Use of sorbs3 as a target for the specific labeling and identification of secondary, cumulus or follicle cells

Abstract

The application discloses application of SORBS3 as a target point in specific labeling and identification of secondary oocytes and follicles. When micro-proteomics detection is carried out on oocytes at various development stages of human beings, it is found that the expression of some proteins of the oocytes has development stage specificity. Screening of the stage-specific expression proteins of the oocytes at various stages finds that the SORBS3 protein is highly expressed at the secondary oocyte stage, is verified by comparison with various databases, and is verified by immunohistochemical verification of ovary sections, and it is confirmed that the SORBS3 protein is specifically expressed in the cytoplasm and cell membrane of the oocytes of the secondary follicle and the follicle. The application proposes that the SORBS3 molecule can be used as a target point for specific labeling and targeted intervention of the secondary oocyte and the follicle, assists researchers in identifying and judging the development stage of the oocyte, and provides an effective molecular target for targeted intervention or development and function-related research of the secondary oocyte and the follicle at the stage.

Description

Technical Field

[0001] This invention relates to the field of reproductive technology, specifically to the application of SORBS3 as a target in the specific labeling and recognition of secondary, antral 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 grading of follicles is currently the recognized and widely used method, ambiguous follicle morphology and inconsistent staging remain issues in clinical practice and scientific research. For example, in pathological sections, different section positions can affect the morphology of the sinus cavity of larger antral follicles, thus influencing the grading. When oocytes lack the assistance of granulosa cells, it is difficult to identify their developmental stage based solely on morphology. Furthermore, the subjectivity of the evaluator is an unavoidable problem in oocyte morphological grading. Because follicle development is continuous, different evaluators often classify the same follicle into different grades. Therefore, molecular markers specifically expressed at specific developmental stages hold promise for solving these problems. Researchers can not only grade follicles or oocytes with ambiguous morphology using specific molecular markers, but also screen or target oocytes or follicles at specific developmental stages based on these markers, providing effective targets for targeted intervention. However, due to the difficulty in obtaining human sexually mature ovarian tissue and oocytes, traditional proteomic analysis requires a high protein content, and there is currently no data on the protein expression characteristics of oocytes at different developmental stages. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the above-mentioned background technology and provide an application of SORBS3 as a target in the specific labeling and recognition of secondary, antral oocytes or follicles. This invention screens and provides a molecule SORBS3 specifically expressed in secondary and antral oocytes 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.

[0005] To achieve the above objectives, this invention provides an application of SORBS3 as a target in the specific labeling and recognition of secondary, antral oocytes or follicles.

[0006] Furthermore, the amino acid sequence of the SORBS3 is shown in SEQ ID NO:1.

[0007] Furthermore, the specific labeling of secondary and antral oocytes with SORBS3 as a target includes using antigen-antibody binding technology, enzyme-linked reaction technology, direct binding technology of fluorescent dyes, genetic modification technology, radionuclide technology, and nano-targeted delivery technology to label secondary and antral oocytes with SORBS3 as a target.

[0008] Furthermore, the SORBS3 target for specific labeling of secondary and antral oocytes includes labeling or identifying secondary and antral oocytes in cell suspensions, cell cultures, tissue sections, and tissues / organs by labeling with SORBS3 to distinguish secondary and antral oocytes from oocytes at other developmental stages.

[0009] Furthermore, the secondary oocyte is an oocyte surrounded by multiple layers of granulosa cells, and no follicular cavity is formed in the granulosa cells.

[0010] Furthermore, the sinus oocyte is an oocyte surrounded by multiple layers of granular cells that form sinus cavities between the cells.

[0011] Furthermore, the oocytes are selected from human, mouse, rat, and monkey oocytes; the oocytes include in vivo oocytes and isolated oocytes.

[0012] Furthermore, after SORBS3 is used as a target marker or to identify secondary oocytes, antral oocytes, or follicles, it can be used to identify and screen oocytes or follicles at other developmental stages.

[0013] This invention also provides an application of SORBS3 as a marker for secondary oocytes or antral oocytes in bioinformatics analysis, single-cell sequencing data analysis, and spatial sequencing analysis to assist in cell clustering and localization analysis.

[0014] This invention also provides an application of SORBS3 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 several consecutive stages. Furthermore, by integrating mRNA data, the Human Protein Atlas database, the GEO database, and the PubMed database, tissue-specific protein SORBS3 is screened. 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.

[0015] Compared with the prior art, the present invention has the following advantages:

[0016] Firstly, this invention utilizes oocytes obtained during sex reassignment surgery and fertility preservation surgery at clearly defined developmental stages. Employing state-of-the-art micro-protein detection technology, it analyzes protein expression at each stage of oocyte development at a near-single-cell level. Integrating published single-cell mRNA data, it screens proteins highly expressed in secondary and antral oocytes and, for the first time, discovers that SORBS3 is specifically expressed in the cytoplasm and cell membrane of secondary and antral oocytes. SORBS3 (Sorbin and SH3 domain containing 3, also known as Vinexin) is a crucial component of the protein network for cell adhesion, participating in cell-cell and cell-extracellular matrix adhesion, and ultimately influencing a series of important cellular behaviors such as migration, differentiation, division, and apoptosis.

[0017] Secondly, this invention is the first to propose that SORBS3 is specifically expressed in a specific stage of human follicles. It can be used as a target for experimental techniques related to specific labeling and targeting of secondary and antral oocytes. It has innovative significance from the perspective of molecular staging of oocyte development, innovative significance from the perspective of discovery process, and innovative significance from the perspective of specific labeling and targeted intervention.

