate, 3.36 m M EDTA, 10 m M KC1, 14 m M N a H C Q ; pH 7.35 ) containing porcine relaxin at a final concentration of 1000 ng/ml before being stored at 12_+ 1°C for 5 days. BTS was used in the control semen samples. T M M R was assessed at 0, 3, 6, 12, 36, 48, 72, 84, 96, 108 and 120 h. Effect of relaxin on frozen-thawed sperm Three ejaculates were prepared for freezing as described by Pursel and Johnson (1975). Each 10-ml frozen pellet of semen was stored in a plastic bottle
24
then transferred to liquid nitrogen for storage. After 3 days of storage, each dose of pelleted semen was removed and thawed in 30 ml BTS which was prewarmed to 50°C. The final temperature of semen was 21-24°C. Sperm concentrations in the thawed semen were estimated and adjusted with BTS to 1.01.5 X l0 s sperm/ml. Each semen sample was divided into four aliquots for addition of BTS (2:1, v/v), 3000 n g / m l relaxin (3000 I U / m g ) , 9 m M caffeine (Eastman Kodak, Rochester, NY) and relaxin plus caffeine. The final concentration of relaxin was 1000 n g / m l and caffeine 3 mM. T M M R was assessed at 0, 10, 40, 80 and 120 min after these treatments.
Statistical analysis Data were analyzed by using the analysis of variance and Student's t-test for comparing pairs of means (Snedecor and Cochran, 1980; Spector et al., 1985). RESULTS
The effects of different levels of relaxin on the percent of motile sperm are shown in Fig. 2. Addition of 1000, 333 and 167, but not 83 n g / m l relaxin to porcine semen significantly attenuated ( P < 0 . 0 1 ) the loss of sperm motility during 180 min at room temperature. Relaxin (1000 n g / m l ) improved the motility ( P < 0 . 0 5 ) of porcine sperm stored for 5 days at 12+ I ° C (Fig.3). Antirelaxin serum (1: 9000) markedly inhibited (P < 0.01) porcine sperm motility within 10 min compared with that of porcine semen diluted with normal rabbit serum (Fig. 4). The effect of adding porcine relaxin (1000 n g / m l ) and caffeine (3 m M ) to frozen-thawed porcine semen is presented in Fig. 5. The addition of caffeine 80
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0
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'
' 60
'
TIME
' 80
'
' ' ' ' ' ' ' ' I 100 120 140 160 180
IN MINUTES
Fig. 2. The effect of 1000 ( & ), 333 ( • ), 167 ( I ) , 83 (C) ) and 0 (<~) ng/ml porcine relaxin (3000 IU/mg) on the percentage of motile boar sperm ( n = 6 ) . Motility was significantly (P<0.01) greater in semen containing 1000, 333 and 167 ng/ml relaxin compared with the control (0 ng/ ml) after 80, 120 and 180 min.
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Fig. 3. Percentage of motile sperm in porcine semen containing 1000 ( • ) and 0 ( 0 ) ng/ml relaxin (3000 IU/mg) during a 5-day period at 12 + 1 °C (n= 5 ). After 36 h, motility was significantly (P < 0.05) greater in semen containing 1000 ng/ml relaxin compared with the control (0 ng/ml). 100
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~ 2o 100
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2
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~ 50
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J 80
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I _ I I 100 110 120
IN MINUTES
Fig. 4. Percentage of motile sperm in porcine semen ( n = 3 ) after addition of antirelaxin serum ( ~ ) and normal rabbit serum (1 :9000) ( O ). Within 10 min, sperm motility was significantly reduced by antirelaxin serum (P<0.01).
increased (P < 0.01 ) sperm motility within 10 min and maintained motility for 40 min. Addition of relaxin had little effect on sperm motility during the first 10 min, but steadily increased motility during the next 80 rain (Fig. 5). The effects of relaxin and caffeine on sperm motility were similar at 40 rain (Fig. 5 ), but at 80 min relaxin was more effective (P < 0.01 ) than caffeine at maintaining motility (30 _ 2.9 vs 16 _+0.5% ). The addition of both relaxin and caffeine to sperm after thawing did not significantly improve motility in comparison with relaxin or caffeine alone (Fig. 5). DISCUSSION
Evaluation of sperm motility was based on the transmembrane migration ratio method first developed by Hong et al. (1981, 1985). This is a particularly
26 5O !
