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, Histology of wild-type and RibosomeST-/- 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, Histology of wild-type and RibosomeST-/- 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, Histology of wild-type and RibosomeST-/- 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 × 10−4) and progressive motility (Strain 2: 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). For c, d, e, g, h, i, k, m, n and p, data are mean ± s.e.m. P values were determined using two-tailed Student’s t-tests. n values represent the number of biologically independent animals.
