MOLECULAR REPRODUCTION AND DEVELOPMENT 29150-156 (1991)
Anucleate Fragments of Parthenogenetic Eggs and of Maturing Oocytes Contain Complementary Factors Required for Development of a Male Pronucleus EWA BORSUK Department of Embryology, Institute of Zoology, University of Warsaw, Warsaw, Poland, Station Centrale de Physiologie Animale, I N R A , Jouy-en-Josas, France Tarkowski and Tittenbrun, personal communication; Borsuk and Tarkowski, 1989). However, the capability of activated cytoplasm to promote transformation of the sperm nucleus into the male pronucleus decreases when the time interval between egg activation and sperm penetration is extended. As it was shown by Borsuk and Tarkowski (1989) anucleate fragments of activated oocytes bisected soon after activation (before the female pronucleus is formed) can promote full or partial transformation of sperm nuclei longer than their nucleate counterparts. However, even in the anucleate fragments this capability slowly disappears. In fragments penetrated 8 h post activation sperm nuclei remain in the cytoplasm unchanged or form abnormal nuclei. The morphology of these nuclei suggests that the breakdown of the sperm nuclear envelope has not occurred and that partial decondensation of chromatin takes place inside the old nuclear envelope (Borsuk and Tarkowski, 1989). It follows that factors responsible for nuclear envelope breakdown and chromatin decondensation become eventually inactivated even in the absence of the female pronucleus and that under these circumstances, the extrachromosomal constituents of the GV cannot support normal pronuclear growth. This study was designed to test the hypothesis that development of the male pronuclei requires factors found in both developing and activated oocytes. Spermatozoa can penetrate the cytoplasm of mouse oocytes at the germinal vesicle stage (Iwamatsu and Chang, 1972; Szollosi et al., 1990) but sperm nuclei remain intact as long as the oocyte nucleus stays in Key Words: Mouse oocytes, Cybrids, Pronuclei Fertilinterphase. After initiation of maturation and germiization, Cytoplasmic factors nal vesicle breakdown (GVBD) sperm nuclei undergo decondensation but do not transform into male pronuINTRODUCTION clei. Sperm nuclei behave similarly in anucleate fragIn normal development, the spermatozoon enters the ments produced before GVBD (our unpublished obsermouse oocyte at the metaphase stage of the second vations). These results confirm earlier findings meiotic division and activates it. One of the visible (BaXakier and Czolowska, 1977)that the factors responeffects of activation is the extrusion of the 2nd polar sible for nuclear envelope breakdown and modification body and formation of female and male pronuclei. From earlier observations it is known that sperm nuclei can Received June 19, 1990; accepted December 27, 1990. transform into male pronuclei also in oocytes in which Address reprint requests to Ewa Borsuk, Department of Embryology, the penetration of the sperm was delayed in relation to Institute of Zoology, University of Warsaw, Krakowskie Przedmiescie oocyte activation (Chalmel, 1962; Komar, 1982; 26/28, 00-927 Warsaw 64, Poland. 0 1991 WILEY-LISS, INC.
ABSTRACT Eight-h-old anucleate fragments of artificially activated mouse oocytes produced within 30 min after activation were fused with anucleate fragments of ovarian oocytes bisected at the GV stage and matured in vitro for 4-5 h. The resulting cybrids were inseminated soon after fusion and fixed 3-4 h or 14 h later. After 3-4 h of culture most of the sperm nuclei underwent decondensation. Early male pronuclei were observed sporadically. After overnight culture small or fully formed male pronuclei were present in monospermic and dispermic cybrids.The abortive nuclei, designated as stage Ila (earlier described by Borsuk and Tarkowski, 1989) were also observed and their occurrence was strongly correlated with the rate of polyspermy. In unfused (control) anucleate fragments of maturing and activated oocytes inseminated at the same time as the cybrids, male pronuclei were never observed. These results show that the cytoplasms of activated eggs and of maturing oocytes contain complementary factors required for the transformation of the sperm nucleus into the male pronucleus. The former has lost the capability to break down the sperm nucleus envelope but contains factors required for pronuclear growth. The latter is able only to initiate the process of transformation, i.e., to break down the nuclear envelope and decondense the denuded sperm chromatin. However, the activity of the factor responsible for nuclear envelope breakdown is limited because in cybrids penetrated by more than two spermatozoa, transformation of sperm nuclei was usually abnormal.
SPERM NUCLEI IN CYBRIDS of chromatin structure (decondensation of sperm nuclei and condensation of interphase nuclei) appear autonomously in the cytoplasm of maturing oocytes. The above observations were extended in the present study by the use of anucleate fragments of parthenogenetic eggs and maturing oocytes in the following experimental design. Two types of cytoplasts, an 8h-old parthenogenetic and a maturing one (the first probably carrying factors required for pronuclear growth, the second carrying factors responsible for nuclear envelope breakdown and chromatin decondensation), were fused together and subsequently fertilized to determine whether the cytoplasm of the resulting cybrids would support transformation of the sperm nucleus into a male pronucleus (see Fig. 1).The results of this experiment appear to confirm the hypothesis that the two types of fragments contain complementary factors for the development of pronuclei.
MATERIALS AND METHODS Collection and Handling of Oocytes Ovarian ooc tes. Fl(C57BL x CBA/H) females were injected witrl 5 iu PMSG (Folligon, Intervet) and killed 46-48 h later. The ovaries were placed in M2 medium (medium 16 buffered with HEPES, Fulton and Whittingham, 1978) into which dibutyryl cyclic AMP (dbcAMP, Sigma Chemical Co., St. Louis, MO) in conc. 50 Fgirnl had been added to arrest oocytes in germinal vesicle stage during manipulations. Oocytes were released by puncturing the ovarian follicles with a needle. Most of the oocytes were free of cumulus cells. Cumulus cells which remained attached to some of the oocytes were removed mechanically by pipetting. The zona pellucida was removed with a-chymotrypsin (30 Fg/ml) or with acidic Tyrode's solution (pH 2.5). Zonafree oocytes were placed in medium M2 containing dbcAMP and supplemented with 5 p.g/ml of cytochalasin D (CD) (Sigma Chemical Co., St. Louis, MO) and 0.5 p.g/ml of nocodazol (Sigma Chemical Co., St. Louis, MO). After 30 min, incubation at 37°C the oocytes were transferred into cold medium (5°C) for 15-20 min and then bisected into halves (nucleate and anucleate) with a glass needle on a surface of 1%agar, according to the technique of Tarkowski (1977).Pairs of fragments were washed carefully in medium 16 (Whittingham, 1971) and placed in microdrops of this medium under liquid paraffin. They were cultured at 37.5"C under 5% C02in air. The anucleate fragments of those pairs, which had started maturation (called in this paper maturing cytoplasts), as was judged by the disappearance of the germinal vesicle (GV) in the nucleate fragment, were used as partners for fusion with anucleate fragments of activated oocytes (Fig. 1). Artificially activated oocytes. Metaphase I1 oocytes were recovered from Fl(C57BL x CBAIH) females induced to ovulate with 5 iu PMSG followed 48-54 h later by 5 iu HCG. Females were sacrificed 16 h after HCG administration and the ovulated oocytes were released into M2 medium containing hyaluronidase (200-300 iu/ml). The zona pellucida was removed with a-chymotrypsin. Zona-free oocytes were artificially activated by a 6 min exposure to 8% ethanol
151
GV
MI t
CULTURE
1 1 00
\
/
E LECTROFUS I O N
i
t CULTURE
Fig. 1. Diagramatic representation of the experiment. The lapse of time between activation and insemination of cybrids-8 hours, between the transfer of fragments of GV oocytes into the dbcAMP-free medium and insemination of cybrids-5-6 hours.
