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Mechanisms of Development 99 (2000) 139±142 www.elsevier.com/locate/modo Gene expression pattern Differential expression of vasa homologue gene in the germ cells during oogenesis and spermatogenesis in a teleost ®sh, tilapia, Oreochromis niloticus Tohru Kobayashi, Hiroko Kajiura-Kobayashi, Yoshitaka Nagahama* Department of Developmental Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan Received 17 March 2000; received in revised form 4 August 2000; accepted 1 September 2000 Abstract Vas (a Drosophila vasa homologue) gene expression pattern in germ cells during oogenesis and spermatogenesis was examined using all genetic females and males of a teleost ®sh, tilapia. Primordial germ cells (PGC) reach the gonadal anlagen 3 days after hatching (7 days after fertilization), the time when the gonadal anlagen was ®rst formed. Prior to meiosis, no differences in vas RNA are observed in male and female germ cells. In the ovary, vas is expressed strongly in oogonia to diplotene oocytes and becomes localized as patches in auxocytes and then strong signals are uniformly distributed in the cytoplasm of previtellogenic oocytes, followed by a decrease from vitellogenic to postvitellogenic oocytes. In the testis, vas signals are strong in spermatogonia and decrease in early primary spermatocytes. No vas RNA expression is evident in either diplotene primary spermatocytes, secondary spermatocytes, spermatids or spermatozoa. The observed differences in vas RNA expression suggest a differential function of vas in the regulation of meiotic progression of female and male germ cells. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Germ cells; Vasa homologue (vas) RNA; Differential expression; In situ hybridization; Oogenesis; Spermatogenesis The gene vasa encodes a DEAD (Asp-Glu-Ala-Asp) family of putative RNA helicase and is present in both the polar granules at the posterior end of the oocyte and the nuage structure in the germ cells in Drosophila. It has been shown that zygotic expression is also restricted to the germ lineage (Lasko and Ashburner, 1988; Hay et al., 1990, 1998; Linder et al., 1989; Ephrussi and Lehmann, 1992; Liang et al., 1994). Vasa-like homologues have been cloned in frogs (Komiya et al., 1994), mice (Fujiwara et al., 1994), rats (Komiya and Tanigawa, 1995) and zebra®sh (Yoon et al., 1997; Olsen et al., 1997). In these animals, the vasa homologues were found to be expressed speci®cally in the germ line of older individuals. The localization of vasa homologue in early embryos has been reported in frogs (Ikenishi, 1998) and zebra®sh (Yoon et al., 1997; Olsen et al., 1997). However, there have been no studies in any vertebrate species describing the expression pattern of vasa homologue genes during oogenesis and spermatogenesis. Tilapia, Oreochromis niloticus, is a gonochoristic teleost ®sh and has been extensively used to examine morphological gonadal sex differentiation (Nakamura et * Corresponding author. Tel.: 181-564-55-7550; fax: 181-564-55-7556. E-mail address: [email protected] (Y. Nagahama). al., 1998). Using all genetic males and females of tilapia, we examined the expression patterns of vasa homologue genes during oogenesis and spermatogenesis. 1. Results and discussion 1.1. Isolation and characterization of tilapia vas cDNA We obtained a clone containing a full-length open reading frame which was highly homologous to the vasa genes previously reported in other animals. This cDNA encodes a protein of 645 amino acids. The predicted amino acid sequence of tilapia vasa is 70.0% identical to that of zebra®sh, 56.2% to Xenopus, 60.0% to rat, 61.2% to mouse and 48.1% to Drosophila. Furthermore, this tilapia gene shows a higher degree of similarity to zebra®sh vasa gene (70%) than to zebra®sh PL10 homologue (45%), another member of the DEAD protein family (Olsen et al., 1997). From these ®ndings, we concluded that the clone we isolated encodes the tilapia homologue of vasa and thus was designated the tilapia vasa homologue, vas (DDBJ accession number; AB032467). 