Microtubules forming within the mouse egg during fertilization are required for the movements leading to the union of the sperm and egg nuclei (male and female pronuclei, respectively). In the unfertilized oocyte, microtubules are predominantly found in the arrested meiotic spindle. At the time for sperm incorporation, a dozen cytoplasmic asters assemble, often associated with the pronuclei. As the pronuclei move to the egg center, these asters enlarge into a dense array. At the end of first interphase, the dense array disassembles and is replaced by sheaths of microtubules surrounding the adjacent pronuclei. Syngamy (pronuclear fusion) is not observed; rather the adjacent paternal and maternal chromosome sets first meet at metaphase. The mitotic apparatus emerges from these perinuclear microtubules and is barrelshaped and anastral, reminiscent of plant cell spindles; the sperm centriole does not nucleate mitotic microtubules. After cleavage, monasters extend from each blastomere nucleus. The second division mitotic spindles also have broad poles, though by third and later diisions the spindles are typical for higher animals, with narrow mitotic poles and fusiform shapes. Colcemid, griseofulvin, and nocodazole inhibit the microtubule formation and prevent the movements leading to pronuclear union; the meiotic spindle is disassembled, and the maternal chromosomes are scattered throughout the oocyte cortex. These results indicate that microtubules forming within fertilized mouse oocytes are required for the union of the sperm and egg nuclei and raise questions about the paternal inheritance of centrioles in mammals.Fertilization results in the union of the parental genomes, and in most animals a microtubule-containing cytoskeleton forming within the activated egg participates in the motility necessary for the cytoplasmic migrations of the sperm and egg nuclei (reviewed in ref. 1). The participation of the egg microtubules during mammalian fertilization is less well understood, though microtubule inhibitors (2-4) prevent the completion of meiosis, resulting in polyploidy; microtubules have also been found within fertilized mammalian eggs with electron microscopy (5-8) and during oogenesis with immunofluorescence microscopy (9).To explore the participation of egg cytoplasmic microtubules during mammalian fertilization and early development, we have performed anti-tubulin immunofluorescence and transmission electron microscopy on mouse oocytes and zygotes* throughout fertilization and have studied the effects of microtubule inhibitors. These results indicate that the egg cytoplasmic microtubules, organized by sources other than the sperm centriole, are required during mammalian fertilization. MATERIALS AND METHODSVirgin CD-1 mice (Charles River Breeding Laboratories) were superovulated with 10 international units of pregnant mare serum followed 48 hr later with 10 international units of human chorionic gonadotropin (10) and introduced to experienced males. After mating, fertilized oocytes were collected (11, ...

The forms and locations of centrosomes in mouse oocytes and in sea urchin eggs were followed through the whole course of fertilization and first cleavage by immunofluorescence microscopy. Centrosomes were identified with an autoimmune antiserum to centrosomal material. Staining of the same preparations with tubulin antibody and with the DNA dye Hoechst 33258 allowed the correlation of the forms of the centrosomes with the microtubule structures that they generate and with the stages of meiosis, syngamy, and mitosis. The results with sea urchin eggs conform to Boveri's view on the paternal origin of the functional centrosomes. Centrosomes are seen in spermatozoa and enter the egg at fertilization. Initially, the centrosomes are compact, but as the eggs enter the mitotic cycle the forms of the centrosomes go through a cycle in which they spread during interphase, apparently divide, and condense into two compact poles by metaphase. In anaphase, they spread to form flat poles. In telophase and during reconstitution of the daughter nuclei, the centrosomal material is dposed as hemispherical caps around the poleward surfaces of the nuclei. Mouse sperm lack centrosomal antigen. In the unfertilized mouse oocyte, the meiotic spindle poles are displayed as broad-beaded centrosomes. In addition, centrosomal material is detected in the cytoplasm as particles, about 16 in number, which are foci of small aster-like arrays of microtubules. The length and number of astral microtubules correlate with the size of the centrosomal foci. After sperm incorporation, as the pronuclei develop and more cytoplasmic microtubules assemble, a few of the foci associate with the peripheries of the nuclei. The number of foci multiplies during the first cell cycle. At the end of interphase, all of the centrosomal foci have concentrated on the nuclear peripheries and the cytoplasmic microtubules have disappeared. At prophase, the centrosomes are seen as two irregular clusters, marking the poles which, at metaphase and anaphase, appear as rough bands with foci, and the spindle is typically barrel-shaped. At telophase, the centrosomes are seen as arcs that lie on the nuclear peripheries after cleavage. The ordering of microtubules in all the stages reflects the shapes of the centrosomes. The findings on the sea urchin confirm the classical theory of the paternal origin of centrosomes and contrast with observations tracing the mitotic poles of the mouse egg to maternal centrosomal material. This evidence strengthens the conclusion that mouse centrosomes derive from the oocyte. Mouse and sea urchin fertilization was as described (6). Sea urchin eggs were extracted in a microtubule-stabilization buffer (7), and mouse egg cytoskeletons were stabilized with a similar mixture (4). The cells were affixed to polylysinecoated coverslips (8). Sea urchin eggs were fixed in methanol at -10TC and mouse eggs were fixed in 10 mM ethylene glycol bis(succinimidyl)succinate (9). Autoimmune centrosomal antiserum 5051 was derived from a patient with scleroder...

