Gene panel for detecting follicular granulosa cell developmental stage and applications thereof

Abstract

The application discloses a gene panel for detecting follicular granulosa cell development stages, comprising 24 genes: Tmem184a, Crip3, Espn, Tmem171, Gatm, Wfdc10, Kctd14, Wnt6, Vim, Acta2, Cald1, Col11a1, Timp2, Ntn4, Ghr, Ctsf, Comp, Lhcgr, Cyp11a1, Gabrb2, Cd24, Lrp11, Prlr and Ptgfr. The application also discloses an application of the gene panel in detecting follicular granulosa cell development stages. The gene panel is screened based on ovary single nucleus transcriptome sequencing and subcellular level spatial transcriptome sequencing technology. The application can detect 24 marker genes at one time, and can quickly and accurately identify and judge granulosa cell positioning.

Description

Technical Field

[0001] This invention relates to a gene panel for detecting the developmental stage of follicular granulosa cells and its application, belonging to the field of gene detection technology. Background Technology

[0002] Follicular development is the most important biological event in the ovary, reflecting female reproductive capacity. Follicles can be classified into three stages according to their developmental stage: preantral follicle (PAF), small antral follicle (SAF), and large antral follicle (LAF). Granulosa cells play a crucial role in follicular development and exhibit dynamic transcriptional changes as development progresses. Preantral follicles are insensitive to gonadotropins, and their granulosa cells highly express genes such as Amh and Wt1. With the emergence of the antral cavity, follicular development becomes dependent on gonadotropin stimulation, and the spatial position of granulosa cells changes. Cells surrounding the oocyte are called cumulus granulosa cells, while those closely adhering to the theca membrane are called parietal granulosa cells. Comparatively, the expression of Slc38a3, Amh, and Ar is increased in cumulus granulosa cells, while the expression of Lhcgr, Cyp11a1, and Cd34 is increased in parietal granulosa cells.

[0003] Determining the follicular stage of granulosa cells quickly and accurately based on marker genes remains a challenging technical problem. Current technologies generally employ two methods for screening granulosa cell marker genes. One method involves bulk RNA sequencing of mechanically isolated cells, analyzing differentially expressed genes to obtain corresponding markers. The other relies on single-cell transcriptomics technology, using bioinformatics analysis to obtain markers for different granulosa cell subpopulations. Both methods have significant technical barriers. Mechanical separation struggles to yield highly pure single-cell subpopulations, reducing the accuracy of captured marker genes. Furthermore, while single-cell sequencing can identify different cell subpopulations and corresponding markers, it cannot determine the spatial location of granulosa cells, and the results are susceptible to change depending on the analytical method used. In addition, these two methods exhibit high heterogeneity, are costly, and require complex analyses to reach definitive conclusions. Summary of the Invention

[0004] In view of the above-mentioned prior art, in order to solve the technical problem that it is difficult to identify the follicular development stage of granulosa cells through a single marker gene, the present invention provides a gene panel for detecting the development stage of follicular granulosa cells, which can detect the development stage of follicular granulosa cells in one go.

[0005] This invention is achieved through the following technical solution:

[0006] The gene panel used to detect the developmental stage of follicular granulosa cells includes the following 24 genes: Tmem184a, Crip3, Espn, Tmem171, Gatm, Wfdc10, Kctd14, Wnt6, Vim, Acta2, Cald1, Col11a1, Timp2, Ntn4, Ghr, Ctsf, Comp, Lhcgr, Cyp11a1, Gabrb2, Cd24, Lrp11, Prlr, and Ptgfr.

[0007] The gene panel for detecting the developmental stage of follicular granulosa cells is described, along with its application in detecting this stage and in preparing detection products for this stage. The detection products include detection probes, detection antibodies, detection reagents, detection kits, gene chips, etc.

