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Effects of androgens on fertilizing capacity of human spermatozoa
CONTRACEPTION
EFFECTS OF ANDRCGFNSON FERTILIZINGCAPACIl-Y OF HUMAN SPERMATOZOA
S.Y.W.Cl-m:, L.C.H. Tang, G.W.K.Tang and P.H. Chan Departmentof Obstetricsand Gynecology,Queen Nary Hospital, Universityof Hong Kong, Hong Kong
ABSTRACT
Ihe potential functionsof testosteroneand 5 ot -dihydrotestosterone, the androgensnormally present in human seminal plasma, on human spermatozoa1physiologywere evaluatedby studying the effects of these two steroidhormones on the in vitro fertilizingcapacity of human spermatozoa. Spermatozoacorrected from presumablyfertile men were washed in BW medium and incubatedwith differentconcentrations (0, 100, 250, 500, 1000 pg/ml) of testosteroneor 5 o( -dihydrotestosterone for 5 hr before inseminationof the zona-freehamster ova. Penetration of the zona-freehamster ova was scored 6 hr later and the resultswere analyzed statistically. Both testosteroneand 50( -dihydrotestosterone, at the concentrationstested, significantlydecreased the -in vitro penetrationof the denudedhamster ova in comparisonto the controls (p < 0.05). A dose-dependentresponsewas also observed for the 5 g dihydrotestosterone tested. These findings indicate that exogenous testosteroneand 5 a( -dihvdrotestosterone can inhibit the fertilizing capacity of human spermatbzoa.invitro, and suggest that the androgens normally present in human seminal plasma may serve, in part, to prevent prematurespermatozoa1capacitationbefore the spermatozoareach the site of fertilizationin vivo.
"Author to whom all correspondenceshould be addressed.
Submittedfor publication October 21, 1983 Accepted for publication December 23, 1983
NOVEMBER
1983VOL.28NO.S
481
CONTRACEPTION
INTRODUCTION ‘Ihe presence of androgens, particularly testosterone and 5% dihydrotestosterone, has been demonstrated in human seminal plasma (1,2,3,4,5). Although testosterone and 5 a-dihydrotestosterone are known to play an important role in spermatozoa1 maturation, the possible effects of these androgens on human spermatozoa1 functions are still not clear. The presence of s ecific androgen receptors on human spennatozoa has been suggested (6,7 P ; and testosterone increased the tetracycline binding capacity of human s ermatozoa and inhibited the human spermatozoa1 motility in vitro (8,9 P . Incubation of human spermatozoa in vitro with testosteEn=ibited the spermatozoa1 respiration and -decreased the number of motile and viable spermatozoa during the incubation period (10). Information relating to the effects of 5 cx -dihydrotestosterone on human spermatozoa1 physiology is limited. In addition, there are no renorts available on the effects of testosterone and 5 d, -dihvdrotestosterone on the fertilizing capacity of human spermatozoa, in vitro or j_t~ vivo . ‘lhe zona-free hamster ova penetration assay, which measures the abilitv of human snermatozoa to undergo canacitation. the acrosome reaction, and fusion with the denuded”hamster ova in’vitro (11,12), has been demonstrated to be effective in evaluating thrisro fertilizing Several studies haveynmed a relacapacity of human spermatozoa. tionship between the in vitro fertilizing capacity and male infertility (13,14,15,16). Spermatozoa - from fertile men were found to be capable of penetrating the zona-free hamster ova at a higher rate than those from subfertile or infertile men (13,14,15,16). In this study, spermatozoa collected from presumably fertile men were treated with graded doses of testosterone or 5 c( -dihydrotestosterone before incubation with the zona-free hamster ova for an evaluation of these androgens on the fertilizing capacity of human spermatozoa in vitro. -MATERIALSAND MEXHODS A total of 20 semen samples were collected for this study. These samples were collected from presumably fertile men who had fathered at Specimens were collected by masturbation after at least one child. least 2 days of abstinence and allowed to liquefy in room temperature. General semen analyses were performed within 2 hr after collection. Ten semen samples were randomly selected for testing the effects of testosterone on the -in vitro fertilizing capacity of human spermatozoa. The other 10 samples were tested for the effects of 5 O(-dihydrotestosterone. After liquefaction, aliquots of semen were mixed with 3 volumes of Biggers, Whitten and Whittingham (BW) medium (17), and centrifuged at 600 x g for 5 min. The supematant was discarded. The spermatozoa1 pellets were resuspended in BWWmedium and the washing process was The spermatozoa were resuspended in fresh BW repeated once more.
