Rescue of male infertility through correcting a genetic mutation causing meiotic arrest in spermatogonial stem cells

Abstract

Azoospermia patients who carry a monogenetic mutation that causes meiotic arrest may have their biological child through genetic correction in spermatogonial stem cells (SSCs). However, such therapy for infertility has not been experimentally investigated yet. In this study, a mouse model with an X-linked testis-expressed 11 (TEX11) mutation (Tex11^PM/Y) identified in azoospermia patients exhibited meiotic arrest due to aberrant chromosome segregation. Tex11^PM/Y SSCs could be isolated and expanded in vitro normally, and the mutation was corrected by clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated endonuclease 9 (Cas9), leading to the generation of repaired SSC lines. Whole-genome sequencing demonstrated that the mutation rate in repaired SSCs is comparable with that of autonomous mutation in untreated Tex11^PM/Y SSCs, and no predicted off-target sites are modified. Repaired SSCs could restore spermatogenesis in infertile males and give rise to fertile offspring at a high efficiency. In summary, our study establishes a paradigm for the treatment of male azoospermia by combining in vitro expansion of SSCs and gene therapy.

Keywords: azoospermia; gene therapy; male infertility; meiotic arrest; spermatogonial stem cells; testis-expressed 11.

Figure 1

Male mice carrying an azoospermia patient-derived TEX11 mutation display meiotic arrest. (a) Testis morphology of WT and Tex11^PM/Y mice. Scale bar = 100 mm. (b) Relative testis/body weight of WT and Tex11^PM/Y mice. The average values of three separate experiments are plotted. Error bar: s.d. Significance was determined by two-tailed, unpaired Student’s t-test. (c) Immunostaining of 10 dpp testis sections with an anti-PLZF antibody. Green: PLZF; Blue: Hoechst 33342. Scale bar = 50 μm. (d) Quantification of PLZF⁺ cells in 10 dpp testis sections. A total of 237 and 222 tubules from three WT and Tex11^PM/Y mice were scored. Error bar: s.d. Significance was determined by two-tailed, unpaired Student’s t-test. NS: no significance. (e) Immunostaining of MLH1/SCP3 in spermatocyte spreads prepared from 8-week-old WT and Tex11^PM/Y mice. Green: MLH1; red: SYCP3. Note the absence of MLH1 foci on three synapsed chromosomes (arrowhead) in the Tex11^PM/Y sample shown in the bottom image. (f) Number of MLH1 foci in Tex11^PM/Y and WT spermatogonia. A dramatic reduction in the number of MLH1 foci (green) in Tex11^PM/Y spermatogonia (approximately 15 foci per cell) relative to WT (22 foci per cell). Error bar: s.d. Significance was determined by two-tailed, unpaired Student’s t-test;^*** P < 0.001. (g) TUNEL analysis of Tex11^PM/Y and WT testis. Massive apoptosis (green) shown in Tex11^PM/Y tubules. Scale bar = 100 μm. (h) Analysis of apoptotic cells in Tex11^PM/Y and WT testis. The average values of three separate experiments are plotted. Error bar: s.d. Significance was determined by two-tailed, unpaired Student’s t-test;^*** P < 0.001. TEX11: testis-expressed 11; WT: wild-type; PLZF: promyelocytic leukemia zinc finger; MLH1: mutl homolog 1; SYCP3: synaptonemal complex protein 3; TUNEL: terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling; DAPI: 4’,6-diamidino-2-phenylindole; s.d.: standard deviation; dpp: days postpartum.

Figure 2

SSCs isolated from Tex11^PM/Y mice are normal. (a) Flow cytometric analysis of the ratio of SSCs using CD146 antibody in 10-day-old WT and Tex11^PM/Y testicular cells. Small black boxes represent the positive population of SSCs. (b) The ratio of CD146-positive cells in WT and Tex11^PM/Y testicular cells (three mice per group) was scored. Error bar: s.d. Significance was determined by two-tailed, unpaired Student’s t-test; NS: no significance. (c) Morphology of cultured WT and Tex11^PM/Y SSCs. P2: passage 2. Scale bar = 100 μm. (d) Proliferation rate of WT and Tex11^PM/Y SSCs during in vitro culture. The average values of three separate experiments are plotted. Error bar: s.d. Significance was determined by two-tailed, unpaired Student’s t-test. (e) Expression of marker genes in WT and Tex11^PM/Y SSCs. Id4 and Sohlh2 were used as undifferentiated marker genes, while Kit and Stra8 were used as differentiated marker genes. The av?erage values of three separate experiments are plotted; error bar: s.d. Significance was determined by two-tailed, unpaired Student’s t-test; ***P < 0.001. (f) Immunostaining of PLZF in WT and Tex11^PM/Y SSCs. Scale bar = 200 μm. (g) Scatter plot of log₁₀-transformed average gene expression profiles. Global gene expression profiles of WT and Tex11^PM/Y SSCs were obtained from RNA-seq analysis (r is the Pearson’s correlation coefficient; yellow lines indicate two-fold upregulation and downregulation). (h) Heatmap correlation of SSC self-renewal genes in WT and Tex11^PM/Y SSCs (r is the Pearson’s correlation coefficient). WT: wild-type; Tex11: testis-expressed 11; SSCs: spermatogonial stem cells; s.d.: standard deviation; PLZF: promyelocytic leukemia zinc finger; SSC-A: Side Scatter-area; Id4: inhibitor of DNA binding 4; Sohlh2: spermatogenesis and oogenesis specific basic helix-loop-helix 2; Kit: Kit proto-oncogene; Stra8: stimulated by retinoic acid 8; Ret: ret proto-oncogene; Zbtb16: zinc finger and BTB domain containing 16; Pou5f1: POU class 5 homeobox 1; Etv5: ETS variant transcription factor 5; Gfra1: GDNF family receptor alpha 1; Foxo1: forkhead box O1; Taf4b: TATA-box binding protein associated factor 4b; T: brachyury; Lhx1: LIM homeobox 1; Bcl6b: BCL6B transcription repressor; Cxcr4: C-X-C motif chemokine receptor 4.