solution in M2 (Cuthbertson, 1983). Thirty minutes post activation oocytes were bisected into halves (one with a spindle and the other anucleate). Pairs of fragments were cultured in microdrops of M16 medium and the anucleate counterparts of those nucleate fragments, which had undergone activation (the 2nd polar body extruded within 2 h of bisection, and the female pronucleus formed), were used for fusion (Fig. 1).
Cell Fusion Pairs of cytoplasts [maturing (3-4 h after dbcAMP removal) and activated1 agglutinated previously in
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phytohaemaglutynin (PHA, 300 pg/ml in M2 without BSA, 3-5 min) were washed twice in a 0.3 M glucose solution in 18 MR pure water supplemented with 100 pM Ca ions and 100 pM Mg ions and then treated individually with an electric current (two pulses, 40 V, duration 200 p.s) in the same solution (Kubiak and Tarkowski, 1985; Ozil and Modlinski, 1986). After this treatment they were washed in M2 medium and placed in drops of M16 under liquid paraffin in standard culture conditions. Those pairs which had undergone fusion were fertilized in vitro.
Fertilization In Vitro Fertilization in vitro was carried out in Whittingham’s medium 16 (1971) as modified by Fraser and Drury (1975) and containing 32 mg of crystalline bovine serum albumin (Sigma Chemical Co., St. Louis, MO). The medium had been equilibrated overnight at 37.5”Cin an atmosphere of 5% C02 in air, under liquid paraffin. A sperm suspension was prepared from the caudae epididymides of adult Fl(C57B1 x CBNH) males. Sperm from two caudae was released into 1 ml of medium. Before mixing the gametes, the sperm suspension was preincubated for 1 h at 375°C under standard conditions. Cybrids were placed in 1 ml of medium and 100 p1 of sperm suspension was added (giving the final concentration of about 2 x lo6 sperm/ ml). At the moment of insemination the age of the two components of cybrids was 8 h post activation and 2-3 h after GVBD (5-6 h after dbcAMP removal). Cybrids were kept with spermatozoa for 1-1.5 h. Then they were washed in M16 and left in culture until the moment of fixation. Some of the cybrids were fixed 3-4 h post insemination. The others were cultured overnight and fixed 14 h post insemination.
RESULTS Transformation of Sperm Nuclei in Cybrids Between Activated and Maturing Cytoplasts Out of 228 ovarian oocytes bisected at the GV stage, 217 (95.2%) survived the operation, and 142 (65.4%) started to mature 2-3 h after dbcAMP removal. Four hundred seventy-six zona-free metaphase I1 oocytes were submitted to alcohol treatment and bisected 20 min post activation. Out of these, 418 (87.8%) survived bisection and 354 pairs (82.3%) underwent activation (2nd polar body extruded and the female pronucleus present in the nucleate fragment). Out of 133 pairs of activated and maturing cytoplasts submitted to electric pulses, 95 (84.1%)underwent fusion and were inseminated shortly afterwards. Sixty-two cybrids (65.2%)were fertilized. Polyspermic fertilization predominated. Only 12 cybrids (19.3%)were monospermic. Among polyspermic cybrids dispermic ones were the most common (27 out of 50) (Table 1). Sperm nuclei were classified according to the previously described classification (Borsuk and Tarkowski, 1989; Fig. 2); 3 4 hours post insemination stage I1 of sperm nucleus decondensation predominated (88% of cybrids, Fig. 3A). Early male pronuclei (stage 111, Fig. 3B) or abnormal nuclei denoted as IIa were present sporadically (Table 2). After overnight culture, three stages of sperm nuclei transformation were found with almost the same frequency: stage 111, early male pronucleus; stage IV, fully formed male pronucleus (Fig. 3C), and stage IIa (Fig. 3D) (Table 2). The nuclei designated as IIa were first observed in anucleate fragments of activated oocytes inseminated 8 h post activation (Series A in Borsuk and Tarkowski, 1989). The karyoplasm of these nuclei is homogenous and darkly stained, with characteristic dark patches of more condensed chromatin. They are surrounded by a light sphere in cytoplasm (Fig. 3D). In contrast to the IIa nuclei observed in oocyte fragments, which are oval or tear shaped, those observed in cybrids are in most cases spherical. Results obtained in this experiment, especially after overnight culture, showed that in more than 60% of the cybrids sperm nuclei could transform into nuclei, which, at least at the light microscope level, resembled normal male pronuclei (stages I11 and IV). However, in 32.4% of the cybrids abnormal nuclei (stage IIa) were observed. The ability of cybrids to promote transformation of sperm nuclei into male pronuclei was strongly correlated with the rate of polyspermy. Only in the case of monospermy and dispermy were male pronuclei formed in all cybrids (Table 3). With the increasing degree of polyspermy, formation of male pronuclei decreased, and in highly polyspermic cybrids the nuclei in stage IIa predominated.
Control Experiments 1. Metaphase I1 oocytes were activated, bisected, and cultured in pairs as described in “Artificially activated oocytes.”Pairs of anucleate fragments were fused in an electric field 6-7 h post activation and inseminated 1-2 h later (8 h after activation). They were fixed after the same time intervals as the experimental cybrids ( 3 4 h or 14 h post insemination). 2. Anucleate fragments of activated and maturing oocytes were inseminated and fixed at the same time as both types of cybrids (experimental and control). 3. Ovarian oocytes were obtained and treated as described in “Ovarian oocytes.” After storage in cold medium M2 containing dbcAMP and supplemented with CD and nocodazol, they were washed carefully in medium 16 and cultured (as intact oocytes)for 18-19 h. Those oocytes which had reached the metaphase I1 stage (the first polar body extruded) were fertilized in vitro and fixed 5-6 h post insemination. Transformation of Sperm Nuclei in Activated Cybrids, fragments (activated and maturing), and and Maturing Cytoplasts oocytes were examined in whole mount, haematoxylinstained preparations (Tarkowski and Wroblewska, Out of 68 anucleate fragments of activated oocytes 1967). inseminated 8 h post activation 18 lysed after insemi-
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TABLE 1. Frequency of Monospermy and Polyspermy in Cybrids* Total Type
of cybrid
1
AM AA
1 2 2 7 9 12 3 7
N u m b e r of sperm nuclei 2 3 4 5
7 3
1 4
6 6
-
number of cybrids 62 29
*AM, cybrids between activated and m a t u r i n g cytoplasts; AA, cybrids between two activated cytoplasts.
