Maternal blood PBMC / placental FSTL3 as a warning marker for fetal testicular dysplasia and its application

Abstract

The present invention discloses a maternal blood PBMC / placenta FSTL3 as a warning marker for fetal testicular dysplasia and its application. The present invention proves at the transcriptional level that the epigenetic modification and expression of FSTL3 have the same changes and are significantly correlated in maternal blood PBMC, placenta and offspring fetal testes after prenatal dexamethasone exposure, and the epigenetic modification and expression of FSTL3 can continue to the testes with blood-testis barrier function damage after birth. Therefore, the risk of disease and disease state of offspring testicular dysplasia can be judged by identifying the changes in epigenetic modification and expression of FSTL3 in maternal blood PBMC / placenta. The present invention is reliable and simple, provides a research basis for the early warning technology of testicular dysplasia, and also provides a possibility for the early and effective prevention and treatment of the occurrence of testicular dysplasia.

Description

Technical Field

[0001] The present invention relates to the technical field of gene functions and applications, and particularly relates to a maternal blood PBMC / placenta FSTL3 as a warning marker for fetal testicular dysplasia and its application. Background Art

[0002] Testicular dysgenesis syndrome (TDS) is an increasingly common environmentally related male reproductive developmental disorder, including symptoms of potential diseases such as poor semen quality, testicular cancer, cryptorchidism, and hypospadias. Epidemiological and experimental studies have shown that TDS is the result of disrupted embryonic programming and gonadal development during fetal life. A large number of studies have confirmed that adverse prenatal environments can cause abnormal testicular structure, reduced testosterone synthesis ability, and fertility damage in offspring [1]. Dexamethasone is a common synthetic glucocorticoid that easily crosses the placental barrier and enters the fetal body. It can promote fetal lung maturation and reduce the occurrence of respiratory distress syndrome, so it is widely used in the treatment of related diseases such as clinical threatened premature birth [2]. However, animal studies have shown that prenatal exposure to dexamethasone may have long-term adverse effects on fetal testicular development. However, its mechanism of occurrence and warning markers are still unclear. Sertoli cells are the first differentiating cells recognizable in the fetal testis and can regulate all subsequent events in testicular development. Their number is closely related to testicular cord development, testosterone production, and spermatogenic function. An important and unique physiological function of Sertoli cells is to maintain a microenvironment suitable for spermatogenesis by establishing the blood-testis barrier (BTB) [3]. The BTB is the main barrier for environmental toxins to enter the testis, so its functional damage may be an important factor in the risk of testicular dysplasia. Therefore, the development of fetal testicular Sertoli cells and the integrity of the blood-testis barrier function during the intrauterine period are crucial for testicular development. Therefore, taking dexamethasone as the research object to explore the pathogenesis and prevention and treatment targets of fetal testicular dysplasia caused by prenatal dexamethasone exposure (PDE) has very important practical significance.

[0003] The etiology of fetal testicular dysplasia occurs in utero, but the onset is in adulthood. The pathogenesis is unclear and the targets are not well-defined, making its early prevention and treatment extremely difficult, and targeted research is almost blank. Some viewpoints have been put forward that the placenta is a good alternative tissue for warning of the susceptibility to diseases after the offspring are born. The research on placental markers is conducive to non-invasive assessment of placental health and fetal intrauterine development status, and even has the potential to predict the occurrence and development of fetal-originated diseases [4]. First of all, in mammals, the placenta can connect the fetus with the mother, and the fetus's perception of the intrauterine environment is mediated by the placenta. The placenta creates a suitable intrauterine environment for the fetus through functions such as material transport, endocrine, barrier, and immunity [5]. Secondly, the placenta is an organ originating from the fetus, carrying the same genetic information as the fetus, and may have similar responses to environmental stimuli, often accompanied by changes in indicators that can be objectively and quantitatively detected. These indicators can be used as biomarkers for developmental abnormalities and functional damage.

[0004] A large number of studies have suggested that placental DNA methylation, microRNA expression patterns, etc. are related to adverse pregnancy outcomes in offspring, organ development (lungs, nervous system, heart) and long-term diseases [6-8]. On the other hand, there are some specific changes in the placenta that reflect the development status of the placenta and the fetus, which can enter the maternal blood through blood circulation. These changes in the placenta-derived indicators in maternal blood can also be used as biomarkers. Peripheral blood mononuclear cells (PBMCs) are cells with a single nucleus in peripheral blood, mainly lymphocytes and a small amount of monocytes, which are easily obtained clinical biological samples. The literature suggests that epigenetic modifications of disease-related functional genes in peripheral blood in the early stage of life can be used as early biomarkers for susceptibility to fetal-originated diseases [9]. Existing studies have confirmed that the methylation level of the BDNF promoter region in PBMCs at birth can be used as a biomarker for childhood autism caused by prenatal bisphenol A exposure. In addition, our recent research in the laboratory has clarified the key role and epigenetic regulatory mechanism of YB-1 and P-gp in mediating the opening of the placental GC barrier, and proposed that detecting the expression of P-gp and YB-1 and related epigenetic modification changes in maternal blood exosomes or peripheral blood mononuclear cells (PBMCs) may achieve the warning goal of IUGR

[10] . It is known that FSTL3 is highly expressed specifically in the placenta and testis. It can not only regulate testicular development, but also regulate placental development and metabolism, suggesting that FSTL3 has a high correlation between the testis and the placenta and has the potential to be used as a warning biomarker. Therefore, detecting biomarkers in the placenta or maternal blood helps to non-invasively assist in predicting abnormal development of multiple fetal organs and susceptibility to related fetal-originated diseases. At the same time, finding biomarkers in the placenta and PBMCs has great clinical significance for warning of testicular dysplasia in PDE offspring.

