Application of TSHB protein or coding gene thereof in regulation and control of sheep ovarian granular cell proliferation

Abstract

The invention relates to the technical field of biology, and particularly discloses application of TSHB protein or a coding gene thereof in regulation and control of sheep ovarian granular cell proliferation. Researches find that in in-vitro ovarian granular cells, overexpression of TSHB can promote cell proliferation over time; proliferation is inhibited by interference expression of TSHB; the overexpression significantly up-regulates the expression of Cyclin E, CDK2, Bcl-2, TSHR and FSHR genes, down-regulates the expression of Bax and Caspase3 genes in cells and steroid-related STAR and CYP19A1, down-regulates the levels of E2 estrogen and P4 progestational hormone, and has a regulation effect on the reproductive traits of sheep. The invention further provides application of the TSHB protein or the coding gene thereof or a biological material containing the coding gene thereof in regulation and control of sheep ovarian granular cell proliferation. The invention discloses the effect of the TSHB gene on promoting proliferation and regulating key gene expression in sheep granular cells, and the TSHB gene has important theoretical value and potential application value.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to the application of TSHB protein or its encoding gene in regulating the proliferation of sheep ovarian granulosa cells. Background Technology

[0002] Reproductive capacity is one of the important economic indicators of sheep, directly affecting their economic benefits. The Dolan sheep, a unique dual-purpose (meat and fat) sheep breed, possesses excellent characteristics such as early sexual maturity, early onset of estrus, and strong reproductive capacity. Its female lambs typically experience their first estrus at 6 months of age and can be first mated at 9-10 months. This early estrus trait lays the foundation for high-frequency lambing, significantly improving production efficiency and economic returns.

[0003] The high fertility of sheep stems from the combined effects of multiple factors, including genetic regulation, hormone secretion, environmental stimuli, and nutritional status. Several candidate genes, such as FecB (BMPR-IB) and NCOA1, have been shown to be associated with increased fertility and litter size. Meanwhile, studies on genes like CTSD and Lin28B suggest their potential involvement in regulating the onset of puberty. However, these studies have not yet covered mechanisms of action beyond the hypothalamus, particularly at the molecular level and their function in ovarian granulosa cells, which remains to be explored. Identifying genes related to high fertility in Dolan sheep can improve their reproductive performance, promote the application of high fertility traits in breeding, and facilitate the conservation and utilization of Dolan sheep, thus holding significant importance for their breeding. Summary of the Invention

[0004] One of the objectives of this invention is to provide a new and effective method for improving the proliferation capacity of granulosa cells in sheep ovaries.

[0005] This invention provides the application of TSHB protein or its encoding gene, or biological materials containing its encoding gene, in any of the following aspects: (1) Regulates the proliferation of granulosa cells in sheep ovaries; (2) Select sheep with strong ovarian granulosa cell proliferation ability; (3) Improvement of the proliferative capacity of sheep ovarian granulosa cells; (4) Enhance the proliferation capacity of granulosa cells in sheep ovaries; The application is for purposes other than disease diagnosis or treatment.

[0006] The ovary is fundamental to female reproduction, its main functions being follicle development, ovulation, and hormone secretion. Follicle growth and development are crucial components of female fertility, comprising oocytes and granulosa cells. The essence of follicle growth and development lies in the proliferation and differentiation of granulosa cells. Granulosa cells, as a vital component of the follicle, play a crucial role in follicle development and ovulation, providing hormones and cytokines for ovarian and follicle growth and development. Their proliferation and differentiation, along with signaling interactions with the oocyte, determine the fate of the oocyte and, in turn, influence female reproductive capacity. When granulosa cell proliferation is promoted, follicles receive more metabolic support and steroid synthesis capacity, thereby increasing follicle survival rate, reducing atresia, promoting follicle dominance, and enhancing ovulation rate. Therefore, granulosa cell proliferation is an important cellular basis for improving animal reproductive performance.

