Hereditas 82: 25---28 (1976)
DNA synthesis during male meiotic prophase in the rat KARL-OVE SODERSTROM and MARTTI PARVINEN Institute of Biomedicine, Department of Anatomy, University o j Turku, Finland
S ~ D E R S T RK~ .M - 0, . and PARVINEN, M. 1976. D N A synthesis during male meiotic prophase in the rat. - Hereditas 82: 25-28. Lund, Sweden. ISSN 0018-0661. Received November 12. 1975 DNA synthesis in different stages of the male meiotic prophase of the rat was studied by autoradiography after incubation of small segments of isolated seminiferous tubules with tritiated thymidine. Compared t o incubation of cell suspensions, this technique gives the advantage that the physiological contacts between different cells are preserved, which diminishes the damage of the cells and their DNA. In addition, the recognition accuracy of the meiotic stages is increased as the morphology of the developing acrosomes can be used as an additional criterium for identification of the stages of the spermatogenic epithelial cycle. Highest synthetic rate was found in the mid-pachytene spermatocytes, while moderate o r low rate was observed in leptotene, zygotene, early pachytene, late pachytene and diplotene stages. Martti Parvinen, Instirule of Biomedicine, Department of Anatomy, University of Turku. SF-20520 Turku 52, Finland
It is generally known that the meiotic DNA is synthesized principally in the preleptotene spermatocytes, shortly before the beginning of the meiotic prophase. However, it has been shown in a lily that about 0.4% of the total DNA is synthesized later during the zygotene and pachytene stages of meiosis (HOTTA,et al. 1966). Some controversy still exists, however, about the existence or distribution of DNA synthesis during meiotic prophase in different animal species. On the basis of autoradiographic analysis MUKHERJEE and COHEN(1968) have reported such a synthesis to exist in the mouse, but several other investigators have failed to find such evidence (MONESI1962; CLERMONT and TROTT1969; ODARTCHENKO and PAVILLARD 1970; KOFMAN-ALFARO and CHANDLEY 1970). If in vitro incubations are used, sufficient amounts of radioactivity can be introduced into the cells for detection of the meiotic DNA synthesis by autoradiography (KOFMAN-ALFARO and CHANDLEY 1971; LIMA-DE-FARIA et al. 1968). In these studies, testicular cell suspensions have been incubated in the presence of radioactive DNA precursors. During the preparation of the suspension, the germinal cells loose their normal contacts with the Sertoli cells and their metabolism and possibly also DNA may be damaged.
Therefore, the incorporation of radioactivity may only reflect the repair of this damage and not a physiological DNA synthesis. Another difficulty associated to the use of cell suspensions is that the exact identification of the different stages of meiosis is based only on the morphology of the chromosomes. We have therefore used a different approach of tissue preparation for studies of DNA synthesis during meiotic prophase. Instead of a cell suspension, we have incubated segments of rat seminiferous tubules containing accurately identified cell types with tritiated thymidine. The spermatogenic cells remain in contact with the Sertoli cells throughout the experiment and the damage to the cells is as small as possible. The identification accuracy of the meiotic stages is increased when the morphology of the developing spermatids belonging to the same cellular associations is used as an additional criterium.
Material and methods Cell separation. - For obtaining accurately identified cells of the meiotic prophase, the seminiferous
26
K . - 0 . Sijl)l R S I R 6 M ANI) M. PARVINEN
HOOO v)
3 W J U 3 2 \
In
za
(L
0
60
50
40
30
20
10.
-
-
I
E X X I H Z ~ I X ~jjYB~ E Y i i V j % E X i & J j i j t---L-+t---Z-t P 11-oi Fig. I The D N A synthetic activity during the male meiotic prophase o f t h e rat. The ordinate shows the number of the grains counted per nuclei. On the abscissa the stage\ o f the seminiferous epithelial wave and of the meiotic prophase are indicated. L = Lcptotene. Z = Zbgotene. P = Pachytens. D = Diplotene. The standard debiation o f the grain count\ are indicated by solid circles (nuclear grain counts) or open circles (background). The highest D N A synthesis occurs in pachytene sperinatocytes at stages VI I X .
