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Decreased fertility in male rats administered the 5α-reductase inhibitor, finasteride, is due to deficits in copulatory plug formation
ReproductiveToxicology,Vol. 5, pp. 353-362, 1991 Printed in the U.S.A.
0890-6238/91 $3.00 + .00 Copyright © 1991PergamonPressplc
DECREASED FERTILITY IN MALE RATS ADMINISTERED THE 5ot-REDUCTASE INHIBITOR, FINASTERIDE, IS DUE TO DEFICITS IN COPULATORY PLUG FORMATION M. A. CUKIERSKI,J. L. SINA, S. PRAHALADA,L. D. WISE, J. M. ANTONELLO,* J. S. MACDONALD, R. T. ROBERTSON Departments of Safety Assessment and *Biometrics, Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania Abstract -- Oral administration of 80 mg/kg/day of finasteride, a potent specific inhibitor of 5a-reductuse, to sexually mature male Sprague-Dawley rats for 24 to 38 weeks caused an approximate 30% to 40% decrease in fertility. There were no effects on mating indices or implants per pregnant female. From the mating trials, a selected group of treated males with poor reproductive performance was compared to a selected group of control males with good reproductive performance. Observed matings showed no qualitative effects on mating behavior or ejaculation. However, finasteride-treated males did not form or formed small and improperly positioned copulatory plugs, which are required in rats to transport sperm into the uterus. Intrauterine Insemination of epididymal sperm from males that were nonfertile by natural mating resulted in similar numbers of embryos and unfertilized oocytes recovered from controls and f'masteride-treated males, confirming that there was no effect of finasteride on the ability of sperm to fertilize. Decreased fertility of f'masteride-treated males was due to failure to form copulatory plugs and is related to decreased weight of seminal vesicles and prostate, an expected pharmacologic effect. Testes weight was unaffected. Decreased fertility in male rats after flnasteride administration is considered a species specific effect. The mechanism of the decrease in rats is not likely to be relevant to species that do not form copulatory plugs. Key Words: finasteride; 5cx-reductase inhibition; fertility assessment; copulatory plug.
may represent a primary developmental effect in young animals rather than strictly a decrease in functional ability. Unpublished observations from this laboratory have shown no effects of long-term finasteride administration on testes weight or on the seminiferous epithelium in rats, mice, rabbits, or dogs. An expected pharmacologic effect of 5ot-reductase inhibitors is reduction in weight and secretions of the accessory sex glands, since these structures are known to be dependent on DHT. Previous studies with finasteride in rats have shown an approximate 60% decrease in prostate weight and 80% decrease in seminal vesicle and coagulating gland weights after 32 weeks of dosing at 80 mg/kg/day (1). These decreases in accessory gland weights paralleled the decrease in fertility. The effects on fertility were completely reversible 6 weeks after stopping drug administration at which time the weights of seminal vesicles and prostate returned to near normal values. Therefore, the primary focus of this study was the effect of finasteride treatment on function of the accessory sex glands. Surgical removal of the seminal vesicles was found to cause complete infertility due to inability to form copulatory plugs, which are necessary for transcervical sperm transport in rats (4). This suggested that finasteride, due to its dramatic effects on reduction of acces-
INTRODUCTION Finasteride [N-(1,1-dimethylethyl)3-oxo-4-aza-5ot-androst1-ene-1713-carboxyamide] is a potent and specific inhibitor of 5ot-reductase, the enzyme responsible for the conversion of testosterone to 5ot-dihydrotestosterone (DHT). This compound is currently under development with the primary indication for treatment of benign prostatic hyperplasia. Previous studies in our laboratory have shown decreased fertility in male rats administered finasteride daily when dosing was begun with young sexually immature males (4 to 6 weeks old) and after long-term administration to sexually mature males (1). In sexually immature males, the decreased fertility (approximately 20% to 48% below control animals) was observed after 12 weeks of dosing. In mature males this effect was not seen until after 24 weeks of administration. The age difference is likely a consequence of the dependence on DHT for the development and maturation of portions of the male reproductive tract (2,3) and
Address correspondence to M. A. Cukierski, Department of Safety Assessment, WP 45-1, Merck Sharp & Dohme Research Laboratories, West Point, PA 19486. Received 11 June 1990; Revision received 9 October 1990; Accepted 12 October 1990. 353
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ReproductiveToxicology
Volume5, Number4, 1991 Female
Male
Cervix 2
~ating~....~
S a,-
Secretions 1
Copulatory Plug Formation
Uterine Tube
Sperm "~ Transport
~ Fertilization
/7
Fig. 1. Hypothesis of mechanism of finasteride-decreased fertility in male rats. (1) Altered secretion from sex accessory glands impairs copulatory plug formation. (2) Copulatory plug essential for transport of sperm across cervix in rat.
sory sex gland weights, may have caused decreased fertility by a similar mechanism (Figure 1). The purpose of this study was to assess the reproducibility and the cause of the decreased fertility observed previously after long-term administration of finasteride to sexually mature male rats. MATERIALS AND METHODS
This study was carded out under U.S. Food and Drug Administration Good Laboratory Practice guidelines for nonclinical safety assessment studies.
Animals The male Sprague-Dawley rats were sexually mature (approximately 16 weeks of age) at initiation (Crl: CD(SD) BR obtained from Charles River Breeding Laboratories, Wilmington, MA). Animals were maintained in accordance with standards established by the Federal Animal Welfare Act and the Institute for Laboratory Animal Resources being housed individually in wire bottom cages with free access to Purina Certified Rodent Chow and tap water. Room temperature was maintained at 20 to 27 °C and room lights were set for a 12-h light and 12-h dark cycle. During drug week 30 (after end of second cohabitation), the light cycles were reversed and set for a 10-h dark, 14-h light cycle (lights off 10:00 AM, lights on 8:00 PM).
Drug administration Suspensions of finasteride were prepared daily in 0.5% aqueous methylcellulose. Forty male rats were dosed with 80 mg/kg/day finasteride daily by oral gavage from initiation until sacrifice. An additional 40 males were dosed with the vehicle at the same volume (5 mL/kg). Samples of dosing suspensions were assayed periodically during the study, and all concentrations were at least 90% of desired concentration.
Body weights and physical signs Body weights were recorded at initiation of the study, once during drug week 1, and twice weekly un-
til study termination. All animals were examined daily for mortality and physical signs of a drug effect.
Reproductive performance and fertility A general outline of reproductive assessment is shown in Figure 2. Males were paired singly with an untreated female (11 to 14 weeks of age) on two occasions (beginning in drug weeks 25 and 28) for a maximum of 16 nights each. A vaginal lavage from each female was examined daily to confirm normal estrous cyclicity and for the presence of sperm as evidence of a positive mating (day 0 of gestation). Copulatory plugs in the vagina and cage pans were also noted daily. After finding of sperm in the vagina, the females were removed from the male cage and housed singly. On day 7 of gestation, females were euthanized by CO 2 asphyxiation and checked for pregnancy status; the number of implants were counted. Females that did not have evidence of a positive mating were euthanized 7 days after the 16-night cohabitation period and examined as above.
Reproductive performance and fertility during natural matings Reproductive performance and fertility of males were evaluated by mean day of mating, mating index (mean of l/[day of mating] for mated females plus zero for females that did not mate), mated females/females cohabited, females with copulatory plugs/mated females, fecundity index (pregnant females/mated females), fertility index (pregnant females/females cohabited), and implants/pregnant female.
Selection of males for additional testing After the second cohabitation, a group of 9 finasteride-treated males were selected for further testing. The treated males were selected on the basis of poor reproductive performance, defined as those males that had positive matings in both cohabitations but failed to produce a pregnancy. These treated males were compared to a group of controls with good reproductive perfor-
Mechanism of finasteride-decreasedfertility• M. A. Cur,mRsraETAL Begin dosing at 16 weeks of age
Drug Week 25
Control
355
Finasteride80 mg/kg/day
2 natural matings (16 days each) with untreated females
Identify drug treated males that mated but produced no pregnancies to compare with an equal number of control males that produced 2 pregnancies n=9 n=9 Reverse light cycle
Drug Week 34
Observe mating with LHRH sychronized females + vaginal sperm + copulatory plug fit Uterine sperm counts
Drug Week 35
Intrauterine insemination with epididymal sperm
Flush uterine tubes (day 2) Count embryos and unfertilized oocytes
Drug Week 38 Age 54 weeks
Sacrifice: organ weights testes seminal vesicles & coagulation gland prostate
Fig. 2. Outline of reproductive assessment in male rats administered finasteride. mance (two positive matings and two pregnancies). This selection was made to maximize the chance of demonstrating a drug effect under the most stringent "worst case" conditions. An additional 5 control males with good reproductive performance (two positive matings and two pregnancies) were used for age matched epididymal sperm collection to determine the optimal number of sperm for intrauterine insemination as described below.