[0007] To overcome the incomplete understanding of the puberty regulation mechanism in existing technologies, particularly the lack of analysis of TSHB function in ovarian granulosa cells and different reproductive tissues, this invention provides a TSHB gene regulation tool and method based on tissue expression and cell function verification. Specifically, this invention cloned and sequenced the sheep TSHB gene, obtaining a 498 bp nucleotide sequence. The expression of the TSHB gene in five different gonadal tissues—hypothalamus, pituitary gland, ovary, fallopian tube, and uterus—was detected during three stages before and after puberty, analyzing its expression patterns in the gonadal axis and its regulatory role in the initiation of puberty. The study verified the cellular effects of TSHB by transfecting the constructed TSHB overexpression plasmid and the synthesized TSHB-siRNA into sheep ovarian granulosa cells. The results showed that TSHB gene overexpression in sheep promoted granulosa cell proliferation over time, significantly upregulated the expression of CyclinE, CDK2, Bcl-2, TSHR, and FSHR genes, downregulated the expression of Bax, Caspase3, STAR, and CYP19A1 genes, and inhibited the secretion of E2 and P4. It has a regulatory effect on the reproductive traits (ovarian granulosa cell proliferation traits) of sheep, especially Dolan sheep.

[0008] Specifically, this invention enhances the Cyclin E / CDK2 signaling axis by overexpressing TSHB, promoting G1 / S phase transition and thus driving ovarian granulosa cell proliferation. It also inhibits ovarian granulosa cell apoptosis by regulating the Bcl-2 / Bax balance and inhibiting Caspase 3 activation through TSHB overexpression. Furthermore, it inhibits steroid production by overexpressing TSHB and maintains follicle-stimulating signals by increasing gonadotropin receptor expression. In its application, this invention can promote sheep ovarian granulosa cell proliferation by overexpressing TSHB and inhibit sheep ovarian granulosa cell proliferation by inhibiting TSHB expression.

[0009] When breeding sheep with strong ovarian granulosa cell proliferation capacity, the expression level of TSHB can be detected, and individuals with relatively high TSHB expression levels can be selected to achieve the breeding of sheep with strong ovarian granulosa cell proliferation capacity.

[0010] To improve or enhance the proliferative capacity of sheep ovarian granulosa cells, overexpression of TSHB can be used to promote the proliferation of sheep ovarian granulosa cells, thereby improving / enhancing their proliferative capacity.

[0011] In the application of this invention, the NCBI accession number of the amino acid sequence of the TSHB protein is XP_004002417.2.

[0012] In the application of the present invention, the nucleotide sequence encoding the TSHB protein is as shown in SEQ ID NO.1 or as shown in a sequence that is completely complementary to the nucleotide sequence shown in SEQ ID NO.1.

[0013] In the application of this invention, the biological material is an expression cassette, a vector, or a host cell.

[0014] In the application of this invention, the sheep is a Dolan sheep.

[0015] This invention also provides a method for altering the proliferative capacity of sheep ovarian granulosa cells for non-disease diagnosis or treatment purposes, which involves regulating... TSHB The expression levels of genes and / or TSHB proteins were used to alter the proliferative capacity of sheep ovarian granulosa cells.

[0016] In the method of this invention, when it is necessary to improve the proliferative capacity of sheep ovarian granulosa cells, overexpression TSHB Genes and / or TSHB protein.

[0017] This invention can be controlled TSHB The expression levels of genes and / or TSHB proteins can be used to alter the proliferative capacity of sheep ovarian granulosa cells. Overexpression... TSHB Genes and / or TSHB protein can enhance the proliferative capacity of granulosa cells in sheep ovaries. If interference... TSHB Expression of the TSHB gene and / or TSHB protein can reduce the proliferative capacity of granulosa cells in sheep ovaries. Methods for overexpressing or interfering with the TSHB gene in Dolan sheep can employ conventional methods in this field.

[0018] The beneficial effects of this invention are at least as follows: This invention provides a novel method for regulating the proliferation of granulosa cells in sheep ovaries and discovers that the TSHB gene can serve as a molecular reference for screening candidate individuals and conducting subsequent functional verification, assisting in the development and verification of molecular breeding strategies for Dolan sheep, and providing an innovative molecular target for a deeper understanding of the mechanism of puberty initiation and future molecular breeding strategies. Attached Figure Description

[0019] Figure 1 This is a cloning diagram of TSHB nucleotides in Example 1. In the diagram, M represents DNA Marker 1, and lanes 1-4 represent the PCR amplification products of the TSHB gene.

[0020] Figure 2 This is an analysis of the amino acid sequence similarity of TSHB in Example 1.

[0021] Figure 3 This is the phylogenetic tree of the TSHB amino acid system in Example 1.

[0022] Figure 4 This is a prediction of the hydrophobicity of the TSHB protein in Example 1.

[0023] Figure 5 This is a prediction of the transmembrane site of the TSHB protein in Example 1.