tubules were isolated from the interstitial tissue by a and MASON technique described by CHRISTENSEN (196s). The individual tubules were observed in a preparation microscope and the exact stage of the seminiferous epithelial cycle (LEBLOND and CLRRMONT 1952) was determined by the transillumination technique ( PARVINEN and VANHA-PERTTULA 1972) combined with phase contrast microscopy (SODERSTROM and PARVINEN 1976) of living unstained cells, Each of the stages contain a constant cellular association and the morphology of the developing acrosomes of the spermatids helps greatly in the recognition of the meiotic prophase stages. 2 mm long pieces of seminiferous tubules containing identified cells were cut and the DNA synthesis of this segment was studied. Luhding conditions. - The tubular segments were incubated in 100 pI of Krebs-Ringer glucose solution
containing 10 pCi of methyl-'H-thymidine. After 4 hours of incubation at 31'C in a 5'5;, CO, and 95?, 0, atmosphere the tubules were carefully washed and fixed in Bouin's fluid. Au/orudiogruphj. - The tubules were embedded in paraffin and cut at 5 pm. The autoradiography was performed with the dipping technique using Kodak NTB-3 emulsion. The exposure time was 1 or 4 weeks and the grains over the nuclei were counted at a 1000-fold magnification. In all stages. 25 nuclei of meiotic prophase cells were counted. In each case, the background level was counted over the late spermatids from an area corresponding to the size of the spermatocyte nuclei.
Hereditus 82 (1976)
DNA SYNTHESIS IN MALE RAT
27
4 Fig. 2-4. - Fig. 2. Autoradiograph of the stage XI1 of rat spermatogenesis after incubation of a tubular fragment with 3H-thymidine. Note the increased grain density over the zygotene spermatocytes (arrow). - Fig. 3. Labelling of the stage V after incubation with 3H-thymidine. Early pachytene spermatocytes (arrow) are clearly labelled. Fig. 4. Labelling of the mid-pachytene spermatocytes at stage IX after 3H-thymidine incubation (arrow). The preleptotene spermatocytes at the bottom row are heavily labelled. ~
Results Fig. 1 shows the level of DNA synthesis in the different cell types of the meiotic prophase belonging to the various stages of the seminiferous epithelial cycle. The highest rate of DNA synthesis can be observed in the mid-pachytene spermatocytes in stages VII-Iy, whereas the level of DNA synthesis is lower in early (stages I-VI) and late pachytene spermatocytes (belonging to stages X-XU). Also in the leptotene, zygotene and diplotene stages there is a low level of DNA synthesis, which is, however, significantly higher than the background. This can be seen in Fig. 2 showing the labelling of stage XI1 late pachytene and zygotene spermatocytes.
Fig. 3 shows the relatively low level of DNA synthesis of early pachytene spermatocytes (stage V) and in Fig. 4 from stage IX the rather high level of DNA synthesis of the mid-pachytene spermatocytes is seen. In this stage the preleptotene spermatocytes have a high rate of DNA synthesis.
Discussion Our results support the evidence obtained by other techniques that there exists a low level of DNA synthesis during the meiotic prophase in animals. We have been able to localize a definite peak of
28
Heredirus 82 (1976)
K.-O. SODERSTROM A N D M. PARVINEN
in the evaluation of the genetic toxicity of different DNA synthesis in mid-pachytene spermatocytes chemical compounds. (spermatogenic stages VII-IX). This is somewhat different from the results of KOFMAN-ALFARO and Acknou/rdgmenls, This work was financially supported by CHANDLEY (1971), who noted the highest activity in the Finnish National Research Council for Medical Sciences. early pachytene spermatocytes, using in vitro labelling The authors are grateful to Prof. A. Lima-de-Faria for of testicular cell suspensions of the mouse. stimulating discussions and to Mrs. Leena Simola and Raija Since the mammalian germinal cells fail to differAndersen for excellent technical assistance. entiate through the meiotic prophase in the cell culture, but do so in organ culture (STEINBERGER et al. Literature cited 1970), it is possible that the incubation conditions BRYANT, B. J. 1966. Incorporation of tritium from thymidine with normal contacts of the germ cells with the into proteins of the mouse. ~.J . c'cdl Biol. 29: 29-36 Sertoli cells and each other may give more reliable CHANDLEY, A. C. and KOtMAN-AI.CARO. s. 1971. "Unscheresults resembling more those existing in vivo. duled" DNA synthesis in human germ cells following U.V. irradiation. -- E.rp. Cell R w . 