MOLECULAR REPRODUCTION AND DEVELOPMENT 27:244-248 (1990)
Decondensation of Mouse Sperm Chromatin in Cell-Free Extracts: A Micromethod MAREK MALESZEWSKI Department of Embryology, Institute of Zoology, University of Warsaw, Poland ABSTRACT A micromethod is presented which makes possible the analysis of mouse sperm nucleus decondensation in vitro using very small volumes of cytoplasmic preparations, even smaller than 1pl. We show that cell-free extracts obtained from interphase HeLa cells as well as lysates from mouse eggs and embryos can sustain early stages of mouse sperm nucleus transformation, provided the sperm nuclear envelope is damaged or removed. Key Words:
Spermatozoa, Nucleus, In vitro, Sperm decondensation
INTRODUCTION The nuclear envelope of sperm breaks down very rapidly after fertilization in mammals and the chromatin becomes exposed to the egg cytoplasm. Subsequently the sperm nucleus decondenses, the new nuclear envelope is formed, and the male pronucleus develops. The capacity of a mammalian oocyte to transform a sperm nucleus into a male pronucleus shows developmental stage dependence. An immature oocyte does not promote sperm nuclear decondensation prior to germinal vesicle breakdown (GVBD) (Usui and Yanagimachi, 1976; Thadani, 1979; Balakier and Tarkowski, 1980; Szollosi et al., submitted). The oocyte cytoplasm acquires this ability after GVBD (Usui and Yanagimachi, 1976; Thadani, 1979; Batakier and Tarkowski, 1980; Szollosi et al., submitted), which subsequently disappears a few hours after fertilization or artificial activation (Usui and Yanagimachi, 1976; Perreault et al., 1984; Borsuk and Tarkowski, 1989). On the molecular level several events occur parallel to the morphological remodelling of the sperm nucleus. The most prominent changes are the reduction of disulphide bonds in sperm chromatin and then replacement of protamines by histones (Perreault et al., 1984; Zirkin et al., 1985; Perreault et al., 1988). The molecular mechanisms controlling these events are as yet largely unknown. Several in vitro studies were reported employing cell free extracts from activated or nonactivated amphibian oocytes (Lohka and Masui, 1983a, 1984; Iwao and Katagiri, 1984; Gordon et al., 1985; Lohka and Maller, 1985; Ohsumi et al., 1988). The reaction of sperm nuclei from different species in-
0 1990 WILEY-LISS, INC.
cubated in these extracts resembles normal fertilization events. It is difficult to obtain sufficient quantities of material for similar experiments in mammals. In this study we present a micromethod that makes possible the in vitro analysis of some of these reactions. Very small volumes of cytoplasmic preparations can be used, even smaller than 1 p1. MATERIALS AND METHODS Preparation of Sperm Chromatin Mature Fl(CBA/H x C57BL/10) male mice were killed by cervical dislocation and sperm suspension was prepared from the cauda epididymidis. Two caudae were removed and transferred to a drop of PBS (phosphate-buffered saline). Sperm were released by squeezing and concentrated by centrifugation. The sperm nuclei were prepared as described by Lohka and Masui (1983b). Sperm obtained from two males were washed with 1 ml of nuclear isolation medium (NIM) (Ziegler and Masui, 1973)and subsequently incubated for 5 min in 100 pl of NIM containing 0.1% lysolecithin. After incubation 900 pl of cold NIM + 3% BSA (bovine serum albumin, fraction V) was added to the suspension. Sperm nuclei were filtered through cotton wool and concentrated by centrifugation. The sperm pellet was washed three times with NIM + 0.4% BSA and finally smeared on a cover glass and air dried. In some experiments sperm nuclei were suspended in distilled water to avoid salt residues after air drying. The cover glass with air-dried sperm nuclei was placed in a Petri dish, covered with paraffin oil, and then used for incubation with cytoplasmic extracts a t 37°C.
Preparation of HeLa Cells Extracts HeLa cells were grown as monolayer culture at 37°C in Eagle’s minimal essential medium supplemented
Received February 22, 1990; accepted May 29, 1990. Address reprint requests to Marek Maleszewski, Department of Embryology, Institute of Zoology, University of Warsaw, Krakowskie Przedmiescie 26/28, 00-927 Warsaw 64, Poland.
