Imprinted gene Gnas-based male peripheral precocious precocious intervention target and application thereof

Abstract

The invention discloses a male-source female peripheral precocious precocious intervention target based on an imprinting gene Gnas and application of the male-source female peripheral precocious precocious intervention target. The pregestation caffeine exposure of the male parent has obvious influence on the ovarian generation of the female offspring, and can lead to PPP (Poly-Protein Protein) generation. Through screening and verification, it is found that expression increase of the ovarian granular cell imprinting gene Gnas plays a key role in the parent PPP. The cAMP / PKA / CREB signaling pathway can be up-regulated or down-regulated by overexpression of the ovary Gnas of the female rat or knockout of the ovary Gnas of the female offspring based on a parent PPP model, so that the ovary estrogen synthesis function and PPP generation are induced or inhibited. The invention discloses a key effect of the Gnaas / cAMP / PKA / CREB pathway in regulation and control of ovarian estrogen synthesis, and provides a new biological basis and an early prevention and treatment strategy for targeted therapy of female PPP.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a paternally derived female peripheral precocious puberty intervention target based on the imprinted gene Gnas and its application. Background Technology

[0002] Precocious puberty is one of the most common endocrine disorders in pediatrics. Its global incidence is about 1 / 5,000, with girls having a much higher incidence than boys, 10 times that of boys [1, 2]. Due to repeated exposure to estrogen, complications (such as early menarche and accelerated skeletal maturation) have caused psychological distress in children, reduced height in adults, and increased risk of adult diseases (such as breast cancer, diabetes, and cardiovascular disease), which have become a public health problem and seriously endanger women's physical and mental health. When sex hormone secretion is excessive and independent of gonadotropin secretion, it is called peripheral precocious puberty (PPP), accounting for 50%-60% of all cases of precocious puberty, and the male-to-female ratio is 1:400-1000 [3]. Clinically, elevated serum estradiol (E2) levels and basal serum luteinizing hormone (LH) levels within the normal range or decreased before puberty are used as indicators for initial screening to determine the type of precocious puberty in girls [4]. Traditional views hold that precocious puberty depends on multiple factors, including genetics, environment, metabolism, and race. However, increasing research suggests that precocious puberty has a fetal developmental origin. Studies have shown that paternal disease conditions (such as metabolic syndrome) and adverse environmental exposures (such as high-fat diets and cypermethrin) are associated with the reproductive endocrine health of female offspring after birth [5-7]. Currently, with the increasing severity of environmental pollution, social pressure, and chronic stress, the incidence of precocious puberty in women is rising year by year, and there is still no effective treatment. This may be related to the complex and diverse etiologies and clinical manifestations of precocious puberty.

[0003] For decades, researchers at home and abroad have proposed the "Developmental Origins of Health and Disease" (DOHaD) theory, revealing that many ovarian diseases, including polycystic ovary syndrome, have an embryonic origin [8]. A woman's health and lifestyle before pregnancy have been proven to be an important factor affecting the health of her offspring, but a long-neglected factor is the father's adverse environmental exposure and overall health status before pregnancy. Compared with early embryonic development, the occurrence and maturation of gametes (sperm and egg) takes a longer time and is more susceptible to the potential impact of adverse environmental factors [9]. Therefore, the potential impact of adverse environmental factors experienced during gametogenesis on the long-term health of offspring deserves close attention, especially since research on paternally derived female PPP is still relatively scarce and needs further in-depth exploration.

[0004] Caffeine is a xanthine alkaloid that is widely found in coffee, tea, energy drinks, food, and analgesics. Clinically, it can be used as an adjunct to treat apnea in premature infants

[10] . Epidemiological surveys have found that caffeine exposure is more severe in men than in women and is trending towards younger ages. According to data from the National Health and Nutrition Examination Survey in the United States, the average caffeine intake of men of reproductive age is 240 mg / day, which is about 1.5 times that of women, and can reach up to 300 mg / day [11-13]. Recently, based on the average daily caffeine intake of adult men, this study explored for the first time the effects of paternal pre-conception caffeine exposure (PPCE) on ovarian development and PPP in female offspring. It found that the imprinted gene Gnas plays a key role in regulating ovarian estrogen synthesis and proposed that Gnas can be used as a potential intervention target for paternal female PPP. This provides a theoretical and experimental basis for screening effective drugs for prevention and treatment and developing early intervention technologies for the occurrence and development of paternal PPP.