Synchronization of estrous cycles for observed matings and intrauterine insemination Untreated females used for observed matings and intrauterine insemination had their estrous cycles synchronized with a single 40-1~g subcutaneous dose of the LHRH agonist [DES-GIy 1°, D-Ala 6 Pro N HET] 9 LHRH (Sigma Chem Co., St. Louis, MO) as described previously (4).
Observed matings, estimation of uterine sperm counts, and copulatory plug fit Mating observations were made with rats on re-
versed light cycle during the dark cycle using red light illumination as described previously (4). Briefly, males were placed into plexiglass rodent delivery boxes with bedding (Beta Chip ®) and allowed to explore for approximately 5 min. A single LHRH agonist-synchronized female (14 to 18 weeks of age) was placed with the male, and the pair was allowed to mate to ejaculation. Mating behavior was observed and ejaculation was presumed after observation of typical ejaculatory behavior. Males who failed to mate successfully after trials on three separate days were not tested further for transcervical sperm transport. After presumed ejaculation, the male was removed from the box and the female left undisturbed for approximately 12 min. Previous work has shown that transcervical sperm transport in the rat takes 6 to 10 min after ejaculation (see reference 4 for review). The female was then examined for the presence of a copulatory plug or sperm in the vaginal lavage. If the female had a copulatory plug or sperm in the lavage, then the female was immediately euthanized by CO 2 asphyxiation, the pelvic cavity was opened, and clamps were placed on the uterus just rostral to the cervix and
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at the uterotubal junction of both uterine horns to prevent further sperm transport. To estimate uterine sperm counts each uterine horn was injected with 0.1 mL of a 20% solution of Liquinox detergent in normal saline to prevent clumping of sperm, and the contents of the uterus were withdrawn into the syringe. The contents of both horns were combined, and the volume of fluid recorded. A sample of the sperm suspension was counted in a hemocytometer. After collection of uterine contents for sperm count the vagina was opened to examine for presence and fit of the copulatory plug.
Collection of epididymal sperm Males were euthanized by CO 2 asphyxiation. The cauda epididymides were quickly removed and placed in 5.0 mL of prewarmed ( - 3 7 °C) phosphate-buffered saline (PBS) with 10 mg/mL of bovine albumin (Sigma Chemical Co., Fraction 5, St. Louis, MO). Several cuts were made in the epididymis with a fine scissors, and the sperm were allowed to swim out for approximately 5 rain. The tissue pieces were removed, and an aliquot of the suspension was removed to check motility and sperm concentration by hemocytometer. The suspension was diluted using the PBS/albumin solution to the desired concentration for insemination if necessary.
Estimation of optimal insemination
sperm
concentration for
Five control males that had two positive matings and pregnancies in both cohabitations were used for collection of epididymal sperm during drug week 35. Sperm samples were collected as above and counted by hemocytometer. After counting, the sperm suspensions were diluted to give approximate concentrations of 0.5, 1.0, and 3.0 million sperm per 0.1 mL, and each concentration was used for intrauterine insemination of 6 to 11 LHRH agonist synchronized females. Embryos were flushed as below. As the sperm suspensions cannot be mixed vigorously without possible sperm damage, an aliquot of the sperm suspensions used for each insemination was recounted by hemocytometer to give the actual number inseminated.
Insemination LHRH agonist synchronized females (14 to 18 weeks of age) were anesthetized with an intramuscular injection of --35 mg/kg tilelamine hydrochloride and zolazeparn hydrochloride (Telazol ®, A. H. Robbins, Richmond, VA). The abdomen was shaved and the caudal end of the uterus was exposed through a low midline incision. The sperm suspension (0.1 mL/horn) was injected into each uterine lumen. Rats without fluid filled uterine horns were presumed to have failure of estrus synchronization and were not used. The incision
Volume 5, Number4, 1991 was closed with wound clips. The rats were kept warm during the initial recovery from the anesthesia and then returned to their cages.
Recovery of embryos Approximately 48 h following insemination, the females were euthanized by CO2 asphyxiation. The ovades, uterine tubes, and rostral portion of the uterine horns were removed. The embryos were flushed from the uterine tube by placing a 27-gauge needle in the infundibulum and flushing with PBS. The number of embryos or unfertilized oocytes were counted and embryos examined under a dissecting microscope. Females with only unfertilized oocytes were classified as nonpregnant. Females with at least one embryo were considered pregnant. Females that did not show evidence of ovulation (persistent large follicles) or from which no oocytes or embryos were recovered from the flush were considered anovulatory and were discarded without further analysis.
Male necropsy Males were euthanized by CO 2 asphyxiation during drug week 38 (except males used for epididymal sperm collection, which were sacrificed in drug weeks 36 and 37). Terminal body weight, testes, prostate, and seminal vesicle (including the coagulating gland) weights were recorded.
Statistical methods Reproductive performance of males during natural matings. The analysis of reproductive performance parameters during natural matings was performed separately for each cohabitation and combined over cohabitations. Treatment differences in the fertility, fecundity, mating, and copulatory plug indices were assessed using a randomization test (5). For the combined cohabitation analyses on these parameters, the withinmale response outcomes (0/2, 0/1), 1/2, and (2/2, 1/1) were scored as 0, 0.5, and 1, respectively. Treatment differences in the time-to-mating/mated female, mating index, and number of implants per pregnant female were assessed on the rankit transformed (6, 7) data using a one-way analysis of variance (ANOVA) model for the separate cohabitation analyses, and using an ANOVA model for a nested design for the combined cohabitation analyses. The rankit transformation was applied to better satisfy the assumptions of the ANOVA by reducing the influence of outlying observations.
Mating behavior during further testing. In this portion of the study, treatment differences in the numbers of mated females with plugs, with sperm in vagina, and
Mechanism of finasteride-decreasedfertility • M. A. CtrKmRSKXETAL
357
Table 1. Summary of reproductive performance of males during natural matings
Dose Group
Cohabitation I Control
CohabitationII Control
40 3.0
40 2.4
Males cohabited Mean day of mating (per male) Mating Indexa (per male) Mated females/females cohabited, % (per male) Females with copulatory plugs/mated females, % Fecundity Index: Pregnant females/mated females, % (per mated male) Fertility Index: Pregnant females/females cohabited, % (per male)
0.37
0.51
Finasteride CohabitationI CohabitationII 80 mg/kg/day 80 mg/kg/day 39 3.6Ns 0.32Ns
Results of combined cohabitation analysis
38 3.3Ns 0.36Ns
NS NS
87.5
85
84.6Ns
78.9Ns
NS
94.3
88.2
45.4***
56.7**
***
82.8
79.4
51.5"*
63.3Ns
*
72.5
67.5
43.6Ns
50.0Ns
*
ablating Index = mean of (1/(day of mating)) for mated females + zero for females that did not mate. NS = Not significantly different from control, P > 0.05. *P --< 0.05, **0.01, ***0.001. Significantly different from control.
with sperm in the uterus were assessed using a randomization test.
Embryo recovery data after intrauterine insemination. The one control male with unilateral testicular degeneration was excluded from the statistical analysis of the embryo recovery data. Treatment differences in the number of embryos/pregnant female, unfertilized oocytes/ pregnant female, and total recoveries/pregnant female were assessed on the rankit transformed data using an A N O V A model for a nested design as females are nested within males. The treatment difference in the number o f positive pregnancies was assessed on a per male basis using a randomization test.
Body and organ weights at sacrifice. Treatment differences in body, testes, prostate weights, and seminal vesicle weights were assessed on the rankit transformed absolute weights and as a percent of body weight using a one-way A N O V A model. All statistical tests were two-sided. Hypothesis testing was performed at the 0.05, 0.01, and 0.001 levels of significance.
during the course of the study. There were no treatmentrelated physical signs. There was a treatment-related decrease in mean body weight gain of 17.9% below controls after 37 weeks o f dosing.
Reproductive performance and fertility The results of the natural matings are shown in Tables 1 and 2.
Cohabitation 1, drug week 25 There were no significant effects o f treatment on mean day of mating, on mating index, or on mated females/females cohabited. The percent females with copulatory plugs/mated females in the finasteride-treated group was significantly decreased 51.9% from the control group. The fecundity index (pregnant females/mated females) was significantly decreased 37.8%. The fertility index (pregnant females/females cohabited) was not significantly changed in the finasteride-treated group. There were no effects of treatment on number of implants/pregnant female as compared to controls on day 7 of gestation.
