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.     

Nonequivalence of maternal centrosomes/centrioles in starfish oocytes: selective casting-off of reproductive centrioles into polar bodies

It is believed that in most animals only the paternal centrosome provides the division poles for mitosis in zygotes. This paternal inheritance of the centrosomes depends on the selective loss of the maternal centrosome. In order to understand the mechanism of centrosome inheritance, the behavior of all maternal centrosomes/centrioles was investigated throughout the meiotic and mitotic cycles by using starfish eggs that had polar body (PB) formation suppressed. In starfish oocytes, the centrioles do not duplicate during meiosis II. Hence, each centrosome of the meiosis II spindle has only one centriole, whereas in meiosis I, each has a pair of centrioles. When two pairs of meiosis I centrioles were retained in the cytoplasm of oocytes by complete suppression of PB extrusion, they separated into four single centrioles in meiosis II. However, after completion of the meiotic process, only two of the four single centrioles were found in addition to the pronucleus. When the two single centrioles of a meiosis II spindle were retained in the oocyte cytoplasm by suppressing the extrusion of the second PB, only one centriole was found with the pronucleus after the completion of the meiotic process. When these PB-suppressed eggs were artificially activated to drive the mitotic cycles, all the surviving single centrioles duplicated repeatedly to form pairs of centrioles, which could organize mitotic spindles. These results indicate that the maternal centrioles are not equivalent in their intrinsic stability and reproductive capacity. The centrosomes with the reproductive centrioles are selectively cast off into the PBs, resulting in the mature egg inheriting a nonreproductive centriole, which would degrade shortly after the completion of meiosis.     

Reproductive maternal centrosomes are cast off into polar bodies during maturation division in starfish oocytes

Animal egg inherits a maternal centrosome from the meiosis-II spindle and sperm can introduce another centrosome at fertilization. It has been believed that in most animals only the sperm centrosome provides the division poles for mitosis in zygotes. This uniparental (paternal) inheritance of the centrosome must depend on the loss of the maternal centrosome. In starfish, suppression of polar body (PB) extrusion is a prerequisite for induction of parthenogenesis (Washitani-Nemoto et al. (1994) Dev. Biol. 163, 293-301), suggesting that the centrosomes cast off into PBs have reproducing capacity. Due to the absence of centriole duplication in meiosis II of starfish oocytes, each centrosome of a meiosis-II spindle has only one single centriole, whereas in meiosis I each has a pair of centrioles (Sluder et al. (1989) Dev. Biol. 131, 567-579; Kato et al. (1990) Dev. Growth Differ. 32, 41-49). Hence, the first PB (PB1) has two centrioles, whereas the second PB (PB2) and the mature egg have only one centriole, respectively. The present study examined the reproductive capacity of PB centrosomes by transplanting them into artificially activated eggs, and then the recipient egg nucleus with the surrounding cytoplasm was removed. A transplanted PB2 centrosome with a single centriole formed a monopolar spindle at the first mitosis, followed by formation of a bipolar spindle in the next mitosis, leading to actual cleavage and subsequent development. This proves the reproducing capacity of the single centriole in the PB2 centrosome. The behavior of the transplanted PB1 centrosome was exactly the same as in the PB2 centrosome, in spite of the difference in the number of centrioles. These results clearly show that four maternal centrioles are heterogeneous in duplicating capacity, during meiosis only one centriole in each of the centrosomes of a meiosis-I spindle pole retains duplicating capacity, the reproductive centrioles are successively cast off into PBs, and finally a mature egg inheriting a nonreproductive centriole alone is formed, and the presence of a single reproductive centriole is sufficient condition for embryonic development in starfish.     