IN VITRO SPERM NUCLEAR DECONDENSATION with fetal calf serum (5%), glutamine, and antibiotics (penicillin, streptomycin, and nystatin). Mitotic cells were removed by selective detachment of rounded mitotic cells. The interphase cells that remained firmly attached were harvested by trypsinization. The cells were then washed three times with PBS and resuspended in modified extraction medium originally employed to stabilize maturation promoting factor (MPF) extracted from mouse oocytes (Sorensen et al., 19851, composed of 10 mM Na,HPO,/NaH,PO,, pH7.3, 80 mM p-glycerolophosphate, 20 mM EGTA, and 15 mM MgC1, in a final concentration of 10-20 x lo7 cells/ml. Cell extracts were obtained by hand homogenization in a glass homogenizer. The homogenate was cetrifuged at 2,lOOg for 5 min. Supernatant thus obtained was used for sperm nuclei decondensation assay. Drops of extracts (volumes s 1 ~ 1were ) spotted under the paraffin oil onto the sperm nuclei prepared as described above.
Preparation of Eggs and Embryos Lysates Groups of 10 to 40 mouse ovulated unfertilized oocytes or one-cell embryos were drawn into a capillary in less than 1 p1 of the extraction medium described above. Embryos were lysed by freezing-thawing, which was repeated three times. Lysates were spotted under paraffin oil onto the sperm smear preparation. Cytological Procedure After incubation the overlying paraffin oil was removed with xylen and the cover glass carefully washed with PBS to remove the extract. Then, the smear of sperm nuclei was fixed in alcohol/acetic acid (3:l) and further stained with Giemsa stain. RESULTS The aim of this study was to determine whether the cytoplasmic extract obtained from interphase HeLa cells would support in vitro the decondensation of sperm nuclei whose nuclear membranes were removed. We also wanted to test in this respect the activity of cell-free extracts obtained from mouse oocytes and embryos. Incubation in cytoplasmic extracts resulted regularly in increased stainability of the sperm nuclei with Giemsa stain (Fig. 1B-H) in comparison with control, demembranated sperm nuclei incubated only in lysis buffer (Fig. 1A). In contrast, when sperm cells neither treated with lysolecithin nor dried on the cover glass were incubated in suspension with the interphase HeLa cells extract, they remained intact. Even after prolonged incubation they did not increase stainability with Giemsa stain. However, when sperm cells were not treated with lysolecithin but were dried on the cover glass prior to incubation in cytoplasmic extract, the reaction was similar to demembranated ones. Demembranated sperm nuclei incubated for 2 (Fig. lB), 4 (Fig. lC), and 8 hours (Fig. 1D) increased their
245
stainability with Giemsa stain and showed slight decondensation. Intensive decondensation occurred after 20 hours of incubation in interphase extract (Fig. 1E). However, the degree and the morphological pattern of decondensation varied among experiments. We suspect that these differences could be due to variable degree of inactivation of factors present in the cell extract responsible for sperm chromatin decondensation. To test this hypothesis aliquots of HeLa interphase extract were deposited on the same spot of the smear of sperm nuclei every 2 hours during a n 8-hour period of incubation. This treatment induced fast decondensation and noticeable dispersion of sperm nuclear chromatin (Fig. lF ), far more intensely than during exposition of sperm nuclei to a single dose of extract for 8 hours (Fig. 1D). All sperm nuclei covered by extract reacted generally in the same way, but the strongest reaction was observed in the center of the spot. During incubation the extract spread radially and the time of exposition of nuclei located at the periphery of the spot was shorter than of those which were covered by the extract all the time. Incubation in the lysate prepared from ovulated metaphase I1 mouse oocytes caused in each case an increased stainability with Giemsa stain and in a few experiments (- 10%) decondensation of demembranated sperm nuclei (Fig. 1G). Only a n increased stainability with the Giemsa stain was observed with lysates made of zygotes (Fig. 1H). To rule out the possibility that decondensation of sperm nuclei could be due to a high concentration of salts left on the cover slip after evaporation of the medium, in control experiments sperm nuclei were suspended in distilled water before being dried. We did not observe any difference in the behavor of sperm nuclei prepared in these two ways. The pattern of sperm decondensation observed in our in vitro system resembled the changes observed in sperm nuclei which take place during fertilization. Most often decondensation began in the postacrosomal region and progressed rostrally, resulting in a n enlarged, rounded, or elongated sperm nucleus composed of thick chromatin fibrils. Even in fully decondensed sperm nuclei, deeply stained and more condensed regions were observed. Most often one region was present in the anterior part and the another near the attachment point of the mid-piece (Fig. lD,E,F).