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I 20
I 30
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O
I 50
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I 70
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T I M E IN M I N U T E S
Fig. 5. Effect of relaxin ( 1000 n g / m l ) ( • ), 3 m M caffeine ( • ), relaxin-caffeine ( [] ) and control (BTS) ( 0 ) on percentage of motile sperm in frozen-thawed porcine semen.
useful method when dealing with small volumes (100 H1) of semen. The TMMR method has proved not only to be objective and reproducible, but it has a high correlation with the percent progressive motility as determined by light microscopy. In the present study, the correlations between T M M R and the sperm motility as determined by light microscopy of fresh and frozen-thawed boar semen were 0.94 ( n = 1 5 ) and 0.92 ( n = 2 5 ) , respectively. The coefficient of variation (c.v.) of measurements of sperm motilities ranging from 32 to 100% was 4.5-8.7% c.v. (Hong et al., 1981 ). The present study indicates that porcine relaxin can attenuate the loss of motility of porcine spermatozoa in a dose- and time-dependent manner, whereas the addition of antirelaxin serum markedly decreased motility. The abrupt decrease in porcine sperm motility within 10 min after the addition of antirelaxin serum as shown here corroborates the studies with washed human spermatozoa (Sarosi et al., 1983a), and supports the idea that relaxin is necessary for optimal sperm motility. Porcine relaxin has a positive effect on in vitro fertilization (IVF) of zona-free mouse oocyte models (Pupula et al., 1986). The observation that the addition of relaxin to liquid-stored (12 _+1 ° C, 5 days) boar semen maintains greater sperm motility suggests that relaxin may be effective in maintaining sperm motility or improving pregnancy rates for artificial insemination. Though relaxin significantly increased sperm motility of frozen-thawed porcine semen as shown in this study as well as of frozen-thawed human semen (Lessing et al., 1985), it has no detectable stimulatory effect on sperm motility of fresh- or liquid-stored porcine semen. It is possible that the changes in the structure of the plasma membrane after cryopreservation enhance the effect of relaxin on sperm motility. Caffeine, an inhibitor of phosphodiesterase, increases intracellular concentrations of cAMP and has been shown to stimulate motility of porcine spermatozoa (Amelar et al., 1980; Dacheux and Paquignon, 1980). Relaxin as well as caffeine increase porcine sperm motility, although
27 they have different time-dependent effects, indicating the possibility of different mechanisms of action. Caffeine initially increases porcine sperm motility, but it drops abruptly; relaxin, however, increases sperm motility slowly and maintains greater motility significantly longer t h a n caffeine. The m e c h a n i s m by which relaxin affects sperm motility is unknown. However, relaxin inhibition of myometrial contractile activity m a y be mediated by an increase in cAMP, a decrease in light-chain phosphorylation, an alteration of the kinetic properties of myosin light-chain kinase, and an increased Ca 2+ efflux from myometrial cell (Sanborn et al., 1982; Hsu et al., 1985; Hsu and Sanborn, 1986; Rao and Sanborn, 1986). Because relaxin is a peptide hormone, it may bind to a surface receptor on the sperm and probably increases sperm motility by altering intracellular c A M P and Ca 2+ level and (or) by activating specific protein kinases and phosphorylation reactions in the sperm plasma membrane. ACKNOWLEDGMENTS This study was presented in part at the 20th Annual Meeting of the Society for the Study of Reproduction, Champaign, IL, 1987 (abstract 369). These studies were supported in part by U.S. D e p a r t m e n t of Agriculture, ARS, CSRS and OGPS Competitive Grant 85-CRCR-l-1862. Journal Paper J-13108 of the Iowa Agriculture and H o m e Economics Experiment Station, Ames, IA (Projects 2443, 2444 and 2754). We t h a n k Drs. J.P. Kunesh and P.G. Eness, Ambulatory Clinics, College of Veterinary Medicine, for monitoring the health status of the experimental animals; Dr. D.K. Hotchkiss, D e p a r t m e n t of Statistics, for assistance with statistical analyses; M.E. Shell and C.R. Bohnker for excellent technical assistance. Porcine relaxin antiserum was generously provided by Dr. B.G. Steinetz, New York University Medical Center (Tuxedo, NY).
REFERENCES Amelar, R.D., Dubin, L. and Schoenfeld,C., 1980. Sperm motility. Fertil. Steril., 34: 197-215. Anderson, L.L., 1987. Regulation of relaxin secretion and its role in pregnancy. Adv. Exp. Med. Biol., 219: 421-463. Brenner, S.H., Leasing, B.J., Schoenfeld,C., Amelar, R.D., Dubin, L. and Weiss, G., 1984. Stimulation of human sperm cervical mucus penetration in vitro by relaxin. Fertil. Steril., 42: 9296. Bryant, G.D., 1972. The detection of relaxin in porcine, ovine and human plasma by radioimmunoassay. Endocrinology,91:1113-1117. Btillesbach, E.E. and Schwabe, C., 1985. Naturally occurringporcine relaxin and large-scalepreparation of the B29 hormone. Biochemistry, 24: 7717-7722. Cameron, D.F., Corton, G.L. and Larkin, L.H., 1982. Relaxin-like antigenicity in the armadillo prostate gland. Ann. N.Y. Acad. Sci., 380: 231-240.