Mother centrioles are kicked out so that starfish zygote can grow

Most oocytes eliminate their centrioles during meiotic divisions through unclear mechanisms. In this issue, Borrego-Pinto et al. (2016. J Cell. Biol. http://dx.doi.org/10.1083/jcb.201510083) show that mother centrioles need to be eliminated from starfish oocytes by extrusion into the polar bodies for successful embryo development.Canonical centrosomes contain a pair of centrioles, often made of nine triplets of microtubules and surrounded by the pericentriolar material (PCM). They are the major microtubule organizing centers in most cells, which organize the microtubule spindle required to segregate chromosomes during cell division. Yet, most oocytes get rid of their centrioles. The biological significance of oocyte centriole riddance remains a mystery. Removing centrioles in oocytes could prevent some species, like Xenopus, from undergoing parthenogenetic development (Tournier et al., 1991). Also, eliminating the maternal centrioles is required to prevent the zygote from having an abnormal number of centrioles after fertilization, as sperm contribute two centrioles (motile sperm cells require centriole-based flagellar assembly and must retain their centrioles until fertilization [Manandhar et al., 2005]). In Drosophila, Xenopus, nematode, mouse, and human oocytes, egg centrioles are eliminated during meiotic prophase before oocyte asymmetric divisions (Szollosi et al., 1972; Manandhar et al., 2005; Januschke et al., 2006). Apart from the involvement of a helicase of undefined substrates, the pathway leading to centriole elimination has not been identified (Mikeladze-Dvali et al., 2012).In contrast, starfish oocytes, like sea urchin or mollusk, eliminate their centrioles later in meiotic divisions (Nakashima and Kato, 2001; Shirato et al., 2006). Centrioles are replicated in a semiconservative manner during the S phase of the cell cycle. The old centriole, named the mother, is characterized by the presence of distal and subdistal appendages and serves as a template for the assembly of a new daughter centriole, lacking appendages (Bornens and Gönczy, 2014). However, to become haploid, oocytes undergo two consecutive divisions with no intervening DNA replication. Hence, centrioles are not duplicated between the two meiotic divisions and oocytes keep their number of centrioles limited to four. This also means that starfish oocytes assemble their first meiotic spindle in the presence of a pair of centrioles at each pole (Fig. 1 A). Out of the four centrioles contained in the oocyte, two (one mother and one daughter centriole) are extruded into the first polar body during the first asymmetric division. Subsequently, the second meiotic spindle is formed with only one centriole per pole (Fig. 1 A), and one centriole is extruded in the second polar body. Previous work suggested that the poles of the second meiotic spindle in starfish are not functionally equivalent (Uetake et al., 2002). In this issue, Borrego-Pinto et al. find that the mother centriole retains the ability to nucleate asters but is specifically guided into the second polar body for extrusion, whereas the daughter centriole is inactivated and then eliminated within the oocyte.Open in a separate windowFigure 1.Centriole elimination during meiotic maturation of starfish oocytes. (A) Scheme of starfish oocyte meiotic divisions and early egg development. Oocyte divisions are asymmetric in size; meiotic spindles are off-centered in these large cells; and daughter cells are tiny, tailored to the chromatin mass, and named polar bodies. Microtubules are green, DNA is pink, maternal centrosomes are yellow, and sperm centrosomes are orange. (B) Fate of mother and daughter centrioles during meiotic divisions. Centrosomes are artificially enlarged to emphasize the centrioles. PB1 and PB2, first and second polar body, respectively. During anaphase I, the DNA and centrioles are segregated; one set of chromosomes and one pair of centrioles are extruded into PB1 during anaphase I. The remaining mother centriole separates from its paired daughter and rapidly moves toward the plasma membrane, where it is extruded in the second polar body (PB2) during anaphase II, leaving one set of oocyte chromatids to combine with the sperm chromatids. The remaining oocyte daughter centriole is inactivated and degraded after anaphase II. Therefore, only the sperm centrioles form the first mitotic spindle in the fertilized oocyte. Oocytes forced to retain a mother centriole form a tripolar aster upon fertilization, which stops development.To investigate the mechanism of centriole elimination in the starfish Patiria miniata, Borrego-Pinto et al. (2016) first isolated homologues of centrosomal proteins and constructed fluorescent protein fusions to several centriolar proteins to track centriole fate in 3D time-lapse imaging during oocyte asymmetric divisions. Using specific markers of mother versus daughter centrioles, they established that, in meiosis I, the two spindle poles are equivalent, being constituted of a pair of mother and daughter centrioles. At anaphase I, one pair of mother/daughter centrioles is extruded into the first polar body. Importantly, the authors described an asymmetry in metaphase II, with the second meiotic spindle always having the mother centriole facing the cortex and the daughter centriole deep inside the cytoplasm (Fig. 1 B).Borrego-Pinto et al. (2016) went on to identify the origin of this asymmetry. They show that the mother centriole, but not the daughter one, starts being rapidly transported toward the plasma membrane before completion of meiosis I spindle disassembly in a microtubule- and dynein-dependent manner, as its trafficking could be impaired by the dynein inhibitor ciliobrevin D (Firestone et al., 2012). In a second step, the mother centriole is anchored to the plasma membrane through the second meiotic division. Interestingly, electron microscopy of starfish oocytes revealed electron-dense