DISCUSSION Based on in vivo and in vitro studies it has been suggested that 1)reduction of the disulphide bonds is a n obligatory step for decondensation of the sperm nucleus during fertilization, and 2) cytoplasm of GV-intact oocytes and pronuclear eggs is unable to reduce sufficient numbers of these disulphide bonds to permit sperm decondensation (Perreault e t al., 1984, 1988; Zirkin et al., 1985). However, previous experiments
246
M. MALESZEWSKI
Fig. 1. Morphology of sperm heads demembranated with lysolecithin and stained with Giemsa stain after incubation in cytoplasmic extracts. Sperm chromatin was prepared and incubated as described in Materials and Methods. Control: (A) 20 h of incubation in extraction buffer, sperm chromatin not stained. (B-F) Experimental sperm heads incubated in interphase HeLa cells extract for (B) 2 h, (C) 4 h, (D) 8 h, and (E) 20 h; (F) 8 h of incubation in HeLa extract, fresh
aliquot of extract was deposited on the same spot every 2 hours during 8 hours of incubation. (G)Sperm heads incubated in lysate from ovulated oocytes, with 20 h of incubation. (H) In lysate obtained from mouse zygotes, with 20 h of incubation. Marks show two most often present, deeply stained regions of decondensed sperm nuclei: arrow, anterior region, arrowhead, attachment point with the midpiece. x 1,750.
IN VITRO SPERM NUCLEAR DECONDENSATION from our laboratory have shown that even if the cytoplasm is unable to support decondensation (GV-intact oocyte, late pronuclear egg, or interphase blastomere) the sperm nucleus introduced into it gains stainability with toluidine blue or with Giemsa stain (Borsuk and Tarkowski, 1989; Szollosi et al., submitted; Tarkowski, personal communication). This observation can be interpreted in two ways: first, limited (and insufficient for initiation of decondensation) reduction of disulphide bonds produces positive histochemical reaction and, second, reduction of disulphide bonds is only one of conditions required for sperm nuclei decondensation. In our in vitro studies we have shown that cytoplasmic extract of interphase HeLa cells could support early events of sperm nucleus transformation which normally occur after fertilization. We observed that even relatively short (2 hours) incubation in this extract resulted in acquired stainability with Giemsa stain. In control experiments demembranated sperm nuclei incubated in buffer alone did not stain with Giemsa stain even after prolonged incubation (24 hours) and intact sperm cells did not stain with Giemsa nor did they decondense after incubation in HeLa cytoplasmic extract in suspension. It was shown that increased sperm head stainability with Giemsa stain (Miller and Masui, 1982) or toluidine blue (Krzanowska, 1982) results from reduction of disulphide bonds present in proteins forming sperm nuclei chromatin. Our results show that the extract from interphase HeLa cells also possesses the ability to increase sperm head stainability, a finding that constitutes indirect evidence that these extracts exhibit disulphide reducing activity. In our in vitro experiments sperm nuclei were pretreated with lysolecithin, a treatment which removes or at least damages the sperm plasma membrane and the nuclear envelope (Lohka and Masui, 1983a). Such denudation of the sperm chromatin was sufficient for its decondensation by interphase somatic cell cytoplasmic extract and occasionally also by the lysate obtained from ovulated oocytes. Recently we have observed that not only lysolecithin treatment but also drying alone of fresh, untreated sperm cells on the cover glass must damage the sperm cell membranes, because during incubation in HeLa cytoplasmic extract the sperm nuclei decondense (data not shown). Thus the ability to decondense sperm chromatin is not restricted to the oocyte which has resumed meiosis but appears to be a n unspecific reaction, provided the sperm nuclear envelope is damaged or removed. In light of our results the cytoplasmic ability to remove this envelope, probably connected with MPF (M-phase promoting factor) (Czdowska et al., 1984; Sorensen et al., 19851, would be a major factor which in vivo restricts the reaction of decondensation of the sperm head to the cytoplasm of M-phase oocyte. We were not able to induce decondensation of demembranated sperm nuclei with the lysate obtained
