Extended Data Fig. 2: The CRISPR-Cas9 knockout strategy and phenotype of RibosomeST-/-mice.
From:A male germ-cell-specific ribosome controls male fertility
a, Schematic of CRISPR-Cas9 and sgRNAs used.b, Sequences of the three mutant strains generated by CRISPR-Cas9.c,d, Relative protein expression levels of RPL39L (pachytene spermatocytes:P = 5.63 × 10−6, spermatids:P = 1.47 × 10−6) (c) and RPL39 (pachytene spermatocytes:P = 1.04 × 10−4, spermatids:P = 1.09 × 10−4) (d) in RibosomeST-/-and wild-type mouse pachytene spermatocytes and spermatids, as evaluated by PRM.n = 3.e, RibosomeST-/-and wild-type testes with the 40S (P = 0.664), 60S (P = 0.664), 80S (P = 1.25 × 10−6), 2-6 (P = 0.712) and 7+ polysome (P = 0.489) peaks annotated and statistically compared.n = 5.f, Testis morphology and size of both RibosomeST-/-and wild-type mice.n = 3. Scale bar, 3 mm.g, Sperm morphology and prevalence of head (P = 7.86 × 10−4) and tail (P = 9.02 × 10−6) defects in RibosomeST-/-and wild-type sperm.n = 4. Scale bar, 10 μm.h, Levels of testosterone (P = 0.662), luteinizing hormone (LH) (P = 0.491) and follicle-stimulating hormone (FSH) (P = 0.437) in RibosomeST-/-and wild-type mice.n = 3.i, Average frequency of vaginal plugs per female mouse for RibosomeST-/-or wild-type male mice during fertility tests (P = 0.902,n = 3).j、野生型的组织学和核糖体ST-/-testis showing seminiferous tubules at stage VII-VIII, circled by dashed lines, and amplified in the bottom panels.n = 3. PL: preleptotene spermatocyte, black arrowhead; PS: pachytene spermatocyte, red arrowhead; RS: round spermatid, green arrowhead; ES: elongated spermatid, blue arrowhead; SC: Sertoli cell. Top panels scale bar, 50 μm; Bottom panels scale bar, 10 μm.k、野生型的组织学和核糖体ST-/-testis showing complete testicular section and stage IX sections together with statistics of percentages of stage IX (P = 0.179) and average number of unreleased sperms per stage IX tubule (P = 5.87 × 10−4). The red dotted circle indicates the seminiferous tubules of stage IX, and the black dotted line indicates delay-released sperms. Left panels scale bar, 500 μm; Right panels scale bar, 20 μm.n = 3.l、野生型的组织学和核糖体ST-/-testis showing unreleased sperms at stage IX. Areas marked by dashed lines are shown at high magnification to the bottom.n = 3. L: leptotene spermatocyte, black arrowhead; PS: pachytene spermatocyte, red arrowhead; ES: elongated spermatid, blue arrowhead; SC: Sertoli cell; RB: residual body. Top panels scale bar, 50 μm; Bottom panels scale bar, 10 μm.m, The images of testes and testis/body weight ratio (P = 5.63 × 10−4,n = 6) of the three RibosomeST-/-strains and wild-type mice. Scale bar, 3 mm.n, Epididymal sperm count (Strain 2:P = 0.048, Strain 3:P = 0.045), motility (Strain 2:P = 4.03 × 10−4, Strain 3:P = 4.14 × 10−42)和进步的能动性(压力:P = 3.77 × 10−4, Strain 3:P = 4.05 × 10−4) in two more RibosomeST-/-strains and wild-type mice.n = 3.o, HE staining of the testis and epidydimal sperms from the three RibosomeST-/-strains.n = 3. Scale bar, 10 μm.p, Fertility of female RibosomeST-/-mice when mated with wild-type male mice (P = 0.606,n = 3). Forc,d,e,g,h,i,k,m,nandp, data are mean ± s.e.m.Pvalues were determined using two-tailed Student’st-tests.nvalues represent the number of biologically independent animals.
