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Fig. 1. (A,B) GCNA1 and MVH immunolabeling of germ cells in (A) Bax+/+ and (B) Bax-/- ovaries. The same section was labeled with fluorophore-conjugated antibodies against GCNA1 (green) and MVH (red), and the merged image is shown in the middle. Bars, 200 µm. The adjacent sections were immunostained with DAB for GCNA1 (left) and MVH (right), and shown at a lower magnification. (A) At 12.5 d.p.c., MVH-positive cells are distributed over the ovary. Fewer GCNA1-positive cells are seen in the same area. All germ cells are positive for MVH in their cytoplasm but only some cells are also positive for GCNA1 in their cytoplasm. A small number of cells are positive for GCNA1 in the nucleus (arrowheads). At 14.5 d.p.c., similar numbers of MVH- and GCNA1-positive cells are distributed over the ovary. The majority of cells are positive for both MVH and GCNA1 in the cytoplasm and nucleus, respectively. However, some cells are positive only for MVH (arrowheads). At 18.5 d.p.c., less MVH labeling is seen in many cells over the ovary compared with the labeling at previous developmental stages. Intense labeling is also seen in fewer cells in clusters. GCNA1-positive cells are distributed in similar areas as the cells with intense MVH labeling. Most cells are positive for both MVH and GCNA1. However, GCNA1 labeling often extends into the cytoplasm and overlaps with MVH labeling. Some GCNA1-positive cells without MVH labeling (arrowheads) are seen in the surface epithelium. At 24.5 d.p.c., large MVH-positive cells are prominent in the central area and smaller cells are concentrated in the peripheral area. GCNA1 labeling is weaker and mainly seen in the peripheral area. Most large MVH-positive cells in the central area show either no or only faint GCNA1 labeling (arrowheads). Faint GCNA1 labeling is also seen in the cytoplasm of smaller cells (arrows). (B) At 12.5 d.p.c., MVH-positive cells are seen clustered in the gonad. A fewer GCNA1-positive cells are seen in the same area. Both MVH- and GCNA-positive cells appear to be fewer compared with the Bax+/+ ovary. Double immunolabeling shows three types of germ cells: most cells are positive only for MVH in the cytoplasm whereas fewer cells are also positive for GCNA1 in the cytoplasm or the nucleus (arrowheads). At 14.5 d.p.c., a considerably higher density of both MVH- and GCNA1-positive cells is seen in the Bax-/- ovary compared with the Bax+/+ ovary. Most cells showed both MVH labeling in the cytoplasm and GCNA1 labeling in the nucleus as well as the cytoplasm. A few germ cells showed only MVH labeling (arrowheads). At 18.5 d.p.c., intense MVH labeling is seen in many cells of the Bax-/- ovary. GCNA1-positive cells are also more evenly distributed in the Bax-/- ovary compared with the Bax+/+ ovary. MVH labeling is only seen in the cytoplasm whereas GCNA1 labeling is seen in both the nucleus and cytoplasm. Some cells are positive only for MVH (arrowheads). At 24.5 d.p.c., distribution of MVH- and GCNA1-positive cells was similar to that observed in the Bax+/+ ovary, except that smaller MVH-positive cells were crowded near the peripheral area. GCNA1 labeling diminished in the large MVH-positive cells in the central area (arrowheads). In the Bax-/- ovary a larger number of germ cells with intense GCNA1 labeling in the nucleus was seen in the surface epithelium compared with the Bax+/+ ovary.
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