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Transmeiotic differentiation of zebrafish germ cells into functional sperm in culture

Department of Marine Bioscience and Graduate School of Bioscience and Biotechnology, Fukui Prefectural University, Obama 917-0003, Japan

View larger version (132K): [in a new window]   Fig. 1. Derivation of ZtA6 feeder cells from tumor-like hypertrophied testis. (A) ZtA6 cells included heterogeneous testicular cell types, including epithelial and fibroblast cells. (B) Many cells showed phagocytic activity in terms of the uptake of latex beads, which is characteristic of mammalian Sertoli cell lines (Tokuda et al., 1992; Rassoulzadegan et al., 1993). Scale bars: 50 µm.

View larger version (32K): [in a new window]   Fig. 2. Expression of Sertoli cell and germ cell marker genes in ZtA6 cells by RT-PCR. ZtA6 cells expressed the marker genes of Sertoli cells, zebrafish sox9a and the Wilms’ tumor suppressor wt1, but scarcely any gata1. Germ cell marker vas was detected at low levels.

View larger version (115K): [in a new window]   Fig. 3. Co-culture of dissociated testicular cells on ZtA6 feeder cells. Male germ cells were attached on the feeder cells as a clump. (A-L) The same single clump was observed every day from 3 days to 14 days of culture. (M) On day 15, the clump had disappeared; however, an aggregation of flagellated sperm was found in suspension. (N) Control normal mature sperm. As the germ cells divided, the clump became larger (A-F) until the appearance of flagellated sperm on day 9 (G). It then decreased in size (H-L). The nuclear sizes of flagellated sperm (arrowhead) on days 9 (G), 12 (J) and 13 (K) are similar to those of normal mature sperm (N). Scale bar: 10 µm.

View larger version (34K): [in a new window]   Fig. 4. Competitive PCR analysis of vas and -tubulin expression in a co-culture of dissociated testicular cells with ZtA6 feeder cells and in a culture of dissociated testicular cells alone. Co-culture was used with cDNA from a co-culture of dissociated testicular cells with mitomycin C-treated ZtA6 cells. The control used cDNA from dissociated testicular cells cultured alone plus the same ZtA6 cells as that used in the co-culture cultured separately, making it equivalent to the co-culture. Two hundred times more vas cDNA was used than -tubulin cDNA. As duplicate experiments resulted in the same pattern, this figure shows the results from one experiment. The number below the photos indicates the ratio of sample density to competitor density.

View larger version (112K): [in a new window]   Fig. 5. BrdU incorporation in cultured male germ cells. (A) BrdU-positive cells at day 5 of culture showed large nuclei. (C) At day 8, the positive nuclei of approximately the same size as those of the mature sperm were present (arrowhead). (E) BrdU-positive flagellated sperm were found on day 9. (B,D,F) Control at day 5 (B), at day 8 (D) and normal mature sperm (F) subjected to BrdU detection. Scale bar: 10 µm.

View larger version (138K): [in a new window]   Fig. 6. Immunohistochemistry for BrdU in an embryo at the late four-cell stage fertilized with cultured sperm. (A) Whole-mount immunohistochemistry. Two positives in each cell indicate chromosome division (arrowheads). (B) Section immunohistochemistry: BrdU-positive cells indicate that chromosomes have already divided (arrowheads). The remaining positives were found in other sections (not shown). (C) Control of whole-mount immunohistochemistry subjected to BrdU detection. (D,E) Control of section immunohistochemistry subjected to BrdU detection (D) and to Hematoxylin staining (E). Scale bars: 50 µm in A for A and C and B for B,D,E.