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Vasa identifies germ cells and critical stages of oogenesis in the Asian seabass.

Xu H, Lim M, Dwarakanath M, Hong Y - Int. J. Biol. Sci. (2014)

Bottom Line: By chromogenic and fluorescent in situ hybridization we revealed germ cell-specific Lcvasa expression in both the testis and ovary.Importantly, Lcvasa shows dynamic patterns of temporospatial expression and subcellular distribution during gametogenesis.At different stages of oogenesis, for example, Lcvasa undergoes nuclear-cytoplasmic redistribution and becomes concentrated preferentially in the Balbiani body of stage-II~III oocytes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543.

ABSTRACT
Germ cells produce sperm and eggs for reproduction and fertility. The Asian seabass (Lates calcarifer), a protandrous marine fish, undergoes male-female sex reversal and thus offers an excellent model to study the role of germ cells in sex differentiation and sex reversal. Here we report the cloning and expression of vasa as a first germ cell marker in this organism. A 2241-bp cDNA was cloned by PCR using degenerate primers of conserved sequences and gene-specific primers. This cDNA contains a polyadenylation signal and a full open reading frame for 645 amino acid residues, which was designated as Lcvasa for the seabass vasa, as its predicted protein is homologous to Vasa proteins. The Lcvasa RNA is maternally supplied and specific to gonads in adulthood. By chromogenic and fluorescent in situ hybridization we revealed germ cell-specific Lcvasa expression in both the testis and ovary. Importantly, Lcvasa shows dynamic patterns of temporospatial expression and subcellular distribution during gametogenesis. At different stages of oogenesis, for example, Lcvasa undergoes nuclear-cytoplasmic redistribution and becomes concentrated preferentially in the Balbiani body of stage-II~III oocytes. Thus, the vasa RNA identifies both female and male germ cells in the Asian seabass, and its expression and distribution delineate critical stages of gametogenesis.

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vasa expression in the adult testis by fluorescent ISH. Testicular cryosections after fluorescent ISH (green) were analyzed by microscopy. Nuclei were stained blue with DAPI. (A) Bright field micrograph showing the testicular structure. (B) Merge micrograph between bright field and fluorescent optics. (C-F) Fluorescent micrograph. Large magnification of the area boxed in (A and B), along with (E and F) highlighting spermatogonia at the periphery. Arrow, CB; sg, spermatogonium; sc1 and sc2, primary and secondary spermatocyte; sm, sperm, st, spermatid; ED, efferent ductile; Scale bars, 50 µm in (A-C, E, F) and 25 µm in (D and D').
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Figure 5: vasa expression in the adult testis by fluorescent ISH. Testicular cryosections after fluorescent ISH (green) were analyzed by microscopy. Nuclei were stained blue with DAPI. (A) Bright field micrograph showing the testicular structure. (B) Merge micrograph between bright field and fluorescent optics. (C-F) Fluorescent micrograph. Large magnification of the area boxed in (A and B), along with (E and F) highlighting spermatogonia at the periphery. Arrow, CB; sg, spermatogonium; sc1 and sc2, primary and secondary spermatocyte; sm, sperm, st, spermatid; ED, efferent ductile; Scale bars, 50 µm in (A-C, E, F) and 25 µm in (D and D').

Mentions: Restriction of the vasa transcript to the gonads suggests its germ cell-specific expression. To confirm this, spatial expression was analyzed by ISH in adult gonads. The adult seabass testis is mainly composed of spermatogonia and spermatogenic cells at different stages. Spermatogonia are at the most peripheral region of seminiferous cysts. Spermatogenesis proceeds synchronously within each cyst and cysts containing germ cells at progressively advanced stages of development are closer to the efferent duct. Chromogenic ISH revealed that vasa RNA expression in the testis was exclusively in germ cells, with the signal being strong in spermatogonia and spermatocytes, reduced in spermatids and barely detectable in sperm (Supplementary Material: FIG. S1). By FISH, the hybridization signal was found also strong in spermatogonia and spermatocytes, and weak in spermatids and sperm (FIG. 5A, B, C, E and F). Interestingly, the vasa RNA are distributed not only in the cytoplasm but also in the nucleus (FIG. 5D and F). At higher magnification, Lcvasa was detected in the chromatoid body (CB) of maturing sperm (FIG. 5F). Therefore, Lcvasa expression in the adult testis is limited to germ cells.


