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Organosilicone induced malformation of rat sperms, a histochemical study
Acta histochem. 75, 149-151 (1984)
Institute of Anatomy II and Institute of Organic Chemistry, University of Dusseldorf, FRG
Organosilicone induced malformation of rat sperms, a histochemical study!) By SYED G. HAIDER, DIETER PASSIA, OSKAR STUHL, and I . .EoNHARD BIRKOFER With Plate IV (Received January 12, 1984)
Summary The effects of an organosilicon compound SOC, a derivative of 1,3-dioxa-2-sila-cyclopentane, on rat spermiogenesis were studied with the help of histochemical and enzyme histochemical methods. The rats were injected with SOC i.p. daily for 3 weeks (dosage: 25 mg SOC/kg body weight). Two changes were observed: a) formation of a distinct vacuole within the acrosomal cap of spermatids b) the splitting of the tails of mature sperms.
Zusammenfassung Mit Hilfe von histochemischen und enzymhistochemischen Methoden wurden die vVirkungen einer siliciumorganischen Verbindung SOC, ein Derivat des 1,3-Dioxa-2-sila-cyclopentans, auf die Spermiogenese der Ratte untersucht. Die Ratten wurden 3 Wochen lang taglich i.p. mit SOC behandelt (Dosierung: 25 mg SOC/kg Korpergewicht). 2 Veranderungen wurden beobachtet: a) Bildung einer distinkten Vakuole in der acrosomalen Kappe der Spermatiden b) Spaltung der Schwanzstucke der reifen Spermien.
Introduction Presently the organosilicon compounds are gaining importance in the field of environmental research, therapeutic medicine, and biomedical applications (VORONKOV et al. 1975; ARKLES 1983). HAIDER et al. (1983) reported the histophysiological effects of an organosilicone on rat liver, kidney and testis; this compound was prepared by BIRKOFER and STUHL (1980), the complete name: 2,2-dimethyl-4-(chloromethyl)-1,3dioxa-2-sila-cyclopentane, abbreviated as SOC. This compound induced the splitting of the sperm tails, as described by HAIDER et al. (1983). The aim of the present report was to study the effects of SOC on the spermatids and sperms, employing histochemical and enzyme histochemical techniques.
Material and Methods 42 male Wistar rats, with 190 to 200 g body weight, were distributed in 6 groups. 2 groups were injected i.p. daily for 3 weeks with 25 mg SOC/kg body wt, suspended in 0.15 ml olive oil, 1) Dedicated to Professor Dr. HANS Gi;NTER GOSLAR on the occasion of his 65th birthday.
150
S. G. HAIDER et al.
the other 2 groups received the vehicle, and the remaining 2 groups were regarded as untreated controls (for details of the preparation procedure and for the physical data of SOC see BIRKOFER and STUHL 1980). 1 group of animals from each category was killed I d after the last injection. The rest of the animals was sacrificed 3 weeks after the last injection, in order to study the reo versibility of the induced effects. The testes were taken out, frozen in liquid nitrogen; and the 8 to 10 f~m cryostat sections were employed for following reactions: a) b) c) d)
PAS reaction, combined with haematoxylin Feulgen reaction, lipid staining with Sudan III/IV, combined with haematoxylin the enzyme histochemical reaction for Mg++.activated mitochondrial adenosine triphosphatase (Mg++.ATPase, E. C. 3.6.1.4), according to the method of MEIJER and VLOEDMAN (1980).
The stages of the spermatogenesis were determined according to the definitions, given by LEBLOND and CLERMONT (1952).
Results Two changes were observed in the process of spermiogenesis: 1. formation of a distinct round or oval vacuole within the acrosomal cap (Plate IV: Fig. 1), and 2. the splitting of the tails of matured sperms (Plate IV: Fig. 2). The cryostat sections, stained with P AS-haematoxylin, revealed that the disturbance started at the phase of acrosomal formation, i.e. the stages IX to XI of the cycle of spermatogenesis: the acrosomal cap was frequently distended and swollen, in the later stages of this phase, i.e. XII to XIV, a small round or oval vacuole was observed in the swollen acrosomal cap. The Feulgen reaction confirmed these changes and showed a distinct disturbance of the nuclear condensation. The sudan III/IV reaction showed an increased number of small round lipid droplets in Sertoli cells (Plate IV: Fig. 1) in the basal compartment of the seminiferous tubules with stages X-XIV of spermatogenesis. The enzyme histochemical reaction of Mg++-ATPase confirmed the observation concerning the splitting of the mature sperms at the stage Vln of the spermatogenesis; the enzymatic activity of the split pieces of the sperm tails disappeared, while the intact tail pieces of the sperms showed a moderate enzymatic activity (Plate IV: Fig. 2). Comparing with controls, the Leydig cells of the treated animals did not show any change in shape, size or appearance. The examination of the cryostat sections, from the animals killed 3 weeks after the last injection, revealed only a partial reversibility of the changes, mentioned above.
