Sperm nuclei entering parthenogenetically activated mouse oocytes before the first mitosis transform into pronuclei an ultrastructural study.код для вставкиСкачать
THE ANATOMICAL RECORD 243:516-518 (1995) Sperm Nuclei Entering Parthenogenetically Activated Mouse Oocytes Before the First Mitosis Transform Into Pronuclei AN ULTRASTRUCTURAL STUDY MAREK MALESZEWSKI Department of Embryology, Institute of Zoology, University of Warsaw, Warsaw, Poland ABSTRACT Background: This report is an extension of previous observations (Maleszewski 1992. Mol. Reprod. Dev., 33:215-221) on the behavior of mouse sperm nuclei incorporated into parthenogenetically activated mouse oocytes prior to the first cleavage division and undergoing transformation during mitosis. Method: Artificially activated mouse oocytes were inseminated in vitro and an ultrastructural analysis was performed of sperm-derived nuclei present in two parthenogenetic two-cell embryos. Results: Both chromatin and nuclear envelope of sperm derived-nuclei are structurally identical with those of oocyte-derived nuclei and of the nuclei of blastomeres of normal two-cell embryos. Conclusions: Cytoplasm of the parthenogenote during the first mitotic division has the ability to transform sperm nucleus into a male pronucleus just like the cytoplasm of a metaphase I1 oocyte. Q 1995 Wiley-Liss, Inc. Key words: Fertilization, Sperm Remodelling, Parthenogenetic Embryo, Mitosis, Mouse The ability of a mammalian oocyte to transform the sperm nucleus into a pronucleus varies according to the developmental stage of the oocyte (Usui and Yanagimachi, 1976; Balakier and Tarkowski, 1980; Borsuk and Tarkowski, 1989; Szollosi et al., 1990, 1994). Breakdown of the sperm nuclear envelope (NE) is one of many steps required for remodeling of the sperm nucleus during fertilization (Yanagimachi, 1994). Breakdown of sperm NE takes place during M phase (Szollosi et al., 1990; Maleszewski, 1992) and also during the transition period to the zygote interphase (Szollosi et al., 1994). In a previous paper Maleszewski (1992) demonstrated that when mouse oocytes are activated by ethanol and inseminated during the second half of the first embryonal cell cycle (between 9 and 13 hours after the onset of activation), sperm nuclei remain condensed until the first mitosis. During the first mitotic M phase, sperm nuclei decondense, subsequently recondense, and are passively displaced into the daughter blastomeres. In the two-cell embryos, sperm nuclei form interphase nuclei that are able to replicate DNA and to condense into discrete chromosomes during the M phase of the following division. These observations suggest that the cytoplasm of the parthenogenote during the first mitotic division has the ability to transform the sperm nucleus into a male pronucleus just like the cytoplasm of metaphase I1 oocytes. The present electron microscopic study gives data confirming suggestions coming from a light microscopic study and reports some new information. nol, and then freed of zonae pellucidae (Maleszewski, 1992). Between 9 and 13hours after ethanol activation, zona-free oocytes were inseminated in vitro (Maleszewski, 1992). Three hours after the completion of the first cleavage, two-cell embryos were examined under a n inverted microscope equipped with Nomarski interference optics. Embryos containing sperm-derived nuclei were fixed and processed for electron microscopic observations a s previously described (Szollosi et al., 1994). MATERIALS AND METHODS Received June 5, 1995; accepted July 25, 1995. Address reprint requests to Marek Maleszewski, Dept. of Embryology, Inst. of Zoology, University of Warsaw, 00-927 Warsaw 64 Poland. Metaphase I1 oocytes from F1 (C57BlxCBA/H) females were collected, artificially activated with etha0 1995 WILEY-LISS, INC. RESULTS AND DISCUSSION Ultrastructural examination was performed on two parthenogenetic two-cell embryos that were inseminated before the first cleavage division and were fixed 3 hours after completion of cleavage. In both embryos one blastomere had only one nucleus, while the other contained two nuclei (Fig. 1A). In one embryo we could identify the sperm-derived nucleus because sperm tail components associated with the implantation fossa (structure that attaches sperm tail to the head; Fawcett, 1975) were in close proximity to its NE (Fig. 1B). Furthermore, part of the sperm tail was found in proximity to the mid-body, inside the cytoplasmic bridge that connected the blastomeres (not shown). We found that both nuclei in one blastomere and the SPERM NUCLEI IN CLEAVING PARTHENOGENETIC EGGS Fig. 1. A: Two nuclei present in one of the blastomeres of a two-cell parthenogenetic embryo. x 4,000. B Fragment of the NE of spermderived nucleus. Implantation fossa (IF) is still visible in proximity to NE. BL, bleb. x 40,000. C: Portion of the NE of the sperm-derived nucleus with nuclear lamina (NL) associated with the internal mem- 517 brane of NE. Arrowhead, nuclear pore. x 67,500. D Nucleolus precursor body (NPB) in the sperm-derived nucleus has chromatin (CHI associated with its surface. In the fragment of NE are visible bleb (BL) and nuclear pore (arrowhead). x 37,500. E: Interchromatin granules (IG) present in the sperm-derived nucleus. x 36,000. 