Method for constructing an animal model of paternal chronic stress leading to changes in the fertility of three generations of offspring and applications thereof

Abstract

The application provides a method for constructing an animal model of paternal chronic stress leading to changes in the fertility of three generations of the paternal line and application thereof. The chronic stress state of the father is constructed by gavage of male rodents with different doses (15, 30 and 60 mg / kg.d) of caffeine for 8 weeks. After administration, the F0 generation of male mice is mated with normal female mice to obtain the F1 generation, and the latter is mated with female mice to obtain the F2 generation. It is found that the fertility of the three generations of the paternal line changes, which is manifested as a significant decrease in the F0 generation, a decrease in the F1 generation, and a return to normal in the F2 generation. Based on the above-mentioned three-generation fertility change model, it is further found that high glucocorticoids of the F0 generation affect the development of the testis of the F1 generation by changing the methylation of sperm imprinting genes, and the fertility of the three generations is related to the glucocorticoid level of the current generation. The three-generation fertility change model constructed by the application has the characteristics of precision and effectiveness, high success rate, simple operation and strong repeatability, and provides an experimental basis for exploring the mechanism of related diseases and clinical early warning targets.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of animal model construction, and particularly relates to a method for constructing an animal model of paternal chronic stress leading to changes in paternal three-generation fertility and application thereof. BACKGROUND

[0002] Fertility refers to the physiological ability of both partners to produce live-born infants. Among them, male fertility refers to the ability of males to produce sperm and sperm to fertilize. At present, the situation of human male fertility decline is serious. Epidemiological reports show that about 15%-20% of couples are troubled by infertility, of which more than half are male infertility [1] . Chronic stress refers to the nonspecific systemic response of the body to long-term stimulation of various internal and external factors, which affects the normal metabolic process of the body by frequently stimulating the cerebral cortex, activating the hypothalamic-pituitary-adrenal (HPA) stress axis and releasing a large amount of glucocorticoids. In recent years, with the acceleration of life rhythm, the increase of social pressure, environmental pollution and other problems, the influence of chronic stress on male fertility has begun to be valued. A large number of epidemiological investigations and animal experiments have confirmed that the chronic stress state caused by adverse lifestyle (such as smoking, alcoholism, depression) before pregnancy, environmental pollution (such as chromium, bisphenol A exposure) and other factors is an important inducement for the decrease of male reproductive function [2] . Chronic stress can affect male fertility and cause developmental and long-term health problems in offspring. Considering the need to open up the "three-child policy", improve population quality and eugenics, male fertility-related diseases caused by paternal chronic stress have become a public health problem that needs to be solved urgently.

[0003] In recent years, the "Development of Health and Disease (DOHaD)" theory has received widespread attention from researchers. For example, maternal exposure to nicotine and alcohol during pregnancy can cause the anogenital distance of male offspring to be shortened, and male fetuses to exhibit feminizing characteristics; the use of certain drugs during pregnancy (such as synthetic glucocorticoids and non-steroidal anti-inflammatory drugs) can inhibit the testosterone synthesis function of offspring testes. However, with the rise of the "Paternal Origin of Health and Disease (POHaD)" theory, more and more evidence supports that adverse environmental exposure before pregnancy by fathers can cause fertility in offspring. Moreover, a part of animal studies have shown that adverse factor exposure before pregnancy by fathers can produce transgenerational inheritance effects. For example, adult zebrafish exposed to low concentrations of carbamazepine will reduce reproductive output, courtship and aggression, reduce testosterone levels and damage sperm morphology, and will pass these effects to the F4 generation through paternal inheritance [3] . Paternal bisphenol A [5]The adverse effects of adverse environmental exposure can be passed on to the F2 generation and even further. However, there are currently relevant maternal animal models of adverse environmental exposure during pregnancy to study the effects of adverse maternal environment during pregnancy on the in-utero and postnatal growth and development of the fetus, but there is no clear and established animal model of paternal adverse environmental exposure leading to changes in the fertility of multiple generations of offspring. Paternal disease models are fundamentally different from maternal disease models. Exogenous interventions such as chronic stress, viral infection, and toxicant intake in maternal models can directly affect fetal development through the placental barrier, simulating adverse environmental exposure during pregnancy. In contrast, exogenous interventions in paternal models can only indirectly affect the fetus through sperm, highlighting the effects of pre-pregnancy exogenous interventions on fetal growth and development. Therefore, to supplement and expand the range of developmental fertility animal models, there is an urgent need to establish and apply animal models of paternal multi-generational fertility changes.