[0024] Figure 6 This is a prediction of the TSHB protein signal peptide in Example 1.

[0025] Figure 7 Prediction of phosphorylation sites of TSHB protein in Example 1.

[0026] Figure 8 This is a prediction of the secondary structure of the TSHB protein in Example 1.

[0027] Figure 9 This is a prediction of the tertiary structure of the TSHB protein in Example 1.

[0028] Figure 10 Analysis of TSHB protein interactions in Example 1.

[0029] Figure 11 This is the TSHB gene expression profile of Dolan sheep in Example 1. In the figure, different uppercase letters under the same estrus stage indicate significant differences (P<0.05), while the same letter under the same estrus stage indicates no significant differences (P>0.05); different lowercase letters under the same estrus stage indicate significant differences (P<0.05), while the same letter under the same estrus stage indicates no significant differences (P>0.05).

[0030] Figure 12 This image shows the identification of ovarian granulosa cells using the TSHB gene in Example 2. The left image shows DAPI, the middle image shows the FSHR-specific signal, and the right image is a merged image (magnification: 200×). The scale bar in the image is 100 μm.

[0031] Figure 13The images shown are of TSHB gene overexpression vector transfected into granulosa cells of Dolan sheep ovaries in Example 2. The top three images are fluorescence images of empty vector transfection (500 μm), and the bottom three images are fluorescence images of plasmid transfection (200 μm).

[0032] Figure 14 This illustrates the effect of TSHB overexpression and interference on cell proliferation in Example 2.

[0033] Figure 15 This is an analysis of the mRNA expression level, protein level, and grayscale value of TSHB gene overexpression and interference in Example 2.

[0034] Figure 16 The levels of TSHB gene overexpression and interference with the mRNA expression levels of proliferation-related genes (CyclinE, CDK2) and apoptosis-related genes (BAX, Caspase3) and the anti-apoptotic gene (Bcl-2) in Example 2 are shown.

[0035] Figure 17 This study analyzes the levels and grayscale values ​​of proteins encoded by TSHB gene overexpression and interference with proliferation-related genes (CyclinE, CDK2) and apoptosis-related genes (BAX, Caspase3), as well as the anti-apoptotic gene (Bcl-2) in Example 2.

[0036] Figure 18 The expression levels of TSHB gene overexpression and interference on related genes (FSHR, TSHR) and steroid secretion-related genes (STAR, CYP19A1) in sheep granular cells are shown in Example 2.

[0037] Figure 19 This study analyzes the levels and grayscale values ​​of proteins encoded by TSHB gene overexpression and interference in related genes (FSHR, TSHR) and steroid secretion-related genes (STAR, CYP19A1) in sheep granular cells, as well as their corresponding proteins, in Example 2.

[0038] Figure 20 This shows the secretion of estrogen E2 and progesterone P4 after TSHB overexpression and interference in Example 2.

[0039] In all figures (if any), ns represents no significant difference. This means P < 0.05. This means P < 0.01. Detailed Implementation

[0040] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available or prepared according to conventional methods in the art.

[0042] Example 1: Cloning, sequence identification, and tissue differential expression analysis of the TSHB gene 1. Collection of experimental animals and samples All experimental sheep were from the Animal Breeding and Experiment Station of Tarim University. Female Dolan sheep of similar size and raised in the same environment were selected. The estrus process was determined using both the ram estrus detection method (twice daily at 10:00 AM and 6:00 PM, starting from 3 months of age) and vulvar observation. Five sheep were selected for each of the following stages: pre-pubertal (3-month-old lambs), pubertal (wolves actively approaching rams and standing still to accept mounting, accompanied by vulvar discharge and redness, tail wagging, bleating, and decreased appetite), and post-pubertal (10 days after the first estrus cessation). Immediately after slaughter, samples of five tissues—thalamus, pituitary gland, ovary, oviduct, and uterus—were collected. These samples were cut into approximately 2cm pieces, stored in cryovials, quickly transferred to liquid nitrogen, and then stored at -80°C for RNA extraction.

[0043] 2. Extracting RNA from tissues RNA was extracted from the five reproductive-related tissues of Dolan sheep using the TRIzol method. The integrity of the RNA bands was detected by 1.5% agarose gel electrophoresis. The concentration and purity of the RNA were determined by a nucleic acid protein detector. The RNA was reverse transcribed into cDNA and stored in a freezer at -20°C.

