Application of C-terminal truncated mutation of CHK1 in regulating male sterility

Abstract

The application discloses application of a C-terminal truncation mutation of CHK1 in regulation of male sterility, wherein the C-terminal truncation mutation refers to that a Chk1 gene is mutated, a C-terminal domain of CHK1 coded by the Chk1 gene is prematurely terminated, a conserved sequence of the C-terminal of the CHK1 protein is destroyed, the C-terminal is truncated, and preferably a heterozygous frameshift mutation p.F441fs*16 is generated. The application also discloses use of the C-terminal truncation mutation of the Chk1 gene as a diagnostic marker of mammalian male sterility, and application of specific primers or kits for detecting the C-terminal truncation mutation of the Chk1 gene in preparation of a mammalian infertility screening product. Researches show that the heterozygous frameshift mutation p.F441fs*16 can seriously impair the male fertility of mice, and lead to NOA or oligospermia. The heterozygous frameshift mutation p.F441fs*16 has potential as a diagnostic marker of male sterility, and potential as a therapeutic target.

Description

Technical Field

[0001] This invention relates to the application of C-terminal truncation mutations in CHK1 in regulating male infertility, and belongs to the field of mutation or genetic engineering technology. Background Technology

[0002] Infertility is a disease affecting fertility, with male factors accounting for approximately 50% of infertility cases. Clinical manifestations of male infertility typically include decreased sperm count (azoospermia or oligospermia), decreased sperm motility (asthenospermia), or increased abnormal sperm morphology (teratospermia). Non-obstructive azoospermia (NOA) is caused by impaired spermatogenesis, resulting in the absence of sperm in the semen. Genetic factors are a significant cause of male infertility, and the incidence of genetic abnormalities increases significantly with decreasing sperm concentration. Notably, men with azoospermia have a higher risk of carrying genetic factors. Traditional clinical genetic testing is limited to karyotype analysis and Y-chromosome AZF microdeletion screening. In recent years, next-generation sequencing and functional studies in mouse models have identified numerous gene mutations associated with non-obstructive azoospermia and oligospermia; however, the contribution of each gene to the etiology is limited, and our understanding of the genetic causes of male infertility remains insufficient.

[0003] Cell cycle checkpoint kinase 1 (CHK1) is a key regulator of DNA damage response and cell cycle checkpoints, ensuring genome stability. It plays a crucial role in reproductive biology; CHK1 knockout in mouse ovaries leads to female infertility, while overexpression increases the number of follicles in aged female mice. CN 116457457 A, a previous study of this invention, showed that heterozygous gain-of-function mutations in the C-terminal domain of CHK1 lead to zygotic arrest and female infertility, but the relationship between CHK1 heterozygous gain-of-function mutations and spermatogenesis and male fertility remains unclear. Interestingly, CHK1 is also essential for male fertility; conditional complete knockout of CHK1 in mouse testicular germ cells disrupts spermatogonial proliferation and impairs DNA damage repair during meiosis. Summary of the Invention

[0004] In response to the aforementioned prior art, this invention investigated the relationship between CHK1 gain-of-function mutations and spermatogenesis and male fertility, and provided the application of C-terminal truncated mutations of CHK1 in regulating male infertility.

[0005] This invention is achieved through the following technical solution: The application of C-terminal truncated mutations in regulating male sterility is manifested as follows: the presence of a heterozygous genotype with a C-terminal truncated mutation in the Chk1 gene of male mammals leads to male sterility; or / and: the expression of a C-terminal truncated mutation in the Chk1 gene of male mammals leads to male sterility. The C-terminal truncation mutation refers to a mutation in the Chk1 gene that causes premature termination of the C-terminal domain of the CHK1 protein it encodes, disrupting the conserved sequence at the C-terminus of the CHK1 protein and resulting in C-terminal truncation.

[0006] Preferably, the mammal species is selected from humans and mice.

[0007] Preferably, the C-terminal truncation mutation is a heterozygous frameshift mutation p.F441fs*16, which, compared to the wild type, terminates prematurely after a 15-amino acid frameshift in the C-terminal domain, resulting in a 21-amino acid truncation. The nucleotide coding sequence of the human Chk1 gene is shown in SEQ ID NO.1, and the amino acid sequence of human CHK1 is shown in SEQ ID NO.2; the nucleotide coding sequence of the heterozygous frameshift mutation p.F441fs*16 of the human Chk1 gene is shown in SEQ ID NO.3, and the amino acid sequence of the heterozygous frameshift mutation p.F441fs*16 of human CHK1 is shown in SEQ ID NO.4. The nucleotide coding sequence of the mouse Chk1 gene is shown in SEQ ID NO.5, and the amino acid sequence of mouse CHK1 is shown in SEQ ID NO.6; the nucleotide coding sequence of the heterozygous frameshift mutation p.F441fs*16 of the mouse Chk1 gene is shown in SEQ ID NO.7, and the amino acid sequence of the heterozygous frameshift mutation p.F441fs*16 of mouse CHK1 is shown in SEQ ID NO.8.

