Journal Article

mTORC1/rpS6 signaling complex that modifies BTB transport function—An in vivo study using the adjudin model

Studies have shown that the mTORC1/rpS6 signaling cascade regulates Sertoli cell blood-testis barrier (BTB) dynamics. For instance, specific inhibition of mTORC1 by treating Sertoli cells with rapamycin promotes the Sertoli cell barrier, making it "tighter." However, activation of mTORC1 by overexpressing a full-length rpS6 cDNA clone (i.e., rpS6-WT, wild type) in Sertoli cells promotes BTB remodeling, making the barrier "leaky." Also, there is an increase in rpS6 and p-rpS6 (phosphorylated and activated rpS6) expression at the BTB in testes at stage VIII-IX of the epithelial cycle, and coincides with BTB remodeling to support the transport of preleptotene spermatocytes across the barrier, illustrating rpS6 is a BTB modifying signaling protein. Herein, we used a constitutively active, quadruple phosphomimetic, mutant of rpS6, namely p-rpS6-MT of p-rpS6-S235E/S236E/S240E/S244E wherein Ser (S) was converted to Glu (E) at amino acid residues 235, 236, 240 and 244 from the N-terminus by site-directed mutagenesis, for its overexpression in rat testes in vivo using the Polyplus in vivo jet-PEI transfection reagent with high transfection efficiency. Overexpression of this p-rpS6-MT was capable of inducing BTB remodeling, making the barrier "leaky". This thus promoted the entry of the non-hormonal male contraceptive adjudin into the adluminal compartment in the seminiferous epithelium to induce germ cell exfoliation. Combined overexpression of p-rpS6-MT with a male contraceptive (e.g., adjudin) potentiated the drug bioavailability by modifying the BTB. This approach thus lowers intrinsic drug toxicity due to a reduced drug dose, further characterizing the biology of BTB transport function.