TGF-?3 Regulates The Blood-testis Barrier And The Lactate Secretion Of Sertoli Cells In Rats Through Lgl2

Objective: In the mammallian seminiferous epithelium,Sertoli cells play an important role in spermatogenesis.Adjacent Sertoli cells form blood-testis barrier(BTB)by cell junctions.BTB divides the seminiferous epithelium into basal compartment and adluminal compartment,creating an independent environment for the spermatocyte differentiation.In stage VIII of the seminiferous epithelial cycle,the movement of preleptotene spermatocyte from the basal compartment into the adluminal compartment involves disassembling of the old BTB ahead and rebuilding of the new BTB behind.Transforming growth factor-?3(TGF-?3)secreted by Sertoli cells and germ cells has been proved to induce BTB disruption via different ways in order to facilitate the migration of spermatocyte.However,the melacular mechanism of the regulation of BTB by TGF-?3 has not been fully understood.Our group has previously screened out those differentially expressed miRNAs in Sertoli cells before and after TGF-?3 treatment by miRNA array,amount which miR-142-3p has been reported to participate in TGF-?-mediated signaling pathways in various of cellular activities.We have predicted using bioinformatic softwares that one of the mammallian homologs of Lethal giant larvae(Lgl)Lgl2 might be a direct target of miR-142-3p,and thus speculated that miR-142-3p might affect the TGF-b3-induced BTB disruption by targeting Lgl2.In the first part of the present study we sought to explore the role of miR-142-3p in the regulation of BTB by TGF-b3 and investigate its melacular mechanism.Lactate is the main energy substrate of male germ cells and plays a critical role in germ cell energy metabolism and spermatogenesis.In addition to the formation of BTB,Sertoli cells are also the main source of lactate in the testis.The glucose metabolism pattern in Sertoli cells is similar to the "Warburg effect" which is prevalent in tumor cells.Sertoli cells secrete large amounts of lactate through aerobic glycolysis with a high flux,providing energy and nutritions for germ cells.TGF-b promotes aerobic glycolysis and lactate secretion in a variety of tumor and non-tumor cells,but its molecular mechanism remains largely uninvestigated.In the second part of the present study,based on the results of the first part,we sought to investigate the regulation effect of TGF-b3 on glucose metabolism and lactate secretion and explore the underlying melacular mechanisms.Methods: 1.The rat seminiferous tubule peripheral cell layers at different stages of seminiferous epithelial cycle were collected using laser capture microdissection(LCM)and the expression levels of TGF-b3,miR-142-3p and Lgl2 were detected by real-time quantitative PCR(qPCR)and Western blot.2.Rat primary Sertoli cells were cultured and miRNA mimic was used to overexpress miR-142-3p followed by a treatment with recombinant human TGF-b3 for 24 h.The trans-epithelial resistance and the sodium fluorescein permeability of the Sertoli cells were measured.The expression levels of Cdc42,Cdc42-GTP,occludin and the type I receptor of TGF-b3 were measured by immunoprecipitation and Western blot.The protein expression of occludin was also detected by immunofluorescence.3.AgomiRNA mimic was injected in the rat testes to overexpress miR-142-3p followed by the injection of recombinant human TGF-b3.The BTB integrity was assessed using Biotin as an indicator.The expression levels of miR-142-3p and occludin were detected by qPCR and Western blot,respectively.4.Mutations were generated at the predicted miR-142-3p binding site in Lgl2 gene sequence and the binding of miR-142-3p to Lgl2 was testified by luciferase reporter assay.Then the protein and mRNA levels of Lgl2 after enhancing or inhibiting the expression of miR-142-3p in the rat primary Sertoli cells were measured using Western blot and qPCR,respectively.5.Rat primary Sertoli cells were cultured and miRNA mimic was used to overexpress miR-142-3p followed by a treatment with recombinant human TGF-b3 for 24 h.The mRNA and protein levels of Lgl2 were detected by qPCR and Western blot,respectively.6.Rat primary Sertoli cells were cultured and the expression of Lgl2 was silenced using siRNA followed by a treatment with recombinant human TGF-b3 for 24 h.The trans-epithelial resistance and the sodium fluorescein permeability of the Sertoli cells were