FERTILITY AND SrERILITY Copyright 0 1975 The American Fertility Society
Vol. 26, No. 2, February 1975 Printed in U.S.A.
ACTION OF CYPROTERONE ACETATE ON THE ACCESSORY ORGANS OF REPRODUCTION IN PREPUBERTAL AND SEXUALLY MATURE RATS* M. RAJALAK.SHMI, M.Sc., PH.D.,
AND
M. R. N. PRASAD, PH.D.
Department of Zoology, University of Delhi, Delhi-110007, India
Microquantities of cyproterone acetate, released from subcutaneously implanted Silastic capsules, cause transient infertility in rats by selectively inhibiting epididymal function. Consequently, sperm motility and viability are lost, while other accessory glands, spermatogenesis, and libido remain normal. 1 •2 Cyproterone acetate (CA) in large doses (100 to 200 mgl day) has also been used in the management of aberrative or increased sexuality in men. 3•5 The purpose of this study was to study the effects of CA on the epididymis and accessory glands of immature and adult rats. Such data could be useful when this compound is evaluated in men. MATERIALS AND METHODS
Prepubertal and adult sexually mature rats of the Holtzman strain were maintained under standard husbandry conditions. The prepubertal rats were divided into three groups and were treated as follows: group 1, immature, 40-day-old rats, administered 2 mglday/rat of CA subcutaneously from days 40 to 50; group 2, immature, 30-day-old rats administered 2 mglday/rat of CA subcutaneously from days 30 to 50; and group 3, untreated, 50-day-old rats (controls). All animals were killed on day 50. Received July 16, 1973. *Supported by The Ford Foundation, the Ministry of Health and Family Planning, Government of India, and the Indian Council of Medical Research.
In another experiment, adult, sexually mature male rats (three and one-half months old) were administered 5 mglday/ rat of CA subcutaneously for 15 days and were autopsied on day 16. Untreated animals of the same age served as controls. At autopsy, the testes and accessory glands were removed and weighed on a torsion balance. A smear of the cauda epididymidis in saline was examined for the presence and motility pattern of spermatozoa. Citric acid was estimated in the ventral prostate (VP) and seminal vesicles (SV). Fructose was estimated in the dorsolateral prostate (DLP) and coagulating glands. s-s Sialic acid, 9 protein, 10 DNA, 11 and RNA 12 were estimated in the caput and cauda epididymides and ventral prostate. The caput and cauda epididymides of the immature animals were fixed in Bouin's fluid and processed for histology. Sections, cut at 5 J.L, were stained with hematoxylin-eosin and PAS-hematoxylin. Incorporation studies. Each adult control and CA-treated rat was injected with 50 microcurie ofL-phenylalanine-3H (specific activity, 7 curie/millimol), uridine-5 3 H (specific activity, 20.9 curie/millimol), and thymidine-6 3 H (specific activity, 9.1 curie/millimol) through the jugular vein while under ether anesthesia; the animals were autopsied by cervical dislocation three hours (phenylalanine), two hours (thymidine), and one hour (uridine) after the injection. The accessory organs were removed, weighed, and homogenized
137
138
RAJALAKSHMI AND PRASAD
FIG. 1. Caput epididymidis of a control 50day-old rat (x 400).
in a glass Teflon homogenizer with 0.5 M perchloric acid. The homogenate was processed for counting of radioactivity according to the method of Reel and Gorski. 13 The dried protein pellet was dissolved in formic acid. For counting of radioactivity, aliquots were put in vials to which 1 ml of isoamyl alcohol and 10 ml of scintillation fluid (5 gm of 2-5-diphenyl oxazole, PPO, and 50 mg 1,4-bis-2-[5-phenyl oxazole, POPOPJ in 1 liter of scintillation grade .. toluene) ' were added. The radioactivity was counted in a Packard Tricarb liquid scintillation spectrometer with a 45% efficiency for tritium. The counts were corrected for background activity and quenching. The amount of radioactivity was expressed in terms of specific activity of the protein, RNA, or DNA. All radiochemicals were purchased from Schwarz/Mann, Dickinson & Co, New York. RESULTS
Histologic changes. The histology of the
February 1975
FIG. 2. Caput epididymidis of a 50-day-old rat treated with cyproterone acetate (2 mg!day) for 20 days (x 400).
