The maturation divisions of the parthenogenetic stick insect Carausius morosus Br. (Orthoptera,Phasmidae)

The meiotic stages in the maturation of the egg of the parthenogenetic stick insect Carausius morosus Br. is described. The meiosis consists of two equational divisions and parthenogenesis is thus apomictic. The first prometaphase is formed between 5.8–5.5 days before oviposition; the first metaphase lasts until oviposition; the meiosis ends between 14 and 24 hours after oviposition. An extra chromosome doubling occurs before first anaphase, causing the first metaphase chromosomes to resemble bivalents and requiring that meiosis consists of two divisions instead of one. A terminal affinity between the daughter chromosomes influences chromosome movement during first and second metaphase and anaphase. The first and second polar bodies degenerate. The first polar body divides amitotically during pycnosis. Meiosis takes place ventrally in the egg, the germ anlage development dorsally. The pronucleus divides mitotically in the ventral part of the egg during its migration from ventral to dorsal, enabling blastoderm development to take place both ventrally and dorsally.     

Reproduction and the mechanism of meiotic restitution in the parthenogenetic lizard Cnemidophorus uniparens

Gross details of the reproductive cycle and the cytology of oogenesis were studied in 155 egg clutches produced by 69 captive individuals of the triploid parthenogenetic lizard Cnemidophorus uniparens. The mean clutch cycle lasted 23 days. The mean number of ova per clutch was 3.3, and the mean number of oocytes per right and left ovaries was 1.65 and 1.70, respectively. Comparison of the size of the oocytes at ovulation (9–10 mm) with the estimated mean duration of vitellogenesis (8.8 days) gave an average of approximately 1 mm yolk deposition per day. The mean time for the retention of eggs in the oviducts was 9.3 days. The germinal disc of the oocyte consists of a series of layers formed by the arrangement of various cytoplasmic and yolk particles in the polar region. In a mature oocyte the germinal vesicle is located immediately below the vitelline membrane and lies at the center of the germinal disc. The germinal vesicle is characterized by a dense disc-like cluster of diplotene chromosomes. Diplonema extends until near ovulation when the oocytes have attained a size of about 9 mm. Diakinesis and metaphase I occur rapidly and immediately prior to ovulation. Counts of approximately as many bivalents as there are somatic chromosomes were obtained from oocytes at diakinesis and metaphase I. The second division occurs almost immediately before or at the precise moment of ovulation. The chromosomes of the first polar body consist of dyads, of which there are as many as the triploid number of 69. A metaphase II plate obtained in polar view also revealed dyad chromosomes, of which there were approximately as many as the triploid somatic number. The second telophase is normal as evidenced by formation of the second polar body. Chromosomes from the opposing telophase plates show a monad structure. The presence of as many bivalents in the first division as the triploid somatic number of 69 indicates that the 3N condition of C. uniparens was doubled prior to meiosis. This is further supported by the occurrence of two maturation divisions each giving rise to a polar body, by the dyad structure of the chromosomes in the first polar body and the second metaphase, and by the presence of monochromosomes at telophase II. Thus, parthenogenesis in these lizards is of the meiotic type. The somatic number of chromosomes is doubled early in oogenesis presumably by a premeiotic endoduplication, and the 3N level is restored by two subsequent maturation divisions.     

Cytological studies of the two-spotted spider mite Tetranychus urticae Koch (Tetranychidae,trombidiformes). I: Meiosis in eggs

Meiosis in eggs of Tetranychus urticae Koch is described. The two maturation divisions result in (a) a haploid female pronucleus consisting of three karyomeres; — (b) a divided first polar body in which the chromosomes change into karyomeres; — (c) a second polar body, entering a new mitosis which is blocked in metaphase. Irradiation of adult females produced chromosome fragments in the meiotic divisions. The fragments behave as intact chromosomes which proves that during meiosis a diffuse kinetochore is present. The meiotic divisions show the cytologically characteristic features of an inverted meiosis. The presence of such a meiosis is corroborated by observations on eggs heterozygous for chromosome mutations. In both maturation divisions the chromosomes are orientated equatorially. It is suggested that the equatorial orientation is brought about by chiasmata having terminalized to both ends of the dyads. It is argued that in organisms with holokinetic chromosomes during meiosis an axial orientation of the bivalents does not necessarily imply a normal meiosis but can also imply an inverted meiosis.This work was carried out with financial aid of the Institute for Atomic Sciences in Agriculture and the Ministry of Public Health and Environment.     