[0008] Furthermore, in specific applications, the expression of the 24 genes of the aforementioned gene panel in the sample to be tested is detected, and the developmental stage of the follicular granulosa cells is determined based on the expression: high expression of Tmem184a, Crip3, Espn, Tmem171, Gatm, Wfdc10, Kctd14, and Wnt6 indicates granulosa cells in the precavitary follicle stage; high expression of Vim, Acta2, Cald1, Col11a1, Timp2, Ntn4, Ghr, and Ctsf indicates granulosa cells in the small cavitary follicle stage; and high expression of Comp, Lhcgr, Cyp11a1, Gabrb2, Cd24, Lrp11, Prlr, and Ptgfr indicates granulosa cells in the large cavitary follicle stage.

[0009] Furthermore, the substances used to detect the expression of the 24 genes in the gene panel were selected from: specific amplification primers and detection probes for the 24 genes in the gene panel, antibodies that specifically bind to the proteins expressed by the 24 genes in the gene panel, nucleic acid aptamers with nucleophilicity to the proteins expressed by the 24 genes in the gene panel, and molecules that specifically bind to the 24 genes in the gene panel.

[0010] A method for detecting the developmental stage of follicular granulosa cells: The expression levels of 24 genes in the panel gene panel of the follicular granulosa cells to be tested are detected, and the developmental stage of the follicular granulosa cells is determined based on the expression levels: granulosa cells with high expression of Tmem184a, Crip3, Espn, Tmem171, Gatm, Wfdc10, Kctd14, and Wnt6 are granulosa cells in the precavitary follicular stage; granulosa cells with high expression of Vim, Acta2, Cald1, Col11a1, Timp2, Ntn4, Ghr, and Ctsf are granulosa cells in the small cavitary follicle stage; and granulosa cells with high expression of Comp, Lhcgr, Cyp11a1, Gabrb2, Cd24, Lrp11, Prlr, and Ptgfr are granulosa cells in the large cavitary follicle stage.

[0011] Furthermore, the substances used to detect the expression of the 24 genes in the gene panel were selected from: specific amplification primers and detection probes for the 24 genes in the gene panel, antibodies that specifically bind to the proteins expressed by the 24 genes in the gene panel, nucleic acid aptamers with nucleophilicity to the proteins expressed by the 24 genes in the gene panel, and molecules that specifically bind to the 24 genes in the gene panel.

[0012] The gene panel of the present invention for detecting the developmental stage of follicular granulosa cells contains 24 genes, which were screened based on ovarian single-cell nuclear transcriptome sequencing and subcellular spatial transcriptome sequencing technology. First, granulosa cell-related marker genes were screened by single-cell nuclear transcriptome sequencing, and then the spatial transcriptome sequencing technology was used to test mice of the same age and estrous cycle to verify whether the above-discovered markers can accurately match the follicular development stage.

[0013] Detection products for detecting the developmental stage of follicular granulosa cells include any one or more of the following substances: specific amplification primers and detection probes for the 24 genes of a gene panel, antibodies that specifically bind to the proteins expressed by the 24 genes of a gene panel, nucleic acid aptamers with nucleophilicity to the proteins expressed by the 24 genes of a gene panel, and molecules that specifically bind to the 24 genes of a gene panel.

[0014] Furthermore, the detection product may be a detection probe, detection antibody, detection reagent, detection kit, or gene chip.

[0015] The method for detecting the developmental stage of follicular granulosa cells of the present invention uses gene panels to determine the developmental stage of follicular granulosa cells. It can detect 24 marker genes at once, and can quickly and accurately identify and determine the location of granulosa cells. It is highly accurate, convenient and fast, and solves the technical problem in the prior art that it is difficult to accurately identify and locate follicles at different developmental stages using a single gene. Attached Figure Description

[0016] Figure 1 Analysis of marker gene expression in granulosa cells of follicles at different developmental stages.

[0017] Figure 2 : A diagram showing the homogenization of PAF marker expression levels at different developmental stages.

[0018] Figure 3 : A diagram showing the uniformity of SAF marker expression levels at different developmental stages.

[0019] Figure 4 : A diagram showing the homogenization of LAF marker expression levels at different developmental stages.

[0020] Figure 5 Schematic diagram of H&E staining of follicular granulosa cells.

[0021] Figure 6 A visual illustration of a comprehensive atlas of cell types.

