CN113288964A

CN113288964A - Application of novel kidney-warming and sperm-producing drink as medicine for reducing expression level of H3K27me3

Info

Publication number: CN113288964A
Application number: CN202110574145.2A
Authority: CN
Inventors: 潘晓燕; 雷钧涛; 孙艳美; 张万生; 王雪楠
Original assignee: Jilin Medical College
Current assignee: Jilin Medical College
Priority date: 2021-05-25
Filing date: 2021-05-25
Publication date: 2021-08-24

Abstract

The invention belongs to the technical field of biology, and particularly relates to application of a novel kidney-warming and sperm-producing drink in promotion of early embryo development by maintaining a low level of H3K27me3. The invention relates to a new warm kidney spermatogenic drink which is prepared by pouring 30 days of new warm kidney spermatogenic drink after molding sexual maturity male mice cyclophosphamide, mating the treated male mice with superovulation treated female mice, counting the development rate of embryos at each stage, detecting the expression of H3K27me3 in sperms, prokaryotic embryos and 2-cell embryos by an immunofluorescence staining method, detecting the expression of histone demethylase KDM6A and methylation transferase EZH2 in the 2-cell embryos with development retardation by western blotting, analyzing the expression of the zygote genome activating genes (ZSA 4, E1F1AX, HSPA1A, ERV4-2, MYC) in the 2-cell embryos with development retardation by qRT-PCR, and analyzing the results of the data to discover the new use of the new warm kidney spermatogenic drink for promoting the development of the embryos by maintaining low and medium-low H3K27me3 modification levels of sperms and the male males.

Description

Application of novel kidney-warming and sperm-producing drink as medicine for reducing expression level of H3K27me3

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of a novel kidney-warming and sperm-producing drink as a medicine for reducing the expression level of H3K27me3.

Background

The new kidney-warming and spermatogenic decoction is a traditional compound traditional Chinese medicine and is widely used for treating male spermatogenic disorder. The kidney-warming and sperm-producing Decoction (NWSSJD) is prepared from 15 Chinese-medicinal materials including ginseng, pilose antler, cynomorium, astragalus root, epimedium, Chinese angelica root, etc. It can maintain high androgen concentration in testis tissue, promote the development of spermatogenic cells, inhibit apoptosis of spermatogenic cells, and increase the generation rate and maturation rate of sperm. An important criterion for testing drugs for the treatment of male spermatogenic disorders is whether normal offspring can be produced. Therefore, studying the effect of nwsjd on early embryo development is a key step to demonstrate its therapeutic efficacy.

Epigenetic modification is an important gene expression regulation mode, which regulates the replication, transcription and repair of DNA and plays an important role in regulation during the development of sperms and early embryos. Among them, H3K27me3 plays an important regulatory role in sperm and early embryo development, and is involved in the transcriptional repression regulation of genes. H3K27me3 is widely present in testis tissues, polycombin SCML2 promotes H3K27me3 modification of spermatogonium by binding to promoter region of undifferentiated spermatogonium, H3K27me3 participates in regulating meiosis of spermatogonium by regulating expression of meiosis-related genes, and H3K27me3 marker is still retained in mature sperm of mouse, human and zebrafish. H3K27me3 is germ line inherited in mammals, and drosophila embryos can transmit the inhibitory histone marker H3K27me3 to their progeny. H3K27me3 in early embryos can accumulate in the regulatory region of H3K27ac, preventing premature activation of the zygotic genome activator gene; studies also found that 2000 μ g/L BPA increased the expression of DNA demethylase and H3K27me3 demethylase in testicular tissue, thereby interfering with epigenetic modification of H3K27me3 in spermatogenic cells. Therefore, the modification of H3K27me3 in the cell can be used as a marker molecule for epigenetic modification disorder in sperms or early embryos. The development of the drug which influences the expression level of H3K27me3 can control the embryonic development, and even the effect of treating diseases can be achieved.

However, in the prior art, the kidney-warming spermatogenic drink is only used for treating sexual dysfunction and improving the incidence rate and the maturation rate of sperms, the relation between the kidney-warming spermatogenic drink and the expression level of H3K27me3 is not found, and the application of the kidney-warming spermatogenic drink in regulating and controlling the expression level of H3K27me3 is not found.

Disclosure of Invention

In order to solve the technical problems, the invention provides application of a novel kidney-warming and sperm-producing drink as a medicine for reducing the expression level of H3K27me3.

The invention aims to provide application of a novel kidney-warming and sperm-producing drink as a medicine for reducing the expression level of H3K27me3.

Preferably, in the application, the kidney-warming and sperm-producing tea is used for preparing a medicine for reducing the expression level of H3K27me3, improving the sperm quality and improving the survival rate.

Preferably, in the application, the kidney-warming and essence-generating decoction is used for preparing a medicine for reducing the expression level of H3K27me3 and promoting the development of early embryos.

Preferably, in the above application, the early embryo is a prokaryotic embryo and a 2-cell embryo.

Preferably, in the above application, the H3K27me3 is expressed in the prokaryotic embryo and the 2-cell embryo and the sperm, respectively.

Preferably, in the above application, the kidney-warming and essence-producing tea is used for preparing a medicament for reducing the expression level of the methylation transferase EZH 2.

Preferably, in the above application, the kidney-warming and sperm-producing tea is used for preparing a medicament for activating a zygote genome activating gene.

Preferably, in the above application, the zygotic genome activating genes are ZSCAN4, E1F1AX, HSPA1A, ERV4-2 and MYC.

Preferably, in the application, the kidney-warming and essence-generating decoction comprises the following substances in parts by weight: 6-7 parts of ginseng, 9-10 parts of cynomorium songaricum, 10-12 parts of astragalus membranaceus, 4-6 parts of epimedium, 1-3 parts of pilose antler, 9-10 parts of cistanche, 4-6 parts of angelica sinensis, 9-12 parts of flatstem milkvetch seed, 13-15 parts of Chinese yam, 6-8 parts of bighead atractylodes rhizome, 3-5 parts of ligusticum wallichii, 4-6 parts of white peony root, 1-3 parts of cinnamon, 1-3 parts of elecampane and 1-3 parts of fennel.

Preferably, in the application, the kidney-warming and essence-generating decoction comprises the following substances in parts by weight: 6 parts of ginseng, 9 parts of cynomorium songaricum, 12 parts of astragalus membranaceus, 6 parts of epimedium, 1 part of pilose antler, 9 parts of cistanche, 6 parts of angelica, 9 parts of flastem milkvetch seed, 15 parts of Chinese yam, 6 parts of bighead atractylodes rhizome, 3 parts of ligusticum wallichii, 6 parts of white peony root, 1 part of cinnamon, 1.5 parts of costustoot and 3 parts of fennel.

Compared with the prior art, the invention has the following beneficial effects:

1. the animal dysspermia model is prepared by cyclophosphamide, so that the influence of NWSSJD on the development of early embryos is researched, the influence of NWSSJD on epigenetic modification of H3K27me3 in sperms and early embryos after treatment by NWSSJD is discussed, and a certain reference is provided for clarifying the treatment mechanism of NWSSJD on dysspermia. The NWSSJD can remarkably maintain the low H3K27me3 modification level in sperms and male embryo pronuclei of the spermatogenic disorder mice, improve the development rate of early embryos and reduce the development retardation of 2-cell embryos. Therefore, we speculate that the H3K27me3 modification may be an important target for nwsjd. The regulation and control functions of NWSSJD on epigenetic modification in sperms and early embryos are found for the first time, and the NWSSJD plays an important role in developing medicaments influencing the expression level of H3K27me3 so as to achieve the aim of regulating and controlling the development of the embryos and even achieve the effect of treating diseases.

