FGA combined with caspase3 in regulating ovarian granulosa cells

Abstract

This invention discloses the application of FGA binding to Caspase3 in regulating ovarian granulosa cells, belonging to the fields of cell engineering and genetic engineering. Using FGA as the research object, this invention employs molecular and cell biology methods to study its application in ovarian granulosa cells, verifying from multiple levels and perspectives that the FGA gene regulates the growth and development of ovarian follicles and affects the onset of puberty in mice. This invention can provide new reference for the genetic improvement of sow reproductive performance and has good application value.

Description

Technical Field

[0001] This invention belongs to the fields of cell engineering and genetic engineering technology, specifically relating to the application of FGA binding to Caspase3 in regulating ovarian granulosa cells. Background Technology

[0002] In female mammals, the growth and development of follicles determine their reproductive potential, and the function of each follicle is controlled by endocrine and paracrine factors. The total number of ovarian follicles is determined early in life, and their depletion with age leads to reproductive senescence. Follicle development progresses through primordial, primary, and secondary follicle stages before acquiring the antral cavity (tertiary and mature follicles). After the antral phase, follicle development has two fates: most follicles undergo atresia and degenerate, while a minority reach the pre-ovulatory stage under the stimulation of circulating Gn after puberty. Compared to less fertile animal species, more fertile species tend to have a higher number of mature follicles and a higher ovulation rate in their ovaries. However, the vast majority of follicles (over 99%) do not reach the pre-ovulatory stage but cease growth during development, undergoing a degenerative process known as atresia. Extensive atresia of follicles leads to a significant reduction in reproductive performance in mammals. In summary, the growth and development of follicles in sow ovaries is a key factor affecting their reproductive performance and can directly impact the economic benefits of farms. However, the specific mechanisms affecting follicle development still need further in-depth research to provide a reference for improving the current relatively low reproductive efficiency and utilization rate.

[0003] Follicular atresia is primarily caused by apoptosis of granulosa cells (GCs). In actual production, female mammals that fail to come into estrus on time or do not come into estrus directly impact utilization rates and the economic benefits of farms. The main reasons for this are twofold: firstly, immature ovarian follicles cannot secrete sufficient hormones to promote estrus; secondly, excessive apoptosis of granulosa cells within the ovarian follicles leads to follicular atresia or over-activation, accelerating the depletion of primordial follicles and resulting in premature ovarian failure.

[0004] Fibrinogen alpha chain (FGA) is a cell adhesion molecule, normally located intracellularly, with a potential paracrine mechanism on surrounding cells. It can activate the FAK / AKT signaling pathway, promoting cell proliferation, migration, and invasion, and inhibiting apoptosis. Numerous studies have shown that FGA can promote angiogenesis by activating the VEGF-VEGFR2-FAK signaling pathway in endothelial cells. Besides being a key protein in coagulation-related mechanisms such as angiogenesis, FGA also primarily affects cell proliferation, adhesion, migration, and invasion in human immortalized ectopic endometrial epithelial cell line (hEM15A), human hepatocellular carcinoma cell line (HepG2), and gastric cancer cell line (GC-27), thereby inhibiting apoptosis. However, research on the relationship between the FGA gene and follicular growth and development has not yet been reported. Summary of the Invention

[0005] To address these issues, the primary objective of this invention is to provide applications for FGA gene-related biomaterials.

[0006] Another objective of this invention is to provide the application of FGA binding to Caspase3 in regulating ovarian granulosa cells.

[0007] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0008] The application of FGA gene-related biomaterials, wherein the biomaterials are any one or more of the following substances A, B, C, D, E, F, and G:

[0009] A. Full-length genomic DNA of the FGA gene or its homologous nucleic acid molecules;

[0010] B. FGA gene CDS region sequence or its homologous nucleic acid molecules;

[0011] The protein encoded by the C.FGA gene or its homologous amino acids;

[0012] D. Substances that can increase the expression level and / or activity of substance A or B;

[0013] E. Substances that can reduce the expression level and / or activity of substance A or B;

[0014] F. Substances that can increase the expression level and / or activity of substance C;

[0015] G. Substances that can reduce the expression level and / or activity of substance C;

[0016] The application described is any one or more of the following applications I, II, III, IV, and V:

[0017] I. Application in regulating follicle development / preparing products for regulating follicle development;

[0018] II. Application in regulating granulosa cell proliferation / preparation of products that regulate granulosa cell proliferation;

[0019] III. Application in regulating granulocyte apoptosis / preparation of products that regulate granulocyte apoptosis;

[0020] IV. Application in regulating estrogen secretion / preparation of products that regulate estrogen secretion;

[0021] V. Application in regulating initial symptom initiation / preparation of products for regulating initial symptom initiation.

[0022] Furthermore, application I includes any one or more of the following applications 1) and 2):

[0023] 1) Application of substances A, B, C, D, or F in promoting follicle development / preparing products that promote follicle development;

[0024] 2) Application of substance E or G in inhibiting follicle development / preparation of products that inhibit follicle development.

[0025] Furthermore, Application II includes any one or more of the following applications 1) and 2):

[0026] 1) Application of substances A, B, C, D, or F in promoting granulocyte proliferation / preparation of products that promote granulocyte proliferation;

[0027] 2) Application of substance E or G in inhibiting granulocyte proliferation / preparation of products that inhibit granulocyte proliferation.

[0028] Furthermore, application III includes any one or more of the following applications 1) and 2):

[0029] 1) Application of substances A, B, C, D, or F in inhibiting granulocyte apoptosis / preparation of products that inhibit granulocyte apoptosis;

[0030] 2) Application of substances E or G in promoting granulocyte apoptosis / preparation of products that promote granulocyte apoptosis.

[0031] Furthermore, application IV includes any one or more of the following applications 1) and 2):

[0032] 1) The application of substances A, B, C, D, or F in promoting estrogen secretion / in the preparation of products that promote estrogen secretion;

[0033] 2) Application of substance E or G in inhibiting estrogen secretion / preparation of products that inhibit estrogen secretion.

[0034] Furthermore, application V includes any one or more of the following applications 1) and 2):

[0035] 1) The application of substances A, B, C, D, or F in promoting primigravida initiation / in the preparation of products that promote primigravida initiation;

[0036] 2) Application of substance E or G in inhibiting primordia initiation / preparation of products that inhibit primordia initiation.

[0037] Furthermore, in application IV, the estrogen includes estradiol E2.

[0038] Furthermore, the application described is an application in an in vitro environment.

[0039] Furthermore, the application described is in mammals.