A
\
Stage Ila
Stage I
I
Fig. 3. ADecondensing sperm nucleus (stage 11).The posterior part decondensed, the anterior part still condensed and darkly stained. AM-cybrid, 3 h post insemination x 1,000. B: Early male pronucleus (stage 111). AM-cybrid, 14 h post insemination. x 1,000. C: Fully formed male pronucleus (stage IV). AM-cybrid, 14 h post insemination x 1,000. D: Sperm nucleus in stage IIa surrounded by a light zone in the cytoplasm (arrow). AM-cybrid, 14 h post insemination. x 1,000.
overnight culture in the majority of fragments decondensed sperm nuclei (stage 11)were present. The cytoplasts in which sperm nuclei did not undergo any changes probably represent those oocyte fragments Fig. 2. Schematic representation of sperm nucleus transformation which did not initiate the process of maturation. Neiin anucleate fragments of activated oocytes and in cybrids. Stage I: ther male pronuclei nor IIa nuclei were ever observed unchanged sperm head. Stage 11: stage of sperm nucleus decondensa- (Table 4). tion (full or partial). Stage 111: early male pronucleus. Stage IV: fully formed male pronucleus. Stage IIa: abnormal nuclei probably decondensing inside the old nuclear envelope.
nation. Among the remaining 50 cytoplasts, 47 (94%) were fertilized. Male pronuclei (stages I11 and IV) were never observed, and in the majority of fragments abnormal nuclei (stage IIa) were found. Sporadically the sperm nuclei remained unchanged or their transformation was blocked during decondensation (Table 4). The anucleate fragments of maturing oocytes, bisected at the GV stage and cultured for 5-6 h before insemination, promoted decondensation of sperm nuclei; 3-4 h post insemination in 39.3%of the fragments, sperm nuclei began decondensation and in 60.7% remained unchanged (stage I). This suggests that the process of maturation started late and proceeded very slowly. Indeed, the first nucleate fragments which underwent germinal vesicle breakdown were observed not earlier than 3 h after dbcAMP removal. After
Transformation of Sperm Nuclei in Control Cybrids (Between Two Activated Cytoplasts) Forty-five pairs of activated cytoplasts were agglutinated in PHA and subjected to electric pulses 6-7 h post activation. Out of these, 41 (91.1%)underwent fusion and were fertilized in vitro 8 h after activation. Twentynine (70.7%)cybrids survived this treatment and were cultured for 3-4 or 14 h before fixation. Monospermic fertilization was observed in 41.4%of the cases (Table 1). In the majority of cybrids fixed 3-4 h and 14 h post insemination, sperm nuclei underwent transformation into abnormal nuclei denoted as stage IIa (Table 2). However, these nuclei were smaller and more darkly stained than IIa nuclei present in experimental cybrids (between activated and maturing cytoplasts), and were identical with the nuclei found in 8-h-old anucleate parthenogenetic fragments (Borsuk and Tarkowski, 1989 and this paper). They were accompanied by a pair of granules resembling those observed in nucleate and anucleate fragments of activated oocytes (Borsuk and
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TABLE 2. Transformation of Sperm Nuclei in Cybrids*
Type of cvbrid
Time post. insemination (h) 3-4
AM"
14 3-4
AA" 14
Stages of sperm nucleus transformation (%)
I
I1
IIa
I11
IV
Total number of cvbrids
- 88.0 4.0 8.0 - - 32.4 32.4 35.2 - 18.2 81.8
37 11
-
18
-
94.4
5.6
-
25
*Both monospermic and polyspermic cybrids are included. In the case of polyspermic cybrids, the most advanced sperm nucleus served a s a basis for classification. "See footnote to Table 1.
Tarkowski, 1989). Most often the granules occurred in the vicinity of the nuclei, but sometimes they moved away from them. This experiment showed that the enlargement of the amount of activated cytoplasm did not improve sperm nucleus transformation.
Transformation of Sperm Nuclei in Fully Matured Oocytes The ovarian oocytes were treated similarly to those which were used for bisection and then cultured intact for 18-19 h to determine whether the short culture in the presence of dbcAMP, nocodazol, and cytochalasin D did not affect their maturation and whether they could support formation of male and female pronuclei after fertilization. Out of 100 oocytes submitted to such treatment, 65 had reached the metaphase I1 stage. During the process of insemination and later culture 12 oocytes lysed. By 5-6 h after insemination 68.2% (30/44) of the oocytes that had been penetrated by sperm extruded the second polar body and formed a female and one or more male pronuclei. In the rest of penetrated oocytes the 2nd polar body was also emitted but the female chromatin was blocked at the telophase stage. The sperm nuclei remained arrested during decondensation or at the stage of slight condensation usually preceding formation of the male pronucleus. These results show that oocytes which were submitted to short culture in the presence of dbcAMP, nocodazol, and CD could, after removal of the drugs, undergo maturation in vitro and could support formation of male and female pronuclei after fertilization. DISCUSSION The sperm nucleus after its penetration into egg cytoplasm undergoes a number of changes which lead to the formation of the male pronucleus. This process includes reduction of disulphide bonds of protamines, nuclear envelope breakdown, decondensation of chromatin, replacement of protamines by histones (Kopeeny and Pavlok, 19751, and, after a short stage of chromatin condensation (Stefanini et al., 1970; Eck-
TABLE 3. Transformation of Sperm Nuclei in Monospermic and Dispermic AM Cybrids* Time Stages of sperm nucleus Total post transformation number inseminaof (%I tion I1 IIa I11 IV cybrids I 3-4 14
-
78.9
-
-
21.1 45
55
19 20
*See footnote to Table 1.
