- Email: [email protected]
In vitro organ culture of rhesus monkey epididymal tubules
CONTRACEPTION
OF
INVIlsoORSANCULTURE
RUBSUS
HONXEY
BPIDIDXMAL
TUB-s
Jagdeep Kaur, P.R.Ramakrishnan and M.Rajalakshmi Department of Reproductive Biology All India Institute of Medical Sciences, New Delhi-110029, India
ABszRacT
The caput and cauda epididymidal tubules of rhesus monkey were cultured for 5 days using a simple organ culture system. The viability of the tubules in culture was established by assessing: (a) the histology of the epididymis; (b) motility and viability of spermatozoa: and (c) scanning electron microscopic morphology of spermatozoa before and at the end of culture. The efficacy of the culture was evaluated by introducing the antiandrogen, cyproterone acetate, into the culture medium. Cyproterone acetate caused degenerative changes in the histology of the epididymis and coiling of the epididymal spermatozoa which may be due to alterations in epididymal milieu.
The epididymis .is an ideal extragonadal site for control of fertility in the male since alterations inits functions leading to impairment of sperm maturation would not have undesirable effects, particularly to the genetic material. Investigators have attempted the culture of epididymal tubules of different species to understand the physiological relationship of spermatozoa and epididymis and also to evolve a system for testing potential male fertility regulating agents, with action on epididymis. Successful maintenance of epididymal tubules and cells in culture was reported in rat (l-4), hamster (5), rabbit (6-8) and bull (9); few studies have been attempted in human (10) and none in primates. Since rhesus monkey is considered as a suitable animal model for reproductive research, it was of interest to establish culture of monkey epididymal tubules. This system would have the advantage that the time taken to study the effects of a drug on the epididymis and the contained spermatozoa would be much shorter compared to ti viva studies which must take into consideration the duration of time needed for spermatozoa to travel from caput to cauda epididymides (11). Additionally, one monkey epididymis could be used to establish large number of cultures and thus more Submitted for publication Accepted for publication
October January
MARCH 1991 VOL. 43 NO. 3
23, 1990 29,1991
than one drug could be tested simultaneously to generate the This would be more appropriate with required data rapidly. the ethical use of non-human primates in biomedical research. Organ culture was chosen for the present study since this would mimic more closely the in vivo system.
MATERIALS
AHD
HBTEODS
Animals
Six adult male rhesus monkeys (Macaca mulatta, 8-10 kg BW) were caught from jungles approximately 250 kms East of Delhi. They were quarantined in the Primate Research Facility of AIIMS for 3 months where they underwent bimonthly tuberculin tests, deworming and haematological tests and then transfered for 15 days of acclimatisation. to the Experimental Facility The animals were electroejaculated (12), the day after blood collection and semen analyses were done (13). Castration was done under ketamine (Themis Chemicals Ltd. India) anaesthesia (10 mg/kg body weight). The epididymal tubules were cultured (14) using Minimum Essential Medium (MEM: Gibco Laboratories, USA) containing 20 mM HEPES, 0.029% L-glutamine, 10% foetal calf serum, 0.003%' insulin, 0.05% 5 OC -dihydrotestosterone (DHT; Sigma Chemical Co., USA), 10% chick embryo extract, 100 100 ug/ml streptomycin sulphate (Ranbaxy IU/ml penicillin, Lab. India) and 2.5 ug/ml Fungizone (Gibco Lab. USA). The culture vessel (Fig.1) consisted of a glass cup (25 mm in diameter and 15 mm in depth), with a stainless steel grid,
I-l-GLASS
LID
w-
LENS
m---
STAINLESS
a-:::
STEEL
BOARD
ORGAN
CULTURE
VESSEL
placed in a watch glass epididymis was divided
296
MESH
Fig.1. Diagram of Chapekar's organ culture vessel used in the study.