Cohabitation 2, drug week 28 RESULTS
Mortality, physical signs, and body weights There were no treatment-related deaths. Two finasteride-treated males were euthanized for reasons unrelated to drug administration, and one control male died
There were no significant effects of treatment on mean day o f mating, mating index, or mated females/ females cohabited. The percent females with copulatory plugs/mated females was significantly decreased 35.8% compared to concurrent controls. The fecundity index and fertility index were not statistically different from
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Table 2. Summary of laparotomy data
Dose Group
Cohabitation I Control
CohabitationII Control
40 29 7 5
40 27 7 6
386 13.3
379 14.0
Females Total Females Pregnant Not pregnant Not bred Implants Implants/group Implants/pregnant female
Finasteride CohabitationI CohabitationII 80 mg/kg/day 80 mg/kg/day 39 17 16 6 211 12.4Ns
Results of combined cohabitation analysis
39 19 11 8 256 13.5NS
NS
NS = Not significantly different from control, P > 0.05.
controls. Combining the results of both cohabitations showed statistically significant decreases in percent females with copulatory plugs, fecundity, and fertility indices. There were no effects of treatment on average implants/pregnant female between the control and treated groups (Table 2).
Selection of males for further testing after two cohabitations After two cohabitations there were 9 males in the finasteride-treated group and 2 males in the control group that had two positive matings (sperm in the vagina of female) but failed to produce a pregnancy. The 9 finasteride-treated males with poor reproductive performance were selected for testing in direct comparison with 9 control males that had good reproductive performance (two positive matings and two pregnancies).
Observed matings, copulatory plug fit, and uterine sperm counts A summary of observed mating is shown in Table
3. There were no qualitative differences observed in mating behavior between the finasteride-treated and control groups when paired with L H R H agonist synchronized females. One control male and one treated male failed to ejaculate on three separate trials and were not observed further. The remaining 8 males in each group ejaculated as evidenced by sperm in the vagina. Twelve minutes postejaculation the uterus was clamped to prevent further sperm transport and the uterine fluid was collected for total sperm counts; copulatory plug fit was examined. Plugs were found in 7/8 control mated females and 2/8 finasteride-mated females. In the control-mated females the plugs were large, filled the entire circumference of the vagina, and were tightly adhered to the vaginal wall (Figure 3). Of the 7 plugs, 5 were adhered to the entire circumference of the vaginal wall, one plug to half the circumference, and 1 plug was dislodged during examination. All of the copulatory plugs in the control group were lodged against the cervix. In the females mated to treated males, the two plugs observed were extremely small and adhered to a small area of the vaginal wall distal to the cervix (Figure 4).
Table 3. Summary of observed mating behavior Dose group Males observed Mean number of trials/male Number with no ejaculation Number observed with: Plugs Sperm in vagina Sperm in uterusa Mean number uterine spermb
Control
80 mg/kg/day
9 1,4 1
9 I. 3 1
7 8 6 34.65 )<
10 6
2* 8 NS 0* 0
a12 rain post ejaculation. bn = 4; counts range from 23 to 52 million; 2 animals had too few sperm to accurately count by hemocytometer. NS = Not significantly different from control, P > 0.05. *P ~ 0.05. Significantly different from control.
Mechanism of finasteride-decreasedfertility • M. A. Cur.w_~sraEr AL
359
Table 4. Summary of results of sperm dilution and insemination of control males used for determination of optimal sperm dilution
Number intrauterine inseminated
Number pregnant (%)
Number embryos
Average embryos per female
Number unfertilized
Average unfertilized per female
Average number recoveries
1a 10 2b 6 3c 11
10 (100) 6 (100) 9 (82)
102 46 52
10.2 7.7 4.7
7 8 46
.7 1.33 4.2
10.9 9.0 8.9
aAverage Sperm Count 3,050,000/0.1 mL; Range = 1,825,000 to 4,300,000/0.1 mL. bAverage Sperm Count 983,125/0.1 mL; Range = 880,000 to 1,075,000/0.1 mL. ¢Average Sperm Count 584,167/0.1 mL; Range = 352,500 to 695,000/0.1 mL.
In the finasteride-mated females, no sperm were recovered from the uterus. In the control group, spermwere recovered from the uterus in 6 females with copulatory plugs. No uterine sperm were recovered from one control-mated female without a copulatory plug and from one control-mated female with a copulatory plug. In 2 control-mated females, the number of sperm recovered was too small to be accurately counted by hemocytometer (one due to loss of fluid during collection). In the remaining 4 females, a mean of 34.65 million sperm
Fig. 3. Copulatory plug in situ from an untreated female mated to a control male. The copulatory plug (P) is large, fills the entire circumference of the vaginal lumen (V), and is lodged tightly against the cervix (C).
were recovered from the uterus approximately 12 min postejaculation.
Intrauterine insemination Determination of number of sperm to be inseminated. Five control males were used for epididymal sperm collection. The collected sperm were diluted twice to achieve concentrations of approximately 0.5, 1.0, and 3 million sperm per 0.1 mL. Each dilution of
Fig. 4. Copulatory plug in situ from an untreated female mated to a finasteride-treated male. The copulatory plug (P) is small and adhered to a small portion of the vagina (V) distal to the cervix (C).
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Table 5. Summary of embryo recovery data after intrauterine insemination Dose group
Control
Number of males Number of males producing 3 or more pregnancies Number of females inseminated Positive pregnancies Anovulatory females Embryos counted Embryos/pregnant female +__ SD Unfertilized oocytes counted Unfertilized oocytes/ pregnant female ___ SD Total recoveries/female ± SD (embryos + oocytes) Total sperm × 106/0.1 mE, mean +_ SD
9 (8) ~ 9
80 mg/kg/day 9 9
34 33 (30) 1 294 (285) 8.91 -4- 3.39 (9.5 ___ 2.93) 52 (37) 1.58 - 1.9 (1.23 ± 1.57)
36 34 NS 2 330 9.71 4- 3.48 Ns
10.48 -+ 3.13 (10.73 - 3.14)
10.82 ± 3.18 Ns
2.9 --- 1.17 (3.2 +_ 0.35) b
38 1.12 +_ 1.81Ns
3.1
-4- 0.77 ¢
aNumber in parentheses excludes one control male with unilateral testicular degeneration and high percentage of morphologically abnormal sperm. bRange 1.3 to 4.0 × 10 6 sperm/0.1 mL. ORange 2.0 to 4.3 x 10 6 sperln/0.1 mE. NS =Not statistically significant, P > 0.05. Statistical comparisons performed excluding the one control male with testicular degeneration.
s p e r m w a s i n j e c t e d into the u t e r u s o f 6 to 11 L H R H agonist synchronized females, and embryos were recove r e d o n d a y 2 o f g e s t a t i o n ( T a b l e 4). A t a m e a n c o n c e n t r a t i o n o f 3 , 0 5 0 , 0 0 0 s p e r m i n j e c t e d , the p r e g n a n c y rate w a s 1 0 0 % ( 1 0 / 1 0 ) , a m e a n o f 10.2 e m b r y o s / female were recovered, and 0.7 unfertilized oocytes/ females were recovered. At a mean concentration of 9 8 3 , 1 2 5 s p e r m , t h e p r e g n a n c y rate w a s 1 0 0 % (6/6), w i t h a m e a n o f 7 . 7 e m b r y o s a n d 1.33 u n f e r t i l i z e d o o c y t e s p e r f e m a l e . A t a m e a n o f 5 8 4 , 1 6 7 s p e r m , the p r e g n a n c y rate w a s 8 2 % (9/11) w i t h a m e a n n u m b e r o f embryos and unfertilized oocytes per pregnant female of
4 . 7 a n d 4 . 2 , r e s p e c t i v e l y . B a s e d o n t h e s e results, the o p t i m u m n u m b e r o f s p e r m for i n t r a u t e r i n e i n s e m i n a t i o n w a s i d e n t i f i e d as 2 to 4 m i l l i o n s p e r m p e r u t e r i n e h o r n . If the e p i d i d y m a l s p e r m p r e p a r a t i o n h a d a h i g h e r c o n c e n t r a t i o n t h a n a p p r o x i m a t e l y 4 m i l l i o n , it w a s diluted. I f the c o n c e n t r a t i o n w a s less t h a n 2 m i l l i o n the p r e p a r a tion w a s u s e d u n d i l u t e d .
