Application of Tmx2 gene in assisted reproduction screening

Abstract

The invention belongs to the technical field of immunoassay, and particularly relates to application of a Tmx2 gene in assisted reproduction screening. The invention verifies that after the Tmx2 gene is knocked down in the embryo of a mouse, the proliferation of embryonic cells is inhibited, and the apoptosis of the embryonic cells is induced. The Tmx2 gene provided by the invention can be used as a marker for evaluating the development condition of an embryo, meanwhile, normal expression of the Tmx2 gene in the embryo can be ensured, the development rate of the embryo in assisted reproduction can be improved, meanwhile, a medicine for treating preimplantation embryo development retardation and recovering Tmx2 gene activation is prepared, and a new clinical scheme is provided for improving the development rate of an embryo in vitro in assisted reproduction.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunoassay, and specifically relates to the application of the Tmx2 gene in assisted reproductive screening. Background Art

[0002] Although assisted reproductive technology has been applied for decades, the pregnancy rate and delivery rate for each transplantation are still approximately 35% and 25% respectively. In order to improve the success rate of assisted reproductive technology and reduce the risk of having babies with genetic diseases, scholars have made many efforts in embryo screening. Human preimplantation embryos can stop developing at all stages between the zygote and the blastocyst. A large proportion of these embryos stop developing at the 2-cell and 8-cell stages and remain non-implanted, with failure of zygotic genome activation. Existing findings have reported that factors such as age-induced decline in oocyte quality, mutations in subcortical maternal complex (SCMC) genes, chromosomal abnormalities, metabolic abnormalities, and changes in in vitro culture conditions can all lead to early embryo developmental arrest.

[0003] By improving in vitro culture conditions and detecting and screening genes related to embryo arrest, the probability of embryo developmental arrest can be reduced to a certain extent.

[0004] TMX2 (Thioredoxin-related Transmembrane Protein 2) is a gene encoding a member of the endoplasmic reticulum (ER) protein disulfide isomerase family, which plays an important role in protein folding and thiol-disulfide exchange reactions. Research by the team of Xiao Fei at Sun Yat-sen University has shown that TMX2 enhances the viability of liver cancer cells by promoting autophagy and mitophagy, revealing the potential of TMX2 as a prognostic marker and therapeutic target for liver cancer. Other studies have also shown that TMX2 is associated with certain neurodevelopmental disorders, indicating that it may play an important role in nervous system development. Summary of the Invention

[0005] The present invention discovers that after knockdown of the Tmx2 gene, autophagy is downregulated, cell proliferation is inhibited, resulting in apoptosis and thus leading to embryo developmental arrest. Therefore, screening embryos with normal expression of the Tmx2 gene and ensuring the normal expression of the Tmx2 gene during embryo development can improve the developmental rate of preimplantation embryos in assisted reproduction in vitro.

[0006] To achieve the above object, the present invention discloses the application of the Tmx2 gene in assisted reproductive screening.

[0007] The application is any one or more of the following a1) to a4):

[0008] a1) for studying the molecular mechanism of embryo development;

[0009] a2) To construct an embryonic model of abnormal embryonic development;

[0010] a3) As a biomarker for detection to evaluate embryonic status;

[0011] a4) As a drug administration target to improve embryonic development status.

[0012] The knockdown of the Tmx2 gene results in:

[0013] 1) Inhibiting the proliferation of embryonic cells;

[0014] 2) Inducing apoptosis of embryonic cells;

[0015] 3) Aggravating mitochondrial dysfunction and triggering oxidative stress in embryos;

[0016] 4) Inhibiting mitophagy and autophagy of morula-stage embryos.

[0017] In a2), the knockdown of the Tmx2 gene in the embryonic model results in the developmental arrest of mouse embryos from the morula stage to the blastocyst stage.

[0018] In a3), embryos with normal Tmx2 gene expression are selected for culture and transplantation.

[0019] In a4), as a drug composition for administration, it can upregulate the expression or activity of the Tmx2 gene or the protein encoded by it at the protein or gene level.

[0020] The drug composition further comprises a pharmaceutically acceptable carrier.

[0021] The present invention also discloses a product for diagnosing embryonic development status, which comprises a reagent for detecting the expression level of the Tmx2 gene. Optionally, the product is a kit or a chip; optionally, the reagent is one or more of a primer, a probe, an antibody, or a ligand.

[0022] Among them, the reagent is selected from:

[0023] A probe that specifically recognizes Tmx2; or

[0024] A primer that specifically amplifies Tmx2; or

[0025] An antibody or ligand that specifically binds to the protein encoded by Tmx2.