material as well as vesicles between the mother centriole and the plasma membrane, suggesting that the mother centriole’s plasma membrane anchorage occurs via its appendages (Reiter et al., 2012; Stinchcombe et al., 2015). Whether the mother centriole migrates to the cortex with its appendages facing or opposite the plasma membrane has not been addressed. However, it is reasonable to assume that, in a viscous environment such as the oocyte cytoplasm, a motion with the appendages up would be favored (Fig. 1 B). Moreover, whereas the migration of the mother centriole to the plasma membrane requires microtubules, its anchoring does not depend on microtubules or microfilaments, as shown by the continued tight association between the centriole and the membrane in the presence of microtubule- and/or actin-depolymerizing agents. This close anchoring via the centriole’s appendages is reminiscent of the anchoring of centrioles forming cilia or at the immunological synapse in T cells (Stinchcombe et al., 2015). The precise mechanisms involved in mother centriole anchoring to the plasma membrane in starfish might be conserved in other systems that also require proximity between these two structures. It would be interesting to assess whether astral microtubules emanating from the mother centriole progressively depolymerize as the mother centriole approaches the plasma membrane to allow the intimate anchoring of the appendages with the plasma membrane. If so, Katanin, a microtubule-severing enzyme whose activity is regulated during meiotic divisions in the nematode oocyte, would be a good candidate to promote such a progressive destabilization (Srayko et al., 2000).Future work will tell us why the daughter centriole does not experience such a migration event. This strongly argues for a functional asymmetry between the two types of centrioles. From the work of Borrego-Pinto et al. (2016), it appears that the daughter centriole is passively pushed inside the oocyte cytoplasm as a result of meiosis II spindle assembly and elongation. Dynein, which controls the migration of the mother centriole, could specifically associate with this centriole, like it does in Saccharomyces cerevisiae, by localizing preferentially to the spindle pole body (the yeast equivalent of the centrosome) facing the bud (Grava et al., 2006). Centrosome asymmetry has been described in several stem cell types (Roubinet and Cabernard, 2014) and this asymmetry is often rooted in its activity. However, Borrego-Pinto et al. (2016) show that the microtubule nucleation capacity of the daughter and mother centrioles is equivalent up to the metaphase II stage. It is only after fertilization and anaphase II that a difference in activity is detected between the mother and daughter centrioles. Thus, what underlies the asymmetry in behavior between the mother and daughter centrioles at anaphase I remains to be discovered. One possibility is that the presence of appendages in the mother centriole allows the recruitment of specific factors, such as dynein, which in turn regulate mother centriole migration and anchoring.Borrego-Pinto et al. (2016) also discovered that specific anchoring of the mother centriole to the plasma membrane, at which the second polar body will form, is the mechanism by which oocytes get rid of the remaining mother centriole. Importantly, actively removing the mother centriole after anaphase II is essential for zygotic development. Indeed, the researchers used the actin polymerization inhibitor cytochalasin D to prevent extrusion of the second polar body and artificially retain the mother centriole in the oocyte after anaphase II. When a mother centriole is retained, it keeps its microtubule nucleation capacity and participates in the first mitotic spindle pole organization of the fertilized egg, whereas the daughter centriole is inactivated and dismantled after anaphase II. As a consequence, because of the two centrioles contributed by the sperm cell, the mitotic spindle ends up being tripolar in the presence of an additional mother centriole, precluding correct chromosome segregation and further development (Fig. 1 B).The origin of the difference in behavior between mother and daughter centrioles after anaphase II will require further investigation. To explain the loss in nucleation capacity of the daughter centriole, it will be important to check for the presence of various PCM components. Indeed, it is reasonable to assume that the daughter centriole loses its PCM association. PCM size scales with centriole size; thus, appendages of the mother centriole might possess an innate ability to maintain association with the PCM (Bobinnec et al., 1998; Delattre et al., 2004). A possible cell cycle–dependent enzymatic activity appearing after anaphase II might explain the rapid loss in microtubule nucleation capacity of the daughter centriole. It is surprising that the starfish zygote cannot cluster the mother centriole material with the centrioles from the sperm, unlike mouse oocytes, which, like cancer cells, are able to cluster PCM to regulate the total number of microtubule organizing centers (Kwon et al., 2008; Breuer et al., 2010). It will be interesting to determine whether starfish zygotes express proteins such as HURP or HSET, which are major players in extra-centrosome clustering (Kwon et al., 2008; Breuer et al., 2010).Altogether, the results from Borrego-Pinto et al. (2016) address a major unresolved question: why do oocytes lose or inactivate their canonical centrioles during female meiosis? They show for the first time that maternal centrioles must be extruded from or inactivated in the starfish egg before fertilization so that they do not perturb mitotic spindle assembly. This is a very important step in our understanding of female gamete formation. Moreover, this work establishes starfish oocyte meiosis as a novel model system to study both functional and structural centrosome asymmetry, an essential component of asymmetric divisions.     

