CN118685505A - Use of GAD1 or its epigenetic marker as an early marker of susceptibility to HPT axis functional activity disorders - Google Patents

Abstract

The invention provides application of GAD1 or an epigenetic mark thereof as an early marker for susceptibility of HPT shaft functional activity disorder, belonging to the technical field of biological detection. The GAD1 gene or the epigenetic mark thereof is used as an early marker for susceptibility of HPT shaft functional activity disorder, so that early warning of susceptibility of HPT shaft functional activity disorder can be realized, early prevention or treatment of HPT shaft functional activity disorder is facilitated, and infertility rate is reduced.

Description

Use of GAD1 or its epigenetic markers as early markers of susceptibility to HPT axis functional activity disorders

Technical Field

The invention belongs to the technical field of biological detection, and particularly relates to the use of GAD1 or its epigenetic marker as an early marker of susceptibility to HPT axis functional activity disorder.

Background Art

The 2009 "China Infertility Status Survey Report" shows that in the past decade, infertility in my country's childbearing population has been 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 important theoretical basis and early markers for the prevention and treatment of infertility. Therefore, it has become a scientific problem that needs to be solved urgently 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 early warning of HPT axis functional activity disorder, the present invention provides the use of GAD1 or its epigenetic marker as an early marker of susceptibility to HPT axis functional activity disorder. Using the GAD1 gene or its epigenetic marker as an early marker of susceptibility to HPT axis functional activity disorder can achieve early warning of susceptibility to HPT axis functional activity disorder, which is beneficial to the early prevention or treatment of HPT axis functional activity disorder and reduces the infertility rate.

The present invention is achieved through the following technical solutions:

The present invention provides use of GAD1 (L-glutamic acid decarboxylase 1) or its epigenetic marker as an early marker of susceptibility to HPT axis functional activity disorder.

Based on the same inventive concept, the present invention provides an early marker for susceptibility to HPT axis functional activity disorder, wherein the early marker comprises GAD1 or an epigenetic marker thereof.

Based on the same inventive concept, the present invention provides the use of GAD1 gene expression level and/or GAD1 gene promoter region methylation level in preparing a kit for early warning of susceptibility to HPT axis functional activity disorder.

Furthermore, in the hypothalamus, if the expression level of the GAD1 gene is increased and/or the methylation level of the GAD1 gene promoter region is decreased, the risk of suffering from HPT axis functional activity disorder is increased.

Furthermore, the kit comprises a detection reagent for the expression level of the GAD1 gene and/or a detection reagent for the methylation level of the promoter region of the GAD1 gene.

Furthermore, the detection reagent for the expression level of the GAD1 gene includes a primer pair for amplifying the GAD1 gene, and the detection reagent for the methylation level of the promoter region of the GAD1 gene includes a primer pair for detecting the methylation level of the promoter region of the GAD1 gene.

Furthermore, the nucleotide sequences of the primer pair for amplifying the GAD1 gene are shown in SEQ ID NO.1 and SEQ ID NO.2, and the nucleotide sequences of the primer pair for detecting the methylation level of the promoter region of the GAD1 gene are shown in SEQ ID NO.3 and SEQ ID NO.4.

Optionally, SEQ ID NO.1 is specifically CAAGTTCTGGCTGATGTGGA, SEQ ID NO.2 is specifically GCCACCCTGTGTAGCTTTTC, SEQ ID NO.3 is specifically AAATTATTATTTTTGGAAGTTAATAAGG, and SEQ ID NO.4 is specifically CAAAACATTTCAAAATACTCAAAAC.

Based on the same inventive concept, the present invention provides a kit for early warning of susceptibility to HPT axis functional activity disorders, the kit comprising a detection reagent for the GAD1 gene expression level and/or a detection reagent for the methylation level of the GAD1 gene promoter region, the detection reagent for the GAD1 gene expression level comprises a primer pair for amplifying the GAD1 gene, and the detection reagent for the methylation level of the GAD1 gene promoter region comprises a primer pair for detecting the methylation level of the GAD1 gene promoter region.

Optionally, the nucleotide sequence of the primer pair for amplifying the GAD1 gene is shown as SEQ ID NO.1 and SEQ ID NO.2, and the nucleotide sequence of the primer pair for detecting the methylation level of the promoter region of the GAD1 gene is shown as SEQ ID NO.3 and SEQ ID NO.4.

Based on the same inventive concept, the present invention provides use of the GAD1 gene expression level and/or the GAD1 gene promoter region methylation level in preparing a kit for early warning of testicular dysplasia.

Based on the same inventive concept, the present invention also provides a kit for early warning of testicular dysplasia, wherein the kit comprises a detection reagent for the expression level of the GAD1 gene and/or a detection reagent for the methylation level of the promoter region of the GAD1 gene.

