RESUMO
The blood-testis barrier (BTB) separates the seminiferous epithelium into the apical and basal compartments. The BTB has to operate timely and accurately to ensure the correct migration of germ cells, meanwhile maintaining the immunological barrier. Testin was first characterized from primary Sertoli cells, it is a secretory protein and a sensitive biomarker to monitor junctions between Sertoli and germ cells. Till now, the functions of testin on BTB dynamics and the involving mechanisms are unknown. Herein, testin acts as a regulatory protein on BTB integrity. In vitro testin knockdown by RNAi caused significant damage to the Sertoli cell barrier with no apparent changes in the protein levels of several major tight junction (TJ), adhesion junction, and gap junction proteins. Also, testin RNAi caused the diffusion of two TJ structural proteins, occludin and ZO-1, diffusing away from the Sertoli cell surface into the cytoplasm. Association and colocalization between ZO-1 and occludin were decreased after testin RNAi, examined by Co-IP and coimmunofluorescent staining, respectively. Furthermore, testin RNAi induced a dramatic disruption on the arrangement of actin filament bundles and a reduced F-actin/G-actin ratio. The actin regulatory protein ARP3 appeared at the Sertoli cell interface after testin RNAi without its protein level change, whereas overexpressing testin in Sertoli cells showed no effect on TJ barrier integrity. The above findings suggest that besides as a monitor for Sertoli-germ cell junction integrity, testin is also an essential molecule to maintain Sertoli-Sertoli junctions.
Assuntos
Proteína 3 Relacionada a Actina/genética , Barreira Hematotesticular/metabolismo , Proteínas/genética , Proteína da Zônula de Oclusão-1/genética , Citoesqueleto de Actina/genética , Junções Aderentes/genética , Animais , Masculino , Camundongos , Ocludina/genética , Proteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Epitélio Seminífero/crescimento & desenvolvimento , Epitélio Seminífero/metabolismo , Células de Sertoli/metabolismo , Espermatogênese/genética , Junções Íntimas/genéticaRESUMO
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.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Barreira Hematotesticular/metabolismo , Células de Sertoli/metabolismo , Espermatócitos/metabolismo , Espermatogênese/genética , Junções Íntimas/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/antagonistas & inibidores , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Animais , Barreira Hematotesticular/efeitos dos fármacos , Caderinas/genética , Caderinas/metabolismo , Movimento Celular/efeitos dos fármacos , Masculino , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Ocludina/genética , Ocludina/metabolismo , Peptídeos/farmacologia , Cultura Primária de Células , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte Proteico/efeitos dos fármacos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Epitélio Seminífero/citologia , Epitélio Seminífero/efeitos dos fármacos , Epitélio Seminífero/metabolismo , Células de Sertoli/citologia , Células de Sertoli/efeitos dos fármacos , Espermatócitos/citologia , Espermatócitos/efeitos dos fármacos , Espermatogênese/efeitos dos fármacos , Junções Íntimas/efeitos dos fármacos , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Spermiation and BTB restructuring, two critical cellular events that occur across seminiferous epithelium in mammalian testis during spermatogenesis, are tightly coordinated by biologically active peptides released from laminin chains. Our earlier study reported that F5-peptide, synthesized based on a stretch of 50 amino acids within laminin-γ3 domain IV, could reversibly induce the impairment of spermatogenesis, disruption of BTB integrity, and germ cell loss, and thus is a promising male contraceptive. However, how F5-peptide when administered intratesticularly enters seminiferous tubules and exerts effects beyond BTB is currently unknown. Here we demonstrated that Slc15a1, a peptide transporter also known as Pept1, was predominantly present in peritubular myoid cells, interstitial Leydig cells, vascular endothelial cells and germ cells, while absent in Sertoli cells or BTB site. The steady-state protein level of Slc15a1 in adult rat testis was not affected by F5-peptide treatment. Knockdown of Slc15a1 by in vivo RNAi in rat testis was shown to prevent F5-peptide induced disruptive effects on spermatogenesis. This study suggests that Slc15a1 is involved in the transport of synthetic F5-peptide into seminiferous epithelium, and thus Slc15a1 is a novel target in testis that could be genetically modified to improve the bioavailability of F5-peptide as a prospective male contraceptive.