[0005] Main references:

[0006] 1. Skakkebaek NE, Rajpert-De Meyts E, Main KM: Testicular dysgenesissyndrome: an increasingly common developmental disorder with environmentalaspects. Hum Reprod 2001, 16(5):972.

[0007] 2. Haram K, Mortensen JH, Magann EF, Morrison JC: Antenatal corticosteroidtreatment: factors other than lung maturation. J Matern Fetal Neonatal Med2017, 30(12):1437.

[0008] 3. Mruk DD, Cheng CY: The Mammalian Blood-Testis Barrier: Its Biology andRegulation. Endocr Rev 2015, 36(5):564.

[0009] 4. Schroeder DI, Schmidt RJ, Crary-Dooley FK, Walker CK, Ozonoff S, Tancredi DJ, Hertz-Picciotto I, LaSalle JM: Placental methylome analysis from aprospective autism study. Molecular autism 2016, 7:51.

[0010] 5. Paquette AG, Houseman EA, Green BB, Lesseur C, Armstrong DA, Lester B, Marsit CJ: Regions of variable DNA methylation in human placenta associatedwith newborn neurobehavior. Epigenetics 2016, 11(8):603.

[0011] 6. Paquette AG, Lester BM, Lesseur C, Armstrong DA, Guerin DJ, Appleton AA, Marsit CJ: Placental epigenetic patterning of glucocorticoid response genes is associated with infant neurodevelopment. Epigenomics 2015, 7(5): 767.

[0012] 7. CL H, CP M, DB F, TL B: O-GlcNAc transferase (OGT) as a placental biomarker of maternal stress and reprogramming of CNS gene transcription in development. Proceedings of the National Academy of Sciences of the United States of America 2013, 110(13): 5169.

[0013] 8. EB K: Genomic imprinting, action, and interaction of maternal and fetal genomes. Proceedings of the National Academy of Sciences of the United States of America 2015, 112(22): 6834.

[0014] 9. Bartolotti N, Lazarov O: CREB signals as PBMC-based biomarkers of cognitive dysfunction: A novel perspective of the brain-immune axis. Brain Behav Immun 2019, 78: 9.

[0015] 10. Ge C, Xu D, Yu P, Fang M, Guo J, Xu D, Qiao Y, Chen S, Zhang Y, Wang H. P-gp expression inhibition mediates placental glucocorticoid barrier opening and fetal weight loss. BMC Med. 2021, 19(1):311. Summary of the Invention

[0016] In order to overcome the deficiencies of the prior art, the purpose of the present invention is to determine the relationship between the level and expression of H3K9me2 in the promoter region of the FSTL3 gene in maternal blood PBMC / placenta and fetal testicular dysplasia, so as to provide a research basis for the early warning of fetal testicular dysplasia. The aim is to provide a maternal blood PBMC / placenta FSTL3 as a warning marker for fetal testicular dysplasia and its application, that is, the application of epigenetic modification and expression of follistatin-like 3 (FSTL3) in warning fetal testicular dysplasia.

[0017] The purpose of the present invention is achieved by the following technical solutions:

[0018] In the first aspect, the present invention provides a warning marker for fetal testicular dysplasia, characterized in that: the warning marker is maternal blood PBMC / placenta FSTL3; the expression differences of the follistatin-like protein-3, that is, the FSTL3 gene, in normal tissues, peripheral blood mononuclear cells PBMC in maternal blood, placenta and testicular tissues: the expression level of the FSTL3 gene in the placenta and testicular tissues is significantly higher than that in other tissues.

[0019] As a preferred solution, the expression differences of the FSTL3 gene in normal testes and dysplastic testes:

[0020] Detect the differential expression of the FSTL3 gene at the transcriptional level. Compared with normal testes, the expression level of the FSTL3 gene in dysplastic testes is significantly increased;

[0021] Detect the differential level of FSTL3 epigenetics at the transcriptional level. Compared with normal testes, the level of H3K9me2 in the promoter region of FSTL3 in dysplastic testes is significantly decreased.

[0022] In a second aspect, the present invention provides the use of maternal blood PBMC / placental FSTL3 in the preparation of a kit for screening early warning of fetal-origin adult testicular dysplasia, characterized in that: when the expression of the FSTL3 gene is increased in maternal blood PBMC / placenta and the level of H3K9me2 in the promoter region of the FSTL3 gene is decreased, there is a risk of fetal-origin testicular dysplasia.

[0023] As a preferred embodiment, the kit contains reagents for detecting the expression of the FSTL3 gene and reagents for detecting the level of H3K9me2 in the promoter region of FSTL3.

[0024] Further, the reagents for detecting the expression of the FSTL3 gene include primer pairs for amplifying the FSTL3 gene and primer pairs for detecting the level of H3K9me2 in the promoter region of the FSTL3 gene.

[0025] The primer pairs for amplifying the FSTL3 gene are shown as SEQ ID No.11, SEQ ID No.12 and SEQ ID No.15, SEQ ID No.16; the primer pairs for detecting the level of H3K9me2 in the promoter region of the FSTL3 gene are shown as SEQ ID No.17, SEQ ID No.18, SEQ ID No.19, SEQ ID No.20.

[0026] In a third aspect, the present invention provides the use of a reagent for detecting the expression of the FSTL3 gene in the preparation of a tool for warning fetal-origin testicular dysplasia.

[0027] The product includes: products for diagnosing fetal-origin testicular dysplasia by detecting FSTL3 expression through RT-PCR, Western Blot, Chip-PCR; the product for diagnosing fetal-origin testicular dysplasia by RT-qPCR includes at least one pair of primer pairs specifically amplifying the FSTL3 gene; the product for detecting and diagnosing fetal-origin testicular dysplasia by Western Blot includes: specific antibodies against FSTL3 protein; the product for diagnosing fetal-origin testicular dysplasia by Chip-PCR includes: sequences of the promoter region of the FSTL3 gene.

[0028] In a fourth aspect, the present invention provides a tool for warning testicular dysplasia, characterized in that: the tool includes reagents for detecting the expression of the FSTL3 gene; the reagents include primers for detecting the FSTL3 gene and reagents for detecting the level of H3K9me2 in the promoter region of FSTL3.

[0029] Specifically, the application of the H3K9me2 level and expression in the promoter region of the FSTL3 gene in maternal blood PBMC / placenta in the preparation of a kit for early warning of fetal origin adult testicular dysplasia means that when the expression of the FSTL3 gene increases in maternal blood PBMC / placenta and the H3K9me2 level in the promoter region of the FSTL3 gene decreases, there is a risk of fetal origin testicular dysplasia.

[0030] The above kit contains reagents for detecting the expression of the FSTL3 gene and reagents for detecting the H3K9me2 level in the promoter region of FSTL3. The reagents for detecting the expression of the FSTL3 gene contain primer pairs for amplifying the FSTL3 gene and primer pairs for detecting the H3K9me2 level in the promoter region of the FSTL3 gene. The sequences of the primer pairs for amplifying the FSTL3 gene are shown in SEQ ID No.11, SEQ ID No.12, SEQ ID No.15, and SEQ ID No.16; the primer pairs for detecting the H3K9me2 level in the promoter region of the FSTL3 gene are shown in SEQ ID No.17, SEQ ID No.18, SEQ ID No.19, and SEQ ID No.20.

[0031] The technical principle and research process of the present invention are as follows:

[0032] The present invention confirms through research that compared with the control group, the expression of FSTL3 in the fetal testes of the offspring rats in the PDE group increases and persists after birth; compared with the control group, the H3K9me2 level in the promoter region of the FSTL3 gene in the fetal testes of the PDE group decreases significantly and persists after birth; at the same time, it is found that the expression of the FSTL3 gene in maternal blood PBMC / placenta in the PDE group increases and the H3K9me2 level in the promoter region decreases significantly. It is suggested that the epigenetic modification and expression changes of FSTL3 in maternal blood PBMC / placenta and fetal testes in the PDE group are consistent. Then, the present invention finds that during the clinical prenatal application of dexamethasone treatment, compared with the control group, the H3K9me2 level in the promoter region of the FSTL3 gene in maternal blood PBMC / placenta in the PDE group decreases significantly and the expression increases, and it is related to androgens in cord blood.

[0033] In summary, the combination of the increase in the H3K9me2 level and expression in the promoter region of the FSTL3 gene in maternal blood PBMC / placenta can be used for early warning of fetal origin testicular dysplasia.

[0034] The advantages and beneficial effects of the present invention are as follows:

[0035] 1. The present invention discovers a new function of FSTL3, that is, dexamethasone can damage the blood-testis barrier of the offspring testes by upregulating FSTL3.

[0036] 2. Based on the role of FSTL3 in the impairment of the blood-testis barrier in offspring caused by PDE, it provides a theoretical and practical basis for the development of early warning markers for fetal testicular dysplasia.

[0037] 3. The present invention uses the expression and epigenetic modification of FSTL3 in maternal blood PBMC / placenta to early warn of long-term diseases in offspring, which has extremely high clinical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 . Detection of fetal testicular morphology and barrier function in the control group and the PDE group.

[0039] Figure 1 In the figure: A: Fetal testicular morphology of PDE offspring; B, C: Maximum cross-sectional area and longest diameter of fetal testes of PDE offspring; D-G: mRNA expression of genes related to the blood-testis barrier in fetal testes of PDE offspring; F: CX43 immunofluorescence in fetal testes of PDE offspring.

[0040] Figure 2 . Detection of testicular morphology and barrier function in adult PW12 / 28 offspring of the control group and the PDE group.

[0041] Figure 2 In the figure: A, B: Testicular morphology of adult PW12 / 28 offspring of PDE; C: Diameter of seminiferous tubules in adult PW12 / 28 offspring of PDE; D: Sperm motility in adult PW12 / 28 offspring of PDE; E: Thickness of spermatogenic epithelium in adult PW12 / 28 offspring of PDE; F: Sperm count in adult PW12 / 28 offspring of PDE; G, J: CX43 immunofluorescence in adult PW12 / 28 offspring of PDE; H, K: mRNA expression of genes related to the blood-testis barrier in testes of adult PW12 / 28 offspring of PDE; I, L: CX43 protein expression in testes of adult PW12 / 28 offspring of PDE.

[0042] Figure 3 . Changes in epigenetic modification and expression of FSTL3 in testes of offspring before and after birth in the control group and the PDE group.

[0043] Figure 3 In the figure: A-F, I: mRNA expression of KDM1B, FSTL3 and TGF-β signaling in testes of offspring before and after birth in the PDE group; G, H, J, K: Protein expression of KDM1B and FSTL3 in testes of offspring before and after birth in the PDE group; L, N: H3K9me2 level in the promoter region of FSTL3 in testes of offspring before and after birth in the PDE group.

[0044] Figure 4 . FSTL3 mediates the inhibition of blood-testis barrier function in Sertoli cells caused by PDE.

[0045] Figure 4In: A-J: mRNA expression of CX43, N-cadherin, E-cadherin, GR, KDM1B, FSTL3 and TGF-β signaling pathway; K: H3K9me2 level of FSTL3 promoter; L-O: Immunofluorescence of CX43, E-cadherin, GR, FSTL3; P: Protein expression of E-cadherin, GR, FSTL3; Q: Interaction detection between GR and KDM1B.

[0046] Figure 5 . GR / KDM1B / FSTL3 cell intervention experiment.

[0047] Figure 5 In: A-D: The decreased expression of Cx43 / E-cadherin genes and proteins were both reversed; E-G: The inhibition of TGF-β signaling pathway caused by dexamethasone was reversed; H, M, K: Silencing KDM1B could reverse the decreased expression of CX43 / E-cadherin genes and proteins caused by dexamethasone; I, L: The low / high expression of H3K9me2 in the FSTL3 promoter region was reversed; N, P, T: After silencing GR, the increased expression of KDM1B and the interaction between GR / KDM1B in Sertoli cells caused by dexamethasone could be reversed; Q, S: The low and high expression of H3K9me2 in the FSTL3 promoter region were reversed; N, O, R: The inhibition of CX43 / E-cadherin expression was reversed.

[0048] Figure 6 . Epigenetic modification and expression changes of FSTL3 in the placenta of the control group and the PDE group.

[0049] Figure 6 In: A: mRNA expression of KDM1B in the control group and the PDE group; B: mRNA expression of FSTL3 in the control group and the PDE group; C: H3K9me2 level in the FSTL3 promoter region of the control group and the PDE group; D: Correlation between testicular FSTL3 expression and placental KDM1B expression; E: Correlation between testicular FSTL3 expression and placental FSTL3 expression; F: Correlation between H3K9me2 level in the testicular FSTL3 promoter region and H3K9me2 level in the placental STL3 promoter region.

[0050] Figure 7 Epigenetic modification and expression changes of FSTL3 in the placenta and maternal PBMC of the control group and the ADT group and their correlation with neonatal testosterone levels.

[0051] Figure 7Chinese: A, B: mRNA expression of KDM1B; C, D: H3K9me2 level in the promoter region of FSTL3; E: Serum testosterone level; F: Correlation between placental FSTL3 mRNA expression and FSTL3 mRNA expression in PBMC; G: Correlation between placental FSTL3 mRNA expression and serum testosterone level; H: Correlation between FSTL3 mRNA expression in PBMC and serum testosterone level; I: Correlation between H3K9me2 level in the promoter region of placental FSTL3 and H3K9me2 level in the promoter region of FSTL3 in PBMC; J: Correlation between serum testosterone level and H3K9me2 level in the promoter region of placental FSTL3; K: Correlation between serum testosterone level and H3K9me2 level in the promoter region of FSTL3 in PBMC. Detailed implementation manners

[0052] The above content of the present invention will be further described in detail below by specific implementation methods in the form of examples. However, it should not be understood that the scope of the above-mentioned subject matter of the present invention is limited to the following examples. Technologies implemented based on the above content of the present invention all belong to the content of the present invention.

[0053]

Example 1

[0054] 1 Experimental animals and treatment

[0055] SPF-grade Wistar rats were purchased from the Hubei Provincial Center for Disease Control and Prevention. The body weight of female rats was controlled at 200 - 240 g, and that of male rats was 260 - 300 g. This animal experiment protocol was strictly implemented in accordance with the relevant principles in the "Guide for the Care and Use of Laboratory Animals" of the National Institutes of Health and was reviewed and approved by the Medical Ethics Committee of the School of Medicine of Wuhan University (approval number: 14016).

[0056] The animals in this study were adaptively fed with ordinary feed at room temperature of 18 - 22°C and humidity of 40% - 60% for one week. Then, they were caged together every night at 18:00 (female:male = 2:1). The female mice were separated the next morning, and vaginal smears were taken with cotton swabs for pregnancy tests. When sperm were observed under the microscope, it was recorded as gestational day 0 (GD0). The pregnant Wistar mice were randomly divided into a control group (C), a low-dose dexamethasone group [PDE(S)], a medium-dose dexamethasone group [PDE(M)], and a high-dose dexamethasone group [PDE(H)], with n = 10 - 12 in each group. From GD9 to GD20, the PDE groups were given subcutaneous injections of 0.1, 0.2, and 0.4 mg / kg·d of dexamethasone respectively, and the C group was given an equal volume of normal saline by subcutaneous injection. Some pregnant mice were sacrificed after anesthesia at GD20 to obtain the testes and placentas of male fetuses. The remaining pregnant mice gave birth naturally, and the male offspring were separately raised until postnatal week (PW) 12 and PW28 and then sacrificed to obtain the testes and sperm. HE staining was used to observe the testicular tissue morphology of the offspring in utero and after birth, the longest diameter and the largest cross-sectional area of the fetal testes were measured and recorded, and the diameters of the seminiferous tubules and the epithelial thickness of the testes of the offspring after birth were statistically analyzed; the number and motility of sperm of the offspring at PW28 were observed and recorded under the microscope; real-time quantitative polymerase-chain-reaction (RT-qPCR) was used to detect the expressions of FSTL3, connexin 43 (Cx43), and E-cadherin, which are genes regulating the blood-testis barrier function, in the testes of the offspring in utero and after birth, and the mRNA expressions of GR, KDM1b, and FSTL3 in the placenta. Immunofluorescence was used to detect the protein expression of E-cadherin in the testes of the offspring in utero, at PW12, and at PW28; Western Blot was used to detect the protein expressions of FSTL3 and E-cadherin in the testes of the offspring after birth; chromatin immunoprecipitation (Chip) PCR was used to detect the level of H3K9me2 in the promoter region of FSTL3 in the testes and placentas of the offspring before and after birth, and a correlation analysis was performed with the corresponding testicular indicators.

