CN118593516A - A method for constructing an animal model of fetal HPT axis functional disorder and its application - Google Patents

Abstract

The present invention provides a method for constructing an animal model of HPT axis functional disorder and its application, belonging to the field of biotechnology, comprising: dividing pregnant rats into a PDE group and a control group, in late pregnancy, the pregnant rats in the PDE group are subcutaneously injected with dexamethasone daily, and the pregnant rats in the control group are subcutaneously injected with saline daily; selecting part of the pregnant rats from the PDE group and the control group respectively, removing the fetuses by caesarean section in late pregnancy, and detecting the testicular morphology, endocrine and HPT axis related gene expression levels of the male fetuses; another part of the pregnant rats give birth naturally, and the male offspring born naturally after adulthood are killed, and the testicular morphology, endocrine and HPT axis related gene expression levels are detected. The method is an effective method for establishing an animal model of HPT axis functional disorder, providing an animal model that can be used for research for the prevention and treatment of male reproductive endocrine system diseases, and can be used to assist in screening drugs for preventing or treating male reproductive endocrine system diseases.

Description

A method for constructing an animal model of fetal HPT axis functional activity disorder and its application

Technical Field

The invention belongs to the field of biotechnology, and particularly relates to a method for constructing an animal model of fetal HPT axis functional activity disorder and an application thereof.

Background Art

The 2009 "China Infertility Status Survey Report" shows that in the past decade, the infertility rate of my country's childbearing population has increased from 3% to 5% to 10% to 15%, and is showing a trend of getting younger, with the largest number of people aged 25 to 30. In addition to the causes that can be clearly diagnosed, infertility is regulated by multiple genes and environmental factors, and its specific pathogenesis is very complex. To date, most of the causes are still not completely clear, and there is a lack of stable and effective animal research models for the prevention and treatment of infertility. Therefore, it has become a scientific problem that urgently needs to be solved in the clinic.

The hypothalamic-pituitary-testicular (HPT) axis is the regulatory center of the reproductive endocrine system in male individuals. Gonadotropin-releasing hormone (GnRH) is the central regulator of the reproductive endocrine system. GnRH is released by hypothalamic GnRH neurons in a pulsed manner and reaches the anterior pituitary gland to regulate the synthesis and secretion of gonadotropins including luteinizing hormone (LH) and follicle-stimulating hormone (FSH). LH can promote the synthesis and secretion of androgens (mainly testosterone) by interstitial cells of the testis, and FSH has the effect of enhancing LH to stimulate testosterone secretion. The two have a synergistic effect, further promoting spermatogenesis and the development of male reproductive organs, as well as maintaining secondary sexual characteristics and sexual function. Disorders in the activity of the HPT axis may lead to dysfunction of the reproductive endocrine system, such as abnormal testosterone levels, decreased scrotal circumference after puberty, decreased number of germ cells, decreased sperm concentration, and increased abnormal sperm.

Summary of the invention

In order to solve the problem of the lack of animal models for HPT axis functional activity disorder, the present invention provides a method for constructing an animal model of fetal HPT axis functional activity disorder, which is an effective method for establishing an animal model of HPT axis functional activity disorder, provides an animal model that can be used for research for the prevention and treatment of male reproductive endocrine system diseases, and can be used to assist in the screening of drugs for preventing or treating male reproductive endocrine system diseases.

The present invention also provides a method for constructing an animal model of fetal HPT axis functional activity disorder and its application in assisting the screening of drugs for preventing or treating male reproductive endocrine system diseases.

The present invention is achieved through the following technical solutions:

The present invention provides a method for constructing an animal model of fetal HPT axis functional activity disorder, the method comprising:

The pregnant mice were divided into PDE group and control group. In the third trimester, the pregnant mice in PDE group were subcutaneously injected with dexamethasone twice a day, and the pregnant mice in control group were subcutaneously injected with an equal amount of normal saline twice a day.

After the subcutaneous injection, some pregnant mice were selected from the PDE group and the control group, and the fetuses were removed by caesarean section to detect the testicular morphology, endocrine and HPT axis-related gene expression levels of the male fetuses.

Another part of the pregnant female mice in the PDE group and the control group gave birth naturally, and the adult male offspring were killed. The testicular morphology, endocrine system and HPT axis-related gene expression levels of the adult male offspring were detected.

Furthermore, the pregnant mice were divided into a PDE group and a control group. In the late pregnancy, the pregnant mice in the PDE group were subcutaneously injected with dexamethasone twice a day, and the pregnant mice in the control group were subcutaneously injected with an equal amount of normal saline twice a day, specifically including:

The pregnant mice were divided into PDE group and control group. On the 20th to 21st day after conception, the pregnant mice in the PDE group were subcutaneously injected with dexamethasone twice a day, with each injection of dexamethasone being 0.4 mg/kg of the pregnant mouse body weight. The pregnant mice in the control group were subcutaneously injected with an equal amount of normal saline twice a day.

Furthermore, after the subcutaneous injection, some pregnant female mice were selected from the PDE group and the control group, and the fetuses were removed by caesarean section to detect the testicular morphology, endocrine and HPT axis-related gene expression levels of the male fetuses, including:

After the subcutaneous injection, some pregnant female mice were randomly selected from the PDE group and the control group, and the fetuses were removed by cesarean section on the 22nd day after conception. The testicular morphology, endocrine and HPT axis-related gene expression levels of the male fetuses were detected.

The detection of testicular morphology, endocrine and HPT axis-related gene expression levels of male fetal mice specifically includes:

HE staining and TEM analysis were performed on the testes of male fetal mice to observe the testicular morphology;

The expression levels of steroidogenic enzymes StAR, P450scc, 3β-HSD, 17α-HSD1, and 17β-HSD3 in the testis of male fetal mice were detected;

The expression levels of GnRH1, GAD1, Egr-1, GR, DNMT1 and DNMT3a in the hypothalamus of male fetal mice were detected, the methylation level of the GAD1 promoter region was detected, and the activities of Glu neurons and GABA neurons were detected.

Furthermore, another part of the pregnant female mice in the PDE group and the control group gave birth naturally, and the adult male offspring mice were killed to detect the testicular morphology, endocrine and HPT axis related gene expression levels of the adult male offspring mice, specifically including:

Another part of pregnant female mice in the PDE group and the control group gave birth naturally, and the male offspring mice were killed by anesthesia on the 85th day after natural birth. The testicular morphology, endocrine and HPT axis-related gene expression levels of the adult male offspring mice were detected;

Among them, the testicular morphology, endocrine and HPT axis-related gene expression levels of adult male offspring were detected, including:

The testicles of adult male offspring were stained with HE and weighed to observe the testicular morphology;

The serum testosterone concentration, testicular testosterone concentration, serum luteinizing hormone concentration, and serum follicle-stimulating hormone concentration of adult male offspring were tested;

The expression levels of testicular steroidogenic enzymes StAR, P450scc, 3β-HSD, 17α-HSD 1, and 17β-HSD3 in adult male offspring rats were detected;

The expression levels of GnRH1 and GAD1 in the hypothalamus of adult male offspring mice were detected, the methylation level of the GAD1 promoter region was detected, and the activities of Glu neurons and GABA neurons were detected.

Furthermore, another part of the pregnant female mice in the PDE group and the control group gave birth naturally, and the adult male offspring mice were killed to detect the testicular morphology, endocrine and HPT axis related gene expression levels of the adult male offspring mice, specifically including:

Another part of pregnant female mice in the PDE group and the control group gave birth naturally, and the male offspring mice were killed by anesthesia on the 85th day after natural birth. The testicular morphology, endocrine and HPT axis-related gene expression levels of the adult male offspring mice were detected;

Among them, the testicular morphology, endocrine and HPT axis-related gene expression levels of adult male offspring were detected, including:

The testes of adult male offspring were stained with HE, analyzed by TEM and weighed to observe the testicular morphology;

The serum testosterone concentration, testicular testosterone concentration, serum luteinizing hormone concentration, and serum follicle-stimulating hormone concentration of adult male offspring were tested;

The expression levels of testicular steroidogenic enzymes StAR, P450scc, 3β-HSD, 17α-HSD 1, and 17β-HSD3 in adult male offspring rats were detected;

The expression levels of GnRH1, GAD1, Egr-1, GR, DNMT1 and DNMT3a in the hypothalamus of adult male offspring mice were detected, the methylation level of the GAD1 promoter region was detected, and the activities of Glu neurons and GABA neurons were detected.

Furthermore, another part of the pregnant female mice in the PDE group and the control group gave birth naturally, and the male offspring born naturally after adulthood were killed, specifically including:

Another part of pregnant female mice in the PDE group and the control group gave birth naturally, and the male offspring were killed by anesthesia on the 85th day after natural birth;

Before the offspring male mice were anesthetized and killed, a part of the naturally born male mice were randomly selected as the drug-treated group. The male mice in the drug-treated group were injected with the test drug on the 75th and 80th days after birth, and the remaining male mice were injected with an equal volume of HifectinⅡ.

Optionally, the drug to be tested includes GAD1 siRNA.

Optionally, the nucleotide sequence of the sense strand of the GAD1 siRNA is shown as SEQ ID NO.1, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO.2.

Based on the same inventive concept, the present invention provides a method for constructing an animal model of fetal HPT axis functional activity disorder and its application in assisting the screening of drugs for preventing or treating male reproductive endocrine system diseases.

Furthermore, the male reproductive endocrine system disease includes HPT axis functional activity disorder and/or testicular dysplasia.

Furthermore, the male reproductive endocrine system diseases include male reproductive endocrine system diseases in offspring caused by exposure to dexamethasone during pregnancy.

One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

The present invention discloses a method for constructing an animal model of fetal HPT axis functional activity disorder. The method is an effective method for establishing an animal model of fetal HPT axis functional activity disorder, and provides an animal model that can be used for research for the prevention and treatment of offspring male reproductive endocrine system diseases caused by prenatal dexamethasone exposure (PDE). Through the model constructed by the present invention, the intrauterine programming mechanism of offspring rat HPT axis functional activity disorder caused by PDE is explored, and it is found that PDE activates the hypothalamic GR, causes hypomethylation and high expression changes in the GAD1 promoter region, promotes the increase in the conversion of Glu to GABA, and further induces high expression of GnRH1. These programmed changes can continue after birth, and finally mediate offspring HPT axis functional activity disorder. The model can be used to assist in screening drugs for preventing or treating male reproductive endocrine system diseases caused by PDE, thereby reducing the infertility rate.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

Figure 1 is a schematic diagram of the animal handling procedure.

Figure 2 shows the effects of dexamethasone exposure during pregnancy on testicular morphology and spermatogenesis in adult male offspring rats 85 days after birth: (A) testicular weight; (B-D) testicular morphology analysis (H&E staining, 100×): thickness of seminiferous epithelium and diameter of seminiferous tubules. The inter-group comparisons were performed using the t-test, mean ± SD, *P < 0.05, **P < 0.01.