[0026] As an embodiment of the present invention, any one pair of siRNA1-3 is used to knockdown the Tmx2 gene;

[0027] The sequence of siRNA1 is shown in SEQ ID NO: 3-4;

[0028] The sequence of siRNA1 is shown in SEQ ID NO: 5-6;

[0029] The sequence of siRNA1 is shown in SEQ ID NO: 7-8.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0031] In the embryos of mice, the present invention verifies that when the Tmx2 gene is knocked down, the proliferation of embryonic cells will be inhibited and embryonic cell apoptosis will be induced. The present invention proposes that the Tmx2 gene can be used as a marker to evaluate the development of embryos, and at the same time ensure the normal expression of the Tmx2 gene in embryos to improve the embryo development rate in assisted reproduction. At the same time, drugs for treating preimplantation embryo development arrest and restoring the activation of the Tmx2 gene are prepared, providing a new clinical plan for improving the in vitro embryo development rate in assisted reproduction. Brief Description of the Drawings

[0032] Figure 1 Showing the expression and localization of Tmx2 in preimplantation embryos of mice. Among them Figure 1 A is the immunofluorescence staining of TMX2 (red) and nuclear DNA (blue) from GV-stage oocytes to blastocyst-stage embryos; Control group: without primary antibody; GV: germinal vesicle-stage oocytes; MII: metaphase II oocytes; Zy: zygotic embryos; 2C: two-cell-stage embryos; 4C: four-cell-stage embryos; 8C: eight-cell-stage embryos; Mo: morula-stage embryos; Bl: blastocyst-stage embryos. Scale bar, 75 μm. Figure 1 B in it is the fluorescence intensity of TMX2 measured by ImageJ. Figure 1 C in it is the RT-qPCR analysis of the mRNA expression of Tmx2 from GV-stage oocytes to blastocyst-stage embryos. Figure 1 D in it shows that TMX2 protein shows partial co-localization with mitochondria and a small amount of ectopic expression at the morula stage. The relative expression level of Tmx2 is normalized to H2afz.

[0033] Figure 2 Showing the effect of Tmx2 knockdown on embryo development. Among them Figure 2 A in it is the determination of Tmx2 knockdown in blastocyst-stage embryos by RT-qPCR. Figure 2 B in it is a representative immunofluorescence image of TMX2 expression after Tmx2 knockdown at the blastocyst stage. Figure 2 C in it is the morphology of blastocysts in the control group and the Tmx2 knockdown group, scale bar, 200 μm. Figure 2 D in it is the blastocyst formation rate in the Tmx2 knockdown group and the control group. Figure 2 E in it is the hatching product of embryos after Tmx2 knockdown. Figure 2In this, F represents the hatching rate of blastocysts in the Tmx2 knockdown group and the control group. (**: p < 0.01; ****: p < 0.0001).

[0034] Figure 3 It shows that Tmx2 knockdown inhibits cell proliferation. Among them Figure 3 In this, A and C are representative images of embryos in the control group and the Tmx2 knockdown group at E3.0 and E4.0, showing EdU positive signals; scale bar, 75 μm. Figure 3 In this, B and D are the ratios of the number of EdU-positive labeled nuclei to the total number of nuclei (proliferation ability index) in the control group and the Tmx2 knockdown group. Figure 3 In this, E is a representative image of DCFH-DA staining in the control group and the Tmx2 knockdown group; scale bar, 200 μm. Figure 3 In this, F is the quantification of ROS levels (fluorescence intensity) in control and Tmx2 knockdown embryos. Figure 3 In this, G is the RT-qPCR analysis of Sod1 and Sod2 mRNA expression in embryos of the control group and the Tmx2 knockdown group. **p < 0.01, ***p < 0.001, and ****p < 0.0001 are considered to indicate significant differences.

[0035] Figure 4 It shows that Tmx2 knockdown induces embryonic apoptosis. Among them Figure 4 In this, A and C are TUNEL assays performed on embryos of the control group and the Tmx2 knockdown group at E3.0 and E4.0; scale bar, 75 μm. Figure 4 In this, B and D are the quantification of apoptotic signals in embryos of the control group and the Tmx2 knockdown group. Figure 4 In this, E is the RT-qPCR analysis of the abundances of p21 and p53 in embryos of the control group and the Tmx2 knockdown group at E3.0. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 are considered to indicate significant differences.

