Abcb1a and Abcb1b genes function differentially in blood\u2013testis barrier dynamics in the rat

Linlin Su,Bin Zhao,Fangfang Yang,Dahai Hu,Yan C Cheng,Will M Lee,Min Zhang,Yixun Liu,Daishu Han

doi:10.1038/cddis.2017.435

Abstract

During spermatogenesis, immature spermatocytes traverse the blood–testis barrier (BTB) and enter the apical apartment of seminiferous epithelium for further development. This course involves extensive junction disassembly and reassembly at the BTB. P-glycoprotein is known to be coded by two genes in rodents, namely Abcb1a and Abcb1b. Our previous studies showed that simultaneously silencing Abcb1a and Abcb1b genes in Sertoli cells impeded BTB integrity. However, the individual role of Abcb1a and Abcb1b in regulating BTB dynamics remains uninvestigated. Here, single knockdown of Abcb1a by RNAi impeded the in vitro Sertoli cell permeability barrier via redistributing TJ proteins, accelerating endocytosis, and affecting endocytic vesicle-mediated protein transportation that undermined Sertoli cell barrier. F5-peptide model was used to induce cell junction disruption and subsequent restructuring in primary Sertoli cells. F5-peptide perturbed this barrier, but its removal allowed barrier ‘resealing’. Abcb1b knockdown was found to inhibit barrier resealing following F5-peptide removal by suppressing the restore of the expression and distribution of junction proteins at BTB, and reducing the migration of internalized junction proteins back to Sertoli cell interface. In summary, Abcb1a is critical in maintaining BTB integrity, while Abcb1b is crucial for junction reassembly at the BTB.

Highlights

At stage VIII of the seminiferous epithelial cycle in adult rat testis, preleptotene spermatocytes migrate across the blood– testis barrier (BTB) from basal into apical apartment.[1]
We examined the individual role of Abcb1a or Abcb1b on BTB dynamics, especially focused on disassembly and reassembly of Sertoli cell–tight junction (TJ) barrier by using RNAi combined with F5-peptide model, which was found to reversibly disrupt the BTB integrity both in vivo and in vitro.[22]
Afterwards, the reaction mixture was withdrawn, Sertoli cells were washed with plain medium three times, incubated for another 1 to 2 days, and harvested for real-time RCR (Figures 1a and b), immunoblot (Figure 1c), trans-epithelial electrical resistance (TER) measurement (Figure 1d) and immunofluorescence analysis (Figure 1e). qPCR results revealed the efficiency of RNAi transfection, presenting an ~ 80% decrease in Abcb1a mRNA level (Figure 1a) and an ~ 70% decrease in Abcb1b mRNA level (Figure 1b)

Summary

Introduction

At stage VIII of the seminiferous epithelial cycle in adult rat testis, preleptotene spermatocytes migrate across the blood– testis barrier (BTB) from basal into apical apartment.[1] This course involves extensive junction disruption and restructuring at Sertoli cell–cell interface to facilitate germ cell movement.[2] In the the immunological integrity of the BTB has to be maintained at all times in order to separate postmeiotic germ cell antigens from the immune system. P-glycoprotein deletion by cosilencing Abcb1a and Abcb1b in Sertoli cells significantly impaired TJ barrier function, affected occludin phosphorylation by the activation of focal adhesion kinase (FAK), and disturbed the endocytosis of junctional complexes that further destabilized barrier function.[5]. The testis is likely to take a similar way to guide junction reconstruction at Sertoli cell surface since endocytosis was found to be involved in the course of spermiation at the interface of Sertoli cell and late spermatid.[15,16] primary Sertoli cells could form an in vitro BTB that features a functional TJ permeability barrier.[17,18,19,20] this in vitro Sertoli cell system was employed to examine the effects of Abcb1a or Abcb1b knockdown on the kinetics of endocytosis and recycling of integral membrane proteins at the BTB.[21]

Methods

Results

Conclusion