6Y: 45-48 The autoradiographic analyses of in vivo DNA CHRISTENSEN, A. K. and MASON,N. R. 1965. Comparative synthesis during the meiotic prophase have given ability of seminiferous tubules and interstitial tissue of rat conflicting results, and in most experiments the level testes to synthesize androgens from progesterone-4-'"C in of the radioactivity has been so low that accurate Endocrinology 76: 646 - . 656 vitro. Y. and TROTT,M. 1969. Duration of the cycle CLERMONT. grain counting has been difficult. Recently, a bioof the seminiferous epithelium in the mouse and hamster chemical determination of highly purified epididymal determined by means of 'H-thymidine and radioautography. spermatozoan nuclei has been made by MEETRICH - Ferri/irj Steriliry 5: 805 -8 I7 and coworkers ( 1975). They injected tritiated thymiGLF.DHILL. B. L. and DARSZYNKIEWICZ. Z . 1973. Unscheduled synthesis of D N A during mammalian spermatogenesis in dine intratesticularly and analyzed the epididymal J . E.vp. Zoo/. 183: 375-382 response to UV irradiation. spermatozoa after given intervals. Even in this HOTTA,Y., 170, M. and S T ~ R NH.. 1966. Synthesis of DNA study, the level of radioactivity incorporated to the during meiosis. - Proc. NUI. Acud. Sci. 56: 1184-1 191 DNA during the meiotic prophase was near the level KOFMAN-ALFARO, S. and CHANDLIY. A. C. 1970. Meiosis in the Chromomale mouse. An autoradiographic investigation. of detectability, although the liquid scintillation .soniu 31: 404-420 counting in this case is more sensitive than autoS. and CHANIXEY. A. C. 1971. RadiationKOFMAN-AL~ARO. radiography. No clear peak correlating to any given initiated D N A synthesis in spermatogenic cells of the stage of meiotic prophase was observed. E.vp. Cell R e s . 6Y: 33 44 mouse. C. P. and CLERMONT. Y . 1952. Definition of the LEBI.OND, A source of error in our study may be the stages of the cycle of the seminiferous epithelium in the possible incorporation of tritium label from thymiAnn. N. Y . Acud. Sci. 55: 548-573 rat. dine into proteins (BRYANT 1966). This may explain LIMA-DE-FARIA. A,, GERMAN. J..GHATNLKAR, M., MCGOVERN. the relatively high background levels observed in J . and ANDERSON. L. 1968. In vitro labelling of human meiotic chromosomes with "H-thymidine. - Hereditus 60: our study, where samples were incubated in rather 249- 26 1 high specific radioactivity of the medium. We have MEISTRICH, M . L.. REID,8. 0. and BARCELLONA, W. J. 1975. reduced this source of error by counting the backMeiotic D N A synthesis during mouse spermatogenesis. ground values in each case over late spermatids, J . Crll Biol. 64: 21 1-222 MONESI. V. 1962. Autoradiographic study of DNA synthesis having absolutely no DNA synthesis, but an active and the cell cycle in spermatogonia and spermatocytes of protein synthesis. mouse testis using tritiated thymidine. -~~J . Cell Biol. 14: The biological significance of the DNA synthesis I - ~ l 8 during the meiotic prophase remains to be solved in MVKHERJEE, A. B. and COHEN, M . M. 1968. D N A synthesis during meiotic prophase in male mice. - Nurure 2 f 9 : detail. It has been suggested that the DNA synthesis 489 -490 during leptotene and zygotene is a delayed replication M. 1970. Late DNA O D A R T C H ~ N KNO. ,and PAVILLARD, of definite chromosomal regions involved in chroScience replication in male mouse meiotic chromosomes. mosomal pairing and that the DNA synthesis during 167: 1133-1134 T. 1972. Identification P A R V I N ~M. N . and VANHA-PERTTULA, pachytene might represent the repair of DNA and enzyme quantitation of the stages of the seminiferous breaks associated with crossing-over or spontaneous epithelial wave in the rat. Anur. Rec. 174: 435-450 mutations. Repair synthesis of DNA during meiosis E., STEINBERCER, A. and FITHER, M. 1970. STHNBERGER, has been described after treatment with certain Study of spermatogenesis and steroid metabolism in cultures Rec. Progr. Hormone Res. 26: mutagenic agents (CHANDLEY and KOFMAN-ALFARO of mammalian testes. 547-- 588 1971; GLEDHILL and DARSZYNKIEWICZ 1973). Our SBDERSTRAM, K . - 0 . and PARVINEN. M. 1976. R N A synthesis method gives further possibilities for analyzing the in different stages of rat seminiferous epithelial cycle. "unscheduled" DNA synthesis at different accurately Mol. Cell. Endocrinol. (in press) defined stages of meiosis, which has a significant value -