caput and cauda epididymides of immature rats treated with cyproterone acetate for ten days was similar to that of untreated controls. However, CA administration to prepubertal rats for 20 days (days 30 to 50) resulted in degenerative histologic changes in the epididymis. No spermatozoa were present in the lumen of the caput and cauda epididymides (Figs. 1 to 4). The lumen of the caput and cauda epididymides was greatly reduced in diameter and the epithelium was devoid of secretory granules. The lumen of the cauda epididymidis was filled with masses of exfoliated epithelial cells and eosinophilic material (Figs. 3 and 4). Biochemical changes. The biochemical changes in the epididymis of immature rats after CA treatment are shown in Table 1. The amounts of sialic acid and protein in the caput and cauda epididymides of immature rats decreased significantly on CA administration for ten days (group 1); this was more pronounced in
139
CYPROTERONE ACETATE AND ACCESSORY ORGANS
Vol. 26, No.2
FIG. 3. Cauda epididymidis of a control 50day-old rat (X 400).
FIG. 4. Cauda epididymidis of a 50-day-old rat treated with cyproterone acetate (2 mg/day) for 20 days (x 400).
rats treated with CA for 20 days. The content ofDNA in the cauda epididymidis and in the caput epididymidis decreased significantly after CA treatment for 10 and 20 days, respectively. The levels of sialic acid in the Cowper's glands (Table 2) and of fructose and citric acid in the acces-
sory glands (Table 2) also showed a similar decrease pattern after CA administration; the decrease was maximal in group 2. Sialic acid in the caput and cauda epididymides significantly decreased in adult rats treated with 5 mg/day of CA for 15 days (Table 3); however, protein and
TABLE 1.-Cha.nges in the Content• of Sialic Acid, Protein, and DNA in the Epididymides of Prepubertal Rats Treated with Cyproterone Acetateb Sialic acid' (!LmoVorgan) Protein' (mg!organ) DNA' (!Lg!organ) No. Group Treatof Caput Caput Caput Cauda Cauda Cauda no. ment rats epididymidis epididymidis epididymidis epididymidis epididymidis epididymidis 1 CA days 40-50d 2 CAdays 30-50" 3 None (control)
6
0.09±0.0031 O.Q7 ±0.0031
4.05±0.17
2.49± 0.2"
92.5±11.8h
9
0.03±0.0031 0.222±0.0051
1.4 ±0.031
0.95±0.111
25.9± 3.0" 20.6±0.841
6
0.15±0.005
6.2 ± 0.4
3.9 ±0.3
0.12 ±0.013
•Mean±SE. h2 mg!rat/day. con one side only. dFrom Rajalakshmi and Prasad.'" •Spermatozoa were absent from the epididymides of these rats. 'Significantly different from control (P0.05). 8
cessory glands of prepubertal rats to CA appears to depend on the duration of treatment; CA administration for ten days to prepubertal rats markedly decreased secretory activity of the epididymis and accessory glands; CA administration for 20 days further decreased secretory activity. Our results show that CA treatment for 20 days (days 30 to 50) significantly decreased DNA in the caput and cauda epididymides; sperm were absent in the epididymides of these rats. However, CA treatment for ten days (days 40 to 50) significantly reduced DNA in the cauda epididymidis despite the spermatozoa in the lumen. The DNA decreases before the entry of the first wave of spermatozoa into the epididymis 16 probably may be accounted for by the loss in cellularity of the epididymis and/or absence of spermatozoa. Further, a short treatment with CA (ten days) inhibits the secretory activity of the accessory glands, while a longer treatment (20 days) causes decrease in weight and changes in histoarchitecture. CA inhibition of androgen-dependent secretory activity of the accessory organs is less evident in adult rats than in prepubertal rats. Administration of CA to adult rats for 15 days caused no change in the content of protein in the caput and cauda epididymides or of sialic acid in the Cowper's glands; however, sialic acid in the caput and cauda epididymides significantly decreased. Based on the con-
