- Email: [email protected]
The anti-progestin CDB 2914 has no antifertility effect in male rats
The Anti-Progestin CDB 2914 Has No Antifertility Effect in Male Rats Christina Wang$*st Amiya Sinha-Hikim,-f and Andrew Leung*jt This study examines the effect of an anti-progestin (CDB 2914) with antiprogestational potencies similar to RU 484 on spermatogenesis, sperm maturation, and fertility in male rats. Adult male rats of proven fertility were administered the ~ti-prog~t~ (10 rng~kg~day~ or vehicle {eontrol group} for $4, 35, and 70 days to study the possible effect of this compound on epididymal sperm maturation, post-meiotic sperm development, spermatogenesis, and fertility, respectively. Fertility rates of the rats were determined by mating studies. The ~ti-prog~tin, CDB 2914, had no effect on testis or accessory organ weights, epididymal sperm content or motility, testicular sperm count, spermatogenesis, and fertility of male rats. This study suggests that anti-progestins, when administered even at higher doses than those used m humans, have no contraceptive effect in adult male rats. ~ONT~CEP~ON 1995;51: 215-218 INY
WORDS:
male reproduction,
male contraception,
sper-
matogenesis
Introduction The anti-progestin RU 486 (Mifepristone, RouselUCLAF; Romainville, France] has been shown to have very potent anti-progestin activities both in vivo and in vitro.’ A single oral dose of 600 mg of RU 486 followed by the administration of a prostaglandin analog in over 2000 women in France resulted in interruption of pregnancy in 96% of women.’ RU 486 may be potentially useful for contragestion and contraception3 and is also currently being tested for treatment of endometriosis and uterine fibroids.4r5 In addition to its anti-progestational activities, RU 486 has antiglucocorticoid activity and has been used at higher doses for the treatment of Gushing’s syndrome.6 RU 486 also has antiandrogenic activity as demonstrated by inhibiting the effects of testosterone on the increases “Division of Endocrinology, Department of Medicine, Center, Los Angeles, CA; and *Harbor-UCLA Medical USA Address for corresponden e and reprint requests: Medical Clinical Study Center @ox I 1 ), tiarbor-UCLA 90509-2910 Submitted for publication Accepted November 17, 1
62 1995 Elsevier Science Inc. 655 Avenue of the Americas New York,
NY 10010
Cedars-Sinai Medical Center, Torrance, CA, Christina Center,
Wang- M.D., Torrance, CA
in seminal vesicle and prostate weights in castrated rats.,’ In vitro studies have indicated that progesterone directly caused rapid increases in calcium influx into human spermatozoa. 7-P This increase in intracellular calcium is associated with induction in acrosome reaction and hyPeractivated motility in human spermatozoa.7p10 Studies by Blackmore et al.sJ” and Baldi et al.9 showed that the anti-progestins RU 486 or ZK 98299 (10-5M) had minimal antagonistic effects on the action of progesterone on intracellular calcium concentrations in human spermatozoa. Our own study also failed to demonstrate any effect of RU 486 ( 10h5M) on the progesterone-induced hyperactivated motility of human spermatozoa.” However, Yang et al. l2 recently reported that RU 486 induced transient decrease of intracellular calcium and inhibited the progesterone-simulated calcium influx and acrosome reaction. In light of these findings, we studied the effects of an anti-progestin on spermatogenesis as well as on fertility in adult male rats.
