Methods for constructing animal models of paternal testicular hypoplasia, intervention targets and their applications

Abstract

This invention provides a method for constructing an animal model of paternal testicular hypoplasia, its intervention target, and its application. The paternal testicular hypoplasia animal model is obtained by mating 6-week-old male Wistar rats, which were administered different doses of caffeine by gavage for two months, with normal female rats to produce F1 generation male rats before and after birth. The latter exhibited testicular hypoplasia, manifested as abnormal testicular morphology and inhibited testosterone synthesis. Based on the paternal testicular hypoplasia model, the testicular function impairment was effectively corrected after injecting Mest-interfering adeno-associated virus into the seminiferous tubules of 12-week-old male rats. The construction method provided by this invention is simple, has a high success rate, and reduces the influence of human intervention. It provides an animal model basis for the pathogenesis, prevention, and treatment of paternal testicular hypoplasia, and has significant research significance and scientific value.

Description

Technical Field

[0001] This invention relates to the field of animal model construction technology, specifically to a method for constructing an animal model of paternal testicular hypoplasia, intervention targets, and their applications. Background Technology

[0002] Testicular hypoplasia refers to the absence or incomplete development of the testes due to various factors. It is an increasingly common and environment-related developmental disorder of the male reproductive system, mainly including cryptorchidism, hypospadias, low sperm quality, and testicular cancer. Known causes of testicular hypoplasia include congenital and acquired factors. Congenital factors are primarily caused by chromosomal abnormalities, often resulting in delayed puberty, indistinct male secondary sexual characteristics, and feminization such as gynecomastia. The incidence rate in the population is 1 in 1000-1500. Acquired factors mainly include infection, trauma, and endocrine abnormalities. Testicular hypoplasia easily induces male infertility and cardiovascular disease. Based on epidemiological surveys, approximately 15% of couples are affected by infertility, and male factors are considered to play a role in 50% of infertile couples, and are the sole cause of infertility in 20% of cases. In my country, there are currently approximately 40-50 million infertile individuals among the reproductive-age population. Currently, with the increasing severity of environmental pollution, social pressure, chronic stress, and other external factors, the incidence of male testicular hypoplasia 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 testicular hypoplasia. Considering the implementation of my country's "three-child policy," the need to improve population quality, and the importance of eugenics, testicular hypoplasia has become an urgent public health issue that needs to be addressed.

[0003] In recent years, the developmental origin of health and disease (DOHaD) theory has received widespread attention from researchers [1]. For example, maternal exposure to nicotine and alcohol during pregnancy can lead to a shortened anal-genital distance in male offspring and feminization of male fetuses; the use of certain drugs during pregnancy (such as synthetic glucocorticoids, nonsteroidal anti-inflammatory drugs, etc.) can lead to inhibition of testosterone synthesis in offspring. Currently, most studies on testicular dysplasia models focus on maternal adverse environmental exposure [2,3]. However, with the rise of the paternal origin of health and disease (POHaD) theory recently, more and more evidence supports that paternal adverse environmental exposure can lead to testicular dysplasia in offspring [4]. For example, paternal exposure to heavy metals such as bisphenol A and cadmium before pregnancy can lead to a decrease in sperm count and sperm quality in offspring [5,6]. Currently, although some testicular dysplasia syndrome models have been widely used, such as mice with Atg5 / Atg7 knockout in testicular spermatogenic cells exhibiting oligospermia and azoospermia, there is still no model that can completely simulate testicular dysplasia and reflect its development process. Therefore, in order to supplement and expand the scope of models of testicular dysplasia, it is urgent to establish and apply paternal testicular dysplasia models to further explore its pathogenesis, early warning and prevention, so as to benefit mankind.

[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 [7]. 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 U.S. National Health and Nutrition Examination Survey, 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 [8-10]. Previous systematic studies of this invention have found that maternal exposure to caffeine during pregnancy has fetal developmental toxicity, which can cause IUGR, low birth weight, and increased risk of testicular hypoplasia in offspring. Recently, with the attention paid to the role of paternal factors in offspring development, this invention focuses on a rat model of paternal progestational caffeine exposure (PPCE) and confirms that PPCE can cause testicular hypoplasia in offspring, thereby providing a method for constructing an animal model of paternal testicular hypoplasia and its application.

[0005] Main references:

[0006] 1.Suzuki K:The developing world ofDOHaD.J Dev Orig Health Dis 2018,9(3):266-269.

