Application of TRUB1 as a target in primordial germ cell, primary oocyte or follicle specific marker and identification

Abstract

This invention discloses the application of TRUB1 as a target for specific labeling and recognition of primordial, primary oocytes, or follicles. In microproteomic analysis of human oocytes at various developmental stages, this invention revealed that the expression of certain proteins in oocytes exhibits developmental stage specificity. Screening for stage-specific expressed proteins and comparing them with data from the Human Protein Atlas, GEO, PubMed databases, and published mRNA data, it was found that TRUB1 is highly expressed in primary oocytes. Histochemical experiments confirmed that TRUB1 is specifically expressed in primordial and primary follicle oocytes. Therefore, it is proposed that the TRUB1 molecule can be used as a target for specific labeling and recognition of primordial and primary oocytes, assisting researchers in identifying and determining the developmental stage of oocytes, and providing an effective molecular target for primordial, primary oocytes, or follicles to facilitate targeted intervention or related molecular biology research.

Description

Technical Field

[0001] This invention relates to the field of reproductive technology, specifically to the application of TRUB1 as a target in the specific labeling and recognition of primordial, primary oocytes or follicles. Background Technology

[0002] The follicle is the basic biological unit in the female ovarian tissue. As the origin of life and the foundation of reproduction, the development of mammalian follicles involves a series of dynamic changes in morphology and function. Based on these morphological and functional changes, follicles can be classified into primordial follicles, primary follicles, secondary follicles, antral follicles, and mature follicles. Currently, the generally accepted follicle grading is mainly based on follicle morphology: primordial follicles consist of a single oogonium surrounded by a layer of flattened granulosa cells; primary follicles consist of a single oocyte surrounded by a layer of cuboidal granulosa cells; secondary follicles consist of multiple layers of granulosa cells surrounding a single oocyte, but without forming a follicular cavity; during the antral follicle stage, follicular fluid is produced between the granulosa cells, accumulating to form the antral cavity; before ovulation, the follicle volume increases significantly, follicular fluid increases significantly, the oocyte moves to one side, the cumulus oophorus complex forms, and under the influence of the LH surge, the oocyte releases the first polar body. At this point, the follicle matures and is released from the ovary.

[0003] Although morphological follicle grading is the currently accepted and widely used method, in clinical practice and scientific research, there are still cases where follicle morphology is ambiguous, making it difficult to accurately classify follicles into a specific grade. Furthermore, in the study of follicular growth and development mechanisms, many questions remain regarding how primordial follicles are activated from a quiescent state to become growing follicles. Specific markers for small follicles may help researchers quickly and conveniently obtain primordial or primary follicles, thereby further investigating the molecular mechanisms of their growth and development. Therefore, screening and identifying molecules specifically expressed at a particular developmental stage can not only help researchers classify morphologically ambiguous follicles using specific molecular markers, but also facilitate the screening of follicles at specific developmental stages for molecular mechanism investigation.

[0004] In recent years, the development of micro-sequencing and even single-cell sequencing technologies has provided a theoretical basis and data support for understanding the molecular changes at each stage of follicular development. By analyzing the transcriptome and proteome of each stage from primordial follicle to pre-ovulatory follicle, and using data analysis methods such as differential analysis and functional enrichment, molecules significantly expressed at a certain developmental stage can be identified. Through a series of screening and validation processes, molecules specifically expressed at a certain stage can then be screened for application in clinical practice and scientific research. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the above-mentioned background technology and provide an application of TRUB1 as a target for specific labeling and recognition of primordial, primary oocytes or follicles. This invention provides a molecule TRUB1 specifically expressed by primordial and primary oocytes by screening and providing it, so as to facilitate the identification of oocyte developmental stages, the identification and selection of oocytes at specific developmental stages, and to provide theoretical and experimental technical support for the study of the physiological and pathological mechanisms of oocyte development.

[0006] To achieve the above objectives, this invention provides an application of TRUB1 as a target in the specific labeling and recognition of primordial cells, primary oocytes, or follicles.

[0007] Furthermore, the amino acid sequence of TRUB1 is shown in SEQ ID NO:1.

[0008] Furthermore, the specific labeling of primordial and primary oocytes using TRUB1 as a target includes labeling primordial and primary oocytes using antigen-antibody binding technology, enzyme-linked reaction technology, direct binding technology with fluorescent dyes, genetic modification technology, and radionuclide technology with TRUB1 as a target.

[0009] Furthermore, the TRUB1 target for specific recognition of primordial and primary oocytes includes distinguishing primordial oocytes and primary oocytes from oocytes at other developmental stages by labeling TRUB1 in cell suspensions, cell cultures, tissue sections, and tissues and organs.