[0005] Main references: 1. Kim YJ, Kwon A, Jung MK, Kim KE, Suh J, Chae HW, Kim DH, Ha S, SeoGH, and Kim HS. Incidence and prevalence of central precocious puberty in Korea: an epidemiologic study based on a national database. J Pediatr. 2019;208: 221-8. 2. Teilmann G, Pedersen CB, Jensen TK, Skakkebaek NE, and Juul A. Prevalence and incidence of precocious pubertal development in Denmark: anepidemiologic study based on national registries. Pediatrics. 2005; 116(6):1323-8. 3. Sultan C, Gaspari L, Kalfa N, and Paris F, Management ofperipheral precocious puberty in girls, in Frontiers in gynecologicalendocrinology: Volume 4: Pediatric and adolescent gynecologicalendocrinology, C Sultan and A R Genazzani, Editors. 2017, SpringerInternational Publishing: Cham. p. 39-48. 4. Neely EK, Hintz RL, Wilson DM, Lee PA, Gautier T, Argente J, andStene M. Normal ranges for immunochemiluminometric gonadotropin assays. JPediatr. 1995; 127(1): 40-6. 5. Fullston T, Shehadeh H, Sandeman LY, Kang WX, Wu LL, Robker RL,McPherson NO, and Lane M. Female offspring sired by diet induced obese malemice display impaired blastocyst development with molecular alterations totheir ovaries, oocytes and cumulus cells. J Assist Reprod Genet. 2015; 32(5):725-35. 6. Leibel NI, Baumann EE, Kocherginsky M, and Rosenfield RL.Relationship of adolescent polycystic ovary syndrome to parental metabolicsyndrome. J Clin Endocrinol Metab. 2006; 91(4): 1275-83. 7. Xia D, Parvizi N, Zhou Y, Xu K, Jiang H, Li R, Hang Y, and Lu Y.Paternal fenvalerate exposure influences reproductive functions in theoffspring. Reprod Sci. 2013; 20(11): 1308-15. 8. Davies MJ and Norman RJ. Programming and reproductive functioning.Trends Endocrinol Metab. 2002; 13(9): 386-92. 9. Donkin I and Barres R. Sperm epigenetics and influence ofenvironmental factors. Mol Metab. 2018; 14: 1-11. 10. Grosso G, Godos J, Galvano F, Giovannucci EL: Coffee, caffeine,and health outcomes: An umbrella review. Annu Rev Nutr 2017, 37:131-156. 11. Jia H, Liu W, Liu J, Jin Q, Li C, Yu Y, Meng L, Wu G, Zhao R,Zhao Y: [Assessment of caffeine intake in children and adolescents aged 6-17years in Beijing City]. Wei Sheng Yan Jiu 2020, 49(2):220-226. 12. Drewnowski A, Rehm CD: Sources of caffeine in diets of USchildren and adults: Trends by beverage type and purchase location. Nutrients2016, 8(3):154. 13. Fulgoni VL, 3rd, Keast DR, Lieberman HR: Trends in intake and sources of caffeine in the diets of US adults: 2001-2010. Am J Clin Nutr2015, 101(5):1081-1087. Summary of the Invention This invention aims to address the technical problems of unclear pathogenesis, unknown intervention targets, and lack of effective treatment methods in paternal female PPP in the prior art, and provides an intervention target for paternal female PPP based on the imprinted gene Gnas and its application.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: This invention provides a paternal female PPP intervention target based on the imprinted gene Gnas.

[0007] This invention, through systematic animal experiments, molecular biology, and histopathological studies, has clarified the core role of the imprinted gene Gnas in paternal PPP and constructed a three-in-one technical system of "target-formulation-strategy," the specific scheme of which is as follows: 1. Discovery and validation of core intervention targets Target screening: Transcriptome sequencing, RT-qPCR, immunofluorescence and ELISA were used to confirm that PPCE can significantly upregulate the mRNA and protein expression of Gnas in the ovarian tissue of female offspring, thereby promoting the activation of downstream cAMP / PKA / CREB signaling pathways, resulting in increased expression of estrogen synthase system (such as StAR, P450scc, 3β-HSD, CYP19), elevated blood estradiol (E2) levels, inhibition of the hypothalamus-pituitary-ovarian (HPO) axis, premature vaginal opening, and estrous cycle disorder.

[0008] Target function validation: Local Gnas knockout in the ovary was performed using CRISPR-Cas9 gene editing technology: sh-RNAs specifically targeting the exons of the Gnas gene were designed and conditional knockout vectors were constructed to confirm that Gnas is a core target for regulating ovarian estrogen synthesis and PPP.

[0009] 2. Gnas Knockout Regulation Method Local Gnas knockout in the ovary was performed using CRISPR-Cas9 gene editing technology: sh-RNAs specifically targeting Gnas gene exons were designed, and a conditional knockout vector adeno-associated virus (AAV9) was constructed. The AAV9 was transfected into PPCE female offspring at 4 weeks postnatal time (PW4) via local microinjection in the ovary. The above-mentioned ovarian function-related indicators were detected at PW8 to verify the improvement effect of PPP and provide tools for mechanism research and target validation.

[0010] The technical principles and research process of this invention are as follows: 1. The role of Gnas in paternally derived female PPP: Studies have found that Gnas play a crucial role in the development of PPP in PPCE female offspring after birth. Under physiological conditions, local overexpression of Gnas in the ovary can promote estrogen synthesis, secretion, and PPP by upregulating the cAMP / PKA / CREB signaling pathway. Therefore, Gnas play a central role in the occurrence and development of paternally derived female PPP and can become a potential therapeutic target.

[0011] 2. Treatment strategy targeting Gnas: This invention proposes an innovative treatment strategy for paternally derived female PPP based on the imprinted gene Gnas. Local microinjection of AAV9-sh-RNA-Gnas into the ovary effectively inhibits estrogen synthesis and significantly alleviates paternally derived PPP induced by PPCE.

