- Email: [email protected]
Labelling of the chromatoid body by [3H]uridine in rat pachytene spermatocytes
488
Preliminary
notes
centres, since ongoing DNA synthesis accompanied by asymmetric growth of the plastid results in a large organelle with aggregated DNA. I thank Drs S. Bonotto and M. Janowski for critical reading of the text. It would have been impossible to produce figs 2 and 3 without the patient help of Dr W. Baeyens. I thank Mrs Cecile Huysmans for preparing the manuscript. Research was supported by the European Communities and the CENISCK.
References 1. Green, B R, Burton, H, Heilpom, V & Limbosch, S, Biology of Acetabularia (ed J Brachet & S Bonotto) p. 35. Academic Press, New York (1970). 2. Green, B R & Burton, H, Science 168(1970) 981. 3. Woodcock, C L F & Bogorad, L, J cell biol 44 (1970) 361. 4. Bonotto, S, Lurquin, P F, Baeyens, W, Charles, P, Hoursiangou-Neubrun, D, Mazza, A, Tramontano, G & Felluga, B, Protoplasma 83 (1975) 172. 5. Mazza, A, Bonotto, S & Felluga, B, Progress in Acetabularia research (ed C L F Woodcock) p. 123. Academic Press, New York (1977). 6. Mazza, A, Bonotto, S, Felluga, B, Casale, A & Sassone Corsi. P. Develoomental bioloev of Acetabularia (ed S Bonotio, V Kefeli -h S Puiseux-Dao) p. 115. Elsevier/North-Holland Biomedical Press, Amsterdam (1979). 7. Heiluom. V & Limbosch. S. Eur i biochem 22 (197i) 573. 8. Green, B R, Muir, B L & Padmanabhan, U, Progress in Acetabuluria research (ed C L F Woodcock) p. 107. Academic Press, New York (1977). 9. Bedbrook, J R & Kolodner, R, Ann rev plant nhvsiol30 ( 1979)593. 10. Green, B R; Protoplasma 75 (1972) 478. 11. Werz. G & Kellner. G. J ultrastruct res 24 (1968) . , 109. 12. Coleman, A W, J cell bio182 (1979) 299. 13. Bonotto, S, Lurquin, P F & Mazza, A, Adv marine biol 14 (1976) 123. 14. Schnedl, W, Dann, 0 & Schweizer, D, Eur j cell biol20 (1980) 290. 15. Lateur, L, Rev algol 1 (1973) 26. 16. Bonotto, S & Kirchmann, R, Bull sot roy bot belg 103 (1970) 255. 17. Franke, W W, Berger, S, Falk, H, Spring, H, Scheer, U, Herth, W, Trendelenburg, M F & Schweiger, H G, Protoplasma 82 (1974) 249. 18. Shephard, D C, Exp cell res 37 (1965) 93. 19. Puiseux-Dao, S, Acetabufaria and cell biology. Logos Press (1970). 20. Clauss, H, Liittke, A, Hellman, F & Reinert, J, Protoplasma 69 (1970) 313. .
Received October 31, 1980 Accepted October 31, 1980 Exp Cell Res I31 (1981)
Labelling of the chromatoid body by [“Hluridine in rat pachytene spermatocytes KARL-OVE
SGDERSTRGM, Department
ogy, University
of Patholof Turku, SF-20520 Turku 52, Finland
In this study it is shown that a cytoplasmic cell organelle, the chromatoid body, becomes labelled with 13Hluridine in the pachytene spermatocytes. The chromatoid body becomes labelied when the cells are first labelled for 2 h in the presence of r3H]uridine and thereafter chased for 9 h in the presence of unlabelled uridine. This labelling is inhibited by the specific RNA polymerase II inhibitor o-amanitin. Based on this it is suggested that part of the RNA synthesized in the pachytene spermatocytes is stored in the chromatoid body and transported to the postmeiotic spermatids where it is used in the differentiation of the spermatids.
Summary.