[0040] Furthermore, the application is described as an application in humans, pigs, or rats.

[0041] Furthermore, when the application is an application in humans:

[0042] The nucleotide sequence of the full-length genomic DNA described in A is shown in the sequence of NCBI Gene ID: 2243 (NCBI Reference Sequence: NC_000004.12).

[0043] The nucleotide sequence of the CDS region described in section B is shown in NCBI CCDS:CCDS3787.1.

[0044] The amino acid sequence of the protein described in C is shown in NCBI NP_000499.1.

[0045] Furthermore, when the application is in pigs or rats:

[0046] The nucleotide sequence of the full-length genomic DNA described in A is shown in the sequence of NCBI Gene ID:14161 (NCBI Reference Sequence: NM_010196.4).

[0047] The nucleotide sequence of the CDS region described in section B is shown in GenBank:BC005467.1.

[0048] The amino acid sequence of the protein described in C is shown in GenBank:AAH05467.1.

[0049] Furthermore, the homologous nucleic acid molecules mentioned in A refer to nucleic acid molecules that have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with full-length genomic DNA.

[0050] Furthermore, the homologous nucleic acid molecules mentioned in B refer to nucleic acid molecules that have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the CDS region sequence.

[0051] Furthermore, the homologous amino acids mentioned in C refer to amino acids that have at least 60%, at least 65%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the amino acid sequence of the protein.

[0052] Furthermore, the substance D or F is selected from: an overexpression recombinant vector containing the corresponding nucleic acid molecule.

[0053] Furthermore, the overexpression recombinant vector uses pcDNA3.1 as the vector.

[0054] Furthermore, the substance E or G is selected from: siRNA, shRNA, sgRNA or knockout recombinant vectors containing the same for knocking out / knocking down gene expression.

[0055] When the application is in humans, the target sequence corresponding to the siRNA is: 5′-GCTGCAGGATGAAAGGGTT-3′.

[0056] When the application is in humans, the target sequence corresponding to the shRNA is: 5′-GGAACAACAAGGATTCTAA-3′.

[0057] Application of FGA binding to Caspase3 in regulating ovarian granulosa cells: In vitro, the FGA gene binds to the apoptosis-related gene Caspase3, negatively regulating apoptosis in ovarian granulosa cells.

[0058] Furthermore, increasing exogenous FGA gene expression and decreasing Caspase3 mRNA and protein expression levels inhibits apoptosis of ovarian granulosa cells; inhibiting FGA gene expression and increasing Caspase3 mRNA and protein expression levels promotes apoptosis of ovarian granulosa cells.

[0059] Furthermore, the increase in exogenous FGA gene expression is achieved through gene overexpression technology. The gene overexpression vector is prepared as follows: (1) RNA is extracted from ovarian granulosa cells, reverse transcribed into cDNA, and PCR amplification is performed using cDNA as a template to obtain the target fragment; (2) The target fragment is ligated into the pcDNA3.1 vector digested with restriction endonucleases BamHI and NHeI to obtain the gene overexpression vector; the primers used for PCR amplification are as follows:

[0060] FGA F: 5′-CGCGGATCCCTTTCAGCTGGAGGTGCTCCTCA-3′

[0061] FGAR: 5′-CTAGCTAGCAAATGCAAGGGGCCATGGGAA-3′.

[0062] Furthermore, the inhibition of FGA gene expression is achieved through siRNA or shRNA.

[0063] When the application is in humans, the target sequence corresponding to the siRNA is: 5′-GCTGCAGGATGAAAGGGTT-3′.

[0064] When the application is in pigs or mice, the target sequence corresponding to the shRNA is: 5′-GGAACAACAAGGATTCTAA-3′.

[0065] The verification results of this invention are as follows:

[0066] 1. FGA promotes the proliferation of ovarian granulosa cells and cell cycle progression.

[0067] This invention transforms a constructed FGA overexpression vector and a small FGA interfering fragment into GCs. EdU assays revealed that FGA promotes GC proliferation. To further understand the influence of gene expression in GC proliferation-related pathways, qRT-PCR and Western blotting results showed that, compared to the control group, FGA significantly promoted the mRNA expression levels of proliferation-related genes such as STAR, PCNA, IKBA, and SP1 in GCs. The expression of the proliferation marker, proliferating cell nuclear antigen PCNA, was significantly increased at both protein and mRNA levels, confirming the EdU proliferation experiment results. Subsequently, flow cytometry was used to detect cell cycle progression. The results showed that FGA interference significantly decreased the S phase of the GC cell cycle, which is the DNA replication stage, and the decreasing trend was also significant in the G2 / M phase. However, after FGA overexpression, the S phase of the GC cell cycle also showed a decreasing trend, but the inhibition level rebounded compared to the FGA interference group, and the decreasing trend in the G2 / M phase was no longer significant. This indicates that FGA interference can inhibit the cell cycle progression of GCs. Results of qRT-PCR and Western blotting analysis on FGA's effects on cell cycle progression pathways revealed that FGA promotes cell cycle progression in GCs by upregulating the mRNA expression levels of MYC, CCND1, CDK7, CCNE2, and CDK4, as well as the protein expression levels of CCND1 and CCNE2. In summary, FGA can promote GC cell cycle progression and thus upregulate GC proliferation rate by increasing the expression levels of PCNA, CCND1, and CCNE2.

[0068] 2. Effects of FGA on follicular development in sows and age of puberty in mice

[0069] This invention first collects porcine ovaries and divides follicles into large follicles (>7mm) and small follicles (<3mm), collecting GCs separately. Differential expression of FGA mRNA and protein in porcine follicles of different sizes is detected by qRT-PCR and Western blotting. It was found that FGA expression gradually increases with follicle development, thus suggesting that the FGA gene is involved in porcine follicle development. Subsequently, porcine follicles are in vitro infected with lentiviruses that overexpress or knock down FGA, and the effect of FGA on follicle development is examined at the tissue level. The results show that FGA overexpression can delay the disappearance of blood vessels on the follicle surface and delay the turbidity of follicular fluid, and inhibit the apoptosis level of follicular GCs. However, the follicle development and GC apoptosis levels after FGA knockdown are the opposite of those after FGA overexpression. Subsequently, female C57BL / 6J mice were treated with the same concentration of lentivirus three times (once per week). At 42 days of age, both ovaries were harvested, and one ovary was sectioned. HE staining and TUNEL assays revealed that overexpression of FGA significantly increased the number of ovarian follicles and corpora lutea, while also inhibiting apoptosis in ovarian GCs. The results of FGA knockdown were the opposite of those of FGA overexpression. These results indicate that FGA can inhibit apoptosis in ovarian GCs, thereby promoting ovarian follicle development.