[0057] 2 HE staining and immunohistochemistry

[0058] The unilateral testes were fixed in 4% paraformaldehyde for more than 24 h, dehydrated in gradients, and then embedded in paraffin. Sections with a thickness of 4 μm were cut along the horizontal axis. The sections were dewaxed and hydrated with xylene and a series of alcohols, stained with hematoxylin-eosin (H&E), and observed and photographed with an Olympus AH-2 optical microscope (Olympus, Tokyo, Japan) (n = 5). The maximum diameter and area of the fetal testes were measured and recorded; the thickness of the seminiferous epithelium and the diameter of the testes after birth were measured and recorded.

[0059] After dewaxing and rehydrating the sections, microwave treatment was performed for 15 min in a citrate buffer (pH 6.0). Then, they were immersed in 3% H2O2 for 25 min to block catalase. Then, they were blocked with 5% BSA at 37 °C for 30 min, incubated overnight at 4 °C with anti-gr (1:100), anti-fstl3 (1:200), or anti-kdm1b (1:100) antibodies, and then incubated with the corresponding secondary antibody of the appropriate species at 37 °C in the dark for 1 h. After immunofluorescent staining of the sections with DAPI, they were mounted with an anti-fluorescence quencher. TM4 cells were seeded at a density of 1×10 5 / cm 2 and cultured in a six-well plate containing coverslips. The cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 for 20 min, and blocked with 5% BSA at room temperature for 30 min. They were incubated overnight at 4 °C with the primary antibody. After washing with PBS, the corresponding fluorescent secondary antibody of the appropriate species (1:200) was incubated at room temperature for 1 h. Finally, the cell nuclei were stained with DAPI solution, and after mounting with an anti-fluorescence quencher, microscopic examination was performed. The mean integrated optical density (IOD) of each sample in 6 different fields was measured using Image-Pro Plus (version 6.1, Media cybertics, Silver Spring, MD, USA), with 5 different samples in each group.

[0060] Detection of gene expression

[0061] The mRNA sequences of the target gene and the housekeeping gene were downloaded from the NCBI website (http: / / www.ncbi.nlm.nih.gov). The upstream and downstream primers were designed using primer5.0, and nucleotide Blast homology alignment was performed on the NCBI website to confirm the specificity of the amplified fragment. Rat testicular tissues were used for total RNA extraction, which was then reverse-transcribed into cDNA, and quantitative analysis of the expression of related genes was performed by RT-PCR. The primer sequences of each gene are shown in Table 1 below.

[0062] Table 1. Primer sequences of related genes in testicular tissues.

[0063]

[0064] 4 Experimental results

[0065] Compared with the control group, male offspring testes in the PDE group showed morphological abnormalities at different time points before and after birth. In fetal testes, the seminiferous epithelium became thinner, the seminiferous tubules showed abnormal morphology with vacuoles inside, and there were pathological changes such as a decrease in the number of Sertoli cells and germ cells. Moreover, the maximum cross-sectional area and the longest diameter of fetal testes decreased in a dexamethasone exposure dose-dependent manner; in the offspring testes at PW12 and PW28, the diameter of seminiferous tubules and the thickness of seminiferous epithelium were significantly shortened, the number of Sertoli cells decreased and detached, and the number of germ cells at all levels decreased and were disorderly arranged. Further, the present invention observed the effect of PDE on the development of the fetal testis barrier function. The results showed that the fetal testis barrier function genes Cx43, E-cadherin, and N-cadherin in the PDE group decreased in a dexamethasone dose-dependent manner, and Occludin decreased significantly in the low and medium dose groups ( Figure 1 in D), with no obvious change in the high dose group. The present invention detected the changes in the BTB function of the offspring rats after adulthood (PW12 / 28). The results showed that at PW12, the mRNA expressions of CX43, E-cadherin, N-cadherin, and Occludin in the testes of the PDE offspring decreased significantly ( Figure 2 in H); at PW28, the mRNA expressions of CX43 and E-cadherin in the testes of the PDE offspring decreased significantly, and the mRNA expressions of N-cadherin and Occludin showed a decreasing trend ( Figure 2 in H), and consistent results were also obtained by Western Bolt or immunofluorescence detection ( Figure 2 in G, I, J, L). It is suggested that the testicular morphological changes and impaired barrier function caused by PDE can continue from intrauterine to postnatal.

[0066]

Example 2

[0067] 1 Experimental animals and treatments

[0068] The same as in

Example 1

[0069] 2 RT-qPCR experimental procedures: The same as in

Example 1

[0070] 3 Chromatin immunoprecipitation experiment

[0071] Homogenize the tissue or cells and fix them with 1% formaldehyde at 37°C for 15 min to crosslink DNA and its associated proteins. Add glycine (final concentration 0.125 M) and terminate the reaction at 4°C for 10 min. Then shear the DNA to a size of 200 - 800 bp using an ultrasonic lysate. After sonication, centrifuge to collect the sample and add dilution buffer. After mixing, take 10 μL of the supernatant as Input for chromatin normalization. Divide the remaining solution into 200 μL aliquots and add to new Eppendorf tubes. Add Protein G agarose beads resuspended in dilution buffer after treatment, and add 1 μL of anti-H3K9me2 or IgG. Incubate with rotation overnight at 4°C. Centrifuge to collect the immunoprecipitated DNA-protein complex bound to the beads and wash it successively with low-salt, high-salt, LiCl immunocomplex, and Tris-EDTA wash buffers. Elute the DNA-protein complex with the prepared elution buffer (1% SDS, 0.1 M NaHCO3), repeating each elution 2 times. Incubate the samples with 200 μg / mL proteinase K at 65°C overnight, and then purify using a DNA purification kit according to the manufacturer's protocol. Finally, dissolve the purified DNA in 50 μL of elution buffer.