Premeiotic endomitosis produces diploid eggs in the natural clone loach, Misgurnus anguillicaudatus (Teleostei: Cobitidae)

The natural clone loach produces unreduced eggs genetically identical to somatic cells of the mother fish and such diploid eggs normally develop as a clone without genetic contribution of sperm. Following the identification of clonal nature and diploidy of eggs, we conducted cytological studies to determine the mechanisms responsible for this unusual oogenesis. Cytolological observation of full-grown oocytes cultured in vitro revealed that oocytes of both the clone and the control loach underwent two successive meiotic divisions: formation of a bipolar spindle and metaphase in meiosis I and equal segregation of chromosomes, extrusion of the first polar body and the appearance of metaphase of meiosis II. However, spindle size of the clone was larger than that of the control. Bivalent chromosome number of germinal vesicle of oocytes was 25 in the control diploid, whereas 50 in the clone. The results suggest that chromosomes are duplicated by mitosis without cytokinesis before meiosis, i.e. premeiotic endomitosis and then oocytes differentiated from tetraploid oogonia undergo a quasinormal meiosis followed by two successive divisions to produce diploid eggs.     

Centriole behavior during meiosis in oocytes of the sea urchin Hemicentrotus pulcherrimus

Ultrastructural changes in the maturing oocyte of the sea urchin Hemicentrotus pulcherrimus were observed, with special reference to the behavior of centrioles and chromosomes, using oocytes that had spontaneously started the maturation division process in vitro after dissection from ovaries. The proportion of oocytes entering the maturation process differed from batch to batch. In those eggs that accomplished the maturation division, it took ~4.5-5 h from the beginning of germinal vesicle breakdown to the formation of a second polar body. Serial sections revealed that a young oocyte before germinal vesicle breakdown had a pair of centrioles with procentrioles, located between the presumed animal pole and the germinal vesicle and accompanied by amorphous aggregates of moderately dense material and dense granules (granular aggregate). Just before germinal vesicle breakdown, a pair of fully grown centrioles located in the granular aggregate, which is present until this stage and then disappears, had already separated from another pair of centrioles. In meiosis I, each division pole had two centrioles, whereas in meiosis II each had only one. The two centrioles in the secondary oocyte separated into single units and formed the mitotic figure of meiosis II. The first polar body had two centrioles and the second had only one. The two centrioles in the first polar body did not form the mitotic figure nor did they separate at the time of meiosis II. These results indicate that, in sea urchins, duplication of the centrioles does not occur during the two successive meiotic divisions and the egg inherits only one centriole from the primary oocyte, confirming the results previously reported for starfish oocytes.     

The cell cycle control protein cdc25C is present, and phosphorylated on serine 214 in the transition from germinal vesicle to metaphase II in human oocyte meiosis

Cdc25C is a dual specificity phosphatase essential for dephosphorylation and activation of cyclin-dependent kinase 1 (cdk1), a prerequisite step for mitosis in all eucaryotes. Cdc25C activation requires phosphorylation on at least six sites including serine 214 (S214) which is essential for metaphase/anaphase transit. Here, we have investigated S214 phosphorylation during human meiosis with the objectives of determining if this mitotic phosphatase cdc25C participates in final meiotic divisions in human oocytes. One hundred forty-eight human oocytes from controlled ovarian stimulation protocols were stained for immunofluorescence: 33 germinal vesicle (GV), 37 metaphase stage I (MI), and 78 unfertilized metaphase stage II (MII). Results were stage dependent, identical, independent of infertility type, or stimulation protocol. During GV stages, phospho-cdc25C is localized at the oocyte periphery. During early meiosis I (MI), phosphorylated cdc25C is no longer detected until onset of meiosis I. Here, phospho-cdc25C localizes on interstitial microtubules and at the cell periphery corresponding to the point of polar body expulsion. As the first polar body reaches the periphery, phosphorylated cdc25C is localized at the junction corresponding to the mid body position. On polar body expulsion, the interior signal for phospho-cdc25C is lost, but remains clearly visible in the extruded polar body. In atresic or damaged oocytes, the polar body no longer stains for phospho-cdc25C. Human cdc25C is both present and phosphorylated during meiosis I and localizes in a fashion similar to that seen during human mitotic divisions implying that the involvement of cdc25C is conserved and functional in meiotic cells.     