Materials and Methods Animals
Adult, male Sprague-Dawley (60 to 90 days old) rats of proven fertility and adult, virgin, females (60 days old) were obtained from Harlan-Sprague-Dawley (Indianapolis, IN). Rats were housed at the vivarium of Cedars-Sinai Medical Center with 12-hr light and 12-hr dark cycles and were fed with laboratory chow and water ad libitum, At the end of the experiment, the animals were sacrificed with carbon dioxide asphyxia in a gas chamber. The protocol was approved by the Institutional Animal Care and Use Committee. Ma teriah The anti-progestin CDB 2914 (17o-acetoxy- 11&(4-N, N-dimethylaminophenyl)19~norpregna-4,9-diene-3, 20-dione; batch numbers 2.75 gm of CDB-2914 I and 3 11 mg of CDB-2914 K) was supplied by Bioequal (Rockville, MD) obtained through Drs. Gabriel Bialy and Richard Blye, CDB, CPR, NICHD. This antiprogestin has antiprogestational potencies similar to lSSN/ OOIO-7824/95/$950 SSDl/ OOlO-7824(95)09920-S
216
Wang
et ai,
RU 486 (personal communications with Dr. G. Bialy). All male animals were dosed with CDB 2914 at 10 mg/kg/day orally. CDB 2914 was made up every week as a suspension of 20 mg/ml in 5% ethanol in sesame oil. The control animals were dosed daily with the vehicle. Experimental
Protocol
To test for an effect on sperm maturation
in the epididymis, 6 to 8 male animals from each group were treated daily over a 14-day (2-week) period with the vehicle or anti-progestin. To test for post-meiotic action in the testis, male animals were treated for a 35day (5week) period. To determine whether the drug has any effect on spermatogenesis, male animals were dosed for a 70-day (lo-week) period. Spermatogenesis in the Sprague-Dawley rat extends over 4 cycles covering a period of 51.6 days.13 Thus, at the end of 70 days, the effect of CDB 2914, if any, on spermatogenesis can be detected. At the end of 14,35, and 70 days, each male rat was placed with 2 females in late proestrus for 4 days. Mating was checked and confirmed by examination for seminal plugs and sperm in vaginal smears. The day on which a spermpositive smear was found was designated day 1 of gestation. At 13 days of gestation, the females were sacrificed and autopsied to determine fertility status. At the end of the mating period, the males in each group were sacrificed and autopsied. Methods Male animals were sacrificed and weighed on days 36 and 71 after mating. Blood samples were obtained by inferior vena cava puncture for the measurement of serum testosterone and corticosterone. The testes, seminal vesicles, and epididymides were weighed. The cauda epididymides were opened and placed into warmed phosphate-buffered saline at 37’C. The presence or absence of spermatozoa and their motility were rapidly assessed within 5 to 10 minutes under phase contrast microscopy. At autopsy, the liver, kidneys, lungs, heart, and adrenals were examined. One testis was homogenized in phosphate-buffered saline and homogenization-resistant spermatozoa were counted.14 The other testis was cut into small pieces and fixed in 5% glutaraldehyde in 0.05 M cacodylate buffer (pH7.4) for 24 hr, post-fixed in 1% osmium tetroxide, dehydrated in a graded series of ethanol, and embedded in Araldite.15 Semi-thin (1 pm) sections were cut from the Araldite-embedded tissue blocks with a LKB ultramicrotome and were stained with 1% toluidine blue for light microscopic examination. Thirteen days after the pairing with male rats, the females were weighed and autopsied and their uteri
Contraception 1995:51:215-218
and ovaries were removed. The uteri were opened longitudinally and the number of normal and abnormal (dead and/or degenerate] fetuses recorded in mated females. Females which were paired but in which no sperm were present may be pseudopregnant. They were sacrificed on day 13 after pairing and the number of corpora lutea recorded. The uteri were examined for evidence of implantation sites. Serum levels of testosterone and corticosterone were measured by sensitive radioimmunoassays using kits supplied by ICN Laboratories (Costa Mesa, CA). These steroid assays have been validated for studies with rat sera.15 Statistical Analysis Arithmetic means were computed across animals within each group for each variable. The group means for each variable at each time point were compared by ANOVA followed by contrasts to test for trends on dosage levels. Differential effects of the antiprogestagen treatment over time were assessed by using two-way ANOVA and testing for significant interaction between time and treatment.
