Use of phosphoprotein phosphatase related factors for diagnosis and treatment of fertility defects

Abstract

The present invention provides methods for diagnosing or predicting a fertility defect, such as infertility, in a mammalian or human male subject by detecting PPP6R3 or a combination of PPP6R3, EIF3C and EIF4G1. The invention also provides kits and instruments for diagnosing or predicting fertility defects, such as infertility disorders, in mammalian male individuals by detecting said proteins. According to the application, it is found and proved that PPP6R3 is crucial to spermatogenesis for the first time, S39 of EIF3C and S1217 of EIF4G1 are targets for PP6 to regulate spermatogonial differentiation, a new insight is provided for the relation between phosphorylation participated by phosphoprotein phosphatase and spermatogonial differentiation, and deep research on the relation is beneficial to development of a male infertility treatment method.

Description

Technical Field

[0001] The present invention relates to the field of diseases and diagnosis, and in particular to a method and a kit for diagnosing or treating male fertility defects in mammals, especially humans, using phosphoprotein phosphatase-related factors. Background Art

[0002] Two key transitions occur during the highly coordinated and complex process of mammalian spermatogenesis: spermatogonial differentiation and meiotic initiation. The former marks the exit of spermatogonia from the stem cell pool, while the latter marks the end of mitosis and the beginning of meiosis. Spermatogonial stem cells (SSCs) are the foundation of ongoing mammalian spermatogenesis. SSCs not only self-renew to produce daughter stem cells but also give rise to spermatogonial cells (SPCs), which undergo spermatogonial differentiation and meiotic initiation upon stimulation by retinoic acid (RA). In the absence of RA, RA receptors (RARs) heterodimerize with retinoid X receptors (RXRs) and bind to RA-responsive elements. These interactions with co-repressors, such as nuclear receptor corepressors (NCoRs) and silencing mediator of RA and thyroid hormone receptors (SMRTs), further maintain compacted and repressed chromatin, ultimately leading to the silencing of target gene transcription. Conversely, RA binding to RARs alters RAR conformation, leading to the release of the RAR / RXR repressor protein complex and the recruitment of coactivators, including histone acetylases and methyltransferases. At this time, chromatin accessibility facilitates the transcription of target genes such as Stra8 and Kit, promoting spermatogonial differentiation. Accurate gene transcription and protein expression are the foundation and prerequisite for spermatogonial differentiation and the initiation of meiosis, and are regulated by multiple factors such as hormones, growth factors, and epigenetic modifications.

[0003] Phosphorylation is one of the most common and reversible post-translational modifications (PTMs). It participates in the regulation of cell proliferation, differentiation, apoptosis, metabolism and tumorigenesis by changing the conformation, localization, stability and interaction of proteins with other biological molecules. Eukaryotic protein phosphatases can be divided into protein phosphatases (PPP), Mg-phosphatases, and Mg-phosphatases according to substrate specificity, catalytic activity and inhibitor sensitivity. 2+ / Mn 2+The PPPs are involved in the regulation of meiosis, including protein phosphatases (PPMs), aspartate-based protein phosphatases, and phosphotyrosine phosphatases (PTPs). Over 80% of protein phosphatase activity in eukaryotic cells is regulated by the PPPs, which include PP1, PP2A, PP2B, PP4, PP5, PP6, and PP7. Studies have shown that PP2A, PP4, and PP6 are all involved in the regulation of meiosis. PP6 consists of a catalytic subunit (PPP6C), a regulatory subunit (protein phosphatase 6 regulatory subunit, PPP6R, also known as SAPS), and a scaffolding subunit. Using Stra8-cre mice, Lei et al. found that germline-specific deletion of PPP6C leads to male infertility, with spermatocytes blocked during development and accompanied by defects in double-strand break (DSB) repair and crossover formation. Furthermore, Sertoli cell-specific deletion of PPP6C in Amh-cre mice also leads to male infertility, due to the loss of mature sperm mediated by hyperphosphorylation of β-catenin. These findings indicate that PPP6C is essential for spermatogenesis and male fertility.

[0004] However, PPP6C activity is regulated by PPP6Rs by restricting PP6 substrate specificity and determining PP6C's intracellular localization. Three conserved PPP6Rs are known in humans, mice, and rats: PPP6R1, PPP6R2, and PPP6R3. Differences in their protein structure, expression patterns in cells or tissues, and substrate recruitment determine the variation or specificity of PPP6C activity and its response to specific signals. Although PPP6R1 and PPP6R3 are more closely related in sequence than they are to PPP6R2, they exhibit distinct functional specificities. For example, one study demonstrated that knockdown of PPP6R1, but not PPP6R3, promoted the degradation of IκBε in response to TNFα stimulation. IκBε may be a specific substrate of the PPP6R1 / PP6 holoenzyme. Furthermore, Yang et al. found that PPP6R3, but not PPP6R1 or PPP6R2, is a negative regulator of AMPK. A high-fat diet induces upregulation of PPP6R3, which recruits PPP6C to inactivate phosphorylated AMPK, leading to metabolic disorders. Inhibition of PPP6R3 may be a potential therapeutic strategy for the treatment of metabolic syndrome. These studies emphasize that PPP6R plays a key role in signal transduction and maintaining metabolic homeostasis. However, there are currently no reports on whether PPP6R is involved in spermatogenesis.

[0005] Although phosphorylation plays an important role in reproductive biology, including spermatogenesis, the field still needs to study the exact function of phosphorylation in male reproduction in mammals, including humans, and to gain a better understanding of the regulation of spermatogenesis, so as to detect and predict male reproductive disorders. Summary of the Invention

[0006] The inventors discovered and demonstrated for the first time that knockout of protein phosphatase 6 regulatory subunit 3 (PPP6R3) leads to translation failure and complete infertility during spermatogonial differentiation in male mice. The loss of PPP6R3 inhibits the translation, rather than transcription, of multiple known spermatogonial differentiation regulators. In addition, the translation initiation factors EIF3C and EIF4G1 are KIT-mediated regulatory factors. + A specific substrate of the PPP6R3 / PP6 holoenzyme in spermatogonia. After PPP6R3 is lost, the phosphorylation levels of EIF3CS39 and EIF4G1S1217 increase, promoting their degradation, and ultimately leading to translation failure during spermatogonial differentiation. The applicant's discovery provides new and in-depth insights into the relationship between phosphorylation involving protein phosphatase (PPP) and spermatogonial differentiation. The applicant thus provides a method for diagnosing male fertility defects in mammals, including humans. At the same time, the inventor also provides a method for diagnosing or treating male fertility defects in mammals, especially humans.

[0007] Specifically, the present invention provides a method for diagnosing or prognosing fertility defects, such as infertility, in male individuals of mammals and humans, comprising detecting the gene or protein of protein phosphatase 6 regulatory subunit 3 (PPP6R3) in the male individual. In another aspect, the method comprises detecting the following combination of genes or proteins: PPP6R3, EIF3C, and EIF4G1.

[0008] In the present invention, the infertility disorder of the male individual includes but is not limited to teratozoospermia, azoospermia, oligospermia, asthenozoospermia, asthenozoospermia, and the like.

[0009] In the present invention, the mammal can be any mammal, including but not limited to rodents, canines, felines, equines, ovines, bovines, porcines and primates. Generally speaking, mammals also include humans.

[0010] Eukaryotic protein phosphatases (PPPs) include PP1, PP2A, PP2B, PP4, PP5, PP6, and PP7. PP6 is composed of a catalytic subunit (PPP6C), a protein phosphatase 6 regulatory subunit (PPP6R, also known as SAPS), and a scaffolding subunit. Three conserved PPP6Rs are known in humans, mice, and rats: PPP6R1, PPP6R2, and PPP6R3 (i.e., protein phosphatase 6 regulatory subunit 3). The Ppp6r3 gene encoding PPP6R3 has a conserved coding sequence across various mammals. The Gene ID for human Ppp6r3 is 55291. The Gene ID for the mouse Ppp6r3 gene is 52036.

[0011] The present invention also found that eukaryotic translation initiation factor 3 subunit C (eukaryotic translation initiation factor 3 subunit C, EIF3C) and eukaryotic translation initiation factor 4 gamma 1 (eukaryotic translation initiation factor 4 gamma 1, EIF4G1) are specific substrates of PPP6R3 in the phosphorylation involved in PPP6R3. Furthermore, it was found that the phosphorylation levels of EIF3C at the S39 site and EIF4G1 at the S1217 site were significantly upregulated after the loss of PPP6R3. The present invention proves that S39 of EIF3C and S1217 of EIF4G1 are targets of PP6 in regulating spermatogonial differentiation, and that the sites of these two proteins can be used as sites for diagnosing fertility defects or infertility in different human sexes. In related applications (including methods and related kits), abnormalities in S39 of EIF3C and S1217 of EIF4G1 of the test individual can be detected, including comparing the subject's amino acid situation with that of normal people in a general or specific group. If significant changes are found, it is determined that an abnormality exists.

[0012] The genes encoding EIF3C and EIF4G1 have conserved coding sequences across mammals. The GeneID for human EIF3C is 8663. The GeneID for mouse EIF3C is 56347. The GeneID for human EIF4G1 is 1981. The GeneID for mouse EIF4G1 is 208643.

[0013] In one aspect of the present invention, the present invention provides a method and kit for diagnosing fertility defects, including steps and reagents for detecting mutations in amino acids encoding EIF3C and EIF4G1. As previously described, the present invention demonstrates that S39 of EIF3C and S1217 of EIF4G1 are targets of PP6 in regulating spermatogonial differentiation, and that the sites of these two proteins can be used as sites for diagnosing fertility defects or infertility in different human genders. In related applications (including methods and related kits), abnormalities in S39 of EIF3C and S1217 of EIF4G1 can be detected in a subject, including comparing the nucleic acid encoding the amino acids in the subject's genes with those in a general or specific population. If significant changes are found, an abnormality is determined.

[0014] In one aspect of the present invention, the method for diagnosing fertility defects provided by the present invention includes the step of detecting the gene of the target protein.

[0015] The gene detection method used in the present invention is a method commonly used for gene detection in the art and is not particularly limited. Examples thereof include mass spectrometry, microarray methods, sequencing methods, and detection methods using base sequence amplification methods such as PCR (polymerase chain reaction).

[0016] In addition, detection of PCR products obtained when PCR is performed using primers specific for each gene can be performed by any method commonly used for detecting and quantifying PCR products. For example, detection can be performed by electrophoresis, by real-time PCR using a fluorescent intercalator such as SYBR Green, or by single molecule fluorescence analysis.

[0017] The polynucleotide that can be used as the primer or probe for detecting gene is not particularly limited as long as it can be a polynucleotide that hybridizes with the partial region comprising this gene or its complementary strand. The design of polynucleotide can adopt any one of the well-known methods in this technical field to carry out. For example, it is possible to design simply by utilizing known genome sequence data and a universal primer design tool. As this primer design tool, for example, Primer3 that can be utilized on the Internet is arranged. In addition, known genome sequence data can usually be obtained in the NCBI as an international base sequence database.

[0018] In one aspect of the present invention, the method for diagnosing fertility defects provided by the present invention includes the step of detecting PPP6R3 and / or EIF3C and EIF4G1 proteins, such as detecting the expression of the proteins (including whether they exist, and whether there is a decrease in expression relative to normal or standard) or their activity.

[0019] In another aspect of the present invention, the method includes the step of detecting the expression of the target protein by immunoassay. For example, expression can be detected by immunofluorescence or Western blotting using an antibody that specifically recognizes the protein. Another example is detecting the presence or amount of mRNA for the protein, such as by RT-PCR to detect the amount of mRNA encoding the target protein in the sample.

[0020] In yet another aspect of the present invention, the method comprises the step of determining the activity of the protein.

[0021] The present invention also provides a kit or apparatus (including a gene chip) for diagnosing or predicting fertility defects such as infertility in mammalian individuals, which includes a reagent for detecting a target gene in the subject.

[0022] In another aspect of the present invention, the kit or instrument includes reagents for detecting the expression or activity of the target protein. For example, the kit or instrument includes reagents for immunoassays, such as reagents for detecting expression by ELISA or Western blot using antibodies that specifically recognize the protein. For another example, the kit or instrument includes reagents for detecting protein expression by detecting the presence or amount of mRNA for the protein, such as reagents for detecting the amount of mRNA encoding the target protein in a sample by RT-PCR.

[0023] In yet another aspect of the present invention, the kit or apparatus includes reagents for detecting genes.

[0024] In one aspect of the present invention, in the above-mentioned methods, kits or instruments of the present invention, the reagents for detecting genetic markers in samples include primers or probes for detecting genes, or reagents for detecting proteins, such as specific antibodies or reagents for detecting the mRNA of the protein.

[0025] The present invention also provides the use of a reagent for detecting PPP6R3 or the following combination: PPP6R3, EIF3CS39, and EIF4G1S1217, in the preparation of a kit or apparatus for diagnosing or prognosing fertility defects, such as infertility, in a mammalian individual, as described above. The reagent for detecting genetic markers includes primers or probes for detecting genes, or reagents for detecting proteins, such as specific antibodies or reagents for detecting mRNA of the proteins.

[0026] The present invention also provides a method for treating or preventing fertility defects in a mammalian individual, for example, comprising treating an individual having a defect, such as a mutation, in PPP6R3 or a combination of PPP6R3, EIF3CS39, and EIF4G1S1217.

[0027] Throughout this document, protein symbols are not italicized and are capitalized; gene symbols are italicized. For example, PPP6R3 is a protein, and the gene encoding it is written as Ppp6r3. Sometimes, protein symbols are not italicized throughout this document. For example, sometimes "Ppp6r3" is used herein to refer to the PPP6R3 protein. Sometimes, gene symbols are not italicized throughout this document. For example, sometimes "PPP6R3" or "PPP6R3 gene" is used herein to refer to the gene Ppp6r3 that encodes the PPP6R3 protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 PPP6R3 is highly expressed in differentiating spermatogonia and early spermatocytes. (A) Protein abundance of PPP6R1, PPP6R2, PPP6R3, and PPP6C in different germ cell populations based on previously reported quantitative proteomics data from mouse testis. (B) Western blotting of PPP6R3 in wild-type mouse testis at the indicated times. (C) Immunostaining of PPP6R3 and KIT in testicular sections from P12 wild-type mice. Scale bar, 15 μm. (D) Immunostaining of PPP6R3 and SYCP3 in testicular tissue from P12 wild-type mice. Scale bar, 15 μm. (E) Immunostaining of PPP6R3 and PLZF in testicular tissue from P12 wild-type mice. Scale bar, 15 μm. (F) Immunostaining of PPP6R3 and SOX9 in testicular sections from P12 wild-type mice. Scale bar, 15 μm. (G) Quantification of PPP6R3 fluorescence intensity in PLZF+ spermatogonia, KIT+ spermatogonia, early spermatocytes, and SOX9+ Sertoli cells. ***p < 0.001. n, not statistically significant. Data are expressed as mean ± SD. N = 20 technical replicates. (H) Western blot detection of PPP6R3 in THY1+ or KIT+ spermatogonia from P12 wild-type mice and in spermatocytes purified using a cell purification kit or fluorescence-activated cell sorting (FACS). *p < 0.05, ***p < 0.001. Data are expressed as mean ± SD. N = 3 biological replicates.

[0029] Figure 2Germline-specific deletion of PPP6R3 results in male infertility and failure of spermatogonial differentiation. (A) Comparison of testis size between adult wild-type and Ppp6r3-cKO mice. (B) Hematoxylin staining of paraffin-embedded sections of testes and epididymis from adult wild-type and Ppp6r3-cKO mice. (C) Hematoxylin staining of paraffin-embedded testis sections from wild-type and Ppp6r3-cKO mice at the indicated times. Scale bar, 20 μm. (D) Immunostaining of MVH cells in testis sections from wild-type and Ppp6r3-cKO mice at the indicated times. Scale bar, 20 μm. (E) Quantification of the number of MVH+ cells per tubule. *p < 0.05, **p < 0.01, ***p < 0.001. n, not statistically significant. Data are expressed as mean ± SD. N = 3 biological replicates. (F) Immunostaining of PLZF and MVH in testis sections from P9 wild-type or Ppp6r3-cKO mice. Scale bar, 20 μm. ***p < 0.001. n, not statistically significant. Data are expressed as mean ± SD. N = 3 biological replicates. (G) Immunostaining of PLZF, STRA8, and KIT in testicular sections from P9 wild-type or Ppp6r3-cKO mice. Scale bar, 20 μm in the left panel and 15 μm in the right panel. (H) Quantification of the number of PLZF+, STRA8+, or KIT+ cells per tubule. ***p < 0.001. Data are expressed as mean ± SD. N = 3 biological replicates.

[0030] Figure 3 PPP6R3 promotes the translation of mRNAs associated with spermatogonial differentiation. (B) Volcano plot of differentially expressed mRNAs in testis transcriptomics after PPP6R3 depletion. N = 3 biological replicates. (C) qRT-PCR analysis of the indicated mRNAs in the testes of P9 wild-type and Ppp6r3-cKO mice. **p < 0.01. n, not statistically significant. Data are expressed as mean ± SD. N = 3 biological replicates. (D) Volcano plot of differentially expressed proteins in the testis proteomics after PPP6R3 depletion. N = 3 biological replicates. (E) GO term enrichment of proteins downregulated in the testis after PPP6R3 depletion. (F) Western blotting of the indicated proteins in the testes of P9 wild-type or Ppp6r3-cKO mice. (G) (F) Quantification of protein expression, *p < 0.05, ***p < 0.001. n, not statistically significant. Data are presented as mean ± SD. N = 3 biological replicates.

[0031] Figure 4This figure shows decreased translation rates of spermatogonial differentiation-related mRNAs during in vitro differentiation of Ppp6r3-cKO SPCs. Immunostaining of PLZF, STRA8, and KIT during in vitro differentiation of wild-type SPCs at the indicated times. Scale bar, 15 μm. (B) Quantification of the proportions of different spermatogonial cell types during in vitro differentiation of wild-type SPCs. Data are expressed as mean ± SD. N = 3 biological replicates. (C) Immunostaining of PLZF, STRA8, and KIT during in vitro differentiation of Ppp6r3-cKO SPCs. Scale bar, 15 μm. (D) Quantification of the proportions of different spermatogonial cell types during in vitro differentiation of Ppp6r3-cKO SPCs. Data are expressed as mean ± SD. N = 3 biological replicates. (E) Western blotting of PLZF, STRA8, KIT, and PPP6R3 at the indicated differentiation times of SPCs from wild-type or Ppp6r3-cKO mice. N = 3 biological replicates. (F) Translation rates of Plzf, Oct4, and Gfrα1 mRNAs in SPCs from wild-type or Ppp6r3-cKO mice at different differentiation stages. *p < 0.05, **p < 0.01, ***p < 0.001. Data are expressed as mean ± SD. N = 3 biological replicates. (G) Translation rates of Stra8, Kit, Dmrt1, and Ccnd2 mRNAs in SPCs from wild-type or Ppp6r3-cKO mice at the indicated differentiation times. *p < 0.05, **p < 0.01, ***p < 0.001. n, not statistically significant. Data are expressed as mean ± SD. N = 3 biological replicates.

[0032] Figure 5 Demonstrating that EIF3C and EIF4G1 directly interact with PPP6R3 in KIT+ spermatogonia. Volcano plot of proteins significantly enriched with the PPP6R3 antibody in KIT+ spermatogonia. (B) Co-IP analysis of the interaction of PPP6R3 with various translation initiation factors in KIT+ spermatogonia. (C) Western blotting of phosphorylated and non-phosphorylated translation initiation factors purified from KIT+ spermatogonia at P10 wild-type or Ppp6r3-cKO mice. (D) Quantification of the levels of phosphorylated and non-phosphorylated translation initiation factors. ***p < 0.001. n, not statistically significant. Data are expressed as mean ± SD. N = 3 biological replicates.

[0033] Figure 6Phosphorylation of EIF3C and EIF4G1 reduces the differentiation efficiency of Ppp6r3-cKO SPCs by downregulating the translation rate of differentiation-related mRNAs. (A) Volcano plot of differential phosphorylation of testicular phosphoproteomics after PPP6R3 depletion. N = 3 biological replicates. (B) Gene ontology (GO) enrichment terms for proteins significantly upregulated in testis after PPP6R3 depletion. (C) Detailed information on sites significantly upregulated in EIF3C and EIF4G1 phosphorylation after PPP6R3 depletion. (D) Western blot analysis of Flag, EIF3C, and EIF4G1 in Ppp6r3-cKO SPCs indicates overexpression of mutant variants in Ppp6r3-cKO SPCs. N = 3 biological replicates. (E) Immunostaining of PLZF, STRA8, and KIT in Ppp6r3-cKO SPCs after 12 hours of differentiation, indicating overexpression of mutant variants. Scale bar, 15 μm. (F) Quantification of the proportions of different spermatogonia after 12 hours of differentiation in Ppp6r3-cKO SPCs overexpressing the indicated mutants. Data are expressed as mean ± SD. N = 3 biological replicates. (G) Translation rates of Stra8, Kit, Dmrt1, and Ccnd2 mRNAs after 12 hours of differentiation in Ppp6r3-cKO SPCs overexpressing the indicated mutants. *p < 0.05, **p < 0.01, ***p < 0.001. Data are expressed as mean ± SD. N = 3 biological replicates. DETAILED DESCRIPTION

[0034] The essential content and beneficial effects of the present invention will be further illustrated below with reference to examples, which are only used to illustrate the present invention rather than to limit the present invention.

[0035] Example 1 Experimental methods and reagents

[0036] animal

[0037] GemPharmatech Co., Ltd. prepared Ppp6r3flox / flox mice with a C57BL / 6 genetic background. Stra8GFP-cre mice were provided by Professor Tong Minghan from the Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences. To obtain Ppp6r3flox / +Stra8cre mice, 8-week-old Stra8GFP-cre male mice were crossed with 7-week-old Ppp6r3flox / flox female mice. Subsequently, Ppp6r3flox / +Stra8cre males were mated with Ppp6r3flox / flox females to produce Ppp6r3flox / flox Stra8cre mice, referred to as Ppp6r3-cKO mice. Exon 4 of Ppp6r3 was selected for knockout. All mice were maintained under controlled conditions at 25°C with a 12-h light / dark cycle in a specific pathogen-free (SPF) facility with free access to food and water. All procedures followed the Guide for the Care and Use of Laboratory Animals of the National Research Council and were approved by the Animal Ethics Committee of Shandong University School of Medicine.

[0038] Genotype identification

[0039] Genomic DNA was extracted from mouse tails and amplified by PCR using primers specific for the Ppp6r3 mutant and wild-type alleles. The PCR product for wild-type mice was 363 bp, while the PCR product for Ppp6r3-cKO mice was 558 bp.

[0040] Histological analysis

[0041] Testes were collected immediately after euthanasia and fixed in 4% paraformaldehyde or Bouin's solution (Sigma-Aldrich, HT10132). After dehydration, they were embedded in paraffin and sectioned into 5 μm slices. Sections fixed in Bouin's solution were subsequently stained with hematoxylin. Images were captured using an Olympus BX53 fluorescence microscope.

[0042] Immunofluorescence staining

[0043] Cells were fixed with 4% paraformaldehyde for 30 minutes and then treated with 0.5% Triton X-100 for 20 minutes at 25°C. This step can be omitted when staining membrane proteins (e.g., GFRα1, KIT). For immunostaining of testicular sections, 1x boiling sodium citrate antigen retrieval buffer (pH 6.0) (Proteintech, PR30001) was used for 20 minutes. After blocking with 5% BSA in PBS for 1 hour, sections and cells were incubated with primary antibodies overnight at 4°C and then with secondary antibodies for 1 hour at 25°C. Samples were washed three times (10 minutes each) with PBS containing 0.05% Tween 20, and cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Images were acquired using a confocal microscope (Andor Dragonfly spinning disk confocal microscope controlled by Fusion software) and processed using Bitplane Imaris software (version 8.1). For double immunofluorescence staining of PPP6R3 and SOX9 on tissue sections, we used the Dual Fluorescence Immunohistochemistry Mouse / Rabbit Kit (pH 9.0) (ImmunoWay, RS0036) because the primary antibodies for PPP6R3 and SOX9 were both anti-rabbit antibodies.

[0044] Chromosome spreading

[0045] Testes were incubated in a hypotonic solution containing 30 mM Tris, 50 mM sucrose, 17 mM trisodium citrate, 5 mM EDTA, and 0.5 mM DTT for 30–40 minutes. The testes were then transferred to 100 mM sucrose, minced, and placed on slides coated with a solution of 1% PFA and 0.15% Triton X-100. After drying and washing with PBS, the slides were immunostained for SYCP1, SYCP3, and γ-h2ax.

[0046] RT-PCR and qRT-PCR

[0047] Total RNA was extracted from testes or KIT+ spermatogonia isolated from wild-type and Ppp6r3-cKO mouse testes using TRIzol reagent according to the manufacturer's protocol. RNA was then reverse-transcribed into cDNA for RT-PCR and qRT-PCR analysis. RT-PCR and qRT-PCR primer sequences are provided in Table S6.

[0048] Western immunoblotting

[0049] Nuclear and cytoplasmic proteins were extracted from wild-type mouse testes using a frozen / fresh tissue cytoplasmic and nuclear extraction kit. Total protein lysates were obtained using RIPA buffer, and protein concentrations were determined using a BCA protein quantification kit. Equal amounts of protein were separated by SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% milk and incubated with the primary antibody overnight at 4°C. After washing three times with TBS-T for 10 minutes, the secondary antibody was applied for 1 hour at 25°C. Immunoblots were visualized using a Bio-Rad ChemiDoc MP imaging system, and band intensities were quantified using ImageJ software.

[0050] To assess the levels of phosphorylated and non-phosphorylated translation initiation factors, a phosphorus assay acrylamide kit was used, and 5% bovine serum albumin was used instead of 5% milk for blocking.

[0051] KIT+ spermatogonia purification

[0052] KIT+ spermatogonia were isolated and purified using the MojoSort™ Mouse CD177 (c-Kit) Isolation Kit (Biolegend, 480146). Briefly, testes from postnatal day 9 (P9) wild-type mice were digested with 1 mg / mL collagenase IV and 0.05% trypsin. After the suspension was passed through a 40 μm cell strainer to remove undigested material, the cells were sequentially incubated with TruStain FcX™ (anti-mouse CD16 / 32) and PE anti-mouse CD117 (c-Kit) antibodies. After washing with MojoSort™ Buffer, KIT+ spermatogonia were enriched using mouse anti-PE nanoparticles. The purity of the sorted cells was confirmed by KIT immunostaining.

[0053] Transcriptomics

[0054] Testicular samples from P9 wild-type and Ppp6r3-cKO mice were collected for transcriptomic analysis. Total RNA was extracted with TRIzol reagent, and mRNA was enriched using Oligo(dT) magnetic beads. cDNA libraries were generated and then sequenced on an Illumina system. Paired-end clean reads were aligned to the mouse mm10 genome using Hisat2 (v2.0.5). Transcriptomic services were provided by Novogene Co., Ltd. (Beijing, China).

[0055] Proteomics and phosphoproteomics

[0056] Total protein was extracted from the testes of P9 wild-type and Ppp6r3-cKO mice, followed by trypsin digestion and desalting. For proteomics, a portion of the digestion product was separated and analyzed using high-precision mass spectrometry. Data were analyzed using database search software such as ProteomeDiscoverer, Mascot, Spectronaut, and MaxQuant. For phosphoproteomic analysis, phosphorylated peptides were enriched using TiO2 and then eluted. Phosphorylation sites were identified and quantified using liquid chromatography-tandem mass spectrometry (LC-MS / MS) with Mascot software. Proteomics and phosphoproteomic services were provided by the National Center for Protein Science (Beijing, China).

[0057] Immunoprecipitation mass spectrometry (IP-MS)

[0058] Purified KIT+ spermatogonia were lysed using Pierce™ IP Lysis Buffer (Thermo Scientific, 87787) supplemented with protease inhibitors. The lysate was incubated with PPP6R3 and IgG antibodies overnight at 4°C. Protein A / G magnetic beads (Selleck, B23202) were used to capture the protein-antibody complex, and bound proteins were eluted for mass spectrometry analysis. IP-MS services were provided by Nanjing Jiangbei New Area Biomedicine Public Service Platform Co., Ltd. (Nanjing, China).

[0059] Protein stability assay

[0060] To evaluate the changes in the stability of EIF3C and EIF4G1 in KIT+ spermatogonia after PPP6R3 deletion, KIT+ spermatogonia were purified from the testes of P9 wild-type and PPP6R3-cKO mice and cultured in SPC differentiation medium supplemented with 10 ng / mL cycloheximide for 0, 3, 6, and 9 h. The cells were then collected and the degradation rates of EIF3C and EIF4G1 were assessed by Western blotting.

[0061] RIP-qPCR

[0062] The enrichment of EIF3C and EIF4G1 in mRNAs associated with spermatogonial maintenance and differentiation was measured using a RIP kit (BersinBio, Bes5101). Lysates were incubated with EIF3C, EIF4G1, and IgG antibodies overnight at 4°C. RNA bound to the target proteins was then extracted. qRT-PCR was used to quantify the enrichment of target mRNAs associated with EIF3C and EIF4G1, and the results were normalized to 1% of the input.

[0063] Target mRNA translation rate detection

[0064] 36 and 37 wild-type and Ppp6r3-cKO SPCs were harvested after 0, 6, and 12 hours of differentiation, respectively. Cells were lysed using a buffer containing 20 mM Tris (pH 7.4), 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 100 μg / mL cycloheximide, 1% Triton X-100, and 25 U / mL Turbo DNase. To digest extraribosome RNA fragments, the lysate was incubated with 100 U / μL RNase I at 22°C and 1000 rpm on a mixer for 45 minutes. Digestion was stopped by adding RNase inhibitor, followed by RNA extraction and reverse transcription. Quantitative PCR was then performed to normalize the translation rates of multiple spermatogonial maintenance and differentiation-related mRNAs to gapdh.

[0065] Preparation of eIF3C and eIF4G1 mutants

[0066] EIF3C and EIF4G1 mutants (EIF3C S39A, EIF4G1S1217A) were generated by standard overlap PCR. These variants were cloned into the pcDNA3.1 plasmid with a 3× FLAG tag attached to the C-terminus.

[0067] Overexpression of eIF3C and eIF4G1 mutants in SPCs

[0068] The SP100 electroporation system (Celetrix, 11-0103) was used to overexpress EIF3CS39A and EIF4G1S1217A mutants in Ppp6r3-cKO spc, as well as a negative control (pcDNA3.1 plasmid). Ppp6r3-cKO SPCs were first digested with 0.05% trypsin, and 3 million Ppp6r3-cKO cells were resuspended in 100 μL electroporation buffer (equal amounts of buffer A and buffer B) containing 10 μg of plasmid. Electroporation was performed three times at 500 V for 20 ms. After electroporation, the cells were cultured on mitomycin c-inactivated mouse embryonic fibroblasts using SPC medium for 6-8 days. The transfected cells were transferred to a matrix-coated 12-well plate, and 200 μg / mL neomycin was added to the SSC medium to select cells that were successfully transfected.

[0069] Quantification and statistical analysis

[0070] All quantitative data are presented as mean ± SD and are based on at least three biological replicates. Statistical significance was determined using an unpaired, two-tailed Student's t-test. For transcriptomics, proteomics, phosphoproteomics, and IP-MS analyses, a fold change greater than 1.5 and p < 0.05 were considered statistically significant.

[0071] Example 2 PPP6R3 is required for spermatogenesis

[0072] To explore the potential role of PPP6R in spermatogenesis, we analyzed the levels of PPP6R1, PPP6R2, and PPP6R3 based on previously reported quantitative proteomic data from mouse tests and found that in all types of germ cell populations, the level of PPP6R2 was the lowest, while the level of PPP6R3 was always the highest, and the protein abundance of PPP6C remained stable ( Figure 1 A).

[0073] Then, the expression of PPP6R3 in multiple mouse tissues was analyzed. PPP6R3 is highly expressed in wild-type testes, and its expression level increases from postnatal day 8 (P8) compared with P6 ( Figure 1 B) and is located in the cytoplasm. Further immunostaining and Western blotting results showed that the level of PPP6R3 in P12 testicular PLZF+ undifferentiated spermatogonia was similar to that in SOX9+ Sertoli cells, but significantly lower than that in KIT+ differentiated spermatogonia and SYCP3+ spermatocytes ( Figure 1 C-1H).

[0074] These results suggest that PPP6R3 plays a role in spermatogenesis, particularly in spermatogonia differentiation and spermatocyte development.

[0075] To test the potential role of PPP6R3 in spermatogenesis, flox / flox The mice were crossed with Stra8-cre mice to generate lineage-specific PPP6R3 knockout mice (abbreviated as Ppp6r3-cKO). PPP6R3 was specifically knocked out in A1 spermatogonia, and PPP6R3 protein levels were significantly reduced in P6 Ppp6r3-cKO testes compared to controls. Adult Ppp6r3-cKO male mice were infertile, and their testes were smaller than those of controls ( Figure 2 A). Hematoxylin staining of testicular sections showed a significant absence of germ cells in adult Ppp6r3-cKO mice, with only a small number of spermatocytes scattered in the seminiferous tubules. No sperm were observed in the epididymis of Ppp6r3-cKO mice ( Figure 2 B) However, through Ppp6r3 flox / flox Somatic cell (including Sertoli and Leydig cells) knockout of Ppp6r3 obtained by crossing mice with Sf1-cre mice did not affect spermatogenesis or male fertility.

[0076] These results indicate that PPP6R3 is required for male germ cell development and spermatogenesis. PPP6R3 is essential for spermatogenesis by regulating the development of male germ cells, but not somatic cells.

[0077] Example 3 PPP6R3 promotes spermatogonial differentiation

[0078] To determine the abnormal spermatogenesis stage in Ppp6r3-cKO mice, hematoxylin staining was performed on testicular sections of P6, P9, P14, and P21 mice. The testicular tubules of Ppp6r3-cKO mice were similar to those of controls at P6, but the number of germ cells was significantly reduced ( Figure 2 C). At P14 and P21, most germ cells were distributed around the lumen of the Ppp6r3-cKO testicular tubules, with only a small number of spermatocyte-like cells scattered within the lumen of the testicular tubules ( Figure 2 C). The dramatic reduction in germ cell numbers in Ppp6r3-CKO testes is also evident by immunostaining for MVH (also known as DDX4 or VASA), a germ cell marker ( Figure 2 Co-immunostaining of the well-known transcription factors MVH and PLZF (also known as ZBTB16), which are required for spermatogonial self-renewal, showed that the number of PLZF+MVH+ germ cells did not change significantly after PPP6R3 deletion, while the number of PLZF-MVH+ germ cells was significantly reduced ( Figure 2 F), indicating that loss of PPP6R3 function significantly affects spermatogonia differentiation.

[0079] We next investigated whether PPP6R3 is essential for spermatogonial differentiation. Co-immunostaining of PLZF and STRA8 (a marker that distinguishes spermatogonia from preleptotene spermatocytes) revealed that few PLZF+STRA8+ spermatogonia were observed in P9Ppp6r3-cKO testes. Figure 2 G, 2H). The number of KIT+ differentiated spermatogonia (representing A1-A4 spermatogonia) was significantly reduced ( Figure 2 G, 2H). Further PLZF immunostaining showed that the number of PLZF+ spermatogonia in the testes of P28 and P42 Ppp6r3-cKO mice was abnormally increased compared with that of wild-type mice, indicating the accumulation of undifferentiated spermatogonia.

[0080] These results indicate that knockout of PPP6R3 from germ cells impairs spermatogonia differentiation.

[0081] Example 4: Loss of PPP6R3 impairs the transition from mitosis to meiosis in spermatogonia

[0082] Given that spermatogonial differentiation is a prerequisite for the onset of meiosis, and based on the above-mentioned results that PPP6R3 deficiency affects spermatogonial differentiation, it is believed that meiosis will be disrupted in Ppp6r3-cKO mice. Compared with the control group, the number of preleptotene spermatocytes (STRA8+SYCP3+ cells or STRA8+γH2AX+ cells) in the testes of P10Ppp6r3-cKO mice was significantly reduced, and these few preleptotene spermatocytes could only develop into zygotene-like spermatocytes. No pachytene or diplotene spermatocytes were observed in the testes of 3-week-old Ppp6r3-cKO mice.

[0083] These results indicate that loss of PPP6R3 impairs meiotic initiation in differentiated spermatogonia.

[0084] Example 5 PPP6R3 is essential for the translation, but not transcription, of multiple spermatogonial differentiation-related mRNAs

[0085] Transcriptomic and proteomic analyses of testicular samples from P9 wild-type and Ppp6r3-cKO mice were performed to investigate how PPP6R3 regulates spermatogonial differentiation. Ppp6r3 mRNA levels were significantly reduced in Ppp6r3-cKO testes compared with controls; although Stra8 mRNA levels were significantly increased in Ppp6r3-cKO testes, there was no difference in the expression levels of several known spermatogonial maintenance genes (Plzf, Oct4, Id4, Gfrα1, Etv5, Nanos2, and Nanos3) or a group of spermatogonial differentiation-related genes (Kit, Dmrt1, Ccnd2, Sohlh1, and Sohlh2). qPCR analysis of all the above mRNA molecules was consistent with the transcriptomic data ( Figure 3 C), indicating that the loss of PPP6R3 does not affect (or at least does not inhibit) the transcription of spermatogonial differentiation-related genes. However, proteomic analysis showed that the levels of STRA8, CCND2, KIT, DMRT1, SOHLH1, and SOHLH2 were significantly downregulated in Ppp6r3-cKO testes ( Figure 3 These results indicate that PPP6R3 is involved in translation-related processes during spermatogonial differentiation.

[0086] Gene Ontology (GO) term enrichment analysis was performed on 620 down-regulated transcripts from transcriptomics data and 70 down-regulated proteins from proteomics data ( Figure 3D). Downregulated transcripts showed enrichment for “meiosis I” (Brca2, Stag3, Rec8, Meiosin, Meioc, Dmrtc2, Gal3st1, and M1ap), “synaptic complex assembly” (Sycp1, Sycp3, Syce1, and Syce3), and “homologous recombination” (Dmc1, Msh5, Mei1, and Spo11) ( Figure 3 B). In addition, the protein levels of TDRKH, STAG3, M1AP, and TEX12, which are related to "meiosis I", "meiotic cell cycle", and "meiotic chromosome segregation", were significantly decreased in the testes of Ppp6r3-cKO mice ( Figure 3 E).

[0087] These results indicate that abnormal spermatogonia differentiation after PPP6R3 loss leads to the inhibition of transcription and protein expression of meiotic genes.