Both in male and female germ cells there exist cytoplasmic ultrastructurally similar cell organelles which in male mammals have been called the chromatoid body [5, 63. It is first seen during spermatogenesis as a distinct cell organelle in mid-pachytene stage and is present in the meiotic divisions and in the post-meiotic cells, the spermatids [ 1, 5, 6, 181. The chemical composition as well as the function of this cell organelle are unknown. Previous light microscopic histochemical data suggest that it contains RNA [2, 211, but electron microscopic histochemistry has failed to confirm this [4]. However, high resolution autoradiography has shown that the chromatoid body becomes labelled with [3H]uridine in the spermatids of the rat [20]. Because the chromatoid body is a clearly recognizable cell organelle already in the mid-pachytene spermatocytes its labelling with r3H]uridine has been studied in these cells. Materials
and Methods
The rats were killed by a blow on the head. The tubules of the testes were prepared free and pieces containing a complete seminiferous epithelial cycle were isolated using the transillumination method [19]. Printed
in Sweden
Preliminary
notes
489
pachytene spermatocytes. Furthermore, cY-amanitin is a specific inhibitor of RNA polymerase II [8, 91 which is involved in the synthesis of HnRNA [16, 231. Also the RNA synthesis in the nucleus of the pachyamanitin (Siama,St Louis, MO). Thereafterthe tubules tene spermatocytes differs from that in the were c&&d as described ‘above, except that (Y- somatic cells because most of the RNA amanitin (10 ~&nl) was present in all culture media. formed in the pachytene spermatocytes After the cuitire small samplesof the tubules, the stage of which were accurately identified r191,were seems to be HnRNA [lo, 11, 17, 191. Alcut-and preparedfor electron microscopic&t&adiothough there is some rRNA synthesis in the graphy as describedpreviously [20]. pachytene spermatocytes its amount seems to be very iow [7, 221. Results and Discussion The chromatoid material seen in the The chromatoid body is first seen as a dis- pachytene spermatocytes is evenly distinct cytoplasmic cell organelle in mid- tributed in the four spermatids which are pachytene spermatocytes at stage VII of the derived from it during the meiotic divisions seminiferous epithelium. After 2 h culture [18]. That the chromatoid body becomes with [3H]uridine no label can be seen over labelled with [3H]uridine already in the the chromatoid body in the pachytene pachytene spermatocytes implies that the spermatocytes although the nucleus of the genome of the pachytene spermatocyte is cell is heavily labelled. When the 2 h pulse involved in the differentiation of the haploid is followed by a 9 h chase in the presence spermatid. This has been shown to be the of unlabelled uridine the chromatoid body case in many insects, e.g. Drosophila melabecomes labelled (fig. 1). The labelling of nogaster, where the differentiation of the the chromatoid body can be seen already at male gametes is dependent on the diploid stage VII or as soon as the chromatoid body genome and not on the haploid genotype can be recognized as a distinct cell or- of its own nucleus [ 131.Although there is a ganelle in the cytoplasm of the pachytene low level of RNA synthesis in the early round nucleated spermatids of the rat [12, spermatocytes and it persists up to late pachytene. After the chase the nucleus of 15, 201, it is not known whether this RNA the pachytene spermatocyte is clearly less synthesis is sufficient to direct the differlabelled than after the 2 h pulse but at no entiation of the spermatid after RNA synthesis has ceased in step 8 spermatids. At time is there any significant cytoplasmic labelling, except at the chromatoid body. least it is well known that after RNA synWhen the tubules were preincubated with thesis has ceased in step 8 spermatids, proa-amanitin (10 pg/ml) 2 h before the la- tein synthesis is still at a high level in the belling with [3H]uridine no grains could be spermatids for 2 weeks [3]. This protein seen over the chromatoid body even after synthesis obviously requires the existence long incubation times in the presence of of long-lived mRNA molecules, some of which might be stored in the chromatoid unlabelled uridine. body. That the chromatoid body becomes labelAs has been pointed out by Fawcett [5] led with [3H]uridine and that this labelling cytoplasmic structures resembling ultrais inhibited by a-amanitin suggests that the chromatoid body contains RNA in the structurally the chromatoid body of mam-