Intrauterine insemination synchronized females
of
LHRH
Thirty-four LHRH agonist synchronized females had intrauterine inseminations with a mean of approxi-
Table 6. Absolute and relative organ weights of control and finasteride-treated males after 38 weeks of dosing Treatment Group
Organ Body Weight, g Testes, g % B.W. Prostate, g % B.W. Seminal Vesicles, g % B.W.
agonist
Control (248 to 260 doses) n = 34
Finasteride (248 to 260 doses) n = 38
% Change from control
778 3.61 0.47 0.57 0.08 1.33 0.18
730 * 3.63 NS 0.50 * 0.21 *** 0.03 *** 0.30 *** 0.04***
- 6.2 +0.6 +6.0 - 63.2 -62.5 -77.5 -77.8
B.W. = body weight. NS = Not significantly different from control, P > 0.05. *P --< 0.05, **0.01, ***0.001, respectively. Significantly different from control.
Mechanism of finasteride-decreasedfertility• M. A. CtZKmRSlCIEr AL
361
DISCUSSION
Fig. 5. Seminal vesicles (S) and portion of the prostate (P) from a control (left) and a male treated with finasteride for 38 weeks (right), illustrating significant reduction in size of the glands.
mately 3 million epididymal sperm from 9 control males, and 36 females were inseminated with epididymal sperm from 9 finasteride-treated males (inseminations made during drug weeks 36 to 37) (Table 5). One control-inseminated female and 2 finasteride-insemihated females were anovulatory and were discarded without further examination. One control male had unilateral testicular degeneration and a high percentage of morphologically abnormal sperm. In calculating the sperm concentration from this male, only morphologically normal sperm were counted. One additional control male had a sperm count less than the desired concentration of 2 x 106/0.1 mL (1.3 x 106). The pregnancy rate for both treated and control group was 100%. There were no effects of treatment on the number of embryos or unfertilized oocytes recovered per female. All 9 males from both treated and control groups produced 3 to 4 pregnancies each.
Testes, prostate, and seminal vesicle weights Organ weight data are summarized in Table 6. There was no effect of finasteride treatment on absolute testicular weight. Testes weight relative to body weight was significantly increased (6%) in the treated group over controls. This slight increase is likely due to the decreased body weights of the treated group. In drug weeks 36 to 38, the finasteride-treated males had significantly decreased ventral prostatic and seminal vesicular weights; the prostatic and seminal vesicular weights when expressed as a percent of body weight were decreased 62.5% and 77.8, respectively, compared to controls.
Previous fertility studies in sexually mature rats administered high doses (80 mg/kg/day) of finasteride have shown 20% to 48% decrease in fertility after 24 weeks or more of dosing (see reference 1 also for discussion of effects on fertility at lower doses). No significant effects on fertility were observed after 12 weeks of administration, the typical duration of dosing for male fertility studies. There were no effects on testes weight or histomorphology of the seminiferous epithelium in previous studies, and the decreased fertility observed was reversible after a 6-week drug-free recovery period. These decreases in fertility were accompanied by large decreases in the weight of the prostate and seminal vesicles, an expected pharmacologic effect of finasteride administration. The purpose of this study was to confirm the effects on fertility of long-term administration to sexually mature male rats and to investigate the mechanism of decreased fertility. In this study the decreased fertility, decreased weight of prostate and seminal vesicles, and the number of males that mated but failed to produce a pregnancy in two successive cohabitations after 24 weeks of dosing with 80 mg/kg/day finasteride closely reproduced the findings of a previous fertility study (1). To investigate the mechanism of decreased fertility, a group of treated males with poor reproductive performance were directly compared with a group of control males with good reproductive performance. The secretions of the seminal vesicles, coagulating glands, and bulbourethral glands in rats clot after ejaculation in the vagina to form a copulatory plug. This plug plays a critical role in transcervical sperm transport (reference 4 and the references therein). In the current study, and in previous studies with finasteride, decreases were observed in the weights of the prostate and seminal vesicles and in the number of copulatory plugs. In observed matings it was found that mating behavior of treated males was qualitatively normal and they were capable of ejaculating sperm. However, they did not form copulatory plugs or formed very small plugs that were not in contact with the cervix and therefore were nonfunctional. Without proper copulatory plug formation, no sperm were transported into the uterus. Fertilizing ability of epididymal sperm collected from these same males was tested by intrauterine insemination into LHRH agonist synchronized females. Artificial insemination has been proposed as a more sensitive approach to fertility assessment in laboratory animals rather than natural matings, due to the huge excess number of sperm normally produced during natural matings above the minimum number required for fertilization (8). Artificial insemination allows deposi-
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Reproductive Toxicology
tion of a critical number of sperm in the female tract from both treated and control animals for direct comparison. Using epididymal sperm from age matched controls, it was found that sperm concentrations below approximately 1 million per uterine horn were associated with a decreased number of fertilizations. The number of sperm for insemination was selected to be slightly above this level (mean 3 million, range approximately 2 to 4 million) for both treated and control animals. This can be compared to the mean of 34.64 million sperm recovered from the uterus during observed control matings. When approximately equal numbers of epididymal sperm from control and treated males were placed in the uterus, comparable numbers of embryos and unfertilized oocytes were recovered. Epididymal sperm from both treated and control groups were able to produce 3 to 4 pregnancies per male by intrauterine insemination. This demonstrates that sperm from finasteride-treated males is transported normally from the uterus to the site of fertilization and that fertilization occurs at rates equivalent to controls. The data presented here support the hypothesis that the decreased fertility observed in finasteride-treated males is related to an improper copulatory plug formation or fit, which is likely secondary to decreased seminal vesicle and prostate weight. Approximately 24 weeks of treatment are required for reduction of the sex accessory glands in sexually mature rats to the point where plug formation is impaired. The maximum degree of reduction in accessory gland weight obtained with finasteride treatment appears to be at or near the threshold for copulatory plug formation. This explains the long duration of treatment required to decrease fertility and the fact that there is considerable individual animal variation in ability to produce a pregnancy in successive cohabitations (1). Slight fluctuations in amount of secretions may determine whether a copulatory plug may be adequately formed during a particular mating. Previous studies have shown that even very small plugs properly placed can function (9). This variability may have the effect of reducing but not abolishing fertility. Previous studies have examined the effects of short-term administration (11 to 15 days) of the less specific 5et-reductase inhibitor 3-oxo-4-androstene-17[3 carboxylic acid, or its methyl ester, on the ability of epididymal sperm from mice to fertilize. Intrauterine insemination of these epididymal sperm into superovulated female mice resulted in decreased fertilization (10), and
Volume 5, Number 4, 1991
epididymal sperm from androgen supplemented castrate mice had decreased capacity to fertilize superovulated oocytes in vitro (11). As discussed in the accompanying paper by Wise and colleagues (1), the timing of these effects in mice is not comparable to that obtained in rats with finasteride. Additionally, intrauterine insemination of epididymal sperm from finasteride-treated males into nonsuperovulated females did not result in reduced fertilization. As the decreased fertility in male rats administered finasteride is associated with defects in copulatory plug formation and not in the ability of the sperm to fertilize, this decrease in fertility is considered a species specific effect and is not relevant to species that do not rely upon formation of copulatory plugs for transcervical sperm transport.
- - We would like to thank Drs. James W. Overstreet and Barb C. Vanderhayden for helpful discussions during initiation of these studies and Mr. Dave Billman for expert technical assistance. Acknowledgments
REFERENCES 1. Wise LD, Minsker DH, Cukierski MA, et al. Reversible decreased fertility in male Sprague-Dawley rats treated orally with finasteride, a 5c~-reductase inhibitor. Reprod Toxicol. 1991;5:337-346. 2. George FW, Peterson KG. 5a-dihydrotestosterone formation is necessary for embryogenesis of the rat prostate. Endocrinology. 1988;122:1159-64. 3. George FW, Johnson L, Wilson JD. The effect of a 5a-reductase inhibitor on androgen physiology in the immature male rat. Endocrinology. 1989;125:2434-38. 4. Cukierski MA, Sina JL, Prahalada S, Robertson RT. Effects of seminal vesicle and coagulating gland ablation on fertility in rats. Reprod Toxicol. 1991;5:347-352 5. Edgington ES. Randomization tests. New York: Marcel Dekker; 1980. 6. Harter HL. Expected values of normal order statistics. Biometrika. 1961;48:151-65. 7. Conover WJ, Iman RL. Rank transformation as a bridge between parametric and nonparametric statistics. Am Statist. 1981;35:12433. 8. Amann RP. Detection of alterations in testicular and epididymal function in laboratory animals. Environ Health Perspect. 1986;70:149-58. 9. Blandau RJ. On the factors involved in sperm transport through the cervix uteri of the albino rat. Am J Anat. 1945;77:253-72. 10. Lau IF, Saksena SK, Chang MC. Antifertility effect of 3-oxo-4androstene-1713 carboxylic acid in male mice. Arch Androl. 1979;2:179-81. 11. Cohen J, Gores MP, Vreeburg JTM. Reduction of fertilizing capacity of epididymal spermatozoa by 5a-steroid reductase inhibitors. Experientia. 1981; 37:1031-32.