[0010] Furthermore, the primordial oocyte is an oocyte surrounded by a single layer of flattened granular cells.

[0011] Furthermore, the primary oocyte is an oocyte surrounded by a layer of cuboidal granular cells.

[0012] Furthermore, the oocytes include oocytes from humans, mice, rats, and monkeys; the oocytes include in vivo oocytes and isolated oocytes.

[0013] Furthermore, after TRUB1 serves as a target marker or identifies primordial oocytes, primary oocytes, or follicles, it can be used to identify and screen oocytes or follicles at other developmental stages.

[0014] This invention also provides an application of TRUB1 as a marker for primordial or primary oocytes in bioinformatics analysis, single-cell sequencing data analysis, and spatial sequencing analysis to assist in cell clustering and localization analysis.

[0015] This invention also provides an application of TRUB1 as a target in oocyte expression. Microprotein detection technology is used to detect protein expression in oocytes at various stages of follicle development. R software is used to perform differential protein analysis on protein expression in oocytes at different developmental stages, screening for molecules expressed only in one or multiple consecutive stages. By integrating mRNA data, the Human Protein Atlas database, the GEO database, and the PubMed database, the tissue-specific protein TRUB1 is screened out. High tissue specificity refers to being displayed as "tissue-enriched" or "group-enriched" in the database. R software is a complete data processing, computation, and graphing software system. Its functions include: data storage and processing system; array operation tools (especially powerful in vector and matrix operations); complete and coherent statistical analysis tools; excellent statistical graphing functions; and a simple yet powerful programming language: capable of manipulating data input and output, implementing branching and looping, and allowing users to customize functions.

[0016] Compared with the prior art, the present invention has the following advantages:

[0017] Firstly, this invention utilizes oocytes obtained during fertility preservation surgery at clearly defined developmental stages. Microprotein detection technology is used to analyze protein expression at each developmental stage of the oocytes. Integrating published mRNA data and supplementing with histochemical verification, it proposes that TRUB1 is a molecule specifically expressed in primordial and primary follicles. TRUB1 is a pseudouridine synthase. Pseudouridine modification is the earliest discovered and most abundant RNA modification, widely present in various RNAs (tRNA, rRNA, snRNA, etc.). TRUB1 introduces pseudouridine modification at position 55 of the TΨC loop during early tRNA maturation, and it also plays a role in mRNA. This invention, through studying data on the expression and distribution of TRUB1 in follicles at different developmental stages, confirms that TRUB1 can be used as a target for specific labeling and recognition of primordial and primary oocytes.

[0018] Secondly, TRUB1 of the present invention, as a specific marker molecule for primordial and primary oocytes, can assist scholars in identifying and determining the developmental stage of oocytes, screening oocytes or follicles at specific developmental stages for molecular biological research, thereby accelerating the exploration of theoretical knowledge of oocyte development mechanisms and promoting the rapid development of the field of reproductive technology.

[0019] Thirdly, this invention discloses the application of TRUB1 as a target in experimental techniques for specific labeling and recognition of primordial and primary oocytes. When performing microproteomic analysis on oocytes at various developmental stages, this invention discovered that the expression of certain proteins in oocytes exhibits developmental stage specificity. Screening for stage-specific expressed proteins and comparing them with data from the HumanProteinAtlas, GEO, PubMed databases, and published mRNA data revealed that TRUB1 is highly expressed in primary oocytes. Histochemical experiments verified that TRUB1 is specifically expressed in primordial and primary follicle oocytes. This invention proposes that the TRUB1 molecule can be used as a target for specific labeling and recognition of primordial and primary oocytes, assisting researchers in identifying and determining the developmental stage of oocytes, and providing effective molecular targets for primordial, primary oocytes, or follicles to facilitate targeted intervention or related molecular biology research. Attached Figure Description

[0020] Figure 1 A schematic diagram illustrating the working process of using microprotein detection technology to detect protein expression in oocytes at various stages of follicles;

[0021] Figure 2 PCA analysis of oocytes at each stage was performed using microprotein technology;

[0022] Figure 3 The microprotein assay revealed different trends in oocyte protein changes at various developmental stages.

[0023] Figure 4 Differential proteins at different developmental stages of oocytes were obtained through differential protein analysis using R software;

[0024] Figure 5 The amount of proteins specifically expressed at different oocyte developmental stages;

[0025] Figure 6 The expression level of TRUB1 at different developmental stages of oocytes was measured using microprotein technology in Example 1 of this invention.