[0008] The application of C-terminal truncated mutations of the Chk1 gene as a diagnostic marker for male infertility in mammals, and their use in the preparation of mammalian infertility screening products.

[0009] Application of specific primers for detecting C-terminal truncated mutations in the Chk1 gene in the preparation of mammalian infertility screening products.

[0010] Application of a kit for detecting C-terminal truncated mutations in the Chk1 gene in the preparation of mammalian infertility screening products, wherein the kit contains specific primers for detecting C-terminal truncated mutations in the Chk1 gene.

[0011] Furthermore, the C-terminal truncation mutation of the Chk1 gene is a heterozygous frameshift mutation p.F441fs*16.

[0012] Furthermore, in practical applications, the Chk1 gene is used as the detection target to detect the genotype of the Chk1 gene in male mammals. If it is a heterozygous genotype containing a C-terminal truncated mutation, it indicates that the individual has a risk of infertility. This judgment result can be used as one of the bases for doctors' diagnosis or risk assessment.

[0013] Application of C-terminal truncated CHK1 in the preparation of mammalian male infertility agents. Specifically, the C-terminal truncated CHK1 is administered to the testes of male mammals via a drug delivery method (e.g., targeted drug delivery), reducing sperm count and motility, thus causing NOA (non-azoospermia) or oligospermia in male mammals. It can be used for mammalian population control or male contraception.

[0014] Application of a male mouse model with a C-terminal truncated mutation in the Chk1 gene in the screening of drugs for treating male infertility. This male mouse model with a C-terminal truncated mutation in the Chk1 gene can be constructed using CRISPR-Cas9. In specific applications, this mouse model will be used as the research subject to screen for drugs to treat male infertility.

[0015] In previous studies, the heterozygous frameshift mutation p.F441fs*16 was identified as a cause of zygotic cleavage failure in infertile women. This invention, through further research, demonstrates that the heterozygous frameshift mutation p.F441fs*16 also severely impairs male fertility in mice, leading to NOA or oligospermia. Mechanistically, this mutation promotes the formation of phase-separated condensates, which abnormally interact with wild-type CHK1 protein, altering its subcellular localization and overactivating CHK1 kinase activity, ultimately disrupting spermatogenesis. Therefore, the heterozygous frameshift mutation p.F441fs*16 has the potential to serve as a diagnostic biomarker for male infertility and as a therapeutic target.

[0016] The various terms and phrases used in this invention have their general meanings known to those skilled in the art. Attached Figure Description

[0017] Figure 1Fertility assessment results of Chk1 mutant mice. A. A Chk1 heterozygous truncated mutant mouse (F441fs16⁺ / ᵗʳ) was constructed using CRISPR-Cas9 technology, and its efficiency was verified by Sanger sequencing; green nucleotide sequences represent synonymous codons between oligonucleotide donors and gRNA, and red sequences represent mutant codons (NM_007691.5; c.1323delC). B–D. Fertility assessment of wild-type (WT) and Chk1 mutant (F441fs16⁺ / ᵗʳ) male mice over 6 months, including the total number of pups produced per male (B), the total number of litters produced per male (C), and the average litter size per male (D); each male mouse was paired with two wild-type females for mating. E. Representative images showing the development of wild-type oocytes after fertilization with WT or Chk1 mutant (F441fs16⁺ / ᵗʳ) sperm; scale bar: 100 μm. F. Developmental rates of pre-implantation embryos (E), normalized to the initial MII oocyte count; two-tailed Student's t-test; error bars represent mean ± standard error; *p < 0.05, **p < 0.01, ***p < 0.001. G. Volcano plot showing differentially expressed genes between wild-type and CHK1 mutant sperm-fertilized embryos (|log2FC| > 1, p < 0.05). H. Hierarchical clustering of 1032 significantly differentially expressed genes; color intensity represents expression levels after z-score normalization. I. Gene Ontology (GO) enrichment analysis of downregulated differentially expressed genes, highlighting affected biological processes (BP).

[0018] Figure 2Chk1 mutant mice exhibit non-obstructive azoospermia or oligospermia. A. Body weight of 8-week-old wild-type (WT) and Chk1 mutant (F441fs*16⁺ / ᵗʳ) mice. B. Testicular weight of 8-week-old WT and Chk1 mutant mice. C. Testicular weight ratio of WT to Chk1 mutant mice. D. Representative testicular images of 8-week-old WT or Chk1 mutant mice; scale bar: 2 mm. E. Representative images of hematoxylin-eosin stained epididymal tail sections from WT and Chk1 mutant mice. F. Sperm analysis of Chk1 mutant mice showed that one-third of the mutant males exhibited azoospermia; representative images show sperm detected in the remaining two-thirds of the mutant mice by computer-assisted semen analysis (CASA; TOX IOVS II). G–J. CASA-based analysis of the motility of Chk1 mutant sperm (2 months old) showed that, compared with wild-type controls, total sperm count (G), motile sperm count (H), curve velocity (I), and whiplash frequency (J) were significantly reduced. Representative hematoxylin-eosin stained images of testicular sections from K.WT and Chk1 mutant mice at day 4 (PD4), day 10 (PD10), day 14 (PD14), and 2 months (2M) and 6 months (6M) after birth; red arrows indicate non-vacuolated seminiferous tubules in testicular sections from Chk1 mutant mice; two-sided Studentt test; error bars represent mean ± standard error; ns, no significant difference; *p<0.05, **p<0.01, ****p<0.0001.