Results and Discussion Both the treated and control animals were healthy and active. Gross examination of the heart, lung, liver, and kidneys were normal in the treated and control animals. There were no significant differences in the body, testis, epididymis, seminal vesicles, and adrenal weights between CDB 2914-treated animals and the controls after 35 or 70 days. Serum testosterone levels were not significantly different between 1. Body and organ weights (g) in rats after 35 and 70 days treatment with the anti-progestin CDB 2914
Table
35 Days
Body Testis Epididymis Adrenal Seminal vesicles Serum testosterone (rig/ml)
Treatment
Control
(II = 6)
(n = 6)
437.8 k ll.O*
2.01 * 0.03 0.65 2 0.01 47.0 k 1.8 1.38 k 0.08 0.87 * 0.23
459.2 1.88 0.63 50.3
k 10.2 * 0.05 2 0.02
k 1.0 1.43 * 0.13
0.75 2 0.38
70 Days
Treatment (n = 7) Body Testis Epididymis Adrenal Seminal vesicles Serum testosterone (n8/ml) *Mean
AZ SEM
442.7 k 7.4
1.89 * 0.12 0.65 k 0.03 56.8 * 2.5
1.31 & 0.6 1.27 * 0.35
Control (n = 8) 448.6
k 9.9
1.91 * 0.02 0.64 k 0.01 50.6 k 2.6 1.28 ? 0.12
1.07 ? 0.24
the control or treated animals (Table 1). Serum corticosterone levels were 1268 ? 40 rig/ml in the antiprogestin-treated animals, which were significantly (p < 0.01) higher than the levels measured in the control group (400 ? 82 rig/ml). As shown in Table 2, the anti-progestin CDB 2914 had no significant effect on epididymal sperm motility and sperm content. The anti-progestin also did not cause any alteration in total testicular sperm content (even after 70 days of treatment). After 14, 35, and 70 days of treatment, CDB 2914 has no effect on fertility of male rats as shown by the results of the mating studies (Table 3). The pregnancy rate, the number of implantation sites per pregnant animal, and the number of normal fetuses per pregnant animal were not different in female animals mated with the treatment or control group. Histological examination of the testicular tissues collected from the animals after 70 days of treatment with CDB 2914 showed no difference in testicular morphology. Tubular profiles representing each of the fourteen stages of seminiferous epithelial cycle were recognized and each of the stages contained characteristic cell associations consisting of spermatogonia, spermatocyte, and spermatids. Examination of the spermatozoa in the epididymis also showed morphologically normal spermatozoa with intact acrosome. This study clearly demonstrates that the antiprogestin CDB 2914 has no antifertility effect in male rats. The dose chosen (10 mg/kg/day) when compared with the dose of a similar anti-progestin (RU 486) given to women is considered a high dose. CDB 2914 has anti-progestin and antiglucocorticoid potencies similar to RU 486. RU 486 has antiprogestational activity at 1 mg/kg/day, antiglucocorticoidal activity at 10 mg/kg/day, and antiandrogen activity at 30 mg/kg/ Table 2. Effect of CDB 2914 on sperm motility
and con-
centration 35 Days Treatment Epididymal motility (% ) Cauda epididymal sperm content (106 per epididymis) Testicular sperm content (106 per testis)
Control
52.3 k 4.7
68.9 k 2.0
131.2 2 8.8
121.7 2 15.2
328 k 16
388 k 41
60.6 k 4.7
64.8 k 1.2
67.2 k 5.8
70.7 k 7.2
298 k 34
307 5 21
70 Days Epididymal motility (% 1 Cauda epididymal sperm content (106 per epididymis) Testicular sperm conteat (106 per testis)
217
Anti-progestins and Male Reproduction
Contraception 1995;51:215218
Table 3. Effect of CDB 2914 on fertility 14 Days Treatment Pregnancy rate Implantation sites per pregnant animal Normal fetuses per pregnant animal Corpora lutea per animal
lo/l2
(83%)
(83%)
12.4 k 1.0
12.7 XL0.9
12.0 k 1.0
17.3 5 1.2
12.4 k 1.4 Control
12/12 (100%)
12/12 (100%)
14.1 * 0.7
14.4 k 1.0
14.0 k 0.8
13.9 k 1.0
17.2 * 0.9
18.0 2 0.5
70 Days Treatment Pregnancy rate Implantation sites per pregnant animal Normal fetuses per pregnant animal Corpora lutea per animal
lo/l2
14.0 k 0.6
35 Days Treatment Pregnancy rate Implantation sites per pregnant animal Normal fetuses per pregnant animal Corpora lutea per animal
Control
Control
11/14 (79%)
14/16 (87%)
12.0 2 1.2
13.3 k 0.9
11.6 5 1.1
12.8 k 0.9
16.9 * 1.4
17.6 5 1.1
day in rats. The present findings of a significant increase in corticosterone and lack of an effect on serum testosterone levels in the treated rats suggest that even at these high doses, CDB 2914 exhibited only antiglucocorticoid but not antiandrogen effects. Consistent with the lack of antiandrogenic effect, this anti-progestin had no effect on epididymal or testicular sperm count, sperm motility, spermatogenesis, and fertility. Thus, in contrast to the females, anti-progestins are unlikely to have potential as a male contraceptive agent after in vivo administration. However, the effect of anti-progestins in vitro on human spermatozoa12 may still be exploited for testing as contraceptive agents acting against ejaculated sperm in the female reproductive tract.