[0026] Figure 7 This invention provides the expression levels of TRUB1 at different developmental stages of oocytes from published mRNA data for Example 2 of this invention;

[0027] Figure 8 The results of TRUB1 specifically labeling oocytes in the ovary are shown in Embodiment 3 of the present invention. Detailed Implementation

[0028] The following examples illustrate the implementation of the present invention in detail, but they do not constitute a limitation on the invention and are merely illustrative. Furthermore, the advantages of the present invention will become clearer and easier to understand by explaining them.

[0029] The main instruments used in this invention are: Orbitrap Fusion tandem mass spectrometer. TM Lumos TM Tribrid TM (Thermo Fisher Scientific, San Jose, CA), UltiMate 3000UHPLC (Thermo), as well as commonly used laboratory instruments such as low-temperature high-speed centrifuges, electrophoresis apparatus, microtome, ultraviolet spectrophotometer, and microscopes.

[0030] The samples used in this invention are human ovarian tissue and follicles.

[0031] Example 1: Expression of TRUB1 protein in human primary oocytes

[0032] The protein expression of oocytes in follicles at each stage was detected using microprotein detection technology. Differential proteins at each developmental stage were analyzed using R software. Molecules expressed only at a certain stage or several consecutive stages were screened. By combining the Human Protein Atlas database, GEO database, and PubMED database, proteins with low tissue specificity were eliminated. Finally, TRUB1, a protein specifically expressed only in the primary stage, was selected.

[0033] 1. Obtaining follicles at various developmental stages: Ovarian tissue is collected from patients who have undergone total or partial oophorectomy due to factors such as fertility preservation techniques. The tissue is cut into 0.5×0.5×1mm pieces and digested in 0.04mg / ml Liberase DH enzyme for 90 minutes. Follicles at each stage are then separated by mechanical needle puncture under a stereomicroscope.

[0034] 2. Separation of granulosa cells and oocytes from follicles: Place follicles in acutase digestive enzyme for 15 min, and carefully separate oocytes and granulosa cells under a stereomicroscope using an insulin needle. Use a Pasteur pipette to aspirate oocytes and granulosa cells into 200 μL low-absorption EP tubes.

[0035] 3. Protein proteometry: Each oocyte sample consists of 3-10 eggs, and each granulosa cell sample consists of 100 granulosa cells. Protein extraction, proteolysis, and mass spectrometry analysis are performed. The specific steps are as follows:

[0036] (1) Centrifuge 200 μL of low-adsorption EP tube for 2 min, add dithiothreitol to a final concentration of 5 mM, sonicate, add a protease inhibitor to a final concentration of 10 mM and react in the dark for 30 min, then add 1 μL of trypsin for enzymatic digestion.

[0037] (2) The enzymatically digested samples were separated using an UltiMate 3000 UHPLC. The samples were first enriched and desalted using a trap column, and then separated in tandem with a self-packed C18 column at a flow rate of 500 nl / min. The peptides separated by liquid chromatography were ionized using a nanoESI source and then detected by a Fusion Lumos tandem mass spectrometer in DDA mode.

[0038] Data Analysis: Trace protein sample data and library construction sample data were analyzed using the Andromeda engine integrated with MaxQuant, selecting "match between runs" for identification. MaxQuant then analyzed the data based on information such as peptide peak intensity, peak area, and liquid chromatography retention time related to primary mass spectrometry. A series of statistical analyses and quality controls were performed on the trace protein samples, followed by statistical analyses based on the protein identification results. Differential protein analysis was performed using R software. Protein specificity was determined by searching the Human Protein Atlas, GEO, and PubMed databases.

[0039] like Figure 6 As shown, TRUB1 protein is highly expressed during the human primary oocyte stage.

[0040] Example 2: TRUB1 mRNA is highly expressed in human primary oocytes.

[0041] Downloaded published mRNA data of human oocytes at various developmental stages and analyzed TRUB1 expression levels using software such as Graphpad and SPSS.

[0042] 1. Download the mRNA expression matrix of human oocytes at various developmental stages from the GEO database. The download link is https: / / www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc=GSE107746.

[0043] 2. Obtain the expression level of TRUB1 mRNA from the matrix.

[0044] 3. Use software such as Graphpad and SPSS to analyze the expression level of TRUB1 at different developmental stages of oocytes and draw expression level bar charts.

[0045] like Figure 7As shown, TRUB1 mRNA is highly expressed in human primary oocytes.

[0046] Example 3: TRUB1 specifically marks primordial and primary oocytes in human ovarian tissue sections.