[0004] Caffeine is a common source of chronic stress in modern life, with the effects of refreshing the mind and relieving fatigue, commonly found in coffee, tea, chocolate, and compound drugs. The annual growth rate of coffee consumption in China is 15%-20%, which is about 10 times the world average. According to the National Health and Nutrition Survey data, the average caffeine intake of men of reproductive age (240mg / d) is about 1.5 times that of women; the intake of coffee, tea, and other caffeine-rich beverages by men over the age of 18 is much higher than that of women. Studies have shown that caffeine, as a central nervous system stimulant, can cause significant chronic stress in men exposed to it for a long time. The current situation of caffeine intake by men of reproductive age is serious, and it is a common source of chronic stress. Most countries have set safety doses for caffeine intake by pregnant women, such as the United States and Canada, which have stipulated that pregnant women should not consume more than 200mg of caffeine per day, equivalent to one cup of 300ml coffee per day, but there is no clear regulation for men preparing for pregnancy [6,7] . Therefore, caffeine, as a common developmental toxicant in men's daily beverages, is very representative in the construction of paternal chronic stress disease animal models.

[0005] Main references:

[0006] 1. Niederberger C: Male Infertility. J Urol 2022, 207(3):717-718.

[0007] 2. Liu Y, Zhang C, Liu Y, Zhu J, Qu H, Zhou S, Chen M, Xu D, Chen L, Wang H: Paternal Nicotine / Ethanol / Caffeine Mixed Exposure Induces Offspring Rat Dysplasia and Its Potential "GC-IGF1" Programming Mechanism. Int J Mol Sci 2022, 23(23).

[0008] 3. Fraz S, Lee AH, Pollard S, Srinivasan K, Vermani A, David E, Wilson JY: Paternal exposure to carbamazepine impacts zebrafish offspring reproduction over multiple generations. Environ Sci Technol 2019, 53(21): 12734-12743.

[0009] 4. Farah Naquiah MZ, James RJ, Suratman S, Lee LS, Mohd Hafidz MI, Salleh MZ, Teh LK: Transgenerational effects of paternal heroin addiction on anxiety and aggression behavior in male offspring. Behav Brain Funct 2016, 12(1): 23.

[0010] 5. Mao Z, Xia W, Chang H, Huo W, Li Y, Xu S: Paternal BPA exposure in early life alters Igf2 epigenetic status in sperm and induces pancreatic impairment in rat offspring. Toxicol Lett 2015, 238(3): 30-38.

[0011] 6. 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.

[0012] 7. 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 Nutr 2015, 101(5): 1081-1087. SUMMARY

[0013] The technical problem solved by the present application is to provide a method for constructing a paternal chronic stress-induced paternal three-generation fertility change animal model with high success rate, effective reliability, strong repeatability and simple operation. The present application creates a more daily and clinically close paternal pro-gestational caffeine exposure (PPCE) chronic stress animal model, discovers the dynamic changes of paternal three-generation fertility, and further uses the animal model to carry out the mechanism and early warning target research of related diseases. The present application provides a theoretical and experimental basis for further promoting the mechanism research of chronic stress-related diseases and exploring the early warning target of male fertility change.

[0014] To solve the above technical problems, the technical solutions of the present application are as follows:

[0015] In a first aspect, the present application provides a method for constructing a paternal chronic stress-induced paternal three-generation fertility change animal model, characterized by comprising the following steps:

[0016] S1: Select healthy 8-week-old male rodents (mice and rats), and give 15, 30, 60 mg / kg caffeine orally by gavage every day within the time period of 8-16 weeks of age to perform PPCE chronic stress treatment, and free diet during the administration period;

[0017] S2: After the administration is completed, the normal female mice in the fertile period are caged for pregnancy. A part of the obtained pregnant mice are euthanized at 20 days of pregnancy, and F1 generation fetal mice are obtained and the related pregnancy outcome indexes are recorded;

[0018] S3: F0 male rodents after the euthanasia administration, collecting testis and sperm and observing the quality change, combined with the results of S2 to evaluate the F0 male fertility;

[0019] S4: Another part of pregnant mice naturally produce F1 generation, taking the production day as postnatal day 0, selecting the litter number of 12-14 at postnatal day 1, adjusting each litter of 6 male and 6 female offspring for lactation feeding;

[0020] S5: F1 generation is weaned at 4 weeks after birth and separated into male and female cages, part of which is normally fed until 6 weeks, 12 weeks and 32 weeks, and the testis and sperm related indexes are detected at each time point to determine the F1 generation testis development and sperm quality;

[0021] S6: The rest of the adult F1 generation PPCE male mice are co-caged with normal female mice for pregnancy. A part of the obtained pregnant mice are euthanized at 20 days of pregnancy to obtain F2 generation fetal mice and record the related pregnancy outcome indexes, and the results obtained in S5 are used to evaluate the F1 generation male fertility;

[0022] S7: Continue to use similar breeding methods to obtain and evaluate the F2 generation male fertility results, and finally obtain the paternal three-generation fertility change animal model.

[0023] As a preferred solution, in step S1, the rodent is an SPF level, including but not limited to rats and mice.

[0024] As a preferred solution, in step S4, the production day is taken as postnatal day 0, and the litter number of 12-14 is selected at postnatal day 1, and each litter of 6 male and 6 female offspring is adjusted for lactation feeding.