[0007] 2.Pei LG,Zhang Q,Yuan C,Liu M,Zou YF,Lv F,Luo DJ,Zhong S,Wang H:TheGC-IGF1axis-mediated testicular dysplasia caused by prenatal caffeineexposure.J Endocrinol 2019,242(1):M17-M32.

[0008] 3.Dimofski P,Meyre D,Dreumont N,Leininger-Muller B:ConsequencesofPaternal Nutrition on Offspring Health and Disease.Nutrients 2021,13(8).

[0009] 4.SoubryA:POHaD:why we should study future fathers.Environ Epigenet2018,4(2):dvy007.

[0010] 5.Goli P,Yazdi M,Poursafa P,Kelishadi R:Intergenerational influenceof paternal physical activity on the offspring's brain:A systematic reviewand meta-analysis.Int J Dev Neurosci 2021,81(1):10-25.

[0011] 6.McPherson NO,Fullston T,Aitken RJ,Lane M:Paternal obesity,interventions,and mechanistic pathways to impaired health inoffspring.AnnNutr Metab 2014,64(3-4):231-238.

[0012] 7. Grosso G, Godos J, Galvano F, Giovannucci EL: Coffee, Caffeine, and Health Outcomes: An Umbrella Review. Annu RevNutr 2017,37:131-156.

[0013] 8.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 inBeijing City]. Wei Sheng Yan Jiu 2020,49(2):220-226.

[0014] 9. Drewnowski A, Rehm CD: Sources of Caffeine in Diets of US Children and Adults: Trends by Beverage Type and Purchase Location. Nutrients 2016,8(3):154.

[0015] 10. Fulgoni VL, 3rd, Keast DR, Lieberman HR: Trends in intake and sources of caffeine in the diets of US adults: 2001-2010. Am J ClinNutr 2015, 101(5): 1081-1087. Summary of the Invention

[0016] The technical problem to be solved by the present invention is to provide a method for constructing an animal model of paternal testicular hypoplasia that is highly successful, effective, reliable, reproducible, simple and easy to implement.

[0017] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0018] In a first aspect, the present invention provides a method for constructing an animal model of paternal testicular hypoplasia, characterized by comprising the following steps:

[0019] S1: Healthy 8-week-old male Wistar rats were selected and administered 15, 30, and 60 mg / kg of caffeine via intragastric gavage daily during the period from 8 to 16 weeks of age, while having free access to food;

[0020] S2: After administration, the rats were bred together with normal female Wistar rats aged 12 weeks. The resulting pregnant rats gave birth naturally to obtain the F1 generation. The birth date was taken as day 0 after birth. One day after birth, litters with 12 to 14 pups were selected, and each litter was adjusted to have 6 male and 6 female pups for nursing.

[0021] S3: The pups were weaned 4 weeks after birth and separated into male and female cages. Some continued to be fed a normal diet until 6, 12 and 32 weeks. Testicular function-related indicators were tested at each time point to determine the occurrence of testicular hypoplasia. Finally, a paternal testicular hypoplasia animal model was obtained.

[0022] S4: Some offspring were raised until 6 weeks after birth, and then adeno-associated recombinant virus overexpressing Mest was injected locally into the seminiferous tubules of the testes. Two weeks later, testicular function-related indicators were tested to determine whether testicular hypoplasia had been improved.

[0023] Furthermore, in step S1, the normal diet is formulated in the same way as the formula feed for mice and rats specified in the National Standard of the People's Republic of China GB14924.3-2001.

[0024] Furthermore, in steps S3 and S4, the testicular function-related indicators are: testicular morphology, blood testosterone level, endogenous testosterone level in the testes, expression of testosterone synthase (StAR, P450scc, 3β-HSD) system, sperm quality (sperm count, motility, morphology, etc.), and mating success rate.

[0025] Secondly, the present invention provides an application of a paternal testicular hypoplasia animal model in screening parental environment, endocrine disruptors or drugs, characterized in that: the paternal testicular hypoplasia animal model is obtained by any of the above construction methods.

[0026] Thirdly, the present invention provides an application of a paternal testicular dysplasia animal model in screening early warning and intervention targets for testicular dysplasia, characterized in that: the paternal testicular dysplasia animal model is obtained by any of the above construction methods.

[0027] Fourthly, the present invention provides an application of a paternal testicular hypoplasia animal model in screening for the prevention and treatment of testicular hypoplasia, characterized in that: the paternal testicular hypoplasia animal model is obtained by any of the above construction methods.