Figure 3 shows the effects of gestational dexamethasone exposure on serum and testicular testosterone levels and testicular steroidogenic enzyme expression in adult male offspring rats 85 days after birth: (A) qRT-PCR detection of testicular steroidogenic enzyme expression; (B) Immunohistochemistry detection of testicular StAR expression; (C, D) Iodine [125i] testosterone radioimmunoassay kit to measure serum and testicular testosterone levels; (E, F) Blood LH and FSH levels, *P<0.05, **P<0.01.

Figure 4 shows the effect of gestational dexamethasone exposure on the expression of GnRH1 and GAD1 in the hypothalamus of adult male offspring rats 85 days after birth: (A) qRT-PCR was used to detect the expression of GnRH1 and GAD1 in the hypothalamus; (B) BSP method was used to detect the methylation level of the GAD1 promoter region; (C-D) Elisa method was used to detect the tissue content of hypothalamic neurotransmitters Glutamate and GABA; (E-F) Immunohistochemistry was used to detect the expression of GnRH1 and GAD1 in the hypothalamus; (G-H) Immunofluorescence method was used to detect the activity of Glu neurons (Glutamate) and GABA neurons (GAD1) projecting to GnRH neurons in the arcuate nucleus (ARN) of the hypothalamus, *P<0.05, **P<0.01.

Figure 5 shows the effects of dexamethasone exposure during pregnancy on testicular morphology and testicular steroidogenic enzyme expression in male fetal mice at 22 days of gestation: (A) Morphological analysis of testicular tissue by H&E staining; (B) qRT-PCR detection of testicular steroidogenic enzyme expression; (C) Immunohistochemistry detection of StAR expression, *P<0.05, **P<0.01.

Figure 6 shows the effect of dexamethasone exposure during pregnancy on the expression of hypothalamic-related genes in male fetal mice at 22 days of gestation: (A) qRT-PCR was used to detect the expression of hypothalamic GnRH1, GAD1 and DNMT3a; (B) BSP method was used to detect the methylation level of GAD1 promoter region; (C) Histocytological changes of hypothalamic neurons were observed under electron microscopy; (D-E) Immunohistochemistry was used to detect the expression of GnRH1 and GAD1; (F-G) Immunofluorescence was used to detect the activity of Glu neurons (Glutamate) and GABA neurons (GAD1) projecting to GnRH neurons in the arcuate nucleus (ARN) of the hypothalamus; *P<0.05, **P<0.01.

Figure 7 shows the effect of dexamethasone on the expression of genes related to hypothalamic cells and the activity of Glu/GABA neurons in primary male fetal mice: (A-J) qRT-PCR detection of the expression of hypothalamic GnRH1, GAD1, Egr-1, DNMT1, and DNMT3a; (K-L) Immunofluorescence detection of the activity of hypothalamic Glu neurons (Glutamate) and GABA neurons (GAD1), *P<0.05, **P<0.01.

Figure 8 shows the mechanism of the animal model of hypothalamic-pituitary-testis axis functional activity disorder induced by dexamethasone exposure during pregnancy mediated by high expression programming of hypothalamic GAD1.

DETAILED DESCRIPTION

The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

The overall idea of the present invention is as follows:

Dexamethasone (DXMS, chemical formula: C ₂₂ H ₂₉ FO ₅ ) was first synthesized in 1957 and is listed in the World Health Organization's Standard List of Essential Medicines. It is one of the essential drugs for the basic public health system. Like other glucocorticoids, dexamethasone has pharmacological effects such as anti-inflammatory, anti-endotoxin, immunosuppressive, anti-shock and enhanced stress response, so it is widely used in various departments to treat a variety of diseases. Studies have reported that children who received dexamethasone treatment before birth have a higher incidence of intrauterine growth retardation (IUGR). Epidemiological surveys, clinical studies and animal experiments have confirmed that offspring with intrauterine growth retardation (IUGR) have an increased susceptibility to reproductive endocrine system diseases in adulthood, such as HPT axis functional activity disorders, testicular dysplasia and reproductive disorders.

The inventors believe that the correlation between IUGR and reproductive endocrine system diseases in adulthood is related to the "programming" hypothesis, that is, adverse factors during pregnancy will cause permanent changes in the structure and function of fetal tissues, causing dysfunction and diseases of tissues and organs in adulthood. Therefore, the inventors speculate that the main mechanism for the increased susceptibility of IUGR offspring to reproductive endocrine system diseases in adulthood may be related to the functional activity of the hypothalamus-pituitary-testis (HPT) axis and the development and differentiation of the high-level regulatory center hypothalamic gonadotropin-releasing hormone (GnRH) neurons and the intrauterine programming changes of its key regulatory point, L-glutamate decarboxylase 1 (GAD1).

Based on this, the present invention provides a method for constructing an animal model of fetal HPT axis functional activity disorder, which is an effective method for establishing an animal model of fetal HPT axis functional activity disorder, and provides an animal model that can be used for research for the prevention and treatment of male reproductive endocrine system diseases in offspring caused by prenatal dexamethasone exposure (PDE). Through the model constructed by the present invention, the intrauterine programming mechanism of PDE-induced HPT axis functional activity disorder in offspring rats is explored.