[0044] 3. Primer synthesis and amplification sequencing Primers for cloning and genotyping were designed based on the sheep TSHB mRNA sequence (XM_004002368.6) from NCBI (https: / / www.ncbi.nlm.nih.gov). The sheep ACTB gene (NM_001009784.3) was used as an internal reference gene and synthesized by Sangon Biotech (Shanghai) Co., Ltd. (primer sequences are shown in Table 1). Pituitary cDNA from Dolan sheep at puberty was used as a template for amplification, and a PCR (25 μL) system was employed: 9.5 μL deionized water, 1 μL each of forward and reverse primers, 1 μL cDNA, and 12.5 μL of 2×EasyTaq PCR SuperMix (+Dye) (Baori Biotechnology (Beijing) Co., Ltd.). The mixture was gently mixed. The specific PCR procedure included: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 55.8℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 30 cycles; 72℃ extension for 15 min; and storage at 4℃. 4 μL of the amplification product was added to a 1.5% agarose gel for electrophoresis to detect the target fragment. The 498 bp amplified band was purified using a gel extraction kit. The purified product was ligated with the pMD19-T vector, and the ligation product was then transformed into *E. coli* DH5α competent cells. The transformed cells were incubated overnight at 37℃. The next day, white monoclonal colonies were selected as candidates using a blue-white screening method, and these colonies were validated by PCR. Finally, the validated bacterial culture was collected and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing analysis.

[0045] Table 1 Primer Information (SEQ ID NO.30-35) 4. Bioinformatics Analysis The TSHB gene sequence obtained from Dornodula sheep was aligned and assembled using sequence alignment analysis software. The analyzed sequence was deduced into amino acids. Referring to TSHB gene sequences of different species in NCBI, similarity comparisons were performed with sheep XP_004002417.2, goat NP_001274505.1, bovine NP_776630.1, pig NP_999533.1, cat XP_044889158.1, human NP_000540.2, rhesus monkey XP_015004345.1, mouse NP_001159411.1, chicken NP_990394.1, and zebrafish NP_852471.1. A phylogenetic tree was constructed using Mega 7. The physicochemical properties and protein structure were analyzed using online bioinformatics software. STRING analysis was used to identify proteins interacting with TSHB.

[0046] 5. Detect the expression level of TSHB gene in Dolan sheep at different time points and in different tissues. RNA was extracted from the five reproductive-related tissues of Dolan sheep using TRIzol reagent (purchased from Thermo Fisher Scientific). The integrity of the RNA bands was detected by 1.5% agarose gel electrophoresis. The concentration and purity were determined using a NanoDrop 8000 spectrophotometer and PrimeScript. TM RT reagent kit (purchased from Baori Biotechnology (Beijing) Co., Ltd.) was used for reverse transcription to perform quantitative real-time PCR (qPCR) on cDNA. Primers designed according to Table 1 were used, with ACTB as a normalized endogenous control. The expression levels of the TSHB gene in the pre-estrus, estrus, and post-estrus phases of the Dolan sheep gonadal axis (hypothalamus, pituitary, ovary, uterus, fallopian tubes) were detected using the qPCR detection system. The qPCR (15 μL) system consisted of: 5.5 μL deionized water, 0.5 μL forward and reverse primers, 1 μL cDNA, and 7.5 μL SYBR Green Real-time PCR Mix. The qPCR program consisted of 40 cycles: 94 ℃ for 30 s, 94 ℃ for 5 s, 58 ℃ for 15 s, and 72 ℃ for 10 s. Three biological replicates were set up for each sample.

[0047] The results showed that using the synthesized TSHB gene-specific primers and primordial pituitary cDNA as a template, the successfully amplified product fragment was 498 bp in length. Figure 1 As shown, the coding region is 417 bp, and the result is shown in SEQ ID NO.1. Comparison with DNAMAN and NCBI BLAST software showed high homology with the predicted sheep TSHB gene sequence (GenBank accession number: XM_004002368.6).

[0048] After deriving the amino acid sequence, the similarity was compared with that of nine other animals: sheep, goat, cow, pig, cat, human, macaque, mouse, chicken, and zebrafish. The similarities were 100%, 100%, 97.8%, 95.7%, 90.6%, 89.1%, 89.1%, 81.2%, 67.9%, and 34.8%, respectively. Figure 2 As shown in the figure. Phylogenetic analysis reveals that it is closely related to sheep and goats, but more distantly related to zebrafish. The results are as follows... Figure 3 As shown.

