MiR-142-3p Inhibits TGF-β3-Induced Blood-Testis Barrier Impairment by Targeting Lethal Giant Larvae Homolog 2

Abstract

Background/Aims: Transforming growth factor-β3 (TGF-β3) has been proved to perturb the blood-testis barrier (BTB) by accelerating junction protein endocytosis in Sertoli cells (SCs) to accommodate the traversing of preleptotene spermatocytes across the BTB around stage VIII in rat. Yet the molecular network underlying the impairment of TGF-β3 on BTB integrity is not fully elucidated. Our study herein was designed to investigate the participation of microRNA-142-3p (miR-142-3p), which has been reported to affect TGF-β3 signaling via different pathways, during BTB dynamics and the corresponding mechanisms. Methods: MiRNA mimic or agomiRNA was co-administered with or without TGF-β3 in the cultured SCs or in the rat testis. The SC permeability barrier function was reflected by measuring the transepithelial resistance (TER) and the permeability of the sodium fluorescein (Na-F). The BTB integrity was detected by the permeation of biotin. A luciferase reporter assay was used to testify the potential target of miR-142-3p, lethal giant larvae 2 (Lgl2). Laser capture microdissection (LCM) was applied to acquire cell components of different stages of seminiferious tubules, followed by detection of the expression levels of miR-142-3p, TGF-β3, and Lgl2 by qPCR. The SC barrier function was also detected as above in the presence of TGF-β3 after Lgl2 knockdown. Results: We revealed a reversion of TGF-β3-induced BTB impairment after miR-142-3p treatment both in vitro and in vivo. Meanwhile, the activation of Cdc42 and reduction in occludin aroused by TGF-β3 were also reversed by miR-142-3p. The predicted binding of miR-142-3p with 3’-untranslated region (3’-UTR) of Lgl2, was verified by the luciferase assay. Moreover, an increased Lgl2 level in TGF-β3-treated SCs was found and correlated stage-specific expressions of TGF-β3, miR-142-3p, and Lgl2 were revealed. Knockdown of Lgl2 in SCs was shown to partially antagonize the BTB disruption mediated by TGF-β3. Conclusions: Collectively, our results suggest a resistance of miR-142-3p on the BTB impairment caused by TGF-β3 during the seminiferous epithelial cycle by targeting Lgl2.

TGF‐β3 induces lactate production in Sertoli cell through inhibiting the Notch pathway

“…We have previously reported the up-regulation of Lgl2 by TGF-β3 in primary cultured SCs and rat testes. 23 Herein, an increased level of Lgl2 was also detected in both primary SCs and TM4 cells treated by TGF-β3 (Figure 2). Overall, 3-ng/ml TGF-β3 treatment for 24 h had the maximal influence on lactate secretion in SC in vitro, which is the ultimate indicator of glycolysis and what we are most concerned of.…”

“…54,55 Recently, Lgl2 has been reported in our study to play an important role in TGF-β3-mediated regulation of BTB Furthermore, the over-activation of Notch signaling pathway in rat testis by re-Jagged1 was shown in this study to result in resumable hypospermatogenesis. This may take place because the abundantly expressed TGF-β3 at stage VIII of the seminiferous cycle facilitates not only the migration of spermatocyte by inducing BTB rebuilding, 23 but also the lactate production of SCs by restraining Notch signaling pathway. Therefore, the exogenous over-activation of Notch signaling inhibited the lactate production of SCs and thus led to a short supply of energy source for spermatogenesis, resulting in a declined count of mature spermatids.…”

“…Lysates from primary cultured SCs, TM4 cells, or testis homogenate after corresponding treatments were prepared as described earlier 23 and the protein concentrations were measured using a Pierce BCA Protein Assay Kit (Thermo Scientific) according to the manufacturer's manual. After denaturation at 100…”

“…Based on the previous observations in cultured SCs and the known stage-specific expression pattern of TGF-β3, that is, peaking at stages VII-VIII during the seminiferous epithelial cycle, 23,38 7A). Next, we collected SC components of different spermatogenic stages using LCM and detected the expression of Jagged1 by qPCR.…”

Retinoic acid can improve autophagy through depression of the PI3K-Akt-mTOR signaling pathway via RARα to restore spermatogenesis in cryptorchid infertile rats

“…Procedures were conducted as previously described. 22 Briefly, PND 60 rats from different groups ( n = 4, each group) were anesthetized and injected with 40 μL of 10 mg/mL EZ-Link Sulfo–NHS–LC-Biotin (Pierce Biotechnology, Rockford, IL, USA), which was dissolved in PBS containing 1 mM CaCl 2 , into the interstitium of cryptorchid testes in the Flu-only group and the Flu + RA group. The testes were dissected and removed after 30 min, and then frozen in liquid nitrogen.…”