[0072] Detect the purified DNA by qRT-PCR. Using the IgG negative control value as the background, quantify the input value and normalize it to the corresponding value of the immunoprecipitation (IP) sample according to the formula (IP / input = 2^(Ct input DNA - Ct IP DNA)). The primers used are shown in Table 2. Table 2. Primers of follistatin-like protein 3 (FSTL3) used for ChIP-PCR.

[0073]

[0074]

[0075] 4 Experimental results

[0076] 4.1 Changes in KDM1B / FSTL3 in the testes of offspring rats before and after birth caused by PDE and epigenetic alterations in the FSTL3 promoter region

[0077] To explore the mechanism of testicular dysplasia caused by PDE, this invention detected the relevant indicators of the TGF-β signaling pathway and FSTL3 epigenetic alterations in the testes of offspring before and after birth. The results showed that compared with their respective control groups, the mRNA expressions of TGFBR1 and SMAD2 / 3 in the fetal testes of the PDE group decreased in a dose-dependent manner ( Figure 3 in A - C below), and the mRNA expression of FSTL3 increased in a dose-dependent manner ( Figure 3In D). Furthermore, the present invention found that, compared with the control group, the mRNA expressions of TGFBR1 and SMAD2 / 3 in the testes of PW12 / PW28 offspring of the PDE group were significantly decreased ( Figure 3 In F, I), and the mRNA and protein expressions of FSTL3 were significantly increased ( Figure 3 In F-K). It is suggested that the expression of FSTL3 in the testes of PDE offspring continuously increases before and after birth, and the TGF-β signaling pathway is continuously inhibited. Further, the present invention detected the expressions of histone modification enzymes related to fetal testes (including HDACs, etc.) and the changes of histone modification sites related to the FSTL3 promoter region in the offspring. After screening, it was found that the expression of KDM1B in the fetal testes of PDE offspring increased most significantly, and the level of H3K9me2 in the FSTL3 promoter region of the testes at different postnatal and prenatal stages (PW12, PW28) continuously decreased ( Figure 3 In L-N), while no obvious changes occurred in the other acetylation and methylation sites. Moreover, the mRNA and protein expressions of the histone demethylase KDM1B in the testes of PDE before and after birth continuously increased ( Figure 3 In E-K). In summary, it is suggested that PDE can cause the increase of KDM1B expression, the decrease of the H3K9me2 level and the increase of the expression of FSTL3 in the testes of offspring before and after birth, and the inhibition of the TGF-β signaling pathway.

[0078] 4.2 Dexamethasone can cause abnormal function of the blood-testis barrier in Sertoli cells

[0079] In order to further confirm the effect of dexamethasone on Sertoli cells, based on the previous detection in our laboratory, the maternal blood dexamethasone concentration in the PDE model was 0.332 μg / mL (846 nM). In the present invention, TM4 Sertoli cell lines were treated with different concentrations of dexamethasone (0, 20, 100, 500 nM) for 24 h and then relevant detections were carried out. The results of RT-qPCR showed that dexamethasone could cause a concentration-dependent decrease in the expression of BTB-related functional genes Cx43 / E-cadherin ( Figure 4 In A-C); cellular immunofluorescence and WB indicated that dexamethasone could also cause a decrease in the protein expressions of Cx43 / E-cadherin in Sertoli cells ( Figure 4 In K, L, O). It is suggested that dexamethasone can inhibit the expression of Cx43 / E-cadherin and the barrier function in Sertoli cells. Meanwhile, the present invention detected the changes of the indicators related to the mechanism of impaired barrier function after dexamethasone treatment of TM4 cell lines. The results showed that dexamethasone could up-regulate GR in a concentration-dependent manner and promote its activation and nuclear entry ( Figure 4 In D, M, O), increase the gene and protein expressions of KDM1B ( Figure 4 In E, O), and the Co-IP results confirmed that dexamethasone could enhance the protein interaction between GR and KDM1B ( Figure 4in P), accompanied by a decrease in the level of H3K9me2 in the FSTL3 promoter region and an increase in expression ( Figure 4 in F, J, O), and inhibition of the mRNA expression of TGFBR1 / Smad2 / Smad3 in the downstream TGF-β signaling pathway ( Figure 4 in G-I). In summary, these results suggest that dexamethasone can activate GR, enhance the expression and interaction of GR and KDM1B, reduce the level of H3K9me2 in the FSTL3 promoter region and increase its expression, and inhibit the downstream TGF-β pathway and the expression of Cx43 / E-cadherin in Sertoli cells.

[0080] 4.3 The GR / KDM1B / FSTL3 / TGF-β pathway mediates the damage of the blood-testis barrier function in Sertoli cells caused by dexamethasone