:::ss
~-Ham
CHAFEKAR’S
PAPER
and covered with a petri dish. The into initial (1 cm), caput (2.2
MARCH 1991 VOL. 43 NO. 3
CONTRACEPTION and cm) , corpus (2.5 cm) and cauda (3.5 cm) segments. Caput were straightened mechanically cauda epididymidal tubules under a stereomicroscope in plain MEM. Six explants of in length were kept on lens paper and approximately 1 cm placed on the grid in the glass cup which contained the supplemented medium. The vessel was covered with petri dish, sealed with melted paraffin to form a closed culture system and was incubated at 34 +l C . The medium was changed after 48 hours of explantation. Two explants from each culture vessel were removed 24,72 and 120 hours after initiation of culture and were processed for morphology of epididymis and sperm structure including types of coiling of tail (I5), sperm motility and viability. Studies using cyproterone
acetate
(CA)
Initially, dose response studies were done using 50, 100 and 200 uM of CA to determine the effective dose. CA was dissolved in ethanol and added to the culture medium (
Morphology
of cultured
caput and cauda epididymidal
tubules
Initial experiments showed that DHT was essential to maintain cultures viably since in its absence, the principal cells showed degenerative morphological changes. In the
MARCH 1991 VOL. 43 NO. 3
297
CONTRACEPTION
presence of DHT and other supplements, the cell types in caput and cauda epididymides maintained normal histology in 85-95% of explants for 5 days when the culture was terminated. The caput epididymidal epithelium on day 5 of culture is composed mainly of tall columnar principal cells with nuclei in the basal third of the cell (Figs. 2,3). occasional The nucleus, oval or spindle shaped, shows infoldings and has distinct nucleolus. The supranuclear region has empty spaces. The apical region has fine granules, pinocytotic vesicles and stereocilia. Basal cells and apical cells are also present. Principal cells of cauda epididymides are similar to that of caput except for :(a) few vacuoles in the supranuclear region; (b) highly infolded nuclei; and (c) presence of intraepithelial crypts (Figs. 4,5). Basal cells
Figs.2,3. Caput epididymidal (CT) tubule on day 5 of culture. Figs.4,5. Cauda epididymidal (CD) tubule on day 5 of culture. Magnification: Figs.2,4 (x 250); Figs.3,5 (x 1000); B- basal cell: C- intraepithelial crypt: P- principal cell. are more numerous while apical cells are lesser in number compared to caput epididymides. The morphology of caput and cauda epididymidal epithelia and their spermatozoa on different days of culture (Figs. 6,7) were similar to that of controls. Coiled tails were seen in 63% of caput and
298
MARCH 1991 VOL. 43 NO. 3
Fig. 6. CT spermatozoa Fig. 7. CD spermatozoa
on day 5 of culture: on day 5 of culture:
x 2190. x 1560.
29% of cauda epididymidal spermatozoa on day 5 of culture which was not significantly different from day 0 values. Based on the complexity of coiling of sperm tail, epididymal spermatozoa were classified in an earlier study into 5 types were : type A - only endpiece (15) - These different types coiled: type B - endpiece and distal principal piece involved in whorl-like coil: type C - endpiece and entire principal piece of sperm were coiled: type D - coiling involved the endpiece, principal piece and midpiece; and type E - the head', midpiece, principal piece and endpiece were involved in coiling. In samples taken prior to and on day 5 of culture, caput epididymidal spermatozoa showed mainly complex type D coiling (15) while caudal sperm showed mainly A and B types of coiling (Figs. 8,9). On day 3 of culture, spermatozoa of
TYPE
A
TYPE
B
TYPE
C
TYPE
D
TVPE
E
Fig.8. Percent spermatozoa from CT tubules prior to culture on day 4 of culture in presence of DHT (O), DHT + 50 (_), (m) or DHT + 200 uM CA (mm), ~4 CA (/SQ), DHT + 100 uM CA showing types A,B,C,D and E coiling.