2. The invention discovers that Cyclophosphamide (CPA) can abnormally increase the modification level of H3K27me3 in a sperm or a male pronucleus to cause the retardation of 2-cell embryonic development, and discovers that the expression of histone methyltransferase EZH2 is remarkably increased in a 2-cell embryo with the retardation of development, the modification level of H3K27me3 is also remarkably increased, the expression of a zygote genome activating gene is reduced, and the retardation of 2-cell embryonic development is possibly related to the activation failure of a zygote genome caused by the abnormal modification of H3K27me3.

Drawings

FIG. 1 shows the expression result of H3K27me3 in mouse sperm;

(A) H3K27me3, H3K27me3 in the immunofluorescence labeled mouse sperm are marked as green, and the sperm nucleus is marked as blue; (B) measuring the average fluorescence intensity of H3K27me3 in sperm using Image J Image analysis software (N10);

FIG. 2 shows the expression results of H3K27me3 in mouse prokaryotic embryos at PN2 stage and PN3 stage;

(A) H3K27me3 and H3K27me3 in the immunofluorescence labeled mouse prokaryotic embryo are labeled as green, and the embryonic cell nucleus is labeled as blue, female pronucleus (female) and male pronucleus (male);

(B) measuring the average fluorescence intensity of H3K27me3 in the male and female pronuclei by using Image J Image analysis software, and calculating the ratio of the average fluorescence intensity of H3K27me3 in the male and female pronuclei (N is 10);

FIG. 3 is an embryo taken from the mouse oviduct 41h to 46h after hCG injection;

(A) embryos obtained from the oviduct of mice 44h after hCG injection, including prokaryotic embryos, 2-cell embryos and lysed embryos;

(B) the ratio of embryos collected at each stage after hCG injection in 41h-42h, 43h-44h and 45h-46h in different experimental groups is counted respectively (N-5);

FIG. 4 is an embryo taken from the oviduct of a mouse 52h after hCG injection;

(A) obtaining embryos at 3-4 cell stage from the oviduct of the mouse 52h after hCG injection;

(B) the development rate of 3-4 cell-stage embryos and the development blocking rate of 2-cell-stage embryos collected 52h after the hCG injection in different experimental groups are counted respectively (N is 5);

FIG. 5 is the results of expression of H3K27me3 in a developmentally arrested 2-cell embryo;

(A) the immunofluorescence marks H3K27me3.H3K27me3 in the normally-developing 2-cell embryo and the development-retarded 2-cell embryo as green, and the embryo nucleus as blue;

(B) measuring the mean fluorescence intensity of H3K27me3 in foetal cell nuclei (N10) using Image J Image analysis software;

FIG. 6 shows the results of expression of demethylases KDM6A and the methyltransferase EZH2 in developmentally arrested 2-cell embryos;

(A) western blotting to detect the expression of demethylase KDM6A and methyltransferase EZH2 in normally-developing 2-cell embryos and development-retarded 2-cell embryos;

(B) image J Image analysis software analyzed the relative expression levels of EZH 2;

FIG. 7 is a qRT-PCR assay for the expression of the zygotic genome activator (ZSCAN4, E1F1AX, HSPA1A, ERV4-2, MYC) in development arrested 2-cell embryos.

Detailed Description

In order that those skilled in the art will better understand the technical solutions of the present invention to be implemented, the present invention will be further described with reference to the following specific embodiments and accompanying drawings.

The methods used in the following examples are conventional unless otherwise specified, and all reagents used are commercially available.

1 materials and methods of treatment

1.1 test animals

Sexually mature Kunming strain male mice at 10 weeks of age and female mice at 8 weeks of age were used for the experiments. Mice were living in a temperature controlled room with a 12h light/12 h dark light cycle. Mice had free access to water and food. The mice were treated as approved by the ethical committee of the gerlin medical institute.

