PP1γ2 (PPP1CC2) is a serine/threonine phosphatase that is highly expressed in testis and the only isoform expressed in mature spermatozoa. It belongs to a group of highly conserved serine/threonine phosphatases known as PP1 and is formed by the alternate splicing of the Ppp1cc gene. PP1 function is regulated by a large number of proteins that either regulate its activity or bind to it and target it to different regions of a cell. Male mice lacking Ppp1cc gene are infertile whereas female mice are normal. This is due to a defect in spermiogenesis.
My first aim involved in analyzing the role of RSPH1, a protein highly expressed in the radial spoke of mature spermatozoa. A previous report showed that RSPH1 is expressed in two isoforms - a larger testicular isoform – P44 and a smaller epididymal isoform – P30. We show that P30 is expressed only in the caput and caudal regions of mouse epididymis and not in the caput. This suggested that P30 could be playing a role in the epididymal maturation of sperm and warranted a closer look at the role of P30 in sperm function. During the course of our studies, we showed by microsequencing that P30 is not an isoform of RSPH1 and is in fact another epididymal protein CRISP1. Considering the phenotypic similarity between Ppp1cc and Rsph1 knockout mice, we wanted to analyze the amounts of PP1γ2 and its binding partners I2, I3 and sds22 in Rsph1 knockout mouse testis and epididymis. We observed no difference in levels of these proteins when compared to wild-type mice.
My second aim involved the identification and characterization of testis-specific isoforms of ubiquitously expressed ancient binding partners of PP1 – I2, I3 and sds22. Our laboratory had previously observed I2-like activity in bovine sperm but had never been able to show the physical presence of I2 in sperm. Using specific antibodies against I2, we were able to, for the first time, conclusively provide evidence for the presence of I2 in testis and sperm. We showed the presence of testis-specific transcripts of I2, I3 and sds22. I2 and sds22 isoforms are produced due to alternate splicing whereas the I3 isoform is produced due to an alternate transcription start site. We also showed that the message and proteins of I2, I3 and sds22 are produced post-meiotically, which is similar to the expression pattern of PP1γ2. Analysis of transcript and protein levels from mice lacking PP1γ2 reveals decreased expression levels for I2, I3 and sds22. This suggests that PP1γ2 could be controlling the expression of these proteins at the transcriptional level.
My third aim involved identification of possible PP1γ2 substrates in mouse sperm. The preliminary experiments were carried out using 2D-DIGE wherein, the proteome of sperm from Ppp1cc +/- and Ppp1cc2 rescue sperm were compared. Ppp1cc2 mice undergo spermatogenesis and produce sperm that display gross morphological abnormalities due to decreased expression of PP1γ2. This suggests that PP1γ2 could be controlling the expression levels of sperm structural proteins. Preliminary results from 2D-DIGE revealed that majority of the proteins that were mis-regulated belonged to the glycolytic pathway.