[0081] To further confirm that the GR / KDM1B / FSTL3 / TGF-β pathway mediates the damage of the blood-testis barrier function in Sertoli cells caused by dexamethasone, we treated the TM4 cell line with FSTL3 siRNA, KDM1B siRNA, or GR siRNA in combination with 500 nM dexamethasone for verification. The results showed that when FSTL3 siRNA was given, the inhibition of the TGF-β signaling pathway caused by dexamethasone was reversed ( Figure 5 in 5E-G), and the decrease in the expression of Cx43 / E-cadherin genes and proteins was both reversed ( Figure 5 in A-D); further, silencing KDM1B could reverse the decrease in the expression of CX43 / E-cadherin genes and proteins caused by dexamethasone ( Figure 5 in H, M, K), and the low / high expression of H3K9me2 in the FSTL3 promoter region was reversed ( Figure 5 in I, L); finally, after silencing GR, the increase in the expression of KDM1B and the interaction between GR / KDM1B in Sertoli cells caused by dexamethasone could be reversed ( Figure 5 in N, P, T), the low level and high expression of H3K9me2 in the FSTL3 promoter region were reversed ( Figure 5 in Q, S), and the inhibition of CX43 / E-cadherin expression was reversed ( Figure 5 in N, O, R). In summary, dexamethasone can promote the activation and nuclear entry of GR in Sertoli cells, synergistically enhance the action of KDM1B, induce a decrease in H3K9me2 in the FSTL3 promoter region and an increase in expression, inhibit the TGF-β signal, and ultimately lead to a decrease in the expression of Cx43 / E-cadherin in Sertoli cells.

[0082]

Example 3

[0083] 1 Experimental animals and treatments

[0084] It is consistent with [Example 1].

[0085] 2RT-qPCR detection

[0086] The experimental procedure is consistent with [Example 1].

[0087] 3Clinical sample collection and processing

[0088] Collect cases of cesarean section or natural childbirth in the Department of Obstetrics and Gynecology of Zhongnan Hospital of Wuhan University (from June 2017 to June 2018), including a control group not given dexamethasone and a dexamethasone group with single-course treatment. The clinical study has been approved by the Ethics Committee of Zhongnan Hospital of Wuhan University (approval number No: 2016016); inclusion and exclusion criteria: singleton pregnancy, male fetus, pregnancy at 34-42 weeks with or without dexamethasone treatment. Exclude pregnancies with fetal chromosomal and structural abnormalities during pregnancy.

[0089] Specimen collection method: Randomly take 1×0.5×0.5 cm tissue samples from the middle and four corners of the placenta, quickly freeze and store them in a -80 °C refrigerator. At the same time, collect maternal blood and fetal umbilical cord blood, and record the pregnant woman's name and hospital number.

[0090] Clinical information collection: Collect sample clinical information from the hospital's hes system, including the pregnant woman's age, gestational age, fetal Apgar score, fetal birth weight and height, etc.

[0091] 4Separation of maternal and umbilical sera and collection of PBMC

[0092] For the clinical blood samples collected in anticoagulant tubes, centrifuge at 3000 rpm for 10 min at 4 °C, and take the supernatant to obtain serum; aspirate the white substance in the lower layer of the serum and add it to 2 mL of sample diluent, and pipette and mix well; prepare another 15 mL centrifuge tube containing 5 mL of lymphocyte separation solution, and slowly add the diluent obtained in (2) along the tube wall, centrifuge at 400 g for 30 min at 4 °C to obtain a clearly stratified liquid; take a new centrifuge tube, add 1 mL of cleaning solution, aspirate and add the white flocculent substance in the middle of (3), gently pipette and mix well, centrifuge at 700 g for 10 min at 4 °C to obtain the precipitate, which is PBMC; after marking the information, store it in a -80 °C refrigerator.

[0093] 5Experimental results

[0094] 5.1 Abnormalities in testicular morphology and blood-testis barrier function of offspring rats caused by PDE

[0095] Compared with the control group, male offspring testes in the PDE group showed morphological abnormalities at different time points before and after birth. In fetal testes, the seminiferous epithelium became thinner, the seminiferous tubules had abnormal morphology and vacuoles appeared in the tubules, and there were pathological changes such as a decrease in the number of Sertoli cells and germ cells. Moreover, the maximum cross-sectional area and the longest diameter of fetal testes decreased in a dexamethasone-exposure dose-dependent manner; in the offspring testes at PW12 and PW28, the diameter of seminiferous tubules and the thickness of seminiferous epithelium were significantly shortened, the number of Sertoli cells decreased and exfoliated, and the number of germ cells at all levels decreased and were disorderly arranged. Further, the present invention observed the effect of PDE on the development of the fetal testis barrier function. The results showed that the fetal testis barrier function genes Cx43, E-cadherin, and N-cadherin in the PDE group decreased in a dexamethasone dose-dependent manner, and Occludin decreased significantly in the low- and medium-dose groups ( Figure 1 in D), and there was no obvious change in the high-dose group. The present invention detected the changes in the BTB function of offspring rats after adulthood (PW12 / 28). The results showed that at PW12, the mRNA expressions of CX43, E-cadherin, N-cadherin, and Occludin in the PDE offspring testes were significantly decreased ( Figure 2 in H); at PW28, the mRNA expressions of CX43 and E-cadherin in the PDE offspring testes were significantly decreased, and the mRNA expressions of N-cadherin and Occludin showed a decreasing trend ( Figure 2 in H), and consistent results were also obtained by Western Bolt or immunofluorescence detection ( Figure 2 in G, I, J, L). It is suggested that the testicular morphological changes and impaired barrier function caused by PDE can continue from intrauterine to postnatal.