[0012] 3. Clinical Application Prospects: This invention provides a new approach to the intervention of paternally derived female PPP. Specific inhibition of Gnas expression can effectively prevent or delay the occurrence of paternally derived female PPP. This treatment strategy offers a completely new direction for the precision treatment and individualized intervention of PPP, and has broad clinical application potential.

[0013] The advantages and beneficial effects of this invention are as follows: With a clearly defined core intervention target and significant application value, this invention is the first to demonstrate that the Gnas gene is a key target mediating paternal female precocious puberty (PPP). Interfering with the expression of this gene can effectively improve the precocious puberty phenotype, providing a clear direction for clinical intervention. Furthermore, the intervention target can be widely applied in multiple fields such as adverse environment screening, early warning target discovery, and drug development, possessing both basic research and clinical translational value. Attached Figure Description

[0014] Figure 1 Peripheral precocious puberty occurred in PPCE offspring rats after birth.

[0015] Figure 1In the study: (A) time of vaginal opening after birth in each group; (B) weight gain rate of PW6; (C) ovarian weight of PW6; (D) ovarian index of PW6; (E) estrous cycle of CON group; (F) estrous cycle of PPCE(H) group; (G) estrous cycle duration of each group; (HJ) serum FSH, LH, and E2 levels of PW6; (KM) serum FSH, LH, and E2 levels of PW12.

[0016] Figure 2 The ovarian morphology of PPCE offspring rats was abnormal before and after birth.

[0017] Figure 2 (A) H&E staining of GD20 ovaries (100×, 400×); (B) Maximum cross-sectional area of ​​GD20 ovaries; (C) Number of oocytes in GD20 ovaries; (D, F) H&E staining of PW6 and PW12 ovaries (20×, 40×); (E, F) Number of follicles at each stage in PW6 and PW12 ovaries.

[0018] Figure 3 PPCE offspring rats exhibit enhanced ovarian estrogen synthesis function before and after birth.

[0019] Figure 3 (A) Volcano plot of differentially expressed genes in GD20 ovaries; (B) KEGG functional enrichment analysis of differentially expressed genes in GD20 ovaries; (C) Genes with significant differential expression in GD20 ovaries; (DF) mRNA expression of StAR, P450scc, 3β-HSD1, CYP17A1, 17βHSD1 / 2 and CYP19A1 in GD20, PW6 and PW12 ovaries; (GL) Statistical plot of immunofluorescence and mean gray value of StAR in GD20, PW6 and PW12 ovaries.

[0020] Figure 4 High expression of Gnas mediates peripheral precocious puberty in female offspring induced by PPCE.

[0021] Figure 4 (A) Venn diagram of differentially expressed genes and imprinted gene sets in parental sperm and ovaries; (B) Expression of GD20 ovarian imprinted genes; (C) Detection of whole-genome DNA methylation in parental sperm; (D, E) Methylation sequencing of the GD20 ovarian Gnas imprinted control region; (FH) mRNA expression of GD20, PW6, and PW12 ovarian Gnas; (IN) Immunofluorescence and protein expression of GD20, PW6, and PW12 ovarian Gnas.

[0022] Figure 5 Gnas / cAMP signaling regulates estrogen synthesis in ovarian granulosa cells.

[0023] Figure 5 In: (A) Gnas interacts with the cAMP signaling pathway; (B, D) Representative immunoblot images and statistical analysis graphs of the expression of proteins related to the cAMP signaling pathway in GD20 ovaries; (C, E) Representative immunoblot images and statistical analysis graphs of the expression of proteins related to the cAMP signaling pathway in PW12 ovaries. (F) E2 concentration in the culture medium; (G) mRNA expression of StAR, P450scc, 3β-HSD1, CYP17A1 and CYP19A1 in KGN cells; (H) Representative immunofluorescence images of Gnas and StAR; (I) Expression of Gnas mRNA in cells; (J) cAMP concentration in cells; (K, L) Representative immunoblot images and statistical analysis graphs of the expression of PKA-Cα, CREB and p-CREB proteins in cells; (M) ChIP-qPCR experiment to verify the binding of CREB to the StAR promoter.

[0024] Figure 6 . Intervention on Gnas can reverse peripheral precocious puberty in female offspring caused by PPCE.

[0025] Figure 6 In: (A) Vaginal opening time; (B) Time of each stage of the estrous cycle; (C) Ovary weight; (D) Ovary index; (E-G) Serum FSH, LH and E2 levels; (H) HE staining of PW12 ovaries; (I) Number of follicles at all levels; (J) Gnas mRNA expression; (K) mRNA expression of estrogen synthesis-related genes (StAR, P450scc, 3β-HSD, CYP17A1, 17βHSD1 / 2 and CYP19A1); (L, M) Immunofluorescence and quantification of Gnas and StAR in ovaries. Specific implementation manners

[0026] The technical content of the present invention will be further elaborated in detail below in combination with specific embodiments and the drawings.

