Application of ZPI as a target in specific labeling and identification of mature follicle

Abstract

The application discloses application of ZPI as a target point in specific marking and identification of mature follicle. When micro-proteome detection is carried out on oocytes at various development stages of human, it is found that the expression of some proteins of the oocytes has development stage specificity. After screening of the proteins specifically expressed at the pre-ovulation stage and comparison with the Human Protein Atlas database, the GEO database, the PubMED database and published mRNA data, it is found that ZPI is highly expressed in the oocyte before ovulation, and immunohistochemistry of the ovarian section proves that ZPI is specifically expressed in the pre-ovulation follicle. Therefore, it is proposed that the ZPI molecule can be applied as a target point to specific identification and targeting of mature follicle and other related experimental technologies, to assist scholars in identifying and judging the development period and ovulation period of the oocyte, and to promote the molecular biology research on the physiology and pathology mechanism of the oocyte or follicle maturation.

Description

Technical Field

[0001] This invention relates to the field of reproductive technology, specifically to the application of ZPI as a target in the specific labeling and identification of mature 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 matures, its 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 and is expelled from the ovary.

[0003] In current clinical practice and scientific research, follicle maturation is primarily determined morphologically. For example, the presence of granulosa vulcanization (GVBD) in oocytes indicates maturation, and a rapid increase in follicle diameter to a certain range suggests imminent ovulation. However, relying solely on morphology to determine follicle maturity carries inherent risks, especially in pathological sections where different sections can affect the follicle's morphology and thus the assessment of its developmental stage. Specific expression molecules of mature follicles may assist clinicians and scientists in determining follicle maturity. Furthermore, mature oocytes are fundamental for subsequent fertilization and embryonic development. A deeper understanding of the physiological mechanisms and regulatory factors involved in oocyte maturation is crucial for better utilizing the patterns of oocyte maturation and providing a theoretical basis for assisted reproductive technology (ART). Specific expression molecules of mature follicles can serve as targets for screening and identifying mature oocytes, enabling applications in emerging technologies such as single-cell sequencing and targeted drug delivery, thus contributing to molecular mechanism research in the field of ART. 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 ZPI as a target in the specific labeling and recognition of mature follicles. This invention screens and provides a molecular ZPI specifically expressed by pre-ovulatory 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 ZPI as a target in the specific labeling and recognition of mature follicles.

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

[0007] Furthermore, the specific labeling of mature follicles with ZPI as a target includes labeling mature follicles with ZPI using antigen-antibody binding technology, enzyme-linked reaction technology, direct binding technology with fluorescent dyes, genetic modification technology, radionuclide technology, and nanoparticle targeted recognition technology.

[0008] Furthermore, the ZPI as a target for specific recognition of mature follicles includes distinguishing mature follicles from follicles at other developmental stages by labeling ZPI in cell suspensions, cell cultures, tissue sections, and tissues / organs.

[0009] Furthermore, the mature follicle is a follicle near the time of ovulation, and the cumulus-oocyte complex moves to one side of the follicle.

[0010] Furthermore, the mature follicle includes oocytes, granulosa cells, and follicular fluid.

[0011] Furthermore, the mature follicles include follicles from humans, mice, rats, and monkeys; the mature follicles include both in vivo and in vitro forms.

[0012] Furthermore, after the ZPI is used as a target marker or to identify mature follicles, it can be used to identify and screen follicles at other developmental stages.

[0013] Furthermore, ZPI is used as a marker for pre-ovulatory oocytes or follicles in single-cell sequencing data analysis and spatial sequencing analysis to assist in cell clustering and localization analysis.

[0014] Furthermore, ZPI is used as a marker of follicular maturity in determining whether a follicle is mature and the timing of ovulation.

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

[0016] Firstly, this invention utilizes micro-protein detection technology to detect protein expression at various stages of oocyte development. Through differential protein analysis at each developmental stage, it proposes that ZPI is a molecule specifically expressed in oocytes during pre-ovulation follicles. ZPI (protein Z-dependent protease inhibitor, also known as Serpin family A member 10, SERPINA10) is a serine protease inhibitor that works synergistically with vitamin K-dependent clotting protein Z (Protein Z, PZ) in the presence of phospholipids and calcium ions. ZPI inhibits activated factor X, and PZ exerts its anticoagulant effect by enhancing the effect of ZPI by 1000-fold. PZ and ZPI are closely related to clinical diseases such as arterial and venous thrombosis, cancer, diabetes, adverse pregnancy outcomes, chronic kidney disease, and hemophilia A.

[0017] Secondly, this invention proposes that ZPI molecules are specifically expressed in pre-ovulatory follicles, and can be used as a target for the identification and targeting of mature follicles. This invention is conceptually innovative from the perspective of molecular staging of oocyte development, technologically innovative from the perspective of the discovery process, and innovative in application from the perspective of specific labeling and targeted intervention.

[0018] Thirdly, this invention, in performing microproteomic analysis on oocytes at various developmental stages, discovered that the expression of certain proteins in oocytes exhibits developmental stage specificity. Screening for proteins specifically expressed in the pre-ovulatory stage and comparing them with data from the Human Protein Atlas, GEO, PubMed databases, and published mRNA data revealed that ZPI is highly expressed in pre-ovulatory oocytes. Immunohistochemistry of ovarian sections confirmed that ZPI is specifically expressed in pre-ovulatory follicles. Therefore, it is proposed that ZPI molecules can be used as a target for specific recognition and targeting of mature follicles in related experimental techniques, assisting researchers in identifying and determining the developmental and ovulatory stages of oocytes, and promoting molecular biological research on the physiological and pathological mechanisms of oocyte or follicle maturation. 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 PCA analysis of oocytes at each stage was performed using microprotein technology;

[0021] Figure 3 The microprotein assay revealed different trends in oocyte protein changes at various developmental stages.

