RNase H1 Antibody - #DF12084

Fig. 3. The function of RNase H1 in mouse oocytes is dependent on its RNase H activity. (a) Immunofluorescence analysis of S9.6 (green) was performed in chromosomal spreads of Rnaseh1F/F and Stra8-Rnaseh1-/- MI oocytes. (b) Quantification of the relative mean intensity of the centromeric S9.6 signal normalized to the background signal near centromeres (see Methods) in Rnaseh1F/F and Stra8-Rnaseh1-/- MI oocytes (n, number of oocytes). Data are presented as mean ± SD. Two-tailed Student’s t-test; ****P < 0.0001. (c) Schematic representation of the domain architecture of RNase H1 and its truncation mutants used in this study. The mouse RNase H1 protein consists of a mitochondrial targeting sequence (MTS), a hybrid binding domain (HBD), a connection domain (CD) and an RNase H domain (H-domain) in which the C-terminal RNase H domain is necessary for hydrolysis. (d) Representative images of spindle morphology and chromosome alignment in Stra8-Rnaseh1-/- + NC, Stra8-Rnaseh1-/- + RNase H1WT, Stra8-Rnaseh1-/- + RNase H1D209N and Stra8-Rnaseh1-/- + RNase H1ΔH-domain MI oocytes. The same amount of RNase-free water was injected as negative control (NC). (e) The ratios of oocytes with normal spindle morphology were recorded for the indicated groups (n = 3 independent replicates, total oocytes = 168). Data are presented as mean ± SEM. Two-tailed Student’s t-test; *P < 0.05. ns, non-significant. (f) The ratios of oocytes with misaligned chromosomes were recorded for the indicated groups (n = 3 independent replicates, total oocytes = 168). Data are presented as mean ± SEM. Two-tailed Student’s t-test; *P < 0.05. ns, non-significant. (g) Representative images of spindle morphology and chromosome alignment in control (WT + NC) and RNase H1WT-overexpressed (WT + RNase H1WT) MI oocytes. Mouse oocytes at GV stages were microinjected with a high concentration of Myc-Rnaseh1WT mRNA (about 650 ng/ul) and cultured in vitro for maturation. (h) The proportions of oocytes with normal spindle morphology were recorded for the indicated groups. WT + NC (n = 3 independent experiments, total oocytes = 75); WT + RNase H1WT (n = 3 independent experiments, total oocytes = 56). Data are presented as mean ± SD. Two-tailed Student’s t-test; ***P < 0.001. (i) The proportions of oocytes with misaligned chromosomes were recorded for the indicated groups. WT + NC (n = 3 independent experiments, total oocytes = 75); WT + RNase H1WT (n = 3 independent experiments, total oocytes = 56). Data are presented as mean ± SD. Two-tailed Student’s t-test; **P < 0.01. (j) Representative images of spindle morphology and chromosome alignment in WT + NC and WT + RNase H1WT oocytes. Mouse oocytes at GV stages were microinjected with low concentration of Myc-Rnaseh1WT mRNA (about 300 ng/ul) and cultured in vitro for maturation. (k) Immunofluorescence analysis of 6MYC-RNase H1WT was performed in chromosomal spreads of control and 6MYC-RNase H1WT-overexpressed MI oocytes by immunolabeling with anti-MYC antibody (red). (l) Immunofluorescence analysis of S9.6 (green) was performed in chromosomal spreads of WT + NC and WT + RNase H1WT MI oocytes which were microinjected with a high concentration of Myc-Rnaseh1WT mRNA. (m) Quantification of the relative mean intensity of S9.6 signals at centromeres in WT + NC and WT + RNase H1WT MI oocytes (n, number of oocytes). Data are presented as mean ± SD. Two-tailed Student’s t-test; ****P < 0.0001. (g-m) The same amount of RNase-free water was injected into WT oocytes as negative control (WT + NC).

Figure 1. Germ cell-specific Rnaseh1 knockout impairs mouse spermatogenesis and causes male infertility. (A) Schematic diagram for the construction of the Rnaseh1F/F and Stra8-Rnaseh1−/− mice. The mouse Rnaseh1 locus surrounding exons 2, 3 and 4 is shown in the wild-type allele panel. (B) The genotyping of the Rnaseh1+/+, Rnaseh1F/+, and Rnaseh1F/F mice. (C) The RNase H1 protein levels are decreased in Stra8-Rnaseh1−/−testes. Immunoblotting of RNase H1 is performed in Rnaseh1F/F and Stra8-Rnaseh1−/− testes. H3 and Tubulin serves as the loading control. (D) The quantification of RNase H1 protein levels in (C) (n = 5 independent experiments). Black dots indicate Rnaseh1F/F mice, and red dots indicate Stra8-Rnaseh1−/− mice. Data are presented as means ± SEM. two-tailed Student's t-test; **P < 0.01. (E and F) Germ cell-specific knockout of Rnaseh1 causes male infertility. The fertility assessment experiments are performed in Rnaseh1F/F and Stra8-Rnaseh1−/− male mice. Pregnancy rates (%) of plugged wild-type female mice after mating with Rnaseh1F/F and Stra8-Rnaseh1−/− male mice (n = 3 independent experiments) (E). Average litter sizes were observed with Rnaseh1F/F and Stra8-Rnaseh1−/− male mice (n = 3 independent experiments) (F). Black dots indicate Rnaseh1F/F mice, and red dots indicate Stra8-Rnaseh1−/− mice. Data are presented as means ± SEM. two-tailed Student's t-test; ***P < 0.001. (G) The Stra8-Rnaseh1−/− mouse testis is smaller than that of the Rnaseh1F/F mouse. (H) Ratio of testis weight/body weight in Rnaseh1F/F and Stra8-Rnaseh1−/− mice (n = 4 independent experiments). Black dots indicate Rnaseh1F/F mice and red dots indicate Stra8-Rnaseh1−/− mice. Data are presented as means ± SEM. two-tailed Student's t-test; *P < 0.05. (I) Histological analysis of the seminiferous tubules and caudal epididymis of the Rnaseh1F/F and Stra8-Rnaseh1−/− mice by hematoxylin and eosin staining. Arrowheads indicate dead cells. (J) Sperm counts from the cauda epididymis significantly decreased in the Stra8-Rnaseh1−/− mice compared with Rnaseh1F/F mice. (n = 5 independent experiments). Black dots indicate Rnaseh1F/F mice, and red dots indicate Stra8-Rnaseh1−/− mice.