Microtubule configurations during fertilization, mitosis, and early development in the mouse and the requirement for egg microtubule-mediated motility during mammalian fertilization.

Abstract

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, ...

High incidence of multiple aster formation in vitrified-warmed bovine oocytes after in vitro fertilization

“…A high incidence of oocytes with multiple asters was notable in the vitrified group (Table 2, Fig, 1B). Except for rodents in which multiple cytoplasmic asters function as MTOC [22,23], each paternal centrosome organizes only a single aster and functions as an MTOC in many mammalian species. Conversely, parthenogenesis [39] and Taxol treatment to stabilize microtubules [19,40] can induce formation of cytoplasmic asters.…”

“…Experiment 3 was conducted to investigate whether the observed multiple asters were the cytoplasmic asters (maternal origin) or the fragmentation of sperm-aster (paternal origin). Based on the comparison of aster positioning between 5.5 and 8 hpi samples, the multiple asters frequently observed in the vitrified oocytes were considered not to be typical cytoplasmic asters of maternal origin reported in rodents oocytes [22,23], parthenogenetically activated horse oocytes [39], and Taxol-treated porcine [19] and human [40] oocytes. Because sperm cells lose most of the pericentriolar materials during spermiogenesis, sperm-derived centrosome can function as MTOC after recruiting centrosomal proteins dispersed in oocytes [18,21].…”

“…Conversely, rodent sperm lose their centrioles during spermiogenesis [21]. Hence, the oocytes use their own MTOC dispersed in the cytoplasm for aster formation; that is called cytoplasmic aster [22,23]. Timing of first cleavage in IVF-derived bovine oocytes is important for yield and quality of blastocysts, as oocytes cleaving earlier are more likely to become blastocysts [24,25], and the resulting blastocysts have higher cryosurvival potential [26] and higher pregnancy rates [24] than those cleaving later.…”

Contribution of spermatozoal centrosomes to the microtubule-organizing centre in Antarctic minke whale (Balaenoptera bonaerensis)

“…During fertilization in mammals, the centrosome introduced into the oocyte by the spermatozoon plays a critical role in assembly of the microtubule network that brings both male and female pronuclei to the center of the newly formed zygote, as reported in human (Simerly et al, 1995), rhesus monkey (Hewitson et al, 1996), rabbit (Pinto-Correia et al, 1994) and cattle (Navara et al, 1994). On the other hand, the paternal inheritance of MTOC does not occur in mouse (Schatten et al, 1985(Schatten et al, , 1986) and rat (Woolley & Fawcett, 1973) and the microtubule network developed from multiple cytoplasmic asters, instead of a single sperm aster, is involved in the migration of pronuclei. Rodent spermatozoa are lacking centrioles and the majority of pericentriolar proteins after spermiogenesis (Woolley & Fawcett, 1973;Manandhar et al, 1998Manandhar et al, , 1999.…”

Microtubule assembly crucial to bovine embryonic development in assisted reproductive technologies

“…Centrosome integrity and microtubule network are crucial to the events around fertilization, including the pronuclear development, migration and fusion, and the first mitotic division (Schatten et al 1985). The present review highlights the importance of spermatozoal centrosomes and microtubule assembly for successful fertilization and subsequent embryonic development in cattle.…”