The isolated sernnents of the tubules were incubated in 100 ~1 of Gker 199 culture media containing 10 uCi of r3Hluridine for 2 h as described nreviouslv i19]. Thkre&er the tubules were cultured’in Parke; 199 culture media containing 10 pi/ml of unlabelled uridine for 2, 4, 6, 9, 12 or 18 h. In the control exoeriments the tubules were first cultured for 2 h in Parker 199 culture media containing 10 pi/ml of (Y-
Exp Cell Res 131 (1981~
490
Preliminary
notes
Preliminary
malian spermatids exist in oocytes of many insects. These are the polar granules which have been extensively investigated and have their origin in the nurse cells. Here the RNA after combining with a protein component moves into the oocyte cytoplasm. The polar granules of the oocyte and the mitochondria form contacts which in appearance closely resemble those between the chromatoid material and mitochondria in early pachytene spermatocytes [5, 181. After fertilization the polar granules are known to be transported to the pole cells leading to the germ cell line of the embryo, During this cellular differentiation process the polar granules make contacts with ribosomes and probably mobilize their stored RNA [ 141. This suggestion for the function of the polar granules has many common features with the one suggested for the chromatoid body in this paper. Obviously if homology exists between the polar granules and the chromatoid body it will be revealed only by careful investigations of the functions of these cell organelles.
notes
491
9. Jacob, S T, Sajdel, E M, Muecke, W & Munro, H N, Cold Spring Harbor symp quant biol 35 (1970) 681. 10. Kierszenbaum, A L & Tres, L L, J cell biol 60 (1974) 39. 11. - Ibid 63 (1975) 923. 12. - Ibid 65 (1975) 258. 13. Lindsley, D L & Grell, E H, Genetics 61, suppl. 1 (1969) 69. 14. Mahowald, A P, Results and problems in cell differentiation (ed J Reinert L H Ursprung) vol. 2, p. 158. Springer-Verlag, Berlin (1971). 15. Monesi, V, J cell biol22 (1964) 521. 16. Roeder, R G & Rutter, W J, Proc natl acad sci US 65 (1970) 675. 17. Soderstriim, K-O, Exp cell res 102 (1976) 237. 18. - Z mikrosk-anat Forsch 3 (1978) 417. 19. Soderstrom, K-O & Parvinen, M, Mol cell endocrinol 5 (1976) 181.
20. - J ce’1biol 7o(!976)239. 21. Sud, B N, QuartJ microsc sci 102(1961) 495. 22. Stefanini, M, dehlartino, C, Agostino, A D, Agrestini, A & Monesi, V, Exp cell res 86 (1974) 166. 23. Zylber, E & Penman, S, Proc natl acad sci US 68 (1971) 2861.
ReceivedAugust27 1980 Revised version received November 19, 1980
AcceptedNovember19v19*0
References’ 1. Comings, D E & Okada, T A, J ultrastruct res 39 (1972) 15. 2. Daoust, R & Clermont, Y, Am j anat % (1955)255. 3. Davis, J R & Firlit, C F, Am j physiol 209 (1965) 425. 4. Eddy, E M, Biol reprod 2 (1970) 114. 5. Fawcett, D W, The genetics of the snermatozoon (ed R A Beatty & S-Gluecksohn-Waelsch) p. 37. Edinburgh (1972). 6. Fawcett, D W, Eddy, E M & Phillips, D M, Biol reprod 2 (1970) 129. 7. Galdieri, M & Monesi, V, Exp cell res 85 (1974) 287. 8. Jacob, S T, Sajdel, E M & Munro, H N, Biochem biophys res commun 38 (1970) 765. Fig. I. Autoradiogram of a mid-pachytene spermatocyte from stage VIII of spermatogenesis. The tubules have been labelled with [3H]uridine for 2 h and thereafter chased in the presence of unlabelled uridine for 9 h. In the nucleus (n) a few grains can be seen and the typically vesicular chromatoid body (c) is also labelled. No grains are seen elsewhere in the cytoplasm. x30000. Printed
in Sweden
Exp Cell Res I31 (1981)
Preliminary
notes
centres, since ongoing DNA synthesis accompanied by asymmetric growth of the plastid results in a large organelle with aggregated DNA. I thank Drs S. Bonotto and M. Janowski for critical reading of the text. It would have been impossible to produce figs 2 and 3 without the patient help of Dr W. Baeyens. I thank Mrs Cecile Huysmans for preparing the manuscript. Research was supported by the European Communities and the CENISCK.
