Construction method and application of animal model for male adult offspring fertility reduction caused by prednisone exposure during pregnancy

Abstract

The invention discloses a construction method and application of a rodent model for male adult offspring fertility reduction caused by prednisone exposure during pregnancy. The fertility of male adult offspring is reduced due to prednisone exposure during pregnancy, rodents are subjected to gavage with prednisone in the whole pregnancy process, and the offspring is normally fed for 12 weeks after being born. Male offspring testis or sperms which are 20 days after pregnancy and 12 weeks after birth are obtained, and fetal testis dysplasia and adult fertility reduction are proved. Based on the animal model, it is proved that male adult offspring fertility reduction caused by prednisone exposure during pregnancy can be reversed when genistein is given to pregnant rats at the same time during pregnancy. The animal model established by the invention has the characteristics of novelty, reliability, simplicity and convenience, and is of great significance to elucidating a fertility reduction mechanism of male offspring caused by prednisone exposure during pregnancy and exploring an early prevention and treatment technology of the male offspring.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method for constructing and applying an animal model of reduced fertility in adult male offspring caused by prednisone exposure during pregnancy. Background Technology

[0002] my country's birth policy has been fully implemented, but this is not matched by the fast pace of modern life, increased work pressure, and delayed childbearing, leading to a continuous decline in the reproductive-age population and the quality of offspring. Male fertility refers to a man's ability to produce sperm and fertilize an egg. In today's society, the problem of declining male fertility is increasingly prominent and has become a global public health issue of widespread concern. Epidemiological surveys show that approximately 15% of couples of reproductive age worldwide experience infertility, with male infertility accounting for as much as 50% of these cases. [1,2] A significant decrease in male fertility is related to a sharp decline in sperm count and quality. [3] Therefore, exploring the pathogenesis of male fertility decline and carrying out early prevention and treatment is of great practical and social significance.

[0003] The Developmental Origins of Health and Disease (DOHaD) theory focuses on the impact of adverse parental environments on offspring health. Previous research on declining male fertility primarily focused on various influencing factors such as age, environment, and lifestyle. However, in recent years, an increasing number of studies have begun to focus on the developmental origins of declining male fertility in the fetus. [4] Fetuses are the most sensitive group to environmental changes and are highly susceptible to various adverse factors during pregnancy. In recent years, people have gradually realized that the impact of parents on offspring fertility is far more extensive and profound than previously thought. Therefore, conducting research on the causes and mechanisms of reduced male offspring fertility due to adverse parental environmental exposure, and exploring early prevention and control strategies, has important theoretical and practical significance.

[0004] Prednisone, a synthetic glucocorticoid, is used to treat various conditions during pregnancy, including maternal autoimmune diseases, recurrent miscarriage, and asthma. It is the most commonly used oral glucocorticoid during pregnancy. Epidemiological surveys show that the usage rate of prednisone during pregnancy is 1.3%, with a usage rate of 20%–60% among pregnant women with autoimmune diseases. [5] Meanwhile, due to the widespread use of prednisone, insufficient metabolism, and limitations in wastewater treatment technology, its residual levels in the environment are excessively high, confirming it as a novel environmental pollutant. [6] In summary, pregnant women may inevitably be exposed to prednisone due to medical treatment or environmental pollution.

[0005] References: 1.Eisenberg ML, Esteves SC, Lamb DJ, Hotaling JM, Giwercman A, HwangK, Cheng YS. Male infertility. Nat Rev Dis Primers. 2023; 9(1): 49. 2.Agarwal A, Baskaran S, Parekh N, Cho CL, Henkel R, Vij S, Arafa M,Panner Selvam MK, Shah R. Male infertility. Lancet. 2021; 397(10271): 319. 3.Levine H, Jørgensen N, Martino-Andrade A, Mendiola J, Weksler-DerriD, Jolles M, Pinotti R, Swan SH. Temporal trends in sperm count: a systematicreview and meta-regression analysis of samples collected globally in the 20thand 21st centuries. Hum Reprod Update. 2023; 29(2): 157. 4.Juul A, Almstrup K, Andersson AM, Jensen TK, Jørgensen N, Main KM,Rajpert-De Meyts E, Toppari J, Skakkebæk NE. Possible fetal determinants ofmale infertility. Nat Rev Endocrinol. 2014; 10(9): 553. 5.Palmsten K, Rolland M, Hebert MF, Clowse MEB, Schatz M, Xu R,Chambers CD. Patterns of prednisone use during pregnancy in women with rheumatoid arthritis: GENly and cumulative dose. Pharmacoepidemiol Drug Saf. 2018; 27(4): 430. 6.Weizel A, Schlüsener MP, Dierkes G, Ternes TA. Occurrence ofglucocorticoids, mineralocorticoids, and progestogens in various treated wastewater, rivers, and streams. Environ Sci Technol. 2018; 52(9): 5296. Summary of the Invention To address the shortcomings of existing technologies, this invention focuses on prenatal prednisone exposure (PPE) rats, confirming reduced fertility in their male adult offspring. This provides a method for constructing an animal model of PPE-induced reduced male adult offspring fertility and its application. This invention is significant for elucidating the pathogenesis of PPE-induced reduced male adult offspring fertility and identifying early intervention targets. The technical problem this invention aims to solve is to provide a highly successful, reliable, reproducible, and simple method for constructing a PPE-induced reduced male adult offspring fertility model, and based on this model, proposes the application of genistein to improve PPE-induced reduced male offspring fertility.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: In a first aspect, the present invention provides a method for constructing an animal model in which prednisone exposure during pregnancy leads to reduced fertility in adult male offspring.

[0007] The animal model construction method described in this invention achieves stable induction of a reduced fertility phenotype in male adult offspring by precisely controlling the prednisone exposure dose, exposure time, and offspring rearing conditions. The specific steps are as follows:

[0008] S1: Prednisone exposure treatment during pregnancy: Healthy pregnant rodents were selected as experimental subjects, preferably SPF-grade Wistar rats, SPF-grade SD rats, or Kunming mice; from day 0 of gestation to day 20 of gestation, the pregnant mice were administered prednisone daily via oral gavage at a dose of 0.1-1 mg / kg / day (preferred dose was 0.25 mg / kg / day); during the gavage period, the pregnant mice were allowed free access to food to ensure normal physiological metabolism.

[0009] S2: Sample collection and testing during pregnancy: On day 20 of gestation, a number of pregnant mice were randomly selected and euthanized. Maternal samples and samples of male fetuses in the womb were collected. Testicular development of the male fetuses was tested, and the transcriptome and metabolome changes of their testicular tissue were analyzed to provide early data support for subsequent mechanism studies.

[0010] S3: Offspring Rearing and Environmental Control: Remaining pregnant mice were allowed to give birth naturally to obtain F1 generation offspring, with the day of birth considered as day 0 of the offspring's life. To eliminate the impact of litter size differences on offspring growth and development, the number of pups in each litter was uniformly adjusted to 12, with female and male pups each accounting for 50%. Offspring were weaned at 4 weeks of age and then separated into different cages according to sex. During the rearing period, offspring were provided with a normal diet, the formula of which was consistent with the formula feed for mice and rats specified in the "National Standard of the People's Republic of China GB14924.3-2001". The offspring were continued to be reared until 12 weeks of age, at which point the male offspring reached adulthood and were used for subsequent fertility evaluation.

[0011] S4: Evaluation of fertility of adult male offspring:

[0012] The fertility of male adult offspring aged 12 weeks after birth was evaluated using multi-dimensional indicators, including:

[0013] Testicular-related indicators: testicular index (ratio of testicular weight to body weight), testicular morphology (observed through histopathological sections), and testicular function (with serum testosterone levels as the core evaluation indicator).

[0014] Sperm quality indicators: sperm count, sperm motility, sperm abnormality rate, sperm DNA fragmentation rate;

[0015] Reproductive outcome indicators: number of matings between male offspring and normal female animals, and the conception rate of female animals after mating (i.e., F1 generation pregnancy outcome).

[0016] Secondly, the present invention also provides applications of the above-described construction method.

[0017] The animal model of reduced fertility in male adult offspring due to prednisone exposure during pregnancy described in this invention can be used to screen intervention targets for reduced fertility in male offspring caused by drug exposure during pregnancy (specifically prednisone exposure). Specifically, the model simulates the damage to male offspring fertility caused by prednisone exposure during pregnancy. By detecting the serum metabolic profiles of model animals (including the pregnant mother, male fetus, and male adult offspring), differences in metabolite expression are analyzed to screen potential intervention targets associated with the phenotype of reduced male offspring fertility. This model accurately reflects the pathophysiological process of drug exposure during pregnancy; therefore, the intervention targets screened based on this model have high reliability and practicality.

[0018] Thirdly, the present invention also provides an intervention target for preventing and treating the reduction in male adult offspring fertility caused by PPE.

[0019] The intervention target for preventing decreased fertility in male adult offspring caused by prednisone exposure during pregnancy, as described in this invention, was screened using the aforementioned method of "animal model combined with serum metabolic profile analysis," specifically genistein. Experimental verification showed that after prednisone exposure during pregnancy, the concentration of genistein in the blood of both mothers and male offspring (including the fetal and adult stages) decreased significantly, and the concentration of genistein in the testicular tissue of male offspring also decreased synchronously, suggesting a direct correlation between changes in genistein concentration and decreased fertility in male offspring.

[0020] Thirdly, the present invention also provides an application of the above-mentioned intervention target.

[0021] The intervention target genistein described in this invention can be used to screen or prepare drugs for preventing and treating reduced fertility in adult male offspring caused by prednisone exposure during pregnancy. Further experiments have confirmed that exogenous supplementation with genistein (achieved through oral administration to pregnant mice) during prednisone exposure during pregnancy significantly improves abnormal testicular development in adult male offspring (such as restoration of testicular index and improved morphology), enhances sperm quality (increased sperm count, improved motility, and reduced abnormal sperm rate and DNA fragmentation), and increases conception rates after mating, ultimately effectively reversing the reduced fertility phenotype in male offspring caused by prednisone exposure during pregnancy.

[0022] The technical principles and research process of this invention are as follows: This invention constructs a model for reducing male offspring fertility caused by PPE, thus creating a model foundation for further exploring intervention targets and applications for PPE-induced reduction in male adult offspring fertility.

[0023] Simultaneously, this invention discovered and confirmed, based on transcriptomics, that PPE reduces the fertility of male adult offspring by inhibiting the Rxrα / Ncoa2 / Ep300 / Cmtm4 pathway. Metabolomics analysis revealed that PPE significantly decreased genistein levels in maternal and male fetal blood (especially in fetal blood), and this decrease was also verified in fetal testicular tissue. This suggests that genistein is a potential intervention target.

[0024] Finally, based on the intervention target explored in Example 2, in the animal model of PPE-induced decreased fertility in adult male offspring established by the method in Example 1, after administering exogenous supplementation of genistein to pregnant mice, the Rxrα / Ncoa2 / Ep300 / Cmtm4 pathway in the offspring was reversed, and fertility was restored. This indicates that genistein can reverse PPE-induced decreased fertility in adult male offspring and is an effective intervention target for PPE-induced decreased fertility in adult male offspring.

[0025] In summary, prednisone administration during pregnancy can successfully establish a model of reduced male adult offspring fertility, and genistein can serve as an effective intervention target to prevent PPE-induced reduced male adult offspring fertility.

[0026] Compared with the prior art, the present invention has the following advantages and effects: 1. The animal model constructed in this invention can stably induce a phenotype of reduced fertility in male adult offspring by precisely controlling the dose and time of prednisone exposure and the offspring's rearing conditions. Moreover, the model has good reproducibility and closely reflects clinical practice, providing a reliable experimental tool for studying the mechanism of the effect of prednisone exposure during pregnancy on the fertility of male offspring.

