The Flagellar Protein RSPH1 Is a Candidate Regulator of the Testis and Sperm-Restricted Protein Phosphatase, PP1gamma2.

Abstract

The protein RSPH1 (also known as TSGA2, MEICHROACIDIN, RSP44) is present in the sperm flagellum. Previous analysis has suggested that various isoforms of the protein are expressed differentially in testis and epididymis. Targeted disruption of Rsph1 impairs spermiogenesis resulting in a male sterility phenotype with remarkable similarity to the one observed in Protein Phosphatase 1 gamma (PP1gamma)-null mice. The initial goal of this study was to confirm that two different isoforms of the protein, resulting from alternate splicing, are expressed in testis and epididymis respectively. Testes and epididymides of PP1gamma-null mice, both nearly devoid of spermatozoa, were used for this study. Western blot analyses and fluorescence immuno-cytochemistry were performed with antibodies raised against synthetic peptide antigens corresponding to the N- and C- termini of mouse RSPH1. Column purification, GST pull-down, and tap-tagged fusion proteins expressed transgenically in the testes were used to isolate the binding partners of RSPH1 in testis and sperm extracts. Our results confirmed that RSPH1 is expressed in the mouse epididymis as a 30-kDa protein compared to the 48-kDa testis isoform and that expression of the 30-kDa, N-terminally truncated, isoform is restricted to the corpus and caudal regions of the epididymis.The 30-kDa isoform of RSPH1 appears to be incorporated into maturing spermatozoa during their passage through the epididymis. Protein micro-sequencing, co-elution during column chromatography, and co-immunoprecipitation show that in testis, RSPH1 is bound to 14-3-3 and PPP1R11, proteins known to regulate the catalytic activity of the protein phosphatase, PP1gamma2. Taken together our results suggest that one role of RSPH1 could be to regulate the activity of the testis- and sperm-restricted protein phosphatase, PP1gamma2. (poster)