0890-6238/91 $3.00 + .00 Copyright © 1991PergamonPressplc
DECREASED FERTILITY IN MALE RATS ADMINISTERED THE 5ot-REDUCTASE INHIBITOR, FINASTERIDE, IS DUE TO DEFICITS IN COPULATORY PLUG FORMATION M. A. CUKIERSKI,J. L. SINA, S. PRAHALADA,L. D. WISE, J. M. ANTONELLO,* J. S. MACDONALD, R. T. ROBERTSON Departments of Safety Assessment and *Biometrics, Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania Abstract -- Oral administration of 80 mg/kg/day of finasteride, a potent specific inhibitor of 5a-reductuse, to sexually mature male Sprague-Dawley rats for 24 to 38 weeks caused an approximate 30% to 40% decrease in fertility. There were no effects on mating indices or implants per pregnant female. From the mating trials, a selected group of treated males with poor reproductive performance was compared to a selected group of control males with good reproductive performance. Observed matings showed no qualitative effects on mating behavior or ejaculation. However, finasteride-treated males did not form or formed small and improperly positioned copulatory plugs, which are required in rats to transport sperm into the uterus. Intrauterine Insemination of epididymal sperm from males that were nonfertile by natural mating resulted in similar numbers of embryos and unfertilized oocytes recovered from controls and f'masteride-treated males, confirming that there was no effect of finasteride on the ability of sperm to fertilize. Decreased fertility of f'masteride-treated males was due to failure to form copulatory plugs and is related to decreased weight of seminal vesicles and prostate, an expected pharmacologic effect. Testes weight was unaffected. Decreased fertility in male rats after flnasteride administration is considered a species specific effect. The mechanism of the decrease in rats is not likely to be relevant to species that do not form copulatory plugs. Key Words: finasteride; 5cx-reductase inhibition; fertility assessment; copulatory plug.
may represent a primary developmental effect in young animals rather than strictly a decrease in functional ability. Unpublished observations from this laboratory have shown no effects of long-term finasteride administration on testes weight or on the seminiferous epithelium in rats, mice, rabbits, or dogs. An expected pharmacologic effect of 5ot-reductase inhibitors is reduction in weight and secretions of the accessory sex glands, since these structures are known to be dependent on DHT. Previous studies with finasteride in rats have shown an approximate 60% decrease in prostate weight and 80% decrease in seminal vesicle and coagulating gland weights after 32 weeks of dosing at 80 mg/kg/day (1). These decreases in accessory gland weights paralleled the decrease in fertility. The effects on fertility were completely reversible 6 weeks after stopping drug administration at which time the weights of seminal vesicles and prostate returned to near normal values. Therefore, the primary focus of this study was the effect of finasteride treatment on function of the accessory sex glands. Surgical removal of the seminal vesicles was found to cause complete infertility due to inability to form copulatory plugs, which are necessary for transcervical sperm transport in rats (4). This suggested that finasteride, due to its dramatic effects on reduction of acces-
INTRODUCTION Finasteride [N-(1,1-dimethylethyl)3-oxo-4-aza-5ot-androst1-ene-1713-carboxyamide] is a potent and specific inhibitor of 5ot-reductase, the enzyme responsible for the conversion of testosterone to 5ot-dihydrotestosterone (DHT). This compound is currently under development with the primary indication for treatment of benign prostatic hyperplasia. Previous studies in our laboratory have shown decreased fertility in male rats administered finasteride daily when dosing was begun with young sexually immature males (4 to 6 weeks old) and after long-term administration to sexually mature males (1). In sexually immature males, the decreased fertility (approximately 20% to 48% below control animals) was observed after 12 weeks of dosing. In mature males this effect was not seen until after 24 weeks of administration. The age difference is likely a consequence of the dependence on DHT for the development and maturation of portions of the male reproductive tract (2,3) and
Address correspondence to M. A. Cukierski, Department of Safety Assessment, WP 45-1, Merck Sharp & Dohme Research Laboratories, West Point, PA 19486. Received 11 June 1990; Revision received 9 October 1990; Accepted 12 October 1990. 353
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ReproductiveToxicology
Volume5, Number4, 1991 Female
Male
Cervix 2
~ating~....~
S a,-
Secretions 1
Copulatory Plug Formation
Uterine Tube
Sperm "~ Transport
~ Fertilization
/7
Fig. 1. Hypothesis of mechanism of finasteride-decreased fertility in male rats. (1) Altered secretion from sex accessory glands impairs copulatory plug formation. (2) Copulatory plug essential for transport of sperm across cervix in rat.
sory sex gland weights, may have caused decreased fertility by a similar mechanism (Figure 1). The purpose of this study was to assess the reproducibility and the cause of the decreased fertility observed previously after long-term administration of finasteride to sexually mature male rats. MATERIALS AND METHODS
This study was carded out under U.S. Food and Drug Administration Good Laboratory Practice guidelines for nonclinical safety assessment studies.
Animals The male Sprague-Dawley rats were sexually mature (approximately 16 weeks of age) at initiation (Crl: CD(SD) BR obtained from Charles River Breeding Laboratories, Wilmington, MA). Animals were maintained in accordance with standards established by the Federal Animal Welfare Act and the Institute for Laboratory Animal Resources being housed individually in wire bottom cages with free access to Purina Certified Rodent Chow and tap water. Room temperature was maintained at 20 to 27 °C and room lights were set for a 12-h light and 12-h dark cycle. During drug week 30 (after end of second cohabitation), the light cycles were reversed and set for a 10-h dark, 14-h light cycle (lights off 10:00 AM, lights on 8:00 PM).
Drug administration Suspensions of finasteride were prepared daily in 0.5% aqueous methylcellulose. Forty male rats were dosed with 80 mg/kg/day finasteride daily by oral gavage from initiation until sacrifice. An additional 40 males were dosed with the vehicle at the same volume (5 mL/kg). Samples of dosing suspensions were assayed periodically during the study, and all concentrations were at least 90% of desired concentration.
Body weights and physical signs Body weights were recorded at initiation of the study, once during drug week 1, and twice weekly un-
til study termination. All animals were examined daily for mortality and physical signs of a drug effect.
Reproductive performance and fertility A general outline of reproductive assessment is shown in Figure 2. Males were paired singly with an untreated female (11 to 14 weeks of age) on two occasions (beginning in drug weeks 25 and 28) for a maximum of 16 nights each. A vaginal lavage from each female was examined daily to confirm normal estrous cyclicity and for the presence of sperm as evidence of a positive mating (day 0 of gestation). Copulatory plugs in the vagina and cage pans were also noted daily. After finding of sperm in the vagina, the females were removed from the male cage and housed singly. On day 7 of gestation, females were euthanized by CO 2 asphyxiation and checked for pregnancy status; the number of implants were counted. Females that did not have evidence of a positive mating were euthanized 7 days after the 16-night cohabitation period and examined as above.
Reproductive performance and fertility during natural matings Reproductive performance and fertility of males were evaluated by mean day of mating, mating index (mean of l/[day of mating] for mated females plus zero for females that did not mate), mated females/females cohabited, females with copulatory plugs/mated females, fecundity index (pregnant females/mated females), fertility index (pregnant females/females cohabited), and implants/pregnant female.
Selection of males for additional testing After the second cohabitation, a group of 9 finasteride-treated males were selected for further testing. The treated males were selected on the basis of poor reproductive performance, defined as those males that had positive matings in both cohabitations but failed to produce a pregnancy. These treated males were compared to a group of controls with good reproductive perfor-
Mechanism of finasteride-decreasedfertility• M. A. Cur,mRsraETAL Begin dosing at 16 weeks of age
Drug Week 25
Control
355
Finasteride80 mg/kg/day
2 natural matings (16 days each) with untreated females
Identify drug treated males that mated but produced no pregnancies to compare with an equal number of control males that produced 2 pregnancies n=9 n=9 Reverse light cycle
Drug Week 34
Observe mating with LHRH sychronized females + vaginal sperm + copulatory plug fit Uterine sperm counts
Drug Week 35
Intrauterine insemination with epididymal sperm
Flush uterine tubes (day 2) Count embryos and unfertilized oocytes
Drug Week 38 Age 54 weeks
Sacrifice: organ weights testes seminal vesicles & coagulation gland prostate
Fig. 2. Outline of reproductive assessment in male rats administered finasteride. mance (two positive matings and two pregnancies). This selection was made to maximize the chance of demonstrating a drug effect under the most stringent "worst case" conditions. An additional 5 control males with good reproductive performance (two positive matings and two pregnancies) were used for age matched epididymal sperm collection to determine the optimal number of sperm for intrauterine insemination as described below.