[0049] Predictive analysis was performed on the characteristics of TSHB protein from Dorno sheep, indicating that the molecular formula is C 692 H 1064 N 172 O 197 S 21The total number of atoms is 2146, the molecular weight is 15.61 ku, and the theoretical isoelectric point is 8.20. It encodes 138 amino acids, as shown in Table 2. Cysteine ​​has the highest number of amino acids (13), accounting for 9.4% of the total. Tryptophan was not found (0.0%). Aspartic acid + glutamine carries 10 negatively charged residues, while arginine + lysine carries 13 positively charged residues. TSHB protein is more positively charged than negatively charged proteins, indicating that TSHB protein is a positively charged basic protein. The aliphatic coefficient of TSHB protein is 66.45, and the instability coefficient is 47. The predicted hydrophobicity of TSHB protein is as follows: Figure 4 As shown, this indicates that TSHB protein is an unstable hydrophilic protein. Prediction of the transmembrane domain of the Dolan sheep TSHB protein revealed that it lacks a transmembrane domain, as shown in the figure. Figure 5 As shown in the figure. Prediction of the TSHB protein signal peptide revealed that its cleavage site is located between amino acids 24 and 25, as shown in the figure. Figure 6 As shown in the figure. TSHB protein has a total of 31 phosphorylation sites, including 8 Serine sites, 12 Threonine sites, and 11 Tyrosine sites. The results are as follows. Figure 7 As shown in the figure. Secondary structure prediction of the TSHB protein indicates that it contains 15 α-helices (10.87%), 31 extended strands (22.46%), and 92 irregular coils (66.67%), as shown in the figure. Figure 8 As shown. Tertiary structure prediction revealed that α-helices and irregular coils were prevalent throughout the amino acid chain, with only a few extended chains, as shown in the results. Figure 9 As shown in the figure. The TSHB protein interaction prediction results indicate that it interacts with proteins such as TSHR and FSHR, as shown in the figure. Figure 10 As shown.

[0050] Table 2. Amino acid content and percentage of TSHB protein from Dolan sheep The expression of the TSHB gene in five tissues (hypothalamus, fallopian tube, pituitary gland, ovary, and uterus) of Dolan sheep was detected by qPCR. The results are as follows: Figure 11 As shown, the TSHB gene was expressed in five tissues across three stages: pre-menopause, menopause, and post-menopause. The expression level of the TSHB gene in the pituitary gland was significantly higher than in other tissues across all three stages (P < 0.05). There was no significant change in TSHB gene expression in ovarian and uterine tissues (P > 0.05). The expression level of the TSHB gene in the hypothalamus and fallopian tubes was significantly higher during menopause than before and after menopause (P < 0.05). These results indicate that this gene plays an important role in the initiation of menopause.

[0051] Example 2 An overexpression vector for the TSHB gene and interfering RNA were constructed and transfected into sheep ovarian granulosa cells to analyze the reproductive-related functions of the TSHB gene. 1. Construction of TSHB gene overexpression plasmid vector and interfering RNA The TSHB gene overexpression (pcDNA3.1-TSHB) vector and TSHB gene interference (siRNA) were constructed by Hanheng Biotechnology (Shanghai) Co., Ltd., and the primers used are shown in Table 3.

[0052] Table 3 TSHB gene overexpression and siRNA primer sequences In Table 3, si-TSHB-F, si-TSHB-R, si-NC-F, and si-NC-R represent the RNA sequence shown in SEQ ID NO.4-7 with the addition of TT bases in DNA form.

[0053] The specific construction method is as follows: Using TSHB cDNA as a template, high-fidelity DNA polymerase was used for PCR amplification to obtain the complete TSHB coding sequence. The obtained target fragment was purified by agarose gel electrophoresis and then ligated into the pcDNA3.1-EF1α-MCS-3Flag-CMV-EGFP expression vector, which had been linearized by double digestion with EcoRI and BamHI. The ligation reaction was performed using HB-infusion. TM A one-step homologous recombination system (Hanheng Biotechnology Co., Ltd., Shanghai, China) was completed. After transformation, bacterial PCR, and sequencing verification, a TSHB overexpression plasmid with the correct structure was obtained.

[0054] All siRNAs used in this invention are purified double-stranded siRNAs. Each siRNA is provided as a lyophilized powder, with a concentration of 2.5 nmol per tube. After dissolving in RNase-free DEPC water, a 20 μM stock solution is prepared (i.e., 2.5 nmol dissolved in 125 μL). The dissolved siRNA is aliquoted and stored at -20°C to avoid repeated freeze-thaw cycles.