The inventors focused on analyzing and comparing the functional activity of the HPT axis in male offspring rats before and after birth after exposure to dexamethasone during pregnancy, and further explored the intrauterine programming mechanism of PDE-induced HPT axis functional activity disorder in offspring rats from the perspective of hypothalamic GnRH neuron development and differentiation and the expression of its key regulatory point, L-glutamate decarboxylase 1. It was found that PDE activated the hypothalamic GR, causing hypomethylation and high expression of the GAD1 promoter region, thereby increasing the conversion of Glu to GABA and further inducing high expression of GnRH1. These programmed changes can continue until after birth, ultimately mediating HPT axis functional activity disorder in offspring. This model can be used to assist in screening drugs for preventing or treating PDE-induced male reproductive endocrine system diseases, thereby reducing the infertility rate.

The following will describe in detail the construction method and application of an animal model of fetal HPT axis functional activity disorder in the present application in combination with examples and experimental data.

Example 1

The present embodiment provides a method for constructing an animal model of fetal HPT axis functional disorder, which specifically includes:

1. SPF (specific pathogen-free) Wistar rats (10 weeks old) were housed in an air-conditioned room under standard conditions (room temperature: 18-22°C; humidity: 40%-60%; light cycle: 12-hour light-dark cycle; 10-15 ventilations per hour), with free access to food and water. After all rats were adaptively fed for one week before the experiment, two female rats and one male rat were caged together for overnight mating. The next day, sperm appeared on the vaginal smear to confirm conception, which was defined as gestational day (GD) 0.

2. The pregnant mice were randomly divided into a control group and a PDE group. Figure 1 shows a schematic diagram of the animal treatment procedure. From GD20 to GD21, the pregnant mice in the PDE group were given subcutaneous injections of dexamethasone 0.4 mg/kg (cat. no. H11020538, Wuhan Shuanghe Pharmaceutical Co., Ltd., China) twice a day, and the control group was given an equal amount of normal saline injection.

3. Some pregnant mice in the control group and PDE group were anesthetized and killed in the late pregnancy (GD22), and the fetuses were removed by caesarean section and weighed. The testicular morphology, endocrine and HPT axis-related gene expression levels of the male fetuses were detected. Another part of the pregnant mice gave birth naturally. The PDE group pups were further divided into the PDE+GAD1 siRNA group (administered group) and the PDE group 75 days after birth. On days 75 and 80 after birth, the PDE+GAD1 siRNA group was injected with GAD1 siRNA (6μg GAD1 siRNA dissolved in 1.2ul HifectinⅡ, injected bilaterally) in the bilateral arcuate nucleus (ARN) region to knock down GAD1 expression. The PDE group was injected with an equal volume of HifectinⅡ in the bilateral ARN region. The adult male offspring were anesthetized and killed on postnatal day 85 (PD85), and the testicular morphology, endocrine and HPT axis-related gene expression levels of the adult male offspring were detected.

Example 2

This example is based on the animal model constructed in Example 1 to explore the intrauterine programming mechanism of PDE-induced HPT axis functional activity disorder in offspring rats.

1. Related detection methods:

1.1 Testosterone detection in testicles:

The testosterone content (ng/mg) in tissue extracts was determined according to the iodine testosterone radioimmunoassay kit methodology.

1.2 Hypothalamic hematoxylin-eosin (HE) staining and transmission electron microscopy (TEM) analysis:

Referring to our published article (Lu J, Jiao Z, Yu Y, et al. Programming for increased expression of hippocampal GAD67 mediated the hypersensitivity of the hypothalamic-pituitary-adrenal axis in male offspring rats with prenatalethanol exposure. Cell Death Dis. 2018. 9 (6): 659.), 5 μm sections were observed and photographed using an Olympus AH-2 optical microscope (Olympus, Tokyo, Japan). For TEM analysis, 1 mm3 hypothalamic tissue blocks were placed in a 3% glutaraldehyde solution containing 0.1 M phosphate buffer solution (PBS). The samples were fixed in a 1% osmium tetroxide solution for 1.5 hours, washed in 0.1 M PBS, dehydrated with different concentrations of ethanol, and embedded in Epon 618. The epoxy blocks were sectioned on an ultramicrotome (LKB-v, LKB, Stockholm, Sweden, 70 nm), stained with uranyl acetate and lead citrate, and examined using a Hitachi H600 transmission electron microscope (Hitachi, Co., Tokyo, Japan). Digital images were acquired by computational

1.3 Reverse transcription and RT-qPCR analysis:

For RT-qPCR analysis, total RNA was extracted from hypothalamic tissue (50 mg) and primary hypothalamic cells using TRIzol (Invitrogen Ltd., Canada, USA) according to the manufacturer's protocol. Single-stranded cDNA was prepared from 2 μg of total RNA according to the protocol of the Takara RT kit (Takara Biotechnology Co., Ltd., Dalian, China). Then, RT-qPCR was performed using the SYBRGreen quantitative PCR Master Mix kit and ABI StepOnePlus Cycler (Thermo Fisher Scientific, Waltham, Massachusetts, USA), with an initial denaturation step of 95°C for 5 minutes. Then, each reaction was cycled 40 times, each cycle consisting of 95°C for 5 seconds, followed by an annealing step with different annealing conditions as shown in Table 1, and a final extension step of 30 seconds at 72°C if the annealing temperature was below 60°C. The relative gene expression of each gene was calculated using the 2-Asct method and glyceraldehyde 3-phosphate dehydrogenase for normalization. Table 1 lists the rat primer sequences and annealing temperatures. The rat primer sequences and annealing temperatures are shown in Table 1.