lund and Levine, 1975; Borsuk and Manka, 1988; Wright and Longo, 19881,reformation of a new nuclear envelope. In normal fertilization all these events take place during the transition of the oocyte from metaphase I1 to interphase and seem to be controlled by different cytoplasmic factors. The first steps of sperm nucleus transformation, i.e., reduction of disulphide bonds of protamines, nuclear envelope breakdown, and chromatin decondensation, occur when the oocyte chromosomes are in condensed state (between metaphase I1 and telophase). During this phase the cytoplasm still exhibits strong chromosome condensing activity (CCA, Masui, 1985) and promotes nuclear envelope breakdown and premature chromosome condensation (PCC) in any interphase nucleus introduced at this moment into the oocyte (Balakier, 1978; Czolowska et al., 1984; Szollosi et al., 1986a,b; 1988).The sperm nucleus reacts to this environment differently: its chromatin decondenses. The subsequent events in the process of male pronucleus formation (reformation of nuclear envelope and nuclear swelling) occur when the CCA of the oocyte cytoplasm disappears and is replaced by the chromosome decondensing activity (CDA) promoting the interphase state of the egg cell (Masui, 1985).During the growth of male and female pronuclei, nuclear lamins (Maul and Schatten, 1986; Kubiak, 1988) and probably also extra-chromosomal constituents of the germinal vesicle released after its breakdown are absorbed from the cytoplasm. Studies on the behaviour of sperm nuclei in artificially activated intact oocytes (Komar, 1982; Tarkowski and Tittenbrun, personal communication) and their nucleate and anucleate fragments (Borsuk and Tarkowski, 1989) have shown that the capability of activated cytoplasm to promote male pronucleus formation gradually disappears. This was especially interesting in the case of anucleate fragments of oocytes bisected soon after activation (Series A in Borsuk and Tarkowski, 1989). Although their cytoplasm must be rich in all substances required for pronucleus formation and its growth (because of the lack of a competitive female pronucleus), sperm nuclei which penetrated many hours post activation either remained unchanged or formed abnormal nuclei apparently decondensing inside a persistant nuclear envelope. Results obtained in the present study clearly show that anucleate fragments of activated oocytes produced soon after activation and cultured for 8 h before insem-
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TABLE 4. Transformation of Sperm Nuclei in Anucleate Fragments of Activated Oocytes and Maturing Oocytes Total Time number post Stages of sperm nucleus of Tvve insemitransformation of nation (%o) fragfragments (h) I I1 IIa 111 IV ments 14 7.1 28.6 64.3 Activated 3-4 31 3.2 6.5 90.3 cytoplasts 14 39.3 28 Maturing 3-4 60.7 cytoplasts 14 13.6 86.4 22
-
ination can recover their ability to promote male pronucleus formation after fusion with cytoplasts originating from GV-intact oocytes matured in vitro. In more than 60% of such AM cybrids, small or fully formed male pronuclei were found after 14 h of culture. If unfused, both types of cytoplasts could promote only partial transformation of sperm nuclei. In 86.4% of the cytoplasts obtained from GV-intact oocytes maturing in vitro, sperm nuclei were blocked at stage I1 of chromatin decondensation. Also the prolonged post insemination culture of activated cytoplasts did not improve the development of sperm nuclei; formation of male pronuclei was never observed. However, in comparison with fragments fixed 3 4 h post insemination, more nuclei underwent transformation into stage IIa. This suggests that when the sperm nuclear envelope is not broken down, the process of chromatin decondensation is greatly slowed down. Similar results were obtained after insemination and culture of cybrids between two activated cytoplasts (AA cybrids), which shows that limited, abortive transformation of sperm nuclei in oocyte fragments was not related to the decreased. volume of the cytoplasm. The cytoplasm of AM cybrids acquired all factors and substances essential for the full transformation of the sperm nucleus into the male pronucleus. The cytoplast obtained by bisection of GV-intact oocytes matured in vitro was the source of active MPF necessary for the breakdown of the sperm nucleus envelope. However, by itself such cytoplasm could not promote male pronucleus formation for at least two reasons: 1) at the moment of sperm penetration the process of oocyte maturation was not completed and 2) cytoplasts used in this experiment were obtained before GVBD and they were deprived of the extrachromosomal constituents of GV which are necessary for the growth of the pronucleus (Balakier and Tarkowski, 1980). These two conditions were satisfied by the activated component of the cybrid. In more than 30%of the cybrids sperm nuclei did not transform into male pronuclei and formed abortive nuclei designated as stage IIa. Their occurrence was strongly correlated with the rate of polyspermy. They were not observed in monospermic and dispermic cybrids. Starting from the trispermic ones the frequency of their formation increased and in highly polyspermic
cybrids (six sperm nuclei), this type of nuclei predominated. Retarded development of supernumerary sperm nuclei after in vitro fertilization of ovulated oocytes was reported by Witkowska (1981) and explained by the probable disappearance or inactivation of some cytoplasmic factors responsible for normal transformation of sperm nucleus into the male pronucleus. Results obtained in our earlier experiment (Borsuk and Tarkowki, 1989) and in this study showed that for the development of male pronuclei, the presence of active MPF in the cytoplasm is very important. A certain critical threshold of this factor has to be achieved in every nucleus to promote its nuclear envelope breakdown. The MPF is probably distributed uniformly among all the nuclei present in the cytoplasm (Clarke and Masui, 1986, 1987). In AM cybrids the cytoplasm originating from GV intact oocytes and matured in vitro was the only source of that factor. The volume of maturing cytoplasts was reduced to about 1/2 of the volume of intact oocyte and probably the activity of cytoplasmic MPF present in such fragments was adequate to cause nuclear envelope breakdown in a limited number of nuclei. Furthermore, this activity could have been partly neutralized by an antagonistic activity connected with the interphase component of the cybrid (the 8-h-oldparthenogenetic cytoplast). Such activity was found in G1 somatic cells (Adlakha et al., 1983; Rao and Adlakha, 19851, in growing pig and mouse oocytes (Fulka et al., 1985, 19861, and in mouse zygotes (Balakier and Masui, 1986).The IIa nuclei may have originated from those sperm nuclei which could not undergo nuclear envelope dissolution because of the lack of active MPF in the cytoplasm but which could nevertheless undergo chromatin decondensation. U1trastructural studies in progress will better characterize the morphology of sperm nuclei developing in cybrids, especially those in stage IIa.
ACKNOWLEDGMENTS This work was carried out within the framework of a French-Polish scientific exchange programme. WHO Small Institutional Grant to the Department of Embryology is kindly acknowledged. This investigation was partially financed by a research grant from the Polish Academy of Sciences CPBP 04.01. I wish to thank Professor Andrzej K. Tarkowski and Doctor Dan
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Szollosi for their interest and helpful advises and for their valuable comments on the manuscript. I wish to thank Mr. Jean-Pierre Ozil for helpful assistance in electrofusion.
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Ultrastructure of cell fusion and premature chromosome condensaClarke HJ, Masui Y (1986): Transformation of sperm nuclei to tion (PCC) of thymocyte nuclei in metaphase I1 mouse oocytes. Biol metaphase chromosomes in the cytoplasm of maturing oocytes of the Cell 56239-250. mouse. J Cell Biol 102:1039-1046. Clarke HJ, Masui Y (1987): Dose-dependent relationship between Szollosi D, Czolowska R, Sdtynska MS, Tarkowski AK (1986b): Remodelling of thymocyte nuclei in activated mouse oocytes: An oocyte cytoplasmic volume and transformation of sperm nuclei to ultrastructural study. Eur J Cell Biol 42140-151. metaphase chromosomes. J Cell Biol 104:831-840. Cuthbertson KSR (1983):Parthenogenetic activation of mouse oocytes Szollosi D, Czotowska R, Szollosi MS, Tarkowski AK (1988): Remodelling of mouse thymocyte nuclei depends on the time of their in vitro with ethanol and benzyl alcohol. J Exp Zool 226:311-314. transfer into activated, homologous oocytes. J Cell Sci 91:603-613. Czolowska R, Modlinski JA, Tarkowski AK (1984): Behaviour of thymocyte nuclei in non-activated and activated mouse oocytes. J Szollosi D, Szollosi MS, Czolowska R, Tarkowski AK (1990): Sperm penetration into immature mouse oocytes and nuclear changes Cell Sci 69:19-34. during maturation: An EM study. Biol Cell 6953-64. Ecklund PS, Levine L (1975): Mouse sperm basic nuclear protein: Electrophoretic characterization and fate after fertilization. J Cell Tarkowski AK (1977): In vitro development of haploid mouse embryos produced by bisection of one-cell fertilized eggs. J Embryol Exp Biol 66:251-262. Morphol38:187-202. Fraser LR, Drury LM (1975): The relationship between sperm concentration and fertilization in vitro of mouse eggs. Biol Reprod Tarkowski AK, Wr6blewska J (1967): Development of blastomeres of mouse eggs isolated at the 4- and 8-cell stage. J Embryol Exp 13:513-5 18. Morphol 18:155-180. Fulka J, Motlik J, Fulka J, Crozet N (1985): Inhibition of nuclear maturation in fully grown porcine and mouse oocytes after their Whittingham DG (1971): Culture of mouse ova. J Reprod Fertil (Suppij14:7-21. fusion with growing porcine oocytes. J Exp Zool 235:255-259. Fulka J, Motlik J, Fulka J, Crozet N (1986): Activity of maturation Witkowska A (1981): Pronuclear develoDment and the first cleavage division in polyspermic mouse eggs. jReprod Fertil 62:493-498promoting factors in mammalian oocytes after its dilution by single Wright SJ, Longo F J (1988): Sperm nuclei enlargement in fertilized and multiple fusions. Dev Biol 118:176-181. hamster eggs is related to meiotic maturation of the maternal Fulton BP, Whittingham DG (1978): Activation of mammalian chromatin. J Exp Zool 247:155-165. oocytes by intracellular injection of calcium. Nature 273:149-151.
Anucleate Fragments of Parthenogenetic Eggs and of Maturing Oocytes Contain Complementary Factors Required for Development of a Male Pronucleus EWA BORSUK Department of Embryology, Institute of Zoology, University of Warsaw, Warsaw, Poland, Station Centrale de Physiologie Animale, I N R A , Jouy-en-Josas, France Tarkowski and Tittenbrun, personal communication; Borsuk and Tarkowski, 1989). However, the capability of activated cytoplasm to promote transformation of the sperm nucleus into the male pronucleus decreases when the time interval between egg activation and sperm penetration is extended. As it was shown by Borsuk and Tarkowski (1989) anucleate fragments of activated oocytes bisected soon after activation (before the female pronucleus is formed) can promote full or partial transformation of sperm nuclei longer than their nucleate counterparts. However, even in the anucleate fragments this capability slowly disappears. In fragments penetrated 8 h post activation sperm nuclei remain in the cytoplasm unchanged or form abnormal nuclei. The morphology of these nuclei suggests that the breakdown of the sperm nuclear envelope has not occurred and that partial decondensation of chromatin takes place inside the old nuclear envelope (Borsuk and Tarkowski, 1989). It follows that factors responsible for nuclear envelope breakdown and chromatin decondensation become eventually inactivated even in the absence of the female pronucleus and that under these circumstances, the extrachromosomal constituents of the GV cannot support normal pronuclear growth. This study was designed to test the hypothesis that development of the male pronuclei requires factors found in both developing and activated oocytes. Spermatozoa can penetrate the cytoplasm of mouse oocytes at the germinal vesicle stage (Iwamatsu and Chang, 1972; Szollosi et al., 1990) but sperm nuclei remain intact as long as the oocyte nucleus stays in Key Words: Mouse oocytes, Cybrids, Pronuclei Fertilinterphase. After initiation of maturation and germiization, Cytoplasmic factors nal vesicle breakdown (GVBD) sperm nuclei undergo decondensation but do not transform into male pronuINTRODUCTION clei. Sperm nuclei behave similarly in anucleate fragIn normal development, the spermatozoon enters the ments produced before GVBD (our unpublished obsermouse oocyte at the metaphase stage of the second vations). These results confirm earlier findings meiotic division and activates it. One of the visible (BaXakier and Czolowska, 1977)that the factors responeffects of activation is the extrusion of the 2nd polar sible for nuclear envelope breakdown and modification body and formation of female and male pronuclei. From earlier observations it is known that sperm nuclei can Received June 19, 1990; accepted December 27, 1990. transform into male pronuclei also in oocytes in which Address reprint requests to Ewa Borsuk, Department of Embryology, the penetration of the sperm was delayed in relation to Institute of Zoology, University of Warsaw, Krakowskie Przedmiescie oocyte activation (Chalmel, 1962; Komar, 1982; 26/28, 00-927 Warsaw 64, Poland. 0 1991 WILEY-LISS, INC.
ABSTRACT Eight-h-old anucleate fragments of artificially activated mouse oocytes produced within 30 min after activation were fused with anucleate fragments of ovarian oocytes bisected at the GV stage and matured in vitro for 4-5 h. The resulting cybrids were inseminated soon after fusion and fixed 3-4 h or 14 h later. After 3-4 h of culture most of the sperm nuclei underwent decondensation. Early male pronuclei were observed sporadically. After overnight culture small or fully formed male pronuclei were present in monospermic and dispermic cybrids.The abortive nuclei, designated as stage Ila (earlier described by Borsuk and Tarkowski, 1989) were also observed and their occurrence was strongly correlated with the rate of polyspermy. In unfused (control) anucleate fragments of maturing and activated oocytes inseminated at the same time as the cybrids, male pronuclei were never observed. These results show that the cytoplasms of activated eggs and of maturing oocytes contain complementary factors required for the transformation of the sperm nucleus into the male pronucleus. The former has lost the capability to break down the sperm nucleus envelope but contains factors required for pronuclear growth. The latter is able only to initiate the process of transformation, i.e., to break down the nuclear envelope and decondense the denuded sperm chromatin. However, the activity of the factor responsible for nuclear envelope breakdown is limited because in cybrids penetrated by more than two spermatozoa, transformation of sperm nuclei was usually abnormal.
SPERM NUCLEI IN CYBRIDS of chromatin structure (decondensation of sperm nuclei and condensation of interphase nuclei) appear autonomously in the cytoplasm of maturing oocytes. The above observations were extended in the present study by the use of anucleate fragments of parthenogenetic eggs and maturing oocytes in the following experimental design. Two types of cytoplasts, an 8h-old parthenogenetic and a maturing one (the first probably carrying factors required for pronuclear growth, the second carrying factors responsible for nuclear envelope breakdown and chromatin decondensation), were fused together and subsequently fertilized to determine whether the cytoplasm of the resulting cybrids would support transformation of the sperm nucleus into a male pronucleus (see Fig. 1).The results of this experiment appear to confirm the hypothesis that the two types of fragments contain complementary factors for the development of pronuclei.
