Spermatozoan response to the toad egg matured after removal of germinal vesicle.

The germinal vesicle (GV) was removed from toad oocytes at various times after treatment with progesterone, and enucleated eggs were inseminated under conditions that ensured fertilization of nucleated control eggs. The eggs enucleated before the initiation of GV break-down did not show genuine cleavage. Cytological examinations revealed, however, that spermatozoa enter the eggs enucleated even before the hormone treatment, without subsequent formation of pronuclei or DNA synthesis. The same lack of response was observed when several detergent-pretreated sperm were injected into enucleated eggs. When GV material was injected back into enucleated oocytes, the injected spermatozoa underwent transformation and DNA synthesis, although in variable degrees, in the egg cytoplasm. It is concluded that the egg becomes fertilizable independently of the GV during the hormone-induced maturation process. After entering the egg, however, spermatozoa require GV material for their participation in the developmental process.     

APPEARANCE OF PRONUCLEUS-INDUCING ACTIVITY DURING HORMONALLY INDUCED MEIOTIC MATURATION IN TOAD OOCYTES*

Detergent-pretreated spermatozoa of the toad, Bufo bufo japonicus, transform into pronuclei when injected into progesterone-matured oocytes at 18 hr post-hormone treatment (PHT). These sperm, however, do not show any change when injected into the oocytes at the same age from which the germinal vesicle (GV) has been removed before the progesterone treatment. In an attempt to determine when and how the pronucleus-inducing activity (PIA) develops in hormonally induced maturation process, enucleated oocytes were injected with GV and sperm at various stages after the hormone treatment and electrically stimulated at 18 hr PHT. It was found that sperm pronuclei are induced only in those oocytes receiving GV before 14 hr PHT. The 1 hr pulse-treatment of maturing oocytes with cycloheximide between 8–18 hr PHT and the injection of sperm at 18 hr PHT revealed that PIA does not occur in the oocytes treated with the inhibitor during 10–14 hr PHT. Injection of α-amanitin into maturing oocytes had no effect in this respect. Determination of DNA synthetic activity in vitro of the oocyte extracts from various maturation stages showed that the net increase of the activity occurs before the formation of PIA. The activity of the cycloheximide-treated oocyte extracts utilizing native DNA did not correlate with the sensitivity of oocytes to the inhibitor with respect to PIA in situ. It is concluded that PIA develops, in association with the GV materials, by way of translational events at 10–14 hr PHT, being quiescent during later maturation stages, and commences to function as an activation response of oocytes at 18 hr PHT.     

Germinal vesicle materials are requisite for male pronucleus formation but not for change in the activities of CDK1 and MAP kinase during maturation and fertilization of pig oocytes

In amphibian oocytes, it is known that germinal vesicle (GV) materials are essential for sperm head decondensation but not for activation of MPF (CDK1 and cyclin B). However, in large animals, the role of GV materials in maturation and fertilization is not defined. In this study, we prepared enucleated pig oocytes at the GV stage and cultured them to examine the activation and inactivation of CDK1 and MAP kinase during maturation and after electro-activation. Moreover, enucleated GV-oocytes after maturation culture were inseminated or injected intracytoplasmically with spermatozoa to examine their ability to decondense the sperm chromatin. Enucleated oocytes showed similar activation/inactivation patterns of CDK1 and MAP kinase as sham-operated oocytes during maturation and after electro-stimulation or intracytoplasmic sperm injection. During the time corresponding to MI/MII transition of sham-operated oocytes, enucleated oocytes inactivated CDK1. However, penetrating sperm heads in enucleated oocytes did not decondense enough to form male pronuclei. To determine whether the factor(s) involved in sperm head decondensation remains associated with the chromatin after GV breakdown (GVBD), we did enucleation soon after GVBD (corresponding to pro-metaphase I, pMI) to remove only chromosomes. The injected sperm heads in pMI-enucleated oocytes decondensed and formed the male pronuclei. These results suggest that in pig oocytes, GV materials are not required for activation/inactivation of CDK1 and MAP kinase, but they are essential for male pronucleus formation.     