Table 1 Rat oligonucleotide primers and reaction conditions used in real-time fluorescence quantitative PCR

In Table 1 , 11βHSD, 11-hydroxysteroid dehydrogenase; GAD1, glutamate decarboxylase 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GR, glucocorticoid receptor; DNMT1, DNA methyltransferase 1; DNMT3a, DNA methyltransferase 3a; Egr-1, early growth response transcription factor 1; GnRH1, gonadotropin-releasing hormone 1; P450scc, cholesterol side-chain cleavage enzyme; StAR, steroidogenic acute regulatory protein.

1.4 Immunohistochemical analysis:

According to the instructions of the kit, immunohistochemistry was performed using the streptavidin peroxidase binding method. The primary antibody used was mouse anti-L-GAD1 (1:200 dilution, cat.no.sc-28376, Santa Cruz Biotechnology Inc., Texas, USA), and the negative control group (using specimens confirmed to contain no known antigens as controls, which should be negative results) used non-immune rabbit IgG as the primary antibody. Observation and photography were performed under an optical microscope, and the area of GAD1 staining on each image was calculated using an imaging system (Nikon H550S, Japan). Brown particles in the neuronal cytoplasm are GAD1-positive expression. The average optical density of 5 random areas of each section was detected by optical microscopy and analyzed using HMIAS-2000.

1.5 Analysis of hypothalamic neurotransmitters - glutamate (Glu) and gammaaminobutyric acid (GABA):

The tissue contents of glutamate and GABA in the hypothalamus were detected using ELISA kits (cat. no. E0900Ge, EIAab science Co. Ltd., Wuhan, China) and biochemical analysis kits (cat. no. A074, Jianchen Bio-Tek Inc., Nanjing, China), respectively.

1.6 Immunofluorescence analysis of hypothalamic Glu/GABA neurons:

Referring to our published article (Lu J, Jiao Z, Yu Y, et al. Programming for increased expression of hippocampal GAD67 mediated the hypersensitivity of the hypothalamic-pituitary-adrenal axis in male offspring rats with prenatalethanol exposure. Cell Death Dis. 2018. 9(6): 659.), immunofluorescence was used to detect Glu/GAD1-positive neurons in adjacent brain sections (10 μm) at the same level of the hypothalamus in each group. Primary antibody: mouse anti-glutamate (1:2000 dilutions, cat. no. 22523, ImmunoStar Inc., WI, USA), mouse anti-GAD1 (1:500 dilutions, cat. no. sc-28376, Santa Cruz Biotechnology Inc., Texas, USA), rabbit anti-NeuN antibody (1:500 dilutions, cat. no. ab104225, Abcam Inc., MA, USA), negative control group using 0.01M PBS instead of primary antibody. Secondary antibody: goat anti-rabbit FITC (1:200 dilutions, cat. no. bs-0295G-FITC, Bioss Biotechnology, Beijing, China), goat anti-mouse Cy3 (1:200 dilutions, cat. no. 115-166-003, Jackson ImmunoResearch Laboratories Inc., Baltimore, USA), nuclear dye DAPI (1:500, cat. no. D1306, Thermo Fisher Scientific Inc., USA). The stained sections were photographed and quantitatively analyzed using a Leica fluorescence microscope (Leica, DM5000B; Leica CTR5000; Leica, Germany) and the accompanying advanced fluorescence software (LAS AF, Leica Microsystems, Germany).

1.7 Intraventricular targeted injection of GAD1 siRNA to interfere with GAD1:

(1) Rats were anesthetized with a mixture of isoflurane and air (5% isoflurane for induction and 2.5% isoflurane for maintenance); (2) Rats were placed in a stereotaxic frame on a 37°C heating plate with the incisor bar set 3.5 mm below the interaural line. The scalp was cut after local disinfection; (3) Appropriate hypothalamic coordinates were selected based on the rat brain stereotaxic atlas and previous pilot experiments. GAD1 siRNA (GAD1 siRNA positive chain nucleotide sequence: 5’-3’GCAAACUGUGCAGUUCUUAUU; antisense chain nucleotide sequence: 3’-5’UAAGAACUGCACAGUUUGCUU) was injected into the bilateral hypothalamic ARN regions (anteroposterior, -2.3mm; left and right, ±0.3mm; superior and inferior, -9.97mm) (posterior 2.3mm, lateral ±0.3mm, ventral 9.9mm), and the model control group was injected with an equal volume of HifectinⅡ; (4) After injection, the pipette was placed in place for 5 minutes to prevent the liquid from flowing back through the injection tract, and then slowly withdrawn for 5 minutes; (5) The rats were locally disinfected with penicillin and the skin was sutured, and then placed on an electric blanket to warm up; (6) After the rats woke up, they were moved to the breeding room.

1.8 Bisulfite sequencing PCR (BSP) detection

DNA from hypothalamic tissue and cells was extracted, and CT conversion was performed using a methylation conversion kit (cat. no. D5005, Zymo, USA). The Gad1 primers were: F-AAATTATTATTTTTGGAAGTTAATAAGG, R-CAAAACATTTCAAAATACTCAAAAC. The PCR product was purified using a PCR purification kit, and the purified PCR product was connected to the T vector. The connection product was transferred into the freshly thawed competent cells, and then ice-bathed for 30 minutes, heat-shocked at 42 degrees for 45 seconds, and then ice-bathed for 2 minutes. 500 μL LB medium was added, and incubated at 37°C, 200 rpm for 1 hour, and 100 μL was taken to plate. The competent cells were spread on a plate containing ampicillin for overnight culture, and a single clone was picked and inoculated into a liquid culture medium containing ampicillin for overnight culture. The cultured bacterial solution was tested by colony PCR, and the positive clones were sent to a sequencing company for sequencing.