MATERIALS AND METHODS Collection and Handling of Oocytes Ovarian ooc tes. Fl(C57BL x CBA/H) females were injected witrl 5 iu PMSG (Folligon, Intervet) and killed 46-48 h later. The ovaries were placed in M2 medium (medium 16 buffered with HEPES, Fulton and Whittingham, 1978) into which dibutyryl cyclic AMP (dbcAMP, Sigma Chemical Co., St. Louis, MO) in conc. 50 Fgirnl had been added to arrest oocytes in germinal vesicle stage during manipulations. Oocytes were released by puncturing the ovarian follicles with a needle. Most of the oocytes were free of cumulus cells. Cumulus cells which remained attached to some of the oocytes were removed mechanically by pipetting. The zona pellucida was removed with a-chymotrypsin (30 Fg/ml) or with acidic Tyrode's solution (pH 2.5). Zonafree oocytes were placed in medium M2 containing dbcAMP and supplemented with 5 p.g/ml of cytochalasin D (CD) (Sigma Chemical Co., St. Louis, MO) and 0.5 p.g/ml of nocodazol (Sigma Chemical Co., St. Louis, MO). After 30 min, incubation at 37°C the oocytes were transferred into cold medium (5°C) for 15-20 min and then bisected into halves (nucleate and anucleate) with a glass needle on a surface of 1%agar, according to the technique of Tarkowski (1977).Pairs of fragments were washed carefully in medium 16 (Whittingham, 1971) and placed in microdrops of this medium under liquid paraffin. They were cultured at 37.5"C under 5% C02in air. The anucleate fragments of those pairs, which had started maturation (called in this paper maturing cytoplasts), as was judged by the disappearance of the germinal vesicle (GV) in the nucleate fragment, were used as partners for fusion with anucleate fragments of activated oocytes (Fig. 1). Artificially activated oocytes. Metaphase I1 oocytes were recovered from Fl(C57BL x CBAIH) females induced to ovulate with 5 iu PMSG followed 48-54 h later by 5 iu HCG. Females were sacrificed 16 h after HCG administration and the ovulated oocytes were released into M2 medium containing hyaluronidase (200-300 iu/ml). The zona pellucida was removed with a-chymotrypsin. Zona-free oocytes were artificially activated by a 6 min exposure to 8% ethanol
151
GV
MI t
CULTURE
1 1 00
\
/
E LECTROFUS I O N
i
t CULTURE
Fig. 1. Diagramatic representation of the experiment. The lapse of time between activation and insemination of cybrids-8 hours, between the transfer of fragments of GV oocytes into the dbcAMP-free medium and insemination of cybrids-5-6 hours.
solution in M2 (Cuthbertson, 1983). Thirty minutes post activation oocytes were bisected into halves (one with a spindle and the other anucleate). Pairs of fragments were cultured in microdrops of M16 medium and the anucleate counterparts of those nucleate fragments, which had undergone activation (the 2nd polar body extruded within 2 h of bisection, and the female pronucleus formed), were used for fusion (Fig. 1).
Cell Fusion Pairs of cytoplasts [maturing (3-4 h after dbcAMP removal) and activated1 agglutinated previously in
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phytohaemaglutynin (PHA, 300 pg/ml in M2 without BSA, 3-5 min) were washed twice in a 0.3 M glucose solution in 18 MR pure water supplemented with 100 pM Ca ions and 100 pM Mg ions and then treated individually with an electric current (two pulses, 40 V, duration 200 p.s) in the same solution (Kubiak and Tarkowski, 1985; Ozil and Modlinski, 1986). After this treatment they were washed in M2 medium and placed in drops of M16 under liquid paraffin in standard culture conditions. Those pairs which had undergone fusion were fertilized in vitro.
Fertilization In Vitro Fertilization in vitro was carried out in Whittingham’s medium 16 (1971) as modified by Fraser and Drury (1975) and containing 32 mg of crystalline bovine serum albumin (Sigma Chemical Co., St. Louis, MO). The medium had been equilibrated overnight at 37.5”Cin an atmosphere of 5% C02 in air, under liquid paraffin. A sperm suspension was prepared from the caudae epididymides of adult Fl(C57B1 x CBNH) males. Sperm from two caudae was released into 1 ml of medium. Before mixing the gametes, the sperm suspension was preincubated for 1 h at 375°C under standard conditions. Cybrids were placed in 1 ml of medium and 100 p1 of sperm suspension was added (giving the final concentration of about 2 x lo6 sperm/ ml). At the moment of insemination the age of the two components of cybrids was 8 h post activation and 2-3 h after GVBD (5-6 h after dbcAMP removal). Cybrids were kept with spermatozoa for 1-1.5 h. Then they were washed in M16 and left in culture until the moment of fixation. Some of the cybrids were fixed 3-4 h post insemination. The others were cultured overnight and fixed 14 h post insemination.
RESULTS Transformation of Sperm Nuclei in Cybrids Between Activated and Maturing Cytoplasts Out of 228 ovarian oocytes bisected at the GV stage, 217 (95.2%) survived the operation, and 142 (65.4%) started to mature 2-3 h after dbcAMP removal. Four hundred seventy-six zona-free metaphase I1 oocytes were submitted to alcohol treatment and bisected 20 min post activation. Out of these, 418 (87.8%) survived bisection and 354 pairs (82.3%) underwent activation (2nd polar body extruded and the female pronucleus present in the nucleate fragment). Out of 133 pairs of activated and maturing cytoplasts submitted to electric pulses, 95 (84.1%)underwent fusion and were inseminated shortly afterwards. Sixty-two cybrids (65.2%)were fertilized. Polyspermic fertilization predominated. Only 12 cybrids (19.3%)were monospermic. Among polyspermic cybrids dispermic ones were the most common (27 out of 50) (Table 1). Sperm nuclei were classified according to the previously described classification (Borsuk and Tarkowski, 1989; Fig. 2); 3 4 hours post insemination stage I1 of sperm nucleus decondensation predominated (88% of cybrids, Fig. 3A). Early male pronuclei (stage 111, Fig. 3B) or abnormal nuclei denoted as IIa were present sporadically (Table 2). After overnight culture, three stages of sperm nuclei transformation were found with almost the same frequency: stage 111, early male pronucleus; stage IV, fully formed male pronucleus (Fig. 3C), and stage IIa (Fig. 3D) (Table 2). The nuclei designated as IIa were first observed in anucleate fragments of activated oocytes inseminated 8 h post activation (Series A in Borsuk and Tarkowski, 1989). The karyoplasm of these nuclei is homogenous and darkly stained, with characteristic dark patches of more condensed chromatin. They are surrounded by a light sphere in cytoplasm (Fig. 3D). In contrast to the IIa nuclei observed in oocyte fragments, which are oval or tear shaped, those observed in cybrids are in most cases spherical. Results obtained in this experiment, especially after overnight culture, showed that in more than 60% of the cybrids sperm nuclei could transform into nuclei, which, at least at the light microscope level, resembled normal male pronuclei (stages I11 and IV). However, in 32.4% of the cybrids abnormal nuclei (stage IIa) were observed. The ability of cybrids to promote transformation of sperm nuclei into male pronuclei was strongly correlated with the rate of polyspermy. Only in the case of monospermy and dispermy were male pronuclei formed in all cybrids (Table 3). With the increasing degree of polyspermy, formation of male pronuclei decreased, and in highly polyspermic cybrids the nuclei in stage IIa predominated.