[0027] Example 1: Construction of the paternal female PPP animal model of the present invention 1 Experimental animals SPF-grade healthy Wistar rats were purchased from Hubei Provincial Center for Disease Control and Prevention, and the animal license number is: SCXK (E) 2018-2020. This study was approved by the Ethics Committee of the Medical Department of Wuhan University and was strictly carried out in accordance with the relevant treatment guidelines of the international experimental animal protection certification and evaluation agency.

[0028] The experimental animals were raised in a barrier environment at a temperature of 22-25°C, a humidity of 50%, and a 12-hour day-night cycle.

[0029] 2 Experimental methods Forty male 7-week-old Wistar rats (weighing 260 - 300 g), with free access to water and food, after 7 days of adaptive feeding, were divided into a control group (intragastrically administered with 1 mL / 100 g of normal saline daily) and a PPCE group (administered with 15, 30, and 60 mg / kg·d of caffeine daily). After two months of drug administration, male Wistar rats (6 - 8 weeks old at this time) were caged and mated with normal 12-week-old Wistar rats at a ratio of 1:2 (Note: Each male rat was ensured to have only one successful mating to ensure a one-to-one relationship between the male rat and the offspring). The next morning, vaginal smears were examined. If sperm were observed under the microscope, it was recorded as GD0, and the pregnant female rats were taken out and separately raised. This was continuously done to ensure that at least 12 pregnant rats were produced in each group. At GD20, some pregnant rats were anesthetized and sacrificed with 2% isoflurane, and fetal blood and ovarian tissues were taken. The blood samples of each litter of fetal rats were combined into one sample for subsequent analysis of relevant blood series indicators. The right ovarian tissues of the fetuses were immediately frozen in liquid nitrogen and stored at -80 °C for further RT-qPCR analysis. In addition, five left fetal ovaries from different litters were randomly selected and placed in 4% paraformaldehyde solution overnight, dehydrated with alcohol, and embedded in paraffin for morphological analysis such as HE, immunofluorescence, and immunohistochemistry. During drug administration and pregnancy, pregnant rats in each group had free and normal diet. The feed was purchased from Wuhan Wanqian Jiaxing Biotechnology Co., Ltd., license number: SCXK (E) 2011 - 0011. The feed formula was the same as the mouse and rat formula feed specified in the "National Standard of the People's Republic of China GB14924.3 - 2001".

[0030] The remaining pregnant rats gave birth naturally. A litter size of 8 - 14 at birth was considered qualified, and the sex and number of fetuses were adjusted between different litters to reach 12 per litter with a sex ratio of 1:1. The offspring rats were weaned at PW4 and separated by sex. Samples were taken at PW6 and PW12. The right ovaries were immediately frozen in liquid nitrogen and stored at -80 °C for further RT-qPCR analysis. Five offspring left ovaries from each group after birth were selected and placed in 4% paraformaldehyde solution overnight, dehydrated with alcohol, and embedded in paraffin for morphological analysis such as HE, immunofluorescence, and immunohistochemistry.

[0031] 3 Detection indexes and methods 3.1 HE staining Rat ovaries were fixed in 4% paraformaldehyde solution for 3 days and then embedded in paraffin. Sagittal sections of the ovaries were prepared at 5 μm for morphological staining analysis. The sections were immersed in hematoxylin staining solution for 15 min, rinsed with distilled water for 15 min, separated by 1% hydrochloric acid ethanol for 10 s (until the color lightens from red), rinsed with distilled water for 10 s, inverted with 0.6% ammonia solution, and then rinsed with running water for 10 s. The stained sections were counterstained with 0.5% eosin solution for 2 min. Then, the sections were sequentially immersed in 95% ethanol I for 5 min → 95% ethanol II for 5 min → anhydrous ethanol I for 5 min → anhydrous ethanol II for 5 min → xylene I for 5 min → xylene II for 3 min. After 5 minutes of clearing, remove the sections from the xylene and let them air dry slightly before mounting with neutral resin.

[0032] 3.2 Immunohistochemistry and Immunofluorescence For immunohistochemical staining, rat ovaries were fixed in 4% paraformaldehyde solution for 3 days and then processed using paraffin embedding. Ovaries were sectioned into 5 μm sagittal sections for morphological staining analysis. After dewaxing, fluid replenishment, and antigen retrieval, paraffin sections were treated with EDTA antigen retrieval buffer (pH 8.0). Detection was performed using a DAB and DAPI staining kit (GeneTech Company, Ltd., Shanghai, China) using BSA to block the key estrogen synthesis enzyme StAR and the imprinted gene Gnas primary antibody.

[0033] 3.3 RT-qPCR For RT qPCR was used to isolate total RNA from ovarian tissue using TRIzol reagent. The isolated RNA was then aliquoted and stored in [the appropriate container / system]. At 80℃, 1 μg of purified RNA was reverse transcribed using a cDNA synthesis kit, followed by cDNA amplification. The reaction was repeated for 40 cycles. Using 2... Relative amplicon expression was calculated using the ΔΔCt method. The expression of StAR, P450scc, 3β-HSD, 17α-HSD, 17β-HSD, and GAPDH in RNA was determined. Rat primer sequences are shown in Table 1. All cDNA sequences were obtained from the NCBI Entrez nucleotide database, and primers were designed using Primer Premier 6.0 (Premier Biosoft International, Palo Alto, CA, USA). Each designed primer sequence was queried using the NCBI BLAST database for homology comparison to determine the final primer sequences used. Primer information is shown in Table 1.