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~
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DNA synthesis during male meiotic prophase in the rat KARL-OVE SODERSTROM and MARTTI PARVINEN Institute of Biomedicine, Department of Anatomy, University o j Turku, Finland
S ~ D E R S T RK~ .M - 0, . and PARVINEN, M. 1976. D N A synthesis during male meiotic prophase in the rat. - Hereditas 82: 25-28. Lund, Sweden. ISSN 0018-0661. Received November 12. 1975 DNA synthesis in different stages of the male meiotic prophase of the rat was studied by autoradiography after incubation of small segments of isolated seminiferous tubules with tritiated thymidine. Compared t o incubation of cell suspensions, this technique gives the advantage that the physiological contacts between different cells are preserved, which diminishes the damage of the cells and their DNA. In addition, the recognition accuracy of the meiotic stages is increased as the morphology of the developing acrosomes can be used as an additional criterium for identification of the stages of the spermatogenic epithelial cycle. Highest synthetic rate was found in the mid-pachytene spermatocytes, while moderate o r low rate was observed in leptotene, zygotene, early pachytene, late pachytene and diplotene stages. Martti Parvinen, Instirule of Biomedicine, Department of Anatomy, University of Turku. SF-20520 Turku 52, Finland
It is generally known that the meiotic DNA is synthesized principally in the preleptotene spermatocytes, shortly before the beginning of the meiotic prophase. However, it has been shown in a lily that about 0.4% of the total DNA is synthesized later during the zygotene and pachytene stages of meiosis (HOTTA,et al. 1966). Some controversy still exists, however, about the existence or distribution of DNA synthesis during meiotic prophase in different animal species. On the basis of autoradiographic analysis MUKHERJEE and COHEN(1968) have reported such a synthesis to exist in the mouse, but several other investigators have failed to find such evidence (MONESI1962; CLERMONT and TROTT1969; ODARTCHENKO and PAVILLARD 1970; KOFMAN-ALFARO and CHANDLEY 1970). If in vitro incubations are used, sufficient amounts of radioactivity can be introduced into the cells for detection of the meiotic DNA synthesis by autoradiography (KOFMAN-ALFARO and CHANDLEY 1971; LIMA-DE-FARIA et al. 1968). In these studies, testicular cell suspensions have been incubated in the presence of radioactive DNA precursors. During the preparation of the suspension, the germinal cells loose their normal contacts with the Sertoli cells and their metabolism and possibly also DNA may be damaged.
Therefore, the incorporation of radioactivity may only reflect the repair of this damage and not a physiological DNA synthesis. Another difficulty associated to the use of cell suspensions is that the exact identification of the different stages of meiosis is based only on the morphology of the chromosomes. We have therefore used a different approach of tissue preparation for studies of DNA synthesis during meiotic prophase. Instead of a cell suspension, we have incubated segments of rat seminiferous tubules containing accurately identified cell types with tritiated thymidine. The spermatogenic cells remain in contact with the Sertoli cells throughout the experiment and the damage to the cells is as small as possible. The identification accuracy of the meiotic stages is increased when the morphology of the developing spermatids belonging to the same cellular associations is used as an additional criterium.
Material and methods Cell separation. - For obtaining accurately identified cells of the meiotic prophase, the seminiferous
26
K . - 0 . Sijl)l R S I R 6 M ANI) M. PARVINEN
HOOO v)
3 W J U 3 2 \
In
za
(L
0
60
50
40
30
20
10.
-
-
I
E X X I H Z ~ I X ~jjYB~ E Y i i V j % E X i & J j i j t---L-+t---Z-t P 11-oi Fig. I The D N A synthetic activity during the male meiotic prophase o f t h e rat. The ordinate shows the number of the grains counted per nuclei. On the abscissa the stage\ o f the seminiferous epithelial wave and of the meiotic prophase are indicated. L = Lcptotene. Z = Zbgotene. P = Pachytens. D = Diplotene. The standard debiation o f the grain count\ are indicated by solid circles (nuclear grain counts) or open circles (background). The highest D N A synthesis occurs in pachytene sperinatocytes at stages VI I X .