[0019] Figure 3Chk1 mutant mice showed a significant reduction in germ cells during embryonic development. A. Immunofluorescence images of the testes in wild-type (WT) and Chk1 mutant (F441fs*16⁺ / ᵗʳ) mice at day 4 (PD4), day 10 (PD10), day 14 (PD14), and 6 months (6M) postnatal time. B. Immunofluorescence images of the testes in WT and Chk1 mutant mice at day 16.5 (E16.5), day 17.5 (E17.5), day 18.5 (E18.5), and day 19.5 (E19.5) postnatal time; red signal from GCNA1 indicates germ cells, and green signal from SOX9 indicates supporting cells. C. Immunofluorescence images of testes from WT and Chk1 mutant mice on day 10 postnatal; co-staining with SYCP3 and STRA8 indicates germ cells in the meiotic stage, and co-staining with GCNA1 and PLZF, SOX3, or c-KIT indicates germ cells in the self-renewal, undifferentiated, or differentiated stages, respectively. D. Statistical analysis of the number of germ cells in each seminiferous tubule in testicular sections from WT and Chk1 mutant mice from day 16.5 postnatal to 6 months postnatal. E. Statistical analysis of the number of Sertoli cells in each seminiferous tubule in testicular sections from WT and Chk1 mutant mice from day 16.5 postnatal to 6 months postnatal. F–I. Statistical analysis of the number of germ cells in different developmental stages in testicular sections from WT and Chk1 mutant mice, as shown in Figure C: self-renewal stage (F), undifferentiated stage (G), differentiated stage (H), and meiotic stage (I); two-sided Student's t-test; error bars represent mean ± standard error; ns, no significant difference; ****p < 0.0001.

[0020] Figure 4The study investigated the interaction between mutant CHK1 and wild-type CHK1, which activates cell cycle checkpoint kinase activity. Specifically: A. Localization patterns of CHK1 with germ cells (GCNA1) or supporting cells (SOX9) in testicular sections from wild-type male mice on day 4 (PD4). B. Immunostaining of PD10 testicular sections with two different CHK1 antibodies, showing the relocation of CHK1 from the nucleus to the cytoplasm in mutant testes; Antibody 1: rabbit polyclonal anti-Chk1 (Sigma, SAB4500208), Antibody 2: anti-Chk1 (G-4) (Santa Cruz, sc-8408). C. Fluorescence imaging of HEK-293 cells transfected with EGFP-labeled wild-type CHK1 (EGFP-WT) or co-transfected with EGFP-WT and Flag-labeled mutant CHK1 (Flag-F441fs*16). D. Immunoprecipitation between wild-type and mutant CHK1. E. Western blot analysis of wild-type or Chk1 mutant testes to detect the expression of phosphorylated CDC25C (S216) and CDK1 (T14 and Y15). F. Volcano plots showing differentially expressed genes between wild-type and Chk1 mutant testes (fold change > 2, q < 0.01). G. Deconvolution analysis of testicular RNA-seq data revealing the proportional distribution of cell types in wild-type and Chk1 mutant testes. H. Gene ontology enrichment analysis of differentially expressed genes between wild-type and Chk1 mutant testes, involving biological processes (BP), cellular components (CC), and molecular functions (MF); Up, upregulated differentially expressed genes; down, downregulated differentially expressed genes.

[0021] Figure 5Heterozygous mutant CHK1 enhances CHK1 kinase activity by improving phase separation. Subcellular localization of A. mCherry-tagged mutant CHK1 (mCherry-F441fs16) co-stained with organelle markers; scale bar: 5 μm. B–E. Phase separation analysis of wild-type (WT) CHK1 in HEK-293T cells at low (WT_Low) or high (WT_High) expression levels: total transfected cell number (B), number of spots per cell (C), relative spot intensity (D), and average spot area (E). F–I. Phase separation characteristics of full-length CHK1 (1–476), N-terminal domain (1–265), and C-terminal domain (266–476): number of spots per cell (F), relative spot intensity (G), average spot area (H), and representative image (I). These constructs were mCherry-tagged and transfected into HEK-293T cells; scale bar: 10 μm. J–M. Comparative analysis of EGFP-labeled wild-type CHK1 (EGFP-WT) and heterozygous EGFP-labeled WT / F441fs16: total cell number (J), number of spots per cell (K), relative spot intensity (L), and average spot area (M). Two-tailed Student's t-test; error bars represent mean ± standard error; ns, not significant; p < 0.05; **p < 0.01; p < 0.001; / #### p < 0.0001. N. Constructs encoding EGFP-labeled wild-type CHK1 (EGFP-WT), EGFP-labeled F441fs16 mutant (EGFP-F441fs16), or co-transfection of both (EGFP-WT / F441fs*16) were transfected into HEK-293T cells. After transfection, each group of cells was treated with 1,6-hexanediol for specified times (0, 15, 60 minutes, and 24 hours). Then, the kinase activity of CHK1 in each group was assessed by Western blot analysis.

[0022] Figure 6 Transcriptome analysis of 2-cell embryos derived from sperm carrying the CHK1 p.F441fs16 mutation. A. Schematic diagram of the CHK1 truncated mutation (p.F441fs16) within its functional domains. B. Patriarchy of infertile families carrying the CHK1 mutation and exhibiting a zygotic arrest phenotype (PMID: 33953335). C. Pearson correlation heatmap comparing the transcriptomes of 2-cell embryos fertilized by wild-type (WT) and mutant (Mut) sperm. D–E. Gene set enrichment analysis (GSEA) showed significant downregulation of genes related to endothelial cell proliferation (GO:0001935) and cytoskeleton-dependent cytokinesis (GO:0061640).

[0023] Figure 7Chk1 mutant mice exhibited seminiferous tubule vacuolation despite normal hormone levels and maintenance of some sperm parameters. A–C. Serum hormone levels in wild-type (WT) and Chk1 mutant (F441fs16⁺ / ᵗʳ) male mice: testosterone (A), luteinizing hormone (B), estradiol (C). D. Hematoxylin-eosin staining of sperm from WT or Chk1 mutant male mice; scale bar: 100 μm. E–H. Computer-aided semen analysis (CASA) of sperm motility: mean path velocity (E), linear velocity (F), linearity (G), and lateral swing amplitude (H). I. Figure 2 Quantitative analysis of vacuolated seminiferous tubules in K-type WT and Chk1 mutant testes. Data are expressed as mean ± standard error; two-tailed Student's t-test; ns, not significant; ** p < 0.01, *** p < 0.0001.

[0024] Figure 8 In the Chk1 mutant testes, only a small number of germ cells are capable of spermatogenesis. Immunofluorescence images of the testes of wild-type (WT) and Chk1 mutant (F441fs*16⁺ / ᵗʳ) mice at day 10 (PD10) and day 2 (PD2) after birth are shown. The red signal of GCNA1 indicates germ cells, and the green signal of SOX9 indicates supporting cells. Immunofluorescence analysis of germ cell subsets in the testes of WT and Chk1 mutant mice are shown in C–F. Co-staining with SYCP3 and STRA8 indicates germ cells in the meiotic stage (D), and co-staining with GCNA1 and PLZF (C), SOX3 (E), or c-KIT (F) indicates germ cells in the self-renewal, undifferentiated, or differentiated stages, respectively.

[0025] Figure 9 Expression and distribution of CHK1 in mouse testes. A. CHK1 expression levels at different stages of spermatogenesis (data from the GamesOmics database). B. Immunofluorescence staining of CHK1 showed its primary localization in the cell nucleus. C. Molecular docking of wild-type CHK1 protein (WT) and mutant CHK1 protein (p.F441fs*16) using AlphaFold3. D–G. Batch RNA-seq analysis of testicular tissues from 3-month-old Chk1 mutant (Mut) and wild-type (WT) mice. Pearson correlation analysis confirmed high reproducibility among biological replicates (C). Principal component analysis showed significant separation between groups (D). Heatmap of differentially expressed genes (DEGs) between Mut and WT testes (E). Cell deconvolution analysis (proportional heatmap) showed a significant reduction in spermatogonia and spermatocytes in the Mut testes (F).

[0026] Figure 10The study investigated the reduction of CHK1 expression in Chk1-mutant testes and transfected cells. Specifically: A. Analysis of Chk1 mRNA levels in wild-type (WT) and Chk1-mutant (F441fs16⁺ / ᵗʳ) testes using RNA-seq. B. Verification of Chk1 mRNA levels in WT and Chk1-mutant testes using real-time quantitative PCR. C. Western blot analysis of CHK1 protein expression in WT and Chk1-mutant testes. D. Decreased CHK1 expression in HEK-293T cells transfected with the F441fs16 mutant plasmid compared to WT CHK1. CPT represents camptothecin. E. Western blot analysis of phosphorylated CHK1 (S345) levels in HEK-293T cells transfected with wild-type or mutant CHK1 after treatment with a topoisomerase II inhibitor (etoposide, ETO) or a proteasome inhibitor (MG132) for 18 hours.

[0027] Figure 11 CHK1 possesses the ability to undergo phase separation. Specifically: A. Immunofluorescence staining of endogenous CHK1 in various cell lines using a CHK1-specific antibody (Abcam, ab196520). B. Verification of CHK1 localization patterns in various cell lines using another independent antibody (Sigma, SAB4500208). C–D. Computational phase separation propensity analysis of CHK1: catGRANULE predicts phase separation potential (C) and PONDR score highlights intrinsically disordered regions (D). E. Live-cell imaging of HEK-293 cells, capturing the fusion process of mutant CHK1 (EGFP_F441fs16) aggregates. F. Photobleaching fluorescence recovery (FRAP) showing signal recovery of mutant CHK1 (mCherry_F441fs*16) after photobleaching. Detailed Implementation

[0028] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.

[0029] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0030] Experimental study on the relationship between CHK1 gain-of-function mutations and spermatogenesis and male fertility (1) C-terminal truncation of heterozygous CHK1 leads to severe male infertility in mice. Our previous work found that mutations in the C-terminal regulatory domain of CHK1 are a cause of female infertility due to failed cleavage of fertilized eggs. Among these, the frameshift mutation (GenBank: NM_0012374; c.1323delC, p.F441fs*16) prematurely terminates the C-terminal domain after a 15-amino acid frameshift, resulting in a 21-amino acid truncation, exhibiting the most severe phenotype among all identified mutations. This de novo mutation (absent in the parental genome) showed the strongest overactivation of CHK1 kinase activity, and the corresponding previous research is detailed in CN 116457457 A. This experiment investigates whether kinase-activating CHK1 mutations also affect male fertility.

[0031] First, a heterozygous knock-in mouse model (p.F441fs*16+ / tr) was constructed using CRISPR-Cas9, and the fertility of male mice was assessed. Six months after mating with healthy reproductive-age female mice, we found a significant decrease in fertility in the Chk1 mutant mice, manifested as a significant reduction in total pups, total litter size, and average litter size compared to age-matched wild-type mice. Interestingly, of the six heterozygous Chk1 mutant mice monitored in mating cages, four (2 / 3) were completely infertile, while only two (1 / 3) showed severely reduced fertility. To further characterize this phenotype, we performed in vitro fertilization using sperm from the mutant mice. Consistent with in vivo observations, oocytes fertilized with mutant sperm showed significantly impaired fertilization rates and subsequent embryonic development.

[0032] To assess the developmental capacity of embryos fertilized by mutant sperm, we performed RNA-seq analysis on late 2-cell stage embryos derived from mutant sperm. Although Pearson correlation analysis showed no global transcriptional differences between wild-type and mutant embryos, we identified 1,032 differentially expressed genes (|log2FC|>1, P<0.05), of which 478 were upregulated and 554 were downregulated. Gene ontology analysis of downregulated DEGs showed significant enrichment in cytokine processes and cell proliferation pathways, and gene set enrichment analysis further supported this result. The fertility assessment results of Chk1 mutant mice are as follows: Figure 1 As shown. In summary, Chk1 mutant mice exhibited severely reduced fertility, suggesting that the p.F441fs*16 mutation, which disrupts the C-terminal domain of CHK1, is a potential factor for male infertility.

[0033] (2) Chk1 mutant male mice exhibited non-obstructive azoospermia or oligospermia. To investigate the underlying causes of the observed fertility impairment, we first assessed the systemic and reproductive parameters of male Chk1 mutant mice. Although the mutant and wild-type male mice had comparable body weights, the Chk1 mutant mice exhibited significant testicular atrophy and a significantly reduced testicular-to-body weight ratio. Interestingly, despite these morphological defects, serum testosterone, luteinizing hormone, and estradiol levels remained unchanged. Furthermore, histological analysis of sperm using hematoxylin-eosin staining revealed no significant morphological abnormalities in the mutant male mice.

[0034] To further investigate the causes of reduced fertility, we assessed the quantity and quality of sperm from the epididymal tail. HE staining histological examination revealed a significant reduction in sperm count in the epididymal tail of mutant mice. Furthermore, a comprehensive sperm analysis was performed using computer-aided sperm analysis. Evaluation of sperm from 8-week-old mutant male mice revealed significant defects: one-third of the mice had undetectable sperm by CASA, while the remaining two-thirds had significantly reduced sperm counts compared to wild-type mice. Detailed motility analysis showed significantly impaired key parameters, including total motile sperm count, curve velocity, and whiplash frequency. However, other motility parameters such as mean path velocity, linear velocity, linearity, and lateral swing amplitude were comparable to wild-type mice. These findings suggest that mice carrying the Chk1 mutation have a significantly reduced sperm count accompanied by mild impaired sperm motility. Consistent with these observations, HE staining of the testes of Chk1 mutant mice from day 4 to 6 months of age showed a significant reduction in sperm and marked vacuolated seminiferous tubules, indicating a phenotype consistent with non-obstructive azoospermia or oligospermia. The corresponding results are illustrated in the figures below. Figure 2 As shown.