Discussion SOC seems to induce malformation in the acrosomal phase of spermatid development; the appearance of a vacuole in the acrosomal cap was most prominent in the stages XIII and XIV of spermatogenesis. The vacuole formation, which perhaps causes the disturbance of the chromatin condensation in the nucleus of the spermatids, cannot be explained yet satisfactorily. Furthermore, the organosilicone SOC probably impairs the energy supply essential for the process of sperm motility, as shown by the disappearance of Mg++.ATPase activity in the split pieces of sperm tails. This finding confirms the results of a previous study from our laboratory on the histophy. siological effects of SOC on rat testis (HAIDER et al. 1983). The deposition of lipid droplets in Sertoli cells, as observed here, has been reported by various authors in cases of pathological changes in the process of spermatogenesis (FLICKINGER and LOVING 1976; HAIDER 1980; VIEHBERGER 1983). The observation, that the Leydig cells remained unaffected by SOC treatment, suggests that the androgen synthesis in the Leydig cells is not disturbed by this organosilicone. The ani· mals, 3 weeks after the SOC treatment, did not show complete recovery; some tubules with cer· tain stages of spermatogenesis (XIII, XIV, VI to VIII) still showed the above mentioned signs of malformation.
Organosilicone induced malformation of rat sperms
151
Acknowledgement The authors thank Ms. GABRIELE BERTHOLD and Ms. MONIKA BAAS EN for their excellent technical assistance.
Literature ARKLES, B., Look what you can make out of silicones. Chern. tech. 13,542-555 (1983). BIRKOFER, L., und STUHL, 0., Siliciumorganische Verbindungen LXXII: Substituierte 1,3Dioxa-2-sila-cyclopentane. J. Organometal. Chern. 187,21-29 (1980). FLICKINGER, C. J., and LOVING, C. K., Fine structure of the testis and epididymis of rats treated with cyproterone acetate. ArneI'. J. Anat. 146,359-384 (1976). HAIDER, S. G., Histophysiological effects of an antiandrogen (cyproterone acetate) on the testis of the frog Rana temporaria. Acta Anat. 106,387-391 (1980). STUHL, 0., GOSLAR, H. G., and BIRKOFER, L., Lethal dose estimation and histophysiological effects of an organosilicon compound on rat kidney, liver and testis. Cell. Mol. BioI. 29, 299306 (1983). LEBLOND, C. P., and CLERMONT, Y., Definition of the stages of the cycle of seminiferous epithelium in the rat. Ann. N. Y. Acad. Sci. 55, 548-573 (1952). :JIEIJER, A. E. F. H., and VLOEDMAN, A. H. T., The histochemical characterization of the coupling state of skeletal muscle mitochondria. Histochemistry 69, 217-232 (1980). VIEHBERGER, G., The Sertoli cell - an essential factor in azoospermia (abstr.). International symposium on vertebrate morphology, Giessen, Aug. 1983; G. Fischer Verlag, Stuttgart (in press). YORONKOV, M. G., ZELCHAX, G. 1., und LUKEVITZ, E., Silizium und Leben; Biochemie, Toxikologie und Pharmakologie del' Verbindungen des Siliziums. Akademie Verlag, 'Berlin 1975, 1-
:no. Author's address: Dr. S. G. HAIDER, Anatomisches Institut (Lehrstuhl II) del' Universitat Diisseldorf, Universitatsstra13e 1, Geb. 22.03, D - 4000 Dusseldorf.
Explanation to Plate IV Fig. 1. A cryostat section of the testis of a rat, treated with the organosilicone SOC. Sudan III/IV reaction counterstained with haematoxylin. Note the vacuole formation (arrow) in the acrosomal cap of the sperm (stage XIII of spermatogenesis). Sertoli cells show increased deposition of lipid droplets in the cytoplasm (red colour). X 1440. Fig. 2. A cryostat section of the testis of a rat, treated with SOC. Enzyme histochemical reaction of Mg++-.\TPase (brown colour) counterstained with haematoxylin (blue colour). Stage VIII of the spermatogenesis. The split pieces of sperm tails lack the enzymatic activity (arrow). X 580.