Mechanisms that prevent catastrophic interactions between paternal chromosomes and the oocyte meiotic spindle

Meiosis produces haploid gametes by accurately reducing chromosome ploidy through one round of DNA replication and two subsequent rounds of chromosome segregation and cell division. The cell divisions of female meiosis are highly asymmetric and give rise to a large egg and two very small polar bodies that do not contribute to development. These asymmetric divisions are driven by meiotic spindles that are small relative to the size of the egg and have one pole juxtaposed against the cell cortex to promote polar body extrusion. An additional unique feature of female meiosis is that fertilization occurs before extrusion of the second polar body in nearly all animal species. Thus sperm-derived chromosomes are present in the egg during female meiosis. Here, we explore the idea that the asymmetry of female meiosis spatially separates the sperm from the meiotic spindle to prevent detrimental interactions between the spindle and the paternal chromosomes.     

Holocentric plant meiosis: first sisters,then homologues

Meiosis is a crucial process of sexual reproduction by forming haploid gametes from diploid precursor cells. It involves 2 subsequent divisions (meiosis I and meiosis II) after one initial round of DNA replication. Homologous monocentric chromosomes are separated during the first and sister chromatids during the second meiotic division. The faithful segregation of monocentric chromosomes is realized by mono-orientation of fused sister kinetochores at metaphase I and by bi-orientation of sister kinetochores at metaphase II. Conventionally this depends on a 2-step loss of cohesion, along chromosome arms during meiosis I and at sister centromeres during meiosis II.     

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.     

Localisation of phosphorylated MAP kinase during the transition from meiosis I to meiosis II in pig oocytes

Mitogen-activated protein kinase (MAPK) has been reported to be involved in oocyte maturation in all animals so far examined. In the present study we investigate the expression and localisation of active phosphorylated MAPKs (p44ERK1/p42ERK2) during maturation of pig oocytes. In immunoblot analysis using anti-p44ERK1 antibody which recognised both active and inactive forms of p44ERK1 and p42ERK2, we confirmed that MAPKs were phosphorylated around the time of germinal vesicle breakdown (GVBD) and the active phosphorylated MAPKs (pMAKs) were maintained until metaphase II, as has been reported. On immunofluorescent confocal microscopy using anti-pMAPK antibody which recognised only phosphorylated forms of MAPKs, pMAPK was localised at the spindle poles in pig mitotic cells. On the other hand, in pig oocytes, no signal was detected during GV stage. After GVBD, the area around condensed chromosomes was preferentially stained at metaphase I although whole cytoplasm was faintly stained. At early anaphase I, the polar regions of the meiotic spindle were prominently stained. However, during the progression of anaphase I and telophase I pMAPK was detected at the mid-zone of the elongated spindle, gradually becoming concentrated at the centre. Finally, at the time of emission of the first polar body, pMAPK was detected as a ring-like structure between the condensed chromosomes and the first polar body, and the staining was maintained even after the metaphase II spindle was formed. The inhibition of MAPK activity with the MAPK kinase inhibitor U0126 during the meiosis I/meiosis II transition suppressed chromosome separation, first polar body emission and formation of the metaphase II spindle. From these results, we propose that the spindle-associated pMAPKs play an important role in the events occurring during the meiosis I/meiosis II transition, such as chromosome separation, spindle elongation and cleavage furrow formation in pig oocytes.     

6-Dimethylaminopurine (6-DMAP), a reversible inhibitor of the transition to metaphase during the first meiotic cell division of the mouse oocyte

The first meiotic cell division (meiotic maturation) of dictyate stage mouse oocytes removed from the follicle resumes spontaneously in vitro. We used the puromycin analog 6-dimethylaminopurine (6-DMAP) to test the respective roles of protein synthesis and protein phosphorylation in driving this process. While protein synthesis inhibitors do not block meiosis resumption, 6-DMAP was found to inhibit germinal vesicle breakdown (GVBD), by inhibiting the burst of protein phosphorylation without changing the rate of incorporation of [35S]methionine into proteins. This effect is reversible; it depends both upon drug concentration and the particular female. When added after GVBD and before the emission of the first polar body, 6-DMAP decreases the level of protein phosphorylation and induces decondensation of the chromosomes and reformation of the nuclear envelope. In contrast, 6-DMAP did not trigger these processes in metaphase II oocytes which only produce resting nuclei when treated by protein synthesis inhibitors. From these data, we conclude that (1) the early appearance and stability of mouse MPF in Metaphase I oocytes depend on protein phosphorylation rather than on protein synthesis, and (2) protein synthesis is necessary to maintain the condensation of the chromosomes in metaphase II oocytes.     