[0034] Table 1. Relevant rat primer sequences

[0035] 4. Experimental Results PPCE can induce PPP in offspring rats, specifically manifested as follows: compared with the control group, the PPCE group showed significantly earlier vaginal opening, earlier estrus, and prolonged estrous cycle; a significantly increased weight gain rate, while ovarian weight remained unchanged but the ovarian index was significantly decreased. Further HPO axis activity analysis showed that, compared with the PW6-CON group, the PPCE group had unchanged hypothalamic GnRH mRNA expression, decreased serum LH levels, but increased E2 levels; similar changes were observed in the PW12-PPCE group, characterized by decreased hypothalamic GnRH mRNA expression, decreased serum LH levels, and increased E2 levels. In conclusion, PPCE can induce PPP in female offspring.

[0036] 4.1 PPP occurs in offspring rats after birth This invention observed the effects of PPCE on vaginal opening, estrous cycle, weight gain rate, ovarian weight, and ovarian index in offspring after birth. The results showed that, compared with the CON group, the PPCE group rats had earlier vaginal opening (…). Figure 1 (A) Disorders of the estrous cycle, prolonged estrous period and total estrous cycle ( Figure 1 EG). Meanwhile, at PW6, the weight gain rate of rats in the PPCE group was significantly higher than that in the CON group ( Figure 1 In the middle B group, there was no difference in ovarian weight, but the ovarian index was significantly lower. Figure 1 (C, D). This suggests that PPCE can cause precocious puberty in offspring. To investigate the types of precocious puberty in female offspring caused by PPCE, this invention measured the serum FSH, LH, and E2 levels in the offspring. The results showed that compared to their respective CON groups, the serum FSH and E2 levels in the PPCE group were significantly increased at PW6 and PW12, while the LH level was significantly decreased (…). Figure 1 (HM). In summary, PPCE can lead to PPP in offspring after birth.

[0037] 4.2 Abnormal ovarian morphology in offspring rats before and after birth Results of abnormal ovarian morphology in offspring rats before and after birth: Figure 2 As shown. The results showed that, compared with their respective CON groups, the number of oocytes per unit area in the GD20 ovaries of the PPCE group was significantly reduced ( Figure 2 In PW6, the number of primordial follicles in the ovary is reduced, while the number of antral follicles and corpus luteum is significantly increased. Figure 2 In PW12, both primordial and antral follicles in the ovary were significantly reduced, and multiple cystic follicles were observed. Figure 2 (F, G). The above results suggest that PPCE can lead to abnormal ovarian morphology and development in offspring.

[0038] 4.3 Enhanced ovarian estrogen synthesis in offspring before and after birth Prenatal estrogen synthesis function in the laryngeal ovary of offspring rats, such as Figure 3 As shown. Sequencing analysis results indicated that, compared with the CON group, a total of 453 genes showed differential expression in the fetal ovaries of the PPCE group (as shown). Figure 3 (A). KEGG functional enrichment analysis of these differentially expressed genes (DEGs) revealed significant changes in the ovarian steroid synthesis and secretion signaling pathways. Figure 3 (B) Specifically, it manifests as increased mRNA expression of estrogen synthesis-related genes such as StAR and CYP11A1. Figure 3 (C). Further RT-qPCR was used to detect the expression of ovarian steroid synthases. The results showed that, compared with the CON group, the mRNA expression of key functional enzyme systems for ovarian steroid synthesis (such as StAR, CYP17A1, and 17βHSD2) in the PPCE group at GD20 was significantly increased. Figure 3 In the middle D), at PW6 and PW12, the mRNA expression of ovarian steroid synthase systems (such as StAR, P450scc, 3β-HSD1, CYP17A1, 17βHSD1 / 2) in the PPCE group was significantly increased. Figure 3 E and F). IF staining also revealed significantly increased StAR protein expression in ovarian tissues before and after birth. Figure 3 (GL). These results indicate that PPCE can enhance ovarian estrogen synthesis in offspring rats before and after birth.

[0039] Example 2: Exploration of early warning targets in the paternal PPP animal model of the present invention 1. The experimental animals and experimental methods are consistent with those in Example 1. 2. Detection Indicators and Methods 2.1 Immunohistochemistry and Immunofluorescence For immunohistochemical staining, rat ovaries were fixed in 4% paraformaldehyde solution for 3 days and then processed using paraffin embedding. Ovaries were sectioned into 5 μm sagittal sections for morphological staining analysis. After dewaxing, fluid replenishment, and antigen retrieval, paraffin sections were treated with EDTA antigen retrieval buffer (pH 8.0). Detection was performed using a DAB and DAPI staining kit (GeneTech Company, Ltd., Shanghai, China) using BSA to block the key estrogen synthesis enzyme StAR and the imprinted gene Gnas primary antibody.