Acta histochem. Vol. 75
Plate IV
1
2
Haider et al. VEB GUSTAV FISCHER VERLAG JENA
Institute of Anatomy II and Institute of Organic Chemistry, University of Dusseldorf, FRG
Organosilicone induced malformation of rat sperms, a histochemical study!) By SYED G. HAIDER, DIETER PASSIA, OSKAR STUHL, and I . .EoNHARD BIRKOFER With Plate IV (Received January 12, 1984)
Summary The effects of an organosilicon compound SOC, a derivative of 1,3-dioxa-2-sila-cyclopentane, on rat spermiogenesis were studied with the help of histochemical and enzyme histochemical methods. The rats were injected with SOC i.p. daily for 3 weeks (dosage: 25 mg SOC/kg body weight). Two changes were observed: a) formation of a distinct vacuole within the acrosomal cap of spermatids b) the splitting of the tails of mature sperms.
Zusammenfassung Mit Hilfe von histochemischen und enzymhistochemischen Methoden wurden die vVirkungen einer siliciumorganischen Verbindung SOC, ein Derivat des 1,3-Dioxa-2-sila-cyclopentans, auf die Spermiogenese der Ratte untersucht. Die Ratten wurden 3 Wochen lang taglich i.p. mit SOC behandelt (Dosierung: 25 mg SOC/kg Korpergewicht). 2 Veranderungen wurden beobachtet: a) Bildung einer distinkten Vakuole in der acrosomalen Kappe der Spermatiden b) Spaltung der Schwanzstucke der reifen Spermien.
Introduction Presently the organosilicon compounds are gaining importance in the field of environmental research, therapeutic medicine, and biomedical applications (VORONKOV et al. 1975; ARKLES 1983). HAIDER et al. (1983) reported the histophysiological effects of an organosilicone on rat liver, kidney and testis; this compound was prepared by BIRKOFER and STUHL (1980), the complete name: 2,2-dimethyl-4-(chloromethyl)-1,3dioxa-2-sila-cyclopentane, abbreviated as SOC. This compound induced the splitting of the sperm tails, as described by HAIDER et al. (1983). The aim of the present report was to study the effects of SOC on the spermatids and sperms, employing histochemical and enzyme histochemical techniques.
Material and Methods 42 male Wistar rats, with 190 to 200 g body weight, were distributed in 6 groups. 2 groups were injected i.p. daily for 3 weeks with 25 mg SOC/kg body wt, suspended in 0.15 ml olive oil, 1) Dedicated to Professor Dr. HANS Gi;NTER GOSLAR on the occasion of his 65th birthday.
150
S. G. HAIDER et al.
the other 2 groups received the vehicle, and the remaining 2 groups were regarded as untreated controls (for details of the preparation procedure and for the physical data of SOC see BIRKOFER and STUHL 1980). 1 group of animals from each category was killed I d after the last injection. The rest of the animals was sacrificed 3 weeks after the last injection, in order to study the reo versibility of the induced effects. The testes were taken out, frozen in liquid nitrogen; and the 8 to 10 f~m cryostat sections were employed for following reactions: a) b) c) d)
PAS reaction, combined with haematoxylin Feulgen reaction, lipid staining with Sudan III/IV, combined with haematoxylin the enzyme histochemical reaction for Mg++.activated mitochondrial adenosine triphosphatase (Mg++.ATPase, E. C. 3.6.1.4), according to the method of MEIJER and VLOEDMAN (1980).
The stages of the spermatogenesis were determined according to the definitions, given by LEBLOND and CLERMONT (1952).
Results Two changes were observed in the process of spermiogenesis: 1. formation of a distinct round or oval vacuole within the acrosomal cap (Plate IV: Fig. 1), and 2. the splitting of the tails of matured sperms (Plate IV: Fig. 2). The cryostat sections, stained with P AS-haematoxylin, revealed that the disturbance started at the phase of acrosomal formation, i.e. the stages IX to XI of the cycle of spermatogenesis: the acrosomal cap was frequently distended and swollen, in the later stages of this phase, i.e. XII to XIV, a small round or oval vacuole was observed in the swollen acrosomal cap. The Feulgen reaction confirmed these changes and showed a distinct disturbance of the nuclear condensation. The sudan III/IV reaction showed an increased number of small round lipid droplets in Sertoli cells (Plate IV: Fig. 1) in the basal compartment of the seminiferous tubules with stages X-XIV of spermatogenesis. The enzyme histochemical reaction of Mg++-ATPase confirmed the observation concerning the splitting of the mature sperms at the stage Vln of the spermatogenesis; the enzymatic activity of the split pieces of the sperm tails disappeared, while the intact tail pieces of the sperms showed a moderate enzymatic activity (Plate IV: Fig. 2). Comparing with controls, the Leydig cells of the treated animals did not show any change in shape, size or appearance. The examination of the cryostat sections, from the animals killed 3 weeks after the last injection, revealed only a partial reversibility of the changes, mentioned above.