0925-4773/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(00)00464-0 140 T. Kobayashi et al. / Mechanisms of Development 99 (2000) 139±142 1.2. Expression pattern of tilapia vas during gametogenesis RT-PCR was used to determine the tissue distribution of tilapia vas mRNA. An ampli®ed product of tilapia vas was seen only when testis and ovary RNAs were used. No ampli®ed products were detected in either brain, spleen, liver, heart or kidney (data not shown). In situ hybridization was used to determine the pattern of tilapia vas expression in germ cells during gonadogenesis and gametogenesis of both sexes (fry 0±100 days after hatching, dah). All genetic females and males were used. To identify primordial germ cells (PGC), we also stained germ cells immunohistochemically with SGSA-1 antibody which stains gonial type germ cells speci®cally (Kobayashi et al., 1998). Immediately after hatching, vasa RNA was already present, localized only in SGSA-1-positive PGC which were distinguishable morphologically from somatic cells by their large size and location at the outer layer of the lateral plate mesoderm (Fig. 1a,b,d,e). PGC ®rst reached the gonadal anlagen 3 dah and contained vas RNA (Fig. 1c,f). During these stages, vas RNA was found only in germ cells. In females of tilapia, oogenesis is initiated at 20±25 dah, at which time oogonial proliferation begins to occur. Meiotic germ cells ®rst appear in ovaries of fry 25±30 dah and at these stages auxocytes are also seen. Up to and during these stages, strong vas RNA expression was observed throughout the cytoplasm of oogonia and small oocytes (Fig. 2a,c). No vas RNA was observed in nuclei of germ cells at any stages. There were marked changes in the localization pattern of vas RNA during the early stages of oogenesis (Fig. 3a±f). Patches of strong vas signals were evident in the cytoplasm of early pre-vitellogenic oocytes (Fig. 3a,b,d). As the size of oocytes increased, the signals became much stronger, eventually occupying the entire cytoplasm (Fig. 3d). Vas RNA signals became weaker with the progression of vitellogenesis, probably due to dispersion of RNA throughout the enlarged oocytes (Fig. 3c). Northern blotting analysis Fig. 2. Vas expression in fry gonads. Ovary at 45 dah stained with hematoxylin (a) or hybridized with DIG-labeled vas antisense RNA (c). Auxocytes have strong signals for vas. Testis at 35 dah stained with hematoxylin (b) or hybridized with DIG-labeled vas antisence RNA (d). Strong vas signals are seen in spermatogonia. BV, blood vessel; ED, intratesticular efferent duct; OC, ovarian cavity; GC, spermatogonia. Scale bar, 20 mm. showed that vasa RNA was present even in full-grown oocytes, ovulated eggs and embryos at the 4-cell stage (Fig. 4). This continuous presence of vas RNA suggests that vas transcript can be supplied maternally. In male tilapia, A-type spermatogonia including stem type cells are the only germ cells present in gonads during testicular differentiation. Active spermatogonial proliferation is not observed until 20±35 dah. At these stages, vas RNA signals were seen in spermatogonia (Fig. 2b,d). The initiation of spermatogenesis does not occur until 70 dah, at which time active spermatogonial proliferation takes place. Spermatozoa ®rst appear in testes of tilapia about 100 dah. Male germ cells were positive for vas RNA, at stages from spermatogonia to early primary spermatocytes (Fig. 5). However, the signals were weaker in primary spermatocytes, compared to spermatogonia, and were absent in diplotene spermatocytes (Figs. 5b,c,e,f). No vas RNA was observed in secondary spermatocytes, spermatids or spermatozoa (Fig. 5d). Fig. 1. Vas expression in germ cells. Immunohistochemistry using anti-SGSA-1 antibody (a±c) and in situ hybridization using DIG-labeled antisense RNA (d± f) were performed on larval sections ®xed at 0 dah (a,d), 2 dah (b,e) and 3 dah (c,f). Arrows indicate the vas expressing cells. PGC, primordial germ cells; LPM, lateral plate mesoderm; G, gut; ND, nephric duct; CC, coelomic cavity. Scale bar, 20 mm. T. Kobayashi et al. / Mechanisms of Development 99 (2000) 139±142 141 2. Experimental procedures 2.1. Animals Tilapia, Oreochromis niloticus, were kept in re-circulating freshwater tanks with a capacity of 500 l at 268C until use. To obtain fry, arti®cial fertilization was performed by mixing sperm suspension and eggs. All genetic females (XX) and males (XY) were obtained by arti®cial fertilization