[0035] (3) **Chk1 mutant testes have a large number of depleted germ cells during the embryonic period. To further investigate the root causes of abnormal sperm development, we used GCNA1 (a germ cell marker) and SOX9 (a Sertoli cell marker) to perform immunofluorescence staining on the testes of Chk1 mutant mice of different ages. From day 4 to 6 months after birth, the number of germ cells in the testes of mutant mice was significantly reduced, with only a few seminiferous tubules containing these cells, while the number of Sertoli cells remained unchanged. Interestingly, examination of the testes at different stages of embryonic development, from 16.5 to 19.5 days, showed a similar trend: a significant reduction in the number of germ cells, while the level of Sertoli cells remained normal.

[0036] Despite a significant reduction in sperm count, approximately one-third of the Chk1 mutant mice retained residual fertility. To investigate the self-renewal and differentiation potential of the mutant germ cells, we co-stained GCNA1 with PLZF (a marker of undifferentiated spermatogonia), SOX3 (a marker of progenitor spermatogonia), or c-KIT (a marker of differentiated spermatogonia). As expected, the germ cell population in the mutant testes was significantly reduced at day 10 postnatal, and these markers were expressed at extremely low levels. Further examination of the residual germ cells in the mutant testes revealed positive signals for PLZF, SOX3, or c-KIT. Subsequently, we co-stained germ cells at day 14 postnatal for SYCP3 and STRA8 (a marker of meiosis). Encouragingly, the residual germ cells also expressed these meiotic markers. The corresponding results are shown in the attached figures. Figure 3 As shown.

[0037] (4) Mutant CHK1 isolates wild-type CHK1 in the cytoplasm and amplifies CHK1 kinase activity. CHK1 expression levels are high during the early stages of spermatogenesis (from spermatogonial stem cells to leptotene spermatocytes). Immunofluorescence staining using germ cell and Sertoli cell markers GCNA1 and SOX9 revealed that CHK1 primarily co-localizes with germ cells and minimally with Sertoli cells in the testes of wild-type mice, highlighting its crucial role in male germ cells. Subsequently, using two different CHK1 antibodies, we found that CHK1 is primarily localized in the nuclei of wild-type germ cells, while in the testes of Chk1 mutant mice, nuclear localization of CHK1 is completely absent despite the preservation of the wild-type copy. This mislocalization phenotype was reproduced in HEK-293 cells co-transfected with wild-type and mutant CHK1 constructs.

[0038] We hypothesized that the mutant CHK1 protein might interact with the wild-type protein, isolating it in the cytoplasm. Molecular modeling of wild-type and mutant CHK1 proteins using AlphaFold 3 revealed their interaction. This prediction was validated by immunoprecipitation experiments in HEK-293T cells, demonstrating the binding between wild-type and mutant CHK1. Notably, the nucleoplasmic distribution of CHK1 is closely related to its checkpoint kinase activity. Consistently, Western blot analysis of mutant mouse testicular tissue showed increased phosphorylation levels of CHK1 downstream targets CDC25C and CDK1, indicating enhanced CHK1 kinase activity in the mutant testes, which may contribute to cell cycle arrest.

[0039] To further elucidate the molecular mechanisms underlying the observed phenotypes, we performed batch RNA sequencing on testicular tissues from 3-month-old Chk1 mutant mice and wild-type mice. Pearson correlation analysis showed high reproducibility between biological replicates, and principal component analysis revealed significant separation between the two groups. 9,609 differentially expressed genes (|Fold change| > 2, P < 0.01) were identified, including 5,014 upregulated transcripts and 4,595 downregulated transcripts. Cell deconvolution analysis showed a significant reduction in spermatogonia and spermatocytes in the mutant testes, consistent with previous immunofluorescence results of germ cell depletion. Gene ontology enrichment analysis of these DEGs revealed alterations in biological processes such as cell differentiation, cell cycle regulation, signal transduction, and sperm motility. Overall, these results suggest that the CHK1 mutation may isolate the wild-type CHK1 protein in the germ cell cytoplasm, enhancing the overall activity of cell cycle checkpoint kinases and leading to germ cell developmental arrest. The corresponding results are illustrated in the figure below. Figure 4 As shown.

[0040] (5) CHK1 mutations lead to overactivity of checkpoint kinases by enhancing phase separation. Quantitative analysis revealed a significant decrease in Chk1 mRNA levels in mutant testes, along with reduced CHK1 protein expression in heterozygous mutant testes and HEK-293T cells expressing the mutant construct. Notably, treatment with the proteasome inhibitor MG132 increased mutant CHK1 protein levels, indicating its rapid degradation via the ubiquitin-proteasome pathway. This raises the question: how can an unstable mutant protein dominantly isolate wild-type CHK1 and amplify kinase activity? New evidence suggests phase segregation dysregulation in disease-associated mutations, but its role in spermatogenesis remains poorly understood. We observed granular CHK1 aggregates in germ cell nuclei and in various cell lines, including HEK-293, HTR-8, and HUVEC. Computational predictions (catGRANULE and PONDR) indicated a phase-segregating tendency in CHK1, with a distinctly inherent disordered region in its C-terminal domain. High-resolution imaging then confirmed that CHK1 aggregates differ from typical membranous organelles such as vesicles, Golgi apparatus, endosomes, lysosomes, and stress granules. In most cases, the phase-segregating tendency of proteins increased with increasing concentration. Consistently, dose-dependent experiments showed that while the intensity of spot immunofluorescence of mCherry-CHK1 remained unchanged, higher transfection concentrations increased the number and area of ​​spots, further revealing the phase-segregating tendency of CHK1. Furthermore, super-resolution microscopy captured the spontaneous fusion of mutant CHK1 aggregates, while fluorescence recovery experiments after photobleaching confirmed their liquid-like nature.