Acknowledgments Support for this project [CSA-92-107/GSA-93-1271 was provided by the Contraceptive Research and De-
velopment Program, Eastern Virginia Medical School, under a Cooperative Agreement with the United States Agency for International Development (U.S.A.I.D.) (DPE-3044-A-00-6063-00). The views ex-
218 Wang et al.
pressed by the authors do not necessarily reflect the views of U.S.A.I.D. and CONRAD. The authors thank Gabriel Bialy, Ph.D., Center for Population Research, NICHD, for his help and encouragement. We thank Sally Avancena for the preparation of the manuscript. Address for correspondence and reprint requests: Christina Wang, M.D., Clinical Study Center [Box 16), Harbor-UCLA Medical Center, Torrance, CA 90509-2910, USA.
References 1. Philibert D. RU38486: An original multifaceted antihormone in vivo. In: Adrenal Steroid Antagonism, Agarwal, MK, ed. Berlin: Gruyter, 1984:77-101. 2. Silvestre L, Dubois C, Renault M, Rezvani Y, Bauheu EE, Ulman A. Voluntary interruption of pregnancy with mifepristone (RU486) and a prostaglandin analogue. N Engl J Med 1990;322:645-8. 3. Baulieu EE. Contragestion and other clinical applications of RU486, an antiprogesterone at the receptor. Science 1989;245: 13.5l-7. 4. Kettel LM, Murphy AA, Mortola JF, Liu JH, Aulmann A, Yen SSC Endocrine responses to long-term administration of the antiprogesterone RU486 in patients with pelvic endometriosis. Fertil Steril 1991;56:402-7. 5. Murphy AA, Kettel LM, Morales AJ, Roberts U, Yen SSC. Regression of uterine leiomyomata in response to the antiprogesterone RU486. J Clin Endocrinol Metab 1993;76:513-7. 6. Bertagner X, Bertagner C, Luton JP, Hussin JM, Dirard
Contraception 1995;51:21!%218
F. ThL new steroid analog RU486 inhibits glucocorticoid action in man. J Clin Endocrinol Metab 1984;59: 25-8. 7. Osman RA, Andria ML, Jones AD, Meizel S. Steroid induced exocytosis: the human sperm acrosome reaction. Biochem Biophys Res Commun 1989;160:828-33. 8. Blackmore PF, Beebe SJ, Danforth DR, Alexander N. Progesterone and 17-alpha-hydroxyprogesterone: Novel stimulators of calcium influx in human sperm. J Biol Chem 1990;265:1376-80. 9. Baldi E, Casano R, Falsetti C, Krausz C, Maggi M, Forti G. Intracellular calcium accumulation and responsiveness to progesterone in capacitating human spermatozoa. J Androl 1991;12:323-30. 10. Uhler ML, Leung A, Chan SYW, Wang C. Direct effects of progesterone and antiprogesterone on human sperm hyperactivated motility and acrosome reaction. Fertil Steril 1992;58:1191-8. 11. Blackmore PF, Neulen J, Lattanzio F, Beebe SJ.Cell surface-binding sites for progesterone mediate calcium uptake in human sperm. J Biol Chem 1991;266:18655-9. 12. Yang J, Serres C, Philibert D, Robe1 P, Baulieu EE, Jouannet P. Progesterone and RU486: Opposing effects on human sperm. Proc Nat1 Acad Sci USA 1994;91: 529-33. 13, Leblond CP, Clermont Y. Definition of the stages of the cycle of the seminiferous epithelium in the rat. Amr NY Acad Sci 1952;55:548-73. 14, Johnson L, Petty CS, Neaves WB. A comparative study of daily sperm production and testicular composition in humans and rats. Biol Reprod 1980;22: 1233-45. 15. Wang C, Leung A, Sinha-Hikkim A. Reproductive aging in the male Brown-Norway rat: A model for the human. Endocrinology 1993;133:2773-81.