[0028] The technical principles and research process of this invention are as follows:

[0029] This invention constructs a PPCE animal model by simulating the easy accessibility of caffeine in men's daily lives. Male offspring were obtained at different time points before and after birth, and testicular function-related indicators were detected to determine the occurrence and development of testicular hypoplasia, thus establishing a rat model of paternal testicular hypoplasia. This model simulates the disease phenotype of paternal testicular hypoplasia, which is of great significance for elucidating the pathogenesis of paternal testicular hypoplasia and determining early warning and prevention.

[0030] The advantages and beneficial effects of this invention are as follows:

[0031] 1. Simple and reproducible modeling method: The modeling method of this invention is simple. The father is given caffeine before conception and mates with a normal female to obtain male offspring at different time points before and after birth. Testicular morphology, serum testosterone levels, expression of the testicular testosterone synthase system (including StAR, P450scc, and 3β-HSD), endogenous testosterone levels, sperm quality (sperm count, motility, morphology, etc.), and mating success rate are measured to observe changes in testicular function and determine whether testicular hypoplasia has occurred. This model exhibits good stability and high reproducibility, providing a reliable method for paternally induced testicular hypoplasia.

[0032] 2. The model has wide applications and great research significance: The paternal testicular hypoplasia model constructed based on this invention can be used to guide men's healthy diet and lifestyle, and is beneficial for exploring the occurrence and development mechanism of paternal testicular hypoplasia, as well as for early warning and intervention targets. Attached Figure Description

[0033] Figure 1 Morphological changes of the testes in PPCE offspring rats before and after birth.

[0034] Figure 1 In the Chinese section: A: Morphology of the fetal testes of PPCE offspring; B, C: Maximum cross-sectional area and longest diameter of the fetal testes of PPCE offspring; D: Maximum cross-sectional area and longest diameter of the fetal testes of PPCE offspring; E: Volume and morphology of the testes of adult PPCE offspring; F: Weight of the testes of adult PPCE offspring; G1: Thickness of the seminiferous epithelium and diameter and area of ​​the seminiferous tubules of adult PPCE offspring.

[0035] Figure 2 PPCE offspring rats showed inhibition of testosterone synthesis before and after birth.

[0036] Figure 2In the table: A: Fetal blood testosterone level in the PPCE group; B: mRNA expression of genes related to testosterone synthesis in the fetal testes of PPCE offspring; C: StAR protein expression in the fetal testes of PPCE offspring; D: Blood testosterone level in adult offspring of PPCE; E: mRNA expression of genes related to testosterone synthesis in the fetal testes of PPCE offspring; F: StAR protein expression in the fetal testes of PPCE offspring.

[0037] Figure 3 PPCE offspring rats exhibit abnormal spermatogenesis and reduced fertility in adult rats.

[0038] Figure 3 In the PPCE adult offspring at 12 weeks: A: Sperm morphology; B: Sperm count of PPCE adult offspring at 12 weeks; C: Sperm count of PPCE adult offspring at 32 weeks; D: Sperm motility of PPCE adult offspring at 32 weeks; E: Sperm abnormality rate of PPCE adult offspring at 32 weeks; F: Mating success rate of PPCE adult offspring.

[0039] Figure 4 The Mest / Wnt signaling pathway mediates abnormal spermatogenesis and reduced fertility in offspring adult rats.

[0040] Figure 4 In the middle: A: Differentially expressed genes in PPCE progeny; B, C: KEGG and GO enrichment analysis of differentially expressed genes in PPCE progeny; D, E: DNA methylation and expression in the testicular Mest promoter region before and after birth in PPCE progeny; FH: Double fluorescence labeling of testicular Mest and 3β-HSD before and after birth in PPCE progeny; I, J: Gene expression of Wnt signaling pathway before and after birth in PPCE progeny; K: Protein expression of Wnt signaling pathway before and after birth in PPCE progeny.

[0041] Figure 5 The Mest / Wnt signaling pathway regulates testosterone synthesis in testicular interstitial cells.

[0042] Figure 5 In Chinese: AC: Enrichment analysis of differentially expressed genes KEGG and GO pathways after cell intervention; DF: Expression of Wnt signaling genes and proteins after cell intervention; GJ: Expression of testosterone synthesis-related genes and proteins after cell intervention.

[0043] Figure 6 Intervention with Mest can reverse PPCE-induced testicular hypoplasia in offspring.

[0044] Figure 6 In the middle: A: Microinjection of Mest adeno-associated virus into the seminiferous tubules of the testes; B: In vivo imaging of the testes; C: Expression of Wnt signaling genes and proteins after intervention; D: Expression of enzyme genes and proteins related to testosterone synthesis after intervention. Detailed Implementation

[0045] The technical content of the present invention will be further elaborated in detail below in conjunction with specific embodiments and the accompanying drawings.