[0055] In the cell transfection experiment, the working concentration of siRNA was determined to be 20 nM (final concentration) based on the preliminary experimental results, and a negative control (NC siRNA) was set up to exclude non-specific effects.

[0056] 2. Cell preparation before transfection (1) Cryopreserved cell resuscitation The granulosa cells from the ovaries of Dolan sheep were collected from Aksu Dolan Livestock Farm. The primary granulosa cells were cryopreserved in liquid nitrogen after isolation. Before use, the cells were removed from the liquid nitrogen container and immediately placed in a 37°C water bath with rapid agitation to thaw. The thawed cells were then centrifuged at 1000 rpm for 5 minutes. The centrifuged cells were carefully removed, and the supernatant was completely discarded in a clean bench, revealing a white cell precipitate at the bottom. 1 mL of complete culture medium containing 10% FBS was added to the precipitate to resuspend it. The mixture was then transferred to a T25 culture flask and incubated at 37°C in a 5% CO2 incubator.

[0057] (2) Cell passage culture When the cell adhesion rate in the culture flask reaches 70%-80%, discard the culture medium, wash the adherent cells with 2 mL of PBS buffer to remove floating impurities, discard the washing solution, add 1.5 mL of 0.25% trypsin to completely cover the cells, and incubate at 37°C for 2 min. When the cells shrink and become round and begin to flow, add an equal volume of complete culture medium to stop the digestion. Rinse the culture flask with a pipette to completely remove the cells, transfer the entire mixture to a 15 mL centrifuge tube, centrifuge at 1500 rpm for 5 min, and discard the supernatant. Resuspend the cells in 2 mL of complete culture medium and seed them into two new culture flasks (1 for 2) for subculture. After 24 h of culture, the subcultured cells are obtained.

[0058] 3. Identification of granulosa cells in the ovaries of Dolan sheep Cells were fixed in culture plates with 4% paraformaldehyde solution (purchased from Beijing Solarbio Science & Technology Co., Ltd.) for 90 min, and washed three times with phosphate-buffered saline (PBS) for 5 min each time. Cells were then permeabilized with 2 ml of 0.1% Triton X-100 for 10 min. Blocked with 10% goat serum at room temperature for 30 min. Anti-follicle-stimulating hormone receptor (FSHR) (purchased from Wuhan Sanying Biotechnology Co., Ltd.) was added at a 1:200 dilution and incubated overnight at 4°C. Cells were washed three times with PBS for 5 min each time. Goat anti-rabbit IgG (purchased from Wuhan Sanying Biotechnology Co., Ltd.) was added at a 1:200 dilution and incubated at room temperature for 2 h. Cells were washed three times with PBS for 5 min each time. DAPI was added in the dark for covering, and staining was performed at 37°C for 5 min. The slides were then treated with a fluorescence quencher and mounted for later use. Images were then observed using a Nikon inverted fluorescence microscope.

[0059] 4. Plasmid transfection of ovarian granulosa cells (1) 10 hours before plasmid transfection, adherent cells were inoculated with 1×10 5 Seeds were placed into 24-well plates. This ensured a cell count of 2 × 10⁶ cells at plasmid transfection. 5 / hole left and right.

[0060] (2) Remove the culture medium, wash twice with PBS, and add 500 μL of complete culture medium to each well.

[0061] (3) During overexpression transfection, add a complex containing 100 μl Opti-MEM, 0.75 μl Lipo3000, 1.75 μl plasmid DNA and 1 μl P3000 to each well to achieve overexpression of the target gene in the target cells.

[0062] During siRNA transfection, add 50 μl Opti-MEM, 2.5 μl siRNA, and 1 μl Lipofectamine to each well. TM The RNAiMAX complex silences the expression of target genes.

[0063] Blank groups are left unprocessed.

[0064] (4) Continue culturing for 24 hours, and replace the culture medium containing plasmids with fresh culture medium.

[0065] (5) Continue training.

[0066] 5. Analysis of plasmid-transfected Dolan sheep ovarian granulosa cells (1) The plasmid contains fluorescent protein (GFP tag). The transfected cells were observed under a fluorescence microscope. Images of granulosa cells from the ovaries of Dolan sheep 48 hours after transfection with the TSHB gene overexpression vector are shown below. Figure 13 As shown, the first row of images is the fluorescence image of empty vector transfection (500 μm), and the second row of images is the fluorescence image of plasmid transfection (200 μm). The results show that the TSHB overexpression vector was successfully transfected into Dolan sheep ovarian granulosa cells.