Centrosome destined to decay in starfish oocytes

In contrast to the somatic cell cycle, duplication of the centrioles does not occur in the second meiotic cycle. Previous studies have revealed that in starfish each of the two centrosomes in fully-grown immature oocytes consists of two centrioles with different destinies: one survives and retains its reproductive capacity, and the other is lost after completion of meiosis. In this study, we investigated whether this heterogeneity of the meiotic centrioles is already determined before the re-initiation of meiosis. We prepared a small fragment of immature oocyte containing the four centrioles and fused it electrically with a mature egg in order to transfer two sets of the premeiotic centrioles into the mature cytoplasm. Two asters were present in this conjugate, and in each of them only a single centriole was detected by electron microscopy. In the first mitosis of the conjugate artificially activated without sperm, two division poles formed, each of which doubled in each subsequent round of mitosis. These results indicate that only two of the four premeiotic centrioles survived in the mature cytoplasm and that they retained their reproductive capacity, which suggests that the heterogeneity of the maternal centrioles is determined well before re-initiation of meiosis, and that some factor in the mature cytoplasm is responsible for suppressing the reproductive capacity of the centrioles destined to decay.     

Distinct mechanisms eliminate mother and daughter centrioles in meiosis of starfish oocytes

Centriole elimination is an essential process that occurs in female meiosis of metazoa to reset centriole number in the zygote at fertilization. How centrioles are eliminated remains poorly understood. Here we visualize the entire elimination process live in starfish oocytes. Using specific fluorescent markers, we demonstrate that the two older, mother centrioles are selectively removed from the oocyte by extrusion into polar bodies. We show that this requires specific positioning of the second meiotic spindle, achieved by dynein-driven transport, and anchorage of the mother centriole to the plasma membrane via mother-specific appendages. In contrast, the single daughter centriole remaining in the egg is eliminated before the first embryonic cleavage. We demonstrate that these distinct elimination mechanisms are necessary because if mother centrioles are artificially retained, they cannot be inactivated, resulting in multipolar zygotic spindles. Thus, our findings reveal a dual mechanism to eliminate centrioles: mothers are physically removed, whereas daughters are eliminated in the cytoplasm, preparing the egg for fertilization.     