Development of pronuclei from human spermatozoa injected microsurgically into frog (Xenopus) eggs

Human spermatozoa were demembranated with Triton X-100 (TX) and injected into the mature eggs of Xenopus laevis. The nuclei of these spermatozoa decondensed and developed into pronuclei. Chromosomes did not appear in the eggs until the end of a 5-hr incubation period. When the demembranated human spermatozoa were further treated with dithiothreitol (DTT) before they were injected into the eggs, the sperm nuclear decondensation and pronuclear development took place considerably faster than in spermatozoa treated with the detergent alone. By the end of the 5-hr incubation period, decondensed chromatin threads or chromosome-like structures appeared, but none of the eggs cleaved. When human spermatozoa were injected into full-grown ovarian oocytes with intact germinal vesicle (GV) or oocytes which had matured without GV, the nuclei of a proportion of TX-treated and all TX-DTT-treated sperm decondensed but showed no sign of developing into pronuclei. Sperm nuclei injected into maturing oocytes formed condensed chromatin fragments as long as the oocytes were not activated, but they transformed into pronuclei when the oocytes were stimulated with electric shock. These results indicate that the cytoplasmic factors responsible for the decondensation of human sperm nuclei are present in egg cytoplasm independent of GV-materials. We also suggest that the factors controlling development of decondensed sperm nuclei into pronuclei are dependent on GV materials.     

The germinal vesicle material required for sperm pronuclear formation is located in the soluble fraction of egg cytoplasm

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 °C for 16 h but was inactivated by heating to 80 °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 paticulate 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.     

Formation of polar-body-like structures induced in polyspermic starfish oocytes that had been inseminated at the germinal vesicle stage

Immature starfish oocytes, which are arrested at the first meiotic prophase and contain a large nucleus called the germinal vesicle (GV), are known to accept multiple sperm on insemination. We found that if these polyspermic starfish oocytes are induced to mature, they often form small protrusion(s) adjacent to the first polar body emitted shortly earlier. We refer to these protrusion(s) as 'polar-body-like structures (PLS).' Fluorescent staining of PLS indicated that they were not merely cytoplasmic protrusions, but contained some chromatin. Maturing process of these polyspermic oocytes was examined by immnofluorescent staining, which showed that: (i) numerous sperm asters were observed after the onset of GV breakdown; (ii) before the first polar body (PB1) emission, a complex microtubular structure resembling a multipolar spindle was formed; and (iii) several isolated asters were observed after PB1 emission. These results indicate that PLS formation may be induced by interaction of meiosis-I spindle with paternal centrosomes incorporated at GV stage.     

Injection of sperm heads into immature rat oocytes

When sperm heads are injected into rat oocytes at the germinal vesicle stage the sperm heads remain intact until the germinal vesicle breaks down. Then they decondense but do not form pronuclei. This observation agrees with the results reported for in vitro fertilization of immature oocytes from rats, mice and hamsters.     

Dose-dependent relationship between oocyte cytoplasmic volume and transformation of sperm nuclei to metaphase chromosomes

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.     

Intra-oocyte localization of MAD2 and its relationship with kinetochores, microtubules, and chromosomes in rat oocytes during meiosis

