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In most animals, the first polar body is formed at the animal pole and subsequently, the second polar body is formed at the vicinity of the first polar body. At this meiotic process, the haploid female pronucleus is formed in the cytoplasm of the fertilized egg. The fertilized egg contains haploid female and male pronucleus. The fusion of the pronucleus occurs in ordinary animals (Longo 1997).

On the contrary, the hermaphroditic clam, Corbicula fluminea, has a peculiar mode of reproduction. At polar body formation, two polar bodies are formed simultaneously. All maternal chromosomes at metaphase of the first meiosis are extruded as two polar bodies (Komaru et al. 2000, Ishibashi et al. 2002, 2003). Hence, the formation of female pronucleus and fusion of the male and female pronuclei did not occur. One male pronucleus transformed into the chromosomes at metaphase of the first mitosis.

At the first meiosis, the spindle axis of metaphase is oriented parallel to the surface of the zygote (Komaru et al. 2000), whereas in most animals, the spindle axis is perpendicularly oriented. At late anaphase of the first meiosis, two bulges are formed and become two polar bodies that include all chromosomes; however, the process of polar body formation, including the process of meiotic cytokinesis has not been described in detail.

Recently, Pielak et al. (2003, 2004, 2005) showed the process of polar body formation in the surfclam Spisula sollidissima, using confocal microscopy. During the first polar body formation, the actin network along the cortex of fertilized eggs was drastically changed at the site of the polar body. From fertilization to metaphase of the first meiosis, the actin layer was equally distributed over the cortex of zygotes. At late anaphase, the actin distribution at the polar body sites became very sparse and an actin-poor circular region was recognized at the animal pole. At anaphase of the first meiosis, the spindle "docked" in this actin-poor region. A circular contractile ring was then formed in the actin-poor region. This change of actin network distribution may be caused by signals along the meiotic spindle, originating from chromatin. Pielak et al. (2005) suggested Ran, a small GTPase, to be involved in contractile ring formation. At late anaphase of the first meiosis, the first actin bundle accumulated in the actin-poor region as the diameter of the actin ring gradually shrank and cytokinesis was completed. These results suggest that in the actin-poor region, the contractile ring was formed at the level of the actin layer and cytokinesis of the polar body occurred.

To better understand the process of the simultaneous formation of the first two polar bodies in an androgenetic clam, the meiotic process was observed by visualizing cytoskeletal elements, such as microtubules and centrosomes, and distribution of actin. In the present study, the detailed process of cytokinesis during the formation of the first two polar bodies in Corbicula fluminea was demonstrated by confocal microscopy.


Collection and Fixation of Zygotes

The specimens of Corbicula fluminea were collected using a fish net in an irrigation canal of Matsusaka City, Mie Prefecture, Japan. Clams were more than 20 mm in shell length. Spawning was induced with 0.1% serotonin for 60 min, and then, the clams were individually transferred to a small plastic container (350 mL), and the temperature was raised from 20 to 26[degrees]C (Obata et al. 2006). Sperm and eggs were spawned simultaneously. The zygotes were collected 5-20 min after spawning from the bottom of the beaker using a pipette.

The zygotes were fixed with 3% paraformaldehyde and 0.01% Triton X in 100 mM PIPES buffer (pH 6.8) containing 5 mM EDTA and 1 mM Mg[Cl.sub.2] (pH 6.8) at 4[degrees]C (slightly modified from Pielak et al. 2005) for at least 12 h. The zygotes were then transferred to 0.2 M phosphate buffer (pH 7.0) containing 3% fetal bovine albumin and 0.00003% Triton X and kept at 4[degrees]C until fluorescence labeling.

Cytoskeletal Fluorescent Labeling

For cytoskeletal observation, the zygotes were processed according to the following procedures. The zygotes were rinsed with rinse solution, 0.2 M phosphate buffer (pH 7.0) containing 3% fetal bovine albumin and 0.00003% Triton X three times before labeling.

Microtubules and Centrosomes

The zygotes were treated with blocking solution containing 3% fetal bovine albumin, 0.01% Triton X, and 0.1% sodium azide to reduce nonspecific antibody reactions for 1 h at room temperature. The zygotes were incubated with a 2000-fold dilution of [alpha]-tubulin monoclonal antibody for 24 h (T-9026; SIGMA) or a [gamma]-tubulin monoclonal antibody (T-6577; SIGMA) for 48 h in the dark at room temperature (Ishibashi et al. 2002, Obata et al. 2006).

After washing three times with rinse solution, the zygotes were incubated with a 200-fold dilution of secondary antibody and goat polyclonal antimouse IgG conjugated with Alexa Fluor-488 (Funakoshi) for 24 h in the dark at room temperature.

F-actin and Nuclei

To visualize actin filaments, after the secondary antibody incubation, the zygotes were treated with a 200-fold dilution of rhodamine phalloidin (R415; Molecular Probes) by DW for 2 h at room temperature and then rinsed in rinse solution three times. Chromosomes and nuclei were then stained with 0.5 [micro],g/mL DAPI for 30 min at room temperature.

Mounting and Observation by Microscopy

A 0.5-mm thick spacer (P18174, press-to-seal silicon isolator; Molecular Probes) was placed on a glass slide to prevent compression of the specimens with the coverslip. The zygotes were then placed on the glass slide with mounting medium and the coverslip applied (H1200; VECTOR Laboratories). The slides were observed by confocal laser scanning microscopy (LSM710; Carl Zeiss). Digital images were obtained and analyzed by Zen software (Carl Zeiss).


Figure 1A-D shows a zygote at metaphase of the first meiosis viewed from the lateral side. The optical plane showing the meiotic spindle and spermatozoa is shown. At the animal pole, the first meiotic apparatus was bipolar and oriented along the cell cortex of zygotes. The meiotic chromosomes were aligned at the metaphase plate (Fig. 1 A). The long axis of the spindle was parallel to the surface (Fig. IB). Both asters were slightly deformed because of their close location to the egg cortex. The nucleus of the sperm had already entered into the egg cytoplasm. The comet-like sperm aster can be observed in Figure IB, D. As shown in Figure 1C, a thin layer of actin was distributed uniformly around the zygote cortex from animal to vegetal pole. Figure 1E-I also shows the egg at metaphase of the first meiosis viewed from the animal pole. The meiotic apparatus was a typical bipolar structure (Fig. 1F, H). In the center of both asters, fine centrosomes were located (Fig. II).

Figure 1J-N shows the zygote at early anaphase of the first meiosis viewed from the animal pole. Chromosomes were segregated to both poles (Fig. 1 J). At early anaphase, the spindle still showed typical features of a bipolar spindle (Fig. IK). The microtubules between the asters were still visible; however, at the region of the asters (Fig. 1J), circular actin-poor regions were observed (Fig. 1L). At the center of these areas, fibrous actin material was distributed (Fig. 1L). A striking difference between metaphase (Fig. 1C, G) and anaphase (Fig. 1L) was actin distribution at the cortex of the animal pole. This structure was called an actin-poor region (Pielak et al. 2004). As shown in Figure 1L, two circular actin-poor regions were clearly observed in eggs viewed from the animal pole. In the center of each of these two actin-poor regions, a small actin-positive spherical fibrous mass was detected. The merged actin and microtubule image in Figures 1M and 2E showed that the spindle at anaphase was "docked" into the actin-poor region at the cortex. The border of the actin-poor region and the actin network was not clear in Figure 1L.

Figure 2A-E shows zygotes at late anaphase of the first meiosis (Fig. 2A). The microtubules were depolymerized and microtubule extensions became rather short (Fig. 2B) compared with those of metaphase or early anaphase. At late anaphase, the range of microtubule extensions (Fig. 2B) was within the actin-poor regions (Fig. 2C, D). At this stage, the microtubules from the two asters did not overlap. In the center of the two actin-poor regions, actin was observed as a spherical mass (Fig. 2C). These structures became more distinct in shape and brighter than those at early anaphase (Fig. 1L). The actin layer around the actin-poor region became more intensely stained at late anaphase, as shown in Figure 2C.

An overlay image through the animal-vegetal axis at anaphase (Fig. 2E) showed that the microtubules from both poles were not overlapped and were observed like two independent half spindles. Within the bulges, chromosomes, centrosomes, and microtubules were observed.

At telophase (Fig. 2F-J), around the actin-poor region, the thick actin ring was organized and looked like two distinct thick rings (Fig. 2H-J). The signal was intense in the region between the rings and around the rings (Fig. 2H). The diameter of the actin ring, i.e., the contractile ring, became smaller than those at anaphase.