tubules were isolated from the interstitial tissue by a and MASON technique described by CHRISTENSEN (196s). The individual tubules were observed in a preparation microscope and the exact stage of the seminiferous epithelial cycle (LEBLOND and CLRRMONT 1952) was determined by the transillumination technique ( PARVINEN and VANHA-PERTTULA 1972) combined with phase contrast microscopy (SODERSTROM and PARVINEN 1976) of living unstained cells, Each of the stages contain a constant cellular association and the morphology of the developing acrosomes of the spermatids helps greatly in the recognition of the meiotic prophase stages. 2 mm long pieces of seminiferous tubules containing identified cells were cut and the DNA synthesis of this segment was studied. Luhding conditions. - The tubular segments were incubated in 100 pI of Krebs-Ringer glucose solution
containing 10 pCi of methyl-'H-thymidine. After 4 hours of incubation at 31'C in a 5'5;, CO, and 95?, 0, atmosphere the tubules were carefully washed and fixed in Bouin's fluid. Au/orudiogruphj. - The tubules were embedded in paraffin and cut at 5 pm. The autoradiography was performed with the dipping technique using Kodak NTB-3 emulsion. The exposure time was 1 or 4 weeks and the grains over the nuclei were counted at a 1000-fold magnification. In all stages. 25 nuclei of meiotic prophase cells were counted. In each case, the background level was counted over the late spermatids from an area corresponding to the size of the spermatocyte nuclei.
Hereditus 82 (1976)
DNA SYNTHESIS IN MALE RAT
27
4 Fig. 2-4. - Fig. 2. Autoradiograph of the stage XI1 of rat spermatogenesis after incubation of a tubular fragment with 3H-thymidine. Note the increased grain density over the zygotene spermatocytes (arrow). - Fig. 3. Labelling of the stage V after incubation with 3H-thymidine. Early pachytene spermatocytes (arrow) are clearly labelled. Fig. 4. Labelling of the mid-pachytene spermatocytes at stage IX after 3H-thymidine incubation (arrow). The preleptotene spermatocytes at the bottom row are heavily labelled. ~
Results Fig. 1 shows the level of DNA synthesis in the different cell types of the meiotic prophase belonging to the various stages of the seminiferous epithelial cycle. The highest rate of DNA synthesis can be observed in the mid-pachytene spermatocytes in stages VII-Iy, whereas the level of DNA synthesis is lower in early (stages I-VI) and late pachytene spermatocytes (belonging to stages X-XU). Also in the leptotene, zygotene and diplotene stages there is a low level of DNA synthesis, which is, however, significantly higher than the background. This can be seen in Fig. 2 showing the labelling of stage XI1 late pachytene and zygotene spermatocytes.
Fig. 3 shows the relatively low level of DNA synthesis of early pachytene spermatocytes (stage V) and in Fig. 4 from stage IX the rather high level of DNA synthesis of the mid-pachytene spermatocytes is seen. In this stage the preleptotene spermatocytes have a high rate of DNA synthesis.
Discussion Our results support the evidence obtained by other techniques that there exists a low level of DNA synthesis during the meiotic prophase in animals. We have been able to localize a definite peak of
28
Heredirus 82 (1976)
K.-O. SODERSTROM A N D M. PARVINEN
in the evaluation of the genetic toxicity of different DNA synthesis in mid-pachytene spermatocytes chemical compounds. (spermatogenic stages VII-IX). This is somewhat different from the results of KOFMAN-ALFARO and Acknou/rdgmenls, This work was financially supported by CHANDLEY (1971), who noted the highest activity in the Finnish National Research Council for Medical Sciences. early pachytene spermatocytes, using in vitro labelling The authors are grateful to Prof. A. Lima-de-Faria for of testicular cell suspensions of the mouse. stimulating discussions and to Mrs. Leena Simola and Raija Since the mammalian germinal cells fail to differAndersen for excellent technical assistance. entiate through the meiotic prophase in the cell culture, but do so in organ culture (STEINBERGER et al. Literature cited 1970), it is possible that the incubation conditions BRYANT, B. J. 1966. Incorporation of tritium from thymidine with normal contacts of the germ cells with the into proteins of the mouse. ~.J . c'cdl Biol. 29: 29-36 Sertoli cells and each other may give more reliable CHANDLEY, A. C. and KOtMAN-AI.CARO. s. 1971. "Unscheresults resembling more those existing in vivo. duled" DNA synthesis in human germ cells following U.V. irradiation. -- E.rp. Cell R w . 