MARCH 1991 VOL. 43 NO. 3
299
CONTRACEPTION
60 r
TYPE
A
TVPE
6
TVPE
C
TVPE
0
TYPE
E
Fig.9. Percent spermatozoa from CD tubules prior to culture on day 4 of culture in presence of DHT (a), DHT + 50 (_)I DHT + 200 uM CA (m), uM CA (KSQ), DHT + 100 uM CA (m), showing types A,B,C,D and E coiling. both regions were motile and live: on day 5 of culture, though the motility had decreased, the spermatozoa were still live indicating the maintenance of the viability of culture. Determination
of effective
dose of CA
and cauda In the presence of 50 uM CA, the caput epididymidal epithelia were maintained near normal in 80% and 62%, respectively, of the tubules. The percentage of caput and cauda epididymidal spermatozoa showing different types of coiling of tail from tubules (types A,B,C,D, and E) exposed to CA were not different from those obtained from control cultures (Figs. 8,9). No recognisable changes in sperm morphology, as visualised by scanning electron microscopy, were seen. But, 100 uM CA caused degenerative changes and only 14% and 12% of caput and cauda epididymidal tubules, respectively, The degenerative were maintained. changes in principal cells included pycnosis of nuclei, loss in cell limits and vacuolation of cytoplasm (Figs. 10,ll). The cytoplasm of basal cells also showed vacuolation. The percentage of spermatozoa with coiled tails increased significantly (pcO.01) to 95% in caput and 72% in cauda epididymidal tubules and a shift towards more complex types of coiling (types D and E; Figs. 12, 13) was seen. Separation of head and tail of 25% of caudal sperm was also seen. The degenerative changes caused by 200 uM CA on epididymal
300
MARCH 1991 VOL. 43 NO. 3
Fig. 10. pycnosis Fig. 11. pycnosis
Effect of 100 uM CA on CT epithelium. and cytoplasmic vacuolation. x 1000. Effect of 100 uM CA on CD epithelium. and desquamation. x 1000.
Fig. 12. Effect of 100 uM CA on CT spermatozoa. Fig. 13. Effect of 100 uM CA on CD spermatozoa. were histology and sperm morphology compared to that caused by 100 uM CA.
slightly
Note
nuclear
Note
nuclear
x 4230. x 3250. more
severe
DISCUSSIOll
In the present study, caput and cauda epididymidal tubules of rhesus monkey were successfully maintained in organ culture for 5 days: this limited duration of time was chosen since the purpose of the study was to evolve a rapid testing regulating agents. No model for potential male fertility differences were found in epididymal histology and morphology of spermatozoa of caput and cauda epididymidal tubules, prior
MARCH 1991 VOL. 43 NO. 3
301
CONTRACEPTION
to and at the termination of culture, establishing the viability of the system. The rapidity of this system as a drug testing model is evident from the fact that only 3 days of exposure of drug to the tubules was sufficient to cause recognisable alterations in histology. This is in contrast to the time required to test a drug in vivo. Further, a number of drugs can be tested simultaneously and thus decrease further the duration of experimentation and its cost. The number of'animals needed is also drastically reduced,which is more in agreement with the humane use of non-human primates. The efficacy of the organ culture system was tested by evaluating the effects of different doses of CA. CA, at an effective dose of 100 uM, induced degeneration of caput and cauda epididymidal and intense coiling of epithelia spermatozoa; the adverse effects may be due to altered testosterone epididymal milieu consequent on deprivation (15). Immature caput epididymidal spermatozoa are known to be rich in sulphydryl groups unlike caudal spermatozoa which have a high degree of disulfide bonds (16). It is likely that androgen deprivation by CA may have resulted in deficiency in disulphide bonding, thereby inhibiting the straightening of sperm tail which occurs during epididymal maturation of spermatozoa in normal animals (15). The culture system developed in the present study is more simple than the static (1,5-7) and perifusion (4,8) systems which have been used so far for organ culture of epididymal tubules of rodents and human. Since preliminary results showed that cultures degenerated in the absence of androgens, DHT at doses recommended earlier (7) were used and successful maintenance was achieved. Degeneration of epididymal principal cells cultured in the absence of androgen was reported earlier (5).
