A method of activating asxl2-pcr2 axis and applications thereof

Abstract

The application belongs to the technical field of biology, and discloses a method for activating an ASXL2-PRC2 axis, wherein adenovirus is injected into the seminiferous tubule of an organism through an outgoing pipe to activate the expression of endogenous EZH2 and spermatocyte-specific ASXL2 in the organism. The method can be used for reducing impaired sperm motility, increasing sperm penetration and tubulin polymerization, inhibiting abnormal spermatogenesis, down-regulating the transcription level of a TZ marker, increasing the enrichment of SUZ12, EED and H3K27me3 in CEP162 and CEP290 promoters under hypoxic conditions, and slowing down hypoxia-induced abnormal spermatogenesis and spermatocyte activation of the ASXL2-PRC2 axis in the germ cell line of the organism.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for activating the ASXL2-PRC2 axis and its application. Background Technology

[0002] It is well known that hypoxia affects spermatogenesis; however, the underlying mechanisms remain largely unclear. To investigate these mechanisms, we placed 8-week-old male SD rats in a hypobaric chamber with 11.7% oxygen (simulating an altitude of 5800 meters), while age- and sex-matched control rats were placed under normal oxygen concentration (21%) for 10 weeks to study the effects of severe chronic hypoxia on rat spermatogenesis. The results showed that prolonged hypoxia exposure impaired the development of elongated spermatocytes. We also observed that under normal conditions, Asxl2 expression gradually decreased from spermatogonia to spermatocytes, then increased abruptly at the round spermatocyte stage, before gradually decreasing again. However, in hypoxic-treated cells, Asxl2 expression was reduced throughout spermatogenesis, with a significant difference between normal and hypoxic treatments, particularly at the round spermatocyte stage. Subsequent analysis suggested that chronic hypoxia exposure may lead to abnormal spermatogenesis by downregulating the Asxl2 / PRC2 axis and increasing CEP162 expression.

[0003] Meanwhile, we further investigated the relationship between ASXL2-PRC2 and male infertility by screening the Male Infertility Knowledge Base (MKI), finding that ASXL2 and PRC2 jointly regulate the occurrence and development of teratospermia and cryptorchidism. To demonstrate the clinical relevance of ASXL2 and PRC2 in spermatogenesis, we analyzed the correlation between ASXL2 and PcG gene expression using the Sequence Archive Reading (SRA) dataset, which consisted of expression data from 3 non-obstructive azoospermia (NOA) samples, 3 non-obstructive azoospermia samples, 13 normal sperm individuals, and 8 teratospermia individuals. Figure 1 As shown, the analysis revealed that ASXL2 or PRC2 expression was downregulated in patients with teratospermia and NOA.

[0004] In summary, the ASXL2-PRC2 axis is closely related to spermatogenesis abnormalities. Summary of the Invention

[0005] This invention provides a method for activating the ASXL2-PRC2 axis, which aims to activate the ASXL2-PRC2 axis through external methods, reduce the expression of CEP162, and thus prevent sperm abnormalities.

[0006] To achieve the above objectives, the present invention provides a method for activating the ASXL2-PRC2 axis, the specific steps of which are as follows:

[0007] Adenoviruses are injected into the seminiferous tubules of an organism via an efferent tube. The adenoviruses are used to activate the expression of endogenous EZH2 and spermatogenic cell-specific ASXL2 in the organism.

[0008] Furthermore, the adenovirus is either pAAV2-EFS-mAsxl2-3xFLAG-pA virus or pAAV2-EFS-3xFLAG-pa virus.

[0009] This invention also provides an application of a method for activating the ASXL2-PRC2 axis, wherein the method for activating the ASXL2-PRC2 axis is used to downregulate the transcriptional level of TZ markers and to increase the enrichment of SUZ12, EED and H3K27me3 in the CEP162 and CEP290 promoters under hypoxic conditions;

[0010] The TZ markers include CEP162 and CEP290.

[0011] The beneficial effects of this invention are as follows:

[0012] This invention provides a method for activating the ASXL2-PRC2 axis by injecting adenovirus into the seminiferous tubules of an organism via an efferent tube, thereby activating the expression of endogenous EZH2 and spermatogenic cell-specific ASXL2. This method can be used to alleviate impaired sperm motility, increase sperm penetration and microtubule polymerization, inhibit sperm abnormalities, downregulate the transcriptional levels of TZ markers, increase the enrichment of SUZ12, EED, and H3K27me3 in the CEP162 and CEP290 promoters under hypoxic conditions, and slow down hypoxia-induced abnormal spermatogenesis and spermatogenic cell activation of the ASXL2-PRC2 axis in germline organisms.

[0013] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0014] Figure 1 For clinical outcomes;

[0015] Figure 2 This represents exogenous overexpression of ASXL2 / EZH2 in mice;

[0016] Figure 3 This indicates that exogenous activation of the Asxl2-PRC2 axis can significantly reduce sperm tail abnormalities and alleviate hypoxia-induced abnormal spermatogenesis.

[0017] Figure 4 This study analyzes gene expression data for the median of teratospermia / NOA in the GEO / SRA dataset. Detailed Implementation

[0018] To make the technical solutions, advantages, and objectives of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the described embodiments of the present invention without creative effort are within the protection scope of this application.

[0019] This embodiment provides a method for activating the ASXL2-PRC2 axis, including the following steps:

[0020] Adenovirus pAAV2-EFS-mAsxl2-3xFLAG-pA or pAAV2-EFS-3xFLAG-pa virus was injected into the seminiferous tubules of mice via efferent tubes to activate the expression of endogenous EZH2 and spermatogenic cell-specific ASXL2, respectively, and their effects on spermatogenesis were examined.

[0021] Comparative experiment

[0022] Mice were exposed to an oxygen-deficient environment for 10 weeks, during which time they were injected every three weeks.

[0023] Experimental group: Eight-week-old male SD rats were placed in a hypoxic environment (a hypobaric chamber with an oxygen concentration of 11.7% to simulate an altitude of 5800 meters) for 10 weeks. During this period, adenovirus was injected every three weeks, that is, the adenovirus was injected into the seminiferous tubules of the mice through the efferent tube.

[0024] Control group: Eight-week-old male SD rats (control rats) were placed under normal oxygen concentration (21%) for 10 weeks.

[0025] Experimental Analysis

[0026] pAAV2-EFS-mAsxl2-3xflag-pA and pAAV2-CAG-EZH2-EGFP-3xflag-WPRE-pA were used to stain mouse testicular sections 3 weeks after virus injection. Figure 2 It can be seen that the ASXL2-PRC2 axis was successfully activated.

[0027] Figure 2This study illustrates the exogenous overexpression of ASXL2 / EZH2 in mice. A shows the immunostaining of ASXL2 10 weeks after microinjection of AAV2; B shows the immunostaining of EZH2 10 weeks after microinjection of AAV2; C shows the Western blot analysis of ASXL2 expression in wild-type untransplanted and microinjected testes within 10 weeks using AAV2; and D shows the Western blot analysis of EZH2 expression in wild-type untransplanted and microinjected testes within 10 weeks using AAV2.

[0028] Mice were exposed to 11.7% oxygen for 10 weeks and injected with either ASXL2-AAV or EZH2-AAV.

[0029] like Figure 3 As shown in A-3H, activation of the ASXL2-PRC2 axis significantly reduced impaired sperm motility.

[0030] We also found that activation of the ASXL2-PRC2 axis significantly increased sperm penetration. Figure 3 I) and microtubule polymerization, and significantly inhibited sperm abnormalities ( Figure 3 J).

[0031] Next, we examined the expression of free tubulin and polymeric microtubules in spermatogenic cells. Western blot analysis showed that after ASXL2 / PRC2 activation, free tubulin in spermatogenic cells decreased, while polymeric microtubules significantly increased. Figure 3 K).