247
from pronuclear eggs, and stainability with Giemsa stain was the only observed reaction induced by this lysate (Fig. 1).We can not offer a n unequivocal explanation of this fact, but several hypotheses are possible. For instance factors responsible for sperm chromatin decondensation could be unstable in lysate obtained from zygotes by freeze-thawing or the unseparated cellular debris present in the uncentrifuged lysate could interfere with the activity of soluble cytoplasmic factors. However freeze-thawing did not inhibit the decondensing activity of HeLa cells extract (not shown). Also we cannot rule out the possibility that the pronuclear cytoplasm may lack factors required to support sperm nuclear decondensation which are additional to demembranation and reduction of disulphide bonds. We think that the reduction of disulphide bonds (manifested by increased stainability) is not sufficient for sperm head decondensation. We suppose this because sperm chromatin gains stainability with Giemsa stain and toluidine blue when introduced into the cytoplasm of any tested cell unable to support its decondensation. Reduction of disulphide bonds probably can proceed already when only the sperm plasma membrane is removed but the nuclear envelope remains intact or is damaged to a small extent. This incomplete denudation occurs in vivo, when sperm head is introduced into the GV-intact oocyte or pronuclear eggs (Szollosi et al., submitted). On the contrary, when sperm nuclear membrane is removed or damaged, as it was during our in vitro tests, either interphase HeLa cells or M-phase oocyte cytoplasms are able to induce decondensation of sperm chromatin. This suggests that the cytoplasmic ability to break down the sperm nuclear envelope has a regulatory role in transformation of the sperm nucleus into the male pronucleus. The micromethod presented in this paper can be applied to studies of mechanisms of other cellular events because it provides a unique opportunity to work with a cell free system using extremely small volumes. Results described above should be considered for the time being as preliminary. We are trying to improve. the methods of preparation of extracts and the conditions of incubation hoping to extend the application of this method.
ACKNOWLEDGMENTS I wish to thank Professor A.K. Tarkowski for his interest and helpful advice during experimental work and for his valuable comments on the manuscript. I thank Dr. D. Szollosi for the critical reading of the manuscript. I am also grateful to Professor St. Moskalewski for the opportunity to learn cell culture techniques in his laboratory in the Medical School in Warsaw and to Dr. Renata Zahorska from Serum and Vaccine Research Laboratory for providing me with HeLa cells. I thank Miss Aneta Muszynska for her excellent photographic work. This investigation was partially financed by a research grant from the Polish
248
M. MALESZEWSKI
Academy of Sciences (CPBP 04.01.). Support of WHO Small Supplies Programme is kindly acknowledged.
REFERENCES Balakier H, Tarkowski AK (1980): The role of germinal vesicle karyoplasm in the development of male pronucleus in the mouse. Exp Cell Res 128:79-85. Borsuk E, Tarkowski AK (1989): Transformation of sperm nuclei into male pronuclei in nucleate and anucleate fragments of parthenogenetic mouse eggs. Gamete Res 24:471-481. Czoiowska R, Modlinski JA, Tarkowski AK (1984): Behaviour of thymocyte nuclei in nonactivated and activated mouse oocytes. J Cell Sci 69:19-34. Gordon K, Brown DB, Ruddle FH (1985):In vitro activation of human sperm induced by amphibian egg extract. Exp Cell Res 157:409418. Iwao Y, Katagiri C (1984): In vitro induction of sperm nucleus decondensation by cytosol from mature toad eggs. J Exp Zoo1 230:115124. Krzanowska H (1982): Toluidine blue staining reveals changes in chromatin stabilization of mouse spermatozoa during epidydymal maturation and penetration of ova. J Reprod Fertil 64:97-101. Lohka MJ, Maller J L (1985): Induction of nuclear envelope breakdown, chromosome condensation and spindle formation in cell-free extracts. J Cell Biol 101:518-523. Lohka MJ, Masui Y (1983a): Formation in vitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic components. Science 220:719-721. Lohka MJ, Masui Y (198313): The germinal vesicle material required for sperm pronuclear formation is located in the soluble fraction of egg cytoplasm. Exp Cell Res 148:481-491. Lohka MJ, Masui Y (1984): Effects of Ca2+ ions on the formation of