This work would not have been possible but for the guidance and help rendered by Dr. T.N. Chapekar in establishing the organ culture system: we thank him for this help. This work was supported by grants from the Department of Science and Technology, Government of India and the Indian Council of Medical Research.
1. 2.
302
Blaquier, J.A. and Breger, D. The in vitro effects of androgens on RNA synthesis by cultured rat epididymal tubules. Endocr Res Commun 1: 247-260 (1974). Klinefelter, G.R., Amann, R.P. and Hammerstedt, R.H. Culture of principal cells from the rat caput epididymis.
MARCH 1991 VOL. 43 NO. 3
CONTRACEPTION
3.
4. 5. 6. 7.
8.
9.
10.
11.
12. 13.
14. 15. 16.
Biol Reprod 26: 885-901 (1982). White, M.G., Huang, Y.S., Tres, L.L. and Kierszenbaum, A.L. Structural and functional aspects of cultured epididymal epithelial cells of pubertal rats. J Reprod Fertil 66: 475-484 (1982). Klinefelter, G.R. and Hamilton, D.W. Organ culture of rat caput epididymal tubules in a perifusion chamber. J Androl 5: 243-258 (1984). Moore, H.D.M., Hartman, T.D. and Smith, C.A. In vitro culture of hamster epididymal epithelium and induction of sperm motility. J Reprod Fertil 78: 327-336 (1986). Orgebin-Crist, M.C. and Tichenor, P.C. A technique for studying sperm maturation in vitro. Nature (Lond) 239: 227 (1972). Hoffman, -L.H., Jahad, N. 'and Orgebin-Crist, M.C. The effect of testosterone, 5s-dihydrotestosterone, 34androstanediol and 3B-androstanediol on epithelial fine structure of the rabbit epididymis in organ culture. Cell Tiss Res 167: 493-514 (1976). Orgebin-Crist, M.C. and Menezo, Y. A continuous flow method for organ culture of rabbit epididymis: morphology, amino acid utilization, glucose uptake, RNA and protein synthesis. J Androl 1: 287-298 (1980). Joshi, M.S. Isolation and cell culture of the epithelial cells of cauda epididymis of the bull. Biol Reprod 33: 187-200 (1985). Vazquez, M.H., de Larminat,, M.A. and Blaquier, J.A. Effect of antiandrogen on androgen receptors in cultured human epididymis. J Endocrinol 111: 343-348 (1986). Amann, R.P., Johnson, L., Thompson, D.L. Jr. and Pickett, B.W. Daily spermatozoa production, epididymal spermatozoa reserves and transit time of spermatozoa through the epididymis of the rhesus monkey. Biol Reprod 15: 586-592 (1976). Mastroianni, L. Jr. and Manson, W.A. Jr. Collection of monkey semen by electroejaculation. Proc Sot Exp Biol Med 112: 1025-1027 (1963). Belsey, M.A., Eliasson, R., Gallegos, A-J., Moghissi, K.S., Paulsen, C.A. and Prasad, M.R.N. Laboratory manual for the examination of the human semen-cervical mucus interaction. Press Concern, Singapore (1980). Chapekar, T.N. and Ranadive, K.J. In vitro studies on mouse mammary gland response to hormonal treatment. Ind J Exp Biol 1: 167-171 (1963). Kaur, J., Ramakrishnan, P.R. and Rajalakshmi, M. Inhibition of sperm maturation in rhesus monkey by cyproterone acetate. Contraception 42: 349-359 (1990). Cornwall, G.A., Vindivich, D., Elstein, K.H. and Chang, T.S.K. The effect of sulphdryl oxidation on the morphology of immature hamster epididymal spermatozoa induced to acquire motility in vitro. Biol Reprod 39: 141-156 (1988).