[0070] Furthermore, this invention found that, compared with the control group, overexpression of FGA advanced the estrus age in mice. In addition, FGA significantly increased the concentration of E2 in mouse serum. Subsequent studies on the effects of FGA lentivirus overexpression or knockdown on pathways related to apoptosis, proliferation, and cell cycle progression in ovarian follicle GCs revealed that FGA inhibited apoptosis in porcine follicle GCs by suppressing the mRNA and protein expression levels of pro-apoptotic genes P53, BIM, and Caspase9, and activated the mRNA and protein expression of PCNA, STAR, and CCNE1 genes, thereby promoting the proliferation and cell cycle progression of porcine follicle GCs. Using the same method, the effects of FGA on pathways related to apoptosis, proliferation, and cell cycle progression in mouse ovarian follicles were examined. The results showed that FGA inhibited apoptosis in mouse ovarian GCs by suppressing the mRNA and protein expression of the cysteine ​​protease family genes Caspase3 and Caspase9, as well as the pro-apoptotic gene Bim. Furthermore, FGA also regulated cell proliferation and cell cycle progression in mouse ovarian GCs by affecting the expression levels of Cdk4 and Pcna. In summary, FGA can promote the growth and development of ovarian follicles by influencing and regulating the apoptosis, proliferation, and E2 secretion of ovarian GCs, thereby advancing the age of puberty in mice.

[0071] The present invention has the following advantages and effects compared with the prior art:

[0072] This invention focuses on FGA (follicular ovarian follicle gene) and employs molecular and cell biology methods to study its application in ovarian granulosa cells. It verifies, from multiple levels and perspectives, that the FGA gene regulates the growth and development of ovarian follicles and influences the onset of puberty in mice. This invention provides a new reference for the genetic improvement of sow reproductive performance and has significant application value. Attached Figure Description

[0073] Figure 1 Figure 1 shows the results of a study on the effects of FGA overexpression or interference on cell apoptosis. (a) Effect of FGA overexpression on the apoptotic phenotype of GCs; (b) Effect of FGA interference on the apoptotic phenotype of GCs; (c) Effect of FGA overexpression or interference on the mRNA expression level of apoptosis-related genes in GCs; (d) Effect of FGA overexpression or interference on the protein expression level of apoptosis-related genes in GCs.

[0074] Figure 2 Figure 1 shows the results of a study on the effects of FGA overexpression or interference on cell proliferation. (a) Effect of FGA overexpression on GC proliferation; (b) Effect of FGA interference on GC proliferation; (c) Effect of FGA overexpression or interference on the mRNA expression level of GC proliferation-related genes; (d) Effect of FGA overexpression or interference on the protein expression level of GC proliferation-related genes.

[0075] Figure 3 Figure 1 shows the results of a study on the effects of FGA overexpression or interference on cell cycle progression. (a) Effect of FGA overexpression on GC cell cycle progression; (b) Effect of FGA interference on GC cell cycle progression; (c) Effect of FGA overexpression or interference on mRNA expression levels of genes related to cell cycle progression; (d) Effect of FGA overexpression or interference on protein expression levels of genes related to cell cycle progression.

[0076] Figure 4 Figure showing the results of a CoIP experiment to detect the binding levels of FGA and GCs apoptosis-related genes and proteins.

[0077] Figure 5 Figure 1 shows the results of a nucleus-cytoplasm separation experiment to detect the expression level of Caspase3 in the nucleus and cytoplasm of GCs after overexpression or interference with FGA; (a) mRNA expression level of Caspase3 after overexpression or interference with FGA; (b) protein expression level of Caspase3 after overexpression or interference with FGA.

[0078] Figure 6 Figure 1 shows the results of a study on the relative expression level of FGA in porcine ovarian follicle development; (a) shows the mRNA expression level of FGA in porcine follicles; (b) shows the protein expression level of FGA in porcine follicles.

[0079] Figure 7 Figure 1 shows the efficiency of FGA overexpression or knockdown lentivirus infection of porcine follicles; (a) efficiency of FGA overexpression or knockdown lentivirus mRNA level detection; (b) efficiency of FGA overexpression or knockdown lentivirus protein level detection.

[0080] Figure 8 Photographs of pig follicles on days 1, 3, and 5 after FGA lentivirus was overexpressed or knocked down.

[0081] Figure 9 Figure showing the results of a study on the effect of FGA lentiviral overexpression or knockdown on apoptosis of GCs in follicles.

[0082] Figure 10 Figure 1 shows the results of a study on the effects of FGA lentiviral inoculation on genes related to the apoptosis-proliferation cycle pathway. (a) Effect of FGA overexpression or knockdown on the mRNA expression level of follicular apoptosis-related genes; (b) Effect of FGA overexpression or knockdown on the protein expression level of follicular apoptosis-related genes; (c) Effect of FGA overexpression or knockdown on the mRNA expression level of follicular proliferation cycle-related genes; (d) Effect of FGA overexpression or knockdown on the protein expression level of follicular proliferation cycle-related genes.

[0083] Figure 11 Figure 1 shows the results of a study on the effect of FGA overexpression or knockdown on the initiation of estrus in mice. (a) Efficiency of FGA overexpression or knockdown lentivirus on the expression level of FGA mRNA in mouse ovaries; (b) Efficiency of FGA overexpression or knockdown lentivirus on the expression level of FGA protein in mouse ovaries; (c) Effect of FGA overexpression or knockdown on the age of estrus in mice.

[0084] Figure 12 Figure 1 shows the results of a study on the growth and development of ovarian follicles in mice infected with FGA-overexpressing or knockdown lentiviruses. (a) Ovarian sections of mice overexpressing FGA were stained and photographed. CL represents the corpus luteum, and black arrows indicate cavitary follicles. Yellow arrows indicate precavitary follicles. (b) Ovarian sections of mice knockdown FGA were stained and photographed. CL represents the corpus luteum, and black arrows indicate cavitary follicles. Yellow arrows indicate precavitary follicles.

[0085] Figure 13 Figure 1 shows the results of a study on the efficiency of FGA overexpression or knockdown lentivirus infection of porcine follicles; (a) shows the effect of FGA overexpression on apoptosis of GCs in mouse ovarian follicles; (b) shows the effect of FGA knockdown on apoptosis of GCs in mouse ovarian follicles.