28 Colon, J.M., Ginsburg, F., Lessing, J.B., Schoenfeld, C., Goldsmith, L.T., Amelar, R.D., Dubin, L. and Weiss, G., 1986. The effect of relaxin and prostaglandin E2 on the motility of human spermatozoa. Fertil. Steril., 46: 1133-1139. Dacheux, J.L. and Paquignon, M., 1980. Effects of caffeine on ram and boar spermatozoa: influence of their stage of maturation and the medium; initiation of progressive motility oftesticular spermatozoa. In: A. Steinberger and E. Steinberger (Editors), Testicular Development, Structure and Function. Raven Press, New York, NY, pp. 513-532. Dubois, M.P. and Dacheux, J.L., 1978. Relaxin, a male hormone? Immunocytologic localization of a related antigen in the boar testis. Cell Tissue Res., 187: 201-214. Essig, M., Schoenfeld, C., Amelar, R.D., Dubin, L. and Weiss, G., 1982. Stimulation of human sperm motility by relaxin. Fertil. Steril., 38: 339-343. Hong, C.Y., Chaputedsaintonge, D.M. and Turner, P., 1981. A simple method to measure drugs effects on human sperm motility. Br. J. Clin. Pharmacol., 11: 385-387. Hong, C.Y., Chiang, B.N., Ku, J., Wei, Y.H. and Fong, C.Y., 1985. Calcium antagonists stimulate sperm motility in ejaculated human semen. Br. J. Clin. Pharmacol., 19: 45-49. Hsu, C.J. and Sanborn, B.M., 1986. Relaxin treatment alters the kinetic properties of myosin light chain kinase activity in rat myometrial cells in culture. Endocrinology, 118: 499-505. Hsu, C.J., McCormack, S.M. and Sanborn, B.M., 1985. The effect of relaxin on cyclic adenosine 3',5'-monophosphate concentrations in rat myometrial cells in culture. Endocrinology, 116: 2029-2035. Kendall, J.Z., Smith, R.G., Shih, Li-N, Webb, P.D. and Tare, W.H., 1982. Characterization of prostate relaxin. In: M. Bigazzi, F.C. Greenwood and F. Gasparri (Editors), Biology of Relaxin and Its Role in the Human. Elsevier Press, New York, NY, pp. 363-366. Lessing, J.B., Brenner, S.H., Schoenfeld, C., Sarosi, P., Amelar, R., Dubin, L. and Weiss, G., 1984. The effect of an anti-insulin antiserum on human sperm motility. Fertil. Steril., 42: 309-311. Lessing, J.B., Brenner, S.H., Schoenfeld, C., Sarosi, P., Goldsmith, L.T., Amelar, R., Dubin, L. and Weiss, G., 1985. The effect of relaxin on the motility of sperm in frozen-thawed human semen. Fertil. Steril., 44: 406-409. Lessing, J.B., Brenner, S.H., Colon, J.M., Ginsburg, F.W., Schoenfeld, C., Sarosi, P., Goldsmith, L.T., Amelar, R , Dubin, L. and Weiss, G., 1986. Effect of relaxin on human spermatozoa. J. Reprod. Med., 31: 304-309. Pupula, M., Quinn, P. and MacLennan, A., 1986. The effect of porcine relaxin on the fertilization of mouse oocytes in vitro. Clin. Reprod. Fertil., 4: 383-387. Pursel, V.G. and Johnson, L.A., 1975. Freezing of boar spermatozoa: fertilizing capacity with concentration semen and a new thawing procedure. J. Anim. Sci., 40: 99-102. Rao, M.R. and Sanborn, B.M., 1986. Relaxin increases calcium efflux from rat myometrial cells in culture. Endocrinology, 119: 435-437. Sanborn, B.N., Nihikori, K., Weisbrodt, N.W. and Sherwood, O.D., 1982. Relaxin affects uterine myosin phosphorylation and related activities. In: M. Bigazzi, F.C. Greenwood and F. Gasparri (Editors), Biology of Relaxin and Its Role in the Human. Elsevier Press, New York, NY, pp. 114-124. Sarosi, P., Schoenfeld, C., Berman, J., Basch, R., Randolph, G., Amelar, R., Dubin, L., Steinetz, B.G. and Weiss, G., 1983a. Effect of anti-relaxin antiserum on sperm motility in vitro. Endocrinology, 112: 1860-1861. Sarosi, P., Schoenfeld, C., Steinetz, B.G. and Weiss, G., 1983b. The effect of rabbit anti-porcine relaxin antibody on sperm motility in vitro. In: M. Bigazzi, F.C. Greenwood and F, Gasparri (Editors), Biology of Relaxin and Its Role in the Human. Elsevier Press, New York, NY, pp. 361-362. Snedecor, G.W. and Cochran, W.G., 1980. Statistical Methods. Edn. 7. Iowa State University Press, Ames, IA, pp. 258-267.