[0027] 2. The method for screening intervention targets based on this model is simple to operate and highly targeted, and can efficiently identify key molecules related to decreased fertility, solving the problem of difficulty in screening intervention targets in existing technologies; 3. The study identified genistein as the core intervention target, with a clear mechanism of action and definite intervention effect. This provides a clear target direction for the development of drugs to prevent and treat the reduction in male offspring fertility caused by prednisone exposure during pregnancy, and has important clinical translational value. Attached Figure Description

[0028] Figure 1 The development of testes in male rats at 20 days of gestation; Figure 1 In the middle: A, B: fetal mouse weight and testicular index; C, D: testicular morphology; E: testosterone level.

[0029] Figure 2 To evaluate the fertility of male rats at 12 weeks of age; Figure 2In the following categories: A: Testicular index; B: Testicular morphology; C: Testosterone level. DI: Sperm quality; JL: Pregnancy outcome; MP: Mitotic gene expression; QT: Expression levels of marker genes during meiosis and spermatogenesis.

[0030] Figure 3 The Rxrα / Ncoa2 / Ep300 / Cmtm4 pathway mediates reduced fertility in offspring rats; Figure 3 In Chinese: AE: RNA-seq results analysis; FK: testicular tissue at 20 days of gestation, testicular tissue at 12 weeks postnatal, and sperm at 12 weeks postnatal. Cmtm4 Expression status; L, M: Cmtm4 gene expression localization; N, O: Cmtm4 promoter region modification analysis and verification; PV: Cmtm4 epigenetic regulatory mechanism analysis and verification.

[0031] Figure 4 Changes in genistein content in maternal blood, male fetal blood, and fetal testicular tissue; Figure 4 In the middle: A, B: Partial least squares discriminant analysis; CE: Metabolomics interaction analysis; F: Liquid chromatography-mass spectrometry analysis and detection.

[0032] Figure 5 A schematic diagram of the method for supplementing the parent plant with genistein.

[0033] Figure 6 Genistein regulates the Rxrα / Ncoa2 / Ep300 / Cmtm4 pathway in spermatogonia; Figure 6 In the middle: AD: Expression of Rxrα / Ncoa2 / Ep300 / Cmtm4 under different concentrations of genistein; EH: Expression of Rxrα / Ncoa2 / Ep300 / Cmtm4 under ERα inhibitor to simulate a low physiological concentration of genistein environment, followed by administration of the optimal concentration of genistein.

[0034] Figure 7 The effect of maternal supplementation with genistein on the fertility of male offspring at 12 weeks postnatal; Figure 7 In the middle: AB: testicular index and size; CE: sperm quality test; F, G: testicular morphology; HK: pregnancy outcome analysis; L: expression of marker genes during meiosis; M: expression of marker genes during meiosis and spermatogenesis; NR: expression of the Rxrα / Ncoa2 / Ep300 / Cmtm4 pathway. Detailed Implementation

[0035] The following is a further detailed description of the above content of the present invention by means of specific implementation methods in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. Technologies implemented based on the above content of the present invention all belong to the content of the present invention.

[0036] Example 1: Construction of reduced fertility in male adult offspring caused by PPE in the present invention 1. Experimental animals SPF-grade healthy Wistar rats were purchased from the Hubei Center for Disease Control and Prevention, and the animal license number is: SYXK(E)2020-0018. This study was approved by the Institutional Animal Care and Use Committee (IACUC) of the Animal Experiment Center of Wuhan University (IACUC NO. WP20240376) and was strictly carried out in accordance with the relevant treatment guidelines of the international experimental animal protection certification and evaluation agency. 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.

[0037] 2. Experimental methods Twenty-five male Wistar rats (weighing 260-300 g) and sixty female Wistar rats (weighing 200-240 g). They were allowed to drink and eat freely. After 7 days of adaptive feeding, they were caged together at a male:female ratio of 1:2. The next morning, vaginal smears were taken, and rats with sperm found under the microscope were determined to be pregnant rats, which were recorded as day 0 of pregnancy.

[0038] The pregnant rats were randomly divided into two groups: a control group and a prednisone group, with 30 rats in each group. From day 0 to day 20 of pregnancy, the prednisone group was intragastrically perfused with prednisone (prednisone acetate tablets) at a dose of 0.25 mg / kg.d, and the control group was given the same volume of solvent (0.5% sodium carboxymethylcellulose). The administration volume was 1 mL / 100 g for both groups. The pregnant rats in each group were allowed to eat and drink normally. The feed was purchased from the Hubei Center for Disease Control and Prevention, and the license number is: SYXK(E)2020-0018. The feed formula was the same as the mouse and rat formula feed specified in the "National Standard of the People's Republic of China GB14924.3-2001".

[0039] To obtain fetal rat testicular tissue, some pregnant rats were randomly selected from the two groups and sacrificed by isoflurane anesthesia on day 20 of pregnancy. The pregnant rats were laparotomized to remove the fetal rats, and maternal blood and fetal blood were collected respectively. The bilateral testes of male fetal rats were separated for transcriptome sequencing, HE staining, and real-time quantitative PCR (RT-qPCR), etc.

[0040] The remaining female mice gave birth naturally, with each litter adjusted to 12 pups. The day of birth was designated as day 0 after birth. On day 1 after birth, litters of 12-14 pups were selected from each group, and each litter was adjusted to have 6 male and 6 female pups for nursing to ensure balanced nutrition. The pups were weaned at 4 weeks of age and separated into male and female cages. At 12 weeks of age, one female offspring was randomly selected from each pregnant mouse in each group, anesthetized and euthanized using the same method described above. Both testes and epididymis were collected for testicular tissue morphology, sperm quality, and RT-qPCR testing.