6Y: 45-48 The autoradiographic analyses of in vivo DNA CHRISTENSEN, A. K. and MASON,N. R. 1965. Comparative synthesis during the meiotic prophase have given ability of seminiferous tubules and interstitial tissue of rat conflicting results, and in most experiments the level testes to synthesize androgens from progesterone-4-'"C in of the radioactivity has been so low that accurate Endocrinology 76: 646 - . 656 vitro. Y. and TROTT,M. 1969. Duration of the cycle CLERMONT. grain counting has been difficult. Recently, a bioof the seminiferous epithelium in the mouse and hamster chemical determination of highly purified epididymal determined by means of 'H-thymidine and radioautography. spermatozoan nuclei has been made by MEETRICH - Ferri/irj Steriliry 5: 805 -8 I7 and coworkers ( 1975). They injected tritiated thymiGLF.DHILL. B. L. and DARSZYNKIEWICZ. Z . 1973. Unscheduled synthesis of D N A during mammalian spermatogenesis in dine intratesticularly and analyzed the epididymal J . E.vp. Zoo/. 183: 375-382 response to UV irradiation. spermatozoa after given intervals. Even in this HOTTA,Y., 170, M. and S T ~ R NH.. 1966. Synthesis of DNA study, the level of radioactivity incorporated to the during meiosis. - Proc. NUI. Acud. Sci. 56: 1184-1 191 DNA during the meiotic prophase was near the level KOFMAN-ALFARO, S. and CHANDLIY. A. C. 1970. Meiosis in the Chromomale mouse. An autoradiographic investigation. of detectability, although the liquid scintillation .soniu 31: 404-420 counting in this case is more sensitive than autoS. and CHANIXEY. A. C. 1971. RadiationKOFMAN-AL~ARO. radiography. No clear peak correlating to any given initiated D N A synthesis in spermatogenic cells of the stage of meiotic prophase was observed. E.vp. Cell R e s . 6Y: 33 44 mouse. C. P. and CLERMONT. Y . 1952. Definition of the LEBI.OND, A source of error in our study may be the stages of the cycle of the seminiferous epithelium in the possible incorporation of tritium label from thymiAnn. N. Y . Acud. Sci. 55: 548-573 rat. dine into proteins (BRYANT 1966). This may explain LIMA-DE-FARIA. A,, GERMAN. J..GHATNLKAR, M., MCGOVERN. the relatively high background levels observed in J . and ANDERSON. L. 1968. In vitro labelling of human meiotic chromosomes with "H-thymidine. - Hereditus 60: our study, where samples were incubated in rather 249- 26 1 high specific radioactivity of the medium. We have MEISTRICH, M . L.. REID,8. 0. and BARCELLONA, W. J. 1975. reduced this source of error by counting the backMeiotic D N A synthesis during mouse spermatogenesis. ground values in each case over late spermatids, J . Crll Biol. 64: 21 1-222 MONESI. V. 1962. Autoradiographic study of DNA synthesis having absolutely no DNA synthesis, but an active and the cell cycle in spermatogonia and spermatocytes of protein synthesis. mouse testis using tritiated thymidine. -~~J . Cell Biol. 14: The biological significance of the DNA synthesis I - ~ l 8 during the meiotic prophase remains to be solved in MVKHERJEE, A. B. and COHEN, M . M. 1968. D N A synthesis during meiotic prophase in male mice. - Nurure 2 f 9 : detail. It has been suggested that the DNA synthesis 489 -490 during leptotene and zygotene is a delayed replication M. 1970. Late DNA O D A R T C H ~ N KNO. ,and PAVILLARD, of definite chromosomal regions involved in chroScience replication in male mouse meiotic chromosomes. mosomal pairing and that the DNA synthesis during 167: 1133-1134 T. 1972. Identification P A R V I N ~M. N . and VANHA-PERTTULA, pachytene might represent the repair of DNA and enzyme quantitation of the stages of the seminiferous breaks associated with crossing-over or spontaneous epithelial wave in the rat. Anur. Rec. 174: 435-450 mutations. Repair synthesis of DNA during meiosis E., STEINBERCER, A. and FITHER, M. 1970. STHNBERGER, has been described after treatment with certain Study of spermatogenesis and steroid metabolism in cultures Rec. Progr. Hormone Res. 26: mutagenic agents (CHANDLEY and KOFMAN-ALFARO of mammalian testes. 547-- 588 1971; GLEDHILL and DARSZYNKIEWICZ 1973). Our SBDERSTRAM, K . - 0 . and PARVINEN. M. 1976. R N A synthesis method gives further possibilities for analyzing the in different stages of rat seminiferous epithelial cycle. "unscheduled" DNA synthesis at different accurately Mol. Cell. Endocrinol. (in press) defined stages of meiosis, which has a significant value -
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