482
NOVEMBER
1983 VOL. 28 NO. 5
CONTRACEPTION
medium and the final
concentration
was adjusted
to 10 x lo6
sperm/ml.
Testosterone or 5 ti-dihydrotestosterone (Sigma Chemical Co., St. Louis, MO, USA) was dissolved in ethanol to different final concentrations (100,250,500, 1000 pg/ml) and added to glass tubes by the pipetting of 0.4 ml of the respective solutions. The control tubes The solvent in each received the same volume of steroid-free solvent. tube was completely evaporated in a 60°C water bath, and the tubes were cooled down to room temperature before the addition of spermatozoa1 suspensions. Four-tenths of a milliliter of Bw14medium-washed spermatozoa1 suspensions were added to each tube, which contained no testosterone / 50~ -dihydrotestosterone (control) or different amounts (40,100,200,400 pg) of testosterone / 5 oC-dihydrotestosterone The final concentrations of testosterone or 5 Oc(experimental). dihydrotestosterone in experimental tubes were thus calculated to be 100, 250, 500 and 1000 pg/ml, respectively. The test tubes were then incubated at 37°C in a horizontal position under air atmosphere for 5 hr. The percent motility of spermatozoa was monitored at 0, 2, and 5 hr during the incubation period. At the end of the 5-hr incubation period, spermatozoa1 suspensions were diluted with equal volumes of fresh BW medium (37’C) and centrifuged at 300 x g for 3 min to remove the testosterone or 5 a-dihydrotestosterone present in the original medium. The control tubes received the same treatment. The supernatants were aspirated, and the spermatozoal pellets were resu pended in fresh BW medium to give a final concentration of 10 x 10 ?I sperm/ml. The spermatozoa1 suspensions were according then tested for the zona-free hamster ova penetration efficient to the procedure originally described by Yanagimachi et al. (12 i’ and validated in the Department’s laboratory by Chan et aK n8,19,20). --. Penetration was scored after 6 hr of sperm-ova incubation. The penetration rate was defined as the number of ova penetrated / number of ova inseminated x 100 %, and the significance was analyzed by the Chisquare test. RESULTS Results of the -in vitro penetration of the zona-free hamster ova by BW medium-washed human spermatozoa following testosterone or 5m dihydrotestosterone treatment are given in Table I. The penetration rate for the control in the testosterone group was 50.2%, which was significantly higher (p < 0.05) than the penetration rates for spermatozoa after treatment with different concentrations of testosterone (100 g/ml, 29.1%; 250 pg/ml, 30.7%; 500 pg/ml, 31.0%; 1000 pg/ml, 28.6% 7 . 5d -Dihydrotestosterone also decreased the penetration rates of spermatozoa in comparison to the control. The penetration rate for the control in the 5 U-dihydrotestosterone group was 14.0%, which was significantly higher (p < 0.05) than the penetration rates observed for the spermatozoa following 5 W-dihydrotestosterone treatment (100 pg/ml, 11.9%; 250 pg/ml, 8.5%; 500 pg/ml, 6.1%; 1000 pg/ml, 4.9%). A dose-dependent response was observed for the penetration rate following 5 o(-dihydrotestosterone treatment as the penetration rates
NOVEMBER
1983 VOL. 28 NO. 5
483
CONTRACEPTION
Table I. Effects of Testosteroneand 5(x -Dihydrotestosterone on the In Vitro Penetrationof Zona-FreeHamster Ova by Human Spermatozoa -Treatment
Number of experiment
Total number of ova inseminated
None (control)
10
410
50.2
100 pg/ml
10
460
29.1 +:
250 pg/ml
10
436
30.7 +:
500 pg/ml
10
490
31.0 ;'(
1000 pg/ml
10
440
28.6 :';
Penetration rate ( % )
Testosterone
5 W-Dihydrotestosterone None (control)
10
444
14.0
100 pg/ml
10
462
11.9 ;k
250 pg/ml
10
436
8.5 :'(
500 &ml
10
425
6.1 "
1000 pg/ml
10
432
4.9 +:
"Significantlydifferent from the controls,p < 0.05 ((hi-squaretest)
were more significantlydecreased (p < 0.05) after treatmentwith higher concentrationsof the steroid hormone. The dose-dependent responsewas not observedwith the range of testosteroneconcentrations tested in the study. 'Ihevalues of percent motility for the control or the testosteronetreated or 5W-dihydrotestosterone-treatedspermatozoaduring the spermatozoa1preincubationperiod before inseminationof the zona-free hamster ova are shown in Table II. The percent motility decreasedas a functionof time during incubationand there were no significant differencesin the percent motility between the control and the testosterone-treated or the 5 ti-dihydrotestosterone-treated spermatozoa (p > 0.05; Sign test).