[0088] Example 6 Knockout of Ppp6r3 from differentiated spermatogonial progenitor cells blocks translation of differentiation-related mRNAs

[0089] To further determine whether the failure of spermatogonial differentiation after PPP6R3 loss is related to translation inhibition, an in vitro spermatogonial progenitor cell (SPCs) differentiation system was established. First, SPCs were isolated and purified from the testes of a wild-type mouse and a Ppp6r3-cKO mouse using anti-mouse CD90.2 magnetic particles and cultured for 5 months. All SPCs tested expressed MVH and undifferentiated spermatogonial markers (PLZF, OCT4, GFRα1, ETV5 and ID4). Wild-type SPCs were then induced to differentiate with RA. qPCR analysis showed that with the extension of RA induction time, the levels of spermatogonial maintenance-related mRNA (Plzf, Gfrα1, Oct4) gradually decreased, while the levels of spermatogonial differentiation-related mRNA (Stra8, Kit, Dmrt1, Ccnd2) increased significantly after 12 hours of differentiation. In addition, co-immunostaining of PLZF, STRA8 and KIT showed that approximately 95% of undifferentiated SPCs (PLZF + STRA8-KIT - cells) entered the differentiation stage after 6 hours of differentiation (PLZF + STRA8 + KIT - cells), which developed into early differentiated spermatogonia (PLZF) after 12 h of differentiation. - STRA8+KIT + Cells represent A1-A4 spermatogonia) Figure 4 B). No late differentiated spermatogonia were observed (PLZF - STRA8 - KIT+ The cells represent intermediate to B-type spermatogonia) and preleptotene spermatocytes (PLZF - STRA8 + KIT - ), even after 24 hours of differentiation ( Figure 4 B).

[0090] These results indicate that this system can effectively induce the differentiation of wild-type SPCs. However, Ppp6r3-cKO SPCs were mostly PLZF after 12-24 h of differentiation. + STRA8 + KIT - , less than 10% of Ppp6r3-cKO SPCs developed into early differentiated spermatogonia, which was similar to the spermatogonia differentiation in male Ppp6r3-cKO mice ( Figure 4 In addition, Western blotting and immunostaining results showed that PPP6R3 was almost completely eliminated and STRA8 expression was significantly decreased in Ppp6r3-cKO SPCs after 6 hours of differentiation. Figure 4 E). These results demonstrate that this cell model is capable of mimicking the physiological changes caused by PPP6R3 deficiency. This cell model was then used in conjunction with the "targeted RNA translation analysis" method reported in Li, BB et al. (2018). Targeted profiling of RNA translation reveals mTOR-4EBP1 / 2-independent translation regulation of mRNAs encoding ribosomal proteins. Proc Natl Acad Sci USA 115, E9325-E9332 to monitor changes in the translation rates of mRNAs involved in sperm maintenance and differentiation in wild-type and Ppp6r3-cKO SPCs at 0, 6, and 12 hours after induction of differentiation.

[0091] In addition to the significant increase in the translation rate of Plzf mRNA at 6 h of differentiation, the translation rate of spermatogonial maintenance-related mRNAs in wild-type SPCs decreased significantly with the extension of differentiation time ( Figure 4 F). In contrast, at 6 and 12 hours after differentiation of Ppp6r3-cKO SPCs, the translation rates of these mRNAs did not decrease significantly compared with those at 0 hours ( Figure 4 F). For mRNAs related to spermatogonial differentiation, the translation rate was significantly higher at 6 to 12 hours after differentiation of wild-type SPCs than at 0 hours ( Figure 4G). Although the translation rate of spermatogonial differentiation-related mRNAs increased slightly during the differentiation of Ppp6r3-cKOSPCs, it was still significantly lower than that of wild-type SPCs ( Figure 4 G).

[0092] These results indicate that PPP6R3 is required for the translation of spermatogonial differentiation-related mRNAs.

[0093] Example 7 EIF3C and EIF4G1 directly interact with PPP6R3 and are the primary targets of PP6 in KIT + Specific substrates in differentiating spermatogonia

[0094] PPP6R is known to regulate the catalytic activity of PPP6C by restricting the substrate specificity of PP6. To determine which molecules serve as potential substrates of PP6 during spermatogonial differentiation, KIT was purified from the testes of P9 wild-type mice. + Spermatogonia were then immunoprecipitated with PPP6R3 antibodies for mass spectrometry (IP-MS). The researchers found that, in addition to known molecules that interact with PPP6R3, such as the PP6 catalytic subunit (PPP6C), two regulatory subunits (PPP6R1 and PPP6R2), and two scaffolding subunits (ANKRD28 and ANKRD44), the PPP6R3 antibody also pulled down multiple translation initiation factors, including EIF3B, EIF3C, EIF3D, EIF3F, EIF3G, EIF3L, EIF3M, EIF4E, and EIF4G1.

[0095] GO term and KEGG pathway analysis of these molecules that potentially interact with PPP6R3 showed that functional annotations related to ribosome structure assembly, translation regulatory factor activity, and translation initiation factor binding were enriched. + The interaction between PPP6R3 and multiple translation initiation factors was confirmed in spermatogonia, and it was also noted that the interaction between PPP6R3 and EIF3C and EIF4G1 was resistant to RNase A treatment, indicating a direct protein-protein interaction between them. Figure 5 B).

[0096] Next, KIT was detected by immunoblotting. + Levels of non-phosphorylated and phosphorylated translation initiation factors in spermatogonia. Compared with the control group, the non-phosphorylated levels of EIF3C and EIF4G1, which directly interact with PPP6R3, were significantly decreased, and the phosphorylated levels were significantly increased. However, the non-phosphorylated and phosphorylated levels of translation initiation factors (EIF3B, EIF3F, EIF3M, and EIF4E) did not change significantly ( Figure 5C, 5D). These results indicate that eIF3C and eIF4G1 are KIT + Specific substrate of PP6 in spermatogonia.

[0097] The relationship between the reduced translation rate after PPP6R3 loss and EIF3C and EIF4G1 was further clarified. ip-qPCR results showed that both EIF3C and EIF4G1 were significantly enriched in spermatogonial maintenance and differentiation-related mRNAs in wild-type mouse testes. PPP6R3 loss did not affect the enrichment of EIF3C and EIF4G1 for spermatogonial maintenance-related mRNAs, but as their non-phosphorylated levels decreased, their enrichment for spermatogonial differentiation-related mRNAs was significantly weakened. In addition, the increased phosphorylation levels of EIF3C and EIF4G1 led to a decrease in their stability and promoted their degradation. Overall, the experimental results indicate that PPP6R3 loss promotes the phosphorylation of EIF3C and EIF4G1, thereby promoting their degradation. On the other hand, the reduction of EIF3C and EIF4G1 weakened their binding to spermatogonial differentiation-related mRNAs, which is the direct cause of the low translation rate.