[0041] 3. Detection indicators and methods 3.1 HE staining 1) Transfer the sections into hematoxylin and immerse for 8-15 minutes; 2) Transfer the stained sections to water and rinse for about 1-2 minutes to remove excess dye; 3) After rinsing, transfer the sections to 1% hydrochloric acid-alcohol differentiation solution and react for about ten seconds; 4) The sections are then washed with running water for 0.5 to 1 hour (note that the water flow should not be too strong) to perform tissue blueing; 5) The tissue sections were then transferred to eosin solution and stained for 2-5 minutes; 6) After staining, transfer the tissue sections to water to wash away any residual eosin solution on the sections, and carefully wipe away any excess solution with a cotton ball; 7) First, dehydrate the tissue sections with 80% alcohol for 1-2 minutes; 8) Then, continue dehydrating the tissue sections with 90% alcohol for 2-4 minutes; 9) Continue to thoroughly dehydrate the tissue sections with 100% anhydrous ethanol for 4-8 minutes; 10) After dehydration, clear tissue sections with xylene I solution for 3-5 minutes; 11) Then continue clearing tissue sections with xylene II solution for 5-10 minutes; 12) After completing the above steps, remove the tissue section. Wipe away the xylene around the section, and place a drop of resin in the center of the tissue section. Then, quickly place a clean coverslip on the resin, slowly press the coverslip flat, and adjust its position to the center of the section. After the resin has cured, place the section in a constant temperature oven to dry, and then observe and photograph it under a microscope.

[0042] 3.2 Sperm quality testing 1) Prepare 12 / 24-well plates in advance, add 1 mL of PBS to each well, place them in a microplate shaker, and incubate at 37°C.

[0043] 2) Sacrifice the male rat, cut open the scrotal skin, free the testes, carefully separate the testes and epididymis, remove the epididymal adipose tissue, retain the epididymal tail and cut it open, immediately put it into PBS, soak it thoroughly and wait for 10 min.

[0044] 3) After 10 min, the PBS was observed to turn into a milky white turbid liquid. Immediately, 10 μL was aspirated and dropped onto a glass slide, covered with a coverslip, and the sperm count and sperm motility were recorded under a regular microscope.

[0045] 4) Use a red blood cell counting chamber (16×25 squares) to count sperm in a portion of the semen.

[0046] 5) Aspirate the remaining semen into a 1.5 mL EP tube, centrifuge at 5000 g for 5 min, discard the supernatant, add 1.5 mL of paraformaldehyde fixative, gently blow and mix thoroughly, then apply hematoxylin and eosin (HE) staining to calculate sperm abnormalities.

[0047] 3.3 Sperm DNA Fragmentation Rate 1) Thaw the acridine orange staining kit in advance and prepare a 1 / 1000 staining working solution.

[0048] 2) Take 500 μL of sperm fixed in paraformaldehyde for more than 24 h, centrifuge at 5000 g for 5 min, and discard the supernatant.

[0049] 3) Add 1 mL of PBS and gently wash once, centrifuge at 5000 g for min, and discard the supernatant.

[0050] 4) Add 100 μL of staining working solution, stain at 37 ℃ for 2 min, immediately place on ice, add 500~1000 μL of PBS and mix well. (Protect from light, and prepare one tube of unstained sample.) 5) Use flow cytometry for detection. Use SSC / FSC to circle a single cell population. Use excitation light of 488 nm (530 nm emission light, green) / excitation light of 530 nm (640 nm emission light, red) to detect fluorescence levels. Adjust the coordinate axis and excitation light gain so that the unstained sample is located as far as possible in the lower left corner (low fluorescence level), and set the quadrant accordingly.

[0051] 6) Test the samples sequentially. 3.4 Testosterone content detection Serum testosterone levels in male PPE rats were measured using an ELISA kit.

[0052] 1) Equilibrate all reagents (from a 4°C freezer) and serum samples (from a -80°C freezer) to room temperature for 1 hour beforehand; 2) Dilute the 20× concentrate labeled on the ELISA kit with distilled water to 1× for later use; 3) Remove the 96-well plate required for the test from the kit, set one blank well on each plate (without adding any sample or reagent), add 50 μL of each calibrator to the first two rows of the plate in sequence to plot two standard curves, and add 50 μL of the serum to be tested or quality control to each of the remaining test wells; 4) Then add 50 μL of enzyme-labeled antigen and enzyme-labeled antibody to each well (except for the blank control well), affix the sealing film, make the appropriate labels, and incubate in a 37°C constant temperature shaker for about 1 h; 5) After incubation, remove the sealing film, carefully pour out the liquid in the wells, add the above diluent to each well (each well must be filled), let stand for 10 seconds to wash the plate, repeat three times, and then spin dry. 6) After washing the plate, add 50 μL each of colorimetric solution A and colorimetric solution B to each well, shake the plate to mix it, and place it in a 37 ℃ oven in the dark (complete darkness required) for 10 min. Add 50 μL of reaction termination solution to each well. 7) Place the reaction plate into the microplate reader, measure the OD value of each well at 450 nm, plot the standard curve and calculate the corresponding sample concentration.

[0053] 3.5 Pregnancy outcome testing Pregnancy outcomes are comprised of fertility parameters including mating rate, fertility rate, litter size, fetal weight, and intrauterine growth retardation rate. It is important to note that any reported value represents the average of all fetal measurements from pregnant rats. Some results were calculated as follows: Mating rate (%) = Number of rats exhibiting mating behavior / Total number of rats × 100; Fertility rate (%) = Number of pregnant rats / Total number of rats × 100; UGR rate (%) = Number of fetuses with body weight less than two standard deviations below average body weight / Number of live births × 100.