Vasa identifies germ cells and critical stages of oogenesis in the Asian seabass.

Xu H, Lim M, Dwarakanath M, Hong Y - Int. J. Biol. Sci. (2014)

vasa expression in the adult testis by fluorescent ISH. Testicular cryosections after fluorescent ISH (green) were analyzed by microscopy. Nuclei were stained blue with DAPI. (A) Bright field micrograph showing the testicular structure. (B) Merge micrograph between bright field and fluorescent optics. (C-F) Fluorescent micrograph. Large magnification of the area boxed in (A and B), along with (E and F) highlighting spermatogonia at the periphery. Arrow, CB; sg, spermatogonium; sc1 and sc2, primary and secondary spermatocyte; sm, sperm, st, spermatid; ED, efferent ductile; Scale bars, 50 µm in (A-C, E, F) and 25 µm in (D and D').
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Related In: Results  -  Collection

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Figure 5: vasa expression in the adult testis by fluorescent ISH. Testicular cryosections after fluorescent ISH (green) were analyzed by microscopy. Nuclei were stained blue with DAPI. (A) Bright field micrograph showing the testicular structure. (B) Merge micrograph between bright field and fluorescent optics. (C-F) Fluorescent micrograph. Large magnification of the area boxed in (A and B), along with (E and F) highlighting spermatogonia at the periphery. Arrow, CB; sg, spermatogonium; sc1 and sc2, primary and secondary spermatocyte; sm, sperm, st, spermatid; ED, efferent ductile; Scale bars, 50 µm in (A-C, E, F) and 25 µm in (D and D').
Mentions: Restriction of the vasa transcript to the gonads suggests its germ cell-specific expression. To confirm this, spatial expression was analyzed by ISH in adult gonads. The adult seabass testis is mainly composed of spermatogonia and spermatogenic cells at different stages. Spermatogonia are at the most peripheral region of seminiferous cysts. Spermatogenesis proceeds synchronously within each cyst and cysts containing germ cells at progressively advanced stages of development are closer to the efferent duct. Chromogenic ISH revealed that vasa RNA expression in the testis was exclusively in germ cells, with the signal being strong in spermatogonia and spermatocytes, reduced in spermatids and barely detectable in sperm (Supplementary Material: FIG. S1). By FISH, the hybridization signal was found also strong in spermatogonia and spermatocytes, and weak in spermatids and sperm (FIG. 5A, B, C, E and F). Interestingly, the vasa RNA are distributed not only in the cytoplasm but also in the nucleus (FIG. 5D and F). At higher magnification, Lcvasa was detected in the chromatoid body (CB) of maturing sperm (FIG. 5F). Therefore, Lcvasa expression in the adult testis is limited to germ cells.

Bottom Line: By chromogenic and fluorescent in situ hybridization we revealed germ cell-specific Lcvasa expression in both the testis and ovary.Importantly, Lcvasa shows dynamic patterns of temporospatial expression and subcellular distribution during gametogenesis.At different stages of oogenesis, for example, Lcvasa undergoes nuclear-cytoplasmic redistribution and becomes concentrated preferentially in the Balbiani body of stage-II~III oocytes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543.

ABSTRACT
Germ cells produce sperm and eggs for reproduction and fertility. The Asian seabass (Lates calcarifer), a protandrous marine fish, undergoes male-female sex reversal and thus offers an excellent model to study the role of germ cells in sex differentiation and sex reversal. Here we report the cloning and expression of vasa as a first germ cell marker in this organism. A 2241-bp cDNA was cloned by PCR using degenerate primers of conserved sequences and gene-specific primers. This cDNA contains a polyadenylation signal and a full open reading frame for 645 amino acid residues, which was designated as Lcvasa for the seabass vasa, as its predicted protein is homologous to Vasa proteins. The Lcvasa RNA is maternally supplied and specific to gonads in adulthood. By chromogenic and fluorescent in situ hybridization we revealed germ cell-specific Lcvasa expression in both the testis and ovary. Importantly, Lcvasa shows dynamic patterns of temporospatial expression and subcellular distribution during gametogenesis. At different stages of oogenesis, for example, Lcvasa undergoes nuclear-cytoplasmic redistribution and becomes concentrated preferentially in the Balbiani body of stage-II~III oocytes. Thus, the vasa RNA identifies both female and male germ cells in the Asian seabass, and its expression and distribution delineate critical stages of gametogenesis.

Show MeSH
Related in: MedlinePlus