Control Experiments 1. Metaphase I1 oocytes were activated, bisected, and cultured in pairs as described in “Artificially activated oocytes.”Pairs of anucleate fragments were fused in an electric field 6-7 h post activation and inseminated 1-2 h later (8 h after activation). They were fixed after the same time intervals as the experimental cybrids ( 3 4 h or 14 h post insemination). 2. Anucleate fragments of activated and maturing oocytes were inseminated and fixed at the same time as both types of cybrids (experimental and control). 3. Ovarian oocytes were obtained and treated as described in “Ovarian oocytes.” After storage in cold medium M2 containing dbcAMP and supplemented with CD and nocodazol, they were washed carefully in medium 16 and cultured (as intact oocytes)for 18-19 h. Those oocytes which had reached the metaphase I1 stage (the first polar body extruded) were fertilized in vitro and fixed 5-6 h post insemination. Transformation of Sperm Nuclei in Activated Cybrids, fragments (activated and maturing), and and Maturing Cytoplasts oocytes were examined in whole mount, haematoxylinstained preparations (Tarkowski and Wroblewska, Out of 68 anucleate fragments of activated oocytes 1967). inseminated 8 h post activation 18 lysed after insemi-
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TABLE 1. Frequency of Monospermy and Polyspermy in Cybrids* Total Type
of cybrid
1
AM AA
1 2 2 7 9 12 3 7
N u m b e r of sperm nuclei 2 3 4 5
7 3
1 4
6 6
-
number of cybrids 62 29
*AM, cybrids between activated and m a t u r i n g cytoplasts; AA, cybrids between two activated cytoplasts.
A
\
Stage Ila
Stage I
I
Fig. 3. ADecondensing sperm nucleus (stage 11).The posterior part decondensed, the anterior part still condensed and darkly stained. AM-cybrid, 3 h post insemination x 1,000. B: Early male pronucleus (stage 111). AM-cybrid, 14 h post insemination. x 1,000. C: Fully formed male pronucleus (stage IV). AM-cybrid, 14 h post insemination x 1,000. D: Sperm nucleus in stage IIa surrounded by a light zone in the cytoplasm (arrow). AM-cybrid, 14 h post insemination. x 1,000.
overnight culture in the majority of fragments decondensed sperm nuclei (stage 11)were present. The cytoplasts in which sperm nuclei did not undergo any changes probably represent those oocyte fragments Fig. 2. Schematic representation of sperm nucleus transformation which did not initiate the process of maturation. Neiin anucleate fragments of activated oocytes and in cybrids. Stage I: ther male pronuclei nor IIa nuclei were ever observed unchanged sperm head. Stage 11: stage of sperm nucleus decondensa- (Table 4). tion (full or partial). Stage 111: early male pronucleus. Stage IV: fully formed male pronucleus. Stage IIa: abnormal nuclei probably decondensing inside the old nuclear envelope.
nation. Among the remaining 50 cytoplasts, 47 (94%) were fertilized. Male pronuclei (stages I11 and IV) were never observed, and in the majority of fragments abnormal nuclei (stage IIa) were found. Sporadically the sperm nuclei remained unchanged or their transformation was blocked during decondensation (Table 4). The anucleate fragments of maturing oocytes, bisected at the GV stage and cultured for 5-6 h before insemination, promoted decondensation of sperm nuclei; 3-4 h post insemination in 39.3%of the fragments, sperm nuclei began decondensation and in 60.7% remained unchanged (stage I). This suggests that the process of maturation started late and proceeded very slowly. Indeed, the first nucleate fragments which underwent germinal vesicle breakdown were observed not earlier than 3 h after dbcAMP removal. After
Transformation of Sperm Nuclei in Control Cybrids (Between Two Activated Cytoplasts) Forty-five pairs of activated cytoplasts were agglutinated in PHA and subjected to electric pulses 6-7 h post activation. Out of these, 41 (91.1%)underwent fusion and were fertilized in vitro 8 h after activation. Twentynine (70.7%)cybrids survived this treatment and were cultured for 3-4 or 14 h before fixation. Monospermic fertilization was observed in 41.4%of the cases (Table 1). In the majority of cybrids fixed 3-4 h and 14 h post insemination, sperm nuclei underwent transformation into abnormal nuclei denoted as stage IIa (Table 2). However, these nuclei were smaller and more darkly stained than IIa nuclei present in experimental cybrids (between activated and maturing cytoplasts), and were identical with the nuclei found in 8-h-old anucleate parthenogenetic fragments (Borsuk and Tarkowski, 1989 and this paper). They were accompanied by a pair of granules resembling those observed in nucleate and anucleate fragments of activated oocytes (Borsuk and
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TABLE 2. Transformation of Sperm Nuclei in Cybrids*
Type of cvbrid
Time post. insemination (h) 3-4
AM"
14 3-4
AA" 14
Stages of sperm nucleus transformation (%)
I
I1
IIa
I11
IV
Total number of cvbrids
- 88.0 4.0 8.0 - - 32.4 32.4 35.2 - 18.2 81.8
37 11
-
18
-
94.4
5.6
-
25
*Both monospermic and polyspermic cybrids are included. In the case of polyspermic cybrids, the most advanced sperm nucleus served a s a basis for classification. "See footnote to Table 1.
Tarkowski, 1989). Most often the granules occurred in the vicinity of the nuclei, but sometimes they moved away from them. This experiment showed that the enlargement of the amount of activated cytoplasm did not improve sperm nucleus transformation.
Transformation of Sperm Nuclei in Fully Matured Oocytes The ovarian oocytes were treated similarly to those which were used for bisection and then cultured intact for 18-19 h to determine whether the short culture in the presence of dbcAMP, nocodazol, and cytochalasin D did not affect their maturation and whether they could support formation of male and female pronuclei after fertilization. Out of 100 oocytes submitted to such treatment, 65 had reached the metaphase I1 stage. During the process of insemination and later culture 12 oocytes lysed. By 5-6 h after insemination 68.2% (30/44) of the oocytes that had been penetrated by sperm extruded the second polar body and formed a female and one or more male pronuclei. In the rest of penetrated oocytes the 2nd polar body was also emitted but the female chromatin was blocked at the telophase stage. The sperm nuclei remained arrested during decondensation or at the stage of slight condensation usually preceding formation of the male pronucleus. These results show that oocytes which were submitted to short culture in the presence of dbcAMP, nocodazol, and CD could, after removal of the drugs, undergo maturation in vitro and could support formation of male and female pronuclei after fertilization. DISCUSSION The sperm nucleus after its penetration into egg cytoplasm undergoes a number of changes which lead to the formation of the male pronucleus. This process includes reduction of disulphide bonds of protamines, nuclear envelope breakdown, decondensation of chromatin, replacement of protamines by histones (Kopeeny and Pavlok, 19751, and, after a short stage of chromatin condensation (Stefanini et al., 1970; Eck-
TABLE 3. Transformation of Sperm Nuclei in Monospermic and Dispermic AM Cybrids* Time Stages of sperm nucleus Total post transformation number inseminaof (%I tion I1 IIa I11 IV cybrids I 3-4 14
-
78.9
-
-
21.1 45
55
19 20
*See footnote to Table 1.
