Research Projects

1. Protein phosphatase (PP1) in testis and spermatozoa The presence of serine/threonine phosphatase in rat testis, and thereby implication in mammalian spermatozoa, was first reported in 1975. Our laboratory showed PP1γ2 is the predominant isoform of Ser/Thr protein phosphatase in testis and the only detectable isoform of PP1 in spermatozoa. There are three Ppp1c genes encoding four isoforms of protein phosphatase catalytic subunits: PP1α, PP1β, PP1γ1 and PP1γ2. PP1γ1 and PP1γ2, differing only at their extreme C-termini, are alternate splice products of the same gene Ppp1cc. PP1α, PP1β , and PP1γ1 are ubiquitously expressed but PP1γ2 is weakly expressed in brain but predominant in testis. It is notable that the genomes of species other than mammals, including birds, amphibians, reptiles, and invertebrates, do not contain a PP1 orthologue resembling PP1γ2. Sperm from these species contain a protein phosphatase 1 catalytic subunit resembling mammalian PP1α, PP1β, or PP1γ1. PP1γ2 is vital for sperm formation and function in mammals.

2.  Glycogen synthase kinase 3 (GSK3) is a highly conserved protein-serine kinase regulating key cellular functions. In mammals GSK3 is expressed as two isoforms, GSK3α and GSK3β, encoded by distinct genes. The catalytic domains of the two isoforms are 98% identical. In most tissues the two isoforms are functionally interchangeable, except in the developing embryo where GSK3β is essential.  We first identified GSK3 as an enzyme regulating sperm PP1γ2, showed that both GSK3 isoforms are present in bovine, primate, and mouse sperm and, suggested that they may play a role in epididymal initiation and regulation of sperm motility. Using genetic approaches, here we have tested requirement for each of the two GSK3 isoforms in testis and sperm. Both GSK3α and GSK3β are expressed at high levels in testis coincident with the onset of spermatogenesis. Mice harboring a conditional knock out of GSK3β in developing germ cells in testis are normal and fertile.  By contrast, conditional knock out of GSK3α in developing testicular germ cells results in male infertility. Despite overlapping expression and localization of the two isoforms in testis, GSK3β does not substitute for loss of GSK3α. GSK3α is essential and irreplaceable in testis and sperm.

3. Sperm Phospho-Proteins. It has been long known that protein kinase A (PKA) activated by cAMP is essential for sperm motility and fertility. While the role of PKA in sperm function is well established little is known about the protein substrates of PKA.  Determination of the identities of these proteins will enable us to understand mechanisms underlying sperm motility and fertility. We are using a novel chemical-genetic approach to identify sperm PKA substrates.  This approach uses a genetically engineered modification of the ATP binding domain of the catalytic subunit of PKA such that the modified enzyme recognizes specific ATP analogues that are not accepted by other kinases.  Phosphorylation of substrates in the presence of the ATP analog enables their identification with relative ease.  We are also determining the phosphoproteome of sperm from transgenic and knock out mice lacking sAC (soluble adenylyl cyclase), GSK3α, calcineuring, and transgenic mice exoressing PP1γ1 instead of PP1γ2 in testis. Comparison of the the phospho protein from sperm from these mice will enable us to identify substrates for the kinases and phosphatase important in regulating sperm motility and metabolism.