[0036] Figure 5 It shows that Tmx2 knockdown interferes with mitochondrial function and increases ROS in morula-stage embryos. Figure 5 In this, A is an image of mitochondrial abundance in embryos of the control group and the Tmx2 knockdown group in the Mito-Tracker Deep Red FM detection experiment; scale bar, 75 μm. Figure 5 In this, B is the quantification of immunofluorescence images showing morula-stage embryos of the control group and the Tmx2 knockdown group. Figure 5 In this, C is the JC-1 staining of morula-stage embryos of the control group and the Tmx2 knockdown group; scale bar, 75 μm. Figure 5 In this, D is the ratio of JC-1 polymers to monomers, indicating the membrane potential state of embryos in the control group and the Tmx2 knockdown group. Figure 5In panel E are representative images of DCFH-DA staining in the control group and the Tmx2 knockdown group; scale bar, 200 μm. Figure 5 In panel F is the fluorescence intensity quantifying the ROS levels in embryos of the control group and the Tmx2 knockdown group. Figure 5 In panel G is the RT-qPCR analysis of the mRNA expression of Sod1 and Sod2 in embryos of the control group and the Tmx2 knockdown group. **p < 0.01, ***p < 0.001, and ****p < 0.0001 are considered to indicate significant differences.

[0037] Figure 6 Shows that Tmx2 knockdown inhibits mitophagy and autophagy in morula-stage embryos. Among them Figure 6 In panels A-D are immunofluorescence stainings of PINK1 (A), PARKIN (B), MAP1LC3B (C), and LAMP1 (D); scale bar, 75 μm. Figure 6 In panels E-H are the quantitative results of the immunofluorescence images of morula-stage embryos in the control group and the Tmx2 knockout group, showing PINK1 (E), PARKIN (F), MAP1LC3B (G), and LAMP1 (H) in embryos of the control group and the Tmx2 knockout group. **p < 0.01, ***p < 0.001, and ****p < 0.0001 are considered to indicate significant differences.

[0038] Figure 7 Is a schematic diagram of a microinjection dish. Detailed implementation mode

[0039] The present invention will be further described below by way of examples, but the present invention is not limited to the scope of the described examples. For the experimental methods without specific conditions noted in the following examples, they are carried out according to conventional methods and conditions, or selected according to the product specifications. The raw materials and equipment used in the examples are well-known to those skilled in the art and can be purchased, easily obtained, or prepared in the market.

[0040] Example 1

[0041] 1. Collection and culture of mouse pre-implantation embryos at various stages in vivo

[0042] The oocytes used in this experiment were divided into two types: germinal vesicle (GV) oocytes and metaphase II (MII) oocytes. To collect GV oocytes, 6 IU of PMSG solution was intraperitoneally injected into 8-week-old female B6D2F1 mice. Forty hours after injection, the whole ovaries were dissected out and placed in M2 medium. A syringe was used to puncture the follicles to obtain cumulus oocyte complexes (COCs), and the cumulus cells were mechanically removed to obtain GV-stage oocytes. To obtain MII oocytes, 6 IU of PMSG solution was first injected, followed by 6 IU of hCG solution. Thirteen hours after hCG solution injection, the oviducts were dissected and the ampulla was punctured to release ovulated MII oocytes. A 0.1 mg / mL hyaluronidase solution was used to remove the cumulus cells. For the collection of fertilized eggs at each stage in vivo, the general procedure was to first inject 6 IU of PMSG solution and 6 IU of hCG solution to stimulate mouse ovulation and estrus, and then collect embryos at each stage at different time points. The specific operation was that after superovulation, female mice were mated with 9-week-old male B6D2F1 mice. Twenty-two hours after hCG solution injection, the ampulla of the oviduct was torn to obtain pronuclear (PN)-stage embryos; 34 hours after hCG solution injection, the whole oviduct was removed to obtain 2-cell-stage embryos; 58 hours after hCG solution injection, the whole oviduct was removed to obtain 4-cell-stage embryos; 68 hours after hCG solution injection, the whole oviduct was removed to obtain morula (Mo)-stage embryos; 78 hours after hCG solution injection, the whole uterus was removed to obtain blastocyst (Bl)-stage embryos. After removing the cumulus cells, the fertilized eggs were washed three times in M2 medium and KSOM medium, and finally transferred to KSOM medium covered with mineral oil and placed in an incubator at 37 °C and 5% CO2 for in vitro culture. It should be noted that this step needs to minimize the in vitro operation time of the embryos as much as possible, with good environmental conditions to avoid contamination. During culture, it is necessary to pay attention to the appropriate conditions in the incubator and maintain a sterile state.

[0043] 2. Microinjection

[0044] 1) Preparation of microinjection needles and embryo fixation needles

[0045] The needle puller used in this experiment has the best effect at room temperature. If the temperature is low, the air conditioner needs to be turned on in advance to raise the environmental temperature of the needle puller to room temperature, and then the air conditioner is turned off. Then, about 0.2 MPa of CO2 gas was introduced into the needle puller. After adjusting the parameters of the needle puller, the borosilicate glass tube was placed in the needle puller (injection needle: general No. 2; fixation needle: general No. 28) to prepare injection needles and fixation needles.