cept of differential threshold of androgens required to maintain the secretory function of the different accessory glands and epididymis, 17 we hypothesize that the antiandrogen dose used (5 mg/day/rat) blocks the receptors in the androgen-sensitive target organs, causing androgen deprivation. This effect is more drastic in the epididymis (which has a higher threshold requirement of androgen) than in the Cowper's glands (which require much lower levels of androgen for the maintenance of their functional integrity). Our data also show that the degree of response of the accessory organs to antiandrogen depends on the sensitivity of the parameters used in the assay; sialic acid is more sensitive to inhibition of androgen action while changes in protein are relatively less sensitive. Further, the responsiveness of the caput and cauda epididymides of prepubertal rats to antiandrogen indicates that the transition period from the prepubertal to the pubertal state is a condition of heightened sensitivity in the response of the epididymis to alterations in androgen action. 15 The uptake of radioactive phenylalanine and uridine into protein and RNA in the caput and cauda epididymides increased after CA administration. The increase in incorporation of 3 H-uridine into RNA and of 3 H-phenylalanine into protein was higher in the cauda epididymidis than in the caput epididy-
142
February 1975
RAJALAKSHMI AND PRASAD
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midis. The specific activity of protein in the VP was not significantly different from that in control animals. The enhanced protein synthesis in the caput and cauda epididymides may have been due to the synthesis of specific lysosomal enzymes which are involved in autolytic processes. Involutionary processes caused by hormone administration (as in the regressing amphibian taiP 8 ) or caused by hormone withdrawal (as in the castrated rat accessory glands 19) are accompanied by the synthesis of specific lysosomal enzymes. Tata20 demonstrated increased incorporation of 3 H-uridine into RNA and 14C-amino acids into protein during tadpole tail regression induced in organ culture with triiodothyronine; however, no net accumulation of RNA and protein in the regressing tissues occurred. In the present study, the incorporation of3H-uridine into RNA and of3H-phenylalanine into protein is maximal m the cauda epididymis, lesser in the caput epididymidis, and almost insignificant in the ventral prostate. These results may be explained by the differential threshold hypothesis and are in agreement with the observations that the cauda epididymidis regresses much faster than the caput epididymidis, while the ventral prostate shows no involution after CA administration.
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Vol. 26, No. 2, February 1975 Printed in U.S.A.
ACTION OF CYPROTERONE ACETATE ON THE ACCESSORY ORGANS OF REPRODUCTION IN PREPUBERTAL AND SEXUALLY MATURE RATS* M. RAJALAK.SHMI, M.Sc., PH.D.,
AND
M. R. N. PRASAD, PH.D.
Department of Zoology, University of Delhi, Delhi-110007, India
Microquantities of cyproterone acetate, released from subcutaneously implanted Silastic capsules, cause transient infertility in rats by selectively inhibiting epididymal function. Consequently, sperm motility and viability are lost, while other accessory glands, spermatogenesis, and libido remain normal. 1 •2 Cyproterone acetate (CA) in large doses (100 to 200 mgl day) has also been used in the management of aberrative or increased sexuality in men. 3•5 The purpose of this study was to study the effects of CA on the epididymis and accessory glands of immature and adult rats. Such data could be useful when this compound is evaluated in men. MATERIALS AND METHODS
Prepubertal and adult sexually mature rats of the Holtzman strain were maintained under standard husbandry conditions. The prepubertal rats were divided into three groups and were treated as follows: group 1, immature, 40-day-old rats, administered 2 mglday/rat of CA subcutaneously from days 40 to 50; group 2, immature, 30-day-old rats administered 2 mglday/rat of CA subcutaneously from days 30 to 50; and group 3, untreated, 50-day-old rats (controls). All animals were killed on day 50. Received July 16, 1973. *Supported by The Ford Foundation, the Ministry of Health and Family Planning, Government of India, and the Indian Council of Medical Research.
