In mouse testis, PP1γ1 is restricted mainly to somatic cells, while PP1γ2 is primarily located in secondary spermatocytes and germ cells, and the only isoform detectable in mammalian spermatozoa. Targeted deletion of the PP1γ gene results in sterile male mice. Because the most prominent marker of this phenotype is aberrant spermatid morphogenesis, its primary cause may be the absence of PP1γ2 since PP1γ1 is mainly in somatic cells. To better understand the role of PP1γ2 in mammalian testis/sperm, this dissertation was undertaken to explore PP1γ2 interactions with its regulatory proteins in testis/sperm. Wild-type and mutant isoforms of PPP1R11/inhibitor 3 (I3), PPP1R2/inhibitor 2 (I2) and a novel protein similar to I2 were studied.
Testicular/sperm PP1γ2 was found to form a complex with I3, a potent protein phosphatase 1 inhibitor, PPP1R7/Sds22, the mammalian homologue of a yeast protein phosphatase 1 binding protein, and actin by binding assay, chromatography and native gel electrophoresis, and that in the spermatoozoon equivalent of this complex, PP1γ2 is catalytically inactive by phosphatase assay. In PP1γ-null testis, I3 levels drop significantly while increased dramatically when a transgenic source of testicular PP1γ2 is provided. Together with studies showing that PP1γ2 and I3 jointly reach their highest steady state levels during wild-type spermiogenesis, these data make a hypothesis that the formation of a complex containing PP1γ2 and I3 in testis and sperm, while inhibiting PP1γ2 catalytic activity, promotes sperm morphogenesis and subsequent function by stabilizing I3.
To understand the role of a naturally occurring mutant isoform of I3 (t/t-I3) in mice involved in an abnormal sperm motility, curlicue, the properties of t/t-I3 and wt-I3 were compared, and found that they differed only in their phosphorylation in vitro. Finally, the presence of PPP1R2/inhibitor 2 (I2) in testis and sperm were demonstrated, and evidence was provided that a novel protein similar to I2 is transcribed in vivo and translated in vitro by Northern blot, RT-PCR and in vitro expression. These studies provide important insights into the regulation of PP1γ2 in testis and sperm.