[0046]

Example 1

[0047] 1 Experimental animals

[0048] SPF-grade healthy Wistar rats were purchased from the 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 School 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.

[0049] The experimental animals were housed in a barrier environment at a temperature of 22-25 °C, a humidity of 50%, and a 12-hour day-night cycle.

[0050] 2 Experimental methods

[0051] Forty 7-week-old male Wistar rats (body weight 260-300 g) were allowed free access to water and food. After 7 days of adaptive feeding, they were divided into a control group (intragastric administration of 1 mL / 100 g of normal saline daily) and a pre-pregnancy PPCE group (administration of 15, 30, and 60 mg / kg of caffeine daily). After two months of drug administration, the male Wistar rats (16 weeks old at this time) were caged and mated with normal 12-week-old female Wistar rats at a ratio of 1:2 (Note: Ensure that each male rat mates successfully only once to ensure that the male rat and the offspring meet the one-to-one requirement). The next morning, vaginal smears were examined. If sperm were observed under the microscope, the day was recorded as gestational day 0 (GD), and the pregnant female rats were removed and housed separately. Continue like this to ensure that at least 12 pregnant rats are produced in each group. At GD20, some pregnant rats were anesthetized and sacrificed with 2% isoflurane, and fetal blood and testicular tissues were taken. The blood samples of male fetuses in each litter were combined into one sample for subsequent analysis of blood series-related indicators. The right testicular 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 testes 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, the pregnant rats in each group were allowed free and normal diet. The feed was purchased from Wuhan Wanqian Jiaxing Biotechnology Co., Ltd., and the license number is: SCXK(E) 2011-0011. The feed formula is the same as the mouse and rat formula feed specified in the "National Standard of the People's Republic of China GB14924.3-2001".

[0052] The remaining pregnant rats gave birth naturally, with litters of 8-14 pups at birth considered acceptable. The sex and number of pups were adjusted between different litters to achieve 12 pups per litter, a sex ratio of 1:1. Offspring rats were weaned and separated by sex at 4 weeks of age. Testes were collected at 6, 12, and 32 weeks postnatally. The right testis was immediately frozen in liquid nitrogen and stored at -80°C for further RT-qPCR analysis. The left testis of 5 randomly selected offspring were placed overnight in 4% paraformaldehyde solution, dehydrated with alcohol, and embedded in paraffin for morphological analysis including HE, immunofluorescence, and immunohistochemistry.

[0053] 3. Detection Indicators and Methods

[0054] 3.1 HE staining

[0055] Rat testes were fixed in 4% paraformaldehyde solution for 3 days and then embedded in paraffin. Sagittal sections of the testes 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, and then separated by 1% hydrochloric acid ethanol for approximately 10 seconds, until the color lightened from red. They were then rinsed with distilled water for approximately 10 seconds, inverted with 0.6% ammonia solution, and rinsed with running water for approximately 10 seconds. 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 3-5 min to achieve transparency. The sections were then removed from the xylene solution, slightly dried, and mounted with neutral resin.

[0056] 3.2 Immunohistochemistry and Immunofluorescence

[0057] For immunohistochemical staining, rat testes were fixed in 4% paraformaldehyde solution for 3 days and then processed using paraffin embedding. Testes 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). The primary antibody against the key testosterone synthesis enzyme StAR and the imprinted gene Mest was blocked using BSA, and detection was performed using a DAB and DAPI staining kit (GeneTech Company, Ltd., Shanghai, China).

[0058] 3.3 RT-qPCR

[0059] For RT-qPCR detection, total RNA was isolated from testicular tissue using TRIzol reagent. The isolated RNA was aliquoted and stored at -80°C. A cDNA synthesis kit was used to reverse transcribe 1 μg of the purified RNA, followed by cDNA amplification. The reaction was performed for 40 cycles. 2 -ΔΔRelative amplicon expression was calculated using the Ct 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). 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.

[0060] Table 1. Relevant primer sequences

[0061]

[0062]

[0063] 4 Experimental Results

[0064] PPCE can cause testicular hypoplasia in male offspring rats, specifically manifested as abnormal testicular morphology in the PPCE group compared to the control group, including interstitial widening, reduced cell number, and irregular seminiferous tubule morphology. Furthermore, serum testosterone levels were decreased, and the expression of the testicular testosterone synthase system was inhibited, indicating suppressed testosterone synthesis. In addition, PPCE-treated male offspring rats showed reduced sperm count, decreased sperm quality, increased sperm abnormality rate, and decreased mating success rate, indicating abnormal spermatogenesis and reduced fertility.