[0067] (2) For transfected ovarian granulosa cells, cell proliferation was detected using a CCK-8 assay kit (purchased from Shanghai Beyotime Biotechnology Co., Ltd.) at 12h, 24h, 36h, and 48h after plasmid transfection. Overexpression of the TSHB gene promoted ovarian granulosa cell proliferation with increasing time (P<0.01), while interference decreased, as shown in the results. Figure 14 As shown, the left image represents overexpression, and the right image represents interference. This preliminary evidence demonstrates that the TSHB gene can promote the proliferation of ovarian granulosa cells.

[0068] (3) RNA was extracted from cells 48 h after transfection and interference, and reverse transcribed to synthesize cDNA. The expression levels of TSHB-related genes CyclinE (XM_042231919.2) and CDK2 (XM_012158800.5) were detected. The expression levels of TSHB-related genes FSHR (NM_001009410.1) and TSHR (NM_001009410.1) were also detected. The primer sequences are shown in Table 4. Cell supernatant was collected 48 h after transfection using the ELISA kit (purchased from Beijing Solarbio Science & Technology Co., Ltd.). The levels of E2 and P4 in granulosa cells after TSHB overexpression and interference were detected using a microplate reader at 450 nm absorbance.

[0069] Table 4. Primers and related primer sequences for TSHB gene qPCR (SEQ ID NO. 8-29) (4) After 48 hours of transfection, discard the culture medium, wash with PBS, add 1 ml of RIPA lysis buffer (purchased from Wuhan Sai Biotechnology Co., Ltd.), lyse on ice for 30 min, scrape off the remaining cells with a cell scraper, centrifuge at 12,000×g for 10 min at 4℃, collect the supernatant as the total protein sample and store at -80℃. Plot the BCA curve according to the BCA protein concentration assay kit (purchased from Beijing Solarbio Science & Technology Co., Ltd.), and calculate the protein concentration. Add SDS loading buffer (5×) (purchased from Shanghai Beyotime Biotechnology Co., Ltd.) at a protein amount of 20 µg per well. After mixing, heat in a 95℃ metal water bath for 5 min, and store the denatured protein at -80℃. Use the one-step PAGE gel rapid preparation kit (purchased from Wuhan Sai Biotechnology Co., Ltd.). Spot the denatured protein at 20 µg per well, and electrophoresis conditions: 80V for 20 min and 120V for 60 min. After electrophoresis, the activated PVDF membrane was transferred to a gel membrane at 150 mA for 70 min. After transfer, it was incubated at room temperature for 2 h. The membrane was then incubated overnight at 4°C with dilutions of TSHB antibody, β-actin antibody, CDK2 antibody, Cyclin E antibody, Bax antibody, Bcl-2 antibody, Caspase3 antibody, STAR antibody, CYP19A1 antibody, TSHR antibody, and FSHR antibody. The membrane was washed three times with TBST (purchased from Beijing Solarbio Science & Technology Co., Ltd.) for 10 min each time. The membrane was incubated with secondary antibody at room temperature for 1 h, and then wetted with chromogenic solution in the dark for 2 min to develop color. Images of the PVDF membrane were captured using a Tanon 4000SF imaging system and saved. The gray values ​​of the bands were analyzed using ImageJ software, and the ratio of the gray value of the target protein to that of the internal control was calculated as the relative expression level of the target protein. The antibodies used in this experiment are shown in Table 5.

[0070] Table 5. Antibody information related to the experiment The results showed that, to confirm that the isolated cells were granulosa cells, immunofluorescence staining was used to detect the expression of FSHR in the cells (positive cytoplasmic FSHR confirmed granulosa cell identity). Granulosa cells isolated from Dolan sheep ovaries were found to specifically express FSHR. The proportion of positive cells exceeded 95%, indicating that the isolated cells were granulosa cells. Figure 12 As shown.