1.2 preparation of the decoction for warming kidney and producing sperm

The compound traditional Chinese medicine kidney-warming and sperm-producing beverage (NWSSJD) comprises 6g of ginseng, 9g of cynomorium songaricum, 12g of astragalus membranaceus, 6g of epimedium herb, 1g of pilose antler, 9g of cistanche, 6g of angelica sinensis, 9g of flatstem milkvetch seed, 15g of Chinese yam, 6g of bighead atractylodes rhizome, 3g of ligusticum wallichii, 6g of white peony root, 1g of cinnamon, 1.5g of costustoot and 3g of fennel (in the specific embodiment of the invention, 1g is equivalent to one weight part in the claims), and all the medicinal materials are purchased from Beijing Tongrentang in China. Weighing the traditional Chinese medicine materials, putting the traditional Chinese medicine materials into an earthen pot, decocting according to a traditional Chinese medicine decocting method, finally decocting and concentrating the decoction, and preparing 1ml decoction from 2g crude medicines. Refrigerating at 4 deg.C for use. The effective components of the NWSSJD are detected to comprise ginsenoside, icariin, ligustrazine, pilose antler polypeptide and the like through HPLC analysis of the effective components in the NWSSJD.

1.3 drug treatment

Sexually mature 10-week-old male mice were divided into a control group, a nwsjd group, and a cyclophosphamide group (CPA group), and 60 mice were each group. Mice in NWSSJD group and CPA group are injected with 80 mg/(k) in abdominal cavityg.d) Cyclophosphamide, injected continuously for 5 days (Feng B, Shen JH, Yuan BB, Qian ZY. study on stability of oligoospermia model estableshied by intraepithelial injection of cyclophopshamamide in mice. Chinese Journal of android, 2007,13(1):71-72), control mice were injected with an equal volume of physiological saline. Then, the NWSSJD group of mice was drenched with NWSSJD at a dose of 12 g.kg^-1·d^-1The control group and the CPA group were infused with physiological saline of equal volume once a day for 30 days.

Among them, CPA is an alkylating agent, and is widely used for immunosuppression after antitumor and organ transplantation, but researches show that the use of cyclophosphamide in males significantly improves the embryo loss rate, the teratogenesis rate and the occurrence rate of offspring behavioral defects, and the abnormalities can be transmitted to offspring. CPA can cause the micronucleus in the embryo to be obviously increased, the expression pattern of acetylated H4K5 in the 2-cell embryo is obviously changed, the repair capability of zygotes on DNA damage is reduced, abnormal histone epigenetic modification is continuously existed, and the embryogenesis is abnormal.

1.4 statistical analysis of data

Statistical analysis of the test data was performed using SPSS 17.0 software and the data are expressed as mean ± Standard Deviation (SD). The difference between groups is compared by adopting an LSD post hoc detection method of one-way ANOVA, and the difference is obvious when P < 0.05.

2 influence of the decoction on sperm

2.1 sperm Collection

The animal subjects were mice of the control group, CPA group and nwsjd group, and male mice were sacrificed by cervical dislocation. Quickly taking down epididymis, placing the epididymis in a culture dish containing 1ml of physiological saline at 37 ℃, scratching the epididymis tail by using a 26-gauge needle, and incubating for 10min in an incubator at 37 ℃ to ensure that sperms are fully dissociated from the epididymis tail.

2.2 immunofluorescence staining of sperm

The sperm suspension was diluted in distilled water and then smeared onto a cover glass and dried at room temperature for 2 h. Sperm dephlegmation (25mM DTT, 0.2% (v/v) Triton X-100 and 200IU/mL heparin) was added drop-wise to the smears and incubated at 37 ℃ for 15min in an incubator. The sperm was discarded and the gel was removed and fixed in 3.7% (v/v) paraformaldehyde solution for 20 min. PBS wash and 5% (v/v) BSA blocking for 2h at room temperature. Primary antibody incubation smear of rabbit derived anti-H3K 27me3(a2363, ABclonal) for 2H. After three washes with PBS, the goat anti-rabbit FITC labeled secondary antibody (AS011, ABclonal) was incubated with the smears for 1 h. The sperm nucleus was stained with Hoechst 33342(14533, Sigma), and then smeared with an anti-fluorescence quenching mounting solution (AR1109, BOSTER), nail polish mounting. 5 mice were used for each experimental group, 5 sperm smears were made for each mouse, observed under an upright microscope oil microscope with Olympus IX-53, randomly picked 5 high power fields (1000) for each sperm smear using a microscopic Image acquisition system (CellSens Dimension), and analyzed for mean fluorescence intensity values of H3K27me3 in sperm using Image J professional Image analysis software.