[0046] To further prepare the injection needle and the fixation needle, they need to be placed on a needle forging instrument. First, place the injection needle horizontally on the instrument, adjust the focal length of the microscope for easy observation, and ensure that the glass bead is tangent to the position of the injection needle with a diameter of approximately 5 μm. Then, cut the injection needle at the contact point by heating the glass bead, and try to avoid bending the needle tip. Next, place the injection needle perpendicular to the needle forging instrument, readjust the focal length of the microscope and the position of the needle, place the glass bead at the position with a diameter of approximately 100 μm of the injection needle, and bend the injection needle by 15° by heating. Finally, store the prepared injection needle for later use.

[0047] After that, the fixation needle also needs to be processed similarly on the needle forging instrument. Place the fixation needle horizontally and adjust the focal length of the microscope, corresponding to the position of the injection needle, and make the glass bead tangent to the position of the fixation needle with a diameter of approximately 90 - 100 μm. Heat the glass bead to cut the fixation needle at the contact point, and try to keep the fracture surface flat at the same time. Subsequently, adjust the fixation needle to the fracture surface, and heat the glass bead to make the diameter of the fracture surface reach approximately 20 μm. Finally, bend the fixation needle 90° twice in the opposite direction on the alcohol lamp, similar to the "Z" shape. The completed fixation needle can be placed in a needle box for later use.

[0048] 2) Preparation of the microinjection dish

[0049] The siRNA used in this experiment was purchased from GenePharma, and the sequences are shown in Table 1 below:

[0050] Table 1 siRNA sequences

[0051]

[0052] Before using the siRNA, please operate according to the instruction manual. The specific steps are as follows: Place the centrifuge tube containing 1 OD siRNA in a centrifuge at 4 °C and centrifuge at a speed of 12000 rpm for 5 minutes. Subsequently, add 31.2 μL of DEPC water to dissolve the siRNA, and then aliquot it into RNase-free centrifuge tubes at 1.5 μL per tube and store them in a -20 °C refrigerator for later use.

[0053] Take a 60 mm diameter black culture dish (Corning, 430166) and prepare the droplets as Figure 7 shown. Add 15 μL to each 10% PVP droplet and M2 droplet, and add 1.5 μL to each Tmx2 siRNA droplet and control siRNA droplet. Finally, cover all the droplets with mineral oil.

[0054] 3) Specific steps of microinjection

[0055] First, use an insulin syringe to inject about 5 mm of mercury into the rear part of the microinjection needle, and then install the injection needle on the injection arm of the micromanipulation. At the same time, fix the fixing needle bent in a "Z" shape on the fixing arm of the micromanipulation. Subsequently, place the microinjection dish on the stage at 37 °C, adjust the position of the stage so that the first 10% PVP droplet is at the center of the field of view. Next, adjust the operating arm, move the injection needle above the first droplet, and drain the mercury in the needle to the tip, then place the injection needle into the 10% PVP droplet. To clean the inner wall of the injection needle, aspirate and expel the liquid in 10% PVP repeatedly 3 times, and then move the stage so that the injection needle is cleaned in the M2 liquid to remove the residues on the outer wall. After cleaning, adjust the stage so that the injection needle is in the siRNA droplet, rotate the injection oil pump, aspirate the liquid to the bent part of the needle, and let it stand for 1 minute for balance. Then, move the injection needle to the second M2 droplet, and adjust the fixing needle to the same height as the injection needle.

[0056] Carefully take out the culture dish from the incubator, use a mouth pipette to take out the PN-stage embryos to be injected, and wash them 5 times in M2 liquid. Then, use a mouth pipette to transfer the embryos to the M2 droplet where the fixing needle and the injection needle are located, and adjust the focal length so that the embryos, the fixing needle, and the injection needle are in the same plane. In this way, injection can be started. After injection, use a mouth pipette to take out the injected embryos, wash them 5 times in KSOM medium, and transfer them to a KSOM culture dish after washing, and place them in an incubator at 37 °C and 5% CO2 for continued culture.

[0057] 3. Extraction of total RNA and RT-qPCR

[0058] 1) Embryo collection

[0059] Before collecting the total RNA of the embryos, first thaw the previously prepared embryo lysate on ice, and then carefully wash the embryos that need to extract total RNA five times in M2 medium, and then transfer the embryos to the corresponding volume of lysate (the volume of lysate is in the ratio of 1 μL of lysate per embryo). It should be noted that as little M2 liquid as possible is carried during the transfer process (to avoid the influence of M2 liquid on the subsequent quantification results). Subsequently, centrifuge briefly, place it at room temperature for 5 minutes for the lysate to react fully with the embryos, then gently flick the tube body, and then heat it in a 75 °C metal bath for 5 minutes to inactivate the enzyme DNase I. Finally, store the obtained lysate containing the total RNA of the embryos in a -80 °C refrigerator and use it as soon as possible to prevent RNA degradation.