Discussion SOC seems to induce malformation in the acrosomal phase of spermatid development; the appearance of a vacuole in the acrosomal cap was most prominent in the stages XIII and XIV of spermatogenesis. The vacuole formation, which perhaps causes the disturbance of the chromatin condensation in the nucleus of the spermatids, cannot be explained yet satisfactorily. Furthermore, the organosilicone SOC probably impairs the energy supply essential for the process of sperm motility, as shown by the disappearance of Mg++.ATPase activity in the split pieces of sperm tails. This finding confirms the results of a previous study from our laboratory on the histophy. siological effects of SOC on rat testis (HAIDER et al. 1983). The deposition of lipid droplets in Sertoli cells, as observed here, has been reported by various authors in cases of pathological changes in the process of spermatogenesis (FLICKINGER and LOVING 1976; HAIDER 1980; VIEHBERGER 1983). The observation, that the Leydig cells remained unaffected by SOC treatment, suggests that the androgen synthesis in the Leydig cells is not disturbed by this organosilicone. The ani· mals, 3 weeks after the SOC treatment, did not show complete recovery; some tubules with cer· tain stages of spermatogenesis (XIII, XIV, VI to VIII) still showed the above mentioned signs of malformation.
Organosilicone induced malformation of rat sperms
151
Acknowledgement The authors thank Ms. GABRIELE BERTHOLD and Ms. MONIKA BAAS EN for their excellent technical assistance.
Literature ARKLES, B., Look what you can make out of silicones. Chern. tech. 13,542-555 (1983). BIRKOFER, L., und STUHL, 0., Siliciumorganische Verbindungen LXXII: Substituierte 1,3Dioxa-2-sila-cyclopentane. J. Organometal. Chern. 187,21-29 (1980). FLICKINGER, C. J., and LOVING, C. K., Fine structure of the testis and epididymis of rats treated with cyproterone acetate. ArneI'. J. Anat. 146,359-384 (1976). HAIDER, S. G., Histophysiological effects of an antiandrogen (cyproterone acetate) on the testis of the frog Rana temporaria. Acta Anat. 106,387-391 (1980). STUHL, 0., GOSLAR, H. G., and BIRKOFER, L., Lethal dose estimation and histophysiological effects of an organosilicon compound on rat kidney, liver and testis. Cell. Mol. BioI. 29, 299306 (1983). LEBLOND, C. P., and CLERMONT, Y., Definition of the stages of the cycle of seminiferous epithelium in the rat. Ann. N. Y. Acad. Sci. 55, 548-573 (1952). :JIEIJER, A. E. F. H., and VLOEDMAN, A. H. T., The histochemical characterization of the coupling state of skeletal muscle mitochondria. Histochemistry 69, 217-232 (1980). VIEHBERGER, G., The Sertoli cell - an essential factor in azoospermia (abstr.). International symposium on vertebrate morphology, Giessen, Aug. 1983; G. Fischer Verlag, Stuttgart (in press). YORONKOV, M. G., ZELCHAX, G. 1., und LUKEVITZ, E., Silizium und Leben; Biochemie, Toxikologie und Pharmakologie del' Verbindungen des Siliziums. Akademie Verlag, 'Berlin 1975, 1-
:no. Author's address: Dr. S. G. HAIDER, Anatomisches Institut (Lehrstuhl II) del' Universitat Diisseldorf, Universitatsstra13e 1, Geb. 22.03, D - 4000 Dusseldorf.
Explanation to Plate IV Fig. 1. A cryostat section of the testis of a rat, treated with the organosilicone SOC. Sudan III/IV reaction counterstained with haematoxylin. Note the vacuole formation (arrow) in the acrosomal cap of the sperm (stage XIII of spermatogenesis). Sertoli cells show increased deposition of lipid droplets in the cytoplasm (red colour). X 1440. Fig. 2. A cryostat section of the testis of a rat, treated with SOC. Enzyme histochemical reaction of Mg++-.\TPase (brown colour) counterstained with haematoxylin (blue colour). Stage VIII of the spermatogenesis. The split pieces of sperm tails lack the enzymatic activity (arrow). X 580.
Acta histochem. Vol. 75
Plate IV
1
2
Haider et al. VEB GUSTAV FISCHER VERLAG JENA