References 1. Green, B R, Burton, H, Heilpom, V & Limbosch, S, Biology of Acetabularia (ed J Brachet & S Bonotto) p. 35. Academic Press, New York (1970). 2. Green, B R & Burton, H, Science 168(1970) 981. 3. Woodcock, C L F & Bogorad, L, J cell biol 44 (1970) 361. 4. Bonotto, S, Lurquin, P F, Baeyens, W, Charles, P, Hoursiangou-Neubrun, D, Mazza, A, Tramontano, G & Felluga, B, Protoplasma 83 (1975) 172. 5. Mazza, A, Bonotto, S & Felluga, B, Progress in Acetabularia research (ed C L F Woodcock) p. 123. Academic Press, New York (1977). 6. Mazza, A, Bonotto, S, Felluga, B, Casale, A & Sassone Corsi. P. Develoomental bioloev of Acetabularia (ed S Bonotio, V Kefeli -h S Puiseux-Dao) p. 115. Elsevier/North-Holland Biomedical Press, Amsterdam (1979). 7. Heiluom. V & Limbosch. S. Eur i biochem 22 (197i) 573. 8. Green, B R, Muir, B L & Padmanabhan, U, Progress in Acetabuluria research (ed C L F Woodcock) p. 107. Academic Press, New York (1977). 9. Bedbrook, J R & Kolodner, R, Ann rev plant nhvsiol30 ( 1979)593. 10. Green, B R; Protoplasma 75 (1972) 478. 11. Werz. G & Kellner. G. J ultrastruct res 24 (1968) . , 109. 12. Coleman, A W, J cell bio182 (1979) 299. 13. Bonotto, S, Lurquin, P F & Mazza, A, Adv marine biol 14 (1976) 123. 14. Schnedl, W, Dann, 0 & Schweizer, D, Eur j cell biol20 (1980) 290. 15. Lateur, L, Rev algol 1 (1973) 26. 16. Bonotto, S & Kirchmann, R, Bull sot roy bot belg 103 (1970) 255. 17. Franke, W W, Berger, S, Falk, H, Spring, H, Scheer, U, Herth, W, Trendelenburg, M F & Schweiger, H G, Protoplasma 82 (1974) 249. 18. Shephard, D C, Exp cell res 37 (1965) 93. 19. Puiseux-Dao, S, Acetabufaria and cell biology. Logos Press (1970). 20. Clauss, H, Liittke, A, Hellman, F & Reinert, J, Protoplasma 69 (1970) 313. .
Received October 31, 1980 Accepted October 31, 1980 Exp Cell Res I31 (1981)
Labelling of the chromatoid body by [“Hluridine in rat pachytene spermatocytes KARL-OVE
SGDERSTRGM, Department
ogy, University
of Patholof Turku, SF-20520 Turku 52, Finland
In this study it is shown that a cytoplasmic cell organelle, the chromatoid body, becomes labelled with 13Hluridine in the pachytene spermatocytes. The chromatoid body becomes labelied when the cells are first labelled for 2 h in the presence of r3H]uridine and thereafter chased for 9 h in the presence of unlabelled uridine. This labelling is inhibited by the specific RNA polymerase II inhibitor o-amanitin. Based on this it is suggested that part of the RNA synthesized in the pachytene spermatocytes is stored in the chromatoid body and transported to the postmeiotic spermatids where it is used in the differentiation of the spermatids.
Summary.