518 M. MALESZEWSKI solitary nucleus in the second had uniformly distributed nuclear pores throughout the entire NE (Fig. lC,D). The internal membrane of the NE formed blebs (Fig. lB,D) similar to control embryos. Filamentous material (probably nuclear lamina) was associated with the internal membrane of the NE (Fig. 1C). Several nucleolus-precursor bodies (NPBs) (Kopecny et al., 1989; Biggiogera et al., 1994) in the form of typical densely packed masses of fibrillar material were detected in all nuclei (Fig. lA,D). Some partially condensed chromatin was also associated with the surface of these inactive NPBs (Fig. 1D). Granular structures (interchromatin granules, perichromatin granules, and large granules typical of pronuclei and early blastomere nuclei) (Fakan and Odartchenko, 1980) were present in every nucleus (Fig. 1E). The presence of nuclear lamina and nuclear pores throughout the entire surface of the NE distinguishes the NE of the male pronucleus from sperm head NE, which is almost entirely devoid of nuclear pores and lamins (Eddy and O’Brien, 1994). Blebing of the inner leaflet of the NE was described in pronuclei and in the nuclei of two-cell embryos at the beginning of the second cell cycle (Szollosi and Szollosi, 1988).The presence of blebs in our experimental sperm-derived nuclei demonstrates that they can resume this activity following their remodeling in the parthenogenote during the first mitotic M phase. The above structural observations of sperm-derived nuclei strongly suggest that sperm nucleus transformation in the cytoplasm of parthenogenetic mitotic embryos corresponds to the steps of the remodeling of sperm nucleus during normal fertilization. These steps are: breakdown of the original NE, sperm chromatin decondensation followed by a period of recondensation, formation of the new NE, chromatin decondensation, and formation of nucleolus precursor bodies (Adenot et al., 1991; Biggiogera et al., 1994; Yanagimachi, 1994). Sperm chromatin can decondense under certain conditions in its original NE (Borsuk and Tarkowski, 1989; Borsuk, 1991, Szollosi et al., 1994). In such cases recondensation of the chromatin does not take place and the nuclei develop abnormally (Szollosi et al., 1994). It has already been observed by Maleszewski (1992) by light microscopy that chromatin of the sperm nuclei entering parthenogenote at the end of the first embryonal cell cycle decondenses and recondenses, suggesting the removal of its original NE. Thus, the cytoplasm of the mouse parthenogenote during its first mitotic division has all the abilities to remodel freshly entered sperm nucleus into active pronucleus [formation of a new NE and its blebing (present paper); DNA synthesis and ability of sperm-derived chromatin to condense into discrete chromosomes during the M phase of the following division (Maleszewski 1992)], as it does in the cytoplasm of mature oocytes during normal fertilization. ACKNOWLEDGMENTS I a m grateful to Dr. Maria S. Szollosi for her invaluable help during the experimental work and the manuscript preparation. I wish to thank Professor A.K. Tarkowski for his interest and helpful advice and Professor Ryuzo Yanagimachi for his valuable comments on the manuscript. This work was partly financed by a grant from the State Committee for Scientific Research (no. 6.6401.91.02) and was carried out within the framework of French-Polish Scientific Exchange Programme (ATP 12). A WHO Small Institutional Grant to the Department of Embryology is gratefully acknowledged. LITERATURE CITED Adenot, P.G., M.S. Szollosi, M. Geze, J.-P. Renard, and P. Debey 1991 Dynamics of paternal chromatin changes in live one-cell mouse embryo after natural fertilization. Mol. Reprod. Dev., 28:23-34. Balakier, H., and A.K. Tarkowski 1980 The role of germinal vesicle karyoplasm in the development of male pronucleus in the mouse. Exp. Cell. Res., 128t79-85. Biggiogera, M., T.E. Martin, J. 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