WORDS:
male reproduction,
male contraception,
sper-
matogenesis
Introduction The anti-progestin RU 486 (Mifepristone, RouselUCLAF; Romainville, France] has been shown to have very potent anti-progestin activities both in vivo and in vitro.’ A single oral dose of 600 mg of RU 486 followed by the administration of a prostaglandin analog in over 2000 women in France resulted in interruption of pregnancy in 96% of women.’ RU 486 may be potentially useful for contragestion and contraception3 and is also currently being tested for treatment of endometriosis and uterine fibroids.4r5 In addition to its anti-progestational activities, RU 486 has antiglucocorticoid activity and has been used at higher doses for the treatment of Gushing’s syndrome.6 RU 486 also has antiandrogenic activity as demonstrated by inhibiting the effects of testosterone on the increases “Division of Endocrinology, Department of Medicine, Center, Los Angeles, CA; and *Harbor-UCLA Medical USA Address for corresponden e and reprint requests: Medical Clinical Study Center @ox I 1 ), tiarbor-UCLA 90509-2910 Submitted for publication Accepted November 17, 1
62 1995 Elsevier Science Inc. 655 Avenue of the Americas New York,
NY 10010
Cedars-Sinai Medical Center, Torrance, CA, Christina Center,
Wang- M.D., Torrance, CA
in seminal vesicle and prostate weights in castrated rats.,’ In vitro studies have indicated that progesterone directly caused rapid increases in calcium influx into human spermatozoa. 7-P This increase in intracellular calcium is associated with induction in acrosome reaction and hyPeractivated motility in human spermatozoa.7p10 Studies by Blackmore et al.sJ” and Baldi et al.9 showed that the anti-progestins RU 486 or ZK 98299 (10-5M) had minimal antagonistic effects on the action of progesterone on intracellular calcium concentrations in human spermatozoa. Our own study also failed to demonstrate any effect of RU 486 ( 10h5M) on the progesterone-induced hyperactivated motility of human spermatozoa.” However, Yang et al. l2 recently reported that RU 486 induced transient decrease of intracellular calcium and inhibited the progesterone-simulated calcium influx and acrosome reaction. In light of these findings, we studied the effects of an anti-progestin on spermatogenesis as well as on fertility in adult male rats.
Materials and Methods Animals
Adult, male Sprague-Dawley (60 to 90 days old) rats of proven fertility and adult, virgin, females (60 days old) were obtained from Harlan-Sprague-Dawley (Indianapolis, IN). Rats were housed at the vivarium of Cedars-Sinai Medical Center with 12-hr light and 12-hr dark cycles and were fed with laboratory chow and water ad libitum, At the end of the experiment, the animals were sacrificed with carbon dioxide asphyxia in a gas chamber. The protocol was approved by the Institutional Animal Care and Use Committee. Ma teriah The anti-progestin CDB 2914 (17o-acetoxy- 11&(4-N, N-dimethylaminophenyl)19~norpregna-4,9-diene-3, 20-dione; batch numbers 2.75 gm of CDB-2914 I and 3 11 mg of CDB-2914 K) was supplied by Bioequal (Rockville, MD) obtained through Drs. Gabriel Bialy and Richard Blye, CDB, CPR, NICHD. This antiprogestin has antiprogestational potencies similar to lSSN/ OOIO-7824/95/$950 SSDl/ OOlO-7824(95)09920-S
216
Wang
et ai,
RU 486 (personal communications with Dr. G. Bialy). All male animals were dosed with CDB 2914 at 10 mg/kg/day orally. CDB 2914 was made up every week as a suspension of 20 mg/ml in 5% ethanol in sesame oil. The control animals were dosed daily with the vehicle. Experimental