484
NOVEMBER
1983VOL.28NO.S
CONTRACEPTION
Effects of Testosterone and 5% -Dihydrotestosterone Table II. Percent Motility of Human Spermatozoa during the Preincubation Percent
Treatment 0 hr
on the Period
Motility 2 hr
5 hr
Testosterone None (control)
48 + 1:‘:
45 + 4
36 + 5
100 pg/ml
48 + 3
44 + 4
33 2 6
250 pg/ml
49 + 3
47 2 3
32 k 6
500 pg/ml
47 * 5
45 t 5
32 + 6
1000 pg/ml
50 + 4
45 + 6
31 f 6
None (control)
53 + 4
51 It 4
45 f 4
100 pg/ml
52 + 3
56 + 4
43 + 6
250 pg/ml
56 t 3
55 f 5
44 f 3
500 pg/ml
55 f 4
51 + 4
39 f 6
1000 pg/ml
55 f 3
52 + 5
38 f 6
5 U-Dihydrotestosterone
“Mean + SEM (standard
error
of
the mean) of results
from 10 experiments
DISCUSSION The roles of seminal plasma androgens in human spermatozoa1 fertilization process, & vitro or in vivo, have not been fully investigated. The findingxthe present study indicate that exogenous testosterone or 5~ -dihydrotestosterone, at the concentrations tested, are capable of inhibiting the --in vitro penetration of zona-free hamster ova by washed spermatozoa. The mechanisms for the inhibitory effects of exogenous testosterone or 5 a-dihydrotestosterone on the by&~ fertilizing capacity of human spermatozoa are presently unknown. There were no significant differences for the percent motility between the control and the testosterone-treated
NOVEMBER
1983 VOL. 28 NO. 5
485
CONTRACEPTION
or the 5 U-dihydrotestosterone-treated spermatozoa1samples during the preincubationperiod and thus the observeddecrease in penetration rates followingsteroidhormone treatmentdo not appear to be related to the effects on human spermatozoa1motility. Since spermatozoa1 capacitationis one prerequisitefor fertilizationin some maaunalian species, includingthe human (12,21,22),the exogenoustestosterone or 5 cx -dihvdrotestosterone might have affected the human spermatozoa1 capacitationprocess in vitro, and resulted in the decreasedpenetration rates. It has been renorted that a loss of tetracyclinebinding by spermatozoais associatedwith the spermatozoa1capacitationin-the rabbit (23) and Briggs (8) observed an increasein the tetracycline binding capacity of washed human spermatozoafollowingexogenous testosteronetreatment. It is known that the whole human seminal plasma revents the fusion of human spermatozoawith zona-freehamster ova (24t;. The testosteroneand 5 W-dihydrotestosterone,normally present in human seminal plasma, may be part of a mechanism to prevent nrematuresnermatozoalcapacitationbefore the spermatozoareach the 'siteof fertilizationin iivo. Further studiesare necessary to ascertainthe role of seminal plasma androgensin the human spermatozoa1 capacitationand fertilizationprocesses. ACKNOWLEDGMENT This project was supportedin part by a grant from the Committee on Research and ConferenceGrants of the Universityof Hong Kong. REFERENCES
1. Biswas, S., Ferguson,K.M., Stedronska,J., Baffoe, G., Mansfield, M. and Kosbab, M. Fructose and hormone levels in semen: their correlationswith sperm counts and motility. Fertil. Steril. 30: 200-204 (1978) 2. Lannou, D.Le., Massart, C., Chambon,Y., Nicol, M. and Allannic,H. Testosteroneand 5 OC-dihydrotestosterone concentrationsin human seminal plasma. Int. J. Androl. 3: 502-506 (1980) 3. Moreno, B.J. Ridley, A.J. and Blasco, L. Free testosteronevalues in human seminal fluid. Biol. Reprod. (Suppl. 1) 22: 91A (1980) 4. Purvis, K., Iandgren,B.M., Cekan, Z. and Diczfalusy,E. Indices of gonadal function in the human male. II-Seminalplasma levels of steroids in normal and pathologicalconditions. Clin. Endocrinol. 4: 247-258 (1975) 5. Schoenfeld,C., Amelar, R.D., Dubin, L. and Numeroff,M. Folliclestimulatinghormone, luteinizinghormone and testosteronelevels found in human seminal plasma. Fertil. Steril. 29: 69-71 (1978) 6. Hyne, R.V. and Boettcher,B. The selectivebinding of steroidsby human spermatozoa. Contraception15: 163-174 (1977)
486
NOVEMBER
1983VOL.28NO.S
CONTRACEPTION
7.