[0071] TSHB gene expression was measured at 24h, 36h, and 48h. Transfected Dolan sheep ovarian granulosa cells were observed using a Nikon fluorescence inverted microscope. The presence of abundant green fluorescent protein indicated successful in vitro overexpression transfection and high expression of the TSHB gene in the granulosa cells. The results are as follows: Figure 13As shown in the figure. CCK-8 assays revealed that cell proliferation increased after overexpression, while decreased after interference. The optimal effect was observed at 48 hours, and subsequent experiments were conducted within this timeframe. Specific results are shown in the figure. Figure 14 As shown. Figure 15 As shown in the figure, pcDNA3.1 represents the overexpression empty vector group, and pcDNA3.1-TSHB represents the TSHB overexpression transfection group; si-NC represents the interference empty vector group, and si-TSHB represents the TSHB interference transfection group. GAPDH and β-actin are internal controls.

[0072] In ovarian granulosa cells overexpressing TSHB for 48 hours, qPCR analysis revealed significantly higher mRNA levels of Cyclin E and CDK2 compared to the empty vector control (P<0.01). This suggests that TSHB upregulation may drive cell proliferation by enhancing the Cyclin E / CDK2 signaling axis and promoting G1 / S phase transition. In TSHB-overexpressing ovarian granulosa cells, the mRNA expression of pro-apoptotic molecules Bax and Caspase 3 was significantly lower than in the empty vector control (P<0.01), while the expression of the anti-apoptotic factor Bcl-2 was significantly increased (P<0.01). Protein levels were consistent with these results. Figure 16 and Figure 17 As shown, TSHB may inhibit apoptosis by regulating the Bcl-2 / Bax balance and inhibiting Caspase3 activation.

[0073] In ovarian granulosa cells overexpressing and interfering with TSHB gene, qPCR was used to detect acute regulatory steroid protein (STAR) and cytochrome P450 family 19 subfamily A member 1 (CYP19A1). The results showed that STAR and CYP19A1 mRNA levels were significantly lower than in the empty vector (P<0.01), and increased after interference. Related genes TSHR and FSHR mRNA levels were significantly higher than in the empty vector (P<0.05), and decreased after interference. Protein levels were consistent with these findings. Figure 18 and Figure 19 As shown in the figure. Furthermore, overexpression resulted in a decrease in the secretion of both E2 and P4 (P<0.05), as indicated by the results. Figure 20 As shown in the figure. The results indicate that TSHB gene overexpression inhibits steroid production and maintains follicle-stimulating signaling by increasing gonadotropin receptor expression.

[0074] In summary, the results show that TSHB overexpression can promote granulosa cell proliferation over time. The TSHB gene can significantly upregulate the expression of Cyclin E, CDK2, Bcl-2 and related genes TSHR and FSHR, downregulate the expression of Bax and Caspase3 genes in cells, as well as steroid-related STAR and CYP19A1, and inhibit the secretion of E2 and P4.

[0075] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. Application of TSHB protein or its coding gene or biological material containing the coding gene of TSHB protein in regulating proliferation of ovine ovarian granulosa cells, which is for non-disease diagnosis or treatment purpose.

2. Application of TSHB protein or its coding gene or biological material containing the coding gene of TSHB protein in breeding ovine sheep with strong proliferation ability of ovarian granulosa cells, which is for non-disease diagnosis or treatment purpose.

3. Application of TSHB protein or its coding gene or biological material containing the coding gene of TSHB protein in improving the trait of proliferation ability of ovine ovarian granulosa cells, which is for non-disease diagnosis or treatment purpose.

4. Application of TSHB protein or its coding gene or biological material containing the coding gene of TSHB protein in improving the proliferation ability of ovine ovarian granulosa cells, which is for non-disease diagnosis or treatment purpose.

5. Use according to any one of claims 1 to 4, characterized in that, The NCBI accession number of the amino acid sequence of the TSHB protein is XP_004002417.

2.

6. Use according to any one of claims 1 to 4, characterized in that, The nucleotide sequence encoding the TSHB protein is shown in SEQ ID NO. 1 or a sequence fully complementary to the nucleotide sequence shown in SEQ ID NO.

1.

7. Use according to any one of claims 1 to 4, characterized in that, The biological material is an expression cassette, a vector or a host cell.

8. Use according to any one of claims 1 to 4, wherein The ovine sheep is Duolang sheep.

9. A method of altering the proliferative capacity of ovine granulosa cells for non-disease diagnostic or therapeutic purposes, characterized by, By modulating TSHB the expression level of the genes and / or TSHB protein to change the proliferation ability of sheep ovarian granulosa cells.

10. The method of claim 9, wherein, When it is necessary to enhance the proliferative capacity of sheep ovarian granulosa cells, overexpression TSHB Genes and / or TSHB protein.

Images