[0041] When comparing the C-terminal domain (amino acids 266-476) and the kinase domain (amino acids 1-265) using mCherry-labeled constructs in HEK-293T cells, the latter showed a stronger ability to form aggregates, suggesting that the kinase domain may play a central role in mediating the phase-separation ability of CHK1. Then, to compare the phase-separation ability of wild-type CHK1 and heterozygous mutant CHK1, we transfected HEK-293T cells with wild-type CHK1 labeled with EGFP alone (labeled EGFP_WT), or co-transfected wild-type CHK1 labeled with EGFP and F441fs*16 labeled with EGFP (labeled EGFP_WT / F441fs*16). We observed that heterozygous CHK1 formed smaller but more numerous aggregates, indicating an enhanced tendency for phase separation despite its proteolytic instability.

[0042] To test whether the hyperactivation of the mutant CHK1 kinase depends on phase-separation properties, we pharmacologically disrupted phase-separation aggregates using 1,6-hexanediol (a fatty alcohol known to disrupt weak hydrophobic interactions in phase-separated aggregates). HEK-293T cells were transfected with constructs encoding EGFP-labeled wild-type CHK1 (EGFP-WT), the EGFP-labeled F441fs*16 mutant (EGFP-F441fs*16), or co-transfected with both. Following transfection, each group of cells was treated with 1,6-hexanediol for different time periods (0, 15, 60 min, and 24 h). Notably, in the CHK1 mutant group, kinase activity, indicated by p.CDC25C(S216) levels, was significantly reduced after phase-separation inhibition, especially at 24 h. This suggests that the enhanced kinase activity of the mutant CHK1 requires its enhanced phase-separation ability. The corresponding results are illustrated in the figure below. Figure 5 As shown.

[0043] Figures showing some of the results from the above study are shown below. Figures 6 to 11 As shown.

[0044] Furthermore, analysis of multiple datasets showed significantly elevated CHK1 expression in spermatogonia and early spermatocytes of NOA patients, further emphasizing its crucial functional role in spermatogenesis and establishing its status as a potential therapeutic target. The corresponding results are shown in Table 1.

[0045] Table 1 CHK1 expression data from male infertility patients in the SMARTdb database

[0046] The antibodies used in this study are shown in Table 2.

[0047] Table 2

[0048] The primers used in this study are shown in Table 3, as shown in SEQ ID NO.9 to SEQ ID NO.16.

[0049] Table 3

[0050] (6) Discussion CHK1 is a recognized cancer risk gene, and its dysfunction is associated with genomic instability and tumorigenesis. However, mounting evidence suggests a crucial but underrecognized role in human reproduction. Our previous work identified pathogenic CHK1 mutations in female infertility, while this study significantly expands our clinical and mechanistic understanding of the role of CHK1 in human fertility by demonstrating that heterozygous truncated CHK1 mutations lead to male infertility, particularly non-obstructive azoospermia and oligospermia. Importantly, we discovered a novel pathophysiological mechanism: abnormal phase segregation of mutant CHK1 leads to condensation, isolating wild-type CHK1, disrupting its subcellular localization, and overactivating CHK1 kinase activity, ultimately resulting in spermatogenesis failure. These findings not only establish CHK1 as a genetic diagnostic marker for male infertility but also reveal that phase segregation is a key driver of its pathogenic effects, opening new avenues for therapeutic intervention.

[0051] Our previous work found that CHK1 missense mutations (such as p.R379Q and p.R442Q) can cause female infertility, but can be transmitted by fertile men. In contrast, the truncated mutation (p.F441fs*16) described in this invention leads to severe spermatogenesis failure. Phenotypic differences may depend on the specific effects of different mutation types on structure and function. Truncating may cause more severe protein errors compared to missense mutations.

[0052] The above embodiments are provided to those skilled in the art to fully disclose and describe how the claimed implementations can be carried out and used, and are not intended to limit the scope of the disclosure herein. Modifications that will be obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. The application of C-terminal truncation mutations in CHK1 in regulating male sterility, characterized by: Male sterility is caused by the presence of a heterozygous genotype with a C-terminal truncated mutation in the Chk1 gene of male mammals; or / and: male sterility is caused by the expression of a C-terminal truncated mutation in the Chk1 gene of male mammals. The C-terminal truncation mutation refers to a mutation in the Chk1 gene that causes premature termination of the C-terminal domain of the CHK1 protein it encodes, disrupting the conserved sequence at the C-terminus of the CHK1 protein and resulting in C-terminal truncation.