[0018] Thirdly, SORBS3 of the present invention, as a specific marker molecule for oocytes in the secondary and antral follicle stages, 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. Attached Figure Description

[0019] 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;

[0020] Figure 2 Differential proteins at different developmental stages of oocytes were obtained through differential protein analysis using R software;

[0021] Figure 3 The amount of proteins specifically expressed at different oocyte developmental stages;

[0022] Figure 4 To provide a micro-protein technique for measuring the expression level of SORBS3 at different developmental stages of oocytes in Example 1 of the present invention;

[0023] Figure 5 This invention provides the expression levels of SORBS3 at different developmental stages of oocytes from published mRNA data for Example 2 of this invention;

[0024] Figure 6 The results of SORBS3 specifically labeling oocytes in the ovary are shown in Embodiment 3 of the present invention. Detailed Implementation

[0025] 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.

[0026] 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.

[0027] The samples used in this invention are human ovarian tissue and follicles.

[0028] Example 1: Expression of SORBS3 protein in human secondary oocytes

[0029] 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, SORBS3, a protein specifically expressed only in the primary stage, was selected.

[0030] 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 sex reassignment surgery or fertility preservation techniques. The tissue is cut into 0.5×0.5×1mm pieces, digested in 0.04mg / ml Liberase DH enzyme for 90 minutes, and then follicles at various stages are separated by mechanical needle acupuncture under a stereomicroscope.

[0031] 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.

[0032] 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:

[0033] (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.

[0034] (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.

[0035] 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 a series of 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.

[0036] like Figure 4 As shown, SORBS3 protein is highly expressed during the human secondary oocyte stage.

[0037] Example 2: SORBS3 mRNA expression in human primary oocytes.

[0038] Downloaded published mRNA data of human oocytes at various developmental stages and analyzed the expression level of SORBS3 using software such as Graphpad and SPSS.

[0039] 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.

[0040] 2. Obtain the expression level of SORBS3 mRNA from the matrix.

[0041] 3. Use software such as Graphpad and SPSS to analyze the expression level of SORBS3 at different developmental stages of oocytes and draw expression level bar charts.

[0042] like Figure 5 As shown, SORBS3 mRNA is expressed in human primary oocytes and beyond.

[0043] Example 3: SORBS3 expression in the cytoplasm and cell membrane of secondary and antral oocytes in human ovarian tissue sections.

[0044] Immunohistochemical staining of human ovarian tissue sections was performed using SORBS3 antibody, and cells expressing SORBS3 were located using DAB chromogenic agent. The specific implementation method is as follows:

[0045] 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 sex reassignment surgery or 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.

[0046] 2. Immunohistochemical staining: Ovarian tissue sections were subjected to immunohistochemical staining for SORBS3 protein. The SORBS3 protein antibody was purchased from Invitrogen. The specific procedures are as follows:

[0047] (1) Baking: Place slices of human ovarian tissue in a 65℃ oven and bake for 60 minutes;

[0048] (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.

[0049] (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.

[0050] (4) Remove endogenous peroxidase: Incubate the slices in 3% hydrogen peroxide for 20 min;

[0051] (5) Wash with PBS 3 times for 5 min each time for histochemistry;

[0052] (6) Block the slices with ready-to-use goat serum for 1 hour;

[0053] (7) Remove the blocking solution, incubate with SORBS3 primary antibody, place the humidified box in a 4°C refrigerator overnight, and the SORBS3 concentration is 1:1000.

[0054] (8) Remove the humidified chamber, allow it to warm to room temperature for 30 minutes, and then wash with PBS 3 times for histochemistry.

[0055] (9) Incubate with HRP-labeled secondary antibody for 1 h, then wash 3 times with PBS;

[0056] (10) After DAB development for 1 min 30 s, the sections were placed in tap water to stop the process.

[0057] (11) Stain the nucleus with hematoxylin for 5 minutes, then rinse with tap water until the water is clear;

[0058] (12) Immerse in the differentiation solution for 3 seconds, then rinse with tap water;

[0059] (13) Soak in the blueing solution for 10 seconds, then rinse with tap water;

[0060] (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;

[0061] (15) After the fume hood is dried, seal the sheet with neutral resin;

[0062] (16) Observe and photograph under a microscope.

[0063] like Figure 6 As shown, although SORBS3 is expressed in the nucleus of small follicles, it is also significantly expressed in the cytoplasm and cell membrane of secondary and antral oocytes, and can be used to label and target oocytes or follicles at this developmental stage.

[0064] 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 SORBS3 protein as a target for specific labeling or recognition in human secondary oocytes or human secondary follicles.

2. The application according to claim 1, characterized in that: The amino acid sequence of the SORBS3 protein is shown in SEQ ID NO:

1.

3. The application according to claim 1, characterized in that: The specific labeling of secondary oocytes using SORBS3 protein as a target includes labeling secondary oocytes with SORBS3 protein using antigen-antibody binding technology, enzyme-linked reaction technology, direct binding technology with fluorescent dyes, genetic modification technology, radionuclide technology, or nano-targeted delivery technology.

4. The application according to claim 1, characterized in that: The SORBS3 protein is used as a target for specific recognition of secondary oocytes, including distinguishing secondary oocytes from oocytes at other developmental stages by labeling the SORBS3 protein in cell suspensions, cell cultures, tissue sections, or tissues and organs.

5. The application according to claim 4, characterized in that: The secondary oocyte is an oocyte surrounded by multiple layers of granulosa cells, and no follicular cavity is formed in the granulosa 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 SORBS3 protein is used as a target marker or to identify secondary oocytes or follicles, it can be used to identify and screen oocytes or follicles at other developmental stages.

Images