[0022] Figure 4 Differential proteins at different developmental stages of oocytes were obtained through differential protein analysis using R software;

[0023] Figure 5 The amount of proteins specifically expressed at different oocyte developmental stages;

[0024] Figure 6 The expression level of ZPI in oocytes at different developmental stages was measured using microprotein technology in Example 1 of the present invention.

[0025] Figure 7 This provides a result diagram of ZPI specifically expressed in pre-ovulatory follicles in ovarian tissue sections for Embodiment 2 of the present invention. Detailed Implementation

[0026] 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.

[0027] The main instruments used in this invention include: Orbitrap Fusion™ Lumos™ Tribrid™ tandem mass spectrometer (Thermo Fisher Scientific, San Jose, CA), UltiMate 3000 UHPLC (Thermo), as well as commonly used laboratory instruments such as low-temperature high-speed centrifuge, electrophoresis apparatus, microtome, ultraviolet spectrophotometer, and microscope.

[0028] The samples used in this invention are human ovarian tissue and follicles.

[0029] Example 1: Expression of ZPI protein in human preovulatory oocytes

[0030] 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, the protein ZPI, which is specifically expressed only in the primary stage, was screened out.

[0031] 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 0.5×0.5×1 mm pieces, digested in 0.04 mg / ml Liberase DH enzyme for 90 min, and then follicles at various stages are separated by mechanical needle acupuncture under a stereomicroscope.

[0032] 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.

[0033] 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:

[0034] (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.

[0035] (2) The enzymatically digested samples were separated by UltiMate 3000 UHPLC. The samples were first enriched and desalted in a trap column, and then separated in series with a self-packed C18 column at a flow rate of 500 nl / min. The peptides separated by liquid chromatography were ionized by a nanoESI source and then detected by a Fusion Lumos tandem mass spectrometer in DDA mode.

[0036] 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.

[0037] like Figure 6 As shown, ZPI protein is highly expressed in human preovulatory oocytes.

[0038] Example 2: ZPI is specifically expressed in pre-ovulatory follicles in human ovarian tissue sections.

[0039] Immunohistochemical staining of human ovarian tissue sections was performed using ZPI antibody, and cells expressing ZPI were located using DAB chromogenic agent. The specific implementation method is as follows:

[0040] 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.

[0041] 2. Immunohistochemical staining: Ovarian tissue sections were subjected to immunohistochemical staining for ZPI protein. ZPI protein antibody was purchased from Invitrogen. The specific procedures are as follows:

[0042] (1) Baking: Place human ovarian tissue slices in a 65℃ oven and bake for 60 minutes;

[0043] (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.

[0044] (3) Antigen retrieval: Boil the antigen retrieval solution on high heat for 15 minutes, place the slide in the boiling antigen retrieval solution, boil on medium heat for 3 minutes, remove and cool in an ice-water complex;

[0045] (4) Removal of endogenous peroxidase: Incubate the slices in 3% hydrogen peroxide for 20 min;

[0046] (5) Wash with PBS 3 times for 5 min each time for histochemistry;

[0047] (6) Block the slices with ready-to-use goat serum for 1 hour;

[0048] (7) Remove the blocking solution, incubate with ZPI primary antibody, place the wet box in a 4°C refrigerator overnight, and the ZPI concentration is 1:1000;

[0049] (8) Remove the humidified chamber, allow it to warm to room temperature for 30 minutes, and then wash it three times with PBS for histochemistry.

[0050] (9) Incubate with HRP-labeled secondary antibody for 1 h, then wash 3 times with PBS;

[0051] (10) After DAB development for 1 min 30 s, the slides were placed in tap water to stop the process;

[0052] (11) Stain the nucleus with hematoxylin for 5 minutes, then rinse with tap water until the water is clear;

[0053] (12) Immerse in the differentiation solution for 3 seconds, then rinse with tap water;

[0054] (13) Soak in the blueing solution for 10 seconds, then rinse with tap water;

[0055] (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;

[0056] (15) After the fume hood is dried, seal the sheet with neutral resin;

[0057] (16) Observe and photograph under a microscope.

[0058] like Figure 7 As shown, ZPI is specifically expressed in mature oocytes in ovarian tissue and can be used to label and identify oocytes or follicles at this developmental stage.

[0059] 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 ZPI (Z-dependent protease inhibitor) protein as a target for specific labeling or recognition of human mature follicles.

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

1.

3. The application according to claim 1, characterized in that: The ZPI protein is used as a target for specific labeling of mature follicles, including using antigen-antibody binding technology, enzyme-linked reaction technology, direct binding technology of fluorescent dyes, genetic modification technology, radionuclide technology or nanoparticle targeted recognition technology to label mature follicles with ZPI protein as the target.

4. The application according to claim 1, characterized in that: The ZPI protein is used as a target for specific recognition of mature follicles, including distinguishing mature follicles from follicles at other developmental stages by labeling ZPI 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 mature follicle is the follicle near ovulation, and the cumulus-oocyte complex moves to one side of the follicle.

6. The application according to claim 3 or 4, characterized in that: The mature follicle includes oocytes, granulosa cells, and follicular fluid.

7. The application according to claim 3 or 4, characterized in that: The mature follicles are in vitro.

8. The application according to claim 1, characterized in that: The ZPI protein, after being used as a target marker or to identify mature follicles, can then be used to identify and screen follicles at other developmental stages.

9. The application of ZPI (Z-dependent protease inhibitor) protein as a marker of human follicle maturation in determining whether a follicle is mature or the timing of ovulation.

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