Prenatal dexamethasone exposure impairs rat blood-testis barrier function and sperm quality in adult offspring via GR/KDM1B/FSTL3/TGFβ signaling

Abstract

Both epidemiological and animal studies suggest that adverse environment during pregnancy can change the offspring development programming, but it is difficult to achieve prenatal early warning. In this study we investigated the impact of prenatal dexamethasone exposure (PDE) on sperm quality and function of blood-testis barrier (BTB) in adult offspring and the underlying mechanisms. Pregnant rats were injected with dexamethasone (0.1, 0.2 and 0.4 mg·kg^-1·d^-1, s.c.) from GD9 to GD20. After weaning (PW4), the pups were fed with lab chow. At PW12 and PW28, the male offspring were euthanized to collect blood and testes samples. We showed that PDE significantly decreased sperm quality (including quantity and motility) in male offspring, which was associated with impaired BTB and decreased CX43/E-cadherin expression in the testis. We demonstrated that PDE induced morphological abnormalities of fetal testicle and Sertoli cell development originated from intrauterine. By tracing to fetal testicular Sertoli cells, we found that PDE dose-dependently increased expression of histone lysine demethylases (KDM1B), decreasing histone 3 lysine 9 dimethylation (H3K9me2) levels of follistatin-like-3 (FSTL3) promoter region and increased FSTL3 expression, and inhibited TGFβ signaling and CX43/E-cadherin expression in offspring before and after birth. These results were validated in TM4 Sertoli cells following dexamethasone treatment. Meanwhile, the H3K9me2 levels of FSTL3 promoter in maternal peripheral blood mononuclear cell (PBMC) and placenta were decreased and its expression increased, which was positively correlated with the changes in offspring testis. Based on analysis of human samples, we found that the H3K9me2 levels of FSTL3 promoter in maternal blood PBMC and placenta were positively correlated with fetal blood testosterone levels after prenatal dexamethasone exposure. We conclude that PDE can reduce sperm quality in adult offspring rats, which is related to the damage of testis BTB via epigenetic modification and change of FSTL3 expression in Sertoli cells. The H3K9me2 levels of the FSTL3 promoter and its expression in the maternal blood PBMC can be used as a prenatal warning marker for fetal testicular dysplasia.

Keywords: FSTL3; H3K9me2; blood-testis barrier; prenatal dexamethasone exposure; prenatal warning markers; testicular dysplasia.

Fig. 1. Changes of testicular morphology and barrier function at PW12 and PW28 caused by PDE.

a Sperm mobility; b Sperm counts; c Abnormal sperm rate; d Sperm morphology; e, f Testicular morphology (H&E, 100×, 200×); g Testicular diameter; h Spermatogenic epithelial thickness; i, l The mRNA expression of CX43, E-cadherin, N-cadherin, and Occludin; j, m CX43 protein expression (CX43 stained by green, 200×); k, n, o E-cadherin protein expression by Western blotting. Mean ± SEM. ^*P < 0.05, ^**P < 0.01 vs. Con. PDE prenatal dexamethasone exposure, PW postnatal week, CX43 connexin 43, Con control.

Fig. 2. Changes in fetal testicular morphology and barrier function caused by PDE.

a Testicular morphology (H&E, 100×, 200×); b Testicular diameter; c Testicular area;　d seminiferous tubule　diameter;　e–h The mRNA expression of CX43, E-cadherin, N-cadherin, and Occludin; i CX43 protein expression (CX43 stained by green, 200×). Mean ± SEM. ^*P < 0.05, ^**P < 0.01 vs. Con. PDE prenatal dexamethasone exposure, CX43 connexin 43, Con control.

Fig. 3. Changes of KDM1B, FSTL3 and TGFβ signal pathway in tests induced by PDE before and after birth.

a–f, i The mRNA expression of KDM1B, FSTL3 and TGFβ pathway; g, h, j, k KDM1B and FSTL3 protein expression; l–n H3K9me2 level of FSTL3 promoter. Mean ± SEM. ^*P < 0.05, ^**P < 0.01 vs. Con. KDM1B histone lysine demethylases, FSTL3 follistatin-like protein 3, TGFβ Transforming growth factor-β, PDE prenatal dexamethasone exposure, H3K9me2 histone3 lysine9 dimethylation, Con control, GD gestational day, PW postnatal week, Con control.