[0086] Figure 14Figure 1 shows the results of a study on the effect of FGA overexpression or knockdown lentiviral infection on the concentration of E2 in mouse serum; (a) E2 concentration level in the serum of FGA overexpressing mice; (b) E2 concentration level in the serum of FGA knockdown mice.

[0087] Figure 15 Figure 1 shows the results of a study on the effects of FGA overexpression or knockdown lentiviral infection on apoptosis, proliferation, and cycle-related genes in mouse ovaries. (a) Effect of FGA overexpression or knockdown on the mRNA expression levels of key genes related to ovarian apoptosis; (b) Effect of FGA overexpression or knockdown on the protein expression levels of genes related to ovarian apoptosis; (c) Effect of FGA overexpression or knockdown on the mRNA expression levels of genes related to ovarian proliferation and cycle; (d) Effect of FGA overexpression or knockdown on the protein expression levels of genes related to ovarian proliferation and cycle. Detailed Implementation

[0088] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions. Unless otherwise stated, all reagents and raw materials used in the present invention are commercially available.

[0089] This invention uses statistical methods to analyze the results of three independent experiments in each embodiment, calculates the "mean ± standard deviation" for each experiment, and uses one-way ANOVA to analyze the significance of the differences (in the figure, "*" indicates P<0.05, and "**" indicates P<0.01).

[0090] The following examples used the human ovarian granulosa cell line KGN, purchased from Wuhan Pronosei Life Sciences Co., Ltd., as a cell model. Ovarian tissue from Large White crossbred gilts from a slaughterhouse in Guangzhou was used for in vitro isolation and culture. 21-day-old C57BL / 6J black female mice, purchased from the Experimental Animal Center of Southern Medical University in Guangdong Province, were used as in vivo experimental models. Overexpression and knockdown of FGA lentivirus, purchased from Guangzhou Dongze Biotechnology Co., Ltd., were used for in vitro culture of porcine ovarian follicles and in vivo experiments in mice. The overexpression lentivirus was obtained by constructing the CDS region sequence (GenBank: BC005467.1) of the mouse FGA gene (NM_010196.4) into the pLVX-C-FLAG-mCMV-ZsGreen-IRES-Puro plasmid through the SpeI+NotI restriction site, and then introducing it into 293T cells to produce a high-titer lentivirus containing the target gene. The knockdown lentivirus was obtained by constructing the shRNA (target sequence: 5'-GGAACAACAAGGATTCTAA-3') for knocking down FGA into the pLVX-shRNA2-Puro plasmid through the BamHI+EcoRI restriction site, and then introducing it into 293T cells to produce a high-titer lentivirus containing the target gene.

[0091] The pMD-18T vector and T4 DNA Ligase used in the following examples were purchased from TaKaRa (Japan), and the pcDNA3.1(+) vector was purchased from Promega (USA).

[0092] Example 1: Study on the effects of FGA on ovarian granulosa cell proliferation and cell cycle progression

[0093] 1. Construct an overexpression vector for the FGA gene.

[0094] (1) Total RNA was extracted from KGN cell line according to the instructions of Shanghai Feijie Biotechnology Total RNA Rapid Extraction Kit and reverse transcribed into cDNA.

[0095] (2) Specific primers for amplifying the CDS region of FGA (Gene ID: 2243) were designed using NCBI, and the extracted cDNA was used as a template for amplification. The amplified fragment was purified, recovered, ligated into the pMD18T vector (purchased from Takara), transformed, screened, and sequenced to confirm its correctness before extracting ordinary plasmids. The specific primer sequences are as follows:

[0096] (2) Analysis using BioEdit software revealed that the CDS region sequence of the FGA gene lacked both BamHI and NHeI restriction endonuclease sites, while the pcDNA3.1 vector contained both BamHI and NHeI restriction sites. BamHI and NHeI restriction site sequences were added to the upstream and downstream primers, respectively. PCR amplification was performed using the recombinant pMD18T plasmid of the FGA CDS region as a template. The fragment was purified, recovered, double-digested, ligated into the pcDNA3.1 vector, transformed, screened, and sequenced to confirm its correctness. An endotoxin-free plasmid (an endotoxin-free plasmid mini-extraction kit was purchased from Magen, USA) was then extracted and named pcDNA3.1-FGA.

[0097] The primer sequences are as follows:

[0098] FGA F:5′- CGC GGATCCCTTTCAGCTGGAGGTGCTCCTCA-3′

[0099] FGA R:5′- CTA GCTAGCAAATGCAAGGGGCCATGGGAA-3′.

[0100] 2. Transfer the constructed FGA overexpression vector and FGA small interfering fragment into GCs.

[0101] After successful construction of the FGA overexpression vector was verified by double enzyme digestion, GCs were seeded in 24-well cell culture plates to verify the overexpression efficiency of pcDNA3.1-FGA and the interference efficiency of si-FGA. pcDNA3.1 was used as a control for pcDNA3.1-FGA, and 100 ng, 200 ng, and 500 ng of pcDNA3.1 and pcDNA3.1-FGA vector plasmids were transfected, with three replicates per group. RNA was extracted after 24 h. The results showed that the mRNA expression level of FGA significantly increased with increasing transfection concentration (P < 0.001). Since different concentrations of the overexpression vector all had good transfection efficiency, 100 ng of pcDNA3.1-FGA was selected for subsequent experiments. Simultaneously, si-NC was used as a control for si-FGA (5′-GCTGCAGGATGAAAGGGTT-3′), and 50 nM and 100 nM of si-NC and si-FGA were transfected into GCs, with three replicates per group. The results showed that qRT-PCR indicated that si-FGA exhibited the best interference efficiency at 50 nM (P < 0.001). Therefore, 100 ng of pcDNA3.1-FGA and 50 nM of si-FGA were selected for subsequent experiments.

[0102] Cell transfection (6-well plate): Observe the cell confluence under an optical microscope. The transfection effect is best when the confluence reaches 70-80%. Discard the culture medium in the culture plate, add PBS (containing 1% penicillin and bismuth subsalicylate) along the wall to wash the cells, repeat the washing once, and then add 1800 μL of incomplete culture medium. Take a 1.5 or 2 mL sterile centrifuge tube, prepare the transfection mixture according to the transfection concentration described above, and refer to the transfection instructions of Lipofectamine 3000. Incubate at room temperature for 10-15 min. Add 200 μL of transfection mixture to each well of the 6-well plate, gently shake in a cross shape to mix, and return to the incubator for culture. After 24-48 h of transfection, collect the cells for subsequent experiments. (2) TSA treatment: Observe the cell confluence under an optical microscope. The treatment effect is best when the confluence reaches 70-80%. According to the TSA drug instructions, weigh the TSA drug, dissolve it in an appropriate amount of DMSO solvent and dilute it to 0.5 μM. Discard the culture medium in the culture plate, add PBS (containing 1% penicillin and bismuth subsalicylate) along the wall to wash the cells, repeat the washing once, add incomplete culture medium, and add DMSO or TSA to each well respectively. DMSO is used as the control group. Repeat three wells for each group. Gently shake the cells in a cross shape to mix them evenly, put them back into the cell culture incubator for culture, and collect the cells for subsequent experiments 24 h after transfection.

