Fertilization and Early Embryogenesis
(W318) MALE MICE LACKING SPERIOLIN PRODUCE DEFECTIVE SPERM AND ABNORMAL ZYGOTES.
Goto, Masuo1, 2, Goulding, Eugenia2, Eddy, Edward2, O'Brien, Deborah1, 1 University of North Carolina School of Medicine, Chapel Hill, NC2 National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC
ABSTRACT- In most mammals the sperm centriole is required for spindle formation in the first division of the zygote, but in mice the centrioles are lost during sperm maturation. However, we have identified a male germ cell-specific protein which might contribute to zