- Email: [email protected]
Expression pattern of vasa in gonads of sea cucumber Apostichopus japonicus during gametogenesis and reproductive cycle
Gene Expression Patterns 13 (2013) 171–176
Contents lists available at SciVerse ScienceDirect
Gene Expression Patterns journal homepage: www.elsevier.com/locate/gep
Expression pattern of vasa in gonads of sea cucumber Apostichopus japonicus during gametogenesis and reproductive cycle Meng Yan a, Juan Sui b, Wanqiang Sheng c, Mingyu Shao a, Zhifeng Zhang a,⇑ a
Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, Qingdao, PR China Chinese Academy of Fishery Sciences, Yellow Sea Fishery Research Institute, Qingdao, PR China c Department of Biological Sciences, National University of Singapore, Kent Ridge, Singapore b
a r t i c l e
i n f o
Article history: Received 8 December 2012 Received in revised form 23 January 2013 Accepted 15 March 2013 Available online 27 March 2013 Keywords: Apostichopus japonicus vasa Gonad Gametogenesis Reproductive cycle
a b s t r a c t The vasa gene is a reliable germline marker to study the origin and development of germ cells and gonads, although the gene product (mRNA or protein) varies between different species. However, there has been little study on vasa genes in holothuroids to date. Here we determined the expression characteristics of the Apostichopus japonicus vasa gene (Aj-vasa) during gametogenesis in the ovary and testis using in situ hybridization and immunohistochemistry. During oogenesis, the expression pattern of Aj-vasa coincided at the mRNA and protein levels. Intensive signals in oogonia decreased gradually with the development of oocytes. Interestingly, the pattern was different during spermatogenesis. The Aj-vasa mRNA level was the highest in spermatogonia, reduced in spermatocytes, low in spermatids and absent in spermatozoa, but the Aj-VASA protein was restricted to spermatogonia and early spermatocytes. These expression characteristics of Aj-vasa persisted in both male and female gonads throughout the reproductive cycle. Our findings show that Aj-vasa mRNA is a good marker for studying the origin and migration of germline cells; moreover, Aj-VASA is a useful tool to identify spermatogonia in A. japonicus. Our findings indicate that Aj-vasa is vital in the development and differentiation of germ cells. Ó 2013 Elsevier B.V. All rights reserved.
The vasa gene was first identified in Drosophila, where it is restricted to germ cells during embryogenesis (Schüpbach and Wieschaus, 1986). It is a valuable marker for tracing the origin of PGCs in zebrafish (Yoon et al., 1997) and has been used widely as a credible germline marker molecule in about 20 vertebrate and invertebrate organisms (Gustafson and Wessel, 2010; Wang et al., 2012a,b; Xu et al., 2005). It has been cloned from more than 70 species among the cnidaria, tunicates, nematodes, platyhelminths, molluscs, annelids, crustaceans, insects, echinoderms and vertebrates (Gustafson and Wessel, 2010; Wang et al., 2012a,b), but its expression characteristics vary between species. In Drosophila, the VASA protein is initially localized in the polar granules of the posterior pole as soon as it becomes detectable in the oocyte, and vasa transcripts are maintained at a uniform distribution up to the early embryo stage. Thus VASA, but not vasa mRNA, has been confirmed as a germline marker in Drosophila, with a specific locaAbbreviations: Aj-vasa, Apostichopus japonicus vasa gene; Aj-VASA, protein of Ajvasa; PGCs, primordial germ cells; sqRT-PCR, semi-quantitative reverse transcription and polymerase chain reaction; H&E, hematoxylin and eosin; ISH, in situ hybridization; IHC, immunohistochemistry. ⇑ Corresponding author. Address: College of Marine Life Sciences, Ocean University of China, 5 Yushan Road, Qingdao, Shandong, PR China. Tel./fax: +86 532 82031647. E-mail address: [email protected] (Z. Zhang). 1567-133X/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gep.2013.03.001
tion in germ cells (Hay et al., 1988a; Liang et al., 1994). Similarly, VASA as a germline marker has also been verified in the amphibian Xenopus laevis (Komiya et al., 1994; Ikenishi and Tanaka, 1997), the mouse Mus musculus (Toyooka et al., 2000), the chicken Gallus domesticus (Tsunekawa et al., 2000) and the human Homo sapiens (Castrillon et al., 2000; Anderson et al., 2007; Albamonte et al., 2008). By contrast, vasa mRNA is a better germline marker in some species such as the zebrafish Danio rerio (Yoon et al., 1997; Olsen et al., 1997; Knaut et al., 2000), trout Oncorhynchus mykiss (Yoshizaki et al., 2000), tilapia Oreochromis niloticus (Kobayashi et al., 2000), medaka Oryzias latipes (Shinomiya et al., 2002), oyster Crassostrea gigas (Fabioux et al., 2004) and silkworm Bombyx mori (Nakao, 1999). In zebrafish oogenesis, the VASA protein is lost after germinal vesicle breakdown while the vasa mRNA remains in the cortex. Furthermore, the maternal vasa mRNA is restricted to the animal pole of the first two cleavage furrows in 4-cell embryos and is located in the four cells that are the precursors of zebrafish PGCs at the blastula stage (Knaut et al., 2000). The expression level of vasa varies between the ovary and testis, and the location of vasa also varies at mRNA and protein levels. In human gametogenesis, the expression of vasa mRNA is less marked in the fetal testis than in the fetal ovary (Anderson et al., 2007). In the gibel carp Carassius auratus gibelio both vasa mRNA and VASA persist throughout oogenesis with dynamic expression from
172
M. Yan et al. / Gene Expression Patterns 13 (2013) 171–176
oogonia to maturing oocytes. In contrast, vasa expression products are detectable in spermatogonia and spermatocytes, but disappear in spermatids and spermatozoa (Xu et al., 2005). There are also differential and dynamic expression patterns of vasa between the ovary and testis is also discovered in the mouse (Toyooka et al., 2000), tilapia (Kobayashi et al., 2000), and catfish Clarias gariepinus (Raghuveer and Senthilkumaran, 2010). Although vasa has been proven to be a germline marker in many animals, recent studies indicate that vasa is also expressed in multipotent stem cells in the flatworm Macrostomum lignano and cnidarian Hydractinia echinata (Pfister et al., 2008; Rebscher et al., 2008). However, RNA interference (RNAi) knockdown of vasa expression had no effect on the somatic neoblasts in M. lignano (Pfister et al., 2008). The function of VASA in somatic cells is unknown in most animals and needs to be studied further. The sea cucumber Apostichopus japonicus, a species of the Holothuroldea in the phylum Echinodermata is an important commercial species in China. There have been numerous studies on its reproductive biology, such as morphology of the reproductive system, reproductive behavior and larval development (Sui et al., 1985; Pang et al., 2006; Kato et al., 2009). However, basic knowledge of the molecular biology of gametogenesis and gonadogenesis is limited in this species. In this study, we examined vasa expression in the gonads of A. japonicus during gametogenesis at the mRNA and protein levels. These data will provide a basic understanding of the expression of vasa in the development of A. japonicus germ cells.
1. Results 1.1. Specific expression of vasa mRNA in gonads Results from sqRT–PCR showed that Aj-vasa mRNA was expressed specifically in the ovary and testis of adult A. japonicus; however, no vasa mRNA appeared in any of the somatic organs examined, namely the respiratory tree and intestine (Fig. 1).
1.2. Germ cell-specific location of Aj-vasa mRNA and Aj-VASA protein during gametogenesis Gonads of A. japonicus are elongate, slender, branching tube-like structures. Germ cells in the growing stage ovary are composed of aggregated oogonia with a high nucleus-to-cytoplasm ratio; 5– 8 lm in diameter and primary oocytes 15–100 lm in diameter; primary oocytes are 110–130 lm in diameter when mature. The testis at this stage contains spermatogonia with a high nucleusto-cytoplasm ratio measuring 5–7 lm in diameter with clear nuclear material. It also contains spermatocytes with a dense nucleus 4–6 lm in diameter; round spermatids with no flagellum 3–4 lm in diameter; and spermatozoa with elliptical heads 2.5–3 lm long and an acidophilic flagellum. These elements are arranged from the
Fig. 1. Spatial expression of Aj-vasa mRNA in adult A. japonicus tissues analyzed by sqRT-PCR.
periphery to the center of the seminiferous tubule, respectively (Sui et al., 1985; Pang et al., 2006) (Fig. 2A and B). The expression and distribution of Aj-vasa mRNA and Aj-VASA during oogenesis and spermatogenesis were detected using ISH and IHC. In the ovary, both mRNA and Aj-VASA were present in germ cells throughout oogenesis. The Aj-vasa mRNA signal was most intense in the cytoplasm of oogonia and early primary oocytes, and declined with oocyte development (Fig. 2C). The locations of Aj-VASA and changes in its signal intensity in the ovary were similar to those of Aj-vasa mRNA (Fig. 2E and G). No signal was observed in somatic cells of the ovary (Fig. 2G). In the testis, Aj-vasa mRNA was expressed in the cytoplasm of spermatogonia, spermatocytes and spermatids. The signal intensity was the highest in spermatogonia, decreased gradually with spermatocyte development, was weak in spermatid and absent from the mature spermatozoa (Fig. 2D). The expression locations differed between Aj-vasa mRNA and Aj-VASA during spermatogenesis, in that AjVASA was only located in the cytoplasm of spermatogonia and early spermatocytes, and not present in late spermatocytes, spermatids or spermatozoa (Fig. 2F and H). No expression was found in somatic cells of the testis (Fig. 2H). 1.3. Expression of Aj-VASA in gonads during the reproductive cycle An abundance of Aj-VASA was located in oogonia and oocytes of the ovary at the growing stage (Fig. 3A and C). It declined in developing oocytes in the ovary at the growing stage. The expression of Aj-VASA was weak in oocytes of the mature ovary and in residual oocytes of the ovary at the post-spawning stage (Fig. 3E and G). In the testis, Aj-VASA expression was concentrated in spermatogonia and early spermatocytes during the early growing stage, and restricted to spermatogonia at the later growing and mature stages (Fig. 3D and F). It was absent from the testis at the post-spawning stage (Fig. 3H). 2. Discussion 2.1. Expression of the vasa gene and potential application as a marker for A. japonicus germ cells The vasa gene has been reported as a molecular marker of germline in many invertebrates such as the Chinese shrimp Fenneropenaeus chinensis (Zhou et al., 2010; Feng et al., 2011), oyster C. gigas (Fabioux et al., 2004) and Chinese mitten crab Eriocheir sinensis (Wang et al., 2012a). Like most species, the expression of Aj-vasa detected by sqRT–PCR was also specific for the A. japonicus gonads (Fig. 1). Furthermore, ISH and IHC revealed that both Aj-vasa mRNA and Aj-VASA were restricted to the germ cells, and no signal was detected in somatic cells of the ovary or testis. Thus the expression pattern of Aj-vasa in the germ cells of A. japonicus was similar to that of equivalent genes in other organisms examined to date. The VASA protein, but not vasa mRNA, has been demonstrated to be a germ cell marker in species such as Drosophila and Xenopus with an initial restriction to germline cells (Hay et al.,1988b; Komiya et al., 1994). However, in the zebrafish, vasa mRNA is more specific as a germline marker than VASA protein during gametogenesis and embryogenesis (Knaut et al., 2000; Braat et al., 2000). Here we found that Aj-vasa mRNA was expressed in a wider range of germ cells including oogonia, oocytes, spermatogonia, spermatocytes and spermatids, but was absent from somatic cells of the gonads. This suggests that the Aj-vasa mRNA might be a better germ cell marker than Aj-VASA to study the origination and migration of PGCs and differentiation of germ cells in A. japonicus. In the male specimens, Aj-VASA expression was restricted to spermatogonia and few early spermatocytes. These distribution
M. Yan et al. / Gene Expression Patterns 13 (2013) 171–176
173
Fig. 2. Location of Aj-vasa mRNA and Aj-VASA detected by ISH and IHC in A. japonicus gonad at the growing stage. (A) Histology of the ovary stained by H&E; the insert is a magnification of the square region. (B) Histology of the edge of the testis stained by H&E. (B’) Histology of the central region of the testis stained by H&E. (C and D) Aj-vasa mRNA expression demonstrated with an antisense probe in the ovary (C) and testis (D). (E and F) Aj-VASA expression demonstrated by IHC with an anti-Aj-VASA antibody in the ovary (E) and testis (F); the insert in (E) is a magnified view of oogonium and an early oocyte in the wall of the gonadal tube. (G) Magnified view of oogonium and developing oocytes on a serial section around (E). (H) Magnified view of spermatogonium, spermatocytes, spermatids and spermatozoa on a serial section around (F). The insert in (H) shows the central part of the testis at the same scale. (I, I’) Hybridization with sense probe of the ovary (I) and testis (I’) as negative control for ISH. (J, J’) Immunostaining with preimmune rabbit serum of the ovary (J) and testis (J’) as negative controls for IHC. Positive signals with an antisense probe for Aj-vasa mRNA detected by ISH are shown in blue and positive signals for Aj-VASA are shown as reddish-brown by IHC. After ISH and IHC sections were counterstained with neutral red and haematoxylin respectively. Abbreviations: N, nucleus; Og, oogonium; Oo, oocyte; Sc, spermatocyte; Sg, spermatogonium; SoC, somatic cell; St, spermatid; Sz, spermatozoon. Scale bars = 25 lm.
174
M. Yan et al. / Gene Expression Patterns 13 (2013) 171–176
Fig. 3. Differential expression of Aj-VASA detected by IHC in gonads of A. japonicus at various stages. (A) Ovary at the early growing stage. (B) Testis at the early growing stage. (C) Ovary at the growing stage. (D) Testis at the growing stage. (E) Ovary at the mature stage. (F) Testis at the mature stage. (G) Ovary at post-spawning stage, the insert in (G) is a negative control. (H) Testis at the post-spawning stage. Positive IHC signals for Aj-VASA are reddish-brown. Abbreviations: OC, ovarian cavity; Og, oogonium; Oo, oocyte; Sc, spermatocyte; Sg, spermatogonium; SoC, somatic cell; Sz, spermatozoon; TC, testis cavity. Scale bars = 25 lm.
characteristics limit its use as a general marker of germline cells; however, this specificity could be used to distinguish and isolate spermatogonia in vitro. In the sea urchin Strongylocentrotus nudus—another echinoderm species—Yakovlev et al. has isolated oogonia from the ovary using density gradient centrifugation. Because intense signals of the sea urchin VASA could be detected in the perinuclear area of oogonia and gave uniform signals in oocytes, oogonia could be isolated and identified with a 70–75% positive rate (Yakovlev et al., 2010). To study the differentiation of male gametes, it will necessary to identify and isolate spermatogonia accurately, but little research on this has been done in invertebrates. In the present study, Aj-VASA was expressed specifically in spermatogonia and early spermatocytes. Thus, it might serve as a probe for studying the origination and differentiation of spermatogonial stem cells in the Holothurioidea.
2.2. The role of VASA in maintaining the development of gametes In A. japonicus, the expression of VASA was different between oogenesis and spermatogenesis: in the testis it was only present in spermatogonia and a few early spermatocytes, but in the ovary it was expressed steadily in all stages of oogenesis. This indicates that the function of Aj-VASA is different between genders. Thus, it participates throughout oogenesis, but only during the early stages of spermatogenesis. The timing of VASA expression during spermatogenesis has been reported in other organisms such as the tilapia (Kobayashi et al., 2000), gibel carp (Xu et al., 2005) and mouse (Toyooka et al., 2000; Tanaka et al., 2000). In the mouse, the Mvh gene (mouse vasa homologue) appears necessary for the completion of spermatogenesis but not for oogenesis. Lack of a mouse VASA protein function causes germ cells to cease differenti-
M. Yan et al. / Gene Expression Patterns 13 (2013) 171–176
ation into pachytene spermatocytes and to undergo apoptotic cell death (Tanaka et al., 2000). However, in Drosophila vasa appeared to have an essential function in female gametogenesis, but not in male gametogenesis. In vasa-null Drosophila ovaries, germaria are atrophied, the number of oocytes declines or even disappears in the egg chambers, and the oocyte nucleus appears more diffuse than that of wild-type flies (Styhler et al., 1998; Tomancak et al., 1998). In the oyster C. gigas, both the amount of mRNA and protein encoded by Oyvlg (oyster vasa-like gene) decreased during spermatogenesis and oogenesis when the Oyvlg expression was knocked down by RNAi. The knockdown phenotype females halted their gametogenesis at prophase I of meiosis before vitellogenesis. In males with Oyvlg knockdown phenotype, development of the germ cells stopped at the spermatocyte stage (Fabioux et al., 2009). These results demonstrate that VASA has an essential role in germ cell maintenance, and probably has functions that differ between male and female gametogenesis. In conclusions, the expression pattern of the Aj-vasa is consistent with the idea that the vasa gene is the most reliable molecular marker for studies on germ cell development in most species. Our findings suggest that the expression level of Aj-vasa varies between ovary and testis, and the location of vasa also varies at mRNA and protein levels. Moreover, Aj-vasa mRNA is a good marker for studying the origin and migration of germline cells and Aj-VASA is a useful tool to identify spermatogonia in A. japonicus. 3. Experimental procedures 3.1. Animal and Tissue preparation Adult A. japonicus were purchased from the aquatic product market of Qingdao, PR China. Tissue samples (ovary, testis, respiratory tree and alimentary tract) were dissected out, frozen immediately in liquid nitrogen and stored at 80 °C for RNA isolation. Part of the fresh gonad was fixed in Bouin’s solution for histology. The other samples were fixed in 4% paraformaldehyde (PFA) for 16 h before being stored in 100% methanol at 20 °C for in situ hybridization (ISH) and immunohistochemistry. According to the histological characteristics described by Sui et al. (Sui et al., 1985), the gonads of adult A. japonicus were divided into three groups. First was the growing stage in which the amounts of germ cells increase. These are composed of aggregated oogonia and developing oocytes (15–100 lm in diameter) in the ovary and various stages, including spermatogonia, spermatocytes, spermatids and mature spermatozoa in the testis. Second was the maturation stage, in which the ovary is filled with mature oocytes 110–130 lm in diameter and the testis is full of mostly mature spermatozoa. Third was the post-spawning stage, in which both the ovary and testis are empty with only a few residual gametes remaining. 3.2. Semi-quantitative reverse transcription polymerase chain reaction (sqRT–PCR) Total RNA was extracted from the ovary, testis, respiratory tree and alimentary tract at the gonadal growing stage using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The concentration and purity of total RNA were determined spectrophotometrically (Bio-Rad, Hercules, CA, USA). First-strand cDNA was synthesized using an ImProm-II™ Reverse Transcription system (Promega, Madison, WI, USA). Tissue distributions of the Aj-vasa in A. japonicus were analyzed using sqRT–PCR with primers VF3 (5-GGTCAGCCAACCGAAGAAAA-3) and VR3 (5-CGTGAACAGAGGGAAAGAGA-3) according to the Aj-vasa cDNA sequence (GenBank Accession No. EU273885) to amplify the Aj-vasa fragment of 556 bp. A fragment (369 bp) of the
175
reference gene (A. japonicus 18S rRNA) was amplified with primers 18F1 (5-GCAAATGCTTTCGCTGTCG-3) and 18F2 (5’-CTCGTAGTCGGATTTCTGG-3) designed according to the 18S rRNA sequence (GenBank Accession No. EU821599). The reaction conditions were 94 °C for 5 min, 94 °C for 30 s, 58 °C for 30 s, 72 °C for 30 s with 30 cycles for the Aj-vasa fragment; 16 cycles for the reference gene and finally extension at 72 °C for 5 min. 3.3. Histology Fixed gonads were dehydrated in an ascending ethanol series, cleared with xylene and embedded in paraffin wax. Sections (5 lm) were stained with hematoxylin and eosin (H&E) and microscopy and digital image capture were done using a Nikon E80i microscope. 3.4. In situ hybridization (ISH) Digoxigenin (DIG)-labeled RNA sense and anti-sense probes were synthesized from the clone of an Aj-vasa 512 bp fragment using a DIG RNA Labeling Kit (SP6/T7, Roche Applied Science, Indianapolis, IN, USA). The fragment was amplified using two specific primers (sense 5-CAAGCTTGGTGGT CGTGATCGACYGTG-3; antisense 5-GGAATTCGACGTTCTCACCGACGCTG-3), based on the fulllength sequence of Aj-vasa cDNA (GenBank Accession No. EU273885). Ovary and testis were embedded in paraffin wax, and the sections (5 lm thick) were affixed to microscope slides with 2% polylysine for 10 h at 37 °C. The sections were rehydrated through a descending series of methanol solutions and phosphatebuffered saline with 0.1% Tween-20 (PBST). Samples were then digested for 30 min at 37 °C using protease K (2 lg/ml), prehybridized at 50 °C in hybridization solution (HS, 50% formamide, 5% SSC, 5 mM EDTA, 1.5% blocking reagent, 0.1% Tween-20, 100 lg/ ml ribonucleic acid (Sigma–Aldrich, St. Louis, MO, USA)) for 6 h, and then hybridized in HS with the Aj-vasa RNA probes for 16 h. The samples were washed at 50 °C for 130 min and at room temperature two times for 10 min in maleic acid buffer (0.1 M maleic acid, 0.15 M NaCl, 0.1% Tween-20, pH 7.5). Finally, hybridization signals were detected using alkaline phosphatase-conjugated anti-DIG antibody and NBT/BCIP (DIG Nucleic Acid Detection Kit, Roche Applied Science) before being counterstained with neutral red. Microscopy and digital image capture were done using a Nikon E80i microscope. 3.5. Immunohistochemistry (IHC) A partial cDNA fragment encoding 88 amino acids of the AjVASA N-terminus was amplified using Ex Taq DNA polymerase (Takara Bio Inc., Shiga, Japan) with primers 5-GAATTCATGG GACACTTCGCTAGAGA-3’ (EcoRI site underlined) as the sense primer and 5-CTCGAGCGCTCTCCTGACGTTCTCAC-3 (XhoI site underlined) as the antisense primer. The PCR product was subcloned into a pMD18-T vector (Takara) followed by digestion with EcoRI and XhoI and then subcloned into the expression vector pET32 (Novagen, Madison, WI, USA). The recombinant pET32–VASA plasmid was transformed into Escherichia coli BL21 (DE3) pLysS. Aj-VASA expression was induced by adding isopropyl -D-thiogalactoside to LB medium at a final concentration of 1.0 mM and incubating at 37 °C for 6 h. The recombinant Aj-VASA was purified using Ni– NTA affinity chromatography following the manufacturer’s instructions (Novagen) and the purity of eluted samples analyzed by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis stained with Coomassie brilliant blue R-250. An emulsion of equal volumes of 200 lg of purified Aj-VASA and Freund’s complete adjuvant (Sigma–Aldrich) was injected subcutaneously at multiple sites on two New Zealand White rabbits.
176
M. Yan et al. / Gene Expression Patterns 13 (2013) 171–176
Two booster injections of 100 lg antigen mixed with Freund’s incomplete adjuvant (Sigma–Aldrich) were administered subcutaneously at 2-week intervals. One week after the final booster, blood was collected and serum prepared. The antibody titer was determined by enzyme-linked immunosorbent assay and the antisera were aliquoted before being stored at 80 °C. Testes and ovaries fixed in 4% PFA were processed as described in Section 3.4 before digestion with protease K. After two 5-min washes in PBST, the endogenous peroxidase activity in the sections was quenched by incubation in methanol with 3% H2O2 (v/v) at room temperature for 10 min, followed by two 5-min washes in PBST. Subsequently, the sections were incubated in 3% bovine serum albumin solution for 30 min, and then incubated with rabbit anti-Aj-VASA antibody (diluted 1:800) for 1 h before incubation with peroxidase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) (diluted 1:5,000) for 1 h. A chromogenic reaction was achieved by the addition of 0.05% Diaminobenzidine (w/v) containing 0.01% H2O2 (v/v) in PBST and maintained in the dark for 5 min. Sections were then washed in PBST, counterstained with hematoxylin, dehydrated through an ascending ethanol series, cleared in xylene, mounted in neutral balsam and observed and photographed using a Nikon E80i microscope. Acknowledgment This work was supported by the Specialized Research Fund for the Doctoral Program of Higher Education, PR China (No. 200804230015) and the National High Technology Research and Development Program of China (863 program) (No. 2012AA10A402). References Albamonte, M.S., Willis, M.A., Albamonte, M.I., Jensen, F., Espinosa, M.B., Vitullo, A.D., 2008. The developing human ovary: immunohistochemical analysis of germ-cell-specific VASA protein, BCL-2/BAX expression balance and apoptosis. Hum. Reprod. 23, 1895–1901. Anderson, R., Fulton, N., Cowan, G., Coutts, S., Saunders, P., 2007. Conserved and divergent patterns of expression of DAZL, VASA and OCT4 in the germ cells of the human fetal ovary and testis. BMC Dev. Biol. 7, 136. Braat, A.K., van de Water, S., Goos, H., Bogerd, J., Zivkovic, D., 2000. VASA protein expression and localization in the zebrafish. Mech. Dev. 95, 271–274. Castrillon, D.H., Quade, B.J., Wang, T.Y., Quigley, C., Crum, C.P., 2000. The human VASA gene is specifically expressed in the germ cell lineage. Proc. Natl. Acad. Sci. USA 97, 9585–9590. Fabioux, C., Huvet, A., Lelong, C., Robert, R., Pouvreau, S., Daniel, J.Y., Minguant, C., Le Pennec, M., 2004. Oyster vasa-like gene as a marker of the germline cell development in Crassostrea gigas. Biochem. Biophys. Res. Commun. 320, 592– 598. Fabioux, C., Corporeau, C., Quillien, V., Favrel, P., Huvet, A., 2009. In vivo RNA interference in oyster -vasa silencing inhibits germ cell development. FEBS J. 276, 2566–2573. Feng, Z.F., Zhang, Z.F., Shao, M.Y., Zhu, W., 2011. Developmental expression pattern of the Fc-vasa-like gene, gonadogenesis and development of germ cell in Chinese shrimp, Fenneropenaeus chinensis. Aquaculture 314, 202–209. Gustafson, E.A., Wessel, G.M., 2010. Vasa genes: emerging roles in the germline and in multipotent cells. Bioessays 32, 626–637. Hay, B., Ackerman, L., Barbel, S., Jan, L.Y., Jan, Y.N., 1988a. Identification of a component of Drosophila polar granules. Development 703, 825–840. Hay, B., Jan, L.Y., Jan, Y.N., 1988b. A protein component of Drosophila polar granules is encoded by vasa and has extensive sequence similarity to ATP-dependant helicases. Cell 55, 577–587. Ikenishi, K., Tanaka, T.S., 1997. Involvement of the protein of Xenopus vasa homolog (Xenopus vasa-like gene 1, XVLG1) in the differentiation of primordial germ cells. Dev. Growth Differ. 39, 625–633.
Kato, S., Tsurumaru, S., Taga, M., Yamane, T., Shibata, Y., Ohno, K., Fujiwara, A., Yamano, K., Yoshikuni, M., 2009. Neuronal peptides induce oocyte maturation and gamete spawning of sea cucumber, Apostichopus japonicus. Dev. Biol. 326, 169–176. Knaut, H., Pelegri, F., Bohmann, K., Schwarz, H., Nusslein-Volhard, C., 2000. Zebrafish vasa RNA but not its protein is a component of the germ plasm and segregates asymmetrically before germline specification. J. Cell Biol. 149, 875–888. Kobayashi, T., Kajiura-Kobayashi, H., Nagahama, Y., 2000. Differential expression of vasa homologue gene in the germ cells during oogenesis and spermatogenesis in a teleost fish, tilapia, Oreochromis niloticus. Mech. Dev. 99, 139–142. Komiya, T., Itoh, K., Ikenishi, K., Furusawa, M., 1994. Isolation and characterization of a novel gene of the DEAD box protein family which is specifically expressed in germ cells of Xenopus laevis. Dev. Biol. 162, 354–363. Liang, L., Diehl-Jones, W., Lasko, P., 1994. Localization of vasa protein to the Drosophila pole plasm is independent of its RNA-binding and helicase activities. Development 120, 1201–1211. Nakao, H., 1999. Isolation and characterization of a Bombyx vasa-like gene. Dev. Genes Evol. 209, 312–316. Olsen, L.C., Aasland, R., Fjose, A., 1997. A vasa-like gene in zebrafish identifies putative primordial germ cells. Mech. Dev. 66, 95–105. Pang, Z.G., Sun, H.L., Yan, J.P., Yu, D.X., Fang, J.G., Jiang, M., 2006. Ultrastructural studies on spermatogenesis of sea cucumber Apostichopus japonicus. Marine Fish. Res. 27, 27–31. Pfister, D., De Mulder, K., Hartenstein, V., Kuales, G., Borgonie, G., Marx, F., Morris, J., Ladurner, P., 2008. Flatworm stem cells and the germ line: developmental and evolutionary implications of macvasa expression in Macrostomum lignano. Dev. Biol. 319, 146–159. Raghuveer, K., Senthilkumaran, B., 2010. Cloning and differential expression pattern of vasa in the developing and recrudescing gonads of catfish, Clarias gariepinus. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 57, 79–85. Rebscher, N., Volk, C., Teo, R., Plickert, G., 2008. The germ plasm component vasa allows tracing of the interstitial stem cells in the cnidarian Hydractinia echinata. Dev. Dyn. 237, 1736–1745. Schüpbach, T., Wieschaus, E., 1986. Germline autonomy of maternal-effect mutations altering the embryonic body pattern of Drosophila. Dev. Biol. 113, 443–448. Shinomiya, A., Tanaka, M., Kobayashi, T., Nagahama, Y., Hamaguchi, S., 2002. The vasa-like gene, olvas, identifies the migration path of primordial germ cells during embryonic body formation stage in the medaka, Oryzias latipes. Dev. Growth Differ. 42, 317–326. Styhler, S., Nakamura, A., Swan, A., Suter, B., Lasko, P., 1998. Vasa is required for GURKEN accumulation in the oocyte, and is involved in oocyte differentiation and germline cyst development. Development 125, 1569–1578. Sui, X.L., Liu, Y.X., Liu, Y.F., Shang, L.B., Hu, Q.M., 1985. A study on the reproductive cycle of sea cucumber. J. Fish. Chin. 9, 303–311. Tanaka, S.S., Toyooka, Y., Akasu, R., 2000. The mouse homolog of Drosophila Vasa is required for the development of male germ cells. Genes Dev. 14, 841–853. Tomancak, P., Guichet, A., Zavorszky, P., Ephrussi, A., 1998. Oocyte polarity depends on regulation of gurken by Vasa. Development 125, 1723–1732. Toyooka, Y., Tsunekawa, N., Takahashi, Y., Matsui, Y., Satoh, M., Noce, T., 2000. Expression and intracellular localization of mouse VASA-homologue protein during germ cell development. Mech. Dev. 93, 139–149. Tsunekawa, N., Naito, M., Sakai, Y., Nishida, T., Noce, T., 2000. Isolation of chicken vasa homolog gene and tracing the origin of primordial germ cells. Development 127, 2741–2750. Wang, Q., Fang, D.A., Sun, J.L., Wang, Y., Wang, J., Liu, L.H., 2012a. Characterization of the vasa gene in the Chinese mitten crab Eriocheir sinensis: a germline molecular marker. Insect Physiol. 58, 960–965. Wang, Y.L., Chen, Y.D., Han, K.H., Zou, Z.H., Zhang, Z.P., 2012b. A vasa gene from green mud crab Scylla paramamosain and its expression during gonadal development and gametogenesis. Mol. Biol. Rep. 39, 4327–4335. Xu, H.Y., Gui, J.F., Hong, Y.H., 2005. Differential expression of vasa RNA and protein during spermatogenesis and oogenesis in the gibel carp (Carassius auratus gibelio), a bisexually and gynogenetically reproducing vertebrate. Dev. Dyn. 233, 872–882. Yakovlev, K.V., Battulin, N.R., Serov, O.L., Odintsova, N.A., 2010. Isolation of oogonia from ovaries of the sea urchin Strongylocentrotus nudus. Cell Tissue Res. 342, 479–490. Yoon, C., Kawakami, K., Hopkins, N., 1997. Zebrafish vasa homologue RNA is localized to the cleavage planes of 2- and 4-cell-stage embryos and is expressed in the primordial germ cells. Development 124, 3157–3166. Yoshizaki, G., Sakatani, S., Tominaga, H., Takeuchi, T., 2000. Cloning and characterization of a vasa-like gene in rainbow trout and its expression in the germ cell lineage. Mol. Reprod. Dev. 55, 364–371. Zhou, Q.R., Zhang, Z.F., Shao, M.Y., Kang, K.H., 2010. Cloning, characterization and expression analysis of the DEAD-box family genes, Fc-vasa and Fc-PL10a, in Chinese shrimp (Fenneropenaeus chinensis). CSOL 28, 37–45.
Contents lists available at SciVerse ScienceDirect
Gene Expression Patterns journal homepage: www.elsevier.com/locate/gep
Expression pattern of vasa in gonads of sea cucumber Apostichopus japonicus during gametogenesis and reproductive cycle Meng Yan a, Juan Sui b, Wanqiang Sheng c, Mingyu Shao a, Zhifeng Zhang a,⇑ a
Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, Qingdao, PR China Chinese Academy of Fishery Sciences, Yellow Sea Fishery Research Institute, Qingdao, PR China c Department of Biological Sciences, National University of Singapore, Kent Ridge, Singapore b
a r t i c l e
i n f o
Article history: Received 8 December 2012 Received in revised form 23 January 2013 Accepted 15 March 2013 Available online 27 March 2013 Keywords: Apostichopus japonicus vasa Gonad Gametogenesis Reproductive cycle
a b s t r a c t The vasa gene is a reliable germline marker to study the origin and development of germ cells and gonads, although the gene product (mRNA or protein) varies between different species. However, there has been little study on vasa genes in holothuroids to date. Here we determined the expression characteristics of the Apostichopus japonicus vasa gene (Aj-vasa) during gametogenesis in the ovary and testis using in situ hybridization and immunohistochemistry. During oogenesis, the expression pattern of Aj-vasa coincided at the mRNA and protein levels. Intensive signals in oogonia decreased gradually with the development of oocytes. Interestingly, the pattern was different during spermatogenesis. The Aj-vasa mRNA level was the highest in spermatogonia, reduced in spermatocytes, low in spermatids and absent in spermatozoa, but the Aj-VASA protein was restricted to spermatogonia and early spermatocytes. These expression characteristics of Aj-vasa persisted in both male and female gonads throughout the reproductive cycle. Our findings show that Aj-vasa mRNA is a good marker for studying the origin and migration of germline cells; moreover, Aj-VASA is a useful tool to identify spermatogonia in A. japonicus. Our findings indicate that Aj-vasa is vital in the development and differentiation of germ cells. Ó 2013 Elsevier B.V. All rights reserved.
The vasa gene was first identified in Drosophila, where it is restricted to germ cells during embryogenesis (Schüpbach and Wieschaus, 1986). It is a valuable marker for tracing the origin of PGCs in zebrafish (Yoon et al., 1997) and has been used widely as a credible germline marker molecule in about 20 vertebrate and invertebrate organisms (Gustafson and Wessel, 2010; Wang et al., 2012a,b; Xu et al., 2005). It has been cloned from more than 70 species among the cnidaria, tunicates, nematodes, platyhelminths, molluscs, annelids, crustaceans, insects, echinoderms and vertebrates (Gustafson and Wessel, 2010; Wang et al., 2012a,b), but its expression characteristics vary between species. In Drosophila, the VASA protein is initially localized in the polar granules of the posterior pole as soon as it becomes detectable in the oocyte, and vasa transcripts are maintained at a uniform distribution up to the early embryo stage. Thus VASA, but not vasa mRNA, has been confirmed as a germline marker in Drosophila, with a specific locaAbbreviations: Aj-vasa, Apostichopus japonicus vasa gene; Aj-VASA, protein of Ajvasa; PGCs, primordial germ cells; sqRT-PCR, semi-quantitative reverse transcription and polymerase chain reaction; H&E, hematoxylin and eosin; ISH, in situ hybridization; IHC, immunohistochemistry. ⇑ Corresponding author. Address: College of Marine Life Sciences, Ocean University of China, 5 Yushan Road, Qingdao, Shandong, PR China. Tel./fax: +86 532 82031647. E-mail address: [email protected] (Z. Zhang). 1567-133X/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gep.2013.03.001
tion in germ cells (Hay et al., 1988a; Liang et al., 1994). Similarly, VASA as a germline marker has also been verified in the amphibian Xenopus laevis (Komiya et al., 1994; Ikenishi and Tanaka, 1997), the mouse Mus musculus (Toyooka et al., 2000), the chicken Gallus domesticus (Tsunekawa et al., 2000) and the human Homo sapiens (Castrillon et al., 2000; Anderson et al., 2007; Albamonte et al., 2008). By contrast, vasa mRNA is a better germline marker in some species such as the zebrafish Danio rerio (Yoon et al., 1997; Olsen et al., 1997; Knaut et al., 2000), trout Oncorhynchus mykiss (Yoshizaki et al., 2000), tilapia Oreochromis niloticus (Kobayashi et al., 2000), medaka Oryzias latipes (Shinomiya et al., 2002), oyster Crassostrea gigas (Fabioux et al., 2004) and silkworm Bombyx mori (Nakao, 1999). In zebrafish oogenesis, the VASA protein is lost after germinal vesicle breakdown while the vasa mRNA remains in the cortex. Furthermore, the maternal vasa mRNA is restricted to the animal pole of the first two cleavage furrows in 4-cell embryos and is located in the four cells that are the precursors of zebrafish PGCs at the blastula stage (Knaut et al., 2000). The expression level of vasa varies between the ovary and testis, and the location of vasa also varies at mRNA and protein levels. In human gametogenesis, the expression of vasa mRNA is less marked in the fetal testis than in the fetal ovary (Anderson et al., 2007). In the gibel carp Carassius auratus gibelio both vasa mRNA and VASA persist throughout oogenesis with dynamic expression from
172
M. Yan et al. / Gene Expression Patterns 13 (2013) 171–176
oogonia to maturing oocytes. In contrast, vasa expression products are detectable in spermatogonia and spermatocytes, but disappear in spermatids and spermatozoa (Xu et al., 2005). There are also differential and dynamic expression patterns of vasa between the ovary and testis is also discovered in the mouse (Toyooka et al., 2000), tilapia (Kobayashi et al., 2000), and catfish Clarias gariepinus (Raghuveer and Senthilkumaran, 2010). Although vasa has been proven to be a germline marker in many animals, recent studies indicate that vasa is also expressed in multipotent stem cells in the flatworm Macrostomum lignano and cnidarian Hydractinia echinata (Pfister et al., 2008; Rebscher et al., 2008). However, RNA interference (RNAi) knockdown of vasa expression had no effect on the somatic neoblasts in M. lignano (Pfister et al., 2008). The function of VASA in somatic cells is unknown in most animals and needs to be studied further. The sea cucumber Apostichopus japonicus, a species of the Holothuroldea in the phylum Echinodermata is an important commercial species in China. There have been numerous studies on its reproductive biology, such as morphology of the reproductive system, reproductive behavior and larval development (Sui et al., 1985; Pang et al., 2006; Kato et al., 2009). However, basic knowledge of the molecular biology of gametogenesis and gonadogenesis is limited in this species. In this study, we examined vasa expression in the gonads of A. japonicus during gametogenesis at the mRNA and protein levels. These data will provide a basic understanding of the expression of vasa in the development of A. japonicus germ cells.
1. Results 1.1. Specific expression of vasa mRNA in gonads Results from sqRT–PCR showed that Aj-vasa mRNA was expressed specifically in the ovary and testis of adult A. japonicus; however, no vasa mRNA appeared in any of the somatic organs examined, namely the respiratory tree and intestine (Fig. 1).
1.2. Germ cell-specific location of Aj-vasa mRNA and Aj-VASA protein during gametogenesis Gonads of A. japonicus are elongate, slender, branching tube-like structures. Germ cells in the growing stage ovary are composed of aggregated oogonia with a high nucleus-to-cytoplasm ratio; 5– 8 lm in diameter and primary oocytes 15–100 lm in diameter; primary oocytes are 110–130 lm in diameter when mature. The testis at this stage contains spermatogonia with a high nucleusto-cytoplasm ratio measuring 5–7 lm in diameter with clear nuclear material. It also contains spermatocytes with a dense nucleus 4–6 lm in diameter; round spermatids with no flagellum 3–4 lm in diameter; and spermatozoa with elliptical heads 2.5–3 lm long and an acidophilic flagellum. These elements are arranged from the
Fig. 1. Spatial expression of Aj-vasa mRNA in adult A. japonicus tissues analyzed by sqRT-PCR.
periphery to the center of the seminiferous tubule, respectively (Sui et al., 1985; Pang et al., 2006) (Fig. 2A and B). The expression and distribution of Aj-vasa mRNA and Aj-VASA during oogenesis and spermatogenesis were detected using ISH and IHC. In the ovary, both mRNA and Aj-VASA were present in germ cells throughout oogenesis. The Aj-vasa mRNA signal was most intense in the cytoplasm of oogonia and early primary oocytes, and declined with oocyte development (Fig. 2C). The locations of Aj-VASA and changes in its signal intensity in the ovary were similar to those of Aj-vasa mRNA (Fig. 2E and G). No signal was observed in somatic cells of the ovary (Fig. 2G). In the testis, Aj-vasa mRNA was expressed in the cytoplasm of spermatogonia, spermatocytes and spermatids. The signal intensity was the highest in spermatogonia, decreased gradually with spermatocyte development, was weak in spermatid and absent from the mature spermatozoa (Fig. 2D). The expression locations differed between Aj-vasa mRNA and Aj-VASA during spermatogenesis, in that AjVASA was only located in the cytoplasm of spermatogonia and early spermatocytes, and not present in late spermatocytes, spermatids or spermatozoa (Fig. 2F and H). No expression was found in somatic cells of the testis (Fig. 2H). 1.3. Expression of Aj-VASA in gonads during the reproductive cycle An abundance of Aj-VASA was located in oogonia and oocytes of the ovary at the growing stage (Fig. 3A and C). It declined in developing oocytes in the ovary at the growing stage. The expression of Aj-VASA was weak in oocytes of the mature ovary and in residual oocytes of the ovary at the post-spawning stage (Fig. 3E and G). In the testis, Aj-VASA expression was concentrated in spermatogonia and early spermatocytes during the early growing stage, and restricted to spermatogonia at the later growing and mature stages (Fig. 3D and F). It was absent from the testis at the post-spawning stage (Fig. 3H). 2. Discussion 2.1. Expression of the vasa gene and potential application as a marker for A. japonicus germ cells The vasa gene has been reported as a molecular marker of germline in many invertebrates such as the Chinese shrimp Fenneropenaeus chinensis (Zhou et al., 2010; Feng et al., 2011), oyster C. gigas (Fabioux et al., 2004) and Chinese mitten crab Eriocheir sinensis (Wang et al., 2012a). Like most species, the expression of Aj-vasa detected by sqRT–PCR was also specific for the A. japonicus gonads (Fig. 1). Furthermore, ISH and IHC revealed that both Aj-vasa mRNA and Aj-VASA were restricted to the germ cells, and no signal was detected in somatic cells of the ovary or testis. Thus the expression pattern of Aj-vasa in the germ cells of A. japonicus was similar to that of equivalent genes in other organisms examined to date. The VASA protein, but not vasa mRNA, has been demonstrated to be a germ cell marker in species such as Drosophila and Xenopus with an initial restriction to germline cells (Hay et al.,1988b; Komiya et al., 1994). However, in the zebrafish, vasa mRNA is more specific as a germline marker than VASA protein during gametogenesis and embryogenesis (Knaut et al., 2000; Braat et al., 2000). Here we found that Aj-vasa mRNA was expressed in a wider range of germ cells including oogonia, oocytes, spermatogonia, spermatocytes and spermatids, but was absent from somatic cells of the gonads. This suggests that the Aj-vasa mRNA might be a better germ cell marker than Aj-VASA to study the origination and migration of PGCs and differentiation of germ cells in A. japonicus. In the male specimens, Aj-VASA expression was restricted to spermatogonia and few early spermatocytes. These distribution
M. Yan et al. / Gene Expression Patterns 13 (2013) 171–176
173
Fig. 2. Location of Aj-vasa mRNA and Aj-VASA detected by ISH and IHC in A. japonicus gonad at the growing stage. (A) Histology of the ovary stained by H&E; the insert is a magnification of the square region. (B) Histology of the edge of the testis stained by H&E. (B’) Histology of the central region of the testis stained by H&E. (C and D) Aj-vasa mRNA expression demonstrated with an antisense probe in the ovary (C) and testis (D). (E and F) Aj-VASA expression demonstrated by IHC with an anti-Aj-VASA antibody in the ovary (E) and testis (F); the insert in (E) is a magnified view of oogonium and an early oocyte in the wall of the gonadal tube. (G) Magnified view of oogonium and developing oocytes on a serial section around (E). (H) Magnified view of spermatogonium, spermatocytes, spermatids and spermatozoa on a serial section around (F). The insert in (H) shows the central part of the testis at the same scale. (I, I’) Hybridization with sense probe of the ovary (I) and testis (I’) as negative control for ISH. (J, J’) Immunostaining with preimmune rabbit serum of the ovary (J) and testis (J’) as negative controls for IHC. Positive signals with an antisense probe for Aj-vasa mRNA detected by ISH are shown in blue and positive signals for Aj-VASA are shown as reddish-brown by IHC. After ISH and IHC sections were counterstained with neutral red and haematoxylin respectively. Abbreviations: N, nucleus; Og, oogonium; Oo, oocyte; Sc, spermatocyte; Sg, spermatogonium; SoC, somatic cell; St, spermatid; Sz, spermatozoon. Scale bars = 25 lm.
174
M. Yan et al. / Gene Expression Patterns 13 (2013) 171–176
Fig. 3. Differential expression of Aj-VASA detected by IHC in gonads of A. japonicus at various stages. (A) Ovary at the early growing stage. (B) Testis at the early growing stage. (C) Ovary at the growing stage. (D) Testis at the growing stage. (E) Ovary at the mature stage. (F) Testis at the mature stage. (G) Ovary at post-spawning stage, the insert in (G) is a negative control. (H) Testis at the post-spawning stage. Positive IHC signals for Aj-VASA are reddish-brown. Abbreviations: OC, ovarian cavity; Og, oogonium; Oo, oocyte; Sc, spermatocyte; Sg, spermatogonium; SoC, somatic cell; Sz, spermatozoon; TC, testis cavity. Scale bars = 25 lm.
characteristics limit its use as a general marker of germline cells; however, this specificity could be used to distinguish and isolate spermatogonia in vitro. In the sea urchin Strongylocentrotus nudus—another echinoderm species—Yakovlev et al. has isolated oogonia from the ovary using density gradient centrifugation. Because intense signals of the sea urchin VASA could be detected in the perinuclear area of oogonia and gave uniform signals in oocytes, oogonia could be isolated and identified with a 70–75% positive rate (Yakovlev et al., 2010). To study the differentiation of male gametes, it will necessary to identify and isolate spermatogonia accurately, but little research on this has been done in invertebrates. In the present study, Aj-VASA was expressed specifically in spermatogonia and early spermatocytes. Thus, it might serve as a probe for studying the origination and differentiation of spermatogonial stem cells in the Holothurioidea.
2.2. The role of VASA in maintaining the development of gametes In A. japonicus, the expression of VASA was different between oogenesis and spermatogenesis: in the testis it was only present in spermatogonia and a few early spermatocytes, but in the ovary it was expressed steadily in all stages of oogenesis. This indicates that the function of Aj-VASA is different between genders. Thus, it participates throughout oogenesis, but only during the early stages of spermatogenesis. The timing of VASA expression during spermatogenesis has been reported in other organisms such as the tilapia (Kobayashi et al., 2000), gibel carp (Xu et al., 2005) and mouse (Toyooka et al., 2000; Tanaka et al., 2000). In the mouse, the Mvh gene (mouse vasa homologue) appears necessary for the completion of spermatogenesis but not for oogenesis. Lack of a mouse VASA protein function causes germ cells to cease differenti-
M. Yan et al. / Gene Expression Patterns 13 (2013) 171–176
ation into pachytene spermatocytes and to undergo apoptotic cell death (Tanaka et al., 2000). However, in Drosophila vasa appeared to have an essential function in female gametogenesis, but not in male gametogenesis. In vasa-null Drosophila ovaries, germaria are atrophied, the number of oocytes declines or even disappears in the egg chambers, and the oocyte nucleus appears more diffuse than that of wild-type flies (Styhler et al., 1998; Tomancak et al., 1998). In the oyster C. gigas, both the amount of mRNA and protein encoded by Oyvlg (oyster vasa-like gene) decreased during spermatogenesis and oogenesis when the Oyvlg expression was knocked down by RNAi. The knockdown phenotype females halted their gametogenesis at prophase I of meiosis before vitellogenesis. In males with Oyvlg knockdown phenotype, development of the germ cells stopped at the spermatocyte stage (Fabioux et al., 2009). These results demonstrate that VASA has an essential role in germ cell maintenance, and probably has functions that differ between male and female gametogenesis. In conclusions, the expression pattern of the Aj-vasa is consistent with the idea that the vasa gene is the most reliable molecular marker for studies on germ cell development in most species. Our findings suggest that the expression level of Aj-vasa varies between ovary and testis, and the location of vasa also varies at mRNA and protein levels. Moreover, Aj-vasa mRNA is a good marker for studying the origin and migration of germline cells and Aj-VASA is a useful tool to identify spermatogonia in A. japonicus. 3. Experimental procedures 3.1. Animal and Tissue preparation Adult A. japonicus were purchased from the aquatic product market of Qingdao, PR China. Tissue samples (ovary, testis, respiratory tree and alimentary tract) were dissected out, frozen immediately in liquid nitrogen and stored at 80 °C for RNA isolation. Part of the fresh gonad was fixed in Bouin’s solution for histology. The other samples were fixed in 4% paraformaldehyde (PFA) for 16 h before being stored in 100% methanol at 20 °C for in situ hybridization (ISH) and immunohistochemistry. According to the histological characteristics described by Sui et al. (Sui et al., 1985), the gonads of adult A. japonicus were divided into three groups. First was the growing stage in which the amounts of germ cells increase. These are composed of aggregated oogonia and developing oocytes (15–100 lm in diameter) in the ovary and various stages, including spermatogonia, spermatocytes, spermatids and mature spermatozoa in the testis. Second was the maturation stage, in which the ovary is filled with mature oocytes 110–130 lm in diameter and the testis is full of mostly mature spermatozoa. Third was the post-spawning stage, in which both the ovary and testis are empty with only a few residual gametes remaining. 3.2. Semi-quantitative reverse transcription polymerase chain reaction (sqRT–PCR) Total RNA was extracted from the ovary, testis, respiratory tree and alimentary tract at the gonadal growing stage using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The concentration and purity of total RNA were determined spectrophotometrically (Bio-Rad, Hercules, CA, USA). First-strand cDNA was synthesized using an ImProm-II™ Reverse Transcription system (Promega, Madison, WI, USA). Tissue distributions of the Aj-vasa in A. japonicus were analyzed using sqRT–PCR with primers VF3 (5-GGTCAGCCAACCGAAGAAAA-3) and VR3 (5-CGTGAACAGAGGGAAAGAGA-3) according to the Aj-vasa cDNA sequence (GenBank Accession No. EU273885) to amplify the Aj-vasa fragment of 556 bp. A fragment (369 bp) of the
175
reference gene (A. japonicus 18S rRNA) was amplified with primers 18F1 (5-GCAAATGCTTTCGCTGTCG-3) and 18F2 (5’-CTCGTAGTCGGATTTCTGG-3) designed according to the 18S rRNA sequence (GenBank Accession No. EU821599). The reaction conditions were 94 °C for 5 min, 94 °C for 30 s, 58 °C for 30 s, 72 °C for 30 s with 30 cycles for the Aj-vasa fragment; 16 cycles for the reference gene and finally extension at 72 °C for 5 min. 3.3. Histology Fixed gonads were dehydrated in an ascending ethanol series, cleared with xylene and embedded in paraffin wax. Sections (5 lm) were stained with hematoxylin and eosin (H&E) and microscopy and digital image capture were done using a Nikon E80i microscope. 3.4. In situ hybridization (ISH) Digoxigenin (DIG)-labeled RNA sense and anti-sense probes were synthesized from the clone of an Aj-vasa 512 bp fragment using a DIG RNA Labeling Kit (SP6/T7, Roche Applied Science, Indianapolis, IN, USA). The fragment was amplified using two specific primers (sense 5-CAAGCTTGGTGGT CGTGATCGACYGTG-3; antisense 5-GGAATTCGACGTTCTCACCGACGCTG-3), based on the fulllength sequence of Aj-vasa cDNA (GenBank Accession No. EU273885). Ovary and testis were embedded in paraffin wax, and the sections (5 lm thick) were affixed to microscope slides with 2% polylysine for 10 h at 37 °C. The sections were rehydrated through a descending series of methanol solutions and phosphatebuffered saline with 0.1% Tween-20 (PBST). Samples were then digested for 30 min at 37 °C using protease K (2 lg/ml), prehybridized at 50 °C in hybridization solution (HS, 50% formamide, 5% SSC, 5 mM EDTA, 1.5% blocking reagent, 0.1% Tween-20, 100 lg/ ml ribonucleic acid (Sigma–Aldrich, St. Louis, MO, USA)) for 6 h, and then hybridized in HS with the Aj-vasa RNA probes for 16 h. The samples were washed at 50 °C for 130 min and at room temperature two times for 10 min in maleic acid buffer (0.1 M maleic acid, 0.15 M NaCl, 0.1% Tween-20, pH 7.5). Finally, hybridization signals were detected using alkaline phosphatase-conjugated anti-DIG antibody and NBT/BCIP (DIG Nucleic Acid Detection Kit, Roche Applied Science) before being counterstained with neutral red. Microscopy and digital image capture were done using a Nikon E80i microscope. 3.5. Immunohistochemistry (IHC) A partial cDNA fragment encoding 88 amino acids of the AjVASA N-terminus was amplified using Ex Taq DNA polymerase (Takara Bio Inc., Shiga, Japan) with primers 5-GAATTCATGG GACACTTCGCTAGAGA-3’ (EcoRI site underlined) as the sense primer and 5-CTCGAGCGCTCTCCTGACGTTCTCAC-3 (XhoI site underlined) as the antisense primer. The PCR product was subcloned into a pMD18-T vector (Takara) followed by digestion with EcoRI and XhoI and then subcloned into the expression vector pET32 (Novagen, Madison, WI, USA). The recombinant pET32–VASA plasmid was transformed into Escherichia coli BL21 (DE3) pLysS. Aj-VASA expression was induced by adding isopropyl -D-thiogalactoside to LB medium at a final concentration of 1.0 mM and incubating at 37 °C for 6 h. The recombinant Aj-VASA was purified using Ni– NTA affinity chromatography following the manufacturer’s instructions (Novagen) and the purity of eluted samples analyzed by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis stained with Coomassie brilliant blue R-250. An emulsion of equal volumes of 200 lg of purified Aj-VASA and Freund’s complete adjuvant (Sigma–Aldrich) was injected subcutaneously at multiple sites on two New Zealand White rabbits.
176
M. Yan et al. / Gene Expression Patterns 13 (2013) 171–176
Two booster injections of 100 lg antigen mixed with Freund’s incomplete adjuvant (Sigma–Aldrich) were administered subcutaneously at 2-week intervals. One week after the final booster, blood was collected and serum prepared. The antibody titer was determined by enzyme-linked immunosorbent assay and the antisera were aliquoted before being stored at 80 °C. Testes and ovaries fixed in 4% PFA were processed as described in Section 3.4 before digestion with protease K. After two 5-min washes in PBST, the endogenous peroxidase activity in the sections was quenched by incubation in methanol with 3% H2O2 (v/v) at room temperature for 10 min, followed by two 5-min washes in PBST. Subsequently, the sections were incubated in 3% bovine serum albumin solution for 30 min, and then incubated with rabbit anti-Aj-VASA antibody (diluted 1:800) for 1 h before incubation with peroxidase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) (diluted 1:5,000) for 1 h. A chromogenic reaction was achieved by the addition of 0.05% Diaminobenzidine (w/v) containing 0.01% H2O2 (v/v) in PBST and maintained in the dark for 5 min. Sections were then washed in PBST, counterstained with hematoxylin, dehydrated through an ascending ethanol series, cleared in xylene, mounted in neutral balsam and observed and photographed using a Nikon E80i microscope. Acknowledgment This work was supported by the Specialized Research Fund for the Doctoral Program of Higher Education, PR China (No. 200804230015) and the National High Technology Research and Development Program of China (863 program) (No. 2012AA10A402). References Albamonte, M.S., Willis, M.A., Albamonte, M.I., Jensen, F., Espinosa, M.B., Vitullo, A.D., 2008. The developing human ovary: immunohistochemical analysis of germ-cell-specific VASA protein, BCL-2/BAX expression balance and apoptosis. Hum. Reprod. 23, 1895–1901. Anderson, R., Fulton, N., Cowan, G., Coutts, S., Saunders, P., 2007. Conserved and divergent patterns of expression of DAZL, VASA and OCT4 in the germ cells of the human fetal ovary and testis. BMC Dev. Biol. 7, 136. Braat, A.K., van de Water, S., Goos, H., Bogerd, J., Zivkovic, D., 2000. VASA protein expression and localization in the zebrafish. Mech. Dev. 95, 271–274. Castrillon, D.H., Quade, B.J., Wang, T.Y., Quigley, C., Crum, C.P., 2000. The human VASA gene is specifically expressed in the germ cell lineage. Proc. Natl. Acad. Sci. USA 97, 9585–9590. Fabioux, C., Huvet, A., Lelong, C., Robert, R., Pouvreau, S., Daniel, J.Y., Minguant, C., Le Pennec, M., 2004. Oyster vasa-like gene as a marker of the germline cell development in Crassostrea gigas. Biochem. Biophys. Res. Commun. 320, 592– 598. Fabioux, C., Corporeau, C., Quillien, V., Favrel, P., Huvet, A., 2009. In vivo RNA interference in oyster -vasa silencing inhibits germ cell development. FEBS J. 276, 2566–2573. Feng, Z.F., Zhang, Z.F., Shao, M.Y., Zhu, W., 2011. Developmental expression pattern of the Fc-vasa-like gene, gonadogenesis and development of germ cell in Chinese shrimp, Fenneropenaeus chinensis. Aquaculture 314, 202–209. Gustafson, E.A., Wessel, G.M., 2010. Vasa genes: emerging roles in the germline and in multipotent cells. Bioessays 32, 626–637. Hay, B., Ackerman, L., Barbel, S., Jan, L.Y., Jan, Y.N., 1988a. Identification of a component of Drosophila polar granules. Development 703, 825–840. Hay, B., Jan, L.Y., Jan, Y.N., 1988b. A protein component of Drosophila polar granules is encoded by vasa and has extensive sequence similarity to ATP-dependant helicases. Cell 55, 577–587. Ikenishi, K., Tanaka, T.S., 1997. Involvement of the protein of Xenopus vasa homolog (Xenopus vasa-like gene 1, XVLG1) in the differentiation of primordial germ cells. Dev. Growth Differ. 39, 625–633.
Kato, S., Tsurumaru, S., Taga, M., Yamane, T., Shibata, Y., Ohno, K., Fujiwara, A., Yamano, K., Yoshikuni, M., 2009. Neuronal peptides induce oocyte maturation and gamete spawning of sea cucumber, Apostichopus japonicus. Dev. Biol. 326, 169–176. Knaut, H., Pelegri, F., Bohmann, K., Schwarz, H., Nusslein-Volhard, C., 2000. Zebrafish vasa RNA but not its protein is a component of the germ plasm and segregates asymmetrically before germline specification. J. Cell Biol. 149, 875–888. Kobayashi, T., Kajiura-Kobayashi, H., Nagahama, Y., 2000. Differential expression of vasa homologue gene in the germ cells during oogenesis and spermatogenesis in a teleost fish, tilapia, Oreochromis niloticus. Mech. Dev. 99, 139–142. Komiya, T., Itoh, K., Ikenishi, K., Furusawa, M., 1994. Isolation and characterization of a novel gene of the DEAD box protein family which is specifically expressed in germ cells of Xenopus laevis. Dev. Biol. 162, 354–363. Liang, L., Diehl-Jones, W., Lasko, P., 1994. Localization of vasa protein to the Drosophila pole plasm is independent of its RNA-binding and helicase activities. Development 120, 1201–1211. Nakao, H., 1999. Isolation and characterization of a Bombyx vasa-like gene. Dev. Genes Evol. 209, 312–316. Olsen, L.C., Aasland, R., Fjose, A., 1997. A vasa-like gene in zebrafish identifies putative primordial germ cells. Mech. Dev. 66, 95–105. Pang, Z.G., Sun, H.L., Yan, J.P., Yu, D.X., Fang, J.G., Jiang, M., 2006. Ultrastructural studies on spermatogenesis of sea cucumber Apostichopus japonicus. Marine Fish. Res. 27, 27–31. Pfister, D., De Mulder, K., Hartenstein, V., Kuales, G., Borgonie, G., Marx, F., Morris, J., Ladurner, P., 2008. Flatworm stem cells and the germ line: developmental and evolutionary implications of macvasa expression in Macrostomum lignano. Dev. Biol. 319, 146–159. Raghuveer, K., Senthilkumaran, B., 2010. Cloning and differential expression pattern of vasa in the developing and recrudescing gonads of catfish, Clarias gariepinus. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 57, 79–85. Rebscher, N., Volk, C., Teo, R., Plickert, G., 2008. The germ plasm component vasa allows tracing of the interstitial stem cells in the cnidarian Hydractinia echinata. Dev. Dyn. 237, 1736–1745. Schüpbach, T., Wieschaus, E., 1986. Germline autonomy of maternal-effect mutations altering the embryonic body pattern of Drosophila. Dev. Biol. 113, 443–448. Shinomiya, A., Tanaka, M., Kobayashi, T., Nagahama, Y., Hamaguchi, S., 2002. The vasa-like gene, olvas, identifies the migration path of primordial germ cells during embryonic body formation stage in the medaka, Oryzias latipes. Dev. Growth Differ. 42, 317–326. Styhler, S., Nakamura, A., Swan, A., Suter, B., Lasko, P., 1998. Vasa is required for GURKEN accumulation in the oocyte, and is involved in oocyte differentiation and germline cyst development. Development 125, 1569–1578. Sui, X.L., Liu, Y.X., Liu, Y.F., Shang, L.B., Hu, Q.M., 1985. A study on the reproductive cycle of sea cucumber. J. Fish. Chin. 9, 303–311. Tanaka, S.S., Toyooka, Y., Akasu, R., 2000. The mouse homolog of Drosophila Vasa is required for the development of male germ cells. Genes Dev. 14, 841–853. Tomancak, P., Guichet, A., Zavorszky, P., Ephrussi, A., 1998. Oocyte polarity depends on regulation of gurken by Vasa. Development 125, 1723–1732. Toyooka, Y., Tsunekawa, N., Takahashi, Y., Matsui, Y., Satoh, M., Noce, T., 2000. Expression and intracellular localization of mouse VASA-homologue protein during germ cell development. Mech. Dev. 93, 139–149. Tsunekawa, N., Naito, M., Sakai, Y., Nishida, T., Noce, T., 2000. Isolation of chicken vasa homolog gene and tracing the origin of primordial germ cells. Development 127, 2741–2750. Wang, Q., Fang, D.A., Sun, J.L., Wang, Y., Wang, J., Liu, L.H., 2012a. Characterization of the vasa gene in the Chinese mitten crab Eriocheir sinensis: a germline molecular marker. Insect Physiol. 58, 960–965. Wang, Y.L., Chen, Y.D., Han, K.H., Zou, Z.H., Zhang, Z.P., 2012b. A vasa gene from green mud crab Scylla paramamosain and its expression during gonadal development and gametogenesis. Mol. Biol. Rep. 39, 4327–4335. Xu, H.Y., Gui, J.F., Hong, Y.H., 2005. Differential expression of vasa RNA and protein during spermatogenesis and oogenesis in the gibel carp (Carassius auratus gibelio), a bisexually and gynogenetically reproducing vertebrate. Dev. Dyn. 233, 872–882. Yakovlev, K.V., Battulin, N.R., Serov, O.L., Odintsova, N.A., 2010. Isolation of oogonia from ovaries of the sea urchin Strongylocentrotus nudus. Cell Tissue Res. 342, 479–490. Yoon, C., Kawakami, K., Hopkins, N., 1997. Zebrafish vasa homologue RNA is localized to the cleavage planes of 2- and 4-cell-stage embryos and is expressed in the primordial germ cells. Development 124, 3157–3166. Yoshizaki, G., Sakatani, S., Tominaga, H., Takeuchi, T., 2000. Cloning and characterization of a vasa-like gene in rainbow trout and its expression in the germ cell lineage. Mol. Reprod. Dev. 55, 364–371. Zhou, Q.R., Zhang, Z.F., Shao, M.Y., Kang, K.H., 2010. Cloning, characterization and expression analysis of the DEAD-box family genes, Fc-vasa and Fc-PL10a, in Chinese shrimp (Fenneropenaeus chinensis). CSOL 28, 37–45.