[0060] 2) RT-qPCR

[0061] Before RT-qPCR operation, first spray RNase scavenger on the laminar flow bench and then dry it. After that, put the required consumables into the laminar flow bench and irradiate with ultraviolet for 30 min. Place the reagents required in the One Step TB PrimeScript TM PLUS RT-PCR Kit (Perfect Real Time) on an ice box for thawing. All steps during the mixing of the liquid are completed on ice.

[0062] Prepare the reaction system according to the following table, dispense it into an eight-strip tube, centrifuge to remove air bubbles, and then put it into a BIO-RAD quantitative PCR instrument to perform RT-qPCR according to the reaction conditions in the following table.

[0063] The reaction system is shown in Table 2 below:

[0064] Table 2 RT-qPCR reaction system

[0065]

[0066]

[0067] The reaction conditions are shown in Table 3 below:

[0068] Table 3 RT-qPCR reaction conditions

[0069]

[0070] The primer sequences are shown in Table 4 below:

[0071] Table 4 RT-qPCR reaction primers

[0072]

[0073]

[0074] 4. Fluorescent staining

[0075] 1) Immunofluorescent staining

[0076] The specific experimental procedures of immunofluorescence have been described in detail in previous publications. Briefly, embryos were first fixed in 4% paraformaldehyde (PFA) solution at room temperature for at least 30 minutes. After fixation, the embryos were washed 5 times with PBS containing 0.3% PVA to remove the remaining 4% PFA on the cell surface. Next, according to the instructions of the primary antibody reagent, the embryos were permeabilized in PBS containing 0.5% Triton X-100 for 0.5 - 1 hour, and then blocked with PBS containing 2% BSA at room temperature for 1 - 1.5 hours. After blocking, the embryos were transferred to the pre-prepared primary antibody solution and incubated overnight at 4°C. The primary antibodies used in this study included rabbit anti-TMX2 (1:50 dilution), rabbit anti-PINK1 (1:50 dilution), rabbit anti-PARKIN (1:50 dilution), rabbit anti-MAP1LC3B (1:50 dilution), and rabbit anti-LAMP1 (1:100 dilution) antibodies.

[0077] After washing several times with PBS containing 0.1% Tween 20 and 0.01% Triton X-100, the corresponding secondary antibody was added for staining and incubated at 37°C for 1 hour. Then, it was washed several times again with PBS containing 0.1% Tween 20 and 0.01% Triton X-100. Finally, the embryo DNA was stained with an anti-quenching mounting medium containing DAPI, and the slides were observed under a confocal laser scanning microscope. The average fluorescence intensity of positive cells was analyzed using ImageJ software.

[0078] 2) EdU fluorescence staining

[0079] According to the instructions of the Cell-Light EdU Apollo567 in vitro kit, KSOM medium containing EdU was pre-prepared and pre-equilibrated in an incubator at 37°C and 5% CO2 for 2 hours. Subsequently, the embryos were transferred to the equilibrated culture medium drop and incubated under the same conditions for 2 hours. After incubation, the embryos were transferred to 4% paraformaldehyde (PFA) for fixation for at least 30 minutes. After fixation, the embryos were transferred to PBS containing 5% Triton X-100 for permeabilization for 40 minutes. After permeabilization, the embryos were transferred to the pre-prepared EdU staining solution and incubated at room temperature for 35 minutes. Then, the embryos were washed twice with PBS solution containing 0.5% Triton X-100 for 15 minutes each time. Finally, the embryo DNA was stained with an anti-quenching mounting medium containing DAPI, and images were observed and taken under a Leica DMI6000 confocal laser scanning microscope. The average fluorescence intensity of positive cells was analyzed using ImageJ software to evaluate cell proliferation.

[0080] The dye formulation is shown in Table 5 below:

[0081] Table 5 EdU Dye Formulation

[0082]

[0083] 3) TUNEL Fluorescent Staining

[0084] According to the instructions of the one-step TUNEL cell apoptosis assay kit, the embryos were fixed in 4% paraformaldehyde (PFA) at room temperature for at least 30 minutes. Subsequently, the embryos were transferred to PBS containing 0.5% Triton X-100 for permeabilization for 1 hour. After permeabilization, the embryos were transferred to freshly prepared TUNEL detection solution and incubated at 37 °C for 1 hour for labeling apoptotic cells. After labeling, the embryos were washed 3 times with PBS solution containing 0.1% Tween 20 and 0.01% Triton X-100 for 20 minutes each time to remove unbound reagents. Finally, the embryo DNA was stained with DAPI-containing anti-quenching mounting medium and pressed onto a glass slide. Images were captured using a Leica TCS SP5 II confocal microscope with an excitation wavelength of 488 nm, and the average fluorescence intensity of positive cells was analyzed using ImageJ software to evaluate apoptosis.