Synchronization of estrous cycles for observed matings and intrauterine insemination Untreated females used for observed matings and intrauterine insemination had their estrous cycles synchronized with a single 40-1~g subcutaneous dose of the LHRH agonist [DES-GIy 1°, D-Ala 6 Pro N HET] 9 LHRH (Sigma Chem Co., St. Louis, MO) as described previously (4).
Observed matings, estimation of uterine sperm counts, and copulatory plug fit Mating observations were made with rats on re-
versed light cycle during the dark cycle using red light illumination as described previously (4). Briefly, males were placed into plexiglass rodent delivery boxes with bedding (Beta Chip ®) and allowed to explore for approximately 5 min. A single LHRH agonist-synchronized female (14 to 18 weeks of age) was placed with the male, and the pair was allowed to mate to ejaculation. Mating behavior was observed and ejaculation was presumed after observation of typical ejaculatory behavior. Males who failed to mate successfully after trials on three separate days were not tested further for transcervical sperm transport. After presumed ejaculation, the male was removed from the box and the female left undisturbed for approximately 12 min. Previous work has shown that transcervical sperm transport in the rat takes 6 to 10 min after ejaculation (see reference 4 for review). The female was then examined for the presence of a copulatory plug or sperm in the vaginal lavage. If the female had a copulatory plug or sperm in the lavage, then the female was immediately euthanized by CO 2 asphyxiation, the pelvic cavity was opened, and clamps were placed on the uterus just rostral to the cervix and
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ReproductiveToxicology
at the uterotubal junction of both uterine horns to prevent further sperm transport. To estimate uterine sperm counts each uterine horn was injected with 0.1 mL of a 20% solution of Liquinox detergent in normal saline to prevent clumping of sperm, and the contents of the uterus were withdrawn into the syringe. The contents of both horns were combined, and the volume of fluid recorded. A sample of the sperm suspension was counted in a hemocytometer. After collection of uterine contents for sperm count the vagina was opened to examine for presence and fit of the copulatory plug.
Collection of epididymal sperm Males were euthanized by CO 2 asphyxiation. The cauda epididymides were quickly removed and placed in 5.0 mL of prewarmed ( - 3 7 °C) phosphate-buffered saline (PBS) with 10 mg/mL of bovine albumin (Sigma Chemical Co., Fraction 5, St. Louis, MO). Several cuts were made in the epididymis with a fine scissors, and the sperm were allowed to swim out for approximately 5 rain. The tissue pieces were removed, and an aliquot of the suspension was removed to check motility and sperm concentration by hemocytometer. The suspension was diluted using the PBS/albumin solution to the desired concentration for insemination if necessary.
Estimation of optimal insemination
sperm
concentration for
Five control males that had two positive matings and pregnancies in both cohabitations were used for collection of epididymal sperm during drug week 35. Sperm samples were collected as above and counted by hemocytometer. After counting, the sperm suspensions were diluted to give approximate concentrations of 0.5, 1.0, and 3.0 million sperm per 0.1 mL, and each concentration was used for intrauterine insemination of 6 to 11 LHRH agonist synchronized females. Embryos were flushed as below. As the sperm suspensions cannot be mixed vigorously without possible sperm damage, an aliquot of the sperm suspensions used for each insemination was recounted by hemocytometer to give the actual number inseminated.
Insemination LHRH agonist synchronized females (14 to 18 weeks of age) were anesthetized with an intramuscular injection of --35 mg/kg tilelamine hydrochloride and zolazeparn hydrochloride (Telazol ®, A. H. Robbins, Richmond, VA). The abdomen was shaved and the caudal end of the uterus was exposed through a low midline incision. The sperm suspension (0.1 mL/horn) was injected into each uterine lumen. Rats without fluid filled uterine horns were presumed to have failure of estrus synchronization and were not used. The incision
Volume 5, Number4, 1991 was closed with wound clips. The rats were kept warm during the initial recovery from the anesthesia and then returned to their cages.
Recovery of embryos Approximately 48 h following insemination, the females were euthanized by CO2 asphyxiation. The ovades, uterine tubes, and rostral portion of the uterine horns were removed. The embryos were flushed from the uterine tube by placing a 27-gauge needle in the infundibulum and flushing with PBS. The number of embryos or unfertilized oocytes were counted and embryos examined under a dissecting microscope. Females with only unfertilized oocytes were classified as nonpregnant. Females with at least one embryo were considered pregnant. Females that did not show evidence of ovulation (persistent large follicles) or from which no oocytes or embryos were recovered from the flush were considered anovulatory and were discarded without further analysis.
Male necropsy Males were euthanized by CO 2 asphyxiation during drug week 38 (except males used for epididymal sperm collection, which were sacrificed in drug weeks 36 and 37). Terminal body weight, testes, prostate, and seminal vesicle (including the coagulating gland) weights were recorded.
Statistical methods Reproductive performance of males during natural matings. The analysis of reproductive performance parameters during natural matings was performed separately for each cohabitation and combined over cohabitations. Treatment differences in the fertility, fecundity, mating, and copulatory plug indices were assessed using a randomization test (5). For the combined cohabitation analyses on these parameters, the withinmale response outcomes (0/2, 0/1), 1/2, and (2/2, 1/1) were scored as 0, 0.5, and 1, respectively. Treatment differences in the time-to-mating/mated female, mating index, and number of implants per pregnant female were assessed on the rankit transformed (6, 7) data using a one-way analysis of variance (ANOVA) model for the separate cohabitation analyses, and using an ANOVA model for a nested design for the combined cohabitation analyses. The rankit transformation was applied to better satisfy the assumptions of the ANOVA by reducing the influence of outlying observations.
Mating behavior during further testing. In this portion of the study, treatment differences in the numbers of mated females with plugs, with sperm in vagina, and
Mechanism of finasteride-decreasedfertility • M. A. CtrKmRSKXETAL
357
Table 1. Summary of reproductive performance of males during natural matings
Dose Group
Cohabitation I Control
CohabitationII Control
40 3.0
40 2.4
Males cohabited Mean day of mating (per male) Mating Indexa (per male) Mated females/females cohabited, % (per male) Females with copulatory plugs/mated females, % Fecundity Index: Pregnant females/mated females, % (per mated male) Fertility Index: Pregnant females/females cohabited, % (per male)
0.37
0.51
Finasteride CohabitationI CohabitationII 80 mg/kg/day 80 mg/kg/day 39 3.6Ns 0.32Ns
Results of combined cohabitation analysis
38 3.3Ns 0.36Ns
NS NS
87.5
85
84.6Ns
78.9Ns
NS
94.3
88.2
45.4***
56.7**
***
82.8
79.4
51.5"*
63.3Ns
*
72.5
67.5
43.6Ns
50.0Ns
*
ablating Index = mean of (1/(day of mating)) for mated females + zero for females that did not mate. NS = Not significantly different from control, P > 0.05. *P --< 0.05, **0.01, ***0.001. Significantly different from control.
with sperm in the uterus were assessed using a randomization test.
Embryo recovery data after intrauterine insemination. The one control male with unilateral testicular degeneration was excluded from the statistical analysis of the embryo recovery data. Treatment differences in the number of embryos/pregnant female, unfertilized oocytes/ pregnant female, and total recoveries/pregnant female were assessed on the rankit transformed data using an A N O V A model for a nested design as females are nested within males. The treatment difference in the number o f positive pregnancies was assessed on a per male basis using a randomization test.
Body and organ weights at sacrifice. Treatment differences in body, testes, prostate weights, and seminal vesicle weights were assessed on the rankit transformed absolute weights and as a percent of body weight using a one-way A N O V A model. All statistical tests were two-sided. Hypothesis testing was performed at the 0.05, 0.01, and 0.001 levels of significance.
during the course of the study. There were no treatmentrelated physical signs. There was a treatment-related decrease in mean body weight gain of 17.9% below controls after 37 weeks o f dosing.
Reproductive performance and fertility The results of the natural matings are shown in Tables 1 and 2.
Cohabitation 1, drug week 25 There were no significant effects o f treatment on mean day of mating, on mating index, or on mated females/females cohabited. The percent females with copulatory plugs/mated females in the finasteride-treated group was significantly decreased 51.9% from the control group. The fecundity index (pregnant females/mated females) was significantly decreased 37.8%. The fertility index (pregnant females/females cohabited) was not significantly changed in the finasteride-treated group. There were no effects of treatment on number of implants/pregnant female as compared to controls on day 7 of gestation.