[0065] 4.1 Morphological changes of the testes in offspring rats before and after birth

[0066] Morphological changes of the testes in offspring rats before and after birth, such as Figure 1 As shown. Compared with the control group, the PPCE group showed morphological changes in the fetal testes, including interstitial widening and interstitial cell nuclear shrinkage. Figure 1 (See red arrow at point A), and its maximum cross-sectional area and longest diameter are significantly shortened ( Figure 1 In B and C), the seminiferous tubules showed a significant reduction in both maximum cross-sectional area and longest diameter. Figure 1 (D, E); in adult rats (12 weeks old), the testicular volume was significantly reduced. Figure 1 In the middle E), morphological changes such as abnormal seminiferous tubule morphology and reduced sperm count are observed in the testes. Figure 1 (See the red arrow at point E). Simultaneously, there was a decreasing trend in testicular weight. Figure 1 In the middle F), the thickness of the seminiferous epithelium and the diameter and area of ​​the seminiferous tubules were significantly reduced ( Figure 1(Medium GI). This suggests that PPCE can cause abnormal testicular morphology in offspring before and after birth.

[0067] 4.2 Inhibition of testosterone synthesis in offspring rats before and after birth

[0068] The results of changes in testosterone synthesis function in offspring rats before and after birth are as follows: Figure 2 As shown in the figure. The results showed that, compared with the control group, serum testosterone levels were significantly lower in the PPCE group during fetal development. Figure 2 Inhibition of mRNA expression of the testosterone synthase system (including P450scc, StAR, 3β-HSD, 17α-HSD, and 17β-HSD) in the middle A group. Figure 2 (B) and the expression of StAR protein, a key enzyme in testosterone synthesis, is reduced ( Figure 2 (C); After adulthood, the blood testosterone level in offspring continues to decrease (C). Figure 2 In middle D), the mRNA expression of testosterone synthesis-related enzymes is inhibited ( Figure 2 In the middle F), StAR protein expression decreased ( Figure 2 (Middle F). This suggests that PPCE can cause sustained inhibition of testosterone synthesis in offspring rats before and after birth.

[0069] 4.3 Abnormal spermatogenesis and reduced fertility in adult offspring rats

[0070] Changes in spermatogenesis in adult offspring rats, such as Figure 3 As shown in the figure. The results indicated that, compared with the control group, the number of abnormal sperm (including headless and tailless sperm) in the offspring adult rats of the PPCE group was significantly increased at 12 weeks and 32 weeks of age. Figure 3 (A, B). Further observation of sperm quality-related indicators revealed that the sperm count in the PPCE group was significantly lower at 12 weeks of age. Figure 3 (C) Sperm motility is significantly reduced ( Figure 3 (D) The sperm abnormality rate is significantly increased ( Figure 3 (E), and when it mates with a normal female, the mating success rate is found to decrease ( Figure 3 (F). The above results suggest that PPCE can lead to abnormal spermatogenesis in male offspring and reduced fertility in adulthood.

[0071] [Example 2] Exploration of early warning targets in the paternal testicular hypoplasia animal model of the present invention

[0072] The experimental animals and experimental methods section is consistent with that in Example 1.

[0073] 2. Detection Indicators and Methods

[0074] 2.1 Immunohistochemistry and Immunofluorescence

[0075] For immunohistochemical staining, rat testes were fixed in 4% paraformaldehyde solution for 3 days and then processed using paraffin embedding. Testes 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). The primary antibody against the key testosterone synthesis enzyme StAR and the imprinted gene Mest was blocked using BSA, and detection was performed using a DAB and DAPI staining kit (GeneTech Company, Ltd., Shanghai, China).

[0076] 2.2 RT-qPCR

[0077] For RT-qPCR detection, total RNA was isolated from testicular tissue using TRIzol reagent. The isolated RNA was aliquoted and stored at -80°C. A cDNA synthesis kit was used to reverse transcribe 1 μg of the purified RNA, followed by cDNA amplification. The reaction was performed for 40 cycles. 2 -ΔΔ Relative amplicon expression was calculated using the Ct method. The expression of the Mest, Wnt 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.

