Basis for the Essential Role of the Mammal-Specific Protein Phosphatase Isoform PP1γ2 in Sperm

In mammals, testicular sperm exiting the seminiferous tubules are immotile. Mammalian sperm gain motility and the ability to fertilize eggs during their passage through the epididymis and undergo hyperactivation in the female reproductive tract. Several studies show that protein kinases and phosphatases play an essential role in controlling the Serine/threonine protein phosphorylation, which is responsible for the phosphorylation of proteins involved in sperm maturation, sperm motility, and hyperactivation. There are two isoforms of the PP1γ protein phosphatase, PP1γ1, and PP1γ2, alternately spliced transcripts derived from one gene, Ppp1cc. The two isoforms are identical, except that the extreme C-terminus of PP1γ1 contains a eight amino acid sequence, whereas PP1γ2 contains a 22 amino acid sequence. The PP1γ1 isoform is expressed in several tissues, whereas the PP1γ2 isoform is expressed in the testis and sperm. Remarkably PP1γ2 is present only in mammalian sperm, whereas sperm from other species contain PP1γ1 or one of two other isoforms, PP1α or PP1β. The first part of this study focuses on developing a knock-in mouse line expressing PP1γ1 instead of PP1γ2 in developing spermatogenic cells in the testis and sperm. It is known that Ppp1cc knockout male mice (-/-) are infertile due to impaired sperm morphogenesis due to the loss of PP1γ2 in the testis. Transgenic expression of PP1γ2 in the testis rescues sperm function and fertility. Transgenic expression of PP1γ1 driven by a testis-specific promoter was unable to rescue fertility. It was unknown whether this limitation was due to the expression driven by an exogenous promoter or whether PP1γ1 was unable to substitute for the unique biochemical function of PP1γ2 in sperm. We have engineered the Ppp1cc gene by homology-directed repair to remove intron 7 using Crisper/Cas9 system to express only the PP1γ1 isoform. We have successfully established the knock-in mouse line and confirmed the modification of the gene and its message. The result of this study shows that PP1γ1 was expressed in all tissue, including the testis and sperm, and the Ppp1cc gene controlled the expression. We have determined that high levels of PP1γ1, comparable to the levels of PP1γ2 in wild-type testis, are expressed in the knock-in mice. The PP1γ1 protein is also incorporated into sperm. The second part of the study focuses on determining the biological and biochemical consequences of knock-in mice expressing PP1γ1 instead of PP1γ2 in spermatogenic cells in the testis. Also, the result of this study shows that sperm containing PP1γ1 have impaired motility. We found out that the fertility of the gamma1-bearing sperm was impaired. The fertility and sperm motility of the knock-in mice were similar to those seen in the transgenic mice. We found out that PP1γ1 (KI/KI) mice sperm was determined to have a low level of ATP. The results from the knock-in mice will enable the determination of the unique function of PP1γ2 only in mammals. The knock-in approach to produce mice bearing PP1γ1is a definitive means to determine why mammals require PP1γ2 whereas sperm from non-mammalian species usually function with the other PP1 isoforms – PP1γ1, PP1α, or PP1β.