Protocol
To test for an effect on sperm maturation
in the epididymis, 6 to 8 male animals from each group were treated daily over a 14-day (2-week) period with the vehicle or anti-progestin. To test for post-meiotic action in the testis, male animals were treated for a 35day (5week) period. To determine whether the drug has any effect on spermatogenesis, male animals were dosed for a 70-day (lo-week) period. Spermatogenesis in the Sprague-Dawley rat extends over 4 cycles covering a period of 51.6 days.13 Thus, at the end of 70 days, the effect of CDB 2914, if any, on spermatogenesis can be detected. At the end of 14,35, and 70 days, each male rat was placed with 2 females in late proestrus for 4 days. Mating was checked and confirmed by examination for seminal plugs and sperm in vaginal smears. The day on which a spermpositive smear was found was designated day 1 of gestation. At 13 days of gestation, the females were sacrificed and autopsied to determine fertility status. At the end of the mating period, the males in each group were sacrificed and autopsied. Methods Male animals were sacrificed and weighed on days 36 and 71 after mating. Blood samples were obtained by inferior vena cava puncture for the measurement of serum testosterone and corticosterone. The testes, seminal vesicles, and epididymides were weighed. The cauda epididymides were opened and placed into warmed phosphate-buffered saline at 37’C. The presence or absence of spermatozoa and their motility were rapidly assessed within 5 to 10 minutes under phase contrast microscopy. At autopsy, the liver, kidneys, lungs, heart, and adrenals were examined. One testis was homogenized in phosphate-buffered saline and homogenization-resistant spermatozoa were counted.14 The other testis was cut into small pieces and fixed in 5% glutaraldehyde in 0.05 M cacodylate buffer (pH7.4) for 24 hr, post-fixed in 1% osmium tetroxide, dehydrated in a graded series of ethanol, and embedded in Araldite.15 Semi-thin (1 pm) sections were cut from the Araldite-embedded tissue blocks with a LKB ultramicrotome and were stained with 1% toluidine blue for light microscopic examination. Thirteen days after the pairing with male rats, the females were weighed and autopsied and their uteri
Contraception 1995:51:215-218
and ovaries were removed. The uteri were opened longitudinally and the number of normal and abnormal (dead and/or degenerate] fetuses recorded in mated females. Females which were paired but in which no sperm were present may be pseudopregnant. They were sacrificed on day 13 after pairing and the number of corpora lutea recorded. The uteri were examined for evidence of implantation sites. Serum levels of testosterone and corticosterone were measured by sensitive radioimmunoassays using kits supplied by ICN Laboratories (Costa Mesa, CA). These steroid assays have been validated for studies with rat sera.15 Statistical Analysis Arithmetic means were computed across animals within each group for each variable. The group means for each variable at each time point were compared by ANOVA followed by contrasts to test for trends on dosage levels. Differential effects of the antiprogestagen treatment over time were assessed by using two-way ANOVA and testing for significant interaction between time and treatment.