2. The testicular morphology, function, endocrine system and related gene expression levels of adult male offspring rats (85 days after birth) were tested.

2.1 Testicular morphological analysis

Compared with the control group, the testis weight and volume of male offspring rats in the PDE group were reduced (P < 0.01, Figure 2A-B), the interstitial cells were significantly disordered, the diameter of the seminiferous tubules and the thickness of the seminiferous epithelium were significantly reduced (P < 0.01, Figure 2C-E), while there was no significant difference in the PDE+siRNA group. This suggests that PDE can cause morphological changes in the testis of male adult offspring, and postnatal intracerebroventricular injection of GAD1 siRNA can reverse the above changes.

Figure 2. The t-test was used for comparison among the groups, mean ± SD, *P < 0.05, **P < 0.01. PDE: dexamethasone exposure during pregnancy (0.4 mg/kg, twice a day); PDE+siRNA: dexamethasone exposure during pregnancy (0.4 mg/kg, twice a day) and intraventricular injection of GAD1 siRNA (6 μg/time, twice) after birth.

2.2 Testicular testosterone synthesis function detection

The biosynthesis of androgens is a complex process. It uses cholesterol as raw material and is catalyzed by a series of enzymes such as StAR, P450scc, 3β-HSD, 17α-HSD ₁ and 17β-HSD3. Male androgens are mainly secreted by Leydig cells. To further study the changes in testicular endocrine function in adult mice, we detected testosterone levels, luteinizing hormone (LH) levels, follicle-stimulating hormone (FSH) levels, and steroidogenic enzyme expression levels at 85 days after birth (PD85). The results showed that compared with the control group, the serum testosterone concentration, testicular testosterone production, and blood LH and FSH levels of the PDE group were significantly reduced (P<0.05, Figure 3C-F); at the same time, the mRNA expression of steroidogenic enzymes (such as StAR, 3β-HSD and 17α-HSD ₁ ) was reduced (P<0.05, Figure 3A), and the StAR protein expression was also significantly reduced (P<0.05, Figure 3B); there was no significant change in the GAD1 siRNA group. Compared with the PDE group, the GAD1 siRNA group could significantly reverse the changes. This suggests that PDE can lead to a decrease in the testosterone synthesis function of the testis of adult male rats, and postnatal intracerebroventricular injection of GAD1 siRNA can reverse the above changes.

In Figure 3, PDE: dexamethasone exposure during pregnancy (0.4 mg/kg, twice a day); PDE+siRNA: dexamethasone exposure during pregnancy (0.4 mg/kg, twice a day) and intraventricular injection of GAD1 siRNA (6 μg/time, twice) after birth. FSH: follicle stimulating hormone; LH: luteinizing hormone; StAR: steroidogenic acute regulatory protein; 3β-HSD, 3β-hydroxysteroid dehydrogenase; 17α-HSD ₁ , 17α-hydroxysteroid dehydrogenase 1; 17β-HSD ₃ , 17β-hydroxysteroid dehydrogenase 3; P450scc, cytochrome P450 cholesterol side chain cleavage enzyme.

2.3 Detection of hypothalamic GnRH1/GAD1 expression and Glu/GABAergic neuron activity

The test results are shown in Figure 4. Compared with the control group, the mRNA expression levels of GnRH1 and GAD1 in the hypothalamus of adult offspring in the PDE group were significantly upregulated (P<0.01, Figure 4A); the methylation level of the GAD1 promoter region was significantly reduced (P<0.05, Figure 4B); the hypothalamus mainly showed that the protein levels of GnRH1 and GAD1 in the arcuate nucleus (ARN) were significantly increased (P<0.01, Figure 4E-F); the tissue concentration of neurotransmitter Glu was reduced, while the tissue concentration of GABA was increased (P<0.05, Figure 4C-D); the activity of Glu neurons projecting to GnRH neurons in ARN was weakened, while the activity of GABA neurons was enhanced (P<0.01, Figure 4G-H). Compared with the control group, there was no significant change in the GAD1 siRNA group; compared with the PDE group, the GAD1 siRNA group could significantly reverse the changes. These results suggest that PDE can cause hypomethylation in the GAD1 promoter region of the hypothalamus of adult male rats, and further mediate the upregulation of GnRH1\GAD1 expression and the imbalance of Glu\GABA neuron activity. Postnatal intracerebroventricular injection of GAD1 siRNA can reverse the above changes.

In Figure 4, PDE: dexamethasone exposure during pregnancy (0.4 mg/kg, twice a day); PDE+siRNA: dexamethasone exposure in late pregnancy (0.4 mg/kg, twice a day) and intraventricular injection of GAD1 siRNA after birth (6 μg/time, twice); GAD1, L-glutamate decarboxylase 1.

3. Test the testicular morphology, function, endocrine system and related gene expression levels of male offspring mice.

3.1 Testicular morphology and testosterone synthesis function detection

HE staining and TEM analysis were performed on the testes of the offspring fetal mice, and the testosterone synthesis function was detected. HE staining results showed that compared with the control group, the fetal testes in the PDE group underwent morphological changes, with smaller seminiferous tubules, enlarged interstitial areas, and significantly disordered interstitial cell arrangement (Figure 5A). The mRNA expression levels of testicular steroidogenic enzymes StAR, P450scc, 3β-HSD, 17α-HSD 1, and 17β-HSD3 in the PDE group were significantly lower than those in the control group (P<0.05, Figure 5B). Immunohistochemistry results also showed that PDE significantly reduced the expression of StAR protein (P<0.05, Figure 5C). The results showed that PDE could cause morphological changes in the testes of male fetal mice and reduce the expression of genes related to testosterone synthesis function and testosterone production in fetal mice, suggesting that the morphological changes in the testes and the reduction of testosterone synthesis function in PDE offspring have intrauterine origins.