[0025] Further, in step S1, the normal diet formula is the same as the experimental animal formula feed stipulated in the "People's Republic of China National Standard GB14924.1-2001".

[0026] Further, in steps S2 and S6, the pregnancy outcome indexes are: fertility parameters (mating rate, fertility rate, abortion rate), litter size parameters (corpus luteum number, implantation number, implantation rate, live birth number, absorbed and dead fetus number), and fetal parameters (body length, tail length, fetal weight, placenta weight, intrauterine growth retardation rate), and the testis function related indexes are: testis morphology, blood corticosterone level, blood testosterone level, sperm quality (sperm number, motility, morphology, etc.), mating success rate.

[0027] Secondly, the application provides an application of the animal model of paternal three-generation fertility change caused by chronic stress in promoting the mechanism research of chronic stress related diseases, characterized in that: the animal model of paternal three-generation fertility change is obtained by any of the above construction methods.

[0028] In a third aspect, the application provides use of an animal model of changes in paternal three-generation fertility caused by chronic stress in exploring early warning targets for changes in male fertility, characterized in that the animal model of changes in paternal three-generation fertility is obtained by any of the above methods.

[0029] The technical principle and research process of the application are as follows:

[0030] The application simulates the easy availability and other characteristics of caffeine in the daily life of men, and constructs a chronic stress animal model of PPCE. By obtaining F0, F1 and F2 generations and detecting related pregnancy outcomes and sperm quality and other indicators, the dynamic changes in the fertility of the paternal three generations are determined. The model simulates the influence of paternal chronic stress exposure on the fertility of three generations, and is of great significance for promoting the research on the mechanism of chronic stress-related diseases, exploring early warning targets and clinical prevention and treatment.

[0031] The advantages and beneficial effects of the application are as follows:

[0032] 1. Precise and effective: caffeine exposure is used as a chronic stress source, and its chronic stress effect has been widely recognized. The model is closer to life and easy to obtain.

[0033] 2. High success rate: compared with sleep deprivation, circadian rhythm deprivation and water deprivation, giving caffeine solution to rodents causes slight and short-term damage, which is more in line with the "3R principle" of modern ethics;

[0034] 3. Simple operation and strong repeatability: the PPCE model can be established by giving different doses of caffeine to the father through gavage for 8 weeks after the father matures. At the same time, the fertility-related indicators detected in the application have good stability and strong preservation.

[0035] In summary, the application provides a reliable method for establishing an animal model of changes in paternal three-generation fertility caused by paternal chronic stress. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 .Effects of PPCE on blood corticosterone, sperm quality and pregnancy outcomes of F0 generation rats.

[0037] Figure 1 Medium: A: blood ACTH level; B: blood CORT level; C: mRNA expression of testicular testosterone synthesis-related genes; D: blood testosterone level; E: testicular HE staining; F: sperm HE staining; G-J: pregnancy outcomes (in order of mating success rate, fetal weight, fetal length, IUGR rate).

[0038] Figure 2 .Effects of PPCE on blood corticosterone, sperm quality and pregnancy outcomes of F1 generation rats.

[0039] Figure 2 Mice: A: blood CORT level; B: blood testosterone level; C: testis HE staining; D: sperm HE staining; E-H: pregnancy outcome (in turn mating success rate, fetal weight, fetal length, IUGR rate).

[0040] Figure 3 Effect of PPCE on blood CORT level, sperm quality and pregnancy outcome of F2 generation rats.

[0041] Figure 3 Mice: A: blood CORT level; B: fetal weight; C: fetal length; D: testis HE staining; E: sperm HE staining; F: sperm motility; G: sperm deformity rate; H: sperm count.

[0042] Figure 4 Effect of PPCE on the function of steroid synthesis of F0 generation.

[0043] Figure 4 Mice: A: mRNA expression of testicular Caspase3; B: mRNA expression of testicular Ki67; C: blood testosterone level; D: mRNA expression of testicular testosterone synthesis related genes; E: IHC staining of testicular StAR; F: WB protein of testicular StAR.

[0044] Figure 5 Effect of high glucocorticoid level on the function of steroid synthesis of testicular interstitial cells.

[0045] Figure 5 Mice: A, H: mRNA expression of Ki67; B, I: mRNA expression of Caspase3; C, J: mRNA expression of Dax1; D, K: mRNA expression of StAR; E, L: mRNA expression of Cyp17a1; F, M: mRNA expression of Hsd17b1; G, N: IF staining of StAR.

[0046] Figure 6 Effect of PPCE on methylation of imprinting gene Mest and the function of steroid synthesis after birth of F1 generation.

[0047] Figure 6 Mice: A: whole genome methylation profile; B: DNA methylation of imprinting gene Mest promoter region; C: blood CORT level; D: testicular GR expression; E: IF double staining of MEST and StAR.

[0048] Figure 7 Effect of PPCE on whole genome methylation of sperm of F0 / 1 generation.