Both in male and female germ cells there exist cytoplasmic ultrastructurally similar cell organelles which in male mammals have been called the chromatoid body [5, 63. It is first seen during spermatogenesis as a distinct cell organelle in mid-pachytene stage and is present in the meiotic divisions and in the post-meiotic cells, the spermatids [ 1, 5, 6, 181. The chemical composition as well as the function of this cell organelle are unknown. Previous light microscopic histochemical data suggest that it contains RNA [2, 211, but electron microscopic histochemistry has failed to confirm this [4]. However, high resolution autoradiography has shown that the chromatoid body becomes labelled with [3H]uridine in the spermatids of the rat [20]. Because the chromatoid body is a clearly recognizable cell organelle already in the mid-pachytene spermatocytes its labelling with r3H]uridine has been studied in these cells. Materials
and Methods
The rats were killed by a blow on the head. The tubules of the testes were prepared free and pieces containing a complete seminiferous epithelial cycle were isolated using the transillumination method [19]. Printed
in Sweden
Preliminary
notes
489
pachytene spermatocytes. Furthermore, cY-amanitin is a specific inhibitor of RNA polymerase II [8, 91 which is involved in the synthesis of HnRNA [16, 231. Also the RNA synthesis in the nucleus of the pachyamanitin (Siama,St Louis, MO). Thereafterthe tubules tene spermatocytes differs from that in the were c&&d as described ‘above, except that (Y- somatic cells because most of the RNA amanitin (10 ~&nl) was present in all culture media. formed in the pachytene spermatocytes After the cuitire small samplesof the tubules, the stage of which were accurately identified r191,were seems to be HnRNA [lo, 11, 17, 191. Alcut-and preparedfor electron microscopic&t&adiothough there is some rRNA synthesis in the graphy as describedpreviously [20]. pachytene spermatocytes its amount seems to be very iow [7, 221. Results and Discussion The chromatoid material seen in the The chromatoid body is first seen as a dis- pachytene spermatocytes is evenly distinct cytoplasmic cell organelle in mid- tributed in the four spermatids which are pachytene spermatocytes at stage VII of the derived from it during the meiotic divisions seminiferous epithelium. After 2 h culture [18]. That the chromatoid body becomes with [3H]uridine no label can be seen over labelled with [3H]uridine already in the the chromatoid body in the pachytene pachytene spermatocytes implies that the spermatocytes although the nucleus of the genome of the pachytene spermatocyte is cell is heavily labelled. When the 2 h pulse involved in the differentiation of the haploid is followed by a 9 h chase in the presence spermatid. This has been shown to be the of unlabelled uridine the chromatoid body case in many insects, e.g. Drosophila melabecomes labelled (fig. 1). The labelling of nogaster, where the differentiation of the the chromatoid body can be seen already at male gametes is dependent on the diploid stage VII or as soon as the chromatoid body genome and not on the haploid genotype can be recognized as a distinct cell or- of its own nucleus [ 131.Although there is a ganelle in the cytoplasm of the pachytene low level of RNA synthesis in the early round nucleated spermatids of the rat [12, spermatocytes and it persists up to late pachytene. After the chase the nucleus of 15, 201, it is not known whether this RNA the pachytene spermatocyte is clearly less synthesis is sufficient to direct the differlabelled than after the 2 h pulse but at no entiation of the spermatid after RNA synthesis has ceased in step 8 spermatids. At time is there any significant cytoplasmic labelling, except at the chromatoid body. least it is well known that after RNA synWhen the tubules were preincubated with thesis has ceased in step 8 spermatids, proa-amanitin (10 pg/ml) 2 h before the la- tein synthesis is still at a high level in the belling with [3H]uridine no grains could be spermatids for 2 weeks [3]. This protein seen over the chromatoid body even after synthesis obviously requires the existence long incubation times in the presence of of long-lived mRNA molecules, some of which might be stored in the chromatoid unlabelled uridine. body. That the chromatoid body becomes labelAs has been pointed out by Fawcett [5] led with [3H]uridine and that this labelling cytoplasmic structures resembling ultrais inhibited by a-amanitin suggests that the chromatoid body contains RNA in the structurally the chromatoid body of mam-