[0022] Figure 7 : A schematic diagram of the spatial speckle assignment, where the left side is the PAF marker (Tmem184a, Crip3, Espn, Tmem171, Gatm, Wfdc10, Kctd14 and Wnt6), the middle side is the SAF marker (Vim, Acta2, Cald1, Col11a1, Timp2, Ntn4, Ghr and Ctsf), and the right side is the LAF marker (Comp, Lhcgr, Cyp11a1, Gabrb2, Cd24, Lrp11, Prlr and Ptgfr). Detailed Implementation

[0023] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.

[0024] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0025] Example 1: Construction of a gene panel for detecting follicular granulosa cells

[0026] Gene panels were constructed by performing single-cell nuclear transcriptome and spatial transcriptome sequencing on the ovaries of adult female C57BL / 6J mice, as follows:

[0027] (1) Ovarian sample preparation: 12-week-old C57BL / 6J mice were housed at the SPF Experimental Animal Center of Shandong University. Estrogenic cycle was monitored daily. Six mice in the interestrus phase were euthanized and unilateral ovaries were removed and mixed together. The six ovaries were cut into pieces with ophthalmic scissors under a stereomicroscope and placed into homogenization tubes.

[0028] (2) Extraction of cell nuclei using density gradient centrifugation: Ovarian tissue was homogenized using a homogenizer, filtered through a 70 μm cell sieve, and centrifuged at 2500 rpm for 15 minutes. The supernatant was slowly removed, and the remaining precipitate was resuspended in 6 ml of sucrose-calcium chloride solution (sucrose concentration 0.25 mol / L, calcium chloride concentration 0.003 mol / L), centrifuged at 2500 rpm for 15 minutes, and the supernatant was discarded. The remaining liquid was pipetted to form a suspension, and finally, cell viability was observed by trypan blue staining.

[0029] (3) 10×Genomics Single-Cell Isolation: 10×Genomics 3' transcriptomics utilizes short-read sequencing and microfluidic technology to achieve simultaneous transcriptomic expression profiling of 500–10,000 cells per sample. Gel beads containing barcode information were combined with a mixture of cells and enzymes, then encapsulated by oil surfactant droplets in a microfluidic "double cross" system to form GEMs (Gel Beads-In-Emulsions). The GEMs flowed into a reservoir and were collected. The gel beads dissolved, releasing the barcode sequence, and cDNA fragments were reverse transcribed, labeling the samples. The gel beads were then broken up, and the oil droplets were fragmented for PCR amplification using cDNA as a template. All GEM products were mixed to construct a standard sequencing library.

[0030] (4) Sequencing Library Construction and Sequencing: First, cDNA was digested and fragmented into 200-300 bp fragments. Then, the library construction process, including the P5 sequencing adapter and R1 sequencing primer, was performed as per traditional next-generation sequencing. Finally, PCR amplification was conducted to obtain the DNA library. The constructed library was then sequenced using the paired-end sequencing mode of the Illumina sequencing platform. Read 1 contained 16 bp of barcode information and 10 bp of UMI information for cell identification and expression quantification. Read 2 contained cDNA fragments for reference genome alignment to determine the gene corresponding to the mRNA.

[0031] (5) Bioinformatics Analysis: Cell Ranger was used for quality statistics and analysis of the raw data, filtering out low-quality cells. Subsequently, Seurat R was used for further quality control and analysis. After normalizing cell expression levels, principal component analysis and cell cluster analysis were performed to obtain differentially expressed genes in subpopulations. Cell types were annotated using singleR, the MCA database (http: / / bis.zju.edu.cn / MCA / index.html), and by searching published databases.

[0032] (6) Discovery of marker gene combinations expressed by granulosa cells of follicles at different stages: such as Figure 1 As shown, high expression of Tmem184a, Crip3, Espn, Tmem171, Gatm, Wfdc10, Kctd14, and Wnt6 indicates granulosa cells in the precavitary follicle stage; high expression of Vim, Acta2, Cald1, Col11a1, Timp2, Ntn4, Ghr, and Ctsf indicates granulosa cells in the small cavitary follicle stage; and high expression of Comp, Lhcgr, Cyp11a1, Gabrb2, Cd24, Lrp11, Prlr, and Ptgfr indicates granulosa cells in the large cavitary follicle stage. The specific names and NCBI IDs of these 24 genes are shown in Table 1.

[0033] Table 1. Panel gene names and gene IDs in granulocyte detection.

[0034]

[0035]

[0036] The three gene groups were uniformly displayed in each granulocyte, and the results are as follows: Figure 2 , 3As shown in Figure 4, the expression levels of PAF markers were highest in granulosa cells during the PAF stage (Tmem184a, Crip3, Espn, Tmem171, Gatm, Wfdc10, Kctd14, and Wnt6); the expression levels of SAF markers were highest in granulosa cells during the SAF stage (Vim, Acta2, Cald1, Col11a1, Timp2, Ntn4, Ghr, and Ctsf); and the expression levels of LAF markers were highest in granulosa cells during the LAF stage (Comp, Lhcgr, Cyp11a1, Gabrb2, Cd24, Lrp11, Prlr, and Ptgfr).

[0037] (7) Spatial transcriptome sequencing was used to verify the granule cell marker genes found above.

[0038] ① C57BL / 6J mice of the same age, estrous cycle, and background were isolated, embedded using OCT, and sectioned on a cryostat to a thickness of 10 μm. The first 20 sections were used for RNA quality testing, with RNA integrity measured using an Agilent 2100 analyzer. Only samples with high RNA integrity underwent tissue permeation experiments, with permeation times of 3 min, 6 min, and 12 min. The best permeation effect was observed at 6 min, and this 6-min permeation time was ultimately selected for formal mounting, library construction, and sequencing.

[0039] ② Mapping of single-cell nuclear transcriptome data with spatial transcriptome sequencing data: Seurat's "FindTransferAnchors" and "TransferData" functions were used to estimate the probability of anchors existing within a single bin. The cell type with the highest probability among all cell types was identified as the cell type at that point. The comprehensive atlas of cell types was then visualized using the "SpatialDimPlot" and "SpatialFeaturePlot" functions. The results are as follows... Figure 6 As shown, the granulosa cells of the follicles identified by mapping can be completely separated and match H&E staining (e.g., Figure 5 (As shown).

[0040] (8) Validation of 24 marker genes: The eight genes from PAF, SAF, and LAF were treated as a separate geneset. The "AddModuleScore" function in Seurat was used to calculate the signature scoring for each spatial spot; the higher the score, the redder the color. Results are as follows: Figure 7 As shown, Figure 7The previously identified PAF marker, SAF marker, and LAF marker can accurately distinguish different stages of follicle development.

[0041] The above embodiments are provided to those skilled in the art to fully disclose and describe how the claimed implementations can be carried out and used, and are not intended to limit the scope of the disclosure herein. Modifications that will be obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. A gene panel for detecting the developmental stage of follicular granulosa cells, characterized in that, It is composed of the following 24 genes: Tmem184a, Crip3, Espn, Tmem171, Gatm, Wfdc10, Kctd14, Wnt6, Vim, Acta2, Cald1, Col11a1, Timp2, Ntn4, Ghr, Ctsf, Comp, Lhcgr, Cyp11a1, Gabrb2, Cd24, Lrp11, Prlr, and Ptgfr.

2. The use of the gene panel for detecting the developmental stage of follicular granulosa cells as described in claim 1 in the preparation of a detection product for detecting the developmental stage of follicular granulosa cells.

3. The application according to claim 2, characterized in that: The detection products are selected from detection probes, detection antibodies, detection reagents, detection kits, and gene chips.

4. A detection product for detecting the developmental stage of follicular granulosa cells, characterized in that: Includes any one or more of the following substances: specific amplification primers and detection probes for the 24 genes of the gene panel, antibodies that specifically bind to the proteins expressed by the 24 genes of the gene panel, nucleic acid aptamers that are nucleophilic to the proteins expressed by the 24 genes of the gene panel, and molecules that specifically bind to the 24 genes of the gene panel. The gene panel is as described in claim 1.

5. The detection product for detecting the developmental stage of follicular granulosa cells according to claim 4, characterized in that: The detection products are detection probes, detection antibodies, detection reagents, detection kits, or gene chips.

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