29 Spector, P.C., Goodnight, J.H., Sall, J.P. and Sarke, W.S., 1985. SAS User's Guide: Statistics, Edn. 5., SAS Institute, Cary, NC, pp. 433-506. Steinetz, B.G., Beach, V.L. and Kroc, R.L., 1959. The physiology of relaxin in laboratory animals. In: C.W. Lloyd (Editor), Recent Progress in the Endocrinology of Reproduction. Academic Press, New York, NY, pp. 389-427. Yki-Jarvinen, H., Wahlstrom, T., Seppala, M., 1983. Immunohistochemical demonstration of relaxin in the genital tract of men. J. Reprod. Fertil., 69: 693-695.
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E f f e c t of R e l a x i n and A n t i r e l a x i n S e r u m on Porcine Sperm Motility H.H. JUANG, A.I. MUSAH, C. SCHWABE, and L.L. ANDERSON ~
Department of Animal Science, Iowa State University, Ames, IA 50011 "U.S.A.) and ~Department of Biochemistry, Medical University of South Carolina, Charleston, SC 29495 (U.S.A.) (Accepted 14 February 1989)
ABSTRAC2 Juang, H.H., Musah, A.I., Schwabe, C. and Anderson, L.L., 1989. Effect of relaxin and antirelaxin serum on porcine sperm motility. Anim. Reprod. Sci., 20: 21-29. Sperm motility was estimated by a transmembrane migration ratio (TMMR) method, which is based on the proportion of sperm that migrate across a membrane containing micropores with a diameter slightly greater than the width of the sperm nucleus. Relaxin (1000, 333 and 167 ng/ ml) attenuated (P < 0.01) loss of sperm motility during 120 rain (50 vs 40% ). The addition of 1000 ng/ml relaxin to semen improved sperm motility (P < 0.05) stored at 12 °C for 5 days with compared controls (43 vs 32 % ). Antirelaxin serum (R6) and normal rabbit serum ( NRS; 1 : 9000 ) were added to semen and the sperm motility was determined at 0, 10, 40, 80 and 120 rain. Antirelaxin serum abruptly inhibited sperm motility (P < 0.01) within 10 min compared with NRStreated control semen (25 vs 58% ). Caffeine and relaxin stimulated motility (P< 0.01) of frozenthawed porcine semen; however, relaxin maintained greater sperm motility (P< 0.01) than caffeine after 80 min (30 vs 16% ). These results indicate that purified porcine relaxin can maintain or increase sperm motility; in contrast, porcine antirelaxin abruptly inhibits sperm motility.
INTRODUCTION R e l a x i n is a p e p t i d e h o r m o n e o f a b o u t 6000 D f o u n d p r i m a r i l y in r e p r o d u c tive t i s s u e s d u r i n g p r e g n a n c y in s e v e r a l m a m m a l i a n species b u t also in t h e m a l e r e p r o d u c t i v e t r a c t ( A n d e r s o n , 1987). R e l a x i n h a s b e e n localized in t h e r o o s t e r testis ( S t e i n e t z et al., 1959 ); r e l a x i n i m m u n o a c t i v i t y in t h e b l o o d p l a s m a o f t h e r a m , b o a r a n d h u m a n m a l e ( B r y a n t , 1972). R e l a x i n - l i k e i m m u n o c y t o logic r e a c t i o n w a s r e p o r t e d in i n t e r s t i t i a l a n d S e r t o l i cells of t h e b o a r t e s t i s ( D u b o i s a n d D a c h e u x , 1978) a n d g e n i t a l t r a c t o f o t h e r species ( C a m e r o n et al., 1982; K e n d a l l et al., 1982; Y k i - J a r v i n e n et al., 1983). S u b s e q u e n t s t u d i e s i n d i c a t e d t h a t r e l a x i n m a i n t a i n e d m o t i l i t y o f w a s h e d or a g e d u n w a s h e d h u m a n s p e r m a t o z o a ( E s s i g et al., 1982; L e s s i n g et al., 1986). R e l a x i n w a s l a t e r s h o w n
0378-4320/89/$03.50
© 1989 Elsevier Science Publishers B.V.