2.3 results

Mature sperm were obtained from the epididymal tail of the control, nwsjd and CPA group mice, respectively, and were subjected to immunofluorescence staining with H3K27me3 (fig. 1A). H3K27me3 was present in the nucleus of sperm (fig. 1A). The mean fluorescence intensity of sperm H3K27me3 in the nwsjd group was not significantly different from that of the control group (fig. 1B), while the mean fluorescence intensity of sperm H3K27me3 in the CPA group was significantly higher than that of sperm H3K27me3 in the nwsjd group and the control group (P <0.05, fig. 1B). Nwsjd maintained low expression levels of H3K27me3 in sperm.

Influence of the decoction on embryo

3.1 obtaining embryos

Female mice were superdrained, intraperitoneally injected with 10IU pregnant horse serum gonadotropin (PMSG), 48h later with 10IU Human Chorionic Gonadotropin (HCG), and then were housed in 1:1 cages with male mice (animal subjects were mice of the above-mentioned control group, CPA group, and WSSJD group). Mice oviducts were harvested at 21H, 41-42H, 43-44H, 45-46H and 52H after HCG injection, placed in PBS droplets, and prokaryotes, 2-cell embryos and 3-4 cell embryos, respectively, were harvested from the ampulla of the oviducts, which required 0.1% hyaluronidase (H3506, Sigma) to remove cumulus cells.

3.2 immunofluorescence of embryos

The obtained prokaryotic embryo and 2-cell embryo are fixed in 3.7% (v/v) paraformaldehyde solution for 20min, acted in 0.1% Trion X-100 PBS solution for 15min, and sealed for 1h by 5% (v/v) BSA confining liquid. Embryos were incubated for 2H in primary antibodies of rabbit-derived anti-H3K 27me3(a2363, ABclonal). After three washes with PBS, embryos were incubated for 1h with goat anti-rabbit FITC-labeled secondary antibody (AS011, ABclonal). Hoechst 33342(14533, Sigma) stains foetal cell nuclei and the foetus is mounted in a mounting drop (AR1109, BOSTER) which is bleached against fluorescence. Observed with a laser scanning confocal microscope (olympus, FV1000), all pictures were taken under laser irradiation of the same intensity. Mean fluorescence intensity analysis of H3K27me3 was performed on 10 embryos per experimental group using NIH Image J professional Image analysis software.

3.3western blotting

Each time 60 embryos at 2-cell stage were added with 6.5. mu.L of protein lysate (950. mu.L of Laemmli sample buffer + 50. mu.L of beta-mercaptoethanol + 0.5. mu.L of protease inhibitor) and stored at-20 ℃ for later use after sufficient lysis at room temperature. Proteins were transferred to PVDF membranes, blocked with 5% (g/v) skim milk for 1h, KDM6A, EZH2 and Lamin A/C primary antibody incubated membranes at 4 ℃ overnight. After PBST washing of the membrane, the membrane was incubated in goat anti-rabbit HRP-labeled secondary antibody for 2h at room temperature, followed by enhanced chemiluminescent development, and the membrane was scanned using ChemiDOC XRS + imaging systems (Bio-Rad Laboratories, Hercules, Calif., USA). Image J Image analysis software analyzed the relative expression levels of KDM6A and EZH 2.