The present study was designed to investigate subcellular localization of MAD2 in rat oocytes during meiotic maturation and its relationship with kinetochores, chromosomes, and microtubules. Oocytes at germinal vesicle (GV), prometaphase I (ProM-I), metaphase I (M-I), anaphase I (A-I), telophase I (T-I), and metaphase II (M-II) were fixed and immunostained for MAD2, kinetochores, microtubules and chromosomes. The stained oocytes were examined by confocal microscopy. Some oocytes from GV to M-II stages were treated by a microtubule disassembly drug, nocodazole, or treated by a microtubule stabilizer, Taxol, before examination. Anti-MAD2 antibody was also injected into the oocytes at GV stage and the injected oocytes were cultured for 6 h for examination of chromosome alignment and spindle formation. It was found that MAD2 was at the kinetochores in the oocytes at GV and ProM-I stages. Once the oocytes reached M-I stage in which an intact spindle was formed and all chromosomes were aligned at the equator of the spindle, MAD2 disappeared. However, when oocytes from GV to M-II stages were treated by nocodazole, spindles were destroyed and MAD2 was observed in all treated oocytes. When nocodazole-treated oocytes at M-I and M-II stages were washed and cultured for spindle recovery, it was found that, once the relationship between microtubules and chromosomes was established, MAD2 disappeared in the oocytes even though some chromosomes were not aligned at the equator of the spindle. On the other hand, when oocytes were treated with Taxol, MAD2 localization was not changed and was the same as that in the control. However, immunoblotting of MAD2 indicated that MAD2 was present in the oocytes at all stages; nocodazole and Taxol treatment did not influence the quantity of MAD2 in the cytoplasm. Significantly higher proportions of anti-MAD2 antibody-injected oocytes proceeded to premature A-I stage and more oocytes had misaligned chromosomes in the spindles. The present study indicates that MAD2 is a spindle checkpoint protein in rat oocytes during meiosis. When the spindle was destroyed by nocodazole, MAD2 was reactivated in the oocytes to overlook the attachment between chromosomes and microtubules. However, in this case, MAD2 could not check unaligned chromosomes in the recovered spindles, suggesting that a normal chromosome alignment is maintained only in the oocytes without any microtubule damages during maturation.     

Penetration in vitro of bovine oocytes during maturation by frozen-thawed spermatozoa

Bovine immature oocytes cultured for various times in TC-199 medium were inseminated with frozen-thawed spermatozoa in Medium BO with caffeine (5 mM) and heparin (10 micrograms/ml). Very high penetration rates (95-100%) were obtained in all oocytes which had been cultured for 0-20 h. When oocytes cultured for 0 and 4 h were inseminated, 100% of them were penetrated and had a decondensing sperm head and most of the oocytes remained at the stage of condensed germinal vesicle (GV) to telophase-I 20-22 h after insemination. The formation of male and female pronuclei was first observed in oocytes inseminated 8 h after culture. The proportions of polyspermy and average number of spermatozoa in penetrated oocytes gradually decreased as oocyte maturation proceeded. Penetration of at least one spermatozoon with a decondensing head into oocytes at the GV stage (without culture) was almost completed up to 8 h after insemination and at that time most of the penetrated oocytes were still at the stage of GV or condensed GV. These results indicate that maturation of bovine oocytes is not required for sperm penetration into the vitellus or for sperm nuclear decondensation under the in-vitro conditions used.     

The changes in sperm nuclei after penetrating fish oocytes matured without germinal vesicle material in their cytoplasm

The processes occurring from sperm penetration to chromosome formation in the cytoplasm of Oocytes matured in vitro, after removal of the germinal vesicle (GV) and before hormonal stimulation, were observed with electron microscope. The dechorionated oocytes, matured without the participation of the GV material, responded to sperm penetration by initiating a cortical reaction within 20 seconds after insemination. The pentrating sperm nuclei transformed to male pronuclei with vesiculation of the nuclear membrane, chromatin decondensation, and formation of a pronuclear membrane. Before cleavage, however, no chromosome formation was observed in these oocytes. Instead, the fully grown pronuclei change to a picnotic chromatin mass without or with an only fragmented nuclear membrane, then disappeared. On the contrary, sperm nuclei that penetrated into the cytoplasm of naked eggs containing GV material during maturation underwent pronuclear and chromosomal formation. Judging from these observation in Oryzias oocytes, the GV material seems to be unnecessary for the formation of pronucleus from the compact sperm nucleus, but is essential for the process of chromosomal formation.     