[0103] 3. Apoptosis detection

[0104] This invention uses the Annexin V-FITC / PI cell apoptosis detection kit to detect the apoptosis rate of GCs cells.

[0105] 4. Cell proliferation detection

[0106] GC proliferation was detected according to the instructions of Ribobio's Cell-Light EdU Apollo567 In Virto Kit. After GCs were transfected with plasmids or small interfering RNA, they were labeled with EdU, fixed and stained, and examined under a microscope immediately or stored at 4°C in the dark.

[0107] 5. Cell cycle distribution detection

[0108] This invention uses flow cytometry to detect cell cycle progression.

[0109] 6. Western blot detection of target gene protein expression

[0110] Total protein extraction, protein quantification, protein denaturation, SDS-PAGE electrophoresis, membrane transfer, blocking, antibody incubation, color development and grayscale analysis.

[0111] 7. Chromatin Immunoprecipitation

[0112] ChIP testing was performed according to the MIKX Chromatin Immunoprecipitation (ChIP) Kit instruction manual.

[0113] 8. Chromatin accessibility

[0114] Refer to Epigen TEK EpiQuik TM Following the instructions of the Chromatin Accessibility Assay Kit, the following steps were performed: transfection of GCs with NC and HDAC2 followed by buffer and solution preparation; GC treatment; cell lysis and chromatin extraction; chromatin digestion; DNA purification; sample qRT-PCR; and data analysis.

[0115] 9. Immunoprecipitation

[0116] Refer to the ACErProtein A / G Magnetic IP / Co-IP Kit instruction manual.

[0117] 10. Nucleocytoplasmic separation

[0118] Nuclear-cytoplasmic RNA extraction: Inoculate a suitable amount of GCs into a culture plate, transfect with plasmids or small interfering fragments. After 24 h, wash the GCs with PBS (containing 1% penicillin and antibiotics) and repeat once. Add 200 μL of pre-chilled Lysis Buffer J to the culture plate and lyse on ice for 5 min. Transfer the lysate to a new 2 mL RNase-free tube and centrifuge at 16000 rpm for 10 min. Transfer the supernatant (cytoplasmic RNA) to a new 2 mL RNase-free tube; the remaining precipitate is nuclear RNA. Add 200 μL of Buffer SK to the supernatant and vortex for 10 s. Add 200 μL of anhydrous ethanol and vortex for 10 s. Transfer the mixture to a collection column, centrifuge at 6000 rpm for 1 min, discard the filtrate, and retain the adsorption column. Add 400 μL of Buffer SK to the nuclear RNA and vortex for 10 s. Repeat the steps above and retain the adsorption column. Add 400 μL of Wash to the adsorption column. Solution: Centrifuge at 16000 rpm for 1 min and discard the filtrate. Repeat twice, discarding the filtrate each time. Centrifuge at 16000 rpm for 1 min and discard the filtrate to dry completely. Add 50 μL of Elution Buffer E to the sponge in the center of the adsorption column, centrifuge at 2000 rpm for 2 min, then centrifuge at 14000 rpm for 1 min. Collect the nuclear and cytoplasmic RNA samples and store at -80℃.

[0119] Nuclear-cytoplasmic protein extraction: Genetic globulins (GCs) were seeded into culture plates and transfected with plasmids or small interfering fragments. After 24 h, the GCs were washed with PBS (containing 1% penicillin and antibiotics) once. The cell pellet was collected with 0.25% trypsin into a 1.5 mL centrifuge tube. The collected cells were washed twice with PBS. 200 μL of pre-chilled CERⅠ was added, vortexed for 15 s, and incubated on ice for 10 min. 11 μL of pre-chilled CERⅡ was added, vortexed for 5 s, incubated on ice for 1 min, vortexed for 5 s, and centrifuged at 16000 rpm for 5 min. The supernatant (cytoplasmic proteins) was immediately transferred to a new 2 mL RNase-free tube and stored at -80 °C. 100 μL of pre-chilled NER was added to the pellet, vortexed for 15 s, and incubated on ice for 40 min, vortexing for 15 s every 10 min and centrifuged at 16000 rpm for 10 min. The supernatant (nuclear proteins) was immediately transferred to a new 2 mL RNase-free tube. Store in RNase-free tubes at -80°C.

[0120] result:

[0121] (1) FGA inhibits granulocyte apoptosis

[0122] like Figure 1 As shown, flow cytometry analysis revealed that pcDNA3.1-FGA significantly inhibited early apoptosis of GCs compared to the control group (P < 0.01); conversely, si-FGA promoted early apoptosis of GCs. Further investigation was conducted to examine the effect of FGA on the expression levels of key apoptosis-related genes. qRT-PCR results showed that pcDNA3.1-FGA significantly inhibited the mRNA expression levels of apoptosis-related genes such as Bax, Caspase3, Caspase7, Caspase9, CREB1, P53, and BID (P < 0.001), and promoted the mRNA level of BAD; while si-FGA significantly promoted the mRNA expression levels of apoptosis-related genes such as Bax, Caspase7, Caspase9, CREB1, and BAD (P < 0.001). Western blot results showed that pcDNA3.1-FGA significantly inhibited the protein expression levels of Caspase3 and Caspase9 (P < 0.01), while si-FGA significantly increased the protein expression levels of Caspase3 (P < 0.05) and Caspase9 (P < 0.001). These results indicate that FGA can inhibit GC apoptosis by affecting the mRNA and protein levels of apoptosis-related genes such as Caspase3 and Caspase9.