MARCH 1991 VOL. 43 NO. 3
303
OF
INVIlsoORSANCULTURE
RUBSUS
HONXEY
BPIDIDXMAL
TUB-s
Jagdeep Kaur, P.R.Ramakrishnan and M.Rajalakshmi Department of Reproductive Biology All India Institute of Medical Sciences, New Delhi-110029, India
ABszRacT
The caput and cauda epididymidal tubules of rhesus monkey were cultured for 5 days using a simple organ culture system. The viability of the tubules in culture was established by assessing: (a) the histology of the epididymis; (b) motility and viability of spermatozoa: and (c) scanning electron microscopic morphology of spermatozoa before and at the end of culture. The efficacy of the culture was evaluated by introducing the antiandrogen, cyproterone acetate, into the culture medium. Cyproterone acetate caused degenerative changes in the histology of the epididymis and coiling of the epididymal spermatozoa which may be due to alterations in epididymal milieu.
The epididymis .is an ideal extragonadal site for control of fertility in the male since alterations inits functions leading to impairment of sperm maturation would not have undesirable effects, particularly to the genetic material. Investigators have attempted the culture of epididymal tubules of different species to understand the physiological relationship of spermatozoa and epididymis and also to evolve a system for testing potential male fertility regulating agents, with action on epididymis. Successful maintenance of epididymal tubules and cells in culture was reported in rat (l-4), hamster (5), rabbit (6-8) and bull (9); few studies have been attempted in human (10) and none in primates. Since rhesus monkey is considered as a suitable animal model for reproductive research, it was of interest to establish culture of monkey epididymal tubules. This system would have the advantage that the time taken to study the effects of a drug on the epididymis and the contained spermatozoa would be much shorter compared to ti viva studies which must take into consideration the duration of time needed for spermatozoa to travel from caput to cauda epididymides (11). Additionally, one monkey epididymis could be used to establish large number of cultures and thus more Submitted for publication Accepted for publication
October January
MARCH 1991 VOL. 43 NO. 3
23, 1990 29,1991
than one drug could be tested simultaneously to generate the This would be more appropriate with required data rapidly. the ethical use of non-human primates in biomedical research. Organ culture was chosen for the present study since this would mimic more closely the in vivo system.
MATERIALS
AHD
HBTEODS
Animals
Six adult male rhesus monkeys (Macaca mulatta, 8-10 kg BW) were caught from jungles approximately 250 kms East of Delhi. They were quarantined in the Primate Research Facility of AIIMS for 3 months where they underwent bimonthly tuberculin tests, deworming and haematological tests and then transfered for 15 days of acclimatisation. to the Experimental Facility The animals were electroejaculated (12), the day after blood collection and semen analyses were done (13). Castration was done under ketamine (Themis Chemicals Ltd. India) anaesthesia (10 mg/kg body weight). The epididymal tubules were cultured (14) using Minimum Essential Medium (MEM: Gibco Laboratories, USA) containing 20 mM HEPES, 0.029% L-glutamine, 10% foetal calf serum, 0.003%' insulin, 0.05% 5 OC -dihydrotestosterone (DHT; Sigma Chemical Co., USA), 10% chick embryo extract, 100 100 ug/ml streptomycin sulphate (Ranbaxy IU/ml penicillin, Lab. India) and 2.5 ug/ml Fungizone (Gibco Lab. USA). The culture vessel (Fig.1) consisted of a glass cup (25 mm in diameter and 15 mm in depth), with a stainless steel grid,
I-l-GLASS
LID
w-
LENS
m---
STAINLESS
a-:::
STEEL
BOARD
ORGAN
CULTURE
VESSEL
placed in a watch glass epididymis was divided
296
MESH
Fig.1. Diagram of Chapekar's organ culture vessel used in the study.