[0078] 2.3 Determination of testicular tissue protein concentration

[0079] Testicular tissue was thawed from a -80°C freezer. 50 mg of testicular tissue was weighed, added to PBS, and thoroughly ground and mixed. The experiment was conducted 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 standard after dilution is 20 μL. In a 96-well plate, add an appropriate volume of the sample to be tested and PBS to each well, and also set up wells with an equal volume of standard. Add 200 μL of the pre-prepared BCA working solution to each well and incubate at 37°C in a shaker for about half an hour. After completing the above steps, 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 values ​​of each well and calculate the protein concentration of the sample in each well according to the sample dilution ratio.

[0080] 2.4 Protein extraction from testicular tissue

[0081] Weigh 50 mg of testicular tissue into a 1.5 mL centrifuge tube, mince it (be careful not to mix in the testicular fascia), add 500 μL of RIPA lysis buffer containing protease inhibitors, and homogenize using a homogenizer until there is no obvious precipitate; place the testicular tissue homogenate on ice and let it stand for 30 minutes to fully lyse the tissue proteins, then centrifuge at 12000 rpm at 4℃ for 15 minutes, carefully aspirate the supernatant and transfer it to a new 1.5 mL centrifuge tube; determine the testicular 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 and mix well, heat in a metal bath at 100℃ for 5 minutes, cool, and then proceed with subsequent detection and storage.

[0082] 2.5 Western Blot Procedure for Testicular Tissue Proteins

[0083] Clean the 1.5mm 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 1.5mm 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 sample 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 60V. After the sample enters the separating gel from the stacking gel, adjust the voltage to 120V and continue electrophoresis. Transfer: Cut a PVDF membrane to an appropriate size and activate it in methanol for 2 minutes. Stack the gel and membrane in sequence into a "sandwich" shape in the clamp, place it in the electroporation tank, and electroporate at a constant current of 150mA at low temperature for 0.5-1.5 hours. 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 hour. Dilute different primary antibodies according to the instructions of each antibody. 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 minutes each time. Dilute the secondary antibody with 5% skim milk according to the instructions of the secondary antibody, and react the PVDF membrane in the secondary antibody at room temperature for 2-3 hours. 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.

[0084] 3 Experimental Results

[0085] 3.1 Mest / Wnt signaling mediates abnormal spermatogenesis and reduced fertility in adult offspring rats

[0086] To investigate the mechanism of testicular hypoplasia in offspring caused by PPCE, this invention first performed transcriptome sequencing on offspring testicular tissue. The results showed that the expression of multiple mRNAs in the PPCE-treated offspring testes differed from that in the control group, with the imprinted gene Mest showing the most significant alteration. Figure 4 Furthermore, this invention, through KEGG and GO enrichment analysis, found that differentially altered genes in the testes of PPCE group offspring were enriched in steroid hormone synthesis and the Wnt signaling pathway. Figure 4(B, C). Sequencing results suggest that Mest and Wnt signaling may play important roles in PPCE-induced testicular hypoplasia in offspring. This invention validated relevant indicators and found that, compared with the control, the DNA methylation levels at multiple sites in the Mest promoter region of the offspring in the PPCE(H) group were decreased before and after birth (GD20 and PW12), and its mRNA expression level was increased. Figure 4 (D, E). To determine which cells in testicular tissue Mest is enriched in, this invention performed immunofluorescence double labeling of the testes with Mest and 3β-HSD. The results showed that in the PPCE(H) group, Mest expression was increased in the interstitial region (i.e., interstitial cells) of the testes before and after birth. Figure 4 (FH). Subsequently, this invention detected Wnt signaling-related indicators and found that the mRNA expression of Wnt signaling-related indicators (such as Wnt1, Wnt2, etc.) in the testes of offspring in the PPCE(H) group was significantly inhibited before and after birth. Figure 4 In the middle I, J), and the expression of the Wnt signaling effector protein β-catenin was significantly inhibited ( Figure 4 Based on the above sequencing and experimental verification, it can be preliminarily suggested that PPCE can lead to a decrease in DNA methylation level and an increase in expression in the Mest promoter region of offspring testicular interstitial cells, as well as inhibition of Wnt signaling, thereby resulting in PPCE-induced testicular hypoplasia in offspring.