3.4qRT-PCR

Real-time PCR analysis was performed for 60 embryos at 2-cell stage at a time. RNAs from embryos were extracted using the Rneasy Micro Kit (Qiagen, Hilden, Germany) and transcribed into cDNA. Reverse Transcription was performed in a 20. mu.L Reverse Transcription system (1. mu.L random primers, 1. mu.L Oligo dT Primer, 4. mu.L Reverse Transcription buffer, 1IU/mL PrimeScriptTEMRT Enzyme Mix I (TaKaRa, Dalian, China)). Quantitative analysis of gene products was performed by quantitative Real-time PCR using iQ5 Multicolor Real-time PCR Detection System (Bio-RAD) using SYBR Premix Ex Taq (Takara Dalian, China) using specific primers as shown in Table 1. The reaction system of Real-time PCR comprises Premix Ex TaqTM II, forward/reverse pTrimers and cDNA template. The amounts of primers and cDNA were optimized by ordinary PCR, followed by real-time PCR. The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 5s, annealing at 60-62 ℃ (depending on the primers used, see Table 1) for 20s, extension at 72 ℃ for 30s, and 40 cycles. The PCR product was confirmed by agarose gel electrophoresis. The housekeeping gene, GAPDH, was used as an internal control for the experiment. Use 2^-△△CtThe method calculates the relative expression level of the target gene.

TABLE 1 primer sequences and annealing temperatures

3.5 results

3.5.1 Effect of NWSSJD on H3K27me3 expression in mouse PN2 and PN3 embryos

Prokaryotic embryos were collected from the oviducts of mice 21h after HCG injection (fig. 2A), and were divided into PN1-PN 55 stages depending on the position of the male and female pronuclei and the size of the male and female pronuclei. The male and female pronuclei of the PN2 and PN3 pronuclei embryos are enlarged, a certain distance is reserved between the male and female pronuclei, the male pronuclei with larger volume is male pronuclei (male) and the female pronuclei with small volume is female. Prokaryotic embryos of PN2 and PN3 were selected for immunofluorescent staining of H3K27me3 in this experiment (fig. 2A) because the male and female pronuclei are easily visualized. H3K27me3 is mainly expressed in a female pronucleus in an NWSSJD group and a control group, and H3K27me3 is hardly expressed in a male pronucleus; in contrast, in the control group, not only the H3K27me3 was expressed in the female pronuclei, but also the H3K27me3 was expressed in the male pronuclei. To further determine its expression level in the hermaphroditic pronucleus, the mean fluorescence intensity of H3K27me3 in the hermaphroditic pronucleus was determined (fig. 2B). The ratio of the average fluorescence intensity of H3K27me3 in the male and female pronuclei in the NWSSJD group is not obviously different from that in the control group, but is obviously higher than that of the average fluorescence intensity of H3K27me3 in the male and female pronuclei in the CPA group. Nwsjd helps maintain a low H3K27me3 modification pattern in the male pronuclei.

3.5.2 Effect of NWSSJD on embryonic development in mice 41-46h and 52h after HCG injection

Embryos were collected from mouse oviducts at 41-42h, 43-44h, 45-46h and 52h after HCG injection, including prokaryotic embryos, 2-cell stage embryos, 3-4 cell embryos and lysed embryos (FIG. 3A, FIG. 4A), and the development rates of the embryos at each stage were counted (FIG. 3B, FIG. 4B). 41h-42h after HCG injection, the ratio of prokaryotic embryos in the NWSSJD group is remarkably increased compared with that in the control group (P <0.05), and is remarkably reduced compared with that in the CPA group (P < 0.05); the development rate of the NWSSJD group 2-cell embryos is not significantly different from that of the control group, but is significantly higher than that of the CPA group 2-cell embryos; the rate of lysed embryos was significantly higher in the CPA group than in the nwsjd and control groups (P < 0.05). The ratio of procaryotic embryos and the ratio of lysed embryos of the NWSSJD group and the control group are significantly lower than that of the CPA group (P <0.05) 43h-44h after HCG injection, while the development rate of 2-cell embryos is significantly higher than that of the CPA group (P <0.05), and no significant difference exists between the procaryotic embryo development rate, the 2-cell embryo development rate and the ratio of lysed embryos of the NWSSJD group and the control group. After 45-46h after HCG injection, the prokaryotic embryos developed into 2-cell embryos or lysed embryos and were not collected. The ratio of nwsjd group 2-cell embryos and the ratio of lysed embryos were not significantly different from the control group, but the development rate of nwsjd group 2-cell embryos was significantly higher than that of CPA group 2-cell embryos, while the ratio of lysed embryos was significantly lower than that of CPA group. NWSSJD promoted 2-cell embryonic development and reduced embryo degeneration 41-46h after HCG injection.

Embryos collected from the oviducts 52h after HCG injection were 2-cell embryos and 3-4 cell embryos (FIG. 4A). The embryos collected from the control group developed fully to 3-4 cells, while some of the embryos collected from the nwsjd and CPA groups blocked development at the 2-cell stage, with the 3-4 cell embryos from the nwsjd group developing at a significantly higher rate than the CPA group (P <0.05) and the 2-cell embryos developing at a significantly lower rate than the CPA group (P < 0.05). NWSSJD alleviates the arrest of 2-cell embryo development by CPA.

3.5.3 expression of H3K27me3 in developmentally arrested 2-cell embryos

H3K27me3 immunofluorescent staining was performed on development-arrested 2-cell embryos collected 52H after HCG injection and on control-normally-developing 2-cell embryos (fig. 5A), and the mean fluorescence intensity of H3K27me3 in the embryos was determined (fig. 5B). The expression level of H3K27me3 in the development-arrested 2-cell embryos was found to be significantly higher than the expression level of H3K27me3 in the normal development 2-cell embryos (P < 0.05).

3.5.4 expression of the modified enzyme H3K27me3 in developmentally arrested 2-cell embryos

The collected development-arrested 2-cell embryos and control-group normally-developing 2-cell embryos were subjected to western blotting analysis using demethylases KDM6A and methyltransferase EZH2 (FIG. 6A). Expression of the demethylase KDM6A was not detected in both the normal 2-cell embryos and the development-arrested 2-cell embryos, but it was found that expression of the methyltransferase EZH2 was significantly higher in the development-arrested 2-cell embryos than in the normal 2-cell embryos (fig. 6B, P < 0.05). Increased expression of EZH2 resulted in increased expression levels of H3K27me3 in developmentally arrested 2-cell embryos.

3.5.5 expression of the zygotic genome activating Gene in developmentally arrested 2-cell embryos

The collected development arrested 2-cell embryos and control normal developing 2-cell embryos were subjected to qRT-PCR analysis of the zygotic genome activator genes (ZSCAN4, E1F1AX, HSPA1A, ERV4-2, MYC) (FIG. 7). As a result, the expression of the zygotic genome activator genes (ZSCAN4, E1F1AX, HSPA1A, ERV4-2 and MYC) in the development-arrested 2-cell embryo is found to be obviously reduced compared with the expression in the normally-developed 2-cell embryo. 2-cell embryonic development arrest is associated with failure of activation of the zygotic genome.

The results show that the NWSSJD can inhibit spermatogenic cells and spermatozoa from apoptosis, promote sperm maturation, improve sperm quality and the like, has various beneficial effects on maintaining testicular function, and can be widely used for treating male spermatogenic disorders. H3K27me3 is a key epigenetic modification molecule for inhibiting transcription in sperm and embryo cells, and we verify a new role of NWSSJD, which can reduce the developmental block rate of 2-cell embryos and improve the development of early embryos by maintaining the modification level of low H3K27me3 in sperm and male pronuclei. The CPA-caused growth retardation of the 2-cell embryo is considered to be due to the fact that the H3K27me3 methyltransferase EZH2 expression in the embryo cell is increased, so that the H3K27me3 level is increased, the expression of the activation gene of the zygotic genome is inhibited, the activation of the zygotic genome fails, and the embryo development is retarded. The invention provides some reference for the development influence of NWSSJD on the offspring after the treatment of male spermatogenesis dysfunction.