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Nuclear structural changes during fertilization and embryogenesis in mice and in sea urchins have been followed by using antibodies against the nuclear lamins A/C and B and against antigens at the periphery of nuclei and chromosomes. Lamins are found on all pronuclei and nuclei during mouse fertilization, but with a diminished intensity on the second polar body nucleus. On sperm in both systems, lamins are reduced and detected only at the acrosomal and centriolar fossae. In sea urchin eggs, lamins are found on both pronuclei. Unlike in other dividing cells, the mitotic chromosomes of sea urchin eggs and embryos retain an association with lamins. The peripheral antibodies delineate each chromosome and nucleus except the mature mouse sperm nucleus. A dramatic change from the expected lamin distribution occurs during early development. In mouse morulae or blastocysts, lamins A/C are no longer recognized, although lamin B remains. In sea urchins both lamins A/C and lamin B, as detected with polyclonal antibodies, are lost after the blastula stage, although a different lamin A/C epitope emerges as recognized by a monoclonal antibody. These results demonstrate that pronucleus formation in both systems involves a new association or exposure of lamins, that the polar body nucleus is largely restricted from the cytoplasmic pool of lamins, and that mitotic chromosomes in the rapidly proliferating sea urchin egg retain associated lamins. They also suggest that changes in the expression or exposure of different lamins are a common feature of embryogenesis.Fertilization requires several dramatic changes in nuclear organization. The architecture of the nuclear surface (reviewed in refs. 1-4) involves the nuclear lamins, which are typically three proteins subjacent to the inner nuclear membrane (5, 6), and nuclear peripheral proteins referred to as "P1" (7) or "Perichromin" (8), which probably reside between the chromatin and the nuclear lamins. During mitosis in somatic cells, the lamins dissociate from the nuclear envelope at prophase and reappear with the reconstituting envelope at telophase (9). Unlike the behavior of the lamins at mitosis, the peripheral antigens separate from the nuclear periphery and ensheathe the condensing chromosomes before nuclear envelope breakdown and dissolution of the lamins during mitosis (7,8). During spermatogenesis the nuclear lamins are lost or vastly reduced (10-12), whereas during oogenesis the lamina may be comprised of only a single lamin (13-16), which differs from somatic lamins (17).In this study the presence and distribution of nuclear lamins and of the nuclear and chromosomal P1 peripheral antigens are traced during fertilization and embryogenesis in mice and in sea urchins. These two systems represent

Microtubule and microfilament organization in porcine oocytes during maturation in vivo and in vitro was imaged by immunocytochemistry and laser scanning confocal microscopy. At the germinal vesicle stage, microtubules were not detected in the oocyte. After germinal vesicle breakdown, a small microtubule aster was observed near the condensed chromatin. During the prometaphase stage, microtubule asters were found in association with each chromatin mass. The asters then elongated and encompassed the chromatin at the metaphase‐I stage. At anaphase‐I and telophase‐I microtubules were detected in the meiotic spindle. Microtubules were observed only in the second meiotic spindle at the metaphase‐II stage. The meiotic spindle was a symmetric, barrel‐shaped structure containing anastral broad poles, located peripherally and radially oriented. Taxol, a microtubule‐stabilizing agent, did not induce microtubules in oocytes at the germinal vesicle stage. After germinal vesicle breakdown, numerous cytoplasmic foci of microtubules were formed in the entire oocyte when oocytes were incubated in the presence of taxol. Microfilaments were observed as a relatively thick uniform area around the cell cortex and were also found throughout the cytoplasm of oocytes at the germinal vesicle stage. After germinal vesicle breakdown, the microfilaments were concentrated close to the female chromatin. During prometaphase, microfilaments were chromatin moved to the peripheral position. At metaphase‐I, two domains, a thick and a thin microfilament area, existed in the egg cortex. Chromosomes were located in the thick microfilament domain of the cortex. In summary, these results suggest that both micro‐tubules and microfilaments are closely involved with chromosomal dynamics after germinal vesicle breakdown and during meiotic maturation in porcine oocytes. © 1996 Wiley‐Liss, Inc.

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