[0085] The dye formulation is as shown in Table 6 below:

[0086] Table 6 TUNEL Dye Formulation

[0087]

[0088] 4) ROS Fluorescent Staining

[0089] According to the instructions of the ROS detection kit, DCFH-DA was first diluted in KSOM medium at a ratio of 1:300 and incubated at 37 °C under 5% CO2 for 1 hour. Subsequently, the morulae were transferred to a balanced culture droplet and incubated at 37 °C for 20 minutes. After incubation, the embryos were washed 5 times with 0.3% PVA-PBS to remove unbound probes. Then, the embryos were transferred to a confocal dish, and images were captured using a Leica TCS SP5 II confocal microscope with an excitation wavelength of 488 nm. Finally, the images were analyzed using ImageJ software to evaluate the level of intracellular reactive oxygen species (ROS).

[0090] 5) Mito-Tracker Deep Red FM Fluorescent Staining

[0091] According to the manufacturer's instructions, first dilute the Mito-Tracker Deep Red FM dye with KSOM solution to a concentration of 500 nmol / mL, then make drops in a culture dish, and finally place it in an incubator at 37 °C and 5% CO2 for pre-equilibration for 2 hours. After equilibration, transfer the embryos to the culture dish and incubate at 37 °C for 20 minutes. After incubation, wash five times with M2 medium, then stain the embryo DNA with an anti-quenching mounting medium containing DAPI, press it on a glass slide, and observe it under a Leica TCS SP5 II confocal microscope. Or fix it with 4% PFA and co-stain with other dyes.

[0092] 6) JC-1 fluorescence staining

[0093] According to the manufacturer's instructions, incubate the embryos with 20 μg / mL JC-1 supplemented at 37 °C and 5% CO2 for 30 minutes. After incubation, wash five times with M2 medium, then place the embryos in a drop of M2 medium on a confocal dish and observe under a Leica TCS SP5 II confocal microscope. The ratio of the red fluorescence intensity of activated mitochondria (J-polymers) to the green fluorescence intensity of less-activated mitochondria (J-monomers) is calculated as the membrane potential by ImageJ.

[0094] 5. Outgrowth experiment

[0095] First, use the Outgrowth growth medium to form a single droplet in a culture dish. To prevent droplet evaporation, cover the culture dish with mineral oil. Before use, pre-equilibrate the culture dish at 37 °C and 5% CO2 for at least 12 hours. Subsequently, carefully transfer the embryos developed to the blastocyst stage to these culture droplets, place three embryos in each droplet, and return them to the incubator for further incubation. Three days later, evaluate the outgrowth by morphology to judge the development of the embryos. Specifically, if the embryos show a unique inner cell mass (ICM) colony surrounded by a single layer of trophoblast, it is considered normal growth; while the embryo growth that fails to hatch or lacks an ICM colony or a single layer of trophoblast is considered a failure.

[0096] Example 2 Spatiotemporal expression of Tmx2 in each stage of mouse pre-implantation embryos

[0097] According to the observed phenotypic characteristics and lethal time, we systematically evaluated the expression of Tmx2 during pre-implantation and gastrulation stages. We first used RT-qPCR to study the expression profile of Tmx2 during the entire pre-implantation development process. The results showed that the transcriptional level of Tmx2 gradually decreased from GV-stage oocytes to the 2-cell stage, and then turned upregulated from the 4-cell stage, with the highest transcriptional level at the morula stage ( Figure 1In addition, immunofluorescence staining was used to evaluate the expression and localization of TMX2 protein during pre-implantation embryo development. The results showed that TMX2 protein was highly expressed at the 4-cell, 8-cell, and morula stages compared to other stages, and was mainly localized in the cytoplasm outside the nucleus of pre-implantation embryos ( Figure 1 in B and Figure 1 in C). The mitochondrial fluorescent probe Mito-Tracker Deep Red FM was used in this experiment to determine whether TMX2 co-localized with mitochondria. The results showed that TMX2 co-localized with mitochondria at the 2-cell, 4-cell, and 8-cell stages, while TMX2 protein showed partial co-localization and a small amount of ectopic expression with mitochondria at the morula stage ( Figure 1 in D). This indicates that TMX2 anchors to mitochondria and functions at the 2-cell, 4-cell, and 8-cell stages, while TMX2 may also play certain functions in other parts at the morula stage.