of eggs from a normal female (XX) and sperm from a sex-reversed male (XX), and eggs from a normal female (XX) and sperm from a super male (YY), respectively. Fertilized eggs were cultured in re-circulating water at 268C. 2.2. cDNA cloning of tilapia vasa homologue and Northern blot analysis Fig. 3. Distribution of vas RNA during oogenesis. (a±d) Antisense probe; (e,f) Sense probe. (a,b,d) Previtellogenic oocytes; (c,e,f) Mid-vitellogenic oocytes. Patches of strong vas signals are evident in the cytoplasm of early pre-vitellogenic oocytes (b, arrows). As the size of oocytes increases, the signals become much stronger, eventually occupying the whole cytoplasm. Signals become localized in the cortical region of mid-vitellogenic oocytes (c, arrow heads). Og, oogonia. Sections (a) and (d) are of identical magni®cation. Sections (b,c,e,f) are the same magni®cation. Scale bar, 50 mm. In summary, the results presented in this study clearly showed the difference in vas RNA pattern in male and female germ cells during gemetogenesis. These results suggest that vas has a differential role in translational regulation during meiotic progression from zygotene to diplotene stages of both sexes. Further investigations are necessary to determine the differences in vas function during spermatogenesis and oogenesis. A vasa cDNA fragment was PCR-ampli®ed with degenerate primers corresponding to the conserved amino acid sequences of vasa MACAQTG and VLDEADRM. To obtain a full length clone, an adult tilapia ovarian cDNA library (Chang et al., 1997) was screened according to a previous report (Kajiura et al., 1993). Ten micrograms of total RNA was used for Northern blot analysis according to a previous report (Kajiura et al., 1993). 2.3. In situ hybridization Gonads of fry 0±10 dah were dissected attached to the trunk and ®xed in 4% paraformaldehyde in 0.1 M phosphate buffer (PB) (pH 7.4) (4% PFA) at 48C overnight. Testes and ovaries from 10 to 100 dah were removed and ®xed in 4% PFA. After ®xation, gonads were embedded into paraf®n. Cross-sections were cut at 5 mm. Probes of sense and antisense digoxigenin (DIG)-labeled RNA strands were transcribed in vitro from linearized tilapia vas cDNA plasmid, using an RNA labeling kit (Boehringer). In situ hybridization was performed as follows: Sections were deparaf®nized, hydrated and treated with proteinase K (Boehringer, 10 mg/ml) and then hybridized using sense or antisense DIG-labeled RNA probe at 608C for 18±24 h. Hybridization signals were then detected by using alkaline phosphataseconjugated anti-DIG antibody (Boehringer) and NBT as the chromogen. Acknowledgements Fig. 4. Northern blot analysis of vas RNA during oogenesis and after fertilization. LV, late vitellogenic oocytes (full grown oocytes); 4-cells, 4-cell stage after fertilization. This work was supported in part by Grant-in-Aid for Research for the Future (JSPS-RFTF 96 L00401) from the Japan Society for the Promotion of Science, and Bio Design from the Ministry of Agriculture, Forestry and Fisheries, Japan to Y.N. and Grant-in-Aid for Research from the Ministry of Education, Science, Sports and Culture of Japan to T.K. (09740619). 142 T. Kobayashi et al. / Mechanisms of Development 99 (2000) 139±142 Fig. 5. Vas expression during spermatogenesis. Testis at 100 dah. (a,b,d) In situ hybridization; (c,e,f) hematoxylin staining. Sections (e,f) are high magni®cation of section (c). Strong signals for vas are seen in germ cells from A-type spermatogonia to primary spermatocytes but not from secondary spermatocytes to spermatozoa. In section d, vas RNA positive and negative primary spermatocytes are seen. Section (b) is adjacent to section c. Note a marked decrease in vas transcripts in primary spermatocytes with the progression of meiosis. Large arrows, vas-expressing primary spermatocytes (pachytene spermatocytes). Small arrows, non-vas expressing primary spermatocytes (pachytene to diplotene spermatoytes). ED, intratesticular efferent duct; a±g, A-type spermatogonia; b±g, Btype spermatogonia; SC, primary spermatocytes; St, spermatids; Lc, Leydig cells. Scale bar, 50 mm (a±c); 20 mm (d±f). References Chang, X.T., Kobayashi, T., Kajiura, H., Nakamura, M., Nagahama, Y., 1997. 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