metaphase chromosomes and sperm pronuclei in cell-free preparations from unactivated Rana pipiens eggs. Dev Biol 103:434-442. Miller MA, Masui Y (1982): Changes in the stainability and sulfhydry1 level in the sperm nucleus during sperm-oocyte interaction in mice. Gamete Res 5:167-179. Ohsumi K, Katagiri C, Yanagimachi R (1988): Human sperm nuclei can transform into condensed chromosomes in Xenopus egg extract. Gamete Res 2O:l-9. Perreault SD, Barbee RR, Slott VL (1988):Importance of glutathione in the acquisition and maintenance of sperm nuclear decondensing activity in maturing hamsters oocytes. Dev Biol 125181-186. Perreault SD, Wolff RA, Zirkin BR (1984): The role of disulfide bond reduction during mammalian sperm nuclear decondensation in vitro. Dev Biol 101:160-167. Sorensen RA, Cyert MS, Pedersen RA (1985): Active maturation-promoting factor is present in mature mouse oocytes. J Cell Biol 100: 1637-1640. Szollosi D, Szollosi M, Czdowska R, Tarkowski AK: Sperm penetration into immature mouse oocytes and the nuclear changes during maturation: EM study. Biol Cell, submitted. Thadani VM (1979): Injection of sperm heads into immature rat oocytes. J Exp Zoo1 210:161-168. Usui N, Yanagimachi R (1976): Behavior of hamster sperm nuclei incorporated into eggs at various stages of maturation, fertilization and early development. J Ultrastruct Res 57:276-288. Ziegler D, Masui Y (1973): Control of chromosome behavior in amphibian oocytes. I. The activity of maturing oocytes inducing chromosome condensation in transplanted brain nuclei. Dev Biol 35: 283-292. Zirkin BR, Soucek DA, Chang TSK, Perreault SD (1985): In vitro and in vivo studies of mammalian sperm nuclear decondensation. Gamete Res 11:349-365.
Decondensation of Mouse Sperm Chromatin in Cell-Free Extracts: A Micromethod MAREK MALESZEWSKI Department of Embryology, Institute of Zoology, University of Warsaw, Poland ABSTRACT A micromethod is presented which makes possible the analysis of mouse sperm nucleus decondensation in vitro using very small volumes of cytoplasmic preparations, even smaller than 1pl. We show that cell-free extracts obtained from interphase HeLa cells as well as lysates from mouse eggs and embryos can sustain early stages of mouse sperm nucleus transformation, provided the sperm nuclear envelope is damaged or removed. Key Words:
Spermatozoa, Nucleus, In vitro, Sperm decondensation
INTRODUCTION The nuclear envelope of sperm breaks down very rapidly after fertilization in mammals and the chromatin becomes exposed to the egg cytoplasm. Subsequently the sperm nucleus decondenses, the new nuclear envelope is formed, and the male pronucleus develops. The capacity of a mammalian oocyte to transform a sperm nucleus into a male pronucleus shows developmental stage dependence. An immature oocyte does not promote sperm nuclear decondensation prior to germinal vesicle breakdown (GVBD) (Usui and Yanagimachi, 1976; Thadani, 1979; Balakier and Tarkowski, 1980; Szollosi et al., submitted). The oocyte cytoplasm acquires this ability after GVBD (Usui and Yanagimachi, 1976; Thadani, 1979; Batakier and Tarkowski, 1980; Szollosi et al., submitted), which subsequently disappears a few hours after fertilization or artificial activation (Usui and Yanagimachi, 1976; Perreault et al., 1984; Borsuk and Tarkowski, 1989). On the molecular level several events occur parallel to the morphological remodelling of the sperm nucleus. The most prominent changes are the reduction of disulphide bonds in sperm chromatin and then replacement of protamines by histones (Perreault et al., 1984; Zirkin et al., 1985; Perreault et al., 1988). The molecular mechanisms controlling these events are as yet largely unknown. Several in vitro studies were reported employing cell free extracts from activated or nonactivated amphibian oocytes (Lohka and Masui, 1983a, 1984; Iwao and Katagiri, 1984; Gordon et al., 1985; Lohka and Maller, 1985; Ohsumi et al., 1988). The reaction of sperm nuclei from different species in-
0 1990 WILEY-LISS, INC.
