Preferential Bivalent Formation in Tetraploid Male of Pacific Oyster Crassostrea gigas Thunberg

ZHANG Zhengrui, WANG Xinglian, ZHANG Quanqi,, and Standish Allen Jr.

1) College of Marine Life Sciences, Key Laboratory of Marine Genetics and Breeding of Ministry of Education, Ocean University of China, Qingdao 266003, P. R. China

2) Aquaculture Genetics and Breeding Technology Center, Virginia Institute of Marine Science, College of William and Mary, Gloucester Point, VA 23062, U. S. A.

Preferential Bivalent Formation in Tetraploid Male of Pacific Oyster Crassostrea gigas Thunberg

ZHANG Zhengrui1), WANG Xinglian1), ZHANG Quanqi1),*, and Standish Allen Jr.2)

1) College of Marine Life Sciences, Key Laboratory of Marine Genetics and Breeding of Ministry of Education, Ocean University of China, Qingdao 266003, P. R. China

2) Aquaculture Genetics and Breeding Technology Center, Virginia Institute of Marine Science, College of William and Mary, Gloucester Point, VA 23062, U. S. A.

Artificially induced tetraploid Pacific oyster, Crassostrea gigas Thunberg, produces more aneuploid gametes than normal diploid one, although they showed a comparable fecundity to diploidy. The meiotic chromosome configuration of 3 tetraploid and 1 tetraploid/triploid mosaic males were analyzed through direct chromosome observation. A majority of metaphase I spermatocytes contained both bivalents and quadrivalents. The chromosome configuration of these males was characterized by preferential formation of bivalents to quadrivalents. Bivalents appeared in all spermatocytes and consisted of 86% of all chromosome aggregates. In comparison, quadrivalents occurred in 91% spermatocytes and consisted of only 12.6% of all chromosome aggregates. The mean bivalent frequency per spermatocyte varied between 14.4 and 17.2; while that of quadrivalents varied between 2.2 and 2.7. Most quadrivalents were tandemly chained (58%) or circled (39%). The total number of chromosome aggregates per spermatocyte ranged from 13 to 20 with an average of 17.6; while 18 (16 bivalents and 2 quadrivalents) was the most frequent. Univalents and trivalents appeared in very low frequency. Aneuploid (hypotetraploid) spermatocytes were observed in a low frequency. The chromosome configuration of in the mosaic individual was similar to that of tetraploid individuals. The percentage of triploid spermatocytes (2%) of the mosaic individual was significantly lower (χ2=30, P < 0.01) than that of triploid cells (46%) in its somatic tissue.

tetraploid oyster; meiosis; bivalent; quadrivalent; reversion

1 Introduction

Studies on meiotic chromosome behavior of polyploids have considerable practical value in terms of explaining and possibly moderating the undesirable effect of polyploidy on fertility and genetic stability (Evans, 1981; Gillies, 1989). Polyploids and polysomics provide opportunities of analyzing chromosome pairing under competitive situation since more than two potential partners are available.

Analysis of chromosome pairing of polyploids has a long history (Newton and Darlington, 1929; McClintock, 1931); but our knowledge and understanding of chromosome pairing after polyploidization are mainly derived from investigations in polyploid plants. The incidence of polyploidy is relatively low in animals and often relates to gynogenetic and parthenogenetic reproduction (Dawley, 1989). Natural and artificially induced bisexual polyploid animals in which analysis of normal meiotic chromosome pairing are available are scarce (Rasmussen, 1977; Rasmussen and Holm, 1979; Chourrout, 1984; Arai et al., 1991; Guo and Allen Jr., 1994; Xiao et al., 2011; Liu, 2010; Yi et al., 2012). The Pacific oyster (Crassostrea gigas Thunberg) is one of animals in which viable triploid and tetraploid individuals can be induced (Allen Jr. and Downing, 1986; Guo and Allen Jr., 1994). The biological characteristics of this species make it a good material for studying meiotic process (Longo et al., 1993; Guo et al., 1992a, 1992b).

Chromosome segregation in polyploid animals has been studied by analyzing isozyme patterns of progeny (Danzman and Borgart, 1981, 1983; Diter et al., 1988), observing chromosome constitution in progeny (Churrout and Nakayama, 1987) , and direct meiotic chromosome observation (Rasmussen, 1977; Rasmussen and Holm, 1979; Thorgaard and Gall, 1979; Gui et al., 1991, 1992; Kawamura, 1994; Oliveira et al., 1995; Guo and Allen Jr., 1997; Que et al., 1997; Zhang et al., 1998; Morishima et al., 2008, 2012; Zhang et al., 2010a; Li et al., 2011). Even some softwares were developed to simulate the meiotic chromosome structure in tetraploid (Voorrips et al., 2012). The fertility of tetraploid is usually lower than that of diploid, which limits their application as their seeds or gametes are depended for propagation (Dewey, 1980).The low fecundity is partially attributed to meiotic multivalent that results in abnormal chromosome segregation and aneuploid gametes. The fecundity of tetraploid Pacific oyster was not significantly different from that of normal diploid (Guo et al., 1996; Guo and Allen Jr., 1997). However, tetraploid produced significantly more aneuploid gametes than normal diploid (Guo et al., 1996; Guo and Allen Jr., 1997). Although this does not seriously affect their application in the production of triploid seeds for the first generation as oysters can tolerate aneuploidy to some extent (Thiriot-Quievreux et al., 1988; Guo et al., 1996; Guo and Allen Jr., 1997; Wang et al., 1999; Zhang et al., 2010b), it is really difficult to maintain their genetic stability as a biotype or broodstock. Therefore, studies of meiosis in polyploid especially tetraploid oyster are very important both economically and academically.

Guo and Allen Jr. (1997) found that homologous chromosomes synapsed predominantly as trivalents in eggs of triploid and as quadrivalents in eggs of tetraploid. Que et al. (1997) also reported the dominant trivalent formation in eggs of triploid. Similar results were obtained in spermatocytes of triploid male individuals (Zhang et al., 2010a). Synapsis of homologous chromosomes in tetraploid males has not been reported yet. In the present study, we found the preferential bivalent formation instead of quadrivalent formation in spermatocytes of tetraploid male individuals.

2 Materials and Methods

Four individuals of 2-year-old tetraploid Pacific oyster Crassostrea gigas Thunberg were selected for meiotic chromosome observation. These tetraploid were produced by blocking the first polar body release with cytochalasin B in the fertilized eggs with the method described early (Guo and Allen Jr., 1994). The oysters were sacrificed and a piece of gill tissue was dissected for ploidy assessment by flow cytometry (FCM) as described by Guo and Allen Jr. (1994). Gill tissue from a diploid individual was sampled as control of FCM. For chromosome preparation, a small piece of gonad tissue was dissected from each individual. These tissues were treated in 0.02% colchicine dissolved in Eagle’s Minimum Essential Medium (14.5 g per 1000 mL, pH 8.3) at room temperature for 4–5 h. After decantation, the tissues were treated with hypotonic 0.8% sodium citrate solution for 40–50 min. Then hypotonic solution was discarded and the tissues were fixed with fresh chilled Carnoy’s fixative (Glacial acetic acid: absolute ethanol = 1:3) at 5℃, 3 changes, 30 min for the first two and at least 2 h for the third. The prepared samples were preserved at −20℃.

To prepare chromosome spreads, a small piece of sample was taken to a microcentrifuge tube containing several drops of fresh fixative, chopped with sharp forceps and pipetted gently with Pasture pipette. The cell suspension was dropped onto 70% ethanol cleaned slides and air-dried. The slides were stained with 5% Giemsa in pH 6.5 phosphate buffer for 15 min, observed under the microscope and photographed.

Meiotic chromosome configuration at metaphase I was observed and analyzed. The numbers of bivalents, quadrivalents and other chromosome aggregates in good metaphase I spreads were counted. At least 40 spermatocytes in each male were recorded.

3 Results

The gill tissue from 4 experimental oyster individuals and a diploid control were detected by flow cytometry. Compared with the control (Fig.1a), three of the four male individuals were tetraploid which contained predominantly tetraploid cells in their gills (Fig.1b); whereas the other one was a tetraploid-triploid mosaic which contained about 54% tetraploid and 46% triploid cells in its gill tissue (Fig.1c).

Fig.1 Flow-cytometric histogram of a diploid (a), a tetraploid (b) and a tetraploid/triploid mosaic (c) of Pacific oyster Crassostrea gigas. The mosaic is composed of 54% tetraploid and 46% triploid cells.

The chromosome configuration of the 4 male individuals is summarized in Table 1. In 3 tetraploid individuals, amajority of metaphase I spermatocytes contained both bivalent (II) and quadrivalent (IV) varied greatly among spermatocytes (Table 1, Fig.2a–e). A small portion of spermatocytes also contained varying numbers of univalent (I) and/or trivalent (III) (Fig.1f). Bivalent was the most common pairing configuration among all chromosome aggregates, which appeared in all spermatocytes observed and accounted for 86% of all chromosome aggregates. The number of bivalents per spermatocyte varied greatly between 20 and 6, and the mean bivalent frequency per spermatocyte was 17.2, 15.5 and 14.4, respectively, in tetraploid male 1, 2 and 3. Quadrivalents were observed in 91% of spermatocytes examined and consisted of 12.6% of all chromosome aggregates. The number of quadrivalents per spermatocyte varied from 0 to 7 while most spermatocytes contained 0–4 quadrivalents. The mean quadrivalent frequency per spermatocyte was 2.2, 2.4 and 2.7, respectively, in the 3 tetraploid male individuals. Univalent and trivalent appeared at a much lower frequency than bivalent and quadrivalent did. Only a few spermatocytes contained one or two of these kinds of chromosome aggregates. Univalent and trivalent consisted of only 2.4% of all chromosome configurations in total. Spermatocytes involving multivalent formation by more than 5 chromosomes were not observed. The total number of chromosome aggregates per spermatocyte ranged from 13 to 20 with an average of 17.6; while 18 was the most frequent number (Table 1, Fig.2).

Table 1 Frequency of various combinations of bivalent (II) and quadrivalent (IV) in three tetraploid and one tetraploid/ triploid mosaic male individual of Pacific oyster C. gigas

Among all spermatocytes detected, 16 II + 2 IV was the most common chromosome configuration that was found in 28.6% of all spermatocytes, followed by 14 II + 3 IV (in 19.5%) and 18 II + 1 IV (in 14.3%) configuration. Hypotetraploid spermatocytes were observed in male individual 2 and 3, but not in individual 1 (Table 1, Fig.2g). Of the 116 quadrivalents analyzed from 60 spermatocytes, 69 (58%) were tandem chains of 4 homologues, 45 (39%) were circles of 4 tandemly arranged homologous chromosomes, and the remaining 2 (3%) were convergent cross-shaped associations (Fig.1b–g).

Fig.2 Meiotic chromosome configuration in tetraploid and tetraploid/triploid mosaic male individuals of Pacific oyster showing varying numbers of bivalents and quadrivalents (large arrows). a, A spermatocyte containing 20 II; b, A spermatocyte containing 18 II + 1 IV; c, A spermatocyte containing 16 II + 2 IV; d, A spermatocyte containing 14 II + 3 IV; e, A spermatocyte containing 8 II + 6 IV; f, A spermatocyte contaianing trivalents (arrowhead); g, An aneuploid spermatocyte containing 17 II + 1 IV; h, A triploid spermatocyte in the tetraploid/triploid mosaic individual containiang 8 III + 2 II + 2 I (arrowheads, trivalents; small arrows, univalents).

Chromosome configuration in mosaic individuals was similar to that observed in other three tetraploid male individuals. Most spermatocytes were true tetraploidy that showed 20–12 II and 0–6 IV. The mean bivalent and quadrivalent frequency per spermatocyte was 17.8 and 2.5, respectively. Univalent and/or trivalent were observed in a minority of spermatocytes. In 47 spermatocytes at metaphase I, only one (2%) was true triploid (Table 1, Fig.1h). The percentage of triploid spermatocytes observed here was significantly lower (χ2=30, P < 0.01) than that expected from the somatic tissue. In this individual, three (6.4%) spermatocytes were aneuploid, one contained 15 II + 2 IV; while the other two contained 16 II + 1 IV.

4 Discussion

In the present study, we observ ed preferential formation of bivalent to quadrivalent in spermatocytes of tetraploid Pacific oyster. The mean quadrivalent frequency was around 2 per spermatocyte in 4 male individuals. In normal diploid, both male and female individuals formed bivalent during meiosis I, and in triploid, three sets of homologous chromosomes formed predominantly trivalents although univalents, bivalents and complicated multivalents also existed (Guo and Allen Jr., 1994, 1997; Que et al., 1997; Zhang et al., 2010a). The homologous chromosomes in the eggs of tetraploid female individuals paired mostly to quadrivalents, as eggs contained 11 chromosome aggregates in average, which corresponded to 9 quadrivalents and 2 bivalents per egg (Guo and Allen Jr., 1997). However, our tetraploid male individuals contained more than 17 chromosome aggre-gates in average. The present results obviously conflicted with the previously reported.

It was reported that four homologues of tetraploid frog Odontophrynus americanus formed mostly quadrivalents. The formation of a small number of trivalents, bivalents and univalents was believed to be a consequence of precocious separation (Becak et al., 1966, 1967). Such kind of quadrivalent formation was considered as the results of synapsis partner switch at pachytene stage. However, preferential bivalent formation or elimination of multivalents during meiotic prophase has been observed in many polyploid plants as well as animals (Rasmussen, 1977; Rasmussen and Holm, 1979; Hobolth, 1981; Holm, 1986; Loidl, 1986; Gillies, 1989; White et al., 1988; Jenkins et al., 1988; Oliveira et al., 1995). In tetraploid loach, bivalents were also more frequently observed than quadrivalents (Li et al., 2011). Our result indicate that similar multivalent elimination system may also exists in polyploid Pacific oyster. Although both precocious separation and multivalent elimination can explain the formation of some trivalents, bivalents and univalents, neither of them can explain the difference in meiotic chromosome behaviors between male and female individuals of tetraploid Pacific oyster.

We have reported that in pachytene spermatocytes of triploid male individuals some homologous chromosomes formed complete trivalent; while some others formed incomplete trivalent. In the later case, tow of the three homologues synapse along the whole chromosome to form a perfect bivalent, and the third one only associated to the bivalent in one or several regions (Zhang et al., 2010a). In tetraploid male oyster four pairing partners are available. It is extremely possible that the third and the fourth homologous chromosomes would form another bivalent, rather than associate with the first bivalent to form an quadrivalent, or an incomplete trivalent and a univalent. After all, two bivalents are more stable than an incomplete quadrivalent or an incomplete trivalent plus a univalent.

Seed fertility improvement has been correlated with reduced quadrivalent formation and increased bivalent frequency in several polyploid plants (Venkateswaralu and Rao, 1983; Pal and Pandley, 1982). Although tetraploid female Pacific oyster showed comparable fecundity to normal diploid, breeding experiments using tetraploid oyster revealed that crosses between tetraploid females and diploid males had lower survival rate than those between diploid females and tetraploid males (Guo et al., 1996). Chromosome observation found that tetraploid females produced noticeably more aneuploid gametes than tetraploid males did (Guo and Allen Jr., 1997). The results of the present study, together with those of previous reports (Guo et al., 1996; Guo and Allen Jr., 1997), can explain the difference in gametogenesis between males and females in tetraploid Pacific oyster. Bivalents are obviously more stable and much easier to go through balanced segregation than quadrivalents are. The high frequency of bivalents in spermatocytes of tetraploid males enables homologous chromosomes to segregate normally to form balanced gametes. In tetraploid females, however, chromosome segregation is more difficult than in males due to the high frequency of quadrivalents.

In both diploids and triploids, there was no obvious difference between males and females in terms of chromosome pairing during meiosis I (Guo and Allen Jr., 1997; Que et al., 1997; Zhang et al., 2010a). The sex-related difference in meiotic chromosome behavior between males and females in the tetraploid Pacific oyster is a particularly interesting phenomenon. Further genetic analyses on this difference should be conducted.

Triploid males and females formed some multivalents associated by more than four chromosomes (Guo and Allen Jr., 1997; Que et al., 1997; Zhang et al., 2010a). In normal diploids, there was also some multivalent formation (Zhang et al., 2010a). Interestingly, our tetraploid males formed only a small part of quadrivalents. Complicated multivalent associated by more than five chromosomes was not observed.

In Pacific oyster, triploid may lose one set of chromosomes in some of their cells to revert to triploid/diploid mosaics (Allen Jr. et al., 1996; Zhang et al., 2010b). Tetraploid may also revert to tetraploid/triploidy or even tetraploid/triploid/diploid mosaics. One of the four males used in the present study was a tetraploid/triploid mosaic that had 54% tetraploid cells and 46% triploid cells in its gill tissue and was believed to be a revertant individual. Interestingly, we observed fairly higher proportion of aneuploid spermatocytes (7%, 3 in 45) in its gonad, but only one true triploid metaphase I spermatocytes was observed. The proportion of triploid spermatocytes (2%) observed was significantly less (χ2=30, P<0.01) than the expected proportion of triploid cells (46%) in the somatic tissue. This result coincided with those observed in triploid/diploid mosaics, in which diploid cells were recorded in their gills but diploid meiotic cells were not observed (Zhang et al., 2010a, 2010b). Mosaic individuals that are undergoing reversion may have more triploid cells in some tissues and less in some other tissues. The mitotic cells in the gonads of the tetraploid/triploid mosaic as well as those of triploid/diploid mosaics were not detected in the present and previous studies. However, as discussed in the previous study (Zhang et al., 2010a), the possibility that the revertant cells in mosaics are difficult to enter into normal meiosis can not be excluded.

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(Edited by Qiu Yantao)

(Received March 3, 2013; revised May 28, 2013; accepted June 25, 2013)

© Ocean University of China, Science Press and Springer-Verlag Berlin Heidelberg 2014

* Corresponding author. 0086-532-82031806

E-mail: qzhang@ouc.edu.cn