[0049] Figure 7Middle: A: whole genome methylation profile; B: DNA methylation status of major imprinted gene promoter regions. DETAILED DESCRIPTION

[0050] The technical content of the present application will be further elaborated below in combination with specific embodiments and drawings.

[0051]

Example 1

[0052] 1 Experimental animals

[0053] SPF level healthy Wistar rats were purchased from Hubei Provincial Center for Disease Control and Prevention, and the animal license number was SCXK(E)2018-2020. This study was approved by the Ethics Committee of Wuhan University Medical School, and was strictly in accordance with the relevant handling guidelines of the International Experimental Animal Protection Certification Evaluation Agency.

[0054] The experimental animals were raised in a barrier environment with a temperature of 22-25°C, a humidity of 50%, and a 12-hour day-night alternation.

[0055] 2 Experimental methods

[0056] Sixty male 8-week-old Wistar rats (body weight 260-300 g) were given free water and food, and after 7 days of adaptive feeding, they were divided into a control group (daily intragastric administration of normal saline 1 mL / 100 g) and a pre-pregnancy caffeine exposure group (daily administration of 15, 30, and 60 mg / kg of caffeine). After two months of drug administration, male Wistar rats (now 16 weeks old) were mated with normal 12-week-old female Wistar rats at a ratio of 1:2 (note: each male mouse was guaranteed to mate only once to ensure a one-to-one relationship between male mice and offspring). The next morning, vaginal smears were examined, and if sperm were observed under a microscope, it was recorded as gestational day 0 (GD), and the pregnant female mice were removed and raised separately. This continued to ensure that at least 12 pregnant mice were produced in each group. On GD20, some pregnant mice were given 2% isoflurane anesthesia for euthanasia, and fetal blood and testicular tissue were taken. The blood samples of male fetal mice from each litter were combined into one sample for subsequent analysis of blood series-related indicators. The right testicular tissue of the fetus was 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 HE, immunofluorescence, immunohistochemistry, and other morphological analyses. Pregnant mice in each group were given free normal food during drug administration and pregnancy. The feed was purchased from Wuhan Wanqiangxing Biological Technology Co., Ltd., with a license number of SCXK(E)2011-0011. The feed formula was the same as that specified in the "National Standard of the People's Republic of China GB14924.3-2001" for mouse and rat formula feed.

[0057] The rest of the pregnant mice were naturally produced, and 8-14 litter sizes at birth were considered to be qualified, and the fetal gender and number were adjusted between different litters to reach 12 per litter, with a 1:1 gender ratio. The offspring rats were weaned at 4 weeks of age and separated by gender. Sampling was performed at three time points: 6 weeks, 12 weeks, and 32 weeks after birth. The right testicular tissue of the offspring was immediately frozen in liquid nitrogen and stored at -80°C for further RT-qPCR analysis. Five left testes of the offspring were randomly selected and placed in 4% paraformaldehyde solution overnight, dehydrated with alcohol, and embedded in paraffin for HE and other morphological analysis. Part of the adult F1 generation PPCE male mice were mated with normal female mice to obtain F2 generation rats, and the F3 generation fetal rats were obtained in the same way (only used to observe the pregnancy outcome of the F2 generation). Changes in blood testosterone levels, sperm quality, and pregnancy outcomes of adult rats were detected to confirm and establish the rat model of fertility changes in the paternal three generations (including F0, F1, and F2 generations) caused by PPCE.

[0058] 3. Test index and method

[0059] 3.1 Pregnancy outcome

[0060] The pregnancy outcome was composed of fertility parameters (mating rate, fertility rate, abortion rate), litter size parameters (corpus luteum number, implantation number, implantation rate, live birth number, absorbed and dead fetus number), and fetal parameters (body length, tail length, fetal weight, placenta weight, intrauterine growth retardation rate). It should be noted that any value reported represents the average of all fetal measurements of pregnant rats. Some results were calculated as follows: mating rate (%) = number of rats with mating behavior / total number of rats x 100; fertility rate (%) = number of pregnant rats / total number of rats x 100; abortion rate (%) = number of aborted rats / total number of rats x 100; implantation number = live fetus number + dead fetus number + absorbed fetus number; implantation rate (%) = implantation number / corpus luteum number x 100; IUGR rate (%) = number of fetal rats with body weight less than two standard deviations of the average body weight / live birth number x 100.

[0061] 3.2 Sperm quality

[0062] Immediately after euthanasia, the right epididymis was removed, crushed in 3 mL of normal saline, and incubated at 37°C for 5 min to completely release the sperm. Sperm suspension was prepared and smeared to calculate sperm motility. At the same time, part of the semen was counted using a red blood cell counting plate (16 grids x 25 grids). Then, another part of the semen was fixed with 4% paraformaldehyde for 2 h, and then stained with hematoxylin and eosin (HE) to calculate the sperm abnormality rate.

