Synaptonemal complex analysis in spermatocytes of diploid and triploid Chinese shrimp Fenneropenaeus chinensis

Tissue and Cell 40 (2008) 343–350

Synaptonemal complex analysis in spermatocytes of diploid and triploid Chinese shrimp Fenneropenaeus chinensis Yusu Xie a,b , Fuhua Li a,∗ , Chengsong Zhang a,b , Kuijie Yu a , Jianhai Xiang a,∗,1 a

Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, PR China b Graduate School, Chinese Academy of Sciences, Beijing 100039, PR China

Received 29 September 2007; received in revised form 15 March 2008; accepted 17 March 2008 Available online 1 May 2008

Abstract A modified surface spreading technique for synaptonemal complex (SC) analysis was tested to assess the process of chromosome synapsis in spermatocytes of diploid and induced triploid Fenneropenaeus chinensis. Spermatocytes of diploid shrimp showed typical morphological characteristics of eukaryote SC, with complete synapsis of bivalents. No recognizable bivalent associated with sex chromosomes was observed in spermatocytes of diploid shrimp. However, differences in morphology of SC, including unsynapsed univalents, bivalents, totally paired trivalents with non-homologous synapsis, partner switches and triple synapsis were identified at early pachytene stage of triploid spermatocytes. Triple synapsis was especially common at late pachytene stage in spermatocytes of triploid shrimp. The observed abnormal synapsis behavior of chromosomes in spermatocytes indicated that triploid male shrimp may find it difficult to develop normal haploid sperm. © 2008 Elsevier Ltd. All rights reserved. Keywords: Synaptonemal complex; Fenneropenaeus chinensis; Spermatocytes; Diploid; Triploid

1. Introduction The synaptonemal complex (SC), which connects paired homologous chromosomes in most meiotic systems, plays an important role in chromosome pairing and recombination and ensures meiotic chromosome segregation (Page and Hawley, 2004). Additionally, SC analysis has been used in cytogenetic studies to investigate the process of meiotic chromosome pairing (Counce and Meyer, 1973; Moses, 1977; Van Eenennaam et al., 1998; Wallace and Wallace, 2003), to determine the accurate number of chromosomes (Hale et al., 1988) and to study the mechanisms for sex determination (Carrasco et al., 1999), and this technique can be applied in the study of chromatin organization (Cu˜nado et al., 2000) or chromosome identification (Belonogova et al., 2006). SC analysis is a very useful tool in studing the impact of altered fertility ∗

Corresponding authors. Tel.: +86 532 82898571; fax: +86 532 82898578. E-mail addresses: [email protected] (F. Li), [email protected] (J. Xiang). 1 Tel.: +86 532 82898568; fax: +86 532 82898578. 0040-8166/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tice.2008.03.003

´ in carriers of abnormal chromosome complement (Swito´ nski and Stranzinger, 1998; Judis et al., 2004). It is known that fertility in triploids frequently decreases due to abnormal chromosome pairing among the three complete sets of chromosomes during meiosis with the formation of imbalanced and non-viable gametes (Borin et al., 2002). There is no uniform standard of chromosome pairing among different triploid species (Oliveira et al., 1995; Borin et al., 2002). SC was found to be present in bivalents in triploid Rhoeo spathacea (Lin, 1979), triploid Oncorhynchus mykiss (Oliveira et al., 1995) and triploid male Scophthalmus maximus (Cu˜nado et al., 2002) in late pachytene nuclei. However, the formation of triple synapsis was common in triploid Saccharomyces cerevisiae (Loidl, 1995) and triploid female S. maximus (Cu˜nado et al., 2002); frequent formation of univalents was observed in triploid Misgurnus anguillicaudatus (Zhang et al., 2002); partner switches and non-homologous synapsis were reported in triploid Allium sphaerocephalon (Loidl and Jones, 1986) and triploid Trichomycterus davisi (Borin et al., 2002). Due to the large number of chromosomes (>60 chromosomes) and small size

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of individual chromosomes (dot-shape, <4 ␮m) obtaining clearly dispersed chromosome is difficult. To our knowledge, there are no reports of SC analysis in crustaceans. Chromosome set manipulations such as polyploidy and gynogenesis have been widely applied for genetic improvement of marine animals in aquaculture (Xiang et al., 1998). Fenneropenaeus chinensis (Osbeck, 1765) is one of the most commercially important shrimps in China, and triploid F. chinensis has been successfully produced by our group (Xiang et al., 1998, 2006; Li et al., 2003a, 2006). Abnormal sex ratio and gonad developmental characteristics of triploid F. chinensis have been reported by our group (Li et al., 2003b). The sex ratio in triploid F. chinensis population was reported to be 4:1 for females to males, and the development of gonad in triploid females of F. chinensis retarded severely, whereas triploid males could produce certain amount of spermatids and a few sperm-like cells were observed in the vas deferens, which were morphologically abnormal and showed triploidy through flow cytometric analysis (Li et al., 2003b; Xiang et al., 2006). In contrast, Dr. Preston’s group in Australia found that all triploid Penaeus japonicus were female, no triploid males being produced (Sellars et al., 2003, 2004). At present, the mechanism of abnormal sperm development in triploid male F. chinensis is not clear. In this study, surface spreading SC preparation techniques of shrimp testis were developed and SC of diploid and triploid F. chinensis spermatocytes was examined. SC analysis of triploid F. chinensis will be very helpful to analyze the mechanism of gametes development.

resultant pellet was resuspended in 1 mL 0.5% NaCl. After settling down for 10 min, the new cell suspension was gently mixed with 0.5% NaCl and 4% paraformaldehyde (PFA, with 0.03% SDS, pH 8.5) in a proportion of 1:4:6. After the mixed solution was settled down for another 10 min, and 500 ␮L of cell suspension taken from the top layer was dropped onto the convex surface of the slides. Plastic-coated slides with Formvar (0.5% w/v in chloroform) were used to pick up the spermatocytes nuclei from the spreaded drop after 5 min. The slides were air-dried and then fixed in 4% PFA for 10 min, washed by distilled water and then air-dried. Slides were examined by phase contrast microscope, and the wellspread SC structures were selected and stained with 50% silver nitrate in a moist chamber at 55 ◦ C as described by Howell and Black (1980), washed by distilled water and then air-dried. Areas with well-spread SC structures were marked with a waterproof pen under a light microscope. The plastic coating was scored with a sharp razor blade along the edges of the slide and floated off on the surface of distilled water after adding a few drops of 0.2 M hydrofluoric acid, which was applied to detach the plastic film from the glass. 50-mesh copper electron microscope grids were placed on the marks and the film, together with the grids, were carefully removed from the water surface with a piece of plastic-coated paper. After drying, the film was examined and photographed at 75 kV with a HITACHI H-7000 transmission electron microscope.

3. Results and discussion 2. Materials and methods 3.1. Electron micrograph of metaphase spermatogonium Gravid F. chinensis were sampled from the wild population in the Yellow Sea. Fertilized eggs were collected and treated by heat shock to inhibit the release of second polar bodies, which in turn induce triploidy. The treatment was performed following the optimized conditions reported by Li et al. (2003a). Induced triploid and diploid siblings were maintained in the aquarium of our institute. Male shrimps, 4–5 g in body weight at immature stage were selected for the present study. The females were impossible for SC analysis with in vitro meiosis processing of oogenesis. The ploidy of the shrimp was confirmed through flow cytometry (FCM) according to the methods described by Li et al. (2003b). The sex ratio of females to males in triploid F. chinensis used for SC analysis was about 4:1. In total, 6 triploid male F. chinensis or diploid male F. chinensis were used for SC analysis. The SC was analyzed by surface spreading technique modified from the Counce–Meyer method (1973). Briefly, the testis from each male diploid or triploid shrimp was dissected, lysed with 2 mL dispersion fluid (2% citrate acid with 0.5% Tween 20) and carefully minced using a glass rod to form a cell suspension. The 2 mL suspension was let to stand for 10 min to allow remaining debris to settle out. An 800 ␮L aliquot was then removed from the top layer and centrifuged at 1500 rpm for 5 min. The supernatant was removed, and the

During the SC preparations, metaphase spermatogonium of both the diploid (Fig. 1a) and triploid (Fig. 1b) F. chinensis were occasionally observed. 5–8 metaphases taken from each ploidy shrimp (total 6 male triploid F. chinensis and 6 male diploid F. chinensis) were observed and the chromosome numbers were counted. It showed that the number of chromosomes in diploid is around 88 and in triploid is about 132, which is in accordance with the results reported previously (Xiang, 1988; Zhang et al., 2003). Generally, the chromosome number of penaeid is large. 44 pairs of chromosomes were reported in F. chinensis, Penaeus monodon (Xiang et al., 1993) and Litopenaeus vannamei (Ramos, 1997), while 43 pairs of chromosomes in P. japonicus (Hayashi and Fujiwara, 1988). Therefore, cytogenetic studies on penaeid SC have been hampered by the large chromosome number. 3.2. SC morphology of diploid Electron micrographs of diploid shrimp spermatocytes nuclei from zygotene to pachytene were obtained. 44 bivalents were observed in the pachytene stage (Fig. 2), which is consistent with the chromosome number (2n = 88) reported in F. chinensis. In most eukaryotes, SC, which consists of

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Fig. 1. Electron micrographs of silver-stained spermatogonium metaphase in diploid (a) and triploid (b) Fenneropenaeus chinensis obtained from the SC preparations. Bars = 10 ␮m.

the paired lateral elements connected by transverse filaments (Page and Hawley, 2004), initially forms at or near the ends of the homologues at the early zygotene stage, achieves matured structure with a tight continuous association along

the homologues length during the pachtene stage and then disassembles during the diplotene stage of meiotic prophase I (Moses, 1977). SC spreads in diploid F. chinensis showed typical morphology of eukaryote SC, with two homologues

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Fig. 2. Electron micrograph of diploid Fenneropenaeus chinensis spermatocytes in pachytene stage. Bar = 10 ␮m.

synapsed completely from end to end with continuous, evenly spaced lateral elements. The presence of unpaired segment among paired chromosomes is one of the several characteristic features of sexual chromosome during meiosis (Carrasco et al., 1999). However, no bivalent with the behavior of the sexual chromosomes could be recognized in F. chinensis. Owing to the relatively large number of chromosomes in this species, it was exceedingly difficult to locate complete well-spread nuclei for analysis. According to our limited observations on the SC of F. chinensis, meiosis in diploid shrimp conforms to the general sequence of events described in other species (Counce and Meyer, 1973; Moses, 1977; Dresser and Giroux, 1988); synapsis occurs only between homologues. However, there was no apparent sex bivalent when we observed the SC of F. chinensis. Benzie et al. (1995) proposed that penaeids utilize a ZW sex determination system as a result of their skewed gender ratios from P. monodon and Penaeus esculentus hybrids and the Haldene effect. Preston’s group also suggested that there was evidence for the ZW system as their marker was linked on the female map for P. japonicus (Li et al., 2003) and because their triploids were always female for P. japonicus (Sellars et al., 2003, 2004). In a recent report of genetic map of L. vannamei,

sex-linked markers were mapped to the maternal map and so they suggested that the female may be the heterogametic sex in this species (Zhang et al., 2007). If penaeids utilize a ZW sex determination system, the male shrimp might be the homogametic sex. SC analysis is a very useful tool to identify heteromorphic sex bivalents, which has distinguished config´ urations in synapsis (Swito´ nski and Stranzinger, 1998; Van Eenennaam et al., 1998; Carrasco et al., 1999). During SC analysis of F. chinensis, we used testis to prepare the SC, so it is reasonable to find no sex bivalent. The present study has revealed no morphological difference between any homologous pair, although it is difficult to confirm individual SC. Further improvement in SC preparations should be applied in order to obtain well characterized SC karyotype of this species, and other methods such as gynogenesis should be developed to understand the sex determination mechanism in shrimps although it is very difficult at present. 3.3. Synapsis in triploid Compared with the diploid SC, irregular formation of SC was observed in the triploid F. chinensis. Very complex synaptic configurations were present in the early zygotene stage, it was found that unpaired axes were tangled, which

Fig. 3. Synapsis in triploid shrimp. (a) Early zygotene with most unpaired axial elements (indicated by arrows); (b) early pachytene with almost all homologues taking part in synapsis; (c) late pachytene with triple pairing SC formation marked by arrows. Bars = 10 ␮m.

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Fig. 3. (Continued ).

made the SC forming a network (Fig. 3a). However, almost all homologues took part in synapsis during the pachytene stage (Fig. 3b) and triple pairings, the connection of all three homologues in the same region, were common in the late pachytene stage (Fig. 3c) with the remaining unsynapsed axes poorly defined, which suggests that SC does not form exclusively between just two of them but all the three homologues simultaneously take part in synapsis in triploid. Additionally, we identified SC with the formation of irregular synaptic shapes in triploid, such as unsynapsed univalents, bivalents, partner switches (Fig. 4a) in which each chromosome can form a SC with either of the two others and totally paired trivalents with non-homologous synapsis (Fig. 4b). The above observations suggested that unusual synapsis behavior, which is similar to those described for triploid A. sphaerocephalon (Loidl and Jones, 1986) and triploid T. davisi (Borin et al., 2002) existed in triploid F. chinensis. It was interesting that triple synapsis was observed in spermatocytes of triploid shrimp with high frequency, which resembles the situation reported in triploid S. cerevisiae (Loidl, 1995) and triploid females of S. maximus (Cu˜nado et al., 2002). On the contrary, only bivalents were found in late pachytene nuclei of triploid O. mykiss (Oliveira et al., 1995) and male S. maximus (Cu˜nado et al., 2002). There is no uniform pattern of chromosome pairing in

triploids among different species, although with widespread presence of SC connecting chromosomes non-specifically (Loidl and Jones, 1986; Oliveira et al., 1995; Borin et al., 2002; Cu˜nado et al., 2002), suggesting that to be homologous is not an indispensable precondition for SC formation; totally paired trivalents with non-homologous regions pairing among the three homologous chromosomes observed in current study are in accordance with this hypothesis. Triploidy usually has an adverse effect on the fertility of the organism with the production of aneuploid gametes resulting from imbalanced segregation among the three homologues at the first meiotic division (Borin et al., 2002). Ploidy detection of sperm observed in the vas deferens of triploid F. chinensis is 3n (Li et al., 2003b), which is different from other triploids demonstrating between haploid and diploid values (Benfey et al., 1986; Guo and Allen, 1994). Triple pairing showed an overall high degree of saturation of synapsis observed in late pachytene nuclei, suggesting triploid shrimp would have more possibilities to overcome the pachytene and metaphase I checkpoints (Cu˜nado et al., 2002), which maybe critical for the production of 3n sperms observed by Li et al. (2003b). Future monitoring of the viability of the triploid sperms would provide further information on the fertility of triploid F. chinensis.

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Fig. 4. Details of irregular synaptic shapes in triploid. Partner switches (a); totally paired trivalents with non-homologous regions synapsis (b). Bar = 10 ␮m.

Acknowledgements This work was supported by General Program of National Natural Science Foundation of China (30471347, 40676087) and National High-Tech Research and Development Program of China (863 program) (2006AA10A403). The authors would like to thank Mr. Ming Jiang and Ms. Jialin Xie for their help in the electron microscopy.

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