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

The invention discloses the use of GAD1 or its epigenetic marker as an early marker of susceptibility to HPT axis functional activity disorder. The inventors focused on observing the HPT axis functional activity of male offspring rats before and after birth after prenatal dexamethasone exposure (PDE), and further explored the intrauterine programming mechanism of HPT axis functional activity disorder in offspring rats caused by PDE from the perspective of hypothalamic GnRH neuron development and differentiation and its key regulatory point GAD1 expression. It was found that PDE activated the hypothalamic GR, causing hypomethylation and high expression changes in the GAD1 promoter region, promoting increased conversion of Glu to GABA, and further inducing high expression of GnRH1. These programmed changes can continue after birth, leading to HPT axis functional activity disorder. GAD1 siRNA can reverse the HPT axis functional activity disorder in offspring caused by prenatal dexamethasone exposure, illustrating that GAD1 and its epigenetic markers can be used as early markers of susceptibility to HPT axis functional activity disorder caused by PDE, which will provide a theoretical basis and scientific value for the prevention and treatment of male reproductive endocrine system diseases.

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.

FIG. 8 is a diagram showing the mechanism of use of GAD1 or its epigenetic markers as early markers of susceptibility to disorders in the functional activity of the hypothalamus-pituitary-testis axis.

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 inventors focused on observing the HPT axis functional activity of male offspring rats before and after birth after prenatal dexamethasone exposure (PDE), and further explored the intrauterine programming mechanism of HPT axis functional activity disorder in offspring rats caused by PDE from the perspective of hypothalamic GnRH neuron development and differentiation and its key regulatory point L-glutamate decarboxylase 1 expression. It was found that PDE activated the hypothalamic GR, causing hypomethylation and high expression of the GAD1 promoter region, increasing the conversion of Glu to GABA, and further inducing high expression of GnRH1. These programmed changes can continue after birth, leading to HPT axis functional activity disorder. The inventors found through experiments that GAD1 siRNA can reverse the functional activity disorder of the hypothalamic-pituitary-testicular axis in offspring caused by gestational dexamethasone exposure. Elucidating that GAD1 and its epigenetic markers can serve as early markers of susceptibility to hypothalamic-pituitary-testicular axis functional activity disorders caused by dexamethasone exposure during pregnancy will provide a theoretical basis and scientific value for the prevention and treatment of male reproductive endocrine system diseases.

The following will describe in detail the use of GAD1 or its epigenetic marker as an early marker of susceptibility to HPT axis functional activity disorders in combination with the examples and experimental data.

Example 1

This example constructs a model of HTP axis functional activity disorder in offspring caused by dexamethasone exposure during pregnancy.

1. SPF (specific pathogen free) grade 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-h light-dark cycle; 10-15 ventilations per hour) with free access to food and water. After all rats were fed adaptively for one week before the experiment, two female rats and one male rat were co-housed for overnight mating. The next day, the appearance of sperm on the vaginal smear confirmed conception, which was defined as gestational day (GD) 0. The pregnant rats were randomly divided into a control group and a PDE group. Figure 1 shows a schematic diagram of the animal handling procedure. From GD20 to GD21, the pregnant rats 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 saline injection.

2. Some pregnant mice in the control group and PDE group were anesthetized and killed in the late pregnancy (22 days), and the fetuses were removed by caesarean section and weighed. Another part of pregnant mice gave birth naturally. The pups in the PDE group were further divided into the PDE+GAD1 siRNA group and the PDE group 75 days after birth. At 75 and 80 days after birth, the PDE+GAD1 siRNA group was injected with GAD1 siRNA (6μgGAD1 siRNA dissolved in 1.2ul animal in vivo transfection reagent HifectinⅡ, injected bilaterally) in the bilateral arcuate nucleus (ARN) region to knock down GAD1 expression, and the PDE group was injected with an equal volume of Hifectin II in the bilateral ARN region. The pups were anesthetized and killed 85 days after birth (postnatal day 85, PD85).

Example 2

This example verifies that GAD1 siRNA can reverse the functional activity disorder of the HPT axis in offspring caused by exposure to dexamethasone during pregnancy.

1. Related detection methods:

1.1 Testicular testosterone detection:

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 lower than 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 (6 μg GAD1 siRNA dissolved in 1.2 μl HifectionⅡ) was injected into the bilateral hypothalamic ARN regions (anteroposterior, -2.3 mm; left and right, ±0.3 mm; superior and inferior, -9.97 mm) (posterior 2.3 mm, lateral ±0.3 mm, ventral 9.9 mm) (GAD1 siRNA positive chain nucleotide sequence: 5’-3’GCAAACUGUGCAGUUCUUAUU; antisense chain nucleotide sequence: 3’-5’UAAGAACUGCACAGUUUGCUU), 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+GAD1siRNA group. This suggests that PDE can cause morphological changes in the testis of male adult offspring, and postnatal intracerebroventricular injection of GAD1siRNA can reverse the above changes.