In Figure 5, PDE: gestational dexamethasone exposure (0.4 mg/kg, twice a day); StAR: steroidogenic acute regulatory protein; P450scc: cytochrome P450 cholesterol side chain; 3β-HSD1, 3β-hydroxysteroid dehydrogenase 1; 17α-HSD1, 17α-hydroxysteroid dehydrogenase 1; 17β-HSD3, 17β-hydroxysteroid dehydrogenase 3.

3.2 Detection of hypothalamic Glu/GABA neuron activity and related gene expression

The results of the detection of Glu/GABA neuron activity and related gene expression in the fetal hypothalamus are shown in Figure 6. Compared with the control group, the mRNA expressions of GnRH1, GAD1, Egr1 and GR in the PDE group were significantly upregulated (Figure 6A, P<0.05), while the mRNA expressions of DNMT3a and DNMT1 were significantly downregulated (Figure 6A, P<0.05). Electron microscopy results showed that compared with the control group, the hypothalamic neurons in the PDE group showed moderate to severe edema, with blurred and damaged cell membranes, discontinuous areas in some areas, sparse intracellular matrix, and large areas of low electron density edema; the nucleus (N) was nearly oval with depressions, chromatin dissolved, and the perinuclear space slightly widened; mitochondria (M) were moderately swollen, and the matrix dissolved and faded; the rough endoplasmic reticulum (RER) was significantly expanded, and the ribosomes were degranulated; two autophagolysosomes (ASS) structures were visible in this field of view, and the Golgi apparatus (Go) was hypertrophic (Figure 6C). BSP results showed that the methylation level of GAD1 promoter region in the fetal hypothalamus in the PDE group was significantly reduced (Figure 6B, P<0.05). Compared with the control group, the protein expression levels of GnRH1 and GAD1 in the hypothalamic ARN in the PDE group were significantly upregulated (P<0.01, Figure 6D-E); the activity of Glu neurons projecting to the GnRH neurons in the ARN was weakened, while the activity of GABA neurons was enhanced (P<0.05, Figure 6F-G). This suggests that PDE can cause upregulation of GAD1 and GnRH1 expression in the fetal hypothalamus and imbalance of Glu/GABA neuron activity, which may be related to changes in the expression of DNMT3a and hypomethylation of the GAD1 promoter region.

In Figure 6, PDE: gestational dexamethasone exposure (0.4 mg/kg, twice a day); GAD1: L-glutamate decarboxylase 1; DNMT3a: DNA methyltransferase 3a; GnRH1: hypothalamic gonadotropin-releasing hormone 1.

4. Study on the epigenetic regulatory mechanism of dexamethasone increasing GAD1 expression in fetal hypothalamus at the cellular and molecular levels

To investigate the epigenetic regulatory mechanism of dexamethasone-induced GAD1 expression in the fetal hypothalamus at the cellular and molecular levels, we isolated primary hypothalamic cells from male fetal mice at GD20-PD7 and cultured them in Neurobasal medium supplemented with B27 and 4 mM L-glutamine at 37°C until adherence. The cells were then treated with 20, 100, and 500 nM dexamethasone or with 1.5 M mifepristone for 5 days at 37°C and 5% CO _2. The complete medium with or without drugs was replaced every day, and the cells were collected on the 5th day.

In primary male fetal hypothalamic cells cultured in vitro, we found that 10-500 nM dexamethasone treatment of fetal hypothalamic cells can directly upregulate the mRNA expression of GnRH1, GAD1, and Egr-1, and downregulate DNMT1 and DNMT3a, and there is a good dose-effect relationship (Figure 7A-E). At the same time, it was observed that 500 nM dexamethasone can reduce the activity of Glu neurons co-expressed with GnRH neurons, and increase the activity of GABA neurons (Figure 7K-L). The glucocorticoid receptor (GR) antagonist RU486 can reverse the above effects of 500 nM dexamethasone on hypothalamic neurons (Figure 7F-L), suggesting that GR activation mediates these effects of dexamethasone.

In FIG. 7 , Egr-1: early growth response factor 1; DEX: dexamethasone; GAD1: L-glutamate decarboxylase 1; DNMT: DNA methyltransferase; GnRH1: hypothalamic gonadotropin-releasing hormone 1.

As can be seen from the above examples, compared with the control group, the expression of gonadotropin-releasing hormone (GnRH) 1 in the male fetus of the PDE group was upregulated. The functional activity of the hypothalamic-pituitary-testicular (HPT) axis of the offspring after birth was disordered, which was manifested by high expression of hypothalamic GAD1 and GnRH1, significantly reduced testosterone levels in the blood and testes, and blood LH and FSH levels, reduced testicular volume, reduced testicular interstitial cells, reduced mRNA expression of steroidogenic enzymes (such as StAR, 3β-HSD and 17α-HSD1), and significantly reduced StAR protein expression.

In summary, PDE induces hypomethylation and high expression of the GAD1 promoter region, thereby increasing the conversion of Glu to GABA and further upregulating the expression of GnRH1. These programmed changes can continue after birth, leading to disorders in the functional activity of the HPT axis (as shown in Figure 8).

It can be seen from the above examples that the PDE method is an effective method for establishing an animal model of HPT axis functional activity disorder in offspring, and will provide an animal model that can be used for research on the prevention and treatment of male reproductive endocrine system diseases.

Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims (10)

1. A method for constructing an animal model of fetal HPT axis functional disorder, characterized in that the construction method comprises: The pregnant mice were divided into PDE group and control group. In the third trimester, the pregnant mice in PDE group were subcutaneously injected with dexamethasone twice a day, and the pregnant mice in control group were subcutaneously injected with an equal amount of normal saline twice a day. After the subcutaneous injection, some pregnant rats were selected from the PDE group and the control group, and the fetuses were removed by laparotomy to detect the testicular morphology, endocrine and HPT axis-related gene expression levels of the male fetuses. Another part of the pregnant female mice in the PDE group and the control group gave birth naturally, and the adult male offspring were killed. The testicular morphology, endocrine system and HPT axis-related gene expression levels of the adult male offspring were detected. 2. The method for constructing an animal model of fetal HPT axis functional disorder according to claim 1, characterized in that the pregnant mice are divided into a PDE group and a control group, and in the late pregnancy, the pregnant mice in the PDE group are subcutaneously injected with dexamethasone twice a day, and the pregnant mice in the control group are subcutaneously injected with an equal amount of normal saline twice a day, specifically comprising: The pregnant mice were divided into PDE group and control group. On the 20th to 21st day after conception, the pregnant mice in the PDE group were subcutaneously injected with dexamethasone twice a day, with each injection of dexamethasone being 0.4 mg/kg of the pregnant mouse body weight. The pregnant mice in the control group were subcutaneously injected with an equal amount of normal saline twice a day. 3. The method for constructing an animal model of fetal HPT axis functional disorder according to claim 1, characterized in that, after the subcutaneous injection, some pregnant mothers are selected from the PDE group and the control group, and the fetuses are removed by caesarean section to detect the testicular morphology, endocrine and HPT axis-related gene expression levels of the male fetuses, specifically comprising: After the subcutaneous injection, some pregnant female mice were randomly selected from the PDE group and the control group, and the fetuses were removed by cesarean section on the 22nd day after conception. The testicular morphology, endocrine and HPT axis-related gene expression levels of the male fetuses were detected. The detection of testicular morphology, endocrine and HPT axis-related gene expression levels of male fetal mice specifically includes: HE staining and TEM analysis were performed on the testes of male fetal mice to observe the testicular morphology; The expression levels of steroidogenic enzymes StAR, P450scc, 3β-HSD, 17α-HSD1, and 17β-HSD3 in the testis of male fetal mice were detected; The expression levels of GnRH1, GAD1, Egr-1, GR, DNMT1 and DNMT3a in the hypothalamus of male fetal mice were detected, the methylation level of the GAD1 promoter region was detected, and the activities of Glu neurons and GABA neurons were detected. 4. The method for constructing an animal model of fetal HPT axis functional disorder according to claim 1, characterized in that another part of the pregnant female mice in the PDE group and the control group give birth naturally, the male offspring born naturally after adulthood are killed, and the testicular morphology, endocrine and HPT axis-related gene expression levels of the adult male offspring are detected, specifically comprising: Another part of pregnant female mice in the PDE group and the control group gave birth naturally, and the male offspring mice were killed by anesthesia on the 85th day after natural birth. The testicular morphology, endocrine and HPT axis-related gene expression levels of the adult male offspring mice were detected; Among them, the testicular morphology, endocrine and HPT axis-related gene expression levels of adult male offspring were detected, including: The testicles of adult male offspring were stained with HE and weighed to observe the testicular morphology; The serum testosterone concentration, testicular testosterone concentration, serum luteinizing hormone concentration, and serum follicle-stimulating hormone concentration of adult male offspring were tested; The expression levels of testicular steroidogenic enzymes StAR, P450scc, 3β-HSD, 17α-HSD 1, and 17β-HSD3 in adult male offspring rats were detected; The expression levels of GnRH1 and GAD1 in the hypothalamus of adult male offspring mice were detected, the methylation level of the GAD1 promoter region was detected, and the activities of Glu neurons and GABA neurons were detected. 5. The method for constructing an animal model of fetal HPT axis functional disorder according to claim 1, characterized in that another part of the pregnant female mice in the PDE group and the control group give birth naturally, and the male offspring born naturally after adulthood are killed, specifically comprising: Another part of pregnant female mice in the PDE group and the control group gave birth naturally, and the male offspring were killed by anesthesia on the 85th day after natural birth; Before the offspring male mice were anesthetized and killed, a part of the naturally born male mice were randomly selected as the drug administration group. The male offspring in the drug administration group were injected with the test drug on the 75th and 80th days after birth, and the remaining male offspring were injected with an equal volume of HifectinⅡ. 6 . The method for constructing an animal model of fetal HPT axis functional disorder according to claim 5 , wherein the drug to be tested comprises GAD1 siRNA. 7. The method for constructing an animal model of fetal HPT axis functional disorder according to claim 6, characterized in that the nucleotide sequence of the positive strand of the GAD1 siRNA is shown as SEQ ID NO.1, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO.2. 8. Use of the method for constructing an animal model of fetal HPT axis functional disorder as described in claims 1 to 7 in assisting the screening of drugs for preventing or treating male reproductive endocrine system diseases. 9. The use according to claim 8, characterized in that the male reproductive endocrine system disease includes HPT axis functional activity disorder and/or testicular dysplasia. 10. The use according to claim 8, characterized in that the male reproductive endocrine system diseases include male reproductive endocrine system diseases in offspring caused by exposure to dexamethasone during pregnancy.