cubated in these extracts resembles normal fertilization events. It is difficult to obtain sufficient quantities of material for similar experiments in mammals. In this study we present a micromethod that makes possible the in vitro analysis of some of these reactions. Very small volumes of cytoplasmic preparations can be used, even smaller than 1 p1. MATERIALS AND METHODS Preparation of Sperm Chromatin Mature Fl(CBA/H x C57BL/10) male mice were killed by cervical dislocation and sperm suspension was prepared from the cauda epididymidis. Two caudae were removed and transferred to a drop of PBS (phosphate-buffered saline). Sperm were released by squeezing and concentrated by centrifugation. The sperm nuclei were prepared as described by Lohka and Masui (1983b). Sperm obtained from two males were washed with 1 ml of nuclear isolation medium (NIM) (Ziegler and Masui, 1973)and subsequently incubated for 5 min in 100 pl of NIM containing 0.1% lysolecithin. After incubation 900 pl of cold NIM + 3% BSA (bovine serum albumin, fraction V) was added to the suspension. Sperm nuclei were filtered through cotton wool and concentrated by centrifugation. The sperm pellet was washed three times with NIM + 0.4% BSA and finally smeared on a cover glass and air dried. In some experiments sperm nuclei were suspended in distilled water to avoid salt residues after air drying. The cover glass with air-dried sperm nuclei was placed in a Petri dish, covered with paraffin oil, and then used for incubation with cytoplasmic extracts a t 37°C.
Preparation of HeLa Cells Extracts HeLa cells were grown as monolayer culture at 37°C in Eagle’s minimal essential medium supplemented
Received February 22, 1990; accepted May 29, 1990. Address reprint requests to Marek Maleszewski, Department of Embryology, Institute of Zoology, University of Warsaw, Krakowskie Przedmiescie 26/28, 00-927 Warsaw 64, Poland.
IN VITRO SPERM NUCLEAR DECONDENSATION with fetal calf serum (5%), glutamine, and antibiotics (penicillin, streptomycin, and nystatin). Mitotic cells were removed by selective detachment of rounded mitotic cells. The interphase cells that remained firmly attached were harvested by trypsinization. The cells were then washed three times with PBS and resuspended in modified extraction medium originally employed to stabilize maturation promoting factor (MPF) extracted from mouse oocytes (Sorensen et al., 19851, composed of 10 mM Na,HPO,/NaH,PO,, pH7.3, 80 mM p-glycerolophosphate, 20 mM EGTA, and 15 mM MgC1, in a final concentration of 10-20 x lo7 cells/ml. Cell extracts were obtained by hand homogenization in a glass homogenizer. The homogenate was cetrifuged at 2,lOOg for 5 min. Supernatant thus obtained was used for sperm nuclei decondensation assay. Drops of extracts (volumes s 1 ~ 1were ) spotted under the paraffin oil onto the sperm nuclei prepared as described above.
Preparation of Eggs and Embryos Lysates Groups of 10 to 40 mouse ovulated unfertilized oocytes or one-cell embryos were drawn into a capillary in less than 1 p1 of the extraction medium described above. Embryos were lysed by freezing-thawing, which was repeated three times. Lysates were spotted under paraffin oil onto the sperm smear preparation. Cytological Procedure After incubation the overlying paraffin oil was removed with xylen and the cover glass carefully washed with PBS to remove the extract. Then, the smear of sperm nuclei was fixed in alcohol/acetic acid (3:l) and further stained with Giemsa stain. RESULTS The aim of this study was to determine whether the cytoplasmic extract obtained from interphase HeLa cells would support in vitro the decondensation of sperm nuclei whose nuclear membranes were removed. We also wanted to test in this respect the activity of cell-free extracts obtained from mouse oocytes and embryos. Incubation in cytoplasmic extracts resulted regularly in increased stainability of the sperm nuclei with Giemsa stain (Fig. 1B-H) in comparison with control, demembranated sperm nuclei incubated only in lysis buffer (Fig. 1A). In contrast, when sperm cells neither treated with lysolecithin nor dried on the cover glass were incubated in suspension with the interphase HeLa cells extract, they remained intact. Even after prolonged incubation they did not increase stainability with Giemsa stain. However, when sperm cells were not treated with lysolecithin but were dried on the cover glass prior to incubation in cytoplasmic extract, the reaction was similar to demembranated ones. Demembranated sperm nuclei incubated for 2 (Fig. lB), 4 (Fig. lC), and 8 hours (Fig. 1D) increased their
