The Miller Lab



Research in our laboratory focuses on molecular aspects of fertilization. The union of two different gametes is an interesting and unique form of cell adhesion and communication. The receptors and intracellular signaling steps that mediate this process are controversial. A detailed knowledge of gamete receptors and their function is essential to improve fertility for infertile couples and control fertility in agricultural species.

At the site of fertilization, mammalian sperm must navigate the egg coat (zona pellucida) to reach the egg plasma membrane. In order to penetrate the zona pellucida, they must first release the sperm acrosome, a secretory vesicle containing enzymes that may aid in digesting a pathway through the zona pellucida. Once inside the zona pellucida, sperm bind and fuse with the egg plasma membrane, triggering the completion of meiosis along with the first mitotic division of the newly formed zygote. At fertilization, a wave of calcium is released. This wave activates the release of cortical granules, secretory vesicles that line the periphery of the egg. The cortical granule contents are released and remove the sperm-binding properties of the zona.

Our primary research interest is the binding of sperm to egg coat (zona pellucida) proteins. Sperm/oocyte binding is mediated by a glycoprotein in the oocyte zona pellucida called ZP3. The ZP3 glycoprotein interaction with sperm activates pertussis toxin sensitive guanine nucleotide binding proteins (G proteins), inducing a cell signaling cascade that triggers the release of acrosomal contents. The acrosome reaction is mediated by a sperm surface ZP3 receptor. Several known receptors are potential candidates. One receptor that we study is a form of the Golgi enzyme, beta 1,4 galactosyltransferase. Although the predominant form of this enzyme is a Golgi resident protein, a longer form is found on the surface of sperm from many different species. This receptor activates a cascade of sperm cytoplasmic signals by binding a terminal sugar residue on ZP3, leading to the exocytotic release of sperm acrosomal enzymes. Many of these signaling molecules may be similar to those found in somatic cells, including G-proteins and tyrosine kinases. To study signaling pathways, we are using site-directed mutagenesis of beta 1,4 galactosyltransferase. The functional importance of specific pathways can be revealed by removing those pathways in a living system. Because mature sperm are transcriptionally inactive, we alter frog oocytes to express these sperm proteins. These oocytes make an excellent model because of their large size, abundance of exocytotic signaling systems and transcriptional capacity.

Because most mammalian fertilization research centers on mouse gametes, it is important to determine if different species have unique gamete receptors. Our preliminary evidence suggests that, although some receptors are found on sperm from a variety of species, additional receptors may have a redundant function. Nature may provide multiple paths for this vital process.

Another goal of our research is to identify important sperm defects in males with poor fertility. We developed a sensitive assay to identify defects in subfertile sperm. The assay is based on allowing stained sperm from two different males to competitively bind and fertilize the same oocytes (See the photo beside the page title). If we find that most subfertile semen samples have sperm with only a few defects, we may be able to design faster and more accurate laboratory assays to evaluate semen.