[0054] 3.6 RT-qPCR detection Total RNA was extracted from testicular tissue using TRIzol reagent. 1 μg of total RNA was first incubated at 42°C for 2 min to remove genomic DNA according to the cDNA reverse transcription kit instructions. Then, 4 μl of HiScript III Enzyme Mix was added to form a 20 μl system, which was incubated at 37°C for 15 min followed by 85°C for 5 s to generate cDNA. PCR reaction was prepared according to the Taq Pro Universal SYBR qPCRMaster Mix instructions, and real-time PCR amplification was performed on a QuantStudio™ 5 System instrument. Primer sequences are shown in Table 1. The real-time PCR conditions were: 95°C / 30 s, 95°C / 10 s, 62°C / 30 s (40 cycles). Data analysis was performed using the formula Y=2. -△△CtAnalysis and processing. All cDNA sequences were obtained from the NCBI Entrez nucleotide database, and primers were designed using PrimerPremier 6.0 software. The primer sequences for rats are shown in Table 1.

[0055] Table 1. Primer sequences

[0056] 4. Experimental Results PPE can reduce the fertility of male offspring, specifically manifested as follows: compared with the control group, the PPE group showed significantly lower fetal testicular index at 20 days of gestation, significantly reduced number of seminiferous tubules, widened interstitium, and decreased testosterone synthesis. At 12 weeks after birth, PPE-treated offspring exhibited decreased testicular index, abnormal testicular morphology and function (thickness of seminiferous epithelium, reduced sperm count in seminiferous tubules, and decreased blood testosterone levels), arrested spermatogenesis (increased number of cells in mitosis and decreased number of cells in meiosis), reduced sperm quality (decreased sperm count, increased abnormality rate, increased DNA fragmentation rate, decreased number of matings with normal females, and reduced conception rate), and adverse pregnancy outcomes (reduced litter size, decreased fetal weight, and increased rate of intrauterine growth retardation).

[0057] 4.1 Testicular development in male offspring during intrauterine period Figure 1 The image shows testicular development in male offspring during intrauterine periods: compared to the control group, the fetal weight and testicular index of the PPE group were significantly lower. Figure 1 The results of HE analysis showed that, compared with the control group, the PPE group had abnormal testicular morphology and a significantly reduced number of seminiferous tubules. Figure 1 (C, D); Testosterone results showed that, compared with the control group, the serum testosterone concentration in the PPE group was decreased ( Figure 1 (E). This suggests that PPE can cause testicular development disorders in male offspring fetuses in utero.

[0058] 4.2 Fertility of male rats at 12 weeks postnatal age Figure 2 The following table shows the fertility of male rats at 12 weeks of age: Compared with the control group, the testicular index (i.e., testicular weight / body weight ratio) of male rats in the PPE group was significantly lower. Figure 2 (A); HE results showed that, compared with the control group, the PPE group had a decrease in testicular seminiferous epithelium thickness and sperm count in the seminiferous tubules ( Figure 2 (B); Testosterone results showed that, compared with the control group, the serum testosterone concentration in the PPE group was lower ( Figure 2 (C). Sperm quality results showed that, compared with the control group, the sperm count of male mice in the PPE group was reduced ( Figure 2In the middle D, the number of abnormal sperm such as those without a head or tail and the rate of sperm deformity increased ( Figure 2 The sperm DNA fragmentation rate was increased (20.62%) in the middle E and F sections. Figure 2 (G), while the cumulative number of matings and conception rate of male mice decreased (G). Figure 2 (H, I). Pregnancy outcomes showed that the F2 generation mice in the PPE group had fewer litters, lower birth weight, and a significantly higher rate of intrauterine growth retardation (IUGR). Figure 2 (JL). RT-qPCR results showed that, compared with the control group, the PPE group had higher levels of spermatogonial marker genes during mitosis. Plzf and Kit Increased expression level ( Figure 2 (MP), marker genes for meiosis and spermatogenesis. Stra8 , Sycp3 , Hspa2 and Spata16 The expression level was significantly reduced ( Figure 2 (QT). This indicates that male PPE offspring have reduced fertility after reaching adulthood.

[0059] Example 2: Using the model of this invention to explore intervention targets for PPE-induced decreased fertility in adult male offspring 1. Experimental Methods A susceptible animal model of reduced male adult offspring fertility induced by PPE was established according to the method in Example 1. Fetal testes were obtained at 20 days of gestation, 12 weeks after birth, and sperm were used for RNA-seq, RT-qPCR, IHC, and Western blot analysis. Maternal blood, fetal blood, and testicular tissue samples from 20-day-old fetuses were obtained for metabolite concentration detection.

[0060] 2. Detection Indicators and Methods 2.1 RNA-seq of testicular tissue The transcriptome sequencing and analysis of fetal testis tissue were completed with the assistance of Beijing Novogene Co., Ltd. A brief summary of the experimental steps is as follows: 1) After extracting testicular RNA, the integrity, purity and total amount of RNA were first detected using an Agilent 2100 bioanalyzer.

[0061] 2) Library Construction and Quality Inspection A cDNA library was constructed, and the obtained library was initially quantified using a Qubit 2.0 Fluorometer. The library concentration was then diluted to 1.5 ng / μL. The insert size of the diluted library was detected using an Agilent 2100 bioanalyzer. Once the target was met, the effective concentration of the library was accurately determined using RT-qPCR. To ensure quality, the effective concentration of the library should be higher than 2 nM.

[0062] 3) Sequencing After completing the cDNA library quality control, the library was further pooled according to the effective concentration and target data volume accurately determined in the previous step, and then sequenced using Illumina. Illumina sequencing operates on the principle of sequencing while synthesis, capturing the released fluorescence signal to obtain sequencing peaks and thus acquiring the sequence information of the detected fragment.

[0063] 4) Data quality control CASAVA was used to convert high-throughput sequencer image data into FASTQ format reads, which included sequence information and sequencing quality information. The raw data was filtered to obtain clean data, including removing reads with adapters, reads containing the letter N (N indicates that base sequence information cannot be determined), and low-quality reads (reads with Qphred <= 20 accounting for more than 50% of the total read length). After obtaining clean data, Q20, Q30, and GC content were calculated. Subsequent sequencing data analysis in this section is based on the obtained clean data.