[0123] (2) FGA promotes granulocyte proliferation

[0124] like Figure 2As shown, the EdU assay was used to detect the proliferation rate of GCs. Compared with the control group, pcDNA3.1-FGA significantly promoted the proliferation rate of GCs (P < 0.001); while the corresponding si-FGA significantly reduced the proliferation rate of GCs. qRT-PCR results showed that overexpression of FGA significantly promoted the mRNA expression of cell proliferation-related genes such as STAR (P < 0.01), PCNA (P < 0.01), IKBA (P < 0.05), SP1 (P < 0.05), and MCL (P < 0.01), and inhibited the mRNA expression level of GSK3B (P < 0.001). Interference with FGA significantly inhibited the mRNA expression of cell proliferation-related genes such as GSK3B, STAR, SP1, and MCL (P < 0.001). Western blot results showed that pcDNA3.1-FGA significantly promoted the expression levels of GSK3B (P < 0.05) and PCNA (P < 0.001) proteins, while si-FGA significantly reduced the expression levels of GSK3B and PCNA proteins (P < 0.01). These results indicate that FGA can promote GC proliferation by increasing the mRNA and protein expression levels of proliferation-related genes such as PCNA.

[0125] (3) FGA accelerates the cell cycle process of granulosa cells.

[0126] like Figure 3 As shown, flow cytometry results indicated that, compared to the control group, FGA overexpression significantly increased the number of GCs entering G0 / G1 phase (P < 0.05), decreased the number of GCs entering S phase (P < 0.01), while there was no significant difference in the number entering G2 / M phase. Interference with FGA significantly inhibited the number of GCs entering S and G2 / M phases (P < 0.001), indicating that FGA interference arrests GCs in G0 / G1 phase, thus delaying cell cycle progression. In the detection of the effects of FGA overexpression or interference on the mRNA expression levels of cell cycle-related genes, pcDNA3.1-FGA significantly promoted the mRNA expression levels of cell cycle-related genes such as MYC (P < 0.01), CCND1 (P < 0.001), and CCNE2 (P < 0.01), while inhibiting the mRNA expression levels of CDK2 and CCNE1 (P < 0.001). si-FGA significantly suppressed the mRNA expression levels of CCND1, CCNE2, and CDK7 (P < 0.001). Western blotting results showed that overexpression of FGA significantly increased the protein expression levels of CCND1 (P < 0.01) and CCNE2 (P < 0.001), while FGA interference significantly decreased the protein expression level of CCND1 (P < 0.05), with no significant difference in CCNE2 protein expression. These results indicate that FGA can promote the cell cycle progression of GCs by regulating the expression of cell cycle-related genes such as CCND1 and CCNE2 mRNA and protein.

[0127] (4) The interaction between FGA and Caspase3 affects granulocyte apoptosis.

[0128] like Figure 4 As shown, FGA antibody was used to co-precipitate ovarian GC proteins, and the protein complex was collected and denatured. Western blotting was then used to detect the binding of the denatured protein complex to antibodies against key apoptosis-related genes. Comparison with the negative control group (IgG) and the positive control group (Input) revealed that FGA protein bound to Caspase3 protein. To further investigate whether FGA and Caspase3 proteins bind to each other, a CoIP reverse verification experiment was performed. Caspase3 and BIM antibodies were used to co-precipitate ovarian GC proteins. After denaturation of the protein complex, Western blotting was used to detect FGA protein expression. The results showed that, compared with the IgG group, Caspase3 antibody could pull down both Caspase3 and FGA proteins. These results indicate that FGA can bind to Caspase3 protein, thereby regulating GC apoptosis.

[0129] (5) Distribution of Caspase3 in granule cells after FGA binds to Caspase3

[0130] like Figure 5 As shown, nucleocytoplasmic separation assays revealed that overexpression of FGA significantly decreased the mRNA expression level of Caspase 3 in the nucleus and cytoplasm of GCs (P < 0.001), while FGA interference significantly promoted the mRNA expression level of Caspase 3 in both the nucleus and cytoplasm of GCs (P < 0.001). Overexpression of FGA significantly inhibited the protein level of Caspase 3 in the nucleus and cytoplasm of GCs (P < 0.01), while FGA interference significantly promoted the protein expression level of Caspase 3 in both the nucleus and cytoplasm of GCs (P < 0.01). In conclusion, the results indicate that the binding of FGA to the apoptosis-related gene Caspase 3 can significantly inhibit the expression levels of Caspase 3 protein and mRNA in the nucleus and cytoplasm of GCs, thereby inhibiting GC apoptosis.

[0131] Example 2: Study on the effect of FGA on porcine follicle growth

[0132] This invention uses the ovaries of healthy commercial sows as experimental materials and divides them into large follicles (>7mm) and small follicles (<3mm) according to their size. RNA and protein are extracted from the large and small follicles respectively, and the expression pattern of FGA in the large and small follicles is detected. Figure 6As shown, qRT-PCR results indicated that FGA mRNA expression significantly increased with the growth and development of porcine follicles (P < 0.001). Western blot results showed that FGA protein expression was significantly higher in large follicles compared to small follicles (P < 0.05). These results suggest that FGA may play a promoting role in follicle growth and development.

[0133] Ovaries from healthy commercial sows were collected and isolated into uniformly sized single follicles in the laboratory, with as much excess connective tissue around the follicles as possible removed. The follicles were then infected with lentiviruses LV-FGA and sh-FGA, with at least three replicates per group. The follicles were cultured in vitro for 5 days, and photographs were taken on days 1, 3, and 5. RNA and protein were then extracted. Results are as follows: Figure 7 As shown in the qRT-PCR results, LV-FGA significantly promoted FGA mRNA expression (P < 0.001), while sh-FGA significantly inhibited FGA mRNA expression (P < 0.001). The FGA protein expression levels after LV-FGA and sh-FGA treatments were similar to those after mRNA treatment (P < 0.05). These results indicate that overexpression or knockdown of FGA lentiviruses is effective in interfering with follicle development and can be used for subsequent experiments.

[0134] 1. Isolation and culture of porcine follicles and lentivirus infection

[0135] Collected ovarian tissue was stored in pre-chilled PBS (containing 1% penicillin and antibiotics) and quickly transported back to the laboratory. It was then washed several times with PBS (containing 1% penicillin and antibiotics) until the PBS solution was clear and free of blood. The tissue was soaked in 75% alcohol for 30 seconds, then washed again with PBS (containing 1% penicillin and antibiotics), sealed, and transferred to the cell culture room. Follicles of 3-5 mm were dissected using a scalpel under a clean bench, carefully removing excess connective tissue from the follicles. The follicles were washed twice with PBS (containing 1% penicillin and antibiotics), then twice with DMEM / F12 follicle culture medium (containing 1% penicillin and antibiotics). One follicle was placed per well in a 24-well plate containing follicle culture medium and incubated at 38.5℃ with 5% CO2 for 24 hours. The follicles were washed twice with PBS (containing 1% penicillin and antibiotics), and the culture medium was replaced with fresh follicle culture medium. Lentiviral virus (titer 10) was then used. 7 Follicles were treated, and photographs were taken on days 1, 3, and 5 after lentivirus infection for observation.