lund and Levine, 1975; Borsuk and Manka, 1988; Wright and Longo, 19881,reformation of a new nuclear envelope. In normal fertilization all these events take place during the transition of the oocyte from metaphase I1 to interphase and seem to be controlled by different cytoplasmic factors. The first steps of sperm nucleus transformation, i.e., reduction of disulphide bonds of protamines, nuclear envelope breakdown, and chromatin decondensation, occur when the oocyte chromosomes are in condensed state (between metaphase I1 and telophase). During this phase the cytoplasm still exhibits strong chromosome condensing activity (CCA, Masui, 1985) and promotes nuclear envelope breakdown and premature chromosome condensation (PCC) in any interphase nucleus introduced at this moment into the oocyte (Balakier, 1978; Czolowska et al., 1984; Szollosi et al., 1986a,b; 1988).The sperm nucleus reacts to this environment differently: its chromatin decondenses. The subsequent events in the process of male pronucleus formation (reformation of nuclear envelope and nuclear swelling) occur when the CCA of the oocyte cytoplasm disappears and is replaced by the chromosome decondensing activity (CDA) promoting the interphase state of the egg cell (Masui, 1985).During the growth of male and female pronuclei, nuclear lamins (Maul and Schatten, 1986; Kubiak, 1988) and probably also extra-chromosomal constituents of the germinal vesicle released after its breakdown are absorbed from the cytoplasm. Studies on the behaviour of sperm nuclei in artificially activated intact oocytes (Komar, 1982; Tarkowski and Tittenbrun, personal communication) and their nucleate and anucleate fragments (Borsuk and Tarkowski, 1989) have shown that the capability of activated cytoplasm to promote male pronucleus formation gradually disappears. This was especially interesting in the case of anucleate fragments of oocytes bisected soon after activation (Series A in Borsuk and Tarkowski, 1989). Although their cytoplasm must be rich in all substances required for pronucleus formation and its growth (because of the lack of a competitive female pronucleus), sperm nuclei which penetrated many hours post activation either remained unchanged or formed abnormal nuclei apparently decondensing inside a persistant nuclear envelope. Results obtained in the present study clearly show that anucleate fragments of activated oocytes produced soon after activation and cultured for 8 h before insem-
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TABLE 4. Transformation of Sperm Nuclei in Anucleate Fragments of Activated Oocytes and Maturing Oocytes Total Time number post Stages of sperm nucleus of Tvve insemitransformation of nation (%o) fragfragments (h) I I1 IIa 111 IV ments 14 7.1 28.6 64.3 Activated 3-4 31 3.2 6.5 90.3 cytoplasts 14 39.3 28 Maturing 3-4 60.7 cytoplasts 14 13.6 86.4 22
-
ination can recover their ability to promote male pronucleus formation after fusion with cytoplasts originating from GV-intact oocytes matured in vitro. In more than 60% of such AM cybrids, small or fully formed male pronuclei were found after 14 h of culture. If unfused, both types of cytoplasts could promote only partial transformation of sperm nuclei. In 86.4% of the cytoplasts obtained from GV-intact oocytes maturing in vitro, sperm nuclei were blocked at stage I1 of chromatin decondensation. Also the prolonged post insemination culture of activated cytoplasts did not improve the development of sperm nuclei; formation of male pronuclei was never observed. However, in comparison with fragments fixed 3 4 h post insemination, more nuclei underwent transformation into stage IIa. This suggests that when the sperm nuclear envelope is not broken down, the process of chromatin decondensation is greatly slowed down. Similar results were obtained after insemination and culture of cybrids between two activated cytoplasts (AA cybrids), which shows that limited, abortive transformation of sperm nuclei in oocyte fragments was not related to the decreased. volume of the cytoplasm. The cytoplasm of AM cybrids acquired all factors and substances essential for the full transformation of the sperm nucleus into the male pronucleus. The cytoplast obtained by bisection of GV-intact oocytes matured in vitro was the source of active MPF necessary for the breakdown of the sperm nucleus envelope. However, by itself such cytoplasm could not promote male pronucleus formation for at least two reasons: 1) at the moment of sperm penetration the process of oocyte maturation was not completed and 2) cytoplasts used in this experiment were obtained before GVBD and they were deprived of the extrachromosomal constituents of GV which are necessary for the growth of the pronucleus (Balakier and Tarkowski, 1980). These two conditions were satisfied by the activated component of the cybrid. In more than 30%of the cybrids sperm nuclei did not transform into male pronuclei and formed abortive nuclei designated as stage IIa. Their occurrence was strongly correlated with the rate of polyspermy. They were not observed in monospermic and dispermic cybrids. Starting from the trispermic ones the frequency of their formation increased and in highly polyspermic
cybrids (six sperm nuclei), this type of nuclei predominated. Retarded development of supernumerary sperm nuclei after in vitro fertilization of ovulated oocytes was reported by Witkowska (1981) and explained by the probable disappearance or inactivation of some cytoplasmic factors responsible for normal transformation of sperm nucleus into the male pronucleus. Results obtained in our earlier experiment (Borsuk and Tarkowki, 1989) and in this study showed that for the development of male pronuclei, the presence of active MPF in the cytoplasm is very important. A certain critical threshold of this factor has to be achieved in every nucleus to promote its nuclear envelope breakdown. The MPF is probably distributed uniformly among all the nuclei present in the cytoplasm (Clarke and Masui, 1986, 1987). In AM cybrids the cytoplasm originating from GV intact oocytes and matured in vitro was the only source of that factor. The volume of maturing cytoplasts was reduced to about 1/2 of the volume of intact oocyte and probably the activity of cytoplasmic MPF present in such fragments was adequate to cause nuclear envelope breakdown in a limited number of nuclei. Furthermore, this activity could have been partly neutralized by an antagonistic activity connected with the interphase component of the cybrid (the 8-h-oldparthenogenetic cytoplast). Such activity was found in G1 somatic cells (Adlakha et al., 1983; Rao and Adlakha, 19851, in growing pig and mouse oocytes (Fulka et al., 1985, 19861, and in mouse zygotes (Balakier and Masui, 1986).The IIa nuclei may have originated from those sperm nuclei which could not undergo nuclear envelope dissolution because of the lack of active MPF in the cytoplasm but which could nevertheless undergo chromatin decondensation. U1trastructural studies in progress will better characterize the morphology of sperm nuclei developing in cybrids, especially those in stage IIa.
ACKNOWLEDGMENTS This work was carried out within the framework of a French-Polish scientific exchange programme. WHO Small Institutional Grant to the Department of Embryology is kindly acknowledged. This investigation was partially financed by a research grant from the Polish Academy of Sciences CPBP 04.01. I wish to thank Professor Andrzej K. Tarkowski and Doctor Dan
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Szollosi for their interest and helpful advises and for their valuable comments on the manuscript. I wish to thank Mr. Jean-Pierre Ozil for helpful assistance in electrofusion.
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