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

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: late pregnancy dexamethasone exposure (0.4 mg/kg, twice a day); PDE+GAD1 siRNA: late pregnancy dexamethasone exposure (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+GAD1 siRNA: dexamethasone exposure during pregnancy (0.4 mg/kg, twice a day) and intracerebroventricular 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.

It can be seen from the above examples that, compared with the control group, the expression of gonadotropin-releasing hormone (GnRH) 1 in the hypothalamus of male fetuses in the PDE group was upregulated. The functional activity of the hypothalamic-pituitary-testicular (HPT) axis was disturbed in the offspring after birth, as manifested by high expression of GnRH1, significantly decreased levels of testosterone in the blood and testes, and blood levels of LH and FSH, reduced testicular volume, reduced testicular interstitial cells, decreased mRNA expression of steroidogenic enzymes (such as StAR, 3β-HSD and 17α-HSD1), and significantly decreased StAR protein expression. It was further found that the mRNA expression levels of GnRH1 and GAD1 in the hypothalamus of the PDE group before and after birth were significantly upregulated; the methylation level of the GAD1 promoter region was significantly decreased; the hypothalamus was mainly manifested by a significant increase in the protein levels of GnRH1 and GAD1 in the ARN region; the tissue concentration of the neurotransmitter Glu was reduced, while the tissue concentration of GABA was increased; the activity of Glu neurons on the GnRH neurons projecting to the ARN region was weakened, while the activity of GABA neurons was enhanced, and intraventricular injection of siRNA-HIFⅡ could reverse the above changes after birth.

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 functional disorders of the HPT axis (as shown in Figure 8). GAD1 and its epigenetic markers can serve as early markers of susceptibility to functional disorders of the hypothalamus-pituitary-testis axis caused by dexamethasone exposure during pregnancy.

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 additional 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 (11)

1. Use of GAD1 or its epigenetic markers as early markers of susceptibility to HPT axis functional activity disorders. 2. An early marker for susceptibility to HPT axis functional activity disorder, characterized in that the early marker includes GAD1 or its epigenetic marker. 3. Use of the GAD1 gene expression level and/or the GAD1 gene promoter region methylation level in the preparation of a kit for early warning of susceptibility to HPT axis functional activity disorders. 4. The use according to claim 3, characterized in that, in the hypothalamus, the expression level of the GAD1 gene is increased and/or the methylation level of the GAD1 gene promoter region is decreased, and the risk of suffering from HPT axis functional activity disorder is increased. 5 . The use according to claim 3 , characterized in that the kit comprises a detection reagent for the expression level of the GAD1 gene and/or a detection reagent for the methylation level of the promoter region of the GAD1 gene. 6. The use according to claim 5, characterized in that the detection reagent for the expression level of the GAD1 gene comprises a primer pair for amplifying the GAD1 gene, and the detection reagent for the methylation level of the promoter region of the GAD1 gene comprises a primer pair for detecting the methylation level of the promoter region of the GAD1 gene. 7. The use according to claim 6, characterized in that the nucleotide sequences of the primer pair for amplifying the GAD1 gene are shown as SEQ ID NO.1 and SEQ ID NO.2, and the nucleotide sequences of the primer pair for detecting the methylation level of the promoter region of the GAD1 gene are shown as SEQ ID NO.3 and SEQ ID NO.4. 8. A kit for early warning of susceptibility to HPT axis functional activity disorders, characterized in that the kit comprises a detection reagent for GAD1 gene expression level and/or a detection reagent for GAD1 gene promoter region methylation level, the detection reagent for GAD1 gene expression level comprises a primer pair for amplifying the GAD1 gene, and the detection reagent for GAD1 gene promoter region methylation level comprises a primer pair for detecting the methylation level of the GAD1 gene promoter region. 9. A kit for early warning of susceptibility to HPT axis functional activity disorders according to claim 8, characterized in that the nucleotide sequences of the primer pair for amplifying the GAD1 gene are as shown in SEQ ID NO.1 and SEQ ID NO.2, and the nucleotide sequences of the primer pair for detecting the methylation level of the GAD1 gene promoter region are as shown in SEQ ID NO.3 and SEQ ID NO.4. 10. Use of the GAD1 gene expression level and/or the GAD1 gene promoter region methylation level in the preparation of a kit for early warning of testicular dysplasia. 11. A kit for early warning of testicular dysplasia, characterized in that the kit comprises a detection reagent for the expression level of the GAD1 gene and/or a detection reagent for the methylation level of the promoter region of the GAD1 gene.