[0136] 2. Other experiments are described in Example 1.

[0137] result:

[0138] (1) FGA promotes the development of follicular GCs

[0139] Lentiviral infection of porcine follicles in vitro culture for 5 days, such as Figure 8As shown, compared with the control group, FGA overexpression delayed the follicular fluid from becoming cloudy and delayed the disappearance of blood vessels on the follicular surface. However, after FGA knockdown, the follicular fluid began to become cloudy on day 3, and the blood vessels on the follicular surface disappeared on day 5. These results indicate that FGA can promote follicular growth and development.

[0140] (2) FGA inhibits apoptosis of follicular GCs

[0141] After lentivirus-infected porcine follicles were cultured in vitro for 5 days, they were collected and fixed with formaldehyde. The effect of FGA on apoptosis of GCs within the follicles was studied using TUNEL assay. Figure 9 As shown, compared with the control group, LV-FGA inhibited apoptosis of intrafollicular GCs, while sh-FGA promoted apoptosis of intrafollicular GCs. These results indicate that FGA can inhibit apoptosis of intrafollicular GCs.

[0142] (3) FGA affects the expression of key genes in the follicle function pathway.

[0143] To investigate the expression regulation of FGA during follicular development, this invention examines the effects of FGA on genes related to apoptosis, proliferation, and cycle progression in follicles. Figure 10As shown, qRT-PCR results indicated that LV-FGA significantly inhibited the mRNA expression levels of BID (P < 0.001), Caspase3 (P < 0.001), Caspase8 (P < 0.001), and BAD (P < 0.01) in follicles, while significantly promoting the expression levels of apoptosis-related gene mRNAs such as Bcl2 (P < 0.001), P53 (P < 0.001), and BAK (P < 0.01). sh-FGA significantly increased the mRNA expression levels of apoptosis-related genes such as BIM, Caspase8, Caspase9, Bcl2, P53, and BAD (P < 0.001). Western blot results showed that overexpression of FGA significantly reduced the protein expression levels of Caspase9 and P53, while there was no significant difference in BIM protein expression. Knockdown of FGA followed by detection of apoptosis-related gene protein expression revealed that sh-FGA significantly promoted the protein expression of BIM, Caspase9, and P53. The same method was used to examine the effects of FGA on genes related to proliferation and cell cycle progression in follicles. Overexpression of FGA significantly promoted the mRNA expression levels of key genes related to the cell cycle, including CDK1, CDK4, CCND1, PCNA, MCL1, CCNB1, and CCND2 (P < 0.001), while there was no significant difference in the mRNA expression level of RRM1. Knockdown of FGA significantly inhibited the mRNA expression levels of genes related to the cell cycle progression, including CDK2 (P < 0.05), CDK4 (P < 0.01), CCNE1 (P < 0.05), PCNA (P < 0.05), MCL1 (P < 0.01), CCNB2 (P < 0.01), CCND2 (P < 0.01), CCNE2 (P < 0.001), and RRM1 (P < 0.001). Western blot (WB) results showed that LV-FGA significantly increased the protein expression levels of proliferation cycle-related genes PCNA, STAR, and CCNE1, while sh-FGA showed the opposite effect, significantly decreasing the protein expression levels of genes such as PCNA, STAR, and CCNE1. These results indicate that FGA can regulate follicular growth and development by modulating the expression levels of mRNA and protein of genes such as Caspase9, P53, PCNA, STAR, and CCNE1 within the follicle.

[0144] Example 3: Study on the effect of FGA on mouse ovarian development

[0145] This invention uses C57BL / 6J female mice as an experimental animal model to study the effects of FGA on ovarian follicle growth and development. Figure 11As shown, compared with the control group, overexpression of FGA lentiviral vector significantly promoted the mRNA expression level of FGA in mouse ovaries (P < 0.001), while knockdown of FGA showed the opposite effect, significantly inhibiting the protein expression level of FGA in mouse ovaries (P < 0.001). Western blot results showed that LV-FGA significantly promoted the protein expression level of FGA in mouse ovaries (P < 0.001), while sh-FGA significantly inhibited the protein expression level of FGA in mouse ovaries (P < 0.01). These results indicate that overexpression or knockdown of FGA lentiviral vector can play a regulatory role in the constructed mouse experimental model and can be used for subsequent experiments.

[0146] 1. Mouse husbandry and lentivirus treatment

[0147] Twenty-six 21-day-old female C57BL / 6J mice were selected and randomly divided into four groups with toe markings: LV-NC group (n=5), LV-FGA group (n=8), sh-NC group (n=5), and sh-FGA group (n=8). After one week of acclimatization at the Experimental Animal Center of South China Agricultural University, mice were injected with lentiviral vector via intraperitoneal injection once a week for three consecutive weeks. The mice were weighed daily, and their estrus status was observed and recorded.

[0148] We observed the estrus patterns of mice daily to investigate the effect of FGA on the age of estrus on mice.

[0149] 3. HE staining

[0150] This invention uses hematoxylin-eosin staining (HE) to detect the development of mouse ovarian follicles. The specific experimental steps are described on the official website of Wuhan Saiweier Biotechnology Co., Ltd.

[0151] 4. Tunel detection of ovarian follicle apoptosis

[0152] This invention uses paraffin sections and fluorescent tubes to detect ovarian follicle apoptosis. The specific experimental steps are described on the official website of Wuhan Saiweier Biotechnology Co., Ltd.

[0153] 5. ELISA detection of estrogen secretion

[0154] The detection of mouse serum estrogen was performed using the estradiol enzyme-linked immunosorbent assay kit. The specific steps were as follows: (1) Add 100 μL of the sample to be tested (and blank wells) to the antibody-coated reaction wells and incubate at 37°C for 2 h; (2) Discard the supernatant, add 300 μL of washing buffer to each well, soak for 2 min and then discard the supernatant; (3) Add 100 μL of biotinylated antibody working solution to each well and incubate at 37°C for 1 h; (4) Same as (2); (5) Add 100 μL of enzyme conjugate working solution to each well and incubate at 37°C for 30 min; (6) Same as (2); (7) Add 100 μL of TMB substrate solution to each well and incubate at 37°C for 30 min in the dark; (8) Add 100 μL of 2M sulfuric acid, and the color changes from blue to yellow; (9) Detect the OD value at 450 mm using an ELISA reader within 10 min and calculate the result.