Figure 3

Correction of Tex11 mutation via CRISPR-Cas9-mediated gene editing in SSCs. (a) Schematic for Tex11 gene correction via HDR induced by CRISPR-Cas9 system and HDR template with WT sequence of Tex11. The mutant nucleotides were marked in red, while the WT nucleotides were in green. (b) Morphology of the three established SSC lines with correct Tex11 sequence (termed HDR-1, 2, and 3). Scale bar = 100 μm. (c) DNA sequencing analysis of SSCs from HDR-1/2/3. Note that the sequences of PCR products amplified from the Tex11 gene show that HDR-1/2/3 carry corrected Tex11 gene. (d) Transcriptional analysis of Tex11 in the corrected SSCs lines. The expression values were normalized to that of WT. The average values of three separate experiments are plotted; error bar: s.d. Significance was determined by two-tailed, unpaired Student’s t-test; ***P < 0.001. (e) DNA methylation analysis of the DMRs of H19, IG, and Snrpn in Tex11^PM/Y and HDR-1 SSCs. Open, filled, and gray circles represent unmethylated, methylated, and uncertain CpG sites, respectively. WT: wild-type; Tex11: testis expressed 11; SSCs: spermatogonial stem cells; s.d.: standard deviation; sgRNA: single-guide RNA; CRISPR-Cas9: clustered regularly interspaced short palindromic repeats-CRISPR-associated endonuclease 9; DMR: differentially methylated regions; H19: H19 imprinted maternally expressed transcript; IG: intergenic differentially methylated region; Snrpn: small nuclear ribonucleoprotein polypeptide N; CpG: cytosine-guanine; HDR: homology-directed repair.

Figure 4

Corrected SSCs restore spermatogenesis in the testes of Kit^W /Kit^WV mice. (a) Diagram showing the repaired SSCs derived from single SSCs without off-target mutations which are selected for transplantation into infertile Kit^W /Kit^WV male mice. Two months later, spermatids are isolated from reconstituted testes and injected into mature oocytes to produce healthy mice carrying corrected Tex11 gene. (b) Testis morphology of Tex11^PM/Y and HDR-1 SSC-transplanted Kit^W /Kit^WV mice. Scale bar = 100 mm. (c) Weight of Tex11^PM/Y and HDR-1 SSC-transplanted Kit^W /Kit^WV testes. The average values of three separate experiments are plotted. Error bar: s.d. Significance was determined by two-tailed, unpaired Student’s t-test;^* P < 0.05. (d) Histological analyses of the Tex11^PM/Y and HDR-1 SSC-transplanted Kit^W /Kit^WV testis. Note successful spermatogenesis in the HDR-1 SSC-transplanted testis. Scale bar = 40 μm. (e) Immunostaining of MVH in Tex11^PM/Y and HDR-1-transplanted Kit^W /Kit^WV testis. Green: MVH; blue: Hoechst. Scale bar = 100 μm. (f) Round spermatids are sorted from testicular cells of reconstituted testes according to DNA content. (g) Morphology of round sperm sorted from HDR-1-transplanted testis. Scale bar = 50 μm. (h) Pups (black coat color) on 12 dpp developed from transferred two-cell embryos generated after the injection of round spermatids from HDR-1 SSCs into mature oocytes. WT: wild-type; Tex11: testis expressed 11; SSCs: spermatogonial stem cells; ROSI: round spermatid injection; s.d.: standard deviation; HDR: homology-directed repair; dpp: days postpartum.

Figure 5

Scheme for PDMX model of male azoospermia therapy. TEX11 mutation has been identified in patients with azoospermia. To validate its pathogenic roles, mouse model with the patient-derived mutation (Tex11^PM/Y mice) was derived, which recapitulated patient’s pathological phenotypes and displayed meiotic arrest. SSCs isolated from the testis of the Tex11^PM/Y mice were expanded in vitro. CRISPR-Cas9-mediated gene correction was employed in Tex11^PM/Y SSCs and repaired SSC lines were derived through single SSC expansion, followed by whole-genome sequence to exclude the off-target mutations. The repaired SSCs were then used for transplantation to restore spermatogenesis in infertility males. PDMX model thus provides proof-of-principle evidence for curing human azoospermia carrying a single mutation that causes meiotic arrest. TEX11: testis-expressed 11; SSCs: spermatogonial stem cells; ROSI: round spermatid injection; CRISPR-Cas9: clustered regularly interspaced short palindromic repeats-CRISPR-associated endonuclease 9; PDMX: patient-derived mutation xenocorrection.