Results and Discussion Both the treated and control animals were healthy and active. Gross examination of the heart, lung, liver, and kidneys were normal in the treated and control animals. There were no significant differences in the body, testis, epididymis, seminal vesicles, and adrenal weights between CDB 2914-treated animals and the controls after 35 or 70 days. Serum testosterone levels were not significantly different between 1. Body and organ weights (g) in rats after 35 and 70 days treatment with the anti-progestin CDB 2914
Table
35 Days
Body Testis Epididymis Adrenal Seminal vesicles Serum testosterone (rig/ml)
Treatment
Control
(II = 6)
(n = 6)
437.8 k ll.O*
2.01 * 0.03 0.65 2 0.01 47.0 k 1.8 1.38 k 0.08 0.87 * 0.23
459.2 1.88 0.63 50.3
k 10.2 * 0.05 2 0.02
k 1.0 1.43 * 0.13
0.75 2 0.38
70 Days
Treatment (n = 7) Body Testis Epididymis Adrenal Seminal vesicles Serum testosterone (n8/ml) *Mean
AZ SEM
442.7 k 7.4
1.89 * 0.12 0.65 k 0.03 56.8 * 2.5
1.31 & 0.6 1.27 * 0.35
Control (n = 8) 448.6
k 9.9
1.91 * 0.02 0.64 k 0.01 50.6 k 2.6 1.28 ? 0.12
1.07 ? 0.24
the control or treated animals (Table 1). Serum corticosterone levels were 1268 ? 40 rig/ml in the antiprogestin-treated animals, which were significantly (p < 0.01) higher than the levels measured in the control group (400 ? 82 rig/ml). As shown in Table 2, the anti-progestin CDB 2914 had no significant effect on epididymal sperm motility and sperm content. The anti-progestin also did not cause any alteration in total testicular sperm content (even after 70 days of treatment). After 14, 35, and 70 days of treatment, CDB 2914 has no effect on fertility of male rats as shown by the results of the mating studies (Table 3). The pregnancy rate, the number of implantation sites per pregnant animal, and the number of normal fetuses per pregnant animal were not different in female animals mated with the treatment or control group. Histological examination of the testicular tissues collected from the animals after 70 days of treatment with CDB 2914 showed no difference in testicular morphology. Tubular profiles representing each of the fourteen stages of seminiferous epithelial cycle were recognized and each of the stages contained characteristic cell associations consisting of spermatogonia, spermatocyte, and spermatids. Examination of the spermatozoa in the epididymis also showed morphologically normal spermatozoa with intact acrosome. This study clearly demonstrates that the antiprogestin CDB 2914 has no antifertility effect in male rats. The dose chosen (10 mg/kg/day) when compared with the dose of a similar anti-progestin (RU 486) given to women is considered a high dose. CDB 2914 has anti-progestin and antiglucocorticoid potencies similar to RU 486. RU 486 has antiprogestational activity at 1 mg/kg/day, antiglucocorticoidal activity at 10 mg/kg/day, and antiandrogen activity at 30 mg/kg/ Table 2. Effect of CDB 2914 on sperm motility
and con-
centration 35 Days Treatment Epididymal motility (% ) Cauda epididymal sperm content (106 per epididymis) Testicular sperm content (106 per testis)
Control
52.3 k 4.7
68.9 k 2.0
131.2 2 8.8
121.7 2 15.2
328 k 16
388 k 41
60.6 k 4.7
64.8 k 1.2
67.2 k 5.8
70.7 k 7.2
298 k 34
307 5 21
70 Days Epididymal motility (% 1 Cauda epididymal sperm content (106 per epididymis) Testicular sperm conteat (106 per testis)
217
Anti-progestins and Male Reproduction
Contraception 1995;51:215218
Table 3. Effect of CDB 2914 on fertility 14 Days Treatment Pregnancy rate Implantation sites per pregnant animal Normal fetuses per pregnant animal Corpora lutea per animal
lo/l2
(83%)
(83%)
12.4 k 1.0
12.7 XL0.9
12.0 k 1.0
17.3 5 1.2
12.4 k 1.4 Control
12/12 (100%)
12/12 (100%)
14.1 * 0.7
14.4 k 1.0
14.0 k 0.8
13.9 k 1.0
17.2 * 0.9
18.0 2 0.5
70 Days Treatment Pregnancy rate Implantation sites per pregnant animal Normal fetuses per pregnant animal Corpora lutea per animal
lo/l2
14.0 k 0.6
35 Days Treatment Pregnancy rate Implantation sites per pregnant animal Normal fetuses per pregnant animal Corpora lutea per animal
Control
Control
11/14 (79%)
14/16 (87%)
12.0 2 1.2
13.3 k 0.9
11.6 5 1.1
12.8 k 0.9
16.9 * 1.4
17.6 5 1.1
day in rats. The present findings of a significant increase in corticosterone and lack of an effect on serum testosterone levels in the treated rats suggest that even at these high doses, CDB 2914 exhibited only antiglucocorticoid but not antiandrogen effects. Consistent with the lack of antiandrogenic effect, this anti-progestin had no effect on epididymal or testicular sperm count, sperm motility, spermatogenesis, and fertility. Thus, in contrast to the females, anti-progestins are unlikely to have potential as a male contraceptive agent after in vivo administration. However, the effect of anti-progestins in vitro on human spermatozoa12 may still be exploited for testing as contraceptive agents acting against ejaculated sperm in the female reproductive tract.
