Fig. 4

Developing gonads of brca2 mutants lack perinucleolar oocytes and develop testes that contain pyknotic and apoptotic cells.

In transitioning gonads of wild types at 21dpf, ovary-like gonads (A) contained perinucleolar oocytes (early stage IB), while testis-like gonads (B) contained gonia, some early oocytes, and a few pyknotic cells. (C) Gonads of 21dpf brca2 mutants contained a few early oocytes and many pyknotic cells. (D) By 27dpf, perinucleolar oocytes had enlarged in wild-type ovaries. (E) Wild-type 27dpf testes possessed gonia. (F) 27dpf brca2 mutant gonads contained gonia, some early oocytes (judging from cell size and the location and number of nucleoli), showed testis-like morphology, and had many pyknotic cells. (G) At 32dpf, wild-type immature ovaries contained growing perinucleolar oocytes that had reached diplotene stage (late stage IB). (H) Wild-type 32dpf testes showed germ cells at all stages of spermatogenesis: spermatogonia, spermatocytes, spermatids, and sperm. (I) Homozygous brca2 mutant gonads at 32dpf possessed spermatogonia and spermatocytes, and tubules filled with pyknotic cells (dashed area) but lacked spermatids and sperm and had instead empty tubules (*). In transitioning gonads at 21–27dpf, we use the term ‘gonia’ (g) to represent cells that show the histological characteristics of spermatogonia (central nucleolus) and in differentiating testes at 32 dpf, we used the term ‘spermatogonia’ (sg) to represent such cells (Figure 4H′, 4I′). (J) Anti-active-Caspase-3 staining did not label cells in immature 60dpf wild-type testes. (K) In contrast, anti-active-Caspase-3 stained cohorts of cells in immature 60dpf mutant testes. (L,M) Staining the same sections with hematoxylin and eosin showed that cells staining for anti-active-Caspase-3 were pyknotic (arrows). Oocyte staging according to [49]. Magnification bar for A-I shown in A and for J-M shown in J. Abbreviations: asterisks (*), empty testis tubules; eo, early oocyte; pc, pyknotic cells; po, perinucleolar oocyte; sc, spermatocytes; sd, spermatids; sg, spermatogonia; sp, sperm.