Figure 2K-0 shows the zygotes just after polar body formation viewed from the lateral side. Finally, the actin ring disappeared, and the two polar bodies were formed (Fig. 2K-0). All chromosomes were included in the two polar bodies.

Figure 3A shows a schematic representation of polar body formation observed perpendicular to the animal pole. Figure 3B shows a schematic representation of actin distribution in eggs viewed from the animal pole. The meiotic spindle was parallel to the egg surface. At metaphase I, actin was distributed uniformly along the cortex. At anaphase, actin-poor regions appeared and each spindle was docked within the actin-poor regions. At telophase, the actin signal around the actin-poor region became more intense and formed clear rings. After polar body formation, the actin rings were not observed.


Interestingly, in oocytes of invertebrates, such as starfish, fruit fly, jelly fish, oligochaetes, and surf clam, during polar body formation, the actin cortical layer is very thin or almost absent at the animal pole (Shimizu 1997, Pielak et al. 2004, 2005, Hamaguchi et al. 2007, and Maddox et al. 2012). The protrusion, called the bulge in oocytes of starfish, oligochaetes, and bivalves, is formed before polar body formation. The bulge formation and actin rearrangement may be closely related. The mechanism of protrusion of the oocyte cortex in mouse and invertebrate species is obviously different with respect to the actin arrangement at the animal pole. The migration and rotation of the meiotic spindle in mouse depends on actin (Sun & Schatten 2006 for review). In contrast, in invertebrates, such as bivalves, oligochaetes, and starfish, the migration of the meiotic spindle depends on microtubules (Shimizu 1997, Pielak et al. 2004, 2005, Hamaguchi et al. 2007). These differences may be due to the presence or absence of centrosomes in the meiotic spindle.

The present study demonstrated that at metaphase of the first meiosis, the actin layer was almost equally distributed along the cortex of fertilized eggs. At anaphase, the two distinct actin-poor regions appeared as described by Pielak et al. (2004, 2005), who showed one actin-poor region at anaphase in the animal pole of surf clam oocytes. This regional disassembly of actins in the cortex at the bulge site may be essential to create the bulge at the animal pole in invertebrates. The cortical disassembly and assembly of the actin layer beneath the plasma membrane of the bulge (Aoki et al. 2016) may occur at late anaphase in the Cubicula clam. The detailed mechanism of this fast actin disassembly in bivalve oocytes is unknown. Pielak et al. (2005) suggested that a Rho family GTPase was a candidate with respect to actin ring signaling. The close association of both meiotic asters, and of a centrosome induced experimentally by chemical treatment, resulted in a dual actin ring in the surf clam (Pielak et al. 2005). Therefore, the spindle at the cortex can transmit a signal to organize a thick actin layer and induce dual actin rings derived from the meiotic spindle during meiosis in Corbicula as predicted by Pielak et al. (2005).

Two possibilities can be proposed to explain the sudden appearance of the actin-poor region. The first possibility is physical force from astral microtubules. At anaphase, because of the force created at astral microtubule disassembly by the cortical motor protein, dynein, the astral spindle was pulled toward the cortex. The aster pulled by the force generated at anaphase disassembly of astral microtubules connecting cortex and centrosome may clear the actin network like a drill.

The second possibility is fast actin filament disassembly by proteins such as ADF/cofilin and Aipl (Bernstein & Bamburg 2010, Brieher 2013, Gressin et al. 2015, Ydenberg et al. 2015). Within a short period, the actin layer should disassemble at late anaphase. Therefore, fast disassembly of the cortical actin layer may induce the actin-poor region. Small GTPases, Rho and Rac, both act in the actin polymerization pathway and also in the inhibitory pathway (Sullivan et al. 1999, Burridge & Wennerberg 2004). It is likely that signaling proteins might be part of a pathway that induces the actin-poor region and then organizes the thick contractile rings during meiosis in molluscan eggs.

At bulge formation at the animal pole, the surface area of the bulge should increase rapidly. In the present study, a very thin layer of actin filament along the bulge surface was detected. Of interest was the spherical actin mass that formed in the center of the actin-poor region at early anaphase. This structure was formed around both centrosomes. The actin then became more distinct and accumulated at telophase near the centrosomes as Shimizu (1997) observed in Tubifex. This change of actin materials at the spindle poles suggests a role of astral microtubules in transport. The minus end, which directs the motor protein dynein on microtubules, may play a role in transporting the disassembled actin to the periphery of the centrosomes. Finally, actin accumulated at the minus end of microtubules around the centrosomes and appeared as dots near the centrosomes.