[0040] 2.2 RT-qPCR For RT qPCR was used to isolate total RNA from ovarian tissue using TRIzol reagent. The isolated RNA was then aliquoted and stored in [the appropriate container / system]. At 80℃, 1 μg of purified RNA was reverse transcribed using a cDNA synthesis kit, followed by cDNA amplification. The reaction was repeated for 40 cycles. Using 2... Relative amplicon expression was calculated using the ΔΔCt method. The expression of Gnas, cAMP signaling pathway, and GAPDH in RNA was determined. Rat primer sequences are shown in Table 1. All cDNA sequences were obtained from the NCBI Entrez nucleotide database, and primers were designed using Primer Premier 6.0 (Premier Biosoft International, Palo Alto, CA, USA). The NCBI BLAST database was used to query each designed primer sequence for homology comparison to determine the final primer sequences used. Primer information is shown in Table 1.

[0041] 2.3 Determination of protein concentration in ovarian tissue Ovarian tissue was thawed from a -80°C freezer, 50 mg of ovarian tissue was weighed, PBS was added, and the tissue was thoroughly ground and mixed. The experiment was performed according to the BCA kit instructions. To prepare the BSA working solution, mix reagent A and reagent B at a ratio of 50:1 according to the required total volume. Note that the BSA working solution should be prepared fresh before use. Dilute the protein standard solution (25 mg / mL) with PBS in a gradient to 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL, respectively, so that the final volume of each diluted standard is 20 µL. Add an appropriate volume of the sample to be tested and PBS to each well of a 96-well plate, and set up an equal volume of standard wells. Add 200 µL of the pre-prepared BCA working solution to each well and mix in a 37°C shaker for about half an hour. After the above steps are completed, place the 96-well plate in a microplate reader and read the absorbance of each well at a wavelength of 570 nm. Finally, plot a standard curve based on the absorbance of each well and calculate the protein concentration of the sample in each well according to the sample dilution ratio.

[0042] 2.4 Protein extraction from ovarian tissue Weigh 50 mg of ovarian tissue into a 1.5 mL centrifuge tube, cut it into small pieces (avoiding ovarian fascia), add 500 µL of RIPA lysis buffer containing protease inhibitors, and homogenize using a homogenizer until no obvious precipitate remains. Place the ovarian tissue homogenate on ice and let it stand for 30 min. After sufficient lysis of tissue proteins, centrifuge at 12000 rpm at 4℃ for 15 min. Carefully aspirate the supernatant and transfer it to a new 1.5 mL centrifuge tube. Determine the ovarian tissue protein concentration using the BCA method according to the above steps, and adjust all samples to a uniform concentration using PBS based on the results. Add an appropriate amount of 5× loading buffer to the tissue protein sample, mix thoroughly, heat in a metal bath at 100 ℃ for 5 min, cool, and then proceed with subsequent detection and storage.

[0043] 2.5 Ovarian tissue protein Western Blot procedure Clean the 1.5 mm glass plate with distilled water, and dry it with a hairdryer after cleaning. After assembly, add distilled water to check for leaks. Prepare the separating gel and stacking gel according to the gel kit instructions, and insert 1.5 mm 10-well or 15-well combs. After the stacking gel solidifies, slowly pull the comb vertically upwards (be careful not to damage the gel). Sample loading: Remove the solidified gel from the gel holder and install it in the electrophoresis tank. Add the pre-prepared electrophoresis buffer to avoid air bubbles in the wells. Then slowly add the processed protein samples into the comb wells (try not to leak out of the wells), and set the protein marker wells at the same time. Electrophoresis: Set the electrophoresis conditions to a constant voltage of 60 V. After the samples transfer from the stacking gel to the separating gel, adjust the voltage to 120 V and continue electrophoresis. Transfer: Cut a PVDF membrane to an appropriate size and activate it in methanol for 2 min. Stack the gel and membrane in sequence into a "sandwich" shape in a clamping plate, place it in an electroporation tank, and electroporate at a constant current of 150 mA at low temperature for 0.5-1.5 h. After electroporation, cut the PVDF membrane and place it in 5% skim milk blocking solution, then place it on a shaker at 4°C for 1 h. Dilute different primary antibodies according to the instructions of each antibody using primary antibody dilution buffer. Completely immerse the cut PVDF membrane in the primary antibody, ensuring that the primary antibody completely covers the PVDF membrane, and incubate it overnight on a shaker at 4°C. Remove the PVDF membrane and place it in TBST solution, ensuring that the membrane is completely immersed in the TBST solution, and wash it three times on a shaker for 5 min each time. Dilute the secondary antibody with 5% skim milk according to the secondary antibody instructions, and react the PVDF membrane in the secondary antibody at room temperature for 2-3 h. Repeat step 8 to wash the membrane. Mix solutions A and B from the ECL chemiluminescence kit in equal proportions to prepare the working solution, then add it to the PVDF membrane and completely cover the membrane. After reacting for several minutes, acquire and save the image using a chemiluminescence imaging system.