A cinematographic study of meiosis in salamander spermatocytes in vitro

Summary Salamander spermatocytes were isolated in a modified Eagle's medium in Rose Chambers. The behavior of the spermatocytes during meiosis was recorded on a time lapse, phase contrast film. The two meiotic divisions progressed without visible irregularities in freshly isolated spermatocytes. Times required for the various meiotic events were obtained. Spermatocytes four days in vitro carried out the first meiotic division, but there were many abnormalities and the second meiotic division did not occur. At first meiotic metaphase, whole bivalent oscillations were accompanied by a relatively higher frequency oscillatory movement of the two homologous kinetochore regions. Oscillations of the kinetochore region were independently variable in magnitude and frequency. A system is proposed by which the metaphase bivalent movements are explained in terms of two pulling forces acting with variable intensity and frequency in opposite directions at the two homologous kinetochores. Meiosis in heavily compressed spermatocytes was blocked at the first meiotic metaphase, apparently because of the absence of a bipolar meiotic apparatus. In compressed spermatocytes, the centrosome divided but the two resulting centrosomes failed to reach their definitive polar positions. After about two hours of separation, the two centrosomes reversed their movement and fused to form a single centrosome from which a unipolar half-spindle radiated.This investigation was supported by grant GB-15 from the National Science Foundation and by Public Health Service Research Grant GB 12431-02 from the Division of General Medical Sciences.Deceased June 17, 1964.     

A metaphase pause: Hormone-induced maturation progresses through a long pause at the first meiotic metaphase in oocytes of the starfish, Pisaster ochraceus

In several species of starfish, it has been reported that the meiotic divisions in fertilized oocytes occur precociously compared to those in unfertilized oocytes. The nature of the 'acceleration' of meiosis was studied using Pisaster ochraceus oocytes. The extent of the acceleration of first polar body formation was found to be completely dependent on the time of fertilization (or artificial activation); fertilization at about 100 min after 1–methyladenine application accelerated meiosis I the most, while earlier or later fertilization resulted in a smaller extent of accelerations of meiosis I. Observation of isolated meiotic spindles and fluorescent visualization of meiotic spindles in whole oocytes showed that progression of meiosis I in Pisaster oocytes pauses transiently at metaphase I for more than 40min unless they are activated. The activation shortened the duration of metaphase I, which resulted in the acceleration of first polar body formation. A new term 'metaphase pause' is proposed to define this long duration of metaphase I in starfish oocytes.     

The behaviour and structure of the sex chromosomes in spermatocytes of Microtus oeconomus

The meiotic behaviour and structure of the sex chromosomes of Microtus oeconomus (2n=30) in Giemsa stained preparations are described. The X-Y pair appears as a sex vesicle at late zygotene. At late pachytene an unfolded sex vesicle is visible. A condensed sex vesicle appears during pre-diffuse diplotene and starts to unfold again during post-diffuse diplotene. At diakinesis and metaphase I the X and Y chromosomes can be recognized in an end-to-end association. During anaphase I, interkinesis and metaphase II the sex chromosomes are heteropycnotic and can therefore easily be recognized during the final stages of meiosis. During spermiogenesis the X and Y chromosomes can be identified in Giemsa stained preparations until the stage of spermatid elongation.     

Study on oocyte maturation and activation of the common prawn palaemon serratus (Pennant): Relationship between oocyte maturation and the molt cycle cytological aspects

A detailed chronology of the cytological events related to maturation that take place within the reproduction molt cycle has been established. It has been shown that oocytes, initially arrested at prophase I, resume meiosis when approaching stage D₁? of the molt cycle, ie, 4–5 days before molting. The following steps characterize this premolt period of oocyte maturation: nuclear envelope folding, nucleolar dissociation, condensation of the chromosomes, and beginning of the breakdown of the nuclear envelope (GVBD). At the ultrastructural level, it has been confirmed that GVBD actually takes place at the D₁??D₂ stage transition, when the germinal vesicle still occupies a central position in the oocyte. The migration of the chromosome takes only a few hours and begins approximately 4 hr before molting. It is only 1–2 hr before molting that the divalent chromosomes that are not yet organized in a metaphase plate become visible at the surface of the oocyte. They lay in a nucleoplasmic area no longer limited by the nuclear envelope. Metaphase I is reached a few minutes after molting. A second meiotic block appears at this stage, which persists until spawning, ie, for about 24 hr. Fertilization occurs at the moment of spawning. In vitro fertilization experiments demonstrated that fertilization normally triggers the release of the second meiotic block. Extrusion of the two polar bodies can be easily observed using a method for clearing and staining the oocytes in toto.     