[0063] Sperm motility can be divided into progressive (PR), non-progressive (NP) and immotile (IM), the ratio of PR+NP out of 200 sperm in each sample was calculated as the sperm motility rate. Sperm count (per mL) = average sperm count per small grid on the counting plate x 4 x 10 6 The head, neck and tail abnormalities were described as abnormal sperm, the ratio of abnormal sperm out of 200 sperm in each sample was calculated as the sperm deformity rate.

[0064] 3.3 HE staining

[0065] Rat testes were fixed in 4% paraformaldehyde solution for 3 days and treated with paraffin embedding technique. The testes were sectioned at 5 pm sagittal for morphological staining analysis. The sections were placed in hematoxylin staining solution for 15 min, distilled water immersion for 15 min, 1% hydrochloric acid ethanol for about 10 s, the color of the section changed from red to light, distilled water immersion for about 10 s, 0.6% ammonia water counterstaining, and then rinsed with running water for about 10 s. The stained sections were placed in 0.5% eosin staining solution for 2 min. Then the sections were placed in 95% alcohol I 5 min→ 95% alcohol II 5 min→ anhydrous ethanol I 5 min→ anhydrous ethanol II 5 min→ xylene I 5 min→ xylene II transparent, the sections were taken out of xylene and slightly dried, neutral resin was used for mounting.

[0066] Examination and evaluation were performed by another experimenter who was blinded to the experimental treatment. Five random fields of each section were observed under a microscope, and the average value of all measurements of each section was considered as one data (n = 5). This study observed and measured the area of seminiferous tubules, the thickness of seminiferous epithelium and Johnson score. The area of seminiferous tubules was measured with the muscle-like cells around the seminiferous tubules as the boundary, the muscle-like cells as the bottom and the round spermatids as the top, the thickness of seminiferous epithelium was measured, and the Johnson score was measured on the basis of previous studies.

[0067] 3.4 Detection of blood testosterone and corticosterone

[0068] The serum testosterone and corticosterone concentrations were determined by ELISA kit. All steps followed the protocol provided by the manufacturer.

[0069] 3.5 RT-qPCR detection

[0070] 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 1 μg sample of purified RNA was reverse transcribed using a cDNA synthesis kit, followed by cDNA amplification. The reaction was performed for 40 cycles. Relative amplicon expression was calculated using the 2-ΔΔCt method. The expression of StAR and GAPDH in the RNA was determined. 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 the sequence of each designed primer for homology comparison to determine the final primer sequences used.

[0071] 3.6 Statistical Analysis

[0072] Data was analyzed and processed using GraphPad Prism software (6.5). All numerical results are calculated as mean ± standard error. Student... ′ The Stokes test was used to analyze significant differences between two groups, and one-way ANOVA was used to compare multiple groups. P < 0.05 indicated that the difference was statistically significant.

[0073] 4 Experimental Results

[0074] 4.1 Effects of PPCE on serum corticosterone, sperm quality, and pregnancy outcome in F0 generation rats

[0075] This invention involves orally administering different doses of caffeine to male reproductive rats (F0 generation) for 8 weeks (covering a complete spermatogenesis cycle), while a normal control group is given an equal volume of physiological saline. The rats are then mated with normal female rats to obtain F1 generation rats. Some F1 generation PPCE adult male rats are mated with normal female rats to obtain F2 generation rats. The same method (paternal inheritance) is used to produce F3 generation fetal rats, thereby establishing a rat model of paternal fertility changes across three generations caused by PPCE.

[0076] The results showed that, compared with the control group, serum ACTH and corticosterone levels were significantly increased in the PPCE group. Figure 1 In patients with A and B, the expression of adrenal steroid synthase systems (such as StAR, P450scc, and 3β-HSD) was significantly increased. Figure 1 (C). Next, this invention examined changes in indicators related to reproductive function and pregnancy outcomes in the F0 generation, finding that serum testosterone levels were decreased in the PPCE group ( Figure 1Abnormal testis morphology (including reduced thickness of seminiferous epithelium, widened interstitial space, reduced sperm number in seminiferous tubule lumen, etc.) Figure 1 Abnormal sperm increase Figure 1 Reduced pregnancy rate Figure 1 Significant reduction in F1 generation birth weight and body length Figure 1 Significant increase in IUGR rate Figure 1 General, J). In summary, PPCE can cause F0 generation HPA axis activation and significantly reduced fertility.