Cohabitation 2, drug week 28 RESULTS
Mortality, physical signs, and body weights There were no treatment-related deaths. Two finasteride-treated males were euthanized for reasons unrelated to drug administration, and one control male died
There were no significant effects of treatment on mean day o f mating, mating index, or mated females/ females cohabited. The percent females with copulatory plugs/mated females was significantly decreased 35.8% compared to concurrent controls. The fecundity index and fertility index were not statistically different from
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Reproductive Toxicology
Volume5, Number 4, 1991
Table 2. Summary of laparotomy data
Dose Group
Cohabitation I Control
CohabitationII Control
40 29 7 5
40 27 7 6
386 13.3
379 14.0
Females Total Females Pregnant Not pregnant Not bred Implants Implants/group Implants/pregnant female
Finasteride CohabitationI CohabitationII 80 mg/kg/day 80 mg/kg/day 39 17 16 6 211 12.4Ns
Results of combined cohabitation analysis
39 19 11 8 256 13.5NS
NS
NS = Not significantly different from control, P > 0.05.
controls. Combining the results of both cohabitations showed statistically significant decreases in percent females with copulatory plugs, fecundity, and fertility indices. There were no effects of treatment on average implants/pregnant female between the control and treated groups (Table 2).
Selection of males for further testing after two cohabitations After two cohabitations there were 9 males in the finasteride-treated group and 2 males in the control group that had two positive matings (sperm in the vagina of female) but failed to produce a pregnancy. The 9 finasteride-treated males with poor reproductive performance were selected for testing in direct comparison with 9 control males that had good reproductive performance (two positive matings and two pregnancies).
Observed matings, copulatory plug fit, and uterine sperm counts A summary of observed mating is shown in Table
3. There were no qualitative differences observed in mating behavior between the finasteride-treated and control groups when paired with L H R H agonist synchronized females. One control male and one treated male failed to ejaculate on three separate trials and were not observed further. The remaining 8 males in each group ejaculated as evidenced by sperm in the vagina. Twelve minutes postejaculation the uterus was clamped to prevent further sperm transport and the uterine fluid was collected for total sperm counts; copulatory plug fit was examined. Plugs were found in 7/8 control mated females and 2/8 finasteride-mated females. In the control-mated females the plugs were large, filled the entire circumference of the vagina, and were tightly adhered to the vaginal wall (Figure 3). Of the 7 plugs, 5 were adhered to the entire circumference of the vaginal wall, one plug to half the circumference, and 1 plug was dislodged during examination. All of the copulatory plugs in the control group were lodged against the cervix. In the females mated to treated males, the two plugs observed were extremely small and adhered to a small area of the vaginal wall distal to the cervix (Figure 4).
Table 3. Summary of observed mating behavior Dose group Males observed Mean number of trials/male Number with no ejaculation Number observed with: Plugs Sperm in vagina Sperm in uterusa Mean number uterine spermb
Control
80 mg/kg/day
9 1,4 1
9 I. 3 1
7 8 6 34.65 )<
10 6
2* 8 NS 0* 0
a12 rain post ejaculation. bn = 4; counts range from 23 to 52 million; 2 animals had too few sperm to accurately count by hemocytometer. NS = Not significantly different from control, P > 0.05. *P ~ 0.05. Significantly different from control.
Mechanism of finasteride-decreasedfertility • M. A. Cur.w_~sraEr AL
359
Table 4. Summary of results of sperm dilution and insemination of control males used for determination of optimal sperm dilution
Number intrauterine inseminated
Number pregnant (%)
Number embryos
Average embryos per female
Number unfertilized
Average unfertilized per female
Average number recoveries
1a 10 2b 6 3c 11
10 (100) 6 (100) 9 (82)
102 46 52
10.2 7.7 4.7
7 8 46
.7 1.33 4.2
10.9 9.0 8.9
aAverage Sperm Count 3,050,000/0.1 mL; Range = 1,825,000 to 4,300,000/0.1 mL. bAverage Sperm Count 983,125/0.1 mL; Range = 880,000 to 1,075,000/0.1 mL. ¢Average Sperm Count 584,167/0.1 mL; Range = 352,500 to 695,000/0.1 mL.
In the finasteride-mated females, no sperm were recovered from the uterus. In the control group, spermwere recovered from the uterus in 6 females with copulatory plugs. No uterine sperm were recovered from one control-mated female without a copulatory plug and from one control-mated female with a copulatory plug. In 2 control-mated females, the number of sperm recovered was too small to be accurately counted by hemocytometer (one due to loss of fluid during collection). In the remaining 4 females, a mean of 34.65 million sperm
Fig. 3. Copulatory plug in situ from an untreated female mated to a control male. The copulatory plug (P) is large, fills the entire circumference of the vaginal lumen (V), and is lodged tightly against the cervix (C).
were recovered from the uterus approximately 12 min postejaculation.
Intrauterine insemination Determination of number of sperm to be inseminated. Five control males were used for epididymal sperm collection. The collected sperm were diluted twice to achieve concentrations of approximately 0.5, 1.0, and 3 million sperm per 0.1 mL. Each dilution of
Fig. 4. Copulatory plug in situ from an untreated female mated to a finasteride-treated male. The copulatory plug (P) is small and adhered to a small portion of the vagina (V) distal to the cervix (C).
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Table 5. Summary of embryo recovery data after intrauterine insemination Dose group
Control
Number of males Number of males producing 3 or more pregnancies Number of females inseminated Positive pregnancies Anovulatory females Embryos counted Embryos/pregnant female +__ SD Unfertilized oocytes counted Unfertilized oocytes/ pregnant female ___ SD Total recoveries/female ± SD (embryos + oocytes) Total sperm × 106/0.1 mE, mean +_ SD
9 (8) ~ 9
80 mg/kg/day 9 9
34 33 (30) 1 294 (285) 8.91 -4- 3.39 (9.5 ___ 2.93) 52 (37) 1.58 - 1.9 (1.23 ± 1.57)
36 34 NS 2 330 9.71 4- 3.48 Ns
10.48 -+ 3.13 (10.73 - 3.14)
10.82 ± 3.18 Ns
2.9 --- 1.17 (3.2 +_ 0.35) b
38 1.12 +_ 1.81Ns
3.1
-4- 0.77 ¢
aNumber in parentheses excludes one control male with unilateral testicular degeneration and high percentage of morphologically abnormal sperm. bRange 1.3 to 4.0 × 10 6 sperm/0.1 mL. ORange 2.0 to 4.3 x 10 6 sperln/0.1 mE. NS =Not statistically significant, P > 0.05. Statistical comparisons performed excluding the one control male with testicular degeneration.
s p e r m w a s i n j e c t e d into the u t e r u s o f 6 to 11 L H R H agonist synchronized females, and embryos were recove r e d o n d a y 2 o f g e s t a t i o n ( T a b l e 4). A t a m e a n c o n c e n t r a t i o n o f 3 , 0 5 0 , 0 0 0 s p e r m i n j e c t e d , the p r e g n a n c y rate w a s 1 0 0 % ( 1 0 / 1 0 ) , a m e a n o f 10.2 e m b r y o s / female were recovered, and 0.7 unfertilized oocytes/ females were recovered. At a mean concentration of 9 8 3 , 1 2 5 s p e r m , t h e p r e g n a n c y rate w a s 1 0 0 % (6/6), w i t h a m e a n o f 7 . 7 e m b r y o s a n d 1.33 u n f e r t i l i z e d o o c y t e s p e r f e m a l e . A t a m e a n o f 5 8 4 , 1 6 7 s p e r m , the p r e g n a n c y rate w a s 8 2 % (9/11) w i t h a m e a n n u m b e r o f embryos and unfertilized oocytes per pregnant female of
4 . 7 a n d 4 . 2 , r e s p e c t i v e l y . B a s e d o n t h e s e results, the o p t i m u m n u m b e r o f s p e r m for i n t r a u t e r i n e i n s e m i n a t i o n w a s i d e n t i f i e d as 2 to 4 m i l l i o n s p e r m p e r u t e r i n e h o r n . If the e p i d i d y m a l s p e r m p r e p a r a t i o n h a d a h i g h e r c o n c e n t r a t i o n t h a n a p p r o x i m a t e l y 4 m i l l i o n , it w a s diluted. I f the c o n c e n t r a t i o n w a s less t h a n 2 m i l l i o n the p r e p a r a tion w a s u s e d u n d i l u t e d .
Intrauterine insemination synchronized females
of
LHRH
Thirty-four LHRH agonist synchronized females had intrauterine inseminations with a mean of approxi-
Table 6. Absolute and relative organ weights of control and finasteride-treated males after 38 weeks of dosing Treatment Group
Organ Body Weight, g Testes, g % B.W. Prostate, g % B.W. Seminal Vesicles, g % B.W.
agonist
Control (248 to 260 doses) n = 34
Finasteride (248 to 260 doses) n = 38
% Change from control
778 3.61 0.47 0.57 0.08 1.33 0.18
730 * 3.63 NS 0.50 * 0.21 *** 0.03 *** 0.30 *** 0.04***
- 6.2 +0.6 +6.0 - 63.2 -62.5 -77.5 -77.8
B.W. = body weight. NS = Not significantly different from control, P > 0.05. *P --< 0.05, **0.01, ***0.001, respectively. Significantly different from control.