measured.The expression levels of Cdc42,Cdc42-GTP,occludin and Lgl2 were measured by immunoprecipitation and Western blot.The protein expression of occludin was also detected by immunofluorescence.7.Rat primary Sertoli cells and TM4 mouse Sertoli cell lines were cultured and treated with recombinant human TGF-b3 with different concentrations for 6-24 h.The amount of lactate secretion,glucose and Glutamine(Gln)consumption,and the activity of Lactate dehydrogenase(LDH),Glutaminase(Gls)and mitochondria were detected.The expression of proteins related to glucose metabolism and main signaling pathways were detected by Western Blot.8.Rat primary Sertoli cells and TM4 cells were cultured and the expression of Lgl2 was silenced using siRNA followed by a treatment with recombinant human TGF-b3 for 24 h.The amount of lactate secretion,glucose and Gln consumption,and the activity of LDH,Gls and mitochondria were detected.The expression of proteins related to glucose metabolism and main signaling pathways were detected by Western Blot.9.Rat primary Sertoli cells and TM4 cells were cultured and the expression of Lgl2 was silenced using siRNA followed by a treatment with DAPT for 48 h.The amount of lactate secretion and the activity of LDH and mitochondria were detected.The expression of proteins related to glucose metabolism and Notch signaling pathway were detected by Western Blot.The trans-epithelial resistance of the rat primary Sertoli cells was measured.10.The expression of Jagged1 at different stages of rat seminiferous epithelial cycle was detected by immunohistochemistry.The rat seminiferous tubule peripheral cell layers at different stages of seminiferous epithelial cycle were collected using LCM and the expression level of Jagged1 mRNA was detected by qPCR.11.Recombinant rat Jagged1 was injected in the rat testes once a day for three days and the spermatogenesis was detected by HE stain on day 3,5,10 and 20 after injection.Results: 1.TGF-?3 and miR-142-3p have opposite expression trends in all stages of seminiferous epithelial cycle.In stage VII-VIII TGF-?3 reaches the highest expression level,while miR-142-3p expresses minimally in the same period.The expression level of Lgl2 protein decreased significantly after stage VIII followed by a significant reduction of its mRNA level in stage IV-VI.2.The overexpression of miR-142-3p significantly inhibited the barrier function impairment of Sertoli cells,the increase of Cdc42 activity and the reduction of occludin expression induced by TGF-?3.3.The overexpression of miR-142-3p significantly inhibited the barrier function impairment of BTB and the reduction of occludin expression induced by TGF-?3 in vivo.4.miR-142-3p is able to specifically bind to the 3'UTR of Lgl2 and inhibits the protein expression of Lgl2 at translational level.5.TGF-?3 significantly enhanced the protein expression of Lgl2 in Sertoli cells,while miR-142-3p inhibited the increase of Lgl2 protein expression induced by TGF-?3.6.Lgl2 silencing partially antagonized the barrier function impairment of BTB and the reduction of occludin expression in Sertoli cells,but had no significant effect on Cdc42 expression and activity.7.TGF-?3 treatment significantly inhibited the activity of mitochondria,enhanced glycolysis,Gln catabolism and lactate secretion,activated the MAPK signaling pathway and inhibited the Akt and Notch signaling pathways in Sertoli cells.8.The Lgl2 silencing in Sertoli cells antagonized the inhibition of mitochondria and Notch signaling pathway and the enhancement of glycolysis and lactate secretion caused by TGF-?3,but could not alter the effects of TGF-?3 on Gln catabolism and Akt and MAPK signaling pathway.9.The inhibition of Notch signaling pathway in Sertoli cells antagonized the increase of mitochondrial activity and the reduction of glycolysis and lactate secretion caused by Lgl2 silencing,but it has no effect on the permeability of Sertoli cells.10.The expression trend of Jagged1 was opposite to that of TGF-?3 in all stages of seminiferous epithelial cycle.11.The activation of Notch signaling pathway induced by recombinant Jagged1 led to reversible spermatogenesis deficiency.Conclusion: 1.miR-142-3p inhibits the BTB disruption induced by TGF-?3 through targeting Lgl2.2.TGF-?3 inhibits the activity of Notch signaling pathway via enhancing the expression of Lgl2 in order to promote the aerobic glycolysis and lactate secretion in Sertoli cells.