Fig. 4. Dexamethasone inhibits the blood-testis barrier function of TM4 cells.

a–j The mRNA expression of CX43, E-cadherin, N-cadherin,GR, KDM1B, FSTL3, and TGFβ pathway,  H3K9me2 level of the FSTL3 promoter; k-n  CX43, E-cadherin, GR, FSTL3 protein expression (CX43, E-cadherin, FSTL3 stained by green; GR stained by red, 200×); o E-cadherin, KDM1B, GR, FSTL3 protein expression; p Combination degree of GR and KDM1B. Mean ± SEM. ^*P < 0.05, ^**P < 0.01 vs. Con. Cx43 connexin 43, GR glucocorticoid receptor, KDM1B histone lysine demethylases, FSTL3 follistatin-like protein 3, TGFβ transforming growth factor β, H3K9me2 histone 3 lysine 9 dimethylation, DEX dexamethasone, IF immunofluorescence, Con control.

Fig. 5. GR/KDM1B/FSTL3/TGFβ mediates dexamethasone-induced inhibition of CX43 and N-cadherin expression in TM4 cells.

a, h, o The mRNA expression of CX43; b The mRNA expression of N-cadherin; e–g, j The mRNA expression of TGFβ pathway; i, q The mRNA expression of FSTL3; p The mRNA expression of KDM1B; c, m, n The protein expression of E-cadherin, KDM1B or FSTL3; l, s H3K9me2 level of the FSTL3 promoter; t Combination degree of GR and KDM1B; d, k, r The protein expression of CX43 and FSTL3 (200×). Mean ± SEM. ^*P < 0.05, ^**P < 0.01 vs. Con. GR glucocorticoid receptor, Cx43 connexin 43, KDM1B histone lysine demethylases, FSTL3 follistatin-like protein 3, TGFβ transforming growth factor-β, H3K9me2 histone3 lysine9 dimethylation, DEX dexamethasone, IF immunofluorescence, Con control.

Fig. 6. Changes of KDM1B, FSTL3 in the placenta, maternal PBMC and Bewo cell after dexamethasone treatment (ADT).

a, c The gene expression of KDM1B; b, d, g The gene expression of FSTL3; e, f, h H3K9me2 level of the FSTL3 promoter; i Correlation of FSTL3 mRNA expression between the placenta and corresponding testis; j Correlation of H3K9me level in FSTL3 promoter between the placenta/PBMC and corresponding testis; k Correlation of FSTL3 mRNA expression between the maternal PBMC and corresponding testis; l Correlation of H3K9me level in FSTL3 promoter between the maternal PBMC and corresponding testis. Mean ± SEM. ^*P < 0.05, ^**P < 0.01 vs. Con. KDM1B histone lysine demethylases, FSTL3 follistatin-like protein 3, PDE prenatal dexamethasone exposure, H3K9me2 histone 3 lysine 9 dimethylation, Con control.

Fig. 7. Placenta and maternal PBMC FSTL3 promoter H3K9me2 and expression level and male neonatal plasma testosterone and the correlations between them respectively.

a, b The gene expression of FSTL3; c, d H3K9me2 level of the FSTL3 promoter; e Serum testosterone level; f Correlation of FSTL3 gene expression between placental and maternal PBMC; g Correlation of FSTL3 promoter H3K9me2 level between placental and maternal PBMC; h Correlation between placental FSTL3 gene expression and fetal plasma testosterone; i Correlation between maternal PBMC FSTL3 gene expression and fetal plasma testosterone level; j Correlation between placental FSTL3 promoter H3K9me2 level and fetal plasma testosterone; k Correlation between maternal PBMC FSTL3 promoter H3K9me2 level and fetal plasma testosterone level. Mean ± SEM. ^*P < 0.05, ^**P < 0.01 vs. Con. PBMC peripheral blood mononuclear cell, FSTL3 follistatin-like protein 3, H3K9me2 histone3 lysine9 dimethylation, ADT antenatal dexamethasone treatment, Con control.

Fig. 8. Molecular mechanism of impaired blood-testis barrier function induced by prenatal dexamethasone exposure.

GR glucocorticoid receptor, KDM1B histone lysine demethylases, FSTL3 follistatin-like protein 3, H3K9me2 histone 3 lysine 9 dimethylation, TGFβ Transforming growth factor-β, CX43 connexin 43.