[0032] Currently, it is generally believed that the components of cilia, including transciliary tract markers (TZs) such as CEP162 and CEP290, play an important role in spermatogenesis, and that multicombs are involved in regulating the transition region. Therefore, we investigated whether activation of the ASXL2-PRC2 axis alters the levels of TZ components. Mice were subjected to the same viral injection to activate the ASXL2-PRC2 axis. Indeed, qPCR analysis showed that under hypoxic conditions, activation of the ASXL2-PRC2 axis significantly downregulated the transcriptional levels of TZ markers, including CEP162 and CEP290. Figure 3 L and 3M, Figure 4 Furthermore, ChIP-qPCR analysis showed that activation of the ASXL2-PRC2 axis increased the enrichment of SUZ12, EED, and H3K27me3 in the CEP162 and CEP290 promoters under hypoxic conditions. Figure 3 In summary, these results indicate that activation of the ASXL2-PRC2 axis significantly rescued H3 histone 27 lysine methyltransferase activity and TZ transcription, and significantly reversed abnormal changes in TZ components, thus mitigating hypoxia-induced abnormal spermatogenesis and spermatogenic cell activation of the ASXL2-PRC2 axis in mouse germline cells.

[0033] Figure 3 This indicates that exogenous activation of the Asxl2-PRC2 axis can significantly reduce sperm tail deformities and alleviate hypoxia-induced abnormal spermatogenesis. Figure 3 In the study, (A) sperm survived for 10 weeks at 11.7% oxygen (n=12). Sperm motility parameters included (B) forward motility (n=12) and (C) linear velocity (n=12). Figure 3 D-3H represent the sperm motility parameters detected by an automated sperm analyzer after overexpression of ASXL2 and EZH2 in mouse sperm cells under hypoxic conditions, including curve velocity (D), average path velocity (E), forward trajectory (F), whipping frequency (G), and head lateral swing amplitude (H). (I) Acetic acid-magenta staining was used to observe the entry of spermatogonia into oocytes, and the percentage of sperm entering the oocyte nucleus (%) was calculated (n=12). (J) Quantitative analysis of abnormalities between WT and ASXL2-AAV and EZH2-AAV (n=12). (K) Immunoblot analysis of free tubulin and polymerized microtubules in spermatogenic cells (n=10). (L) qPCR analysis of ciliary assembly-related genes in primary spermatogenic cells between the ASXL2-AAV group and the control group (n=6). (M) qPCR was also used to compare the mRNA levels of CEP162 and CEP290 in GC2 cells between the ASXL2 overexpression treatment group, the EZH2 overexpression treatment group, and the control group. The mRNA expression values ​​of the treatment group were normalized to the expression values ​​of β-actin and expressed as the ratio of normalized mRNA expression value to mRNA expression value in control cells (n=10). Figure 3 After overexpressing ASXL2 and EZH2 in G2 cells (N-3P), respectively, Cut&tag-qPCR was used to detect the binding of SUZ12 (N), EED (O), and H3K27me3 (P) to the CEP162 and CEP290 promoters. In G2 cells treated with the control group, AD-ASXL2, or AD-EZH2, the binding of (N)SUZ12 (N=10) and (O)EED (N=10) to the CEP162 (left) or CEP290 (right) promoters was measured. Data are expressed as mean ± SEM. **P<0.01, ***P<0.001, ****P<0.0001, determined by two-tailed, unpaired Student's multiple t-test (L), one-way ANOVA, and Tukey's post-hoc test (AK, MP).

[0034] Figure 4 This study analyzed gene expression data of median teratospermia / NOA in the GEO / SRA dataset, including statistics of patients identified as having (A) teratospermia or (B) high or low expression of NOA-related genes.

[0035] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the protection scope of the present invention.

Claims

1. A recombinant adenovirus, characterized in that: The recombinant adenovirus is pAAV2-EFS-mAsxl2-3xFLAG-pA virus or pAAV2-EFS-3xFLAG-pA virus; the recombinant adenovirus can target and activate the expression of mouse endogenous EZH2 and spermatogenic cell-specific ASXL2, and under hypoxic conditions, it increases the enrichment of SUZ12, EED and H3K27me3 in the promoters of CEP162 and CEP290, and downregulates the transcription of the TZ marker CEP162 / CEP290.

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