[0044] 3. Experimental Results 3.1 High expression of Gnas hypermethylation mediates PPP in female offspring rats To investigate the mechanism of PPCE-induced PPP in female offspring, this invention first examined changes in the prenatal transcriptome expression profiles of paternal sperm and offspring rats to screen for commonly differentially expressed imprinted genes as potential toxicity targets. Sequencing analysis showed that, compared to the CON group, the PPCE group exhibited altered expression of multiple imprinted genes in the paternal epididymis and offspring ovaries. Figure 4 (Among them, A), there are 4 imprinted genes that are commonly differentially expressed ( Figure 4 (A), especially Gnas, showed the most significant changes ( Figure 4 (B) Whole-genome methylation sequencing of F0 generation sperm showed that the methylation level of the Gnas imprinting control region (ICR), i.e., the differentially methylated region (DMR) of exon 1A, was higher in the PPCE group than in the CON group. Figure 4 (C); F1 generation intrauterine GD20 ovarian BSP detection also found that the Gnas ICR methylation level in the PPCE group was significantly higher than that in the control group ( Figure 4 (D, E). This suggests that PPCE can lead to increased Gnas ICR methylation levels in F0 generation sperm and F1 generation ovaries. RT-qPCR and immunofluorescence results showed that, compared with the CON group, the PPCE group at GD20, PW6, and PW12 significantly increased ovarian Gnas mRNA and protein expression. Figure 4 (Middle FN). These results indicate that PPCE can increase the methylation level of GnasICR in paternal sperm, which can persist to offspring, leading to increased Gnas expression in the ovaries before and after birth. This suggests that Gnas may be a potential toxic target mediating PPCE-induced PPP in female offspring.

[0045] 3.2 Gnas / cAMPPKA / CREB signaling regulates estrogen synthesis in ovarian granulosa cells This invention further elucidates the molecular mechanism by which Gnas high expression mediates enhanced estrogen synthesis in PPCE progeny rats. First, ovarian RNA-seq data analysis suggests an interaction between ovarian steroid synthesis, the cAMP signaling pathway, and Gnas. Figure 5 (A). And at the overall level, it was found that at GD20 and PW6, the protein expression of PKA-Cα and p-CREB in the cAMP signaling pathway in the PPCE group was significantly higher than that in the CON group. Figure 5(BE). Further, Gnas overexpression or interference was performed by transfecting OE-Gnas or sh-Gnas plasmids into human ovarian granulosa cell (KGN) cell lines to confirm the regulatory role of Gnas in KGN estrogen synthesis. The results showed that, compared with the OE-NC group, Gnas overexpression increased the mRNA expression of estrogen synthase genes (StAR, P450scc, 3β-HSD1, CYP17A1, and CYP19A1) and the expression of StAR protein. Figure 5 (F,G), and increase E2 concentration ( Figure 5 (H). Interference with Gnas can produce the opposite effect. Finally, this invention confirms that Gnas exerts its estrogen synthesis regulation function through the cAMP / PKA / CREB signaling pathway. The results show that overexpression of Gnas can significantly increase cAMP concentration (H). Figure 5 Expression of PKA-Cα and p-CREB proteins (I), PKA-Cα, and p-CREB proteins (I) Figure 5 In the case of J and K), interference with Gnas significantly reversed the above changes. Simultaneously, inhibition of PKA (H-89) or CREB (666-15) respectively showed no change in cAMP concentration. Figure 5 In the middle L), the level of p-CREB protein was significantly reduced ( Figure 5 The mRNA expression levels of the M, N), estrogen synthase system (StAR, P450scc, 3β-HSD1, CYP17A1, and CYP19A1) and StAR protein expression were significantly reduced, and E2 concentration was also decreased. Figure 5 (OQ). ChIP-qPCR experiments also confirmed the interaction between CREB and the StAR promoter ( Figure 5 (R, S). In summary, these results indicate that the cAMP signaling pathway enhances the ovarian steroid synthesis function in PPCE female offspring induced by Gnas overexpression.

[0046] Example 3: Validation of the intervention target in the paternal PPP animal model of the present invention 1. The experimental animals and methods are the same as in [Example 1]. In addition, recombinant adeno-associated virus was injected into the ovaries of PW4 progeny rats in the PPCE group for four weeks. Blood and ovarian tissues from the corresponding groups (empty vector group and Gnas intervention group) were obtained for further testing.

[0047] 2. Detection Indicators and Methods 2.1 Immunohistochemistry and Immunofluorescence Consistent with [Example 2].

[0048] 2.2 RT-qPCR Consistent with Example 1. Primer information is shown in Table 1.

[0049] 2.3 Determination of protein concentration in ovarian tissue Consistent with [Example 2].

[0050] 2.4 Protein extraction from ovarian tissue Consistent with [Example 2].

[0051] 2.5 Ovarian tissue protein Western Blot procedure Consistent with [Example 2].