[0047] Immunohistochemical staining of human ovarian tissue sections was performed using TRUB1 antibody, and cells expressing TRUB1 were located using DAB chromogenic agent. The specific implementation method is as follows:

[0048] 1. Preparation of paraffin sections from human ovarian tissue: Ovarian tissue was collected from patients who underwent total or partial oophorectomy due to factors such as fertility preservation techniques. After fixation in paraformaldehyde for 24 hours, the tissue was dehydrated in a dehydrator. After paraffin embedding and fixation, 4µm paraffin sections were cut using a microtome, mounted, and baked at 65°C for 20 minutes.

[0049] 2. Immunohistochemical staining: Ovarian tissue sections were subjected to immunohistochemical staining for TRUB1 protein. The TRUB1 protein antibody was purchased from Invitrogen. The specific procedures are as follows:

[0050] (1) Baking: Place slices of human ovarian tissue in a 65℃ oven and bake for 60 minutes;

[0051] (2) Dewaxing: The sections were dewaxed in the following order: 20 min of environmentally friendly dewaxing solution → 20 min of environmentally friendly dewaxing solution → 5 min of anhydrous ethanol → 5 min of 95% ethanol → 5 min of 85% ethanol → 5 min of 75% ethanol → 3 × 5 min of PBS for histochemistry.

[0052] (3) Antigen retrieval: Boil the antigen retrieval solution on high heat for 15 minutes, place the slice in the boiling antigen retrieval solution, boil on medium heat for 3 minutes, remove and cool in an ice-water complex.

[0053] (4) Remove endogenous peroxidase: Incubate the slices in 3% hydrogen peroxide for 20 min;

[0054] (5) Wash with PBS 3 times for 5 min each time for histochemistry;

[0055] (6) Block the slices with ready-to-use goat serum for 1 hour;

[0056] (7) Remove the blocking solution, incubate with TRUB1 primary antibody, place the humidified box in a 4°C refrigerator overnight, and the TRUB1 concentration is 1:100;

[0057] (8) Remove the humidified chamber, allow it to warm to room temperature for 30 minutes, and then wash with PBS 3 times for histochemistry.

[0058] (9) Incubate with HRP-labeled secondary antibody for 1 h, then wash 3 times with PBS;

[0059] (10) After DAB development for 1 min 30 s, the sections were placed in tap water to stop the process.

[0060] (11) Stain the nucleus with hematoxylin for 5 minutes, then rinse with tap water until the water is clear;

[0061] (12) Immerse in the differentiation solution for 3 seconds, then rinse with tap water;

[0062] (13) Soak in the blueing solution for 10 seconds, then rinse with tap water;

[0063] (14) Reverse alcohol gradient: 75% ethanol 5 min → 85% ethanol 5 min → 95% ethanol 5 min → anhydrous ethanol 5 min → environmentally friendly dewaxing solution 10 min → environmentally friendly dewaxing solution 10 min;

[0064] (15) After the fume hood is dried, seal the sheet with neutral resin;

[0065] (16) Observe and photograph under a microscope.

[0066] like Figure 8 As shown, TRUB1 is specifically expressed in primordial and primary oocytes in ovarian tissue and can be used to label and identify oocytes or follicles at this developmental stage.

[0067] The above are merely specific embodiments of the present invention. It should be noted that any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the protection scope of the present invention. Any other aspects not described in detail are prior art.

Claims

1. Application of TRUB1 protein as a target for specific labeling or recognition in human primary oocytes or human primary follicles.

2. The application according to claim 1, characterized in that: The amino acid sequence of the TRUB1 protein is shown in SEQ ID NO:

1.

3. The application according to claim 1, characterized in that: The TRUB1 protein is used as a target for specific labeling of primary oocytes, including labeling primary oocytes with TRUB1 protein using antigen-antibody binding technology, enzyme-linked reaction technology, direct binding technology with fluorescent dyes, genetic modification technology, or radionuclide technology.

4. The application according to claim 1, characterized in that: The TRUB1 protein is used as a target for specific recognition of primary oocytes, including distinguishing primary oocytes from oocytes at other developmental stages by labeling the TRUB1 protein in cell suspensions, cell cultures, tissue sections, or tissues and organs.

5. The application according to claim 3 or 4, characterized in that: The primary oocyte is an oocyte surrounded by a layer of cuboidal granular cells.

6. The application according to claim 5, characterized in that: The oocytes mentioned are isolated oocytes.

7. The application according to claim 1, characterized in that: After the TRUB1 protein is used as a target marker or to identify primary oocytes or primary follicles, it can be used to identify and screen oocytes or follicles at other developmental stages.

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