22
to be necessary for proper penetration of cervival mucus by human spermatozoa (Brenner et al., 1984). The addition of antirelaxin serum, but not antiinsulin serum, to washed human spermatozoa causes marked inhibition of motility (Sarosi et al., 1983ab; Lessing et al., 1984). The addition of relaxin to frozen-thawed human semen significantly increased sperm motility, although adding relaxin before freezing the semen, has no effect on sperm motility after thawing (Lessing et al., 1985). Prostaglandin E2 (PGE2) and relaxin antagonize each other's action on sperm motility (Colon et al., 1986). These results indicate that porcine relaxin has a specific stimulatory effect on the motility of human spermatozoa, though the mechanism of action is unknown. The objective of this study was to determine the effect of purified porcine relaxin and antirelaxin serum on the motility of fresh, 12 ° C stored, and frozenthawed boar sperm by using the transmembrane migration ratio (TMMR) method. MATERIALS AND METHODS
Ejaculates were collected from three fertile Yorkshire boars. The semen was filtered through cheese-cloth and assessed immediately for motility and sperm concentration.
Estimation of sperm motility The transmembrane migration ratio (TMMR) apparatus (Fig. 1 ) was modified from Hong et al. (1981, 1985). This method is based on the proportion of sperm that migrate from an inner well across a membrane containing micropores with a diameter slightly greater than the width of the sperm nucleus. The upper chambers were fabricated from a plastic sperm chamber (Durawel, Taipei,
!ii! t upperchamber .]
Rubber Ring 0.1 ml Semen Nuclepore Membrane
t
~
--Culture-Well
•
1
i
Ixm
Lower Chamber - - 1 ml PBS Buffer
8
m
I
8 ~m
Fig. 1. The apparatus for measuring the transmembrane migration ratio of porcine sperm.
23 Taiwan). The lower part of the upper chamber consisted of a sheet (13 mm diameter, 10/~m thickness) of polycarbonate membrane (8~m pore, Nuclepore, Pleasanton, CA) bonded with methylene chloride. The upper chamber was inserted into a 24-well multicell culture plate (Fisher Scientific, Pittsburg, PA) containing 1 ml phosphate buffer saline (PBS; 136 m M NaC1, 2.68 m M KC1, 4.29 ~Y/Na2HPO4, 1.47 m M KH2PO4) forming the lower chamber. An aliquot of prepared boar semen (100 zl) was pipetted into the chamber and the apparatus incubated in a water bath for 3 h at 37°C. After incubation, the upper chamber was removed, and the concentration of sperm in the lower chamber was estimated (n = 4) by hemocytometer counts. The proportion of sperm crossing the membrane into the lower chamber was highly correlated with the percentage of progressively moving sperm and the results are expressed in this form. There is a 0.96 correlation between T M M R and percent of sperm which show progressive motility as evaluated by light microscopy (Hong et al., 1981, 1985). It is thus a simple quantitative and reproducible method which requires small aliquots of semen to evaluate the effects of hotmones and drugs on sperm motility. Effect of relaxin on fresh sperm Six ejaculates were diluted in tubes with PBS (pH 7.3 ) to 1.0-1.5 × 10s sperm/ ml, and porcine relaxin (3000 IU/mg; Biillesbach and Schwabe, 1985) in PBS was added in three volumes (1 : 2, v/v) to give final concentrations of 1000, 333, 167, 83 and 0 ng/ml. Aliquots were taken at 0, 40, 80, 120 and 180 min for T M M R measurement. Effect of antirelaxin on fresh sperm Antirelaxin serum (Dual Rabbit R6; Dr. B.G. Steinetz, New York University, Tuxedo, NY) and normal rabbit serum (NRS; Antibodies Inc., Davis, CA) were diluted in PBS and added (1:2, v/v) to three boar semen samples. The final concentration was 1 : 9000. T M M R was assessed at 0, 10, 40, 80 and 120 rain. Effect of relaxin on sperm stored at 12 ° C Five ejaculates were treated as in the first experiment except that they were diluted (2:1) with Beltsville Thawing Solution (BTS) (205 m M glucose, 2.04 m M trisodium citrate, 3.36 m M EDTA, 10 m M KC1, 14 m M N a H C Q ; pH 7.35 ) containing porcine relaxin at a final concentration of 1000 ng/ml before being stored at 12_+ 1°C for 5 days. BTS was used in the control semen samples. T M M R was assessed at 0, 3, 6, 12, 36, 48, 72, 84, 96, 108 and 120 h. Effect of relaxin on frozen-thawed sperm Three ejaculates were prepared for freezing as described by Pursel and Johnson (1975). Each 10-ml frozen pellet of semen was stored in a plastic bottle
24
then transferred to liquid nitrogen for storage. After 3 days of storage, each dose of pelleted semen was removed and thawed in 30 ml BTS which was prewarmed to 50°C. The final temperature of semen was 21-24°C. Sperm concentrations in the thawed semen were estimated and adjusted with BTS to 1.01.5 X l0 s sperm/ml. Each semen sample was divided into four aliquots for addition of BTS (2:1, v/v), 3000 n g / m l relaxin (3000 I U / m g ) , 9 m M caffeine (Eastman Kodak, Rochester, NY) and relaxin plus caffeine. The final concentration of relaxin was 1000 n g / m l and caffeine 3 mM. T M M R was assessed at 0, 10, 40, 80 and 120 min after these treatments.