In another experiment, adult, sexually mature male rats (three and one-half months old) were administered 5 mglday/ rat of CA subcutaneously for 15 days and were autopsied on day 16. Untreated animals of the same age served as controls. At autopsy, the testes and accessory glands were removed and weighed on a torsion balance. A smear of the cauda epididymidis in saline was examined for the presence and motility pattern of spermatozoa. Citric acid was estimated in the ventral prostate (VP) and seminal vesicles (SV). Fructose was estimated in the dorsolateral prostate (DLP) and coagulating glands. s-s Sialic acid, 9 protein, 10 DNA, 11 and RNA 12 were estimated in the caput and cauda epididymides and ventral prostate. The caput and cauda epididymides of the immature animals were fixed in Bouin's fluid and processed for histology. Sections, cut at 5 J.L, were stained with hematoxylin-eosin and PAS-hematoxylin. Incorporation studies. Each adult control and CA-treated rat was injected with 50 microcurie ofL-phenylalanine-3H (specific activity, 7 curie/millimol), uridine-5 3 H (specific activity, 20.9 curie/millimol), and thymidine-6 3 H (specific activity, 9.1 curie/millimol) through the jugular vein while under ether anesthesia; the animals were autopsied by cervical dislocation three hours (phenylalanine), two hours (thymidine), and one hour (uridine) after the injection. The accessory organs were removed, weighed, and homogenized
137
138
RAJALAKSHMI AND PRASAD
FIG. 1. Caput epididymidis of a control 50day-old rat (x 400).
in a glass Teflon homogenizer with 0.5 M perchloric acid. The homogenate was processed for counting of radioactivity according to the method of Reel and Gorski. 13 The dried protein pellet was dissolved in formic acid. For counting of radioactivity, aliquots were put in vials to which 1 ml of isoamyl alcohol and 10 ml of scintillation fluid (5 gm of 2-5-diphenyl oxazole, PPO, and 50 mg 1,4-bis-2-[5-phenyl oxazole, POPOPJ in 1 liter of scintillation grade .. toluene) ' were added. The radioactivity was counted in a Packard Tricarb liquid scintillation spectrometer with a 45% efficiency for tritium. The counts were corrected for background activity and quenching. The amount of radioactivity was expressed in terms of specific activity of the protein, RNA, or DNA. All radiochemicals were purchased from Schwarz/Mann, Dickinson & Co, New York. RESULTS
Histologic changes. The histology of the
February 1975
FIG. 2. Caput epididymidis of a 50-day-old rat treated with cyproterone acetate (2 mg!day) for 20 days (x 400).
caput and cauda epididymides of immature rats treated with cyproterone acetate for ten days was similar to that of untreated controls. However, CA administration to prepubertal rats for 20 days (days 30 to 50) resulted in degenerative histologic changes in the epididymis. No spermatozoa were present in the lumen of the caput and cauda epididymides (Figs. 1 to 4). The lumen of the caput and cauda epididymides was greatly reduced in diameter and the epithelium was devoid of secretory granules. The lumen of the cauda epididymidis was filled with masses of exfoliated epithelial cells and eosinophilic material (Figs. 3 and 4). Biochemical changes. The biochemical changes in the epididymis of immature rats after CA treatment are shown in Table 1. The amounts of sialic acid and protein in the caput and cauda epididymides of immature rats decreased significantly on CA administration for ten days (group 1); this was more pronounced in
139
CYPROTERONE ACETATE AND ACCESSORY ORGANS
Vol. 26, No.2
FIG. 3. Cauda epididymidis of a control 50day-old rat (X 400).
FIG. 4. Cauda epididymidis of a 50-day-old rat treated with cyproterone acetate (2 mg/day) for 20 days (x 400).
rats treated with CA for 20 days. The content ofDNA in the cauda epididymidis and in the caput epididymidis decreased significantly after CA treatment for 10 and 20 days, respectively. The levels of sialic acid in the Cowper's glands (Table 2) and of fructose and citric acid in the acces-
sory glands (Table 2) also showed a similar decrease pattern after CA administration; the decrease was maximal in group 2. Sialic acid in the caput and cauda epididymides significantly decreased in adult rats treated with 5 mg/day of CA for 15 days (Table 3); however, protein and
TABLE 1.-Cha.nges in the Content• of Sialic Acid, Protein, and DNA in the Epididymides of Prepubertal Rats Treated with Cyproterone Acetateb Sialic acid' (!LmoVorgan) Protein' (mg!organ) DNA' (!Lg!organ) No. Group Treatof Caput Caput Caput Cauda Cauda Cauda no. ment rats epididymidis epididymidis epididymidis epididymidis epididymidis epididymidis 1 CA days 40-50d 2 CAdays 30-50" 3 None (control)
6
0.09±0.0031 O.Q7 ±0.0031
4.05±0.17
2.49± 0.2"
92.5±11.8h
9
0.03±0.0031 0.222±0.0051
1.4 ±0.031
0.95±0.111
25.9± 3.0" 20.6±0.841
6
0.15±0.005
6.2 ± 0.4
3.9 ±0.3
0.12 ±0.013
•Mean±SE. h2 mg!rat/day. con one side only. dFrom Rajalakshmi and Prasad.'" •Spermatozoa were absent from the epididymides of these rats. 'Significantly different from control (P0.05). 8
cessory glands of prepubertal rats to CA appears to depend on the duration of treatment; CA administration for ten days to prepubertal rats markedly decreased secretory activity of the epididymis and accessory glands; CA administration for 20 days further decreased secretory activity. Our results show that CA treatment for 20 days (days 30 to 50) significantly decreased DNA in the caput and cauda epididymides; sperm were absent in the epididymides of these rats. However, CA treatment for ten days (days 40 to 50) significantly reduced DNA in the cauda epididymidis despite the spermatozoa in the lumen. The DNA decreases before the entry of the first wave of spermatozoa into the epididymis 16 probably may be accounted for by the loss in cellularity of the epididymis and/or absence of spermatozoa. Further, a short treatment with CA (ten days) inhibits the secretory activity of the accessory glands, while a longer treatment (20 days) causes decrease in weight and changes in histoarchitecture. CA inhibition of androgen-dependent secretory activity of the accessory organs is less evident in adult rats than in prepubertal rats. Administration of CA to adult rats for 15 days caused no change in the content of protein in the caput and cauda epididymides or of sialic acid in the Cowper's glands; however, sialic acid in the caput and cauda epididymides significantly decreased. Based on the con-
cept of differential threshold of androgens required to maintain the secretory function of the different accessory glands and epididymis, 17 we hypothesize that the antiandrogen dose used (5 mg/day/rat) blocks the receptors in the androgen-sensitive target organs, causing androgen deprivation. This effect is more drastic in the epididymis (which has a higher threshold requirement of androgen) than in the Cowper's glands (which require much lower levels of androgen for the maintenance of their functional integrity). Our data also show that the degree of response of the accessory organs to antiandrogen depends on the sensitivity of the parameters used in the assay; sialic acid is more sensitive to inhibition of androgen action while changes in protein are relatively less sensitive. Further, the responsiveness of the caput and cauda epididymides of prepubertal rats to antiandrogen indicates that the transition period from the prepubertal to the pubertal state is a condition of heightened sensitivity in the response of the epididymis to alterations in androgen action. 15 The uptake of radioactive phenylalanine and uridine into protein and RNA in the caput and cauda epididymides increased after CA administration. The increase in incorporation of 3 H-uridine into RNA and of 3 H-phenylalanine into protein was higher in the cauda epididymidis than in the caput epididy-
142
February 1975
RAJALAKSHMI AND PRASAD
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midis. The specific activity of protein in the VP was not significantly different from that in control animals. The enhanced protein synthesis in the caput and cauda epididymides may have been due to the synthesis of specific lysosomal enzymes which are involved in autolytic processes. Involutionary processes caused by hormone administration (as in the regressing amphibian taiP 8 ) or caused by hormone withdrawal (as in the castrated rat accessory glands 19) are accompanied by the synthesis of specific lysosomal enzymes. Tata20 demonstrated increased incorporation of 3 H-uridine into RNA and 14C-amino acids into protein during tadpole tail regression induced in organ culture with triiodothyronine; however, no net accumulation of RNA and protein in the regressing tissues occurred. In the present study, the incorporation of3H-uridine into RNA and of3H-phenylalanine into protein is maximal m the cauda epididymis, lesser in the caput epididymidis, and almost insignificant in the ventral prostate. These results may be explained by the differential threshold hypothesis and are in agreement with the observations that the cauda epididymidis regresses much faster than the caput epididymidis, while the ventral prostate shows no involution after CA administration.
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