RNA- binding protein Stau2 is important for spindle integrity and meiosis progression in mouse oocytes

Staufen2 (Stau2) is a double-stranded RNA-binding protein involved in cell fate decision by regulating mRNA transport, mRNA stability, translation, and ribonucleoprotein assembly. Little is known about Stau2 expression and function in mammalian oocytes during meiosis. Herein we report the sub-cellular distribution and function of Stau2 in mouse oocyte meiosis. Western blot analysis revealed high and stable expression of Stau2 in oocytes from germinal vesicle (GV) to metaphase II (MII). Immunofluorescence showed that Stau2 was evenly distributed in oocytes at GV stage, and assembled as filaments after germinal vesicle breakdown (GVBD), particularly, colocalized with spindle at MI and MII. Stau2 was disassembled when microtubules were disrupted with nocodazole, on the other hand, when MTs were stabilized with taxol, Stau2 was not colocalized with the stabilized microtubules, but aggregated around the chromosomes array, indicating Stau2 assembly and colocalization with microtubules require both microtubule integrity and its normal dynamics. During interphase and mitosis of BHK and MEF cells, Stau2 was not distributed on microtubules, but colocalized with cis-Golgi marker GM130, implying its association with Golgi complex but not the spindle in fully differentiated somatic cells. Specific morpholino oligo-mediated Stau2 knockdown disrupted spindle formation, chromosome alignment and microtubule-kinetochore attachment in oocytes. The majority oocytes were arrested at MI stage, with bright MAD1 at kinetochores, indicating activation of spindle assembly checkpoint (SAC). Some oocytes were stranded at telophase I (TI), implying suppressed first polar body extrution. Together these data demonstrate that Stau2 is required for spindle formation and timely meiotic progression in mouse oocytes.     

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.     

Changes at the hamster oocyte surface from the germinal vesicle stage to ovulation

Immature and ovulated hamster oocytes were studied with the scanning electron microscope. Immature oocytes at the germinal vesicle stage have their surface uniformly covered by microvilli. When meiosis has progressed to the first meiotic metaphase the overlying surface shows the differentiation of a circular area 19 μm in diameter with a low density of microvilli. Later, from this region the first polar body emerges, and the oocyte surface at the point from which it was extruded shows a cluster of cytoplasmic, conical projections. When the zona-free oocytes are cultured at 37°C for 5 minutes these projections disappear and the oocyte surface at that point becomes smooth. However, when the oocytes remain in the oviduct for several hours after ovulation these projections remain unchanged. The in vitro interactions of capacitated hamster sperm with the immature oocyte was always seen at microvillus surfaces and never associated with the differentiated regions.     

Cyclic assembly-disassembly of cortical microtubules during maturation and early development of starfish oocytes

An extensive array of cortical microtubules in oocytes of the starfish Pisaster ochraceus undergoes multiple cycles of disappearance and reappearance during maturation and early development. These events were studied in isolated fragments of the oocyte cortex stained with antitubulin antibodies for indirect immunofluorescence. The meshwork of long microtubules is present in the cortex (a) of immature oocytes, i.e., before treatment with the maturation-inducing hormone 1-methyladenine, (b) for 10-20 min after treatment with 1-methyladenine, (c) after formation of the second polar body (in reduced numbers in unfertilized oocytes), and (d) in the intermitotic period between first and second cleavage divisions. The array of cortical microtubules is absent in oocytes (a) undergoing germinal vesicle breakdown, (b) during the two meiotic divisions (polar body divisions), and (c) during mitosis of the first and, perhaps, subsequent cleavage divisions. The cycle of assembly-disassembly of cortical microtubules is synchronized to the cycle of nuclear envelope breakdown and reformation and to the mitotic cycle; specifically, cortical microtubules are present when a nucleus is intact (germinal vesicle, female pronucleus, zygote nucleus, blastomere nucleus) and are absent whenever a meiotic or mitotic spindle is present. These findings are discussed in terms of microtubule organizing centers in eggs, possible triggers for microtubule assembly and disassembly, the eccentric location of the germinal vesicle, and the regulation of oocyte maturation and cell division.