[0064] 5) Sequence alignment to the reference genome An index of the reference genome was constructed using HISAT2 v2.0.5, and paired-end clean reads were aligned with the reference genome.

[0065] 6) Quantitative analysis of gene expression levels FPKM can be used to compare the relative expression levels of different genes in different samples, taking into account gene length and the total number of mapped reads. The feature counts software (1.5.0-p3) was used to calculate the reads mapped to each gene, and the FPKM of each gene was calculated based on the gene length.

[0066] 7) Differential expression analysis Differential expression analysis was performed using DESeq2 software (v1.20.0). To control for the false detection rate, the calculated values ​​were adjusted using the Benjamini and Hochberg methods. P Value. Genes with an adjusted p < 0.05 were considered to be differentially expressed genes.

[0067] 2.2 RT-qPCR detection Detection of the Rxrα / Ncoa2 / Ep300 / Cmtm4 pathway in fetal testes at 20 days of gestation, in testicular tissue, and in sperm at 12 weeks after birth Cmtm4 The expression and primer sequences are shown in Table 1 of Example 1.

[0068] 2.3 Western blot detection The expression of Cmtm4 in the Rxrα / Ncoa2 / Ep300 / Cmtm4 pathway in the fetal testes at 20 days of gestation, in testicular tissue, and in sperm at 12 weeks after birth was detected.

[0069] 1) Clean the 1.5 mm glass plate with distilled water, dry it with a hair dryer after cleaning, and add distilled water to check for leaks after assembly; 2) Prepare the separating gel and stacking gel according to the gel kit instructions, and insert a 1.5 mm 15-well comb. After the stacking gel solidifies, slowly pull the comb out vertically upwards (be careful not to damage the gel); 3) Sample loading: Remove the solidified gel from the gel holder and install it into the electrophoresis tank. Add the pre-prepared electrophoresis buffer, avoiding air bubbles in the wells. Then, slowly add the processed protein sample into the comb wells (trying not to leak out of the wells), and simultaneously set up the protein marker wells; 4) Electrophoresis: Set the electrophoresis conditions to a constant voltage of 60 V. After the sample moves from the stacking gel to the separating gel, adjust the voltage to 120 V and continue electrophoresis. 5) Transfer: Cut a PVDF membrane to a suitable size and activate it in methanol for 2 min. Stack the gel and membrane in sequence into a "sandwich" shape in the clamping plate, place it in the electrotransfer bath, and electrotransfer at a low temperature and constant current of 300 mA for 40 min; 6) After the PVDF membrane has been electroporated, cut it and place it in a rapid sealing solution, then place it on a shaker and seal it at room temperature for 20 minutes. 7) Dilute different primary antibodies according to the instructions for each antibody using the primary antibody dilution buffer. Completely immerse the cut PVDF membrane in the primary antibody, ensuring that the primary antibody completely covers the PVDF membrane, and incubate overnight at 4°C on a shaker. 8) Remove the PVDF membrane and place it in TBST solution, ensuring the membrane is completely submerged. Wash it three times on a shaker for 5 minutes each time. 9) Dilute the secondary antibody with 5% skim milk according to the instructions, and place the PVDF membrane in the secondary antibody at room temperature for 2-3 hours. 10) Repeat step 8 to wash the membrane; 11) Mix solution A and solution B from the ECL chemiluminescence kit in equal proportions to prepare a working solution. Then add the solution to the PVDF membrane and completely cover the membrane. After reacting for several minutes, use a chemiluminescence imaging system to acquire and save the image.

[0070] 2.4 IHC detection Prepare paraffin sections of testicular tissue according to the following steps: 1) Place the tissue sections into freshly prepared dewaxing solution in three separate applications, each for 10 minutes. After completion, soak the sections in anhydrous ethanol I, II, and III for 5 minutes each, and then wash them with distilled water. 2) Place the testicular tissue sections into a repair container containing EDTA antigen repair solution and place the container in a microwave oven. The specific repair procedure is as follows: heat on medium for 8 minutes, then stop and let stand for 8 minutes, followed by heat on medium-low for 7 minutes. Care should be taken to monitor the amount of buffer solution in the repair container, ensuring it does not evaporate too quickly to avoid drying out the tissue sections. After removing the sections from the microwave oven, allow them to equilibrate to room temperature and place them in a washing container containing PBS. Wash three times, adjusting the shaker speed, for 5 minutes each time. Then treat with 3% hydrogen peroxide. 3) After slightly drying the tissue sections, use a marker to draw circles around the testicular tissue, being careful not to touch the tissue to prevent the antibodies from flowing away in subsequent steps; 4) Add BSA to the tissue section and react for 0.5 h to block the reaction; 5) Remove the BSA blocking solution from the slide, then drop the pre-prepared primary antibody onto the tissue section to ensure complete coverage of the entire testicular tissue. Place the slide flat in a humidified chamber and incubate overnight at 4°C. 6) Subsequently, place the tissue sections in a washing box, add PBS to the washing box, and rinse three times on a shaker for 5 minutes each time. After rinsing, spin-dry the sections, add secondary antibody of the same species as the primary antibody into the indentation, completely cover the tissue, and incubate at room temperature for about 1 hour; 7) Wash 3 times with PBS, 5 min each time, and develop with DAB for 5-10 min. Monitor the degree of staining under a microscope. 8) Rinse with PBS or tap water for 10 min, counterstain with hematoxylin for 2 min, and differentiate with hydrochloric acid alcohol; 9) Rinse with tap water for 10-15 minutes. Dehydrate, clear, mount, and examine under a microscope.

[0071] 2.5 Non-target metabolic profiling detection Changes in non-target metabolic profiles in maternal blood and male fetal blood serum were detected, and the concentration changes of genistein in fetal testicular tissue were verified: 100 mg of tissue sample was ground in liquid nitrogen, and 500 μl of 80% methanol aqueous solution was added. After shaking and mixing, the sample was incubated on ice for 5 min, and then centrifuged at 15000 g and 4℃ for 20 min. The supernatant was collected, diluted with mass spectrometry grade water to a methanol concentration of 53%, and centrifuged again to collect the supernatant for LC-MS analysis.

[0072] 3. Experimental Results 3.1 The Rxrα / Ncoa2 / Ep300 / Cmtm4 pathway mediates decreased fertility in offspring rats Figure 3 This invention first investigates the mechanism by which PPE reduces the fertility of male offspring. RNA-seq results showed that, compared with the control group, the transcriptional levels of multiple genes in the testicular tissue of the PPE group were altered (…). Figure 3 (A, B) The PPE group showed significant changes in the mRNA levels of 13 common genes before and after birth, among which... Cmtm4 The most obvious change ( Figure 3 (CE); RT-qPCR results showed that compared with the control group, the PPE group had significantly higher levels of cervical mucus in testicular tissue at 20 days of gestation, and in testes and sperm at 12 weeks after birth. Cmtm4 mRNA levels were significantly reduced ( Figure 3 Western blot results showed that, compared with the control group, the levels of CMTM4 protein in testicular tissue at 20 days of gestation, testes at 12 weeks after birth, and sperm were significantly lower in the PPE group. Figure 3 IHC results showed that, compared with the control group, the PPE group had significantly lower levels of CMTM4 protein in testicular tissue at 20 days of gestation and 12 weeks after birth. Figure 3 (L, M). The possible epigenetic modifications and sites of Cmtm4 were analyzed using the CistromeDB website. The results suggest that the most likely modifications and sites in the Cmtm4 promoter region include H3K4me2, H3K27me3, and H3K27ac, etc. Figure 3 This invention uses ChIP-qPCR to detect the H3K27 level in the Cmtm4 promoter region of fetal testes, confirming that the H3K27ac level in the Cmtm4 promoter region of PPE fetal testes is significantly reduced (N). Figure 3 (O). Transcription factors that may regulate Cmtm4 expression in the fetal testis were predicted and cross-analyzed with fetal testis transcriptome sequencing data. Results indicated that Rxrα and Ep300 showed the most significant differential changes ( Figure 3 (P,Q). The study found that Rxrα, Ncoa2, and Ep300 interact and regulate Cmtm4 expression. The expression and nuclear translocation of Rxrα, Ncoa2, and Ep300 were detected, confirming that the expression of Rxrα, Ncoa2, and Ep300 in the fetal testes of the PPE group was decreased, and their nuclear translocation was reduced. Figure 3 (RV). This suggests that PPE can reduce the expression and interaction of Rxrα, Ncoa2, and Ep300 in the testes, thereby inhibiting the H3K27ac promoter region of Cmtm4, ultimately leading to reduced fertility in male offspring of PPE.

[0073] 3.2 Changes in genistein content in maternal blood, male fetal blood, and fetal testicular tissue Figure 4The figure shows the changes in genistein content in maternal blood, male fetal blood, and fetal testicular tissue. Non-targeted metabolic profiling results showed that, compared with the control group, the metabolic profiles of maternal blood and male fetal blood changed at day 20 of gestation in the PPE group. Specifically, genistein levels decreased in both maternal blood and fetal blood in the PPE group, with the most significant decrease observed in fetal blood. Figure 4 (Middle AE). Meanwhile, this invention used liquid chromatography-mass spectrometry to detect the concentration of genistein in fetal testicular tissue, and found that the concentration of genistein in fetal testicular tissue decreased in the PPE group ( Figure 4 (Middle F). The results showed that, compared with the control group, the metabolic profiles of maternal blood and male fetal blood serum in the PPE group were significantly altered, especially the content of genistein in maternal blood, female fetal rats, and fetal testis tissues was decreased. This suggests that genistein may be a potential intervention target for PPE-induced reduction in male adult offspring fertility.

[0074] Example 3: Application of genistein in PPE-induced decreased fertility of adult male offspring 1. Experimental Methods Based on the intervention target explored in Example 2, this invention treats mouse spermatogonial cell line (GC-1) in vitro culture system with different physiological concentrations of genistein or first administers ERα inhibitors to simulate a low physiological concentration of genistein environment, followed by the administration of the optimal concentration of genistein for signal pathway detection. Simultaneously, in establishing the method of prednisone exposure-induced male offspring fertility reduction in Example 1, pregnant rats were randomly divided into three groups: a control group, a prednisone group, and a prednisone + genistein supplementation group. The control group received the same volume of solvent (0.5% sodium carboxymethyl cellulose); the prednisone group received prednisone (prednisone acetate tablets) 0.25 mg / kg / day via intragastric gavage; the prednisone + genistein supplementation group received 0.25 mg / kg / day prednisone + 30 mg / kg / day genistein via intragastric gavage, with a dosage volume of 1 mL / 100g (e.g., Figure 5 (As shown in the image). Testicular samples were obtained at 20 days of gestation, 12 weeks after birth, and sperm samples for fertility testing.

[0075] 2. Detection Indicators and Methods The relevant tests were performed according to the methods in Examples 1 and 2.

[0076] 3. Experimental Results 3.1 Gentiana flavonoids regulate the Rxrα / Ncoa2 / Ep300 / Cmtm4 pathway in spermatogonia. Figure 6Gentiana flavonoids regulate the Rxrα / Ncoa2 / Ep300 / Cmtm4 pathway in spermatogonia. Spermatogonia cell line GC-1 was treated with physiological concentrations (25, 50, and 100 nM), and cells were harvested after 72 h for analysis. Results showed that genistein concentration-dependently increased the mRNA expression of Cmtm4 and its regulated genes (Rxrα, Ncoa2, and Ep300). Figure 6 (AD). Further, the ERα inhibitor Fulvestant (500 nM) was administered to simulate a low physiological concentration of genistein, followed by the optimal concentration (100 nM) of genistein. The results showed that a low physiological concentration of genistein could inhibit the expression of Rxrα, Ncoa2, Ep300, and Cmtm4, while supplementation with the optimal concentration of genistein could reverse the decrease in the expression of these genes.