[0087] 3.2 Mest / Wnt signaling regulates testosterone synthesis in testicular interstitial cells

[0088] To confirm that the Mest / Wnt signaling pathway is involved in regulating testosterone synthesis in testicular interstitial cells, this invention used the rat testicular interstitial cell line R2C as the research subject. After intervention with Mest and Wnt signaling, changes in downstream testosterone synthesis-related indicators were detected. First, transcriptomic sequencing of R2C cells after Mest silencing and overexpression revealed changes in the expression of multiple mRNAs. KEGG and GO enrichment analysis showed that these mRNAs were also enriched in the Wnt signaling pathway, suggesting that Mest may function through Wnt signaling. Figure 5 (AC). Downstream indicators revealed that silencing Mest activated Wnt signaling (increased expression of Wnt, β-catenin, etc.). Figure 5 In the middle E and F groups, the expression of mRNA related to testosterone synthesis was increased, and the expression of StAR protein was increased. Figure 5 (D, F, L); while when Mest is overexpressed, Wnt signal is suppressed ( Figure 5 In the middle E and F), the expression of testosterone synthesis-related enzymes is reduced ( Figure 5(D, F). This indicates that Mest may regulate testosterone synthesis in interstitial cells by negatively regulating Wnt signaling. Furthermore, this invention overexpressed Mest on R2C cells to mimic altered Mest mRNA expression in an animal model, and subsequently co-treated with the Wnt signaling pathway agonist SKL2001 to confirm whether the Wnt pathway is involved in regulating interstitial cell testosterone synthesis. The results showed that the Wnt signaling agonist could reverse the inhibition of testosterone synthesis-related enzymes such as StAR caused by Mest overexpression. Figure 5 (GJ). In summary, this suggests that the Mest / Wnt signaling pathway is involved in regulating testosterone synthesis in testicular interstitial cells.

[0089] [Example 3] Validation of the intervention target in the animal model of paternal testicular hypoplasia of the present invention

[0090] The experimental animals and methods were the same as in [Example 1]. In addition, recombinant adeno-associated virus was injected into the seminiferous tubules of the PW8 offspring rats in the PPCE group for four weeks. Blood and testicular tissue were obtained from the corresponding groups (sham operation group, empty vector group, and Mest intervention group) for further testing.

[0091] 2. Detection Indicators and Methods

[0092] 2.1 Immunohistochemistry and Immunofluorescence

[0093] For immunohistochemical staining, rat testes were fixed in 4% paraformaldehyde solution for 3 days and then processed using paraffin embedding. Testes 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). The primary antibody against the key testosterone synthesis enzyme StAR and the imprinted gene Mest was blocked using BSA, and detection was performed using a DAB and DAPI staining kit (GeneTech Company, Ltd., Shanghai, China).

[0094] 2.2 RT-qPCR

[0095] For RT-qPCR detection, total RNA was isolated from testicular tissue using TRIzol reagent. The isolated RNA was aliquoted and stored at -80°C. A cDNA synthesis kit was used to reverse transcribe 1 μg of the purified RNA, followed by cDNA amplification. The reaction was performed for 40 cycles. 2 -ΔΔRelative amplicon expression was calculated using the Ct method. The expression of testosterone synthesis-related genes, Mest, Wnt 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.

[0096] 2.3 Determination of testicular tissue protein concentration

[0097] Testicular tissue was thawed from a -80°C freezer, 50 mg of testicular 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 standard after dilution is 20 μL. In a 96-well plate, add an appropriate volume of the sample to be tested and PBS to each well, and also set up wells with an equal volume of standard. Add 200 μL of the pre-prepared BCA working solution to each well and incubate at 37°C in a shaker for about half an hour. After completing the above steps, 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 values ​​of each well and calculate the protein concentration of the sample in each well according to the sample dilution ratio.

[0098] 2.4 Protein extraction from testicular tissue

[0099] Weigh 50 mg of testicular tissue into a 1.5 mL centrifuge tube, mince it (be careful not to mix in the testicular fascia), add 500 μL of RIPA lysis buffer containing protease inhibitors, and homogenize using a homogenizer until there is no obvious precipitate; place the testicular tissue homogenate on ice and let it stand for 30 minutes to fully lyse the tissue proteins, then centrifuge at 12000 rpm at 4℃ for 15 minutes, carefully aspirate the supernatant and transfer it to a new 1.5 mL centrifuge tube; determine the testicular 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 and mix well, heat in a metal bath at 100℃ for 5 minutes, cool, and then proceed with subsequent detection and storage.