The isolated sernnents of the tubules were incubated in 100 ~1 of Gker 199 culture media containing 10 uCi of r3Hluridine for 2 h as described nreviouslv i19]. Thkre&er the tubules were cultured’in Parke; 199 culture media containing 10 pi/ml of unlabelled uridine for 2, 4, 6, 9, 12 or 18 h. In the control exoeriments the tubules were first cultured for 2 h in Parker 199 culture media containing 10 pi/ml of (Y-
Exp Cell Res 131 (1981~
490
Preliminary
notes
Preliminary
malian spermatids exist in oocytes of many insects. These are the polar granules which have been extensively investigated and have their origin in the nurse cells. Here the RNA after combining with a protein component moves into the oocyte cytoplasm. The polar granules of the oocyte and the mitochondria form contacts which in appearance closely resemble those between the chromatoid material and mitochondria in early pachytene spermatocytes [5, 181. After fertilization the polar granules are known to be transported to the pole cells leading to the germ cell line of the embryo, During this cellular differentiation process the polar granules make contacts with ribosomes and probably mobilize their stored RNA [ 141. This suggestion for the function of the polar granules has many common features with the one suggested for the chromatoid body in this paper. Obviously if homology exists between the polar granules and the chromatoid body it will be revealed only by careful investigations of the functions of these cell organelles.
notes
491
9. Jacob, S T, Sajdel, E M, Muecke, W & Munro, H N, Cold Spring Harbor symp quant biol 35 (1970) 681. 10. Kierszenbaum, A L & Tres, L L, J cell biol 60 (1974) 39. 11. - Ibid 63 (1975) 923. 12. - Ibid 65 (1975) 258. 13. Lindsley, D L & Grell, E H, Genetics 61, suppl. 1 (1969) 69. 14. Mahowald, A P, Results and problems in cell differentiation (ed J Reinert L H Ursprung) vol. 2, p. 158. Springer-Verlag, Berlin (1971). 15. Monesi, V, J cell biol22 (1964) 521. 16. Roeder, R G & Rutter, W J, Proc natl acad sci US 65 (1970) 675. 17. Soderstriim, K-O, Exp cell res 102 (1976) 237. 18. - Z mikrosk-anat Forsch 3 (1978) 417. 19. Soderstrom, K-O & Parvinen, M, Mol cell endocrinol 5 (1976) 181.
20. - J ce’1biol 7o(!976)239. 21. Sud, B N, QuartJ microsc sci 102(1961) 495. 22. Stefanini, M, dehlartino, C, Agostino, A D, Agrestini, A & Monesi, V, Exp cell res 86 (1974) 166. 23. Zylber, E & Penman, S, Proc natl acad sci US 68 (1971) 2861.
ReceivedAugust27 1980 Revised version received November 19, 1980
AcceptedNovember19v19*0
References’ 1. Comings, D E & Okada, T A, J ultrastruct res 39 (1972) 15. 2. Daoust, R & Clermont, Y, Am j anat % (1955)255. 3. Davis, J R & Firlit, C F, Am j physiol 209 (1965) 425. 4. Eddy, E M, Biol reprod 2 (1970) 114. 5. Fawcett, D W, The genetics of the snermatozoon (ed R A Beatty & S-Gluecksohn-Waelsch) p. 37. Edinburgh (1972). 6. Fawcett, D W, Eddy, E M & Phillips, D M, Biol reprod 2 (1970) 129. 7. Galdieri, M & Monesi, V, Exp cell res 85 (1974) 287. 8. Jacob, S T, Sajdel, E M & Munro, H N, Biochem biophys res commun 38 (1970) 765. Fig. I. Autoradiogram of a mid-pachytene spermatocyte from stage VIII of spermatogenesis. The tubules have been labelled with [3H]uridine for 2 h and thereafter chased in the presence of unlabelled uridine for 9 h. In the nucleus (n) a few grains can be seen and the typically vesicular chromatoid body (c) is also labelled. No grains are seen elsewhere in the cytoplasm. x30000. Printed
in Sweden
Exp Cell Res I31 (1981)