:::ss
~-Ham
CHAFEKAR’S
PAPER
and covered with a petri dish. The into initial (1 cm), caput (2.2
MARCH 1991 VOL. 43 NO. 3
CONTRACEPTION and cm) , corpus (2.5 cm) and cauda (3.5 cm) segments. Caput were straightened mechanically cauda epididymidal tubules under a stereomicroscope in plain MEM. Six explants of in length were kept on lens paper and approximately 1 cm placed on the grid in the glass cup which contained the supplemented medium. The vessel was covered with petri dish, sealed with melted paraffin to form a closed culture system and was incubated at 34 +l C . The medium was changed after 48 hours of explantation. Two explants from each culture vessel were removed 24,72 and 120 hours after initiation of culture and were processed for morphology of epididymis and sperm structure including types of coiling of tail (I5), sperm motility and viability. Studies using cyproterone
acetate
(CA)
Initially, dose response studies were done using 50, 100 and 200 uM of CA to determine the effective dose. CA was dissolved in ethanol and added to the culture medium (
Morphology
of cultured
caput and cauda epididymidal
tubules
Initial experiments showed that DHT was essential to maintain cultures viably since in its absence, the principal cells showed degenerative morphological changes. In the
MARCH 1991 VOL. 43 NO. 3
297
CONTRACEPTION
presence of DHT and other supplements, the cell types in caput and cauda epididymides maintained normal histology in 85-95% of explants for 5 days when the culture was terminated. The caput epididymidal epithelium on day 5 of culture is composed mainly of tall columnar principal cells with nuclei in the basal third of the cell (Figs. 2,3). occasional The nucleus, oval or spindle shaped, shows infoldings and has distinct nucleolus. The supranuclear region has empty spaces. The apical region has fine granules, pinocytotic vesicles and stereocilia. Basal cells and apical cells are also present. Principal cells of cauda epididymides are similar to that of caput except for :(a) few vacuoles in the supranuclear region; (b) highly infolded nuclei; and (c) presence of intraepithelial crypts (Figs. 4,5). Basal cells
Figs.2,3. Caput epididymidal (CT) tubule on day 5 of culture. Figs.4,5. Cauda epididymidal (CD) tubule on day 5 of culture. Magnification: Figs.2,4 (x 250); Figs.3,5 (x 1000); B- basal cell: C- intraepithelial crypt: P- principal cell. are more numerous while apical cells are lesser in number compared to caput epididymides. The morphology of caput and cauda epididymidal epithelia and their spermatozoa on different days of culture (Figs. 6,7) were similar to that of controls. Coiled tails were seen in 63% of caput and
298
MARCH 1991 VOL. 43 NO. 3
Fig. 6. CT spermatozoa Fig. 7. CD spermatozoa
on day 5 of culture: on day 5 of culture:
x 2190. x 1560.
29% of cauda epididymidal spermatozoa on day 5 of culture which was not significantly different from day 0 values. Based on the complexity of coiling of sperm tail, epididymal spermatozoa were classified in an earlier study into 5 types were : type A - only endpiece (15) - These different types coiled: type B - endpiece and distal principal piece involved in whorl-like coil: type C - endpiece and entire principal piece of sperm were coiled: type D - coiling involved the endpiece, principal piece and midpiece; and type E - the head', midpiece, principal piece and endpiece were involved in coiling. In samples taken prior to and on day 5 of culture, caput epididymidal spermatozoa showed mainly complex type D coiling (15) while caudal sperm showed mainly A and B types of coiling (Figs. 8,9). On day 3 of culture, spermatozoa of
TYPE
A
TYPE
B
TYPE
C
TYPE
D
TVPE
E
Fig.8. Percent spermatozoa from CT tubules prior to culture on day 4 of culture in presence of DHT (O), DHT + 50 (_), (m) or DHT + 200 uM CA (mm), ~4 CA (/SQ), DHT + 100 uM CA showing types A,B,C,D and E coiling.