Statistical analysis Data were analyzed by using the analysis of variance and Student's t-test for comparing pairs of means (Snedecor and Cochran, 1980; Spector et al., 1985). RESULTS
The effects of different levels of relaxin on the percent of motile sperm are shown in Fig. 2. Addition of 1000, 333 and 167, but not 83 n g / m l relaxin to porcine semen significantly attenuated ( P < 0 . 0 1 ) the loss of sperm motility during 180 min at room temperature. Relaxin (1000 n g / m l ) improved the motility ( P < 0 . 0 5 ) of porcine sperm stored for 5 days at 12+ I ° C (Fig.3). Antirelaxin serum (1: 9000) markedly inhibited (P < 0.01) porcine sperm motility within 10 min compared with that of porcine semen diluted with normal rabbit serum (Fig. 4). The effect of adding porcine relaxin (1000 n g / m l ) and caffeine (3 m M ) to frozen-thawed porcine semen is presented in Fig. 5. The addition of caffeine 80
50
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20
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IN MINUTES
Fig. 2. The effect of 1000 ( & ), 333 ( • ), 167 ( I ) , 83 (C) ) and 0 (<~) ng/ml porcine relaxin (3000 IU/mg) on the percentage of motile boar sperm ( n = 6 ) . Motility was significantly (P<0.01) greater in semen containing 1000, 333 and 167 ng/ml relaxin compared with the control (0 ng/ ml) after 80, 120 and 180 min.
25 80
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IN HOURS
Fig. 3. Percentage of motile sperm in porcine semen containing 1000 ( • ) and 0 ( 0 ) ng/ml relaxin (3000 IU/mg) during a 5-day period at 12 + 1 °C (n= 5 ). After 36 h, motility was significantly (P < 0.05) greater in semen containing 1000 ng/ml relaxin compared with the control (0 ng/ml). 100
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IN MINUTES
Fig. 4. Percentage of motile sperm in porcine semen ( n = 3 ) after addition of antirelaxin serum ( ~ ) and normal rabbit serum (1 :9000) ( O ). Within 10 min, sperm motility was significantly reduced by antirelaxin serum (P<0.01).
increased (P < 0.01 ) sperm motility within 10 min and maintained motility for 40 min. Addition of relaxin had little effect on sperm motility during the first 10 min, but steadily increased motility during the next 80 rain (Fig. 5). The effects of relaxin and caffeine on sperm motility were similar at 40 rain (Fig. 5 ), but at 80 min relaxin was more effective (P < 0.01 ) than caffeine at maintaining motility (30 _ 2.9 vs 16 _+0.5% ). The addition of both relaxin and caffeine to sperm after thawing did not significantly improve motility in comparison with relaxin or caffeine alone (Fig. 5). DISCUSSION
Evaluation of sperm motility was based on the transmembrane migration ratio method first developed by Hong et al. (1981, 1985). This is a particularly
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T I M E IN M I N U T E S
Fig. 5. Effect of relaxin ( 1000 n g / m l ) ( • ), 3 m M caffeine ( • ), relaxin-caffeine ( [] ) and control (BTS) ( 0 ) on percentage of motile sperm in frozen-thawed porcine semen.