Mechanism of finasteride-decreasedfertility• M. A. CtZKmRSlCIEr AL
361
DISCUSSION
Fig. 5. Seminal vesicles (S) and portion of the prostate (P) from a control (left) and a male treated with finasteride for 38 weeks (right), illustrating significant reduction in size of the glands.
mately 3 million epididymal sperm from 9 control males, and 36 females were inseminated with epididymal sperm from 9 finasteride-treated males (inseminations made during drug weeks 36 to 37) (Table 5). One control-inseminated female and 2 finasteride-insemihated females were anovulatory and were discarded without further examination. One control male had unilateral testicular degeneration and a high percentage of morphologically abnormal sperm. In calculating the sperm concentration from this male, only morphologically normal sperm were counted. One additional control male had a sperm count less than the desired concentration of 2 x 106/0.1 mL (1.3 x 106). The pregnancy rate for both treated and control group was 100%. There were no effects of treatment on the number of embryos or unfertilized oocytes recovered per female. All 9 males from both treated and control groups produced 3 to 4 pregnancies each.
Testes, prostate, and seminal vesicle weights Organ weight data are summarized in Table 6. There was no effect of finasteride treatment on absolute testicular weight. Testes weight relative to body weight was significantly increased (6%) in the treated group over controls. This slight increase is likely due to the decreased body weights of the treated group. In drug weeks 36 to 38, the finasteride-treated males had significantly decreased ventral prostatic and seminal vesicular weights; the prostatic and seminal vesicular weights when expressed as a percent of body weight were decreased 62.5% and 77.8, respectively, compared to controls.
Previous fertility studies in sexually mature rats administered high doses (80 mg/kg/day) of finasteride have shown 20% to 48% decrease in fertility after 24 weeks or more of dosing (see reference 1 also for discussion of effects on fertility at lower doses). No significant effects on fertility were observed after 12 weeks of administration, the typical duration of dosing for male fertility studies. There were no effects on testes weight or histomorphology of the seminiferous epithelium in previous studies, and the decreased fertility observed was reversible after a 6-week drug-free recovery period. These decreases in fertility were accompanied by large decreases in the weight of the prostate and seminal vesicles, an expected pharmacologic effect of finasteride administration. The purpose of this study was to confirm the effects on fertility of long-term administration to sexually mature male rats and to investigate the mechanism of decreased fertility. In this study the decreased fertility, decreased weight of prostate and seminal vesicles, and the number of males that mated but failed to produce a pregnancy in two successive cohabitations after 24 weeks of dosing with 80 mg/kg/day finasteride closely reproduced the findings of a previous fertility study (1). To investigate the mechanism of decreased fertility, a group of treated males with poor reproductive performance were directly compared with a group of control males with good reproductive performance. The secretions of the seminal vesicles, coagulating glands, and bulbourethral glands in rats clot after ejaculation in the vagina to form a copulatory plug. This plug plays a critical role in transcervical sperm transport (reference 4 and the references therein). In the current study, and in previous studies with finasteride, decreases were observed in the weights of the prostate and seminal vesicles and in the number of copulatory plugs. In observed matings it was found that mating behavior of treated males was qualitatively normal and they were capable of ejaculating sperm. However, they did not form copulatory plugs or formed very small plugs that were not in contact with the cervix and therefore were nonfunctional. Without proper copulatory plug formation, no sperm were transported into the uterus. Fertilizing ability of epididymal sperm collected from these same males was tested by intrauterine insemination into LHRH agonist synchronized females. Artificial insemination has been proposed as a more sensitive approach to fertility assessment in laboratory animals rather than natural matings, due to the huge excess number of sperm normally produced during natural matings above the minimum number required for fertilization (8). Artificial insemination allows deposi-
362
Reproductive Toxicology
tion of a critical number of sperm in the female tract from both treated and control animals for direct comparison. Using epididymal sperm from age matched controls, it was found that sperm concentrations below approximately 1 million per uterine horn were associated with a decreased number of fertilizations. The number of sperm for insemination was selected to be slightly above this level (mean 3 million, range approximately 2 to 4 million) for both treated and control animals. This can be compared to the mean of 34.64 million sperm recovered from the uterus during observed control matings. When approximately equal numbers of epididymal sperm from control and treated males were placed in the uterus, comparable numbers of embryos and unfertilized oocytes were recovered. Epididymal sperm from both treated and control groups were able to produce 3 to 4 pregnancies per male by intrauterine insemination. This demonstrates that sperm from finasteride-treated males is transported normally from the uterus to the site of fertilization and that fertilization occurs at rates equivalent to controls. The data presented here support the hypothesis that the decreased fertility observed in finasteride-treated males is related to an improper copulatory plug formation or fit, which is likely secondary to decreased seminal vesicle and prostate weight. Approximately 24 weeks of treatment are required for reduction of the sex accessory glands in sexually mature rats to the point where plug formation is impaired. The maximum degree of reduction in accessory gland weight obtained with finasteride treatment appears to be at or near the threshold for copulatory plug formation. This explains the long duration of treatment required to decrease fertility and the fact that there is considerable individual animal variation in ability to produce a pregnancy in successive cohabitations (1). Slight fluctuations in amount of secretions may determine whether a copulatory plug may be adequately formed during a particular mating. Previous studies have shown that even very small plugs properly placed can function (9). This variability may have the effect of reducing but not abolishing fertility. Previous studies have examined the effects of short-term administration (11 to 15 days) of the less specific 5et-reductase inhibitor 3-oxo-4-androstene-17[3 carboxylic acid, or its methyl ester, on the ability of epididymal sperm from mice to fertilize. Intrauterine insemination of these epididymal sperm into superovulated female mice resulted in decreased fertilization (10), and
Volume 5, Number 4, 1991
epididymal sperm from androgen supplemented castrate mice had decreased capacity to fertilize superovulated oocytes in vitro (11). As discussed in the accompanying paper by Wise and colleagues (1), the timing of these effects in mice is not comparable to that obtained in rats with finasteride. Additionally, intrauterine insemination of epididymal sperm from finasteride-treated males into nonsuperovulated females did not result in reduced fertilization. As the decreased fertility in male rats administered finasteride is associated with defects in copulatory plug formation and not in the ability of the sperm to fertilize, this decrease in fertility is considered a species specific effect and is not relevant to species that do not rely upon formation of copulatory plugs for transcervical sperm transport.
- - We would like to thank Drs. James W. Overstreet and Barb C. Vanderhayden for helpful discussions during initiation of these studies and Mr. Dave Billman for expert technical assistance. Acknowledgments
REFERENCES 1. Wise LD, Minsker DH, Cukierski MA, et al. Reversible decreased fertility in male Sprague-Dawley rats treated orally with finasteride, a 5c~-reductase inhibitor. Reprod Toxicol. 1991;5:337-346. 2. George FW, Peterson KG. 5a-dihydrotestosterone formation is necessary for embryogenesis of the rat prostate. Endocrinology. 1988;122:1159-64. 3. George FW, Johnson L, Wilson JD. The effect of a 5a-reductase inhibitor on androgen physiology in the immature male rat. Endocrinology. 1989;125:2434-38. 4. Cukierski MA, Sina JL, Prahalada S, Robertson RT. Effects of seminal vesicle and coagulating gland ablation on fertility in rats. Reprod Toxicol. 1991;5:347-352 5. Edgington ES. Randomization tests. New York: Marcel Dekker; 1980. 6. Harter HL. Expected values of normal order statistics. Biometrika. 1961;48:151-65. 7. Conover WJ, Iman RL. Rank transformation as a bridge between parametric and nonparametric statistics. Am Statist. 1981;35:12433. 8. Amann RP. Detection of alterations in testicular and epididymal function in laboratory animals. Environ Health Perspect. 1986;70:149-58. 9. Blandau RJ. On the factors involved in sperm transport through the cervix uteri of the albino rat. Am J Anat. 1945;77:253-72. 10. Lau IF, Saksena SK, Chang MC. Antifertility effect of 3-oxo-4androstene-1713 carboxylic acid in male mice. Arch Androl. 1979;2:179-81. 11. Cohen J, Gores MP, Vreeburg JTM. Reduction of fertilizing capacity of epididymal spermatozoa by 5a-steroid reductase inhibitors. Experientia. 1981; 37:1031-32.