In Tubifex oocytes, at the animal pole, an actin-bright spot was recognized at anaphase of meiosis II (Shimizu 1990). This bright aggregation of actin may be formed during bulge formation as a result of actin filament reorganization. In the process of bulge formation, the distribution of cortical actin was altered. As shown by Shimizu (1990), actin filaments were absent from the sides of the bulge. The bright aggregation of actin may form after actin disassembly and accumulation on microtubules by the dynein astral force. During these dynamic changes of actin architecture in Tubifex meiosis, the involvement of a protein kinase was suggested (Shimizu 1997).

At cytokinesis in the Spisula clam (Pielak et al. 1994, 1995), Tubifex (Shimizu 1997), and Corbicula clams (this study), the actin rings thicken around the bulge base. This accumulation of actin and myosin around the base of the bulge, in an actin-poor region, may be induced by a common mechanism in invertebrates. In the present study, the actin cortex layer around the dual contractile ring became thicker between anaphase to telophase compared with that at metaphase, as described by Pielak et al. (2004, 2005) in the surf clam. As the diameter of the actin ring decreased, the actin signal became more intense. These observations indicate that actin accumulation around the contractile ring occurred at polar body formation during cytokinesis.

In the androgenetic clam, two actin-poor regions were simultaneously formed at anaphase. Both asters oriented (docked) to the actin-poor regions. The centrosomes were at the top of the two bulges and the late anaphase spindle docked to the actin-poor region. Overlapping of microtubules was not present after anaphase. The astral half spindles at anaphase were docked to the two bulges. The centrosomes were located at the top of the bulge (Komaru et al. 2000). The astral rays extended from the top of the bulges to the edge of the actinpoor region. Chromatin-derived signals for assembling the contractile ring, such as Rho, may be transported by astral microtubules, as suggested by Pielak et al. (2003), from the "half spindle" to the edge of the actin-poor region and organize the thick layer around the bulges at the level of the actin cortex.

The cleavage plane of normal somatic cells is determined by microtubules. Protein complexes containing Ect2, septin, and myosin localize at the overlapping zone of microtubules in the equator of the cells (Green et al. 2012 for general review). This study suggested that in polar body formation, one peripheral aster could determine the cleavage plane and provide the components of the contractile ring. Cleavage occurred at the level of the actin cortex. In mitotic cells, the overlapping of microtubules at the equator is important to construct the contractile ring (Green et al. 2012). But, no overlapping of microtubules from the poles occurred during anaphase in the present study. The present study showed that only signals from one peripheral aster could induce cytokinesis during polar body formation. Canman et al. (2003) experimentally demonstrated in mammalian cultured cells that if half of astral microtubules are experimentally disassembled, cytokinesis still successfully occurs. The present study also suggests that only one peripheral astral microtubule and a plus end directing motor can transmit the signals, which constitute the contractile ring at polar body formation.

In Corbicula fluminea, two polar bodies are formed simultaneously. The centrosomes and asters play a role in determining the polar body sites. In most animals, only one centrosome of the meiotic spindle is oriented close to the oocyte cortex (Longo 1997, Maddox et al. 2012). At metaphase, the meiotic spindle is oriented perpendicular to the egg surface in most animals. In a bisexual species Corbicula sandai, closely related to C.fluminea, only one centrosome attaches at the membrane, so that the spindle is oriented perpendicular to the oocyte surface (Obata et al. 2006); however, in the present study, both asters are oriented very close to the egg membrane at metaphase and anaphase. At the oocyte membrane, there should be two sites where dynein is anchored by dynactin (Schaerer-Brodbeck & Riezman 2000). The two centrosomes may be pulled from the membrane by depolymerization of microtubules by dynein on the cell membrane. The spindle at metaphase is flattened, which might be because of the pulling force by the motor on the surface at metaphase-I (Komaru et al. 2000). Why are both centrosomes of the meiotic spindle attached to the cortex in the Corbicula clam? It has been assumed that both centrosomes, including centrioles, in Corbicula zygotes are equivalent with respect to migration and attachment to the membrane.

