A 22-amino-acid peptide regulates tight junctions through occludin and cell apoptosis

Abstract

Occludin is a structural protein of tight junctions (TJ) in the blood–testis barrier (BTB). A 22-amino-acid peptide (22AA) in the second extracellular loop can reversibly regulate TJ, but its regulatory mechanism is unknown. In this study, a 22AA-induced TJ destruction animal model was constructed to investigate the effect of 22AA on Sertoli cells (SCs) and spermatid counts and cell apoptosis at different time points using a multiplex immunofluorescence technique. The effect of 22AA on the location and distribution of occludin was analyzed via dual confocal fluorescence microscope. Western blotting was used to analyze dynamic changes in occludin expression. Real-time RT-PCR was used to analyze miR-122-5p expression changes. Sperm density counts and mating methods were used to analyze the effect of 22AA on fertility in mice. The results showed that 22AA promoted SC and spermatid apoptosis, downregulated occludin, upregulated miR-122-5p, and decreased sperm density and litter size before 27 days (27D). After 27D, the expression of occludin increased again, miR-122-5p expression decreased again, both sperm density and litter size returned to normal, apoptosis stopped, and spermatogenesis began to recover. Therefore, it can be concluded that 22AA can destroy TJ by downregulating occludin and inducing cell apoptosis. After 27D, TJ and spermatogenesis functions return to normal.

Bibliometric and visual analysis of blood-testis barrier research

“…The BTB loses its integrity when the tight link between Sertoli cells is broken, resulting in spermatogenesis problems. Some important key proteins or components are constantly being discovered, such as PTBP1 ( Yang et al, 2021 ), noncollagenase domain (NC1)-peptide ( Liu et al, 2021 ), a 22-amino-acid peptide (22AA) ( Zhu et al, 2020 ), ubiquitously expressed transcript ( Thomas et al, 2020 ), TBC1D20 ( Thomas et al, 2020 ), SENP3 ( Wu et al, 2017 ), BT-IgSF ( Pelz et al, 2017 ), Pin1 ( Islam et al, 2017 ), ANXA2 ( Chojnacka et al, 2017 ), AKAP9 ( Venkatesh et al, 2016 ), GATA4 ( Schrade et al, 2016 ), and intercellular adhesion molecule-1 ( Mruk, 2016 ), that are required for the structural integrity of the BTB. Regulation of BTB dynamics is also an important research area.…”

miR-122-5p regulates the tight junction of the blood-testis barrier of mice via occludin

“…1 (b). The cells were filled with WT1 which was one of marker protein of SCs [ 26 ]. The results suggested that the isolated cells were SCs.…”

“…The slides were block with peroxidase for 5 min and washed as above. The slides were incubated for 30 min with the primary anti-Wilms tumor protein1(anti-WT1) [ 26 ] (1:100 dilution, abcam, USA). After washed three times with phosphate buffer saline (PBS) for 3 min each, the horseradish peroxidase-labeled secondary antibody (1:800 dilution, abcam, USA) was added dropwise, then incubated at 37 °C for 30 min, followed by the substrate-chromogen solution (3,3′-diaminobenzidine), and finally counter stained with hematoxylin.…”

“…The reaction conditions for qRT-PCR included initial denaturation at 94 °C for 4 min followed by 35 cycles of denaturation at 94 °C for 20 s, annealing at 60 °C for 30 s and extension at 72 °C for 30 s. U6 was used as internal control. The expression ratio was calculated using the 2 - △△Ct method [ 26 ].…”