[0077] 4.2 Effect of PPCE on F1 generation rat serum corticosterone, sperm quality and pregnancy outcome

[0078] The present application detects the changes in serum corticosterone and testosterone levels in F1 generation rats on the above (1) PPCE rat model. Compared with the control group, the serum corticosterone level and testosterone level in the PPCE group were significantly reduced Figure 2 A, B). Then, the present application observed the changes in testicular pathological morphology and sperm quality, and found that the F1 generation testis of the PPCE group showed reduced thickness of seminiferous epithelium, widened interstitial space, reduced sperm number in seminiferous tubule lumen, etc. Figure 2 C), while the number of abnormal morphological sperm such as headless and tailless increased Figure 2 D). Finally, the present application detected the changes in pregnancy outcome related indicators, and found that the F1 generation rats of the PPCE group had reduced pregnancy rate Figure 2 E), reduced F2 generation fetal rat birth weight and body length Figure 2 F, G), and increased IUGR rate Figure 2 H). It is suggested that PPCE causes the reduction of serum corticosterone level in F1 generation rats, while the serum testosterone level, sperm quality and fertility are also reduced, but are less severe than in F0 generation.

[0079] 4.3 Effect of PPCE on F2 generation rat serum corticosterone, sperm quality and pregnancy outcome

[0080] Further, the present application observes the changes in serum corticosterone level and sperm quality in F2 generation rats on the above (1) PPCE rat model. The results show that compared with the control group, the serum corticosterone level in the F2 generation PPCE group of rats has no significant change Figure 3 A). At the same time, the F3 generation fetal weight and fetal body length have no obvious change Figure 3 B, C). Further, the testis morphology Figure 3 D) and sperm number, sperm motility and deformity rate in the PPCE group also have no significant change Figure 3 E-H). It is suggested that PPCE can restore the normal levels of corticosterone and sperm quality in F2 generation.

[0081] 5Research Conclusion

[0082] PPCE can cause the dynamic changes of the fertility of male rats in three generations, which is specifically manifested as follows: PPCE leads to the changes of the fertility of the paternal three generations, which is manifested as the obvious decrease of F0 generation, the recovery of F1 generation, and the recovery of F2 generation.

[0083]

Example 2

[0084] 1Experimental animals

[0085] The experimental animals were the same as in Example 1.

[0086] 2Experimental methods

[0087] The previous animal model construction method was Example 1.

[0088] 3Detection index and method

[0089] 3.1 Immunohistochemistry and immunofluorescence

[0090] For immunohistochemistry, the sections were incubated with the corresponding primary antibody at 4°C for about 12 hours. Then the sections were placed in PBS and washed 3 times by shaking on a destaining shaker for 5 minutes each time. After that, the secondary antibody was incubated for 5 minutes. IHC analysis was performed using a DAB staining kit to determine the expression level of the protein. The staining intensity was determined by measuring the average optical density in five random fields of each section (n=5). For immunofluorescence, the sections were incubated with the corresponding primary antibody at 4°C for about 12 hours. After rewarming for 15 minutes, the sections were placed in PBS and washed 3 times by shaking on a destaining shaker for 5 minutes each time. After that, the corresponding fluorescent secondary antibody (1:400) was added, and the sections were incubated at room temperature in the dark for 1 hour. Nuclear counterstaining (DAPI; Sigma Aldrich) was diluted 1:500 in TBS and incubated for 10 minutes. The number of cells per unit area of interstitial tissue area (10 4 μm 2 ) or protein expression level was calculated by examining five randomly selected sites in five sections of each group. All images were taken using an Olympus AH-2 optical microscope (Olympus, Tokyo, Japan). The staining images were analyzed using Olympus software.

[0091] 3.2 Testicular tissue protein concentration determination

[0092] The testicular tissue was taken from the -80°C refrigerator, weighed 50 mg of testicular tissue, then added PBS and ground thoroughly. The experiment was performed according to the BCA kit instructions. According to the required total volume, mix A reagent with B reagent at a ratio of 50:1 to prepare the BSA working solution. Note that the BSA working solution needs to be prepared immediately before use; dilute the protein standard solution (25 mg / mL) according to the gradient to 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 mg / mL, and make the final volume of each standard after dilution 20 μL; take out the 96-well plate, add appropriate volume of sample to be tested and PBS to each well, and set the standard volume at the same time; add the pre-prepared BCA working solution to each well, 200 μL per well, and mix well on a 37°C constant temperature shaker for about half an hour; after the above steps are completed, place the 96-well plate in the enzyme marker, and read the absorbance value of each well at 570 nm wavelength; finally, draw a standard curve according to the absorbance value of each well, and calculate the protein concentration of each well according to the sample dilution ratio.

[0093] 3.3 Protein extraction of testicular tissue

[0094] Weigh 50 mg of testicular tissue in a 1.5 mL centrifuge tube, cut it into small pieces (make sure not to mix with testicular fascia), add 500 μL of RIPA lysis buffer containing protease inhibitors, and homogenize it with a homogenizer until there is no obvious precipitate; place the testicular tissue homogenate on ice for 30 minutes, centrifuge at 12000 rpm at 4°C for 15 minutes after the tissue protein is fully lysed, and carefully transfer the supernatant to a new 1.5 mL centrifuge tube; determine the protein concentration of the testicular tissue according to the above steps using the BCA method, and adjust the concentration of each sample to a uniform concentration according to the results; mix the tissue protein sample with an appropriate amount of 5x loading buffer, heat at 100°C in a metal bath for 5 minutes, and then cool for subsequent detection and storage.

