FIGURE 4.

Depletion of Eif2s2 in premeiotic germ cells cause failure of homologous recombination and oocytes arrest at the early diplotene stages. (A) Representative images of mouse oocyte nuclei at the zygotene, pachytene, diplotene and dictyate stages of meiosis prophase I. Surface spreads were stained with SYCP3 (green) immunolabelling. During the zygotene stage, chromosomes begin to align, initiating the formation of the synaptonemal complex; pachytene, paired homologous chromosomes undergo maximal shortening and thickening; diplotene, homologous chromosomes separate; dictyate, two to four nucleoli are clearly visible. The percentages of oocytes at various stages were shown at 0, 1, 3 and 5 days postpartum (dpp). 0 dpp: Eif2s2 ^fl/fl N = 152, Eif2s2 ^cKO n = 279. 1 dpp: Eif2s2 ^fl/fl N = 396, Eif2s2 ^cKO n = 327. 3 dpp: Eif2s2 ^fl/fl N = 182, Eif2s2 ^cKO n = 213. 5 dpp: Eif2s2 ^fl/fl N = 156, Eif2s2 ^cKO n = 198. (B, C) Immunofluorescence staining of oocyte nuclear chromosome spreads by anti‐γH2Ax (red, B) and anti‐SYCP3 (green, B) antibodies in Eif2s2 ^fl/fl and Eif2s2 ^cKO mouse ovaries at 0 dpp. Numbers of patches of γH2Ax in Eif2s2 ^fl/fl (n = 30) and Eif2s2 ^cKO (n = 30) mouse ovaries (C). (D, E) Immunofluorescence staining of oocyte nuclear chromosome spreads by anti‐RPA2 (red, left panel of D), anti‐RAD51 (red, right panel of D) and anti‐SYCP3 (green, D) antibodies in Eif2s2 ^fl/fl and Eif2s2 ^cKO mouse ovaries. Numbers of foci of RPA2 and RAD51 in Eif2s2 ^fl/fl and Eif2s2 ^cKO mouse ovaries (E). RPA2: Eif2s2 ^fl/fl N = 45, Eif2s2 ^cKO n = 45. RAD51: Eif2s2 ^fl/fl N = 30, Eif2s2 ^cKO n = 45. (F) Western blotting (WB) analysis of RAD51 and DMC1 expression in Eif2s2 ^fl/fl and Eif2s2 ^cKO mouse ovaries. β‐actin was utilized as an internal control. n = 3. Scale bar: 10 μM. Bars represent the mean ± SD. A two‐sided Student's t‐test was performed to determine p values (*p < 0.05, **p < 0.01 and ***p < 0.001).