[0098] Example 3 Tmx2 knockdown embryo model

[0099] To develop a more effective platform for studying the function of Tmx2 during development, a Tmx2 knockdown (KD) embryo model was established by microinjection and RNAi interference techniques. The knockdown efficiency of Tmx2 siRNAs was detected by RT-qPCR and immunofluorescence staining. As Figure 2 shown in A and B, compared with the control group, siRNA2 and siRNA3 effectively knocked down Tmx2 transcripts and proteins (p<0.0001). These findings indicate that Tmx2-siRNA2 and siRNA3 can reduce Tmx2 expression. Morphological analysis showed that the blastocyst formation rate in the siRNA2 knockdown group was close to zero, and the blastocyst formation rate in the siRNA3 knockdown group was about two-thirds of that in the control group ( Figure 2 in C and D). After determining the decrease in blastocyst formation rate, we then studied the quality of the surviving blastocysts, and we analyzed the hatching ability of these surviving blastocysts. The results of the 72h Outgrowth experiment showed that the control group blastocysts grew obvious inner cell mass (ICM) colonies (red dotted line), surrounded by strongly proliferating trophoblast (TE) cells (blue dotted line); while in the Tmx2 knockdown group (siRNA3), most of the blastocysts in the morula could not hatch from the zona pellucida and could not normally hatch and grow outgrowths ( Figure 2 in E). Through statistical analysis, the successful hatching rate of the control group blastocysts (86.52%) was significantly higher than that of the KD group blastocysts (64.78%) ( Figure 2 in F). These experimental results confirmed that both siRNA2 and siRNA3 can effectively knock down the expression of TMX2 and affect the quality of blastocysts from both morphological and molecular potential aspects.

[0100] Example 4: Knockdown of Tmx2 inhibits cell proliferation

[0101] The decrease in the number of cells in the blastocyst is the main reason for the low implantation rate of the blastocyst. The total cell count results ( Figure 3 in B) showed that the knockdown of Tmx2 might affect blastocyst implantation by inhibiting cell proliferation. Therefore, we used EdU assay to study the effect of Tmx2 knockdown on cell proliferation. The results showed that at E3.0, the intensity of EdU-positive signals in the siRNA2- and siRNA3-knockdown embryos was significantly lower than that in the control embryos, and the intensity of EdU-positive signals in the siRNA2-knockdown embryos was slightly lower than that in the siRNA3-knockdown embryos ( Figure 3 in A and B). This might be because the knockdown efficiency of siRNA3 was lower than that of siRNA2, resulting in a lower degree of influence of siRNA3 knockdown on embryonic development. In addition, we detected the EdU signal intensity of the siRNA3-knockdown embryos at E4.0, and the results showed that the inhibitory effect of Tmx2 knockdown on cell proliferation persisted until the blastocyst stage ( Figure 3 in C and D). These results confirmed that the knockdown of Tmx2 significantly inhibited the proliferation of embryonic cells during the pre-implantation embryonic development stage.

[0102] Example 5: Knockdown of Tmx2 induces cell apoptosis

[0103] The decrease in the total cell number might also be attributed to the increase in cell apoptosis. Therefore, we used TUNEL staining to identify apoptotic cells. As Figure 4 shown in A and B, at E3.0, the ratio of the number of TUNEL-positive signals to the total number of cell nuclei in the siRNA2-knockdown embryos was significantly greater than that in the control embryos. At E3.0, the knockdown of siRNA3 did not affect cell apoptosis. This might be because the knockdown efficiency of siRNA3 was lower than that of siRNA2, resulting in a relatively late effect of siRNA3 knockdown on embryonic development. Therefore, we detected the apoptosis of the siRNA3-knockdown embryos at E4.0, and the results showed that siRNA3 knockdown induced cell apoptosis. These results indicated that Tmx2 knockdown led to cell apoptosis ( Figure 4 in C and D). P21 is a traditional cell cycle inhibitor, and P21 is an important target of P53. The increased p53 transcription triggers cell cycle arrest and cell apoptosis. Therefore, we quantitatively detected the mRNA expression of p53 and p21. As Figure 4 shown in E, the expressions of p53 and p21 were both upregulated in the knockdown group. Moreover, the level of p53 transcripts in the siRNA2-knockdown embryos was higher than that in the siRNA3-knockdown embryos, which was consistent with the TUNEL detection results. The level of p21 transcripts in the siRNA2-knockdown embryos was lower than that in the siRNA3-knockdown embryos, which was consistent with Figure 4There were differences in the EdU detection results between A and B, which might be related to the fact that the degradation of p21 can promote apoptosis. In summary, these results indicate that Tmx2 knockdown induces apoptosis in embryonic cells.