[0100] 2.5 Western Blot Procedure for Testicular Tissue Proteins

[0101] Clean the 1.5mm 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 1.5mm 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 sample 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 60V. After the sample enters the separating gel from the stacking gel, adjust the voltage to 120V and continue electrophoresis. Transfer: Cut a PVDF membrane to an appropriate size and activate it in methanol for 2 minutes. Stack the gel and membrane in sequence into a "sandwich" shape in the clamp, place it in the electroporation tank, and electroporate at a constant current of 150mA at low temperature for 0.5-1.5 hours. 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 hour. Dilute different primary antibodies according to the instructions of each antibody. 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 minutes each time. Dilute the secondary antibody with 5% skim milk according to the instructions of the secondary antibody, and react the PVDF membrane in the secondary antibody at room temperature for 2-3 hours. 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.

[0102] 3 Experimental Results

[0103] 3.1 Intervention with Mest can reverse testosterone synthesis function in testicular interstitial cells.

[0104] This invention involves constructing a silent recombinant adeno-associated virus (AAV) of Mest and administering it via microinjection into the seminiferous tubules of the rat testes. Figure 6 (A) Changes in testosterone synthesis-related indicators in the testes were detected. The results showed that Mest recombinant adeno-associated virus can target the testes locally and has a good intervention effect. Figure 6 (B). After the above intervention was completed, the detection of testosterone synthesis-related indicators revealed that the inhibition of Wnt signaling gene and β-catenin protein expression was reversed after the intervention. Figure 6 (C). Simultaneously, the expression of testosterone synthesis-related mRNAs increased, while StAR protein expression reverted (…). Figure 6(D). In summary, the above experiments show that Mest intervention can reverse testicular hypoplasia in offspring rats, providing a basis for the prevention and treatment strategy of testicular hypoplasia.

[0105] This invention involves administering 15, 30, and 60 mg / kg / day of caffeine to 8-week-old male Wistar rats via gavage for two months prior to pregnancy. Male offspring are then bred with normal female rats. The PPCE male offspring exhibit typical phenotypes of testicular hypoplasia. Through the exploration and application of relevant early warning targets, this demonstrates the successful establishment of a paternal-derived testicular hypoplasia model and the identification of potential therapeutic targets. The modeling method of this invention is simple, and the stability of indicators such as abnormal testicular morphology, decreased testosterone levels, inhibition of testosterone synthesis, and reduced testicular spermatogenesis and fertility is good, indicating that the modeling method of this invention is stable, effective, reliable, and highly reproducible.

Claims

1. A method for constructing an animal model of paternal testicular hypoplasia, characterized in that: Includes the following steps: S1: Normal six-week-old rodents were selected and administered different doses of caffeine (15 mg / kg.d, 30 mg / kg.d, and 60 mg / kg.d) by gavage for two months. During the administration period, animals had free access to food. S2: After administration, the mice were mated with normal female rodents in a paternal inheritance pattern to obtain the corresponding pregnant mice; S3: In step S2 above, fetal blood and fetal testes were collected from some pregnant mice at 20 days of gestation; some pregnant mice gave birth naturally to obtain F1 offspring. Pregnant mice with litters of ≥10 offspring were retained, with 10 offspring per pregnant mouse, half male and half female. The offspring were weaned 4 weeks after birth and separated into male and female cages. Some male offspring continued to be raised on a normal diet until 6 weeks, 12 weeks and 32 weeks of age, and blood and testes were collected from them. S4: After the above steps are completed, testicular function-related indicators of offspring are detected at different time points, namely 20 days of gestation, 6 weeks, 12 weeks and 32 weeks after birth, to comprehensively determine the occurrence of testicular hypoplasia; finally, an animal model of paternal testicular hypoplasia is obtained. In step S1, the rodents are SPF-grade Wistar and SD rats; In steps S1 and S3, the normal diet is formulated in the same way as the formula feed for mice and rats specified in the "National Standard of the People's Republic of China GB14924.3-2001"; In step S4, the relevant indicators for testicular function testing are: blood testosterone level, endogenous testosterone level in the testes, testicular testosterone synthesis function, and sperm quality.

2. The application of a paternally induced testicular hypoplasia animal model in screening for adverse pre-pregnancy environments in fathers, characterized by: The animal model is obtained by the construction method as described in claim 1.

3. The application of an animal model of paternal testicular hypoplasia in screening early warning targets for paternal testicular hypoplasia, characterized in that: The animal model is obtained by the construction method as described in claim 1.

4. The application of an animal model of paternal testicular hypoplasia in screening early intervention targets for paternal testicular hypoplasia, characterized in that: The animal model is obtained by the construction method as described in claim 1.

5. The application of a paternally derived animal model of testicular hypoplasia in screening drugs for the prevention and treatment of testicular hypoplasia, characterized in that: The animal model is obtained by the construction method as described in claim 1.

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