Cheng, C.Y., Boettcher,B., Rose, R.J., Kay, D.J. and Tinneberg,H.R. The binding of sex steroids to human spermatozoa. An autoradiographic study. Int. J. Androl. 4: 1-17 (1981)
a. Brigs, M. Tetracyclineand steroidhormone binding to human spermatozoa. Acta. Endocrinol.75: 785-792 (1974) 9.
Ridley, A.J. and Blasco, L. Testosteroneand gossypol effects on human sperm motility. Fertil. Steril. 36: 638-642 (1981)
10. Trifunac,N.P. and Bernstein,G.S. Effect of steroidhormones on the metabolismof human spermatozoa. Contraception23: 527-533 (1981)
11. Talbot, P. and Chacon, R.S. Ultrastructuralobservationson binding and membrane fusion between human sperm and zona pellucida-free hamster oocytes. Fertil. Steril. 37: 240-248 (1982) 12. Yanagimachi,R., Yanagimachi,H. and Rogers, B.J. The use of
zona-freeanimal ova as a test system for the assessmentof the fertilizingcapacity of human spermatozoa. Biol. Reprod. 15: 471-476 (1976) 13. Barros, C., Gonzaler,J., Herrera, E. and Bustos-Obregon,E. Human sperm penetrationinto zona-freehamster oocytes as a test to evaluate the sperm fertilizingability. Andrologia11: 197-210 (1979) 14. Overstreet,J.W., Yanagimachi,R., Katz, D.F., Hayashi, K. and Hanson, F.W. Penetrationof human spermatozoainto the human zona pellucidaand the zona-freehamster egg: a study of fertile donors and infertilepatients. Fertil. Steril. 33: 534-542 (1980) 15. Rogers, B.J., Van-Campen,H., Ueno, M., Iambert, H., Bronson,R. and Hale, R. Analysis of human spermatozoa1fertilizin ability using zona-freeova. Fertil. Steril. 32: 664-670 (19797 16. Zausner-Guelman,B., Blasco, L. and Wolf, D.P. Zona-freehamster eggs and human sperm penetrationcapacity:a comparativestudy of proven fertile donors and infertilitypatients. Fertil. Steril. 36: 771-777 (1981) 17. Biggers, J.D., Whitten, W.K. and Whittingham,D.F. in Methods of MammalianEmbryology(J.C. Daniel, Editor).Raven Press, New York, 1971 p. 86-116 18. Chan, S.Y.W., Tang, L.C.H. and Ma, H.K. Stimulationof the zonafree hamster ova penetrationefficiencyby human spermatozoaafter 17-8 estradiol treatment. Fertil. Steril. 39: 80-84 (1983) 19. Chan, S.Y.W., Tang, L.C.H. and Ma, H.K. Stimulationof the human spermatozoa1fertilizingability by dibutyryl-3',5'-CAMP and theophylline-in vitro. Arch. Androl. 11: 19-23 (1983)
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1983 VOL. 28 NO. 5
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20. Chan, S.Y.W., Tang, L.C.H., Chan, P.H., Tang, G.W.K. and Ma, H.K. Relationshipsof seminal plasma prolactinwith spermatozoa1 characteristicsand fertilizingcapacity-in vitro. Arch. Androl. In Press. 21. Bedford, J.M. Sperm capacitationand fertilizationin mammals. Biol. Reprod. (Suppl.)2: 128-158 (1970) 22. Bedford, J.M. Significanceof the need for sperm capacitation before fertilizationin eutherianmamnals. Biol. Reprod. 28: 108-121 (1983) 23. Vaidya, R.A., Bedford, J.M., Glass, R.H. and Morris, J.M. Evaluationof the removal of tetracyclinefluorescencefrom spermatozoaas a test for capacitationin the rabbit. J. Reprod. Fertil. 19: 483-489 (1969) 24. Kanwar, C., Yanagimachi,R. and Lopata, A. Effects of human seminal plasma on fertilizin capacity of human spermatozoa. Fertil. Steril. 31: 321-327 71979)
488
NOVEMBER
1983VOL. 28N0.5
EFFECTS OF ANDRCGFNSON FERTILIZINGCAPACIl-Y OF HUMAN SPERMATOZOA
S.Y.W.Cl-m:, L.C.H. Tang, G.W.K.Tang and P.H. Chan Departmentof Obstetricsand Gynecology,Queen Nary Hospital, Universityof Hong Kong, Hong Kong
ABSTRACT
Ihe potential functionsof testosteroneand 5 ot -dihydrotestosterone, the androgensnormally present in human seminal plasma, on human spermatozoa1physiologywere evaluatedby studying the effects of these two steroidhormones on the in vitro fertilizingcapacity of human spermatozoa. Spermatozoacorrected from presumablyfertile men were washed in BW medium and incubatedwith differentconcentrations (0, 100, 250, 500, 1000 pg/ml) of testosteroneor 5 o( -dihydrotestosterone for 5 hr before inseminationof the zona-freehamster ova. Penetration of the zona-freehamster ova was scored 6 hr later and the resultswere analyzed statistically. Both testosteroneand 50( -dihydrotestosterone, at the concentrationstested, significantlydecreased the -in vitro penetrationof the denudedhamster ova in comparisonto the controls (p < 0.05). A dose-dependentresponsewas also observed for the 5 g dihydrotestosterone tested. These findings indicate that exogenous testosteroneand 5 a( -dihvdrotestosterone can inhibit the fertilizing capacity of human spermatbzoa.invitro, and suggest that the androgens normally present in human seminal plasma may serve, in part, to prevent prematurespermatozoa1capacitationbefore the spermatozoareach the site of fertilizationin vivo.
"Author to whom all correspondenceshould be addressed.
Submittedfor publication October 21, 1983 Accepted for publication December 23, 1983
NOVEMBER
1983VOL.28NO.S
481
CONTRACEPTION
INTRODUCTION ‘Ihe presence of androgens, particularly testosterone and 5% dihydrotestosterone, has been demonstrated in human seminal plasma (1,2,3,4,5). Although testosterone and 5 a-dihydrotestosterone are known to play an important role in spermatozoa1 maturation, the possible effects of these androgens on human spermatozoa1 functions are still not clear. The presence of s ecific androgen receptors on human spennatozoa has been suggested (6,7 P ; and testosterone increased the tetracycline binding capacity of human s ermatozoa and inhibited the human spermatozoa1 motility in vitro (8,9 P . Incubation of human spermatozoa in vitro with testosteEn=ibited the spermatozoa1 respiration and -decreased the number of motile and viable spermatozoa during the incubation period (10). Information relating to the effects of 5 cx -dihydrotestosterone on human spermatozoa1 physiology is limited. In addition, there are no renorts available on the effects of testosterone and 5 d, -dihvdrotestosterone on the fertilizing capacity of human spermatozoa, in vitro or j_t~ vivo . ‘lhe zona-free hamster ova penetration assay, which measures the abilitv of human snermatozoa to undergo canacitation. the acrosome reaction, and fusion with the denuded”hamster ova in’vitro (11,12), has been demonstrated to be effective in evaluating thrisro fertilizing Several studies haveynmed a relacapacity of human spermatozoa. tionship between the in vitro fertilizing capacity and male infertility (13,14,15,16). Spermatozoa - from fertile men were found to be capable of penetrating the zona-free hamster ova at a higher rate than those from subfertile or infertile men (13,14,15,16). In this study, spermatozoa collected from presumably fertile men were treated with graded doses of testosterone or 5 c( -dihydrotestosterone before incubation with the zona-free hamster ova for an evaluation of these androgens on the fertilizing capacity of human spermatozoa in vitro. -MATERIALSAND MEXHODS A total of 20 semen samples were collected for this study. These samples were collected from presumably fertile men who had fathered at Specimens were collected by masturbation after at least one child. least 2 days of abstinence and allowed to liquefy in room temperature. General semen analyses were performed within 2 hr after collection. Ten semen samples were randomly selected for testing the effects of testosterone on the -in vitro fertilizing capacity of human spermatozoa. The other 10 samples were tested for the effects of 5 O(-dihydrotestosterone. After liquefaction, aliquots of semen were mixed with 3 volumes of Biggers, Whitten and Whittingham (BW) medium (17), and centrifuged at 600 x g for 5 min. The supematant was discarded. The spermatozoa1 pellets were resuspended in BWWmedium and the washing process was The spermatozoa were resuspended in fresh BW repeated once more.
482
NOVEMBER
1983 VOL. 28 NO. 5
CONTRACEPTION
medium and the final
concentration
was adjusted
to 10 x lo6
sperm/ml.
Testosterone or 5 ti-dihydrotestosterone (Sigma Chemical Co., St. Louis, MO, USA) was dissolved in ethanol to different final concentrations (100,250,500, 1000 pg/ml) and added to glass tubes by the pipetting of 0.4 ml of the respective solutions. The control tubes The solvent in each received the same volume of steroid-free solvent. tube was completely evaporated in a 60°C water bath, and the tubes were cooled down to room temperature before the addition of spermatozoa1 suspensions. Four-tenths of a milliliter of Bw14medium-washed spermatozoa1 suspensions were added to each tube, which contained no testosterone / 50~ -dihydrotestosterone (control) or different amounts (40,100,200,400 pg) of testosterone / 5 oC-dihydrotestosterone The final concentrations of testosterone or 5 Oc(experimental). dihydrotestosterone in experimental tubes were thus calculated to be 100, 250, 500 and 1000 pg/ml, respectively. The test tubes were then incubated at 37°C in a horizontal position under air atmosphere for 5 hr. The percent motility of spermatozoa was monitored at 0, 2, and 5 hr during the incubation period. At the end of the 5-hr incubation period, spermatozoa1 suspensions were diluted with equal volumes of fresh BW medium (37’C) and centrifuged at 300 x g for 3 min to remove the testosterone or 5 a-dihydrotestosterone present in the original medium. The control tubes received the same treatment. The supernatants were aspirated, and the spermatozoal pellets were resu pended in fresh BW medium to give a final concentration of 10 x 10 ?I sperm/ml. The spermatozoa1 suspensions were according then tested for the zona-free hamster ova penetration efficient to the procedure originally described by Yanagimachi et al. (12 i’ and validated in the Department’s laboratory by Chan et aK n8,19,20). --. Penetration was scored after 6 hr of sperm-ova incubation. The penetration rate was defined as the number of ova penetrated / number of ova inseminated x 100 %, and the significance was analyzed by the Chisquare test. RESULTS Results of the -in vitro penetration of the zona-free hamster ova by BW medium-washed human spermatozoa following testosterone or 5m dihydrotestosterone treatment are given in Table I. The penetration rate for the control in the testosterone group was 50.2%, which was significantly higher (p < 0.05) than the penetration rates for spermatozoa after treatment with different concentrations of testosterone (100 g/ml, 29.1%; 250 pg/ml, 30.7%; 500 pg/ml, 31.0%; 1000 pg/ml, 28.6% 7 . 5d -Dihydrotestosterone also decreased the penetration rates of spermatozoa in comparison to the control. The penetration rate for the control in the 5 U-dihydrotestosterone group was 14.0%, which was significantly higher (p < 0.05) than the penetration rates observed for the spermatozoa following 5 W-dihydrotestosterone treatment (100 pg/ml, 11.9%; 250 pg/ml, 8.5%; 500 pg/ml, 6.1%; 1000 pg/ml, 4.9%). A dose-dependent response was observed for the penetration rate following 5 o(-dihydrotestosterone treatment as the penetration rates
NOVEMBER
1983 VOL. 28 NO. 5
483
CONTRACEPTION
Table I. Effects of Testosteroneand 5(x -Dihydrotestosterone on the In Vitro Penetrationof Zona-FreeHamster Ova by Human Spermatozoa -Treatment
Number of experiment
Total number of ova inseminated
None (control)
10
410
50.2
100 pg/ml
10
460
29.1 +:
250 pg/ml
10
436
30.7 +:
500 pg/ml
10
490
31.0 ;'(
1000 pg/ml
10
440
28.6 :';
Penetration rate ( % )
Testosterone
5 W-Dihydrotestosterone None (control)
10
444
14.0
100 pg/ml
10
462
11.9 ;k
250 pg/ml
10
436
8.5 :'(
500 &ml
10
425
6.1 "
1000 pg/ml
10
432
4.9 +:
"Significantlydifferent from the controls,p < 0.05 ((hi-squaretest)
were more significantlydecreased (p < 0.05) after treatmentwith higher concentrationsof the steroid hormone. The dose-dependent responsewas not observedwith the range of testosteroneconcentrations tested in the study. 'Ihevalues of percent motility for the control or the testosteronetreated or 5W-dihydrotestosterone-treatedspermatozoaduring the spermatozoa1preincubationperiod before inseminationof the zona-free hamster ova are shown in Table II. The percent motility decreasedas a functionof time during incubationand there were no significant differencesin the percent motility between the control and the testosterone-treated or the 5 ti-dihydrotestosterone-treated spermatozoa (p > 0.05; Sign test).
484
NOVEMBER
1983VOL.28NO.S
CONTRACEPTION
Effects of Testosterone and 5% -Dihydrotestosterone Table II. Percent Motility of Human Spermatozoa during the Preincubation Percent
Treatment 0 hr
on the Period
Motility 2 hr
5 hr
Testosterone None (control)
48 + 1:‘:
45 + 4
36 + 5
100 pg/ml
48 + 3
44 + 4
33 2 6
250 pg/ml
49 + 3
47 2 3
32 k 6
500 pg/ml
47 * 5
45 t 5
32 + 6
1000 pg/ml
50 + 4
45 + 6
31 f 6
None (control)
53 + 4
51 It 4
45 f 4
100 pg/ml
52 + 3
56 + 4
43 + 6
250 pg/ml
56 t 3
55 f 5
44 f 3
500 pg/ml
55 f 4
51 + 4
39 f 6
1000 pg/ml
55 f 3
52 + 5
38 f 6
5 U-Dihydrotestosterone
“Mean + SEM (standard
error
of
the mean) of results
from 10 experiments
DISCUSSION The roles of seminal plasma androgens in human spermatozoa1 fertilization process, & vitro or in vivo, have not been fully investigated. The findingxthe present study indicate that exogenous testosterone or 5~ -dihydrotestosterone, at the concentrations tested, are capable of inhibiting the --in vitro penetration of zona-free hamster ova by washed spermatozoa. The mechanisms for the inhibitory effects of exogenous testosterone or 5 a-dihydrotestosterone on the by&~ fertilizing capacity of human spermatozoa are presently unknown. There were no significant differences for the percent motility between the control and the testosterone-treated
NOVEMBER
1983 VOL. 28 NO. 5
485
CONTRACEPTION
or the 5 U-dihydrotestosterone-treated spermatozoa1samples during the preincubationperiod and thus the observeddecrease in penetration rates followingsteroidhormone treatmentdo not appear to be related to the effects on human spermatozoa1motility. Since spermatozoa1 capacitationis one prerequisitefor fertilizationin some maaunalian species, includingthe human (12,21,22),the exogenoustestosterone or 5 cx -dihvdrotestosterone might have affected the human spermatozoa1 capacitationprocess in vitro, and resulted in the decreasedpenetration rates. It has been renorted that a loss of tetracyclinebinding by spermatozoais associatedwith the spermatozoa1capacitationin-the rabbit (23) and Briggs (8) observed an increasein the tetracycline binding capacity of washed human spermatozoafollowingexogenous testosteronetreatment. It is known that the whole human seminal plasma revents the fusion of human spermatozoawith zona-freehamster ova (24t;. The testosteroneand 5 W-dihydrotestosterone,normally present in human seminal plasma, may be part of a mechanism to prevent nrematuresnermatozoalcapacitationbefore the spermatozoareach the 'siteof fertilizationin iivo. Further studiesare necessary to ascertainthe role of seminal plasma androgensin the human spermatozoa1 capacitationand fertilizationprocesses. ACKNOWLEDGMENT This project was supportedin part by a grant from the Committee on Research and ConferenceGrants of the Universityof Hong Kong. REFERENCES
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