[0096] 5.2 PDE can cause a decrease in the level of H3K9me2 and an increase in the expression of the FSTL3 promoter region in rat placenta, and it is positively correlated with the corresponding indicators in fetal testes. In order to further explore whether the epigenetic and expression changes of FSTL3 in the placenta of the PDE rat model are consistent with those in the testis, the present invention detected the expressions of KDM1B and FSTL3 in the rat placenta and the histone modification changes in the FSTL3 promoter region. First, the sequencing results showed that compared with the control group, the mRNA expressions of KDM1B and FSTL3 in the placenta of the PDE group were increased. At the same time, the expressions of KDM1B and FSTL3 in the placenta of the PDE group increased in a dose-dependent manner, and the level of H3K9me2 in the FSTL3 promoter region decreased ( Figure 6 in A-C), which was consistent with the changes in the corresponding indicators in testicular tissue. Then, the present invention analyzed the correlation between the changes in the epigenetic and expression regulation-related indicators of FSTL3 in the placenta, maternal blood PBMC, and testicular tissue. The analysis results showed that the level of H3K9me2 in the FSTL3 promoter region and its expression changes in the placenta of the PDE group were significantly positively correlated with the corresponding indicators in the testis, while there was no obvious correlation in the control group.Figure 6 In D, E); meanwhile, the levels of H3K9me2 in the promoter region of FSTL3 and its expression changes in PBMC of maternal blood in the PDE group were significantly positively correlated with the corresponding indicators of the testis, while there was no obvious correlation in the control group. There was no obvious correlation in the control group. Figure 6 In E, F). The above results suggest that PDE can cause a decrease in the level of H3K9me2 in the promoter region of FSTL3 in PBMC of maternal blood and placenta and an increase in its expression in rats, and there is a good correlation with the corresponding indicators of the fetal testis of the offspring.

[0097] 5.3 Prenatal application of dexamethasone can cause epigenetic modification and expression changes of FSTL3 in human placenta and PBMC of maternal blood and is related to umbilical cord blood testosterone

[0098] Previous studies have confirmed that PBMC of maternal blood can be used to predict the development of offspring, and the placenta, as a link between the mother and the fetus, is the best organ for predicting the development of the fetus. Therefore, in order to further confirm whether the level of H3K9me2 in the promoter region of FSTL3 and its expression can be used as early warning markers for fetal testicular dysplasia in the population, the present invention collected PBMC of maternal blood and placenta specimens from clinical ADT. First, the present invention statistically analyzed the clinical characteristics of the included research subjects. Compared with the control group, no obvious differences were found in the indicators of the ADT group (including maternal age, weight, gestational week number; fetal birth weight, body length). Further, the present invention detected the levels of H3K9me2 in the promoter region of FSTL3 and mRNA expression in the placenta and PBMC of maternal blood in the control group and the ADT group, and the testosterone level in umbilical cord blood. The results showed that the umbilical cord blood testosterone level in the ADT group was significantly lower than that in the control group Figure 7 In E), which preliminarily suggests that ADT may cause abnormal development of the fetal testis of the offspring. In addition, the level of H3K9me2 in the promoter region of FSTL3 in the placenta and PBMC of maternal blood in the ADT group decreased / expression increased Figure 7 In A-D). Further, serial correlation analysis also found that the mRNA expression of FSTL3 in the placenta and PBMC of maternal blood in the ADT group was positively correlated, and was negatively correlated with the corresponding umbilical cord blood testosterone respectively Figure 7 In F-H); the levels of H3K9me2 in the promoter region of FSTL3 in the placenta and PBMC of maternal blood in the ADT group were positively correlated, and were both positively correlated with the androgen level in umbilical cord blood Figure 7 In I-K). It is suggested that ADT can reduce the umbilical cord blood testosterone level, cause a decrease in the level of H3K9me2 in the promoter region of FSTL3 in the placenta and PBMC of maternal blood and an increase in its expression, and there is a good correlation between the two.

[0099] In summary, fetal testicular dysplasia may be related to low levels / high expression of H3K9me2 in the promoter region of FSTL3 and originates from the uterus. It has been confirmed by animal and human applications that the changes in the levels and expression of H3K9me2 in the promoter region of FSTL3 in the placenta and maternal blood PBMCs are significantly correlated with the androgen levels in the umbilical cord blood of male fetuses. The present invention has first successfully constructed a model of fetal testicular dysplasia and explored and confirmed possible early warning targets for fetal testicular dysplasia through animal and human applications.

Claims

1. Use of maternal blood PBMC FSTL3 or placental FSTL3 in the preparation of a warning marker for fetal testicular dysplasia under dexamethasone exposure.

2. Use of maternal blood PBMC FSTL3 or placental FSTL3 in the preparation of a kit for screening early warning of fetal-origin adult testicular dysplasia under dexamethasone exposure, characterized in that: If the expression of the FSTL3 gene is elevated in maternal blood PBMC or placenta, and at the same time the level of H3K9me2 in the promoter region of the FSTL3 gene is decreased, there is a risk of fetal testicular dysplasia. The kit contains reagents for detecting the expression of the FSTL3 gene and reagents for detecting the level of H3K9me2 in the promoter region of FSTL3. The reagent for detecting the expression of the FSTL3 gene contains a primer pair for amplifying the FSTL3 gene and a primer pair for detecting the level of H3K9me2 in the promoter region of the FSTL3 gene. The primer pair for amplifying the FSTL3 gene is shown as SEQ ID No.11, SEQ ID No.12, SEQ ID No.15, and SEQ ID No.16; the primer pair for detecting the level of H3K9me2 in the promoter region of the FSTL3 gene is shown as SEQ ID No.17, SEQ ID No.18, SEQ ID No.19, and SEQ ID No.

20.

3. Use of a reagent for detecting the expression of the FSTL3 gene in the preparation of a product for warning fetal testicular dysplasia under dexamethasone exposure, characterized in that: The product includes: a product for diagnosing fetal testicular dysplasia by detecting FSTL3 expression through RT-PCR, Western Blot, or Chip-PCR; the product includes at least a pair of primers for specifically amplifying the FSTL3 gene, or a specific antibody against the FSTL3 protein, or a reagent for detecting the level of H3K9me2 in the promoter region of FSTL3.

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