It should be noted that, when the present invention relates to a numerical range, it should be understood that two endpoints of each numerical range and any value between the two endpoints can be selected, and since the steps and methods adopted are the same as those in the embodiment, in order to prevent redundancy, the present invention describes a preferred embodiment. While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Sequence listing

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Claims

1. Application of the novel kidney-warming and sperm-producing drink as a medicine for reducing the expression level of H3K27me3.

2. The application of the kidney-warming spermatogenic drink as a medicine for reducing the expression level of H3K27me3 in claim 1, wherein the kidney-warming spermatogenic drink is used for preparing the medicine for reducing the expression level of H3K27me3 to improve sperm quality and improve survival rate.

3. The application of the kidney-warming kidney-essence-producing drink as a medicine for reducing the expression level of H3K27me3, which is characterized in that the kidney-warming kidney-essence-producing drink is used for preparing the medicine for reducing the expression level of H3K27me3 and promoting the development of early embryos.

4. The application of the kidney-warming spermatogenic drink as a medicament for reducing the expression level of H3K27me3 in claim 3, wherein the early embryo is a prokaryotic embryo and a 2-cell embryo.

5. The use of the neo-kidney warming spermatogenic drink according to claim 2 or 4 as a medicament for reducing the expression level of H3K27me3, wherein the H3K27me3 is expressed in the prokaryotic embryo, 2-cell embryo and sperm, respectively.

6. The application of the kidney-warming sperm-producing drink as a medicine for reducing the expression level of H3K27me3, wherein the kidney-warming sperm-producing drink is used for preparing the medicine for reducing the expression level of a methylation transferase EZH 2.

7. The application of the kidney-warming and sperm-producing drink as a medicine for reducing the expression level of H3K27me3, wherein the kidney-warming and sperm-producing drink is used for preparing a medicine for activating a zygote genome activating gene.

8. The use of the novel kidney-warming spermatogenic drink as a medicament for reducing the expression level of H3K27me3 as claimed in claim 7, wherein the zygote genome activating genes are ZSCAN4, E1F1AX, HSPA1A, ERV4-2 and MYC.

9. The use of the neorenal warming spermatogenic drink according to any one of claims 1 to 4 and 6 to 8 as a medicament for reducing the expression level of H3K27me3, wherein the neorenal warming spermatogenic drink comprises the following substances in parts by weight: 6-7 parts of ginseng, 9-10 parts of cynomorium songaricum, 10-12 parts of astragalus membranaceus, 4-6 parts of epimedium, 1-3 parts of pilose antler, 9-10 parts of cistanche, 4-6 parts of angelica sinensis, 9-12 parts of flatstem milkvetch seed, 13-15 parts of Chinese yam, 6-8 parts of bighead atractylodes rhizome, 3-5 parts of ligusticum wallichii, 4-6 parts of white peony root, 1-3 parts of cinnamon, 1-3 parts of elecampane and 1-3 parts of fennel.

10. The application of the kidney-warming kidney-essence-producing drink as a medicine for reducing the expression level of H3K27me3 according to claim 9, wherein the kidney-warming kidney-essence-producing drink comprises the following substances in parts by weight: 6 parts of ginseng, 9 parts of cynomorium songaricum, 12 parts of astragalus membranaceus, 6 parts of epimedium, 1 part of pilose antler, 9 parts of cistanche, 6 parts of angelica, 9 parts of flastem milkvetch seed, 15 parts of Chinese yam, 6 parts of bighead atractylodes rhizome, 3 parts of ligusticum wallichii, 6 parts of white peony root, 1 part of cinnamon, 1.5 parts of costustoot and 3 parts of fennel.