2. The application of the C-terminal truncated mutation of CHK1 according to claim 1 in regulating male infertility, characterized in that: The mammal species are selected from humans or mice.

3. The application of the C-terminal truncated mutation of CHK1 according to claim 2 in regulating male infertility, characterized in that: The C-terminal truncated mutation is a heterozygous frameshift mutation p.F441fs*16; the nucleotide coding sequence of the heterozygous frameshift mutation p.F441fs*16 of the human Chk1 gene is shown in SEQ ID NO.3, the amino acid sequence of the heterozygous frameshift mutation p.F441fs*16 of the human CHK1 gene is shown in SEQ ID NO.4; the nucleotide coding sequence of the heterozygous frameshift mutation p.F441fs*16 of the mouse Chk1 gene is shown in SEQ ID NO.7, and the amino acid sequence of the heterozygous frameshift mutation p.F441fs*16 of the mouse CHK1 gene is shown in SEQ ID NO.

8.

4. The use of C-terminal truncated mutations of the Chk1 gene as a diagnostic marker for male infertility in mammals, or their application in the preparation of screening products for infertility in mammals, characterized in that; The C-terminal truncation mutation refers to a mutation in the Chk1 gene that causes premature termination of the C-terminal domain of the CHK1 protein it encodes, disrupting the conserved sequence at the C-terminus of the CHK1 protein and resulting in C-terminal truncation.

5. The application according to claim 4, characterized in that; The mammal species is selected from humans or mice; the C-terminal truncated mutation is a heterozygous frameshift mutation p.F441fs*16; the nucleotide coding sequence of the heterozygous frameshift mutation p.F441fs*16 of the human Chk1 gene is shown in SEQ ID NO.3, the amino acid sequence of the heterozygous frameshift mutation p.F441fs*16 of the human CHK1 gene is shown in SEQ ID NO.4; the nucleotide coding sequence of the heterozygous frameshift mutation p.F441fs*16 of the mouse Chk1 gene is shown in SEQ ID NO.7, and the amino acid sequence of the heterozygous frameshift mutation p.F441fs*16 of the mouse CHK1 gene is shown in SEQ ID NO.

8.

6. The application of specific primers or kits for detecting C-terminal truncated mutations in the Chk1 gene in the preparation of mammalian infertility screening products, characterized in that: The C-terminal truncation mutation refers to a mutation in the Chk1 gene that causes premature termination of the C-terminal domain of the CHK1 protein it encodes, disrupting the conserved sequence at the C-terminus of the CHK1 protein and resulting in C-terminal truncation.

7. The application according to claim 6, characterized in that: The mammalian species are selected from humans or mice; the C-terminal truncated mutation is a heterozygous frameshift mutation p.F441fs*16. The nucleotide coding sequence of the heterozygous frameshift mutation p.F441fs*16 of the human Chk1 gene is shown in SEQ ID NO.3, and the amino acid sequence of the heterozygous frameshift mutation p.F441fs*16 of the human CHK1 gene is shown in SEQ ID NO.4; the nucleotide coding sequence of the heterozygous frameshift mutation p.F441fs*16 of the mouse Chk1 gene is shown in SEQ ID NO.7, and the amino acid sequence of the heterozygous frameshift mutation p.F441fs*16 of the mouse CHK1 gene is shown in SEQ ID NO.

8.

8. The application according to claim 6 or 7, characterized in that: In practical applications, the Chk1 gene is used as the detection target to detect the genotype of the Chk1 gene in male mammals. If it is a heterozygous genotype containing a C-terminal truncated mutation, it indicates that the individual has a risk of infertility.

9. The application of C-terminal truncated mutant CHK1 in the preparation of mammalian male infertility agents, characterized in that: The mammalian species is selected from humans or mice; the C-terminal truncated mutation is a heterozygous frameshift mutation p.F441fs*16; the nucleotide coding sequence of the heterozygous frameshift mutation p.F441fs*16 of the human Chk1 gene is shown in SEQ ID NO.3, the amino acid sequence of the heterozygous frameshift mutation p.F441fs*16 of the human CHK1 gene is shown in SEQ ID NO.4; the nucleotide coding sequence of the heterozygous frameshift mutation p.F441fs*16 of the mouse Chk1 gene is shown in SEQ ID NO.7, and the amino acid sequence of the heterozygous frameshift mutation p.F441fs*16 of the mouse CHK1 gene is shown in SEQ ID NO.

8.

10. The application of a male mouse model with a C-terminal truncated mutation in the Chk1 gene in the screening of drugs for treating male infertility, characterized by: The C-terminal truncated mutation is a heterozygous frameshift mutation p.F441fs*16; the nucleotide coding sequence of the heterozygous frameshift mutation p.F441fs*16 of the mouse Chk1 gene is shown in SEQ ID NO.7, and the amino acid sequence of the heterozygous frameshift mutation p.F441fs*16 of the mouse CHK1 gene is shown in SEQ ID NO.8.

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