[0095] 3.4 Western Blot steps of testicular tissue protein

[0096] 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.

[0097] 3.5 Other methods are the same as in Example 1.

[0098] 4 Experimental Results

[0099] 4.1 Effects of high glucocorticoid levels on F0 generation steroid synthesis and its mechanism

[0100] This invention investigated the effects of PPCE on steroid synthesis in paternal testes. First, testicular proliferation and apoptosis-related indicators were measured. The results showed that, compared with the control group, PPCE had no significant effect on Caspase-3 in parental rat testes. Figure 4 (A), but can significantly inhibit Ki67 expression ( Figure 4 (B) Testosterone synthesis function tests revealed that PPCE significantly reduced serum testosterone levels. Figure 4 (C), while the mRNA expression of the testosterone synthesis enzymes StAR and CYP17a1 was significantly inhibited (C). Figure 4PPCE significantly inhibited the expression of StAR protein in parental testes, but had no significant effect on the expression of 3β-HSD and 17β-HSD. Immunohistochemistry and Western blotting results both confirmed that PPCE also inhibited the expression of StAR protein in parental testes. Figure 4 (E, F)

[0101] To clarify the effects of corticosterone / caffeine on testicular interstitial cells, this invention investigated the effects of different concentrations of corticosterone (0, 250, 500, 1000 nM) and caffeine (0, 0.1, 1, 10 μM) on proliferation, apoptosis, and testosterone synthesis in the rat testicular interstitial cell line R2C. After administration of corticosterone, interstitial cell proliferation was inhibited (…). Figure 5 In the middle A), apoptosis showed no significant change. Figure 5 (B) After examining testosterone synthesis function, it was found that the mRNA expression of Dax1, a negative regulator of testosterone synthesis, increased in a corticosterone-dependent manner. Figure 5 In the middle C), the mRNA expression of testosterone synthesis-related genes StAR and 17β-HSD showed a significant corticosterone-dependent inhibition. Figure 5 Corticosterone (DF) had no significant effect on Cyp17a mRNA expression. Cell smear results showed that corticosterone also inhibited StAR protein expression in mesenchymal cells in a dose-dependent manner. Figure 5 (G), which initially indicates that corticosterone can inhibit the testosterone synthesis function of interstitial cells; however, when caffeine was administered, no significant changes were observed in the proliferation and apoptosis of interstitial cells. Figure 5 The expression of H,I) and testosterone synthase system genes (including StAR, 3β-HDS, Cyp17a1) were also unaffected. Figure 5 The results (JN) indicate that caffeine has no significant effect on the testosterone synthesis function of interstitial cells. Combining the results of whole animal and cell experiments, under PPCE conditions, high corticosterone (but not caffeine) mediates the inhibition of parental testicular interstitial cell proliferation and reduced testosterone synthesis function.

[0102] 4.2 Effects of high glucocorticoid levels on postnatal steroid synthesis in F1 generation mediated by altered Mest gene methylation

[0103] The F0 generation sperm whole-genome DNA methylation results showed that, compared with the control group, the sperm DNA methylation level was increased in the PPCE(L) group, while the sperm DNA methylation level was decreased in the PPCE(M) and PPCE(H) groups. Figure 6 (A) Among them, the DNA methylation level in the Mest promoter region was significantly reduced. Figure 6 (B). Furthermore, this invention found that F0 generation blood corticosterone levels were elevated ( Figure 6 In the middle C), testicular GC expression and nuclear activation increased ( Figure 6Fig. 3D). Immunofluorescence detection found that the expression of StAR in the PPCE group was significantly reduced, and the expression of MEST was increased, and their expressions were co-localized Figure 6 Fig. 3E).

[0104] 5 Research conclusion

[0105] The application further explores the application of the animal model of chronic stress leading to the change of paternal three generations fertility in promoting the mechanism research of chronic stress related diseases, that is, high glucose corticosteroids directly affect the steroid synthesis function of F0 generation, and affect the testis development of F1 generation by changing the methylation of sperm imprinting genes of F0 generation.

[0106]

Example 3

[0107] 1 Experimental animals

[0108] The experimental animals are the same as in Example 1.

[0109] 2 Experimental method

[0110] The previous animal model construction method is Example 1.

[0111] 3 Detection index and method

[0112] 3.1 Whole genome methylation sequencing (second generation)

[0113] DNA methylation is an important epigenetic marker information, and obtaining the methylation level data of all C sites in the whole genome has important significance for the spatiotemporal specificity research of epigenetics. Bisulfite methylation sequencing is based on the new generation of high-throughput sequencing platform, combined with whole genome Bisulfite processing and bioinformatics data analysis technology, to carry out low-cost, high-efficiency, high-accuracy whole genome DNA methylation level mapping.

[0114] 3.2 The other methods are the same as in Example 1 and Example 2.