245
stainability with Giemsa stain and showed slight decondensation. Intensive decondensation occurred after 20 hours of incubation in interphase extract (Fig. 1E). However, the degree and the morphological pattern of decondensation varied among experiments. We suspect that these differences could be due to variable degree of inactivation of factors present in the cell extract responsible for sperm chromatin decondensation. To test this hypothesis aliquots of HeLa interphase extract were deposited on the same spot of the smear of sperm nuclei every 2 hours during a n 8-hour period of incubation. This treatment induced fast decondensation and noticeable dispersion of sperm nuclear chromatin (Fig. lF ), far more intensely than during exposition of sperm nuclei to a single dose of extract for 8 hours (Fig. 1D). All sperm nuclei covered by extract reacted generally in the same way, but the strongest reaction was observed in the center of the spot. During incubation the extract spread radially and the time of exposition of nuclei located at the periphery of the spot was shorter than of those which were covered by the extract all the time. Incubation in the lysate prepared from ovulated metaphase I1 mouse oocytes caused in each case an increased stainability with Giemsa stain and in a few experiments (- 10%) decondensation of demembranated sperm nuclei (Fig. 1G). Only a n increased stainability with the Giemsa stain was observed with lysates made of zygotes (Fig. 1H). To rule out the possibility that decondensation of sperm nuclei could be due to a high concentration of salts left on the cover slip after evaporation of the medium, in control experiments sperm nuclei were suspended in distilled water before being dried. We did not observe any difference in the behavor of sperm nuclei prepared in these two ways. The pattern of sperm decondensation observed in our in vitro system resembled the changes observed in sperm nuclei which take place during fertilization. Most often decondensation began in the postacrosomal region and progressed rostrally, resulting in a n enlarged, rounded, or elongated sperm nucleus composed of thick chromatin fibrils. Even in fully decondensed sperm nuclei, deeply stained and more condensed regions were observed. Most often one region was present in the anterior part and the another near the attachment point of the mid-piece (Fig. lD,E,F).
DISCUSSION Based on in vivo and in vitro studies it has been suggested that 1)reduction of the disulphide bonds is a n obligatory step for decondensation of the sperm nucleus during fertilization, and 2) cytoplasm of GV-intact oocytes and pronuclear eggs is unable to reduce sufficient numbers of these disulphide bonds to permit sperm decondensation (Perreault e t al., 1984, 1988; Zirkin et al., 1985). However, previous experiments
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Fig. 1. Morphology of sperm heads demembranated with lysolecithin and stained with Giemsa stain after incubation in cytoplasmic extracts. Sperm chromatin was prepared and incubated as described in Materials and Methods. Control: (A) 20 h of incubation in extraction buffer, sperm chromatin not stained. (B-F) Experimental sperm heads incubated in interphase HeLa cells extract for (B) 2 h, (C) 4 h, (D) 8 h, and (E) 20 h; (F) 8 h of incubation in HeLa extract, fresh
aliquot of extract was deposited on the same spot every 2 hours during 8 hours of incubation. (G)Sperm heads incubated in lysate from ovulated oocytes, with 20 h of incubation. (H) In lysate obtained from mouse zygotes, with 20 h of incubation. Marks show two most often present, deeply stained regions of decondensed sperm nuclei: arrow, anterior region, arrowhead, attachment point with the midpiece. x 1,750.