Centriolin,a centriole-appendage protein,regulates peripheral spindle migration and asymmetric division in mouse meiotic oocytes

Unlike somatic cells mitosis, germ cell meiosis consists of 2 consecutive rounds of division that segregate homologous chromosomes and sister chromatids, respectively. The meiotic oocyte is characterized by an absence of centrioles and asymmetric division. Centriolin is a relatively novel centriolar protein that functions in mitotic cell cycle progression and cytokinesis. Here, we explored the function of centriolin in meiosis and showed that it is localized to meiotic spindles and concentrated at the spindle poles and midbody during oocyte meiotic maturation. Unexpectedly, knockdown of centriolin in oocytes with either siRNA or Morpholino micro-injection, did not affect meiotic spindle organization, cell cycle progression, or cytokinesis (as indicated by polar body emission), but led to a failure of peripheral meiotic spindle migration, large polar body emission, and 2-cell like oocytes. These data suggest that, unlike in mitotic cells, the centriolar protein centriolin does not regulate cytokinesis, but plays an important role in regulating asymmetric division of meiotic oocytes.     

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.     

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 REPLICATION : A Study of Spermatogenesis in the Snail Viviparus

This paper describes the replication of centrioles during spermatogenesis in the Prosobranch snail, Viviparus malleatus Reeve. Sections for electron microscopy were cut from pieces of testis fixed in OsO₄ and embedded in the polyester resin Vestopal W. Two kinds of spermatocytes are present. These give rise to typical uniflagellate sperm carrying the haploid number of 9 chromosomes, and atypical multiflagellate sperm with only one chromosome. Two centrioles are present in the youngest typical spermatocyte. Each is a hollow cylinder about 160 mµ in diameter and 330 mµ long. The wall consists of 9 sets of triplet fibers arranged in a characteristic pattern. Sometime before pachytene an immature centriole, or procentriole as it will be called, appears next to each of the mature centrioles. The procentriole resembles a mature centriole in most respects except length: it is more annular than tubular. The daughter procentriole lies with its axis perpendicular to that of its parent. It presumably grows to full size during the late prophase, although the maturation stages have not been observed with the electron microscope. It is suggested that centrioles possess a constant polarization. The distal end forms the flagellum or other centriole products, while the proximal end represents the procentriole and is concerned with replication. The four centrioles of prophase (two parents and two daughters) are distributed by the two meiotic divisions to the four typical spermatids, in which they function as the basal bodies of the flagella. Atypical spermatocytes at first contain two normal centrioles. Each of these becomes surrounded by a cluster of procentrioles, which progressively elongate during the late prophase. After two aberrant meiotic divisions the centriole clusters give rise to the basal bodies of the multiflagellate sperm. These facts are discussed in the light of the theory, first proposed by Pollister, that the supernumerary centrioles in the atypical cells are derived from the centromeres of degenerating chromosomes.     