[0052] 3. Experimental Results 3.1 Intervention with Gnas can reverse estrogen synthesis function in ovarian granulosa cells and the occurrence of postnatal PPP. To confirm the regulatory role of Gnas on ovarian estrogen synthesis and PPP in rats, we performed ovarian microinjection of Gnas-overexpressing (OE-Gnas) or empty vector (OE-NC) adeno-associated virus (AAV8) into female offspring rats from the CON group at PW4, and observed them 8 weeks later. The results showed that at PW12, compared with the CON group, rats in the CON+OE-Gnas group showed earlier vaginal opening (…). Figure 6 (A) Disorders of the estrous cycle, prolonged estrous period and total estrous cycle ( Figure 6 In the middle B group, ovarian weight decreased but ovarian index remained unchanged. Figure 6 (C, D). HPO axis-related hormone testing revealed no significant changes in serum FSH and LH levels in the CON+OE-Gnas group compared to the CON+NC group. Figure 6 (E, F), but serum E2 levels were significantly elevated ( Figure 6 (G). This suggests that Gnas overexpression can induce the PPP phenotype in CON group rats. Further morphological examination showed that, compared with the CON+OE-NC group, CON+OE-Gnas rats had a significantly increased number of cystic follicles in their ovaries (indicated by red pentagrams). Figure 6 In the middle H, the number of primordial follicles and antral follicles is significantly reduced ( Figure 6 (I). RT-qPCR results showed that, compared with the CON+NC group, the mRNA expression of Gnas and estrogen synthesis-related genes (StAR, P450scc, 3β-HSD, CYP17A1, 17βHSD1 / 2, and CYP19A1) was significantly increased in the CON+OE-Gnas group. Figure 6 J and K). Meanwhile, the co-localization and fluorescence signal of Gnas and StAR in the ovaries of rats in the CON+OE-Gnas group were significantly increased (J and K). Figure 6 (L, M). These results indicate that Gnas are important regulatory molecules for estrogen synthesis in the ovary, and overexpression of Gnas can lead to enhanced ovarian estrogen synthesis, ultimately resulting in PPP.

[0053] To further confirm that Gnas overexpression programming mediates altered estrogen synthesis in PPCE female offspring, we conducted a reverse intervention at PW4 by microinjecting AAV8-sh-Gnas adeno-associated virus into the ovaries of PPCE female offspring. The results showed that at PW12, Gnas inhibition could reverse the premature vaginal opening and estrous cycle disruption in PPCE offspring rats before and after birth. Figure 6 (A, B). HPO axis-related hormone detection revealed that serum FSH and estrogen levels in female PPCE offspring rats were significantly reduced after Gnas inhibition. Figure 6 (E, G), but serum LH levels showed no significant change ( Figure 6 (Middle F). Histological results showed that the number of cystic follicles in the ovaries of PPCE offspring suppressed by Gnas was significantly reduced ( Figure 6 (H), and the number of antral follicles increases ( Figure 6 (I) Notably, the mRNA expression of ovarian Gnas and estrogen synthesis-related genes (StAR, P450scc, 3β-HSD, CYP17A1, 17βHSD1 / 2, and CYP19A1) in the PPCE+sh-Gnas group was significantly lower than that in the PPCE+sh-NC group. Figure 6 J, K). Meanwhile, in the PPCE+sh-Gnas group, the fluorescence co-localization and fluorescence signal of ovarian Gnas and StAR were significantly reduced ( Figure 6 (L, M). These results indicate that inhibiting Gnas can correct the enhanced ovarian estrogen synthesis before and after birth in PPCE offspring rats, thus inhibiting postnatal PPP. In conclusion, Gnas intervention can reverse PPP in female offspring rats, providing experimental evidence for PPP prevention and control strategies.

Claims

1. An intervention target, characterized in that: The intervention target is a paternally derived female peripheral precocious puberty intervention target based on the imprinted gene Gnas.

2. The intervention target according to claim 1, characterized in that: The intervention target acts on the cAMP / PKA / CREB signaling pathway.

3. The intervention target according to claim 2, characterized in that: Local overexpression of Gnas in ovarian tissue using CRISPR-Cas9 gene editing technology can enhance the cAMPPKA / CREB signaling pathway, thereby promoting ovarian estrogen synthesis and PPP.

4. The intervention target according to claim 2, characterized in that: CRISPR-Cas9 gene editing technology can be used to locally knock out Gnas in ovarian tissue or inhibit the cAMPPKA / CREB signaling pathway, thereby suppressing ovarian estrogen synthesis and PPP.

5. The intervention target according to claim 3 or 4, characterized in that: Gnas are regulatory factors expressed locally by ovarian granulosa cells.

6. The intervention target according to claim 5, characterized in that: By regulating the function of ovarian granulosa cells through the cAMP / PKA / CREB signaling pathway, the occurrence and development of female PPP can be prevented, treated, or delayed.

7. The use of the intervention target as described in claim 6 in screening drugs for the prevention and treatment of female PPP.

8. The application according to claim 7, characterized in that: By screening for candidate drugs that can reduce Gnas expression, the aim is to improve ovarian estrogen synthesis function and precocious puberty phenotype in female PPP.

9. The application of the intervention target as described in claim 6 in the development of early intervention techniques for female PPP. Its characteristics are: By reducing Gnas expression, the ovarian estrogen synthesis function and precocious puberty phenotype of female PPP were improved.

10. The application according to claim 9, characterized in that: CRISPR-Cas9 gene editing technology was used to reduce Gnas expression.

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