IN VITRO SPERM NUCLEAR DECONDENSATION from our laboratory have shown that even if the cytoplasm is unable to support decondensation (GV-intact oocyte, late pronuclear egg, or interphase blastomere) the sperm nucleus introduced into it gains stainability with toluidine blue or with Giemsa stain (Borsuk and Tarkowski, 1989; Szollosi et al., submitted; Tarkowski, personal communication). This observation can be interpreted in two ways: first, limited (and insufficient for initiation of decondensation) reduction of disulphide bonds produces positive histochemical reaction and, second, reduction of disulphide bonds is only one of conditions required for sperm nuclei decondensation. In our in vitro studies we have shown that cytoplasmic extract of interphase HeLa cells could support early events of sperm nucleus transformation which normally occur after fertilization. We observed that even relatively short (2 hours) incubation in this extract resulted in acquired stainability with Giemsa stain. In control experiments demembranated sperm nuclei incubated in buffer alone did not stain with Giemsa stain even after prolonged incubation (24 hours) and intact sperm cells did not stain with Giemsa nor did they decondense after incubation in HeLa cytoplasmic extract in suspension. It was shown that increased sperm head stainability with Giemsa stain (Miller and Masui, 1982) or toluidine blue (Krzanowska, 1982) results from reduction of disulphide bonds present in proteins forming sperm nuclei chromatin. Our results show that the extract from interphase HeLa cells also possesses the ability to increase sperm head stainability, a finding that constitutes indirect evidence that these extracts exhibit disulphide reducing activity. In our in vitro experiments sperm nuclei were pretreated with lysolecithin, a treatment which removes or at least damages the sperm plasma membrane and the nuclear envelope (Lohka and Masui, 1983a). Such denudation of the sperm chromatin was sufficient for its decondensation by interphase somatic cell cytoplasmic extract and occasionally also by the lysate obtained from ovulated oocytes. Recently we have observed that not only lysolecithin treatment but also drying alone of fresh, untreated sperm cells on the cover glass must damage the sperm cell membranes, because during incubation in HeLa cytoplasmic extract the sperm nuclei decondense (data not shown). Thus the ability to decondense sperm chromatin is not restricted to the oocyte which has resumed meiosis but appears to be a n unspecific reaction, provided the sperm nuclear envelope is damaged or removed. In light of our results the cytoplasmic ability to remove this envelope, probably connected with MPF (M-phase promoting factor) (Czdowska et al., 1984; Sorensen et al., 19851, would be a major factor which in vivo restricts the reaction of decondensation of the sperm head to the cytoplasm of M-phase oocyte. We were not able to induce decondensation of demembranated sperm nuclei with the lysate obtained
247
from pronuclear eggs, and stainability with Giemsa stain was the only observed reaction induced by this lysate (Fig. 1).We can not offer a n unequivocal explanation of this fact, but several hypotheses are possible. For instance factors responsible for sperm chromatin decondensation could be unstable in lysate obtained from zygotes by freeze-thawing or the unseparated cellular debris present in the uncentrifuged lysate could interfere with the activity of soluble cytoplasmic factors. However freeze-thawing did not inhibit the decondensing activity of HeLa cells extract (not shown). Also we cannot rule out the possibility that the pronuclear cytoplasm may lack factors required to support sperm nuclear decondensation which are additional to demembranation and reduction of disulphide bonds. We think that the reduction of disulphide bonds (manifested by increased stainability) is not sufficient for sperm head decondensation. We suppose this because sperm chromatin gains stainability with Giemsa stain and toluidine blue when introduced into the cytoplasm of any tested cell unable to support its decondensation. Reduction of disulphide bonds probably can proceed already when only the sperm plasma membrane is removed but the nuclear envelope remains intact or is damaged to a small extent. This incomplete denudation occurs in vivo, when sperm head is introduced into the GV-intact oocyte or pronuclear eggs (Szollosi et al., submitted). On the contrary, when sperm nuclear membrane is removed or damaged, as it was during our in vitro tests, either interphase HeLa cells or M-phase oocyte cytoplasms are able to induce decondensation of sperm chromatin. This suggests that the cytoplasmic ability to break down the sperm nuclear envelope has a regulatory role in transformation of the sperm nucleus into the male pronucleus. The micromethod presented in this paper can be applied to studies of mechanisms of other cellular events because it provides a unique opportunity to work with a cell free system using extremely small volumes. Results described above should be considered for the time being as preliminary. We are trying to improve. the methods of preparation of extracts and the conditions of incubation hoping to extend the application of this method.
ACKNOWLEDGMENTS I wish to thank Professor A.K. Tarkowski for his interest and helpful advice during experimental work and for his valuable comments on the manuscript. I thank Dr. D. Szollosi for the critical reading of the manuscript. I am also grateful to Professor St. Moskalewski for the opportunity to learn cell culture techniques in his laboratory in the Medical School in Warsaw and to Dr. Renata Zahorska from Serum and Vaccine Research Laboratory for providing me with HeLa cells. I thank Miss Aneta Muszynska for her excellent photographic work. This investigation was partially financed by a research grant from the Polish
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M. MALESZEWSKI
Academy of Sciences (CPBP 04.01.). Support of WHO Small Supplies Programme is kindly acknowledged.
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