The spatial and mechanical challenges of female meiosis

Recent work shows that cytokinesis and other cellular morphogenesis events are tuned by an interplay among biochemical signals, cell shape, and cellular mechanics. In cytokinesis, this includes cross-talk between the cortical cytoskeleton and the mitotic spindle in coordination with cell cycle control, resulting in characteristic changes in cellular morphology and mechanics through metaphase and cytokinesis. The changes in cellular mechanics affect not just overall cell shape, but also mitotic spindle morphology and function. This review will address how these principles apply to oocytes undergoing the asymmetric cell divisions of meiosis I and II. The biochemical signals that regulate cell cycle timing during meiotic maturation and egg activation are crucial for temporal control of meiosis. Spatial control of the meiotic divisions is also important, ensuring that the chromosomes are segregated evenly and that meiotic division is clearly asymmetric, yielding two daughter cells - oocyte and polar body - with enormous volume differences. In contrast to mitotic cells, the oocyte does not undergo overt changes in cell shape with its progression through meiosis, but instead maintains a relatively round morphology with the exception of very localized changes at the time of polar body emission. Placement of the metaphase-I and -II spindles at the oocyte periphery is clearly important for normal polar body emission, although this is likely not the only control element. Here, consideration is given to how cellular mechanics could contribute to successful mammalian female meiosis, ultimately affecting egg quality and competence to form a healthy embryo.     

The chromosomal basis of meiotic acentrosomal spindle assembly and function in oocytes

Several aspects of meiosis are impacted by the absence of centrosomes in oocytes. Here, we review four aspects of meiosis I that are significantly affected by the absence of centrosomes in oocyte spindles. One, microtubules tend to assemble around the chromosomes. Two, the organization of these microtubules into a bipolar spindle is directed by the chromosomes. Three, chromosome bi-orientation and attachment to microtubules from the correct pole require modification of the mechanisms used in mitotic cells. Four, chromosome movement to the poles at anaphase cannot rely on polar anchoring of spindle microtubules by centrosomes. Overall, the chromosomes are more active participants during acentrosomal spindle assembly in oocytes, compared to mitotic and male meiotic divisions where centrosomes are present. The chromosomes are endowed with information that can direct the meiotic divisions and dictate their own behavior in oocytes. Processes beyond those known from mitosis appear to be required for their bi-orientation at meiosis I. As mitosis occurs without centrosomes in many systems other than oocytes, including all plants, the concepts discussed here may not be limited to oocytes. The study of meiosis in oocytes has revealed mechanisms that are operating in mitosis and will probably continue to do so.     

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.     

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.     

Suppression of DNA replication via Mos function during meiotic divisions in Xenopus oocytes.

Meiosis is characterized by the absence of DNA replication between the two successive divisions. In Xenopus eggs, the ability to replicate DNA develops during meiotic maturation, but is normally suppressed until fertilization. Here we show that development of the DNA-replicating ability depends on new protein synthesis during meiosis I, and that mere ablation of the endogenous c-mos product Mos allows maturing oocytes to enter interphase and replicate DNA just after meiosis I. Moreover, we demonstrate that during normal maturation cdc2 kinase undergoes precocious inactivation in meiosis I and then premature reactivation before meiosis II; importantly, this premature cdc2 reactivation absolutely requires Mos function and its direct inhibition by a dominant-negative cdc2 mutant also results in nuclear reformation and DNA replication immediately after meiosis I. These findings indicate that suppression of DNA replication during meiotic divisions in Xenopus oocytes is accomplished by the Mos-mediated premature reactivation of cdc2 kinase. We suggest that these mechanisms for suppressing DNA replication may be specific for meiosis in animal oocytes, and that the ultimate biological function, including the well known cytostatic factor activity, of Mos during meiotic maturation may be to prevent undesirable DNA replication or parthenogenetic activation before fertilization.     

Ca(2+)-promoted cyclin B1 degradation in mouse oocytes requires the establishment of a metaphase arrest

