All Rlim germline KO (Δ) embryos were generated by RlimcKO/Δ-SC x RlimΔ/Y crosses. Embryonic stages are indicated, troph = trophoblasts. (A, B) Lack of RLIM in oocytes of RlimcKO/Δ-SC females. Immunohistochemical stainings of representative ovarian sections of adult RlimWT/WT (A) and RlimcKO/Δ-SC (B) females (n = 3, each) using antibodies directed against RLIM. Scale bars, 60 μm. Boxed regions are shown in higher magnification below. Note the lack of RLIM immunoreactivity in nuclei and pronuclei of both somatic cell types and oocytes in RlimcKO/Δ-SC females, respectively. (C, D) Gender determination of embryos in RNA-seq on whole preimplantation embryos WT and KO for Rlim. As example, the distributions of reads at the 8-cell stage normalized to autosomes of Y-linked genes (C) and Xist (D) are shown in box-plots. Note that embryos with high levels of Y-linked genes display low levels of Xist and were therefore categorized as males, whereas those with low and high levels of Y-linked genes and Xist, respectively, were categorized as females. cpm = counts per million mapped reads. (E) Modified from the UCSC Genome Browser: Cumulative mapped raw reads on the Rlim locus of pooled embryos WT/WT or Δ/Δ for Rlim (females only) at all developmental stages (variable scales). Structure of the Rlim gene is shown below in blue with boxed exon regions. Protein coding regions are indicated in thicker stroke. Arrow indicates direction of transcription. Floxed area deleted in the Rlim cKO is indicated. Note the lack of reads in the floxed area of RlimΔ/Δ females. This was also true for male RlimΔ/Y embryos (data not shown). (F) Developmental profile of relative expression of selected single genes in WT embryos. Data representing Oct4/Pou5f1 and Nanog (ES cell markers) and Krt8 (trophoblast) were pooled from WT females and males. Rlim data were collected from WT/WT females only. Reads were normalized to those at stage E3.5, because all of the selected genes are expected to be active at this stage.