[0155] 6. For other experiments, refer to Example 1 or Example 2.

[0156] result:

[0157] (1) FGA promotes the age of puberty in mice

[0158] The results are as follows Figure 11 As shown, mice in the FGA lentiviral vector overexpression group showed estrus at an earlier age than control mice (P < 0.05). Mice in the FGA lentiviral vector knockdown group showed estrus at an average later age than control mice (P < 0.05). These results indicate that FGA can promote the age of estrus onset in mice.

[0159] (2) FGA promotes mouse follicle development

[0160] Hematoxylin and eosin staining were used to detect the growth and development of mouse ovarian follicles, and the results were as follows: Figure 12 As shown, compared with the control group, the number of pre-cavity follicles in the ovaries of mice overexpressing FGA was significantly reduced, while the number of corpora lutea and cavity follicles was significantly increased. The number of pre-cavity follicles in the ovaries of mice knocked down with FGA was significantly increased compared with the control group. These results indicate that FGA can promote the growth and development of ovarian follicles in mice.

[0161] (3) FGA inhibits ovarian apoptosis in mice

[0162] The level of GC apoptosis in mouse ovarian follicles was detected using a TUNEL section assay, and the results are as follows: Figure 13 As shown, compared with the control group, the apoptosis rate of GCs in the ovarian follicles of mice overexpressing FGA was significantly reduced. Conversely, knocking down FGA resulted in a significantly higher level of apoptosis of GCs in the ovarian follicles of mice in the FGA knockdown group compared to the control group. These results indicate that FGA can inhibit the apoptosis level of GCs in the ovarian follicles of mice.

[0163] (4) FGA promotes serum E2 secretion in mice

[0164] Mouse blood was collected, serum was obtained by centrifugation, and the concentration of E2 in mouse serum was detected by ELISA. Results are as follows: Figure 14 As shown, the serum E2 concentration in mice overexpressing FGA was significantly higher than that in the control group (P < 0.01). Compared with the control group, the serum E2 concentration in mice knocked down FGA was significantly lower (P < 0.01). These results indicate that FGA can significantly promote estrogen secretion in mice.

[0165] (5) FGA affects the expression of key genes in the mouse ovarian function pathway.

[0166] This invention uses qRT-PCR and Western blotting to detect the effects of FGA on the mRNA and protein expression levels of key genes in the apoptosis and proliferation cycle-related pathways of mouse ovarian GCs. For example... Figure 15As shown, FGA overexpression significantly inhibited the expression levels of apoptosis-related gene mRNAs such as Creb1, Caspase3, Caspase7, Bax, P53, and Bcl2 (P < 0.001). Conversely, FGA knockdown significantly increased the mRNA expression levels of Creb1 (P < 0.01), Caspase3 (P < 0.01), Caspase7 (P < 0.01), Bax (P < 0.001), P53 (P < 0.01), Bcl2 (P < 0.001), and Bim (P < 0.001), while there was no significant difference in Caspase8 and Caspase9 expression. Compared with the control group, FGA overexpression significantly reduced the protein expression levels of Caspase3 (P < 0.01), Bim (P < 0.01), and Caspase9 (P < 0.05). The protein expression levels of Caspase3 (P < 0.001), Bim (P < 0.001), and Caspase9 (P < 0.01) in the FGA knockdown group were significantly higher than those in the control group. The same method was used to examine the effects of FGA on mouse ovarian cell cycle-related pathways. Compared with the control group, the LV-FGA group significantly promoted the mRNA expression levels of cell cycle-related genes such as Star (P < 0.05), Mcl1 (P < 0.001), Pcna (P < 0.05), P65 (P < 0.01), Cdk4 (P < 0.02), and Ccne1 (P < 0.05), significantly inhibited the expression of Sp1 gene mRNA (P < 0.001), and showed no significant difference in Ccnb2 mRNA expression. The mRNA expression levels of Star (P < 0.01), Mcl1 (P < 0.001), Sp1 (P < 0.001), Pcna (P < 0.01), P65 (P < 0.001), Cdk4 (P < 0.001), and Ccnb2 (P < 0.001) in the sh-FGA group were significantly higher than those in the control group. Protein level results showed that overexpression of FGA significantly increased the protein expression level of the cell cycle-related gene P65 (P < 0.001), while the protein levels of Cdk4 and Pcna showed no significant difference compared to the control group. Conversely, knockdown of FGA significantly inhibited the protein expression levels of P65, Cdk4, and Pcna (P < 0.05). The above results indicate that FGA can inhibit apoptosis of mouse ovarian GCs by suppressing the mRNA and protein expression of Caspase3, Bim and Caspase9; and promote the proliferation rate and GC cycle progression of mouse ovarian GCs by regulating the expression levels of P65, Cdk4 and Pcna.

[0167] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the embodiments described above. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of FGA gene-related biomaterials, characterized by: The biomaterial is any one or more of the following substances A, B, C, and D: A. Full-length genomic DNA of the FGA gene; B. FGA gene CDS region sequence; C. Protein encoded by the FGA gene; D. Recombinant vectors containing overexpression of the corresponding nucleic acid molecules in A or B; The application is any one or more of the following applications I and II: I. Application of FGA gene overexpression in promoting follicle development / preparation of products promoting follicle development; the promotion of follicle development is for non-therapeutic purposes; II. Application of FGA gene overexpression in promoting first puberty / in the preparation of products that promote first puberty.

2. The application of the FGA gene-related biomaterials according to claim 1, characterized in that: The application described is in mammals; The application described is for use in an in vitro environment.

3. The application of the FGA gene-related biomaterials according to claim 2, characterized in that: The application described refers to its use in humans, pigs, or mice.

4. The application of the FGA gene-related biomaterials according to claim 3, characterized in that: When the application is a human application: The nucleotide sequence of the full-length genomic DNA described in section A is shown in the sequence of NCBI Gene ID: 2243; The nucleotide sequence of the CDS region described in section B is shown in the sequence of NCBI CCDS:CCDS3787.1; The amino acid sequence of the protein described in C is shown in NCBI NP_000499.

1. When the application is in pigs or rats: The nucleotide sequence of the full-length genomic DNA described in section A is shown in the sequence of NCBI Gene ID:14161; The nucleotide sequence of the CDS region described in section B is shown in GenBank: BC005467.1; The amino acid sequence of the protein described in C is shown in GenBank: AAH05467.

1.

5. The application of the FGA gene-related biomaterials according to claim 1, characterized in that: The overexpression recombinant vector used is pcDNA3.1.

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