Second Messengers and Cell Regulation
Summary: David Garbers targeted the germline for study. His questions were focused on the molecular basis of fertilization and the development of technology to maintain and manipulate male germ cells in culture. He also studied the mechanisms of regulation of the guanylyl cyclase receptor family.
Our laboratory primarily focuses on research targeting the germline, but we are also interested in the functions of the guanylyl cyclase receptors. These receptors produce an intracellular second messenger that regulates cell behavior, including the regulation of blood pressure and vision. The germline research is important in biomedicine at both a basic and applied level and should significantly advance our ability to intervene at fertilization in animals and humans, to modify genomes in a wide range of species, and to develop new methods to produce and study stem cells.
Unraveling the Molecular Basis of Fertilization
We have taken multiple approaches to identify molecules critical for fertilization in mammals; the most valuable approaches are proving to be screens that do not rely on studying specific behavior in the gametes as a first step. Our methods rely heavily on the use of data from the genome project. One method defines molecules present on the membranes of cells. Since most proteins specifically involved in fertilization are likely expressed on the surface of gametes, this method is particularly attractive for defining candidate regulatory molecules in the fertilization process. We have identified many proteins expressed exclusively on spermatozoa, including a unique divalent cation channel (CatSper), a regulator of intracellular pH called a sperm-specific sodium-hydrogen exchanger (sNHE), and a protein that regulates lipid distribution across the membrane. Disruption of the genes for these proteins results in partial or absolute male infertility. When the gene for CatSper is inactivated, the sperm behavioral defect is a failure to “hyperactivate” motility. Somewhat surprisingly, failure to exhibit this form of motility results in a complete failure to fertilize the egg due to an inability of sperm cells to penetrate the extracellular matrix of the egg. This identification of the primary upstream regulator of hyperactivated motility also establishes that this form of motility is essential for fertilization, and thus a viable target for the development of a male contraceptive.
When a second sperm protein that is critical for regulation of intracellular pH (sNHE) is eliminated from sperm cells, they are normal in morphology and in numbers of sperm cells produced, but the sperm cells lack the ability to swim forward. A number of sperm cell functions appear critically regulated by intracellular pH, making this sperm-specific molecule of particular interest. The sNHE is required for protein phosphorylation that normally occurs during sperm capacitation. In mammals, including the human, sperm cells are not able to fertilize an egg until a period of incubation in the female; this process is termed capacitation. Unexpectedly, both the loss of motility and protein phosphorylation can be rescued by an intracellular second messenger, cyclic AMP, suggesting that cAMP metabolism is impaired in the spermatozoa lacking the sNHE. In fact, measurements of cAMP in sperm lacking the sNHE demonstrate that concentrations of the intracellular messenger are very low.
We have also undertaken research to provide a more encompassing view of the numbers of sperm-specific proteins possibly involved in the fertilization process. We showed that nearly 4 percent of the mouse genome is dedicated to the production of sperm-specific proteins, many of which are likely expressed on mature spermatozoa. We therefore have a large databank of proteins that are potential participants in fertilization. Of greatest interest have been those proteins that may serve as targets for germ celldirected contraception. To identify proteins with the greatest potential for contraceptive development, we have selected transcripts from the microarray analyses that satisfy three basic criteria: (1) highest expression in postmeiotic, haploid germ cells, (2) testis-specific expression, and (3) presence of potential transmembrane and/or signal peptide motifs indicative of cell surface expression.
Targeting the Male Germ Stem Cell
During the past year, we have developed technology to maintain and expand male germ cells in the culture dish. The use of a transgenic line of rats that express enhanced green fluorescent protein (EGFP) exclusively in the germline has allowed us to separate feeder layers and contaminating testis somatic cells from germ cells, resulting in identification of a set of spermatogonial stem cell marker transcripts. With these molecular markers as a guide, we have devised conditions where rat spermatogonial stem cells renew and proliferate in culture, with a doubling time between 3 and 4 days. The marker transcripts increase in relative abundance as a function of time in culture, and the stem cells retain competency to colonize and develop into spermatids after transplantation to the testes of recipient rats. The cells also remain euploid after at least 12 passages. Cell lines can be isolated and cryopreserved and upon subsequent thawing continue to self-renew. Transfection of the spermatogonial stem cells with a plasmid containing the neomycin phosphotransferase (neo) selectable marker resulted in selection of resistant cell lines that effectively colonize recipient testes, suggesting that gene targeting is now feasible in the rat by going through the germline.
Guanylyl Cyclase Receptors
A family of receptors called guanylyl cyclases form cyclic GMP, a chemical messenger inside cells. These receptors detect signals from other cells in the body, and then increase the concentrations of a small-molecule messenger (cyclic GMP) to cause a behavioral change. My laboratory discovered these receptors on spermatozoa. On sperm cells of invertebrate animals, the guanylyl cyclase receptors detect signals from the egg; these signals enable spermatozoa to know in which direction to swim. Subsequently, we found members of the family in most vertebrate cells, including those of mammals. Of the seven mammalian guanylyl cyclase receptors discovered, four remain orphans, in that the presumed molecules that normally bind to these putative receptors remain unknown. Two of the orphans are found in the eye, one in the nose, and one principally in lung or skeletal muscle. Although genes for these seven receptors also exist in the human, recent work by others suggests that both the putative receptor expressed in the nose and the one found in lung or skeletal muscle of rats or mice are not functional genes in the human. The two in the eye are expressed in photoreceptors, where they seem to be primary requisites for proper vision. The one in the nose is likely a receptor for an odorant or pheromone. We have been using various methods to search for the regulators (ligands) of these orphan receptors.
The other three guanylyl cyclases possess known ligands. One of the cyclases is the receptor for a small molecule that is released from the heart to regulate blood pressure. When blood volume increases, the heart releases this hormone, which then travels through the bloodstream to bind to this cyclase receptor, found in the kidney, blood vessels, and adrenal gland. Elevations of the second messenger in these areas result in a marked lowering of blood pressure. Another of the cyclases is the receptor for small peptides produced by pathogenic bacteria that cause "traveler's diarrhea" in adults. The third is the receptor for a small peptide produced in many regions of the body where it likely plays a role in tissue remodeling, such as in wound repair. We are attempting to define the functions of these various receptors.
We have eliminated the expression of four of these cyclases through the disruption of their genes in mice. As an example, elimination of the gene for the receptor that responds to the heart hormone results in a salt-resistant form of hypertension, a behavior displayed by about half of Americans with essential hypertension. Furthermore, lack of this receptor results in a marked increase in the size of the heart, which we have recently shown is due to the apparent regulation of cardiac myocyte size by this receptor, independent of blood pressure. Thus, the receptor becomes a possible target to control cardiac hypertrophy, a common problem as a result of elevated blood pressure.
More recently we have purified a factor in serum that markedly and specifically causes desensitization of one of the cyclase receptors to its normal extracellular ligand. This serum factor, sphingosine-1-phosphate (S1P), is a molecule that appears to possess five different membrane receptors in mammals. S1P often acts to increase cell proliferation, whereas the guanylyl cyclase receptor blocks cell proliferation. Therefore, S1P and the guanylyl cyclase receptor are adversaries with respect to the regulation of cell proliferation. This relationship no doubt plays a significant role in tissue remodeling, including wound healing.
Grants from the National Institutes of Health and the Lalor Foundation provided support for some of this work.