MARCH 1991 VOL. 43 NO. 3
299
CONTRACEPTION
60 r
TYPE
A
TVPE
6
TVPE
C
TVPE
0
TYPE
E
Fig.9. Percent spermatozoa from CD tubules prior to culture on day 4 of culture in presence of DHT (a), DHT + 50 (_)I DHT + 200 uM CA (m), uM CA (KSQ), DHT + 100 uM CA (m), showing types A,B,C,D and E coiling. both regions were motile and live: on day 5 of culture, though the motility had decreased, the spermatozoa were still live indicating the maintenance of the viability of culture. Determination
of effective
dose of CA
and cauda In the presence of 50 uM CA, the caput epididymidal epithelia were maintained near normal in 80% and 62%, respectively, of the tubules. The percentage of caput and cauda epididymidal spermatozoa showing different types of coiling of tail from tubules (types A,B,C,D, and E) exposed to CA were not different from those obtained from control cultures (Figs. 8,9). No recognisable changes in sperm morphology, as visualised by scanning electron microscopy, were seen. But, 100 uM CA caused degenerative changes and only 14% and 12% of caput and cauda epididymidal tubules, respectively, The degenerative were maintained. changes in principal cells included pycnosis of nuclei, loss in cell limits and vacuolation of cytoplasm (Figs. 10,ll). The cytoplasm of basal cells also showed vacuolation. The percentage of spermatozoa with coiled tails increased significantly (pcO.01) to 95% in caput and 72% in cauda epididymidal tubules and a shift towards more complex types of coiling (types D and E; Figs. 12, 13) was seen. Separation of head and tail of 25% of caudal sperm was also seen. The degenerative changes caused by 200 uM CA on epididymal
300
MARCH 1991 VOL. 43 NO. 3
Fig. 10. pycnosis Fig. 11. pycnosis
Effect of 100 uM CA on CT epithelium. and cytoplasmic vacuolation. x 1000. Effect of 100 uM CA on CD epithelium. and desquamation. x 1000.
Fig. 12. Effect of 100 uM CA on CT spermatozoa. Fig. 13. Effect of 100 uM CA on CD spermatozoa. were histology and sperm morphology compared to that caused by 100 uM CA.
slightly
Note
nuclear
Note
nuclear
x 4230. x 3250. more
severe
DISCUSSIOll
In the present study, caput and cauda epididymidal tubules of rhesus monkey were successfully maintained in organ culture for 5 days: this limited duration of time was chosen since the purpose of the study was to evolve a rapid testing regulating agents. No model for potential male fertility differences were found in epididymal histology and morphology of spermatozoa of caput and cauda epididymidal tubules, prior
MARCH 1991 VOL. 43 NO. 3
301
CONTRACEPTION
to and at the termination of culture, establishing the viability of the system. The rapidity of this system as a drug testing model is evident from the fact that only 3 days of exposure of drug to the tubules was sufficient to cause recognisable alterations in histology. This is in contrast to the time required to test a drug in vivo. Further, a number of drugs can be tested simultaneously and thus decrease further the duration of experimentation and its cost. The number of'animals needed is also drastically reduced,which is more in agreement with the humane use of non-human primates. The efficacy of the organ culture system was tested by evaluating the effects of different doses of CA. CA, at an effective dose of 100 uM, induced degeneration of caput and cauda epididymidal and intense coiling of epithelia spermatozoa; the adverse effects may be due to altered testosterone epididymal milieu consequent on deprivation (15). Immature caput epididymidal spermatozoa are known to be rich in sulphydryl groups unlike caudal spermatozoa which have a high degree of disulfide bonds (16). It is likely that androgen deprivation by CA may have resulted in deficiency in disulphide bonding, thereby inhibiting the straightening of sperm tail which occurs during epididymal maturation of spermatozoa in normal animals (15). The culture system developed in the present study is more simple than the static (1,5-7) and perifusion (4,8) systems which have been used so far for organ culture of epididymal tubules of rodents and human. Since preliminary results showed that cultures degenerated in the absence of androgens, DHT at doses recommended earlier (7) were used and successful maintenance was achieved. Degeneration of epididymal principal cells cultured in the absence of androgen was reported earlier (5).