Ultrastructural studies show that animal cells usually contain one centrosome per cell except for the cells at M phase. Centrosomes contain two centrioles, the mother centriole and a younger newly duplicated daughter centriole (Vorobjev & Chentsov 1982, Azimzadeh & Marshall 2010). Only the mother centriole has appendages, not the daughter centriole. The process of obtaining distal and subdistal appendages on centrioles is called maturation (Kobayashi & Dynlacht 2011), and after the maturation process, the daughter centriole can get distal and subdistal appendages, consisting of numerous proteins at the distal end of the centriole (Brito et al. 2012). The centriole with appendages can attach to the plasma membrane and anchor the microtubules (Azimzadeh & Bornens 2007, Debec et al. 2010, Hoyer-Fender 2010, Brito et al. 2012).

In a recent outstanding study on starfish oocytes, Borrengo-Pinto et al. (2016) showed that the mother centriole at the second meiosis is always attached to the oocyte membrane and the spindle orientates perpendicular to the egg cortex. This study clearly demonstrated that the two centrosomes of the meiotic spindle are not equivalent as Uetake et al. (2002) suggested. Only a centrosome with a "mature" mother centriole with appendages and reproductive ability can approach and anchor to the plasma membrane, and then be cast off into the polar body in starfish. Only one centrosome possessing daughter centrioles without reproductive ability remained in the egg cytoplasm. This is a fundamental mechanism to maintain the paternal inheritance of centrioles during meiosis.

With respect to reproduction, the new findings in the present study suggests that in C. fluminea, both centrosomes at each pole of the first meiotic spindle should have equal ability to approach and attach to the plasma membrane of oocyte. The process of formation of actin-poor region and contractile rings during the cytokinesis of polar body was not different at two polar body sites. The altered ability of the centorosome may cause the unusual orientation of both centrosomes induce two actin-poor region and two polar body simultaneously as shown in Figure 4. For example, both centrosomes may have mature distal and subdistal appendages of the centrioles and can attach to the oocyte membrane.

Therefore, the spindle orientation may become parallel to the oocyte surface and induce the unusual process of androgenesis. The alternation of centrioles in the maturation process during gametogenesis should occur in this species. It has been suggested that the cortex of oocytes may be modified so that both centrosomes can attach to the surface (Komaru et al. 2000); however, if the chromatin is artificially injected into mouse oocytes, actin assembly occurs near the injection site (Deng et al. 2007, Yi & Li 2012). It is obvious that chromatin-derived signals such as RhoA (Zhang et al. 2008, Liu 2012) can determine the polar body site, which is not limited to only one restricted site of the oocyte cortex near the animal pole. In starfish oocytes, if the orientation of the meiotic spindle is altered at metaphase, the spindle can attach to a different site (Hamaguchi 2001). Therefore, the unusual orientation of the meiotic spindle in C.fluminea may be due to the intrinsic factors of centrosomes, i.e., centriole function rather than alternation of the cortex.

The hypothesis in this study is speculative at present and is not confirmed by direct evidence of centrosome function. The results of a histological study on the ultrastructure of nonreductional and biflagellar spermatozoa of C.fluminea can only be discussed (Konishi et al. 1998). The ploidy of spermatozoa in androgenetic Corbicula is not haploid but identical to the somatic cells (Ishibashi et al. 2003). The spermatozoa have two flagella of the same length. Between the nucleus and mitochondria, one pair of the centrioles is present. Surprisingly, two similar flagella were generated from distal and proximal centrioles oriented at a right angle to each other (Konishi et al. 1998). These facts suggest that the function of centrioles during oogenesis or spermatogenesis might be modified by mutation of genes or disruption of the process that generates the appendage on the centrioles. Such modification of centriole structure and function may induce the attachment of both centrosomes to the oocyte membrane (Ishibashi et al. 2002) and may cause the unusual two flagellar formation in nonreductional sperm in C.fluminea.


This work was supported by JSPS KAKENHI Grant Number 16K07462.


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Graduate school of Bioresources, Mie University, 1577 Kurimamachiya-cho, Tsu city, Mie Prefecture 514-8507, Japan

(*) Corresponding author. E-mail:

DOI: 10.2983/035.037.0111
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Author:Hotta, Masaru; Komaru, Akira
Publication:Journal of Shellfish Research
Article Type:Report
Date:Apr 1, 2018

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