The induction of reversible and irreversible chromosome decondensation by protein synthesis inhibition during meiotic maturation of mouse oocytes

We investigated the effects of puromycin on mouse oocyte chromosomes during meiotic maturation in vitro. Puromycin treatment for 6 hr at 100 μg/ml almost completely, but reversibly, suppressed [³⁵S]methionine incorporation into oocyte protein at all stages of maturation tested. Nevertheless, oocytes treated at the germinal vesicle stage underwent germinal vesicle breakdown (GVBD) and chromosome condensation. These oocytes completed nuclear maturation to metaphase II (MII) if the inhibitor was withdrawn. Prolonged (24-hr) treatment, however, caused the chromsomes to degenerate. The chromosomes of oocytes treated shortly after GVBD for 6 hr remained condensed, but the oocytes failed to form a polar body. However, 24-hr treatment caused the chromosomes to decondense to form an interphase nucleus. Oocytes treated near MI for 6 hr gave off a polar body during the treatment, and their chromosomes decondensed to form a nucleus, which remained as long as the treatment was continued. However, if the puromycin was withdrawn, the chromosomes recondensed to a state morphologically similar to that at MII. Thus, the chromosome decondensation induced by protein synthesis inhibition at MI was reversible. Oocytes treated at MII, several hours after first polar body formation, also underwent chromosome decondensation to form a nucleus. In the continuous presence of puromycin, the chromosomes remained decondensed, but neither DNA synthesis nor mitosis occurred. However, following puromycin withdrawal, these occytes synthesised DNA and underwent mitosis. Thus, protein synthesis inhibition at MII, by parthenogenetically activating the oocytes, caused irreversible chromosome decondensation. Based on these observations, we discussed the roles of protein synthesis in the regulation of oocyte chromosome behaviour during meiotic maturation.     

Sperm penetration into immature mouse oocytes and nuclear changes during maturation: an EM study

The ultrastructure of oocyte and sperm nuclei was studied in mouse ovarian oocytes inseminated in vitro and cultured for 1 1/2 and 3 h in a medium containing dbcAMP or lacking the maturation inhibitor. In oocytes blocked at the germinal vesicle (GV) stage, certain maturation-linked changes were noted. Sperm apposition and sperm-oocyte fusion were similar to that during fertilization of ovulated oocytes. The sperm nucleus and its nuclear envelope remained intact after penetrating into the ovarian oocyte. One and a half h after removal of the drug (time 0 of maturation) the germinal vesicle (GV) and sperm nucleus remained intact. In oocytes maturing for 3 h, the nuclear envelopes of the GV and sperm nucleus had fragmented. The NE of the oocyte formed quadruple membranes while the NE of the sperm remained as flat vesicles. Oocyte chromatin condensed to form chromosomes, whereas at the same time the sperm chromatin was in the process of decondensation and was surrounded by fragments of the sperm NE. The sperm chromatin, composed of DNA complexed with protamines, consisted of thin fibrils; the individual fibrils measured 3.8 nm in diameter. Near the penetrated spermatozoa only occasional Mts were detected which were not related to the proximal centriole which was recognizable in the neck-piece of the flagellum. Thus in mouse oocytes the introduced sperm centriole is not capable of behaving as a centrosome and organizing microtubules in the form of an aster.     