useful method when dealing with small volumes (100 H1) of semen. The TMMR method has proved not only to be objective and reproducible, but it has a high correlation with the percent progressive motility as determined by light microscopy. In the present study, the correlations between T M M R and the sperm motility as determined by light microscopy of fresh and frozen-thawed boar semen were 0.94 ( n = 1 5 ) and 0.92 ( n = 2 5 ) , respectively. The coefficient of variation (c.v.) of measurements of sperm motilities ranging from 32 to 100% was 4.5-8.7% c.v. (Hong et al., 1981 ). The present study indicates that porcine relaxin can attenuate the loss of motility of porcine spermatozoa in a dose- and time-dependent manner, whereas the addition of antirelaxin serum markedly decreased motility. The abrupt decrease in porcine sperm motility within 10 min after the addition of antirelaxin serum as shown here corroborates the studies with washed human spermatozoa (Sarosi et al., 1983a), and supports the idea that relaxin is necessary for optimal sperm motility. Porcine relaxin has a positive effect on in vitro fertilization (IVF) of zona-free mouse oocyte models (Pupula et al., 1986). The observation that the addition of relaxin to liquid-stored (12 _+1 ° C, 5 days) boar semen maintains greater sperm motility suggests that relaxin may be effective in maintaining sperm motility or improving pregnancy rates for artificial insemination. Though relaxin significantly increased sperm motility of frozen-thawed porcine semen as shown in this study as well as of frozen-thawed human semen (Lessing et al., 1985), it has no detectable stimulatory effect on sperm motility of fresh- or liquid-stored porcine semen. It is possible that the changes in the structure of the plasma membrane after cryopreservation enhance the effect of relaxin on sperm motility. Caffeine, an inhibitor of phosphodiesterase, increases intracellular concentrations of cAMP and has been shown to stimulate motility of porcine spermatozoa (Amelar et al., 1980; Dacheux and Paquignon, 1980). Relaxin as well as caffeine increase porcine sperm motility, although
27 they have different time-dependent effects, indicating the possibility of different mechanisms of action. Caffeine initially increases porcine sperm motility, but it drops abruptly; relaxin, however, increases sperm motility slowly and maintains greater motility significantly longer t h a n caffeine. The m e c h a n i s m by which relaxin affects sperm motility is unknown. However, relaxin inhibition of myometrial contractile activity m a y be mediated by an increase in cAMP, a decrease in light-chain phosphorylation, an alteration of the kinetic properties of myosin light-chain kinase, and an increased Ca 2+ efflux from myometrial cell (Sanborn et al., 1982; Hsu et al., 1985; Hsu and Sanborn, 1986; Rao and Sanborn, 1986). Because relaxin is a peptide hormone, it may bind to a surface receptor on the sperm and probably increases sperm motility by altering intracellular c A M P and Ca 2+ level and (or) by activating specific protein kinases and phosphorylation reactions in the sperm plasma membrane. ACKNOWLEDGMENTS This study was presented in part at the 20th Annual Meeting of the Society for the Study of Reproduction, Champaign, IL, 1987 (abstract 369). These studies were supported in part by U.S. D e p a r t m e n t of Agriculture, ARS, CSRS and OGPS Competitive Grant 85-CRCR-l-1862. Journal Paper J-13108 of the Iowa Agriculture and H o m e Economics Experiment Station, Ames, IA (Projects 2443, 2444 and 2754). We t h a n k Drs. J.P. Kunesh and P.G. Eness, Ambulatory Clinics, College of Veterinary Medicine, for monitoring the health status of the experimental animals; Dr. D.K. Hotchkiss, D e p a r t m e n t of Statistics, for assistance with statistical analyses; M.E. Shell and C.R. Bohnker for excellent technical assistance. Porcine relaxin antiserum was generously provided by Dr. B.G. Steinetz, New York University Medical Center (Tuxedo, NY).
REFERENCES Amelar, R.D., Dubin, L. and Schoenfeld,C., 1980. Sperm motility. Fertil. Steril., 34: 197-215. Anderson, L.L., 1987. Regulation of relaxin secretion and its role in pregnancy. Adv. Exp. Med. Biol., 219: 421-463. Brenner, S.H., Leasing, B.J., Schoenfeld,C., Amelar, R.D., Dubin, L. and Weiss, G., 1984. Stimulation of human sperm cervical mucus penetration in vitro by relaxin. Fertil. Steril., 42: 9296. Bryant, G.D., 1972. The detection of relaxin in porcine, ovine and human plasma by radioimmunoassay. Endocrinology,91:1113-1117. Btillesbach, E.E. and Schwabe, C., 1985. Naturally occurringporcine relaxin and large-scalepreparation of the B29 hormone. Biochemistry, 24: 7717-7722. Cameron, D.F., Corton, G.L. and Larkin, L.H., 1982. Relaxin-like antigenicity in the armadillo prostate gland. Ann. N.Y. Acad. Sci., 380: 231-240.