[0077] 3.2 Protective effect of maternal supplementation with genistein during pregnancy against reduced fertility in male offspring of PPE (perpetual genital fibrosis). Figure 7 The image shows the protective effect of maternal supplementation with genistein during pregnancy against reduced fertility in male offspring of women who underwent PPE treatment. Compared with the PPE control group, the PPE + genistein group showed a significant recovery in testicular index and size. Figure 7 (A, B); Sperm quality results showed that, compared with the PPE group, the PPE + genistein group had increased sperm count in male mice at 12 weeks after birth ( Figure 7 In the middle C), the number of abnormal sperm such as those without a head or tail and the rate of sperm deformity decreased ( Figure 7 (D) Decreased sperm DNA fragmentation rate ( Figure 7 The results of HE analysis showed that, compared with the PPE group, the number of seminiferous tubules increased in the PPE + genistein group at day 20 of gestation. Figure 7 In the middle F group, at 12 weeks after birth, the number of sperm in the seminiferous tubules of the testes increased in the PPE + genistein group. Figure 7 (G); Pregnancy outcomes showed that the pregnancy rate was increased in the PPE + genistein group compared to the PPE group (G). Figure 7 In the middle H generation, the litter size of F2 generation mice increased, birth weight increased, and the rate of intrauterine growth retardation (IUGR) was significantly reduced. Figure 7 (IK); RT-qPCR results showed that, compared with the PPE group, the PPE + genistein group had lower levels of marker genes for meiosis and spermatogenesis at 12 weeks after birth. Stra8 , Sycp3 , Hspa2 and Spata16 Isoexpression level recovery ( Figure 7 In the middle L, M), and the Rxrα / Ncoa2 / Ep300 / Cmtm4 pathway is reversed ( Figure 7(NR). This indicates that genistein can reverse the PPE-induced decrease in male adult offspring fertility and is an intervention target for PPE-induced decrease in male adult offspring fertility.

[0078] In summary, the modeling method of this invention, through prednisone treatment during pregnancy, revealed testicular developmental disorders and reduced fertility in male offspring at 12 weeks postnatally. RNA-seq analysis confirmed that the testicular Rxrα / Ncoa2 / Ep300 / Cmtm4 pathway mediates the reduced fertility in PPE-induced male offspring. Metabolic profiling analysis confirmed a decrease in genistein content in maternal blood, male fetal blood, and testicular tissue. Furthermore, maternal administration of genistein at the whole-body level reversed the testicular Rxrα / Ncoa2 / Ep300 / Cmtm4 pathway in the PPE group, and improved offspring fertility. This demonstrates that this method is an effective approach for establishing a model of PPE-induced reduced fertility in adult male offspring, and can be used to explore intervention targets for PPE-induced reduced fertility in adult male offspring, guiding rational clinical drug use during pregnancy and early prevention and treatment of PPE-induced reduced fertility in adult male offspring.

Claims

1. A method for constructing an animal model of reduced fertility in adult male offspring due to prednisone exposure during pregnancy, characterized in that: Includes the following steps: S1: Select healthy pregnant rodents and administer 0.1-1 mg / kg / day of prednisone orally from day 0 to day 20 of gestation. The pregnant rodents have free access to food. S2: Collect maternal and male fetal mouse transcripts from pregnant mice at 20 days of gestation to detect testicular development and changes in transcriptome and metabolome. S3: The remaining pregnant mice give birth naturally to obtain the F1 generation. The birth date is taken as day 0 after birth. The number of pups in each litter is adjusted to 8-14, with half being female and half being male. The pups are weaned 3-4 weeks after birth and separated into male and female cages. They continue to be fed normally for 8-9 weeks until adulthood. S4: The fertility of adult male offspring is evaluated by measuring testicular index, testicular morphology and function, sperm quality, and F1 pregnancy outcomes.

2. The construction method according to claim 1, characterized in that: Includes the following steps: In step S1: the dose of prednisone administered orally by gavage is 0.25 mg / kg / day; In step S4: testicular morphology and function include structure and blood testosterone levels; sperm quality includes sperm count, motility, abnormality rate, DNA fragmentation rate, number of matings and conception rate; F1 pregnancy outcomes include litter size, fetal weight and IUGR rate.

3. The construction method according to claim 2, characterized in that: The rodents are SPF-grade Wistar, SD rats, or Kunming mice.

4. The construction method according to claim 3, characterized in that: The normal diet in step S3 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.

5. The construction method according to claim 4, characterized in that: The fertility testing indicators in step S4 are: testicular index, testicular morphology and function, sperm quality, and F1 pregnancy outcome.

6. The application of the construction method as described in any one of claims 1 to 4 in screening intervention targets for reduced fertility in male offspring caused by drug exposure during pregnancy.

7. The application according to claim 6, characterized in that: Screening for potential intervention targets using serum metabolic profiles.

8. An intervention target for preventing PPE-induced decreased fertility in adult male offspring, characterized in that: The intervention target was obtained through the application screening of claim 7.

9. Genistein as an intervention target for preventing PPE-induced decreased fertility in adult male offspring.

10. The application of the intervention target as described in claim 9 in screening / preparing drugs for preventing and treating PPE-induced decreased fertility in adult male offspring, characterized in that: The concentration of the target genistein in the blood and testicular tissue is reduced, and exogenous supplementation with genistein during pregnancy can effectively prevent the reduction in male adult offspring fertility caused by prednisone exposure during pregnancy.

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