[0115] 4 Experimental results

[0116] Sperm is the only medium for fathers to affect their sons. WGBS sequencing shows that the whole genome methylation of F0 generation PPCE group sperm has a significant downward trend, and the degree of reduction of F1 generation sperm whole genome methylation is reduced Figure 7 In addition, compared with F0 generation sperm, the methylation level of part of the key imprinting genes of embryo development in F1 generation sperm is reversed Figure 7Conclusion of Example 1 and Example 2, the present application speculates that chronic stress directly damages the testosterone synthesis function of F0 generation interstitial cells and its related fertility indicators on one hand, and on the other hand, changes the whole genome methylation level of sperm to affect the development of F1 generation fetus (such as testis), and then damages the fertility of F1 generation, and the damage mechanism is not consistent with that of F0 generation. In F1 generation, due to the low basal level change of serum GC content opposite to that of F0 generation, it reverses the methylation level of part of related genes in sperm, thereby leading to the gradual normalization of each phenotype of F2 generation.

[0117] 5 Research conclusion

[0118] The present application further explores the application of the animal model of chronic stress leading to the change of paternal three-generation fertility in exploring the early warning target of male fertility change, that is, the change of F0 / F1 / F2 three-generation fertility is related to the level of glucocorticoid, and the change of glucocorticoid level and its downstream related indicators may be the potential early warning target of male fertility change.

[0119] The present application method administers 15, 30, 60 mg / kg.d of caffeine to 8-week-old male rodents by oral gavage before pregnancy and lasts for 8 weeks, and then mates with normal female mice to obtain male offspring, and the typical phenotype of three-generation fertility change after PPCE treatment can be found, which indicates that the animal model of paternal three-generation fertility change caused by paternal chronic stress is successfully established. The present application explores the influence of high glucocorticoid level on the function of testis and sperm of F0 / F1 / F2 generation, which is of great significance for promoting the mechanism research of chronic stress related diseases, exploring the early warning target in clinic and clinical prevention and treatment. The stability of the indexes such as testis morphology, serum testosterone level, sperm quality and pregnancy outcome in the animal model is good, the modeling method of the present application is precise and effective, has high success rate, is simple to operate and has strong repeatability.

Claims

1. A method for constructing an animal model of paternal chronic stress leading to changes in the fertility of three generations of the paternal line, characterized by the fact that: The method comprises the following steps: S1: selecting healthy 8-week-old male rodents, and orally administering 15, 30, or 60 mg / kg of caffeine to the rodents every day within a period of 8-16 weeks to perform chronic stress treatment of paternal pre-pregnancy caffeine exposure, and allowing the rodents to freely eat during the administration period; S2: after the administration is completed, the rodents are mated with normal female rodents in the breeding period to obtain pregnant rodents; a part of the obtained pregnant rodents are euthanized on the 20th day of pregnancy to obtain F1 generation fetal rodents and record related pregnancy outcome indexes; S3: the F0 generation male rodents after the administration are euthanized, testes and sperm are collected, and quality changes of the testes and sperm are observed, and F0 generation male fertility is evaluated in combination with the results of step S2; S4: another part of the pregnant rodents in step S2 naturally give birth to F1 generation, and the day of the birth is taken as postnatal day 0, and on postnatal day 1, litter size is 12-14, and the litter is adjusted to have 6 male and 6 female rodents for breastfeeding; S5: the F1 generation is weaned after 4 weeks of birth, and the male and female rodents are separated, and part of the rodents are normally fed for 6 weeks, 12 weeks, and 32 weeks, and testes and sperm related indexes are detected at different time points to determine F1 generation testes development and sperm quality; S6: the remaining adult F1 generation PPCE male rodents are mated with normal female rodents; a part of the obtained pregnant rodents are euthanized on the 20th day of pregnancy to obtain F2 generation fetal rodents and record related pregnancy outcome indexes, and F1 generation male fertility is evaluated in combination with the results obtained in step S5; S7: the above breeding method is continuously used to obtain and evaluate F2 generation male fertility results, and finally, an animal model of paternal three-generation fertility changes is obtained; In step S1, the rodents are SPF grade mice; In step S1, the caffeine exposure dose and time are 15, 30, or 60 mg / kg·d, and the administration is started from puberty and lasts for 8 weeks; In steps S2 and S6, the pregnancy outcome indexes are fertility rate parameters, litter size parameters, and fetal parameters; and testes function related indexes are testes morphology, blood testosterone and corticosterone levels, sperm quality, and mating success rate.

2. The use of an animal model in which chronic stress causes changes in the fertility of three generations of males in the screening / preparation of drugs for chronic stress-related diseases, characterized in that: The animal model is obtained by the construction method of claim 1.

3. Use of an animal model of chronic stress leading to changes in patrilineal fertility in three generations for screening early warning targets of male fertility changes, characterized in that: The animal model is obtained by the construction method of claim 1.

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