Fer tyrosine kinase is required for germinal vesicle breakdown and meiosis-I in mouse oocytes

The control of microtubule and actin-mediated events that direct the physical arrangement and separation of chromosomes during meiosis is critical since failure to maintain chromosome organization can lead to germ cell aneuploidy. Our previous studies demonstrated a role for FYN tyrosine kinase in chromosome and spindle organization and in cortical polarity of the mature mammalian oocyte. In addition to Fyn, mammalian oocytes express the protein tyrosine kinase Fer at high levels relative to other tissues. The objective of the present study was to determine the function of this kinase in the oocyte. Feline encephalitis virus (FES)-related kinase (FER) protein was uniformly distributed in the ooplasm of small oocytes, but became concentrated in the germinal vesicle (GV) during oocyte growth. After germinal vesicle breakdown (GVBD), FER associated with the metaphase-I (MI) and metaphase-II (MII) spindles. Suppression of Fer expression by siRNA knockdown in GV stage oocytes did not prevent activation of cyclin dependent kinase 1 activity or chromosome condensation during in vitro maturation, but did arrest oocytes prior to GVBD or during MI. The resultant phenotype displayed condensed chromosomes trapped in the GV, or condensed chromosomes poorly arranged in a metaphase plate but with an underdeveloped spindle microtubule structure or chromosomes compacted into a tight sphere. The results demonstrate that FER kinase plays a critical role in oocyte meiotic spindle microtubule dynamics and may have an additional function in GVBD.     

Localization of mitotic arrest deficient 1 (MAD1) in mouse oocytes during the first meiosis and its functions as a spindle checkpoint protein

The present study was designed to investigate the localization of mitotic arrest deficient 1 (MAD1) in mouse oocytes during meiotic maturation and its relationship with kinetochores, chromosomes, and microtubules. Oocytes at various stages during the first meiosis were fixed and immunostained for MAD1, kinetochores, microtubules, and chromosomes. The stained oocytes were examined by confocal microscopy. Some oocytes were treated with nocodazole or Taxol before examination. The anti-MAD1 antibody was injected into the oocytes at the germinal vesicle (GV) stage for examination of chromosome alignment and spindle formation. It was found that MAD1 was present in the oocytes from the GV to prometaphase I stages around the nuclei. When the oocytes reached the metaphase I (M-I) to metaphase II (M-II) stages, MAD1 was mainly localized at the spindle poles. However, MAD1 relocated to the vicinity of the chromosomes when spindles were disassembled by nocodazole or cooling, and the relocated MAD1 moved back to the spindle poles during spindle recovery. Taxol treatment did not affect the MAD1 localization. Although anti-MAD1 antibody injection did not affect nuclear maturation, significantly higher proportions of injected oocytes had misaligned chromosomes when the oocytes reached the M-I to M-II stages. The results of the present study indicate that MAD1 is present in mouse oocytes at all stages during the first meiosis and that it participates in spindle checkpoint during meiosis. However, MAD1 could not check misaligned chromosomes during spindle recovery after the spindles were destroyed by drug or cooling, which caused some chromosomes to scatter in the oocytes.     

PKCβ1 regulates meiotic cell cycle in mouse oocyte

PKCβI, a member of the classical protein kinase C family, plays key roles in regulating cell cycle transition. Here, we report the expression, localization and functions of PKCβI in mouse oocyte meiotic maturation. PKCβI and p-PKCβI (phosphor-PKCβI) were expressed from germinal vesicle (GV) stage to metaphase II (MII) stage. Confocal microscopy revealed that PKCβI was localized in the GV and evenly distributed in the cytoplasm after GV breakdown (GVBD), and it was concentrated at the midbody at telophase in meiotic oocytes. While, p-PKCβI was concentrated at the spindle poles at the metaphase stages and associated with midbody at telophase. Depletion of PKCβI by specific siRNA injection resulted in defective spindles, accompanied with spindle assembly checkpoint activation, metaphase I arrest and failure of first polar body (PB1) extrusion. Live cell imaging analysis also revealed that knockdown of PKCβI resulted in abnormal spindles, misaligned chromosomes, and meiotic arrest of oocytes arrest at the Pro-MI/MI stage. PKCβI depletion did not affect the G2/M transition, but its overexpression delayed the G2/M transition through regulating Cyclin B1 level and Cdc2 activity. Our findings reveal that PKCβI is a critical regulator of meiotic cell cycle progression in oocytes.