[0098] Example 8 Hyperphosphorylation of EIF3CS39 and EIF4G1S1217 after PPP6R3 loss is the cause of the decreased translation rate of differentiation-related mRNAs

[0099] Next, we screened for sites where EIF3C and EIF4G1 phosphorylation changes occurred after PPP6R3 deletion by phosphoproteomics analysis of the testes of P9 wild-type and Ppp6r3-cKO mice. Phosphoproteomics data showed that compared with wild-type mice, the phosphorylation levels of 320 sites in the testes of Ppp6r3-cKO mice were significantly downregulated, and the phosphorylation levels of 64 sites were significantly upregulated ( Figure 6 A). Subsequently, the proteins corresponding to the 320 significantly downregulated phosphorylation sites and the 64 significantly upregulated phosphorylation sites were enriched. The proteins with significantly downregulated phosphorylation were involved in the processes of "meiosis I", "germ cell development", "stem cell differentiation regulation" and "spermatocyte differentiation". However, the proteins with significantly upregulated phosphorylation (including EIF3C and EIF4G1) were involved in multiple translation-related processes, such as "regulation of translation response to stress" and "translation initiation" ( Figure 6 A, 6B). We then focused on the changes in the modification levels of EIF3C and EIF4G1 phosphorylation sites after PPP6R3 deletion and found that the phosphorylation levels of EIF3C at S39 and EIF4G1 at S1217 in the testes of Ppp6r3-cKO mice were significantly upregulated compared with those in wild-type mice ( Figure 6C). However, the phosphorylation status of other serine and threonine sites in EIF3C and EIF4G1, as well as the phosphorylation levels of other translation initiation factors (EIF3B, EIF3F, EIF3M, and EIF4E), did not change significantly ( Figure 6 A). These results suggest that EIF3CS39 and EIF4G1S1217 may be targets of PP6 in regulating spermatogonial differentiation. To test their functions, EIF3C and EIF4G1 mutants (i.e., EIF3CS39A, EIF4G1S1217A) were overexpressed in Ppp6r3-cKO SPCs ( Figure 6 D) Immunostaining showed that PLZF overexpressed EIF3CS39A, EIF4G1S1217A, and EIF3CS39A & EIF4G1S1217A in Ppp6r3-cKO SPCs. + STRA8 + KIT - The cell ratio decreased from 83.31% to 55.75%, 66.35% and 37.94% after 12h of differentiation ( Figure 6 E, 6F). Meanwhile, the proportion of early differentiated spermatogonia increased significantly from 16.04% to 43.45%, 32.70% and 61.93% ( Figure 6 E, 6F). Western blotting results also showed that overexpression of EIF3CS39A, EIF4G1S1217A, and EIF3CS39A & EIF4G1S1217A in Ppp6r3-cKO SPCs promoted the expression of spermatogonial differentiation-related proteins and downregulated the expression levels of spermatogonial maintenance-related proteins. Thus, cells that had been arrested at the differentiation entry stage continued to differentiate. Finally, we investigated whether the restoration of spermatogonial differentiation efficiency after overexpression of EIF3C and EIF4G1 mutant variants was accompanied by changes in translation rates. Compared with the control group, the translation rates of mRNAs related to spermatogonial maintenance (Plzf, Oct4, and Gfrα1) were significantly reduced during differentiation in Ppp6r3-cKO SPCs overexpressing EIF3C and EIF4G1 mutant variants. However, during differentiation of Ppp6r3-cKOSPCs overexpressing EIF3C and EIF4G1 mutant variants, the translation rates of spermatogonial differentiation-related mRNAs (Stra8, Kit, Dmrt1, and Ccnd2) were significantly increased ( Figure 6 G). These results suggest that PP6 promotes the translation of spermatogonial differentiation-related mRNAs by targeting EIF3CS39 and EIF4G1S1217 for dephosphorylation, which are essential for spermatogenesis and male fertility.

[0100] The applicants discovered for the first time that PPP6R3 deficiency blocked spermatogonial differentiation and caused complete animal infertility, demonstrating that PPP6R3 is essential for spermatogenesis. The applicants' discovery provides new insights into the relationship between phosphorylation of phosphoprotein phosphatase (PPP) and spermatogonial differentiation. In-depth research on this relationship will contribute to the development of treatments for male infertility. The applicants have thus provided a method for diagnosing male fertility defects in mammals, including humans, and have provided new ideas and methods for developing new treatments for male infertility individuals affected by gene mutations.

[0101] The above is an explanation of the present invention and should not be regarded as limiting the present invention. Unless otherwise noted, the practice of the present invention will use the conventional techniques of organic chemistry, polymer chemistry, biotechnology, etc., and it is obvious that in addition to being particularly described in the above description and embodiments, the present invention can also be realized in other ways. Other aspects and improvements within the scope of the present invention will be apparent to those skilled in the art. According to the teachings of the present invention, many changes and variations are feasible, and therefore they are within the scope of the present invention.

Claims

1. Use of a reagent for detecting PPP6R3 in a subject for preparing a kit or instrument for diagnosing or predicting fertility defects in mammals or humans, such as male infertility.

2. The use according to claim 1, wherein the reagent is a reagent for detecting a combination of PPP6R3, EIF3C and EIF4G1.

3. The method according to claim 1 or 2, wherein the male infertility disorder is selected from teratospermia, azoospermia, oligospermia, asthenospermia, and asthenospermia.

4. The use according to claim 1 or 2, wherein the reagent is a reagent for detecting a gene, preferably, it also includes a reagent for detecting whether a gene has a mutation, for example, a reagent for detecting whether the nucleotides encoding S39 of EIF3C and S1217 of EIF4G1 have a mutation.

5. The use according to claim 1 or 2, wherein the reagent is a reagent for detecting a protein, preferably, further comprising a reagent for determining the activity of the target protein, more preferably, further comprising a reagent for detecting whether the target protein has a mutation, for example, a reagent for detecting whether S39 of EIF3C and S1217 of EIF4G1 have a mutation.

6. The use according to claim 5, which comprises a reagent for detecting protein expression by immunoassay, such as a reagent for detecting protein expression by ELISA or Western blotting using an antibody that specifically recognizes the protein; or These include reagents for detecting the protein by detecting the presence or amount of mRNA, such as reagents for detecting the amount of mRNA encoding a target protein in a sample by RT-PCR.

7. A kit for diagnosing fertility defects in mammals or humans, such as male infertility, comprising a reagent for detecting PPP6R3 or the following combination: PPP6R3, EIF3CS39, and EIF4G1S1217 in a subject.

8. The kit according to claim 7, wherein the male infertility disorder is selected from teratozoospermia, azoospermia, oligozoospermia, asthenozoospermia, and asthenozoospermia.

9. The kit according to claim 7 or 8, comprising a reagent for detecting genes of a test individual.

10. The kit according to claim 7 or 8, comprising a reagent for detecting a protein in a test subject, preferably, further comprising a reagent for determining the activity of a target protein.

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