28 Colon, J.M., Ginsburg, F., Lessing, J.B., Schoenfeld, C., Goldsmith, L.T., Amelar, R.D., Dubin, L. and Weiss, G., 1986. The effect of relaxin and prostaglandin E2 on the motility of human spermatozoa. Fertil. Steril., 46: 1133-1139. Dacheux, J.L. and Paquignon, M., 1980. Effects of caffeine on ram and boar spermatozoa: influence of their stage of maturation and the medium; initiation of progressive motility oftesticular spermatozoa. In: A. Steinberger and E. Steinberger (Editors), Testicular Development, Structure and Function. Raven Press, New York, NY, pp. 513-532. Dubois, M.P. and Dacheux, J.L., 1978. Relaxin, a male hormone? Immunocytologic localization of a related antigen in the boar testis. Cell Tissue Res., 187: 201-214. Essig, M., Schoenfeld, C., Amelar, R.D., Dubin, L. and Weiss, G., 1982. Stimulation of human sperm motility by relaxin. Fertil. Steril., 38: 339-343. Hong, C.Y., Chaputedsaintonge, D.M. and Turner, P., 1981. A simple method to measure drugs effects on human sperm motility. Br. J. Clin. Pharmacol., 11: 385-387. Hong, C.Y., Chiang, B.N., Ku, J., Wei, Y.H. and Fong, C.Y., 1985. Calcium antagonists stimulate sperm motility in ejaculated human semen. Br. J. Clin. Pharmacol., 19: 45-49. Hsu, C.J. and Sanborn, B.M., 1986. Relaxin treatment alters the kinetic properties of myosin light chain kinase activity in rat myometrial cells in culture. Endocrinology, 118: 499-505. Hsu, C.J., McCormack, S.M. and Sanborn, B.M., 1985. The effect of relaxin on cyclic adenosine 3',5'-monophosphate concentrations in rat myometrial cells in culture. Endocrinology, 116: 2029-2035. Kendall, J.Z., Smith, R.G., Shih, Li-N, Webb, P.D. and Tare, W.H., 1982. Characterization of prostate relaxin. In: M. Bigazzi, F.C. Greenwood and F. Gasparri (Editors), Biology of Relaxin and Its Role in the Human. Elsevier Press, New York, NY, pp. 363-366. Lessing, J.B., Brenner, S.H., Schoenfeld, C., Sarosi, P., Amelar, R., Dubin, L. and Weiss, G., 1984. The effect of an anti-insulin antiserum on human sperm motility. Fertil. Steril., 42: 309-311. Lessing, J.B., Brenner, S.H., Schoenfeld, C., Sarosi, P., Goldsmith, L.T., Amelar, R., Dubin, L. and Weiss, G., 1985. The effect of relaxin on the motility of sperm in frozen-thawed human semen. Fertil. Steril., 44: 406-409. Lessing, J.B., Brenner, S.H., Colon, J.M., Ginsburg, F.W., Schoenfeld, C., Sarosi, P., Goldsmith, L.T., Amelar, R , Dubin, L. and Weiss, G., 1986. Effect of relaxin on human spermatozoa. J. Reprod. Med., 31: 304-309. Pupula, M., Quinn, P. and MacLennan, A., 1986. The effect of porcine relaxin on the fertilization of mouse oocytes in vitro. Clin. Reprod. Fertil., 4: 383-387. Pursel, V.G. and Johnson, L.A., 1975. Freezing of boar spermatozoa: fertilizing capacity with concentration semen and a new thawing procedure. J. Anim. Sci., 40: 99-102. Rao, M.R. and Sanborn, B.M., 1986. Relaxin increases calcium efflux from rat myometrial cells in culture. Endocrinology, 119: 435-437. Sanborn, B.N., Nihikori, K., Weisbrodt, N.W. and Sherwood, O.D., 1982. Relaxin affects uterine myosin phosphorylation and related activities. In: M. Bigazzi, F.C. Greenwood and F. Gasparri (Editors), Biology of Relaxin and Its Role in the Human. Elsevier Press, New York, NY, pp. 114-124. Sarosi, P., Schoenfeld, C., Berman, J., Basch, R., Randolph, G., Amelar, R., Dubin, L., Steinetz, B.G. and Weiss, G., 1983a. Effect of anti-relaxin antiserum on sperm motility in vitro. Endocrinology, 112: 1860-1861. Sarosi, P., Schoenfeld, C., Steinetz, B.G. and Weiss, G., 1983b. The effect of rabbit anti-porcine relaxin antibody on sperm motility in vitro. In: M. Bigazzi, F.C. Greenwood and F, Gasparri (Editors), Biology of Relaxin and Its Role in the Human. Elsevier Press, New York, NY, pp. 361-362. Snedecor, G.W. and Cochran, W.G., 1980. Statistical Methods. Edn. 7. Iowa State University Press, Ames, IA, pp. 258-267.
29 Spector, P.C., Goodnight, J.H., Sall, J.P. and Sarke, W.S., 1985. SAS User's Guide: Statistics, Edn. 5., SAS Institute, Cary, NC, pp. 433-506. Steinetz, B.G., Beach, V.L. and Kroc, R.L., 1959. The physiology of relaxin in laboratory animals. In: C.W. Lloyd (Editor), Recent Progress in the Endocrinology of Reproduction. Academic Press, New York, NY, pp. 389-427. Yki-Jarvinen, H., Wahlstrom, T., Seppala, M., 1983. Immunohistochemical demonstration of relaxin in the genital tract of men. J. Reprod. Fertil., 69: 693-695.