The germinal vesicle material required for sperm pronuclear formation is located in the soluble fraction of egg cytoplasm.
ABSTRACT The chromatin of Xenopus laevis sperm nuclei was induced to decondense, swell and form mitotic chromosomes following its injection into mature Rana pipiens oocytes. In contrast, the sperm chromatin did not decondense or form mitotic chromosomes when injected into oocytes from which the germinal vesicle (GV) was removed prior to the initiation of maturation. Injection into enucleated oocytes of the material extracted from manually-isolated GVs restored their ability to decondense sperm nuclei. This soluble GV material was stable at 18 degrees C for 16 h but was inactivated by heating to 80 degrees C for 10 min. We examined the distribution of this GV material in a cytoplasmic preparation from activated eggs which can induce sperm pronuclear formation in vitro. The cytoplasmic preparation was separated into soluble and particulate fractions by centrifugation and then each fraction was injected into enucleated eggs to determine whether or not it restored the ability to decondense sperm nuclei. We found that the soluble, but not the particulate fraction could restore the ability to decondense sperm nuclei to enucleated oocytes. This result clearly indicates that the soluble fraction contains most of the GV material required for chromatin decondensation. However, since the soluble fraction fails to decondense sperm chromatin in vitro in the absence of material from the particulate fraction, sperm pronuclear formation appears to require both the soluble material derived from the GV and particulate material which can develop in the oocyte cytoplasm in the absence of the GV.
Article: Dose-dependent relationship between oocyte cytoplasmic volume and transformation of sperm nuclei to metaphase chromosomes.[show abstract] [hide abstract]
ABSTRACT: We have studied the chromosome condensation activity of mouse oocytes that have been inseminated during meiotic maturation. These oocytes remain unactivated, and in those penetrated by up to three or four sperm, each sperm nucleus is transformed, without prior development of a pronucleus, into metaphase chromosomes. However, those penetrated by more than four sperm never transform any of the nuclei into metaphase chromosomes (Clarke, H. J., and Y. Masui, 1986, J. Cell Biol. 102:1039-1046). We report here that, when the cytoplasmic volume of oocytes was doubled or tripled by cell fusion, up to five or eight sperm nuclei, respectively, could be transformed into metaphase chromosomes. Conversely, when the cytoplasmic volume was reduced by bisection of oocytes after the germinal vesicle (GV) had broken down, no more than two sperm could be transformed into metaphase chromosomes. Thus, the capacity of the oocyte cytoplasm to transform sperm nuclei to metaphase chromosomes was proportional to its volume. The contribution of the nucleoplasm of the GV and the cytoplasm outside the GV to the chromosome condensation activity was investigated by bisecting oocytes that contained a GV and then inseminating the nucleate and anucleate fragments. The anucleate fragments never induced sperm chromosome formation, indicating that GV nucleoplasm is required for this activity. In the nucleate fragments, the capacity to induce sperm chromosome formation was reduced as compared with whole oocytes, in spite of the fact that the fragments contained the entire GV nucleoplasm. This implies that non-GV cytoplasmic material also was required for chromosome condensation activity. When inseminated oocytes were incubated in the presence of puromycin, the sperm nuclei were transformed into interphase-like nuclei, but no metaphase chromosomes developed. However, when protein synthesis resumed, the interphase nuclei were transformed to metaphase chromosomes. These results suggest that the transformation of sperm nuclei to metaphase chromosomes in the cytoplasm of mouse oocytes requires both the nucleoplasm of the GV and non-GV cytoplasmic substances, including proteins synthesized during maturation.The Journal of Cell Biology 05/1987; 104(4):831-40. · 10.26 Impact Factor
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ABSTRACT: Ninety-five metaphase II human oocytes, aged in vitro for either one day or for two days, and five fresh immature oocytes with no visible germinal vesicle nucleus were partitioned into small cytoplasts after removal of the zona pellucida and exposure to cytochalasin B. Seventy-one metaphase II and four immature oocytes were used as intact zona-free controls. The cytoplasts derived from each partitioned oocyte and all the zona-free whole oocytes were exposed to normal or subfertile donor sperm and later assessed for signs of male pronucleus development. A total of 711 fragments (an average of 8 fragments per partitioned egg) with a mean diameter of about 50 microns were produced from the 100 partitioned oocytes. When exposed to normal sperm, 76% of the 1-day-old metaphase II fragments, 67% of the 2-day-old metaphase II fragments, and 83% of the immature oocyte fragments were fertilized. The mean number of decondensing nuclei per partitioned oocyte was 11.9 for the 1-day-old metaphase II oocytes, 6.4 for the 2-day-old metaphase II oocytes, and 13 for the immature oocytes. The mean number of decondensing nuclei per a whole zona-free oocyte was 5.6 for the 1-day-old metaphase II oocytes (p < 0.05), 6.7 for the 2-day-old metaphase II oocytes (NS), and 13 for the immature oocytes. When exposed to subfertile sperm, 54% of the 1-day-old metaphase II fragments and 28% of the 2-day-old metaphase II fragments were fertilized.(ABSTRACT TRUNCATED AT 250 WORDS)Biology of Reproduction 07/1995; 53(1):209-13. · 4.01 Impact Factor
Article: Nuclear envelope formation by chromatin-mediated reorganization of the endoplasmic reticulum.[show abstract] [hide abstract]
ABSTRACT: The formation of the nuclear envelope (NE) around chromatin is a major membrane-remodelling event that occurs during cell division of metazoa. It is unclear whether the nuclear membrane reforms by the fusion of NE fragments or if it re-emerges from an intact tubular network of the endoplasmic reticulum (ER). Here, we show that NE formation and expansion requires a tubular ER network and occurs efficiently in the presence of the membrane fusion inhibitor GTPgammaS. Chromatin recruitment of membranes, which is initiated by tubule-end binding, followed by the formation, expansion and sealing of flat membrane sheets, is mediated by DNA-binding proteins residing in the ER. Thus, chromatin plays an active role in reshaping of the ER during NE formation.Nature Cell Biology 11/2007; 9(10):1160-6. · 19.49 Impact Factor