Identification of protein tyrosine phosphatases and dual specificity phosphatases in mammalian spermatozoa and their role in sperm motility and protein tyrosine phosphorylation

Abstract

Protein tyrosine kinases have important roles in spermatozoa; however, little is known about the presence and regulation in these cells of their counterparts in signaling, namely, protein tyrosine phosphatases (PTPs) and dual-specificity phosphatases (DSPs). The objectives of the present study were to identify PTPs and DSPs in boar, stallion, and dog spermatozoa; to characterize their subcellular distribution; and to investigate the roles of tyrosine phosphatases in maintenance of protein tyrosine phosphorylation level and in sperm motility. Using Western blotting with specific antibodies in boar and stallion sperm lysates, we unequivocally identified two PTPs (PTPRB and PTPN11) and two DSPs (DUSP3 and DUSP4). In dog sperm lysates, only PTPN11, DUSP3, and DUSP4 were detected. In all these species, we did not detect the specific signal with anti- PTPRC (CD45), CDKN3, DUSP1, DUSP2, DUSP6, DUSP9, PTPN1, PTPN3, PTPN6, PTPN7, PTPN13, PTPRA, PTPRG, PTPRJ, PTPRK, or PTPRZ antibodies. Positive matches were further investigated by indirect immunofluorescence and confocal microscopy. Results showed that PTPRB was associated with the plasma membrane in the head and tail of boar and stallion spermatozoa. In agreement with Western blotting results, PTPRB antibodies did not show immunoreactivity in dog sperm analyzed by immunofluorescence. In the three species, DUSP4 was mainly found in the tail of spermatozoa, with little or no immunoreactivity in the head. PTPN11 was mainly located in the postacrosomal region in the head, whereas DUSP3 immunoreactivity was extended within the acrosome. PTPN11 and DUSP3 showed immunoreactivity in the tail that was restricted to the midpiece. Finally, we incubated boar, stallion, and dog spermatozoa with pervanadate and sodium orthovanadate, two PTP inhibitors, and analyzed overall protein tyrosine phosphorylation and assessed sperm motility. Sodium orthovanadate and pervanadate showed concentration-dependent inhibition of sperm motility that was rapid and reversible.

Las proteínas tirosina quinasas desempeñan funciones importantes en los espermatozoides; sin embargo, se sabe poco sobre la presencia y regulación en estas células de sus homólogas en la señalización, a saber, las proteínas tirosina fosfatasas (PTPs) y las fosfatasas de doble especificidad (DSPs). Los objetivos del presente estudio fueron identificar PTPs y DSPs en espermatozoides de jabalí, semental y perro; caracterizar su distribución subcelular; e investigar el papel de las tirosina fosfatasas en el mantenimiento del nivel de fosforilación de la tirosina proteína y en la motilidad espermática. Utilizando Western blotting con anticuerpos específicos en lisados de esperma de verraco y semental, identificamos inequívocamente dos PTPs (PTPRB y PTPN11) y dos DSPs (DUSP3 y DUSP4). En los lisados de esperma de perro, sólo se detectaron PTPN11, DUSP3 y DUSP4. En todas estas especies, no detectamos la señal específica con anticuerpos anti-PTPRC (CD45), CDKN3, DUSP1, DUSP2, DUSP6, DUSP9, PTPN1, PTPN3, PTPN6, PTPN7, PTPN13, PTPRA, PTPRG, PTPRJ, PTPRK, o PTPRZ. Las coincidencias positivas se investigaron más a fondo mediante inmunofluorescencia indirecta y microscopía confocal. Los resultados mostraron que PTPRB estaba asociada a la membrana plasmática en la cabeza y la cola de los espermatozoides de verracos y sementales. En concordancia con los resultados de Western blotting, los anticuerpos PTPRB no mostraron inmunoreactividad en espermatozoides de perro analizados por inmunofluorescencia.