Abbreviations: PKC, protein kinase C; COC, cumulus-oocyte complexes; GV, germinal vesicle; GVBD, germinal vesicle breakdown; Pro-MI, first pro-metaphase; MI, first metaphase; Tel I, telophase I; MII, second metaphase; PB1, first polar body; SAC, spindle assembly checkpoint     

Quantitative studies of changes in cortical granule number and distribution in the mouse oocyte during meiotic maturation

Cortical granules (CGs) undergo a substantial change in distribution in the mouse oocyte cortex during meiotic maturation. In order to determine the mechanism of their change in distribution near the time of ovulation, CG density, total number per oocyte, and domain areas were quantitated. CGs were visualized microscopically by Lens culinaris agglutinin-biotin and Texas red-strepavidin fluorescence as well as by electron microscopy. Immature germinal vesicle stage (GV) oocytes from adult mice had a continuous cortical localization with some interior granules. Mature oocytes had an asymmetric cortical distribution with a CG-free domain, overlying the meiosis II metaphase spindle, occupying 40% of the cortex. The mean CG densities of the granule-occupied cortex of mature oocytes and the entire cortex of GV oocytes were 43 and 34 CGs/100 micron 2, respectively. The mean total numbers of CGs/oocyte were 4127 (mature) and 7440 (GV), and staining was absent in fertilized oocytes with two pronuclei. Calcium ionophore (A23187)-activated mature oocytes had a mean total number of 1235 CGs, some of which may have been in the process of exocytosis. The first polar body had few CGs, and thus was unlikely to account for the difference in CG number between GV and mature oocytes. The smaller total number and higher density of CGs in mature mouse oocytes suggests that both exocytosis and redistribution are plausible mechanisms for the development of the CG-free domain. Prefertilization exocytosis could account for the locus of sperm penetration which others have reported to occur in the hemisphere opposite the meiotic spindle in the mouse.     

The cohesion stabilizer sororin favors DNA repair and chromosome segregation during mouse oocyte meiosis

Maintenance and timely termination of cohesion on chromosomes ensures accurate chromosome segregation to guard against aneuploidy in mammalian oocytes and subsequent chromosomally abnormal pregnancies. Sororin, a cohesion stabilizer whose relevance in antagonizing the anti-cohesive property of Wings-apart like protein (Wapl), has been characterized in mitosis; however, the role of Sororin remains unclear during mammalian oocyte meiosis. Here, we show that Sororin is required for DNA damage repair and cohesion maintenance on chromosomes, and consequently, for mouse oocyte meiotic program. Sororin is constantly expressed throughout meiosis and accumulates on chromatins at germinal vesicle (GV) stage/G2 phase. It localizes onto centromeres from germinal vesicle breakdown (GVBD) to metaphase II stage. Inactivation of Sororin compromises the GVBD and first polar body extrusion (PBE). Furthermore, Sororin inactivation induces DNA damage indicated by positive γH2AX foci in GV oocytes and precocious chromatin segregation in MII oocytes. Finally, our data indicate that PlK1 and MPF dissociate Sororin from chromosome arms without affecting its centromeric localization. Our results define Sororin as a determinant during mouse oocyte meiotic maturation by favoring DNA damage repair and chromosome separation, and thereby, maintaining the genome stability and generating haploid gametes.     

Injection of isolated centriolar complex to induce mitosis in starfish eggs

When centriolar complex isolated from starfish spermatozoa was injected into starfish immature oocytes (fully grown, germinal vesicle stage) followed by treating the latter with 1-methyladenine, mitotic events such as condensation and division of chromosomes of the female pronucleus and cytokinesis following completion of meiosis were observed. No cortical reaction detected in the oocytes. Essentially the same was noted for mature oocytes (pronuclear stage) into which the centriolar complex had been injected. The oocytes that had received sperm tail fraction or buffer alone did not initiate cleavage. It would thus appear that sperm centriolar complex is significantly essential to the initiation of cleavage.