[0104] Example 6 Knockdown of Tmx2 impairs mitochondrial function and increases the ROS level in arrested morula-stage embryos

[0105] As Figure 1 shown in D of Figure 5 At the 2-cell to 8-cell stage, TMX2 co-localizes with mitochondria, which are the main endogenous producers of ROS. Therefore, we inferred that Tmx2 knockdown might affect mitochondrial function and oxidative stress. We first determined the number and membrane potential level of mitochondria to evaluate mitochondrial function. As Figure 5 shown in A of Figure 5 we observed a significant decrease in the fluorescence density of Mito-Tracker Deep Red FM in Tmx2 knockdown embryos, indicating a decrease in the number and membrane potential of mitochondria. We also measured the mitochondrial membrane potential using JC-1 dye. As Figure 5 shown in B of

[0106] Example 7 Tmx2 knockdown inhibits mitophagy and autophagy

[0107] TMX2 has been shown to promote cytoprotective mitophagy and autophagy during oxidative stress. Therefore, we hypothesized that Tmx2 knockdown could inhibit mitophagy and autophagy in pre-implantation embryos. To test our hypothesis, we performed immunofluorescence staining for PINK1 and PARKIN, which are the main mitophagy-related proteins. The protein expression levels of PINK1 and PARKIN in Tmx2 knockdown embryos were significantly lower than those in control embryos Figure 6 shown in A-B of Figure 6In (E-F). To further monitor autophagy and mitophagy inhibition, we monitored the expression of MAP1LC3B, which is crucial for the formation of mature autophagosomes. The expression of MAP1LC3B was reduced in Tmx2 knockdown embryos, indicating a decrease in autophagosome formation ( Figure 6 In (C) and Figure 6 In (G). Since autophagosomes containing abnormal intracellular proteins or damaged organelles fuse with lysosomes for hydrolysis, we measured the lysosome content in embryos by immunostaining with LAMP1, a type I transmembrane glycoprotein mainly located in lysosomes and late endosomes. The results showed that the LAMP1 positive signal was significantly downregulated ( Figure 6 In (D) and Figure 6 In (H), indicating that Tmx2 knockout inhibited mitophagy and autophagy in morula-stage embryos.

[0108] It should be noted that the above are only the preferred specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, makes equivalent substitutions or changes, and should be covered by the protection scope of the present invention.

Claims

1. Application of the Tmx2 gene in assisted reproductive screening.

2. Use of the Tmx2 gene according to claim 1 in assisted reproductive screening, characterized in that, The application is any one or more of the following a1) to a4): a1) For studying the molecular mechanism of embryonic development; a2) For constructing an embryonic model with abnormal embryonic development; a3) As a detection marker for evaluating embryonic status; a4) As a drug administration target for improving embryonic development status.

3. The application of the Tmx2 gene according to claim 2 in assisted reproductive screening, characterized in that, Knockdown of the Tmx2 gene results in: 1) Inhibition of the proliferation of embryonic cells; 2) Induction of apoptosis of embryonic cells; 3) Aggravation of mitochondrial dysfunction and triggering of oxidative stress in embryos; 4) Inhibition of mitophagy and autophagy in morula-stage embryos.

4. Use of the Tmx2 gene according to claim 2 in assisted reproductive screening, characterized in that, In a2), knockdown of the Tmx2 gene in the embryonic model causes the development of mouse embryos to be blocked from the morula stage to the blastocyst stage.

5. Use of the Tmx2 gene according to claim 2 in assisted reproductive screening, characterized in that, In a3), embryos with normal Tmx2 gene expression are selected for culture and transplantation.

6. Use of the Tmx2 gene according to claim 2 in assisted reproductive screening, characterized in that, In a4), as a pharmaceutical composition for drug administration, it can up-regulate the expression or activity of the Tmx2 gene or the protein encoded by it at the protein or gene level.

7. Use of the Tmx2 gene according to claim 6 in assisted reproductive screening, characterized in that, The pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

8. A product for diagnosing the embryonic development status, characterized in that, It contains a reagent for detecting the expression level of the Tmx2 gene. Optionally, the product is a kit or a chip; optionally, the reagent is one or more of a primer, a probe, an antibody, or a ligand.

9. The product according to claim 8, wherein The reagent is selected from: A probe that specifically recognizes Tmx2; or A primer that specifically amplifies Tmx2; or An antibody or ligand that specifically binds to the protein encoded by Tmx2.

10. The application of the Tmx2 gene according to claim 3 in assisted reproductive screening, characterized in that, Knockdown of the Tmx2 gene using any one pair of siRNA1-3; wherein the sequence of siRNA1 is shown as SEQ ID NO: 3-4; or, the sequence of siRNA1 is shown as SEQ ID NO: 5-6; or, the sequence of siRNA1 is shown as SEQ ID NO: 7-8.

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