This work would not have been possible but for the guidance and help rendered by Dr. T.N. Chapekar in establishing the organ culture system: we thank him for this help. This work was supported by grants from the Department of Science and Technology, Government of India and the Indian Council of Medical Research.
1. 2.
302
Blaquier, J.A. and Breger, D. The in vitro effects of androgens on RNA synthesis by cultured rat epididymal tubules. Endocr Res Commun 1: 247-260 (1974). Klinefelter, G.R., Amann, R.P. and Hammerstedt, R.H. Culture of principal cells from the rat caput epididymis.
MARCH 1991 VOL. 43 NO. 3
CONTRACEPTION
3.
4. 5. 6. 7.
8.
9.
10.
11.
12. 13.
14. 15. 16.
Biol Reprod 26: 885-901 (1982). White, M.G., Huang, Y.S., Tres, L.L. and Kierszenbaum, A.L. Structural and functional aspects of cultured epididymal epithelial cells of pubertal rats. J Reprod Fertil 66: 475-484 (1982). Klinefelter, G.R. and Hamilton, D.W. Organ culture of rat caput epididymal tubules in a perifusion chamber. J Androl 5: 243-258 (1984). Moore, H.D.M., Hartman, T.D. and Smith, C.A. In vitro culture of hamster epididymal epithelium and induction of sperm motility. J Reprod Fertil 78: 327-336 (1986). Orgebin-Crist, M.C. and Tichenor, P.C. A technique for studying sperm maturation in vitro. Nature (Lond) 239: 227 (1972). Hoffman, -L.H., Jahad, N. 'and Orgebin-Crist, M.C. The effect of testosterone, 5s-dihydrotestosterone, 34androstanediol and 3B-androstanediol on epithelial fine structure of the rabbit epididymis in organ culture. Cell Tiss Res 167: 493-514 (1976). Orgebin-Crist, M.C. and Menezo, Y. A continuous flow method for organ culture of rabbit epididymis: morphology, amino acid utilization, glucose uptake, RNA and protein synthesis. J Androl 1: 287-298 (1980). Joshi, M.S. Isolation and cell culture of the epithelial cells of cauda epididymis of the bull. Biol Reprod 33: 187-200 (1985). Vazquez, M.H., de Larminat,, M.A. and Blaquier, J.A. Effect of antiandrogen on androgen receptors in cultured human epididymis. J Endocrinol 111: 343-348 (1986). Amann, R.P., Johnson, L., Thompson, D.L. Jr. and Pickett, B.W. Daily spermatozoa production, epididymal spermatozoa reserves and transit time of spermatozoa through the epididymis of the rhesus monkey. Biol Reprod 15: 586-592 (1976). Mastroianni, L. Jr. and Manson, W.A. Jr. Collection of monkey semen by electroejaculation. Proc Sot Exp Biol Med 112: 1025-1027 (1963). Belsey, M.A., Eliasson, R., Gallegos, A-J., Moghissi, K.S., Paulsen, C.A. and Prasad, M.R.N. Laboratory manual for the examination of the human semen-cervical mucus interaction. Press Concern, Singapore (1980). Chapekar, T.N. and Ranadive, K.J. In vitro studies on mouse mammary gland response to hormonal treatment. Ind J Exp Biol 1: 167-171 (1963). Kaur, J., Ramakrishnan, P.R. and Rajalakshmi, M. Inhibition of sperm maturation in rhesus monkey by cyproterone acetate. Contraception 42: 349-359 (1990). Cornwall, G.A., Vindivich, D., Elstein, K.H. and Chang, T.S.K. The effect of sulphdryl oxidation on the morphology of immature hamster epididymal spermatozoa induced to acquire motility in vitro. Biol Reprod 39: 141-156 (1988).
MARCH 1991 VOL. 43 NO. 3
303