CDK1-cyclin B1 is a universal cell cycle kinase required for mitotic/meiotic cell cycle entry and its activity needs to decline for mitotic/meiotic exit. During their maturation, mouse oocytes proceed through meiosis I and arrest at second meiotic metaphase with high CDK1-cyclin B1 activity. Meiotic arrest is achieved by the action of a cytostatic factor (CSF), which reduces cyclin B1 degradation. Meiotic arrest is broken by a Ca2+ signal from the sperm that accelerates it. Here we visualised degradation of cyclin B1::GFP in oocytes and found that its degradation rate was the same for both meiotic divisions. Ca2+ was the necessary and sufficient trigger for cyclin B1 destruction during meiosis II; but it played no role during meiosis I and furthermore could not accelerate cyclin B1 destruction during this time. The ability of Ca2+ to trigger cyclin B1 destruction developed in oocytes following a restabilisation of cyclin B1 levels at about 12 h of culture. This was independent of actual first polar body extrusion. Thus, in metaphase I arrested oocytes, Ca2+ would induce cyclin B1 destruction and the first polar body would be extruded. In contrast to some reports in lower species, we found no evidence that oocyte activation was associated with an increase in 26S proteasome activity. We therefore conclude that Ca2+ mediates cyclin B1 degradation by increasing the activity of an E3 ubiquitin ligase. However, this stimulation occurs only in the presence of the ubiquitin ligase inhibitor CSF. We propose a model in which Ca2+ directly stimulates destruction of CSF during mammalian fertilisation.     

Rac activity is polarized and regulates meiotic spindle stability and anchoring in mammalian oocytes

Mammalian meiotic divisions are asymmetrical and generate a large oocyte and two small polar bodies. This asymmetry results from the anchoring of the meiotic spindle to the oocyte cortex and subsequent cortical reorganization, but the mechanisms involved are poorly understood. We investigated the role of Rac in oocyte meiosis by using a fluorescent reporter for Rac-GTP. We find that Rac-GTP is polarized in the cortex overlying the meiotic spindle. Polarization of Rac activation occurs during spindle migration and is promoted by the proximity of chromatin to the cortex. Inhibition of Rac during oocyte maturation caused a permanent block at prometaphase I and spindle elongation. In metaphase II-arrested oocytes, Rac inhibition caused the spindle to detach from the cortex and prevented polar body emission after activation. These results demonstrate that Rac-GTP plays a major role in oocyte meiosis, via the regulation of spindle stability and anchoring to the cortex.     

Specific deletion of Cdc42 does not affect meiotic spindle organization/migration and homologous chromosome segregation but disrupts polarity establishment and cytokinesis in mouse oocytes

Mammalian oocyte maturation is distinguished by highly asymmetric meiotic divisions during which a haploid female gamete is produced and almost all the cytoplasm is maintained in the egg for embryo development. Actin-dependent meiosis I spindle positioning to the cortex induces the formation of a polarized actin cap and oocyte polarity, and it determines asymmetric divisions resulting in two polar bodies. Here we investigate the functions of Cdc42 in oocyte meiotic maturation by oocyte-specific deletion of Cdc42 through Cre-loxP conditional knockout technology. We find that Cdc42 deletion causes female infertility in mice. Cdc42 deletion has little effect on meiotic spindle organization and migration to the cortex but inhibits polar body emission, although homologous chromosome segregation occurs. The failure of cytokinesis is due to the loss of polarized Arp2/3 accumulation and actin cap formation; thus the defective contract ring. In addition, we correlate active Cdc42 dynamics with its function during polar body emission and find a relationship between Cdc42 and polarity, as well as polar body emission, in mouse oocytes.     

Assembly and persistence of primary cilia in dividing Drosophila spermatocytes

Basal bodies are freed from cilia and transition into?centrioles to organize centrosomes in dividing cells. A mutually exclusive centriole/basal body existence during cell-cycle progression has become a widely accepted principle. Contrary to this view, we?show here that cilia assemble and persist through?two meiotic divisions in Drosophila spermatocytes. Remarkably, all four centrioles assemble primary cilia-centriole complexes that transit from the plasma membrane encased in a packet of membrane, recruit centrosomal material into microtubule-organizing centers, and persist at the spindle poles through division. Thus, spermatocyte centrioles organize centrosomes and cilia simultaneously at cell division. These findings challenge the prevailing view that cilia antagonize cell-cycle progression and raise the possibility that cilium retention at cell division may occur in diverse organisms and cell types.