Acknowledgments Support for this project [CSA-92-107/GSA-93-1271 was provided by the Contraceptive Research and De-
velopment Program, Eastern Virginia Medical School, under a Cooperative Agreement with the United States Agency for International Development (U.S.A.I.D.) (DPE-3044-A-00-6063-00). The views ex-
218 Wang et al.
pressed by the authors do not necessarily reflect the views of U.S.A.I.D. and CONRAD. The authors thank Gabriel Bialy, Ph.D., Center for Population Research, NICHD, for his help and encouragement. We thank Sally Avancena for the preparation of the manuscript. Address for correspondence and reprint requests: Christina Wang, M.D., Clinical Study Center [Box 16), Harbor-UCLA Medical Center, Torrance, CA 90509-2910, USA.
References 1. Philibert D. RU38486: An original multifaceted antihormone in vivo. In: Adrenal Steroid Antagonism, Agarwal, MK, ed. Berlin: Gruyter, 1984:77-101. 2. Silvestre L, Dubois C, Renault M, Rezvani Y, Bauheu EE, Ulman A. Voluntary interruption of pregnancy with mifepristone (RU486) and a prostaglandin analogue. N Engl J Med 1990;322:645-8. 3. Baulieu EE. Contragestion and other clinical applications of RU486, an antiprogesterone at the receptor. Science 1989;245: 13.5l-7. 4. Kettel LM, Murphy AA, Mortola JF, Liu JH, Aulmann A, Yen SSC Endocrine responses to long-term administration of the antiprogesterone RU486 in patients with pelvic endometriosis. Fertil Steril 1991;56:402-7. 5. Murphy AA, Kettel LM, Morales AJ, Roberts U, Yen SSC. Regression of uterine leiomyomata in response to the antiprogesterone RU486. J Clin Endocrinol Metab 1993;76:513-7. 6. Bertagner X, Bertagner C, Luton JP, Hussin JM, Dirard
Contraception 1995;51:21!%218
F. ThL new steroid analog RU486 inhibits glucocorticoid action in man. J Clin Endocrinol Metab 1984;59: 25-8. 7. Osman RA, Andria ML, Jones AD, Meizel S. Steroid induced exocytosis: the human sperm acrosome reaction. Biochem Biophys Res Commun 1989;160:828-33. 8. Blackmore PF, Beebe SJ, Danforth DR, Alexander N. Progesterone and 17-alpha-hydroxyprogesterone: Novel stimulators of calcium influx in human sperm. J Biol Chem 1990;265:1376-80. 9. Baldi E, Casano R, Falsetti C, Krausz C, Maggi M, Forti G. Intracellular calcium accumulation and responsiveness to progesterone in capacitating human spermatozoa. J Androl 1991;12:323-30. 10. Uhler ML, Leung A, Chan SYW, Wang C. Direct effects of progesterone and antiprogesterone on human sperm hyperactivated motility and acrosome reaction. Fertil Steril 1992;58:1191-8. 11. Blackmore PF, Neulen J, Lattanzio F, Beebe SJ.Cell surface-binding sites for progesterone mediate calcium uptake in human sperm. J Biol Chem 1991;266:18655-9. 12. Yang J, Serres C, Philibert D, Robe1 P, Baulieu EE, Jouannet P. Progesterone and RU486: Opposing effects on human sperm. Proc Nat1 Acad Sci USA 1994;91: 529-33. 13, Leblond CP, Clermont Y. Definition of the stages of the cycle of the seminiferous epithelium in the rat. Amr NY Acad Sci 1952;55:548-73. 14, Johnson L, Petty CS, Neaves WB. A comparative study of daily sperm production and testicular composition in humans and rats. Biol Reprod 1980;22: 1233-45. 15. Wang C, Leung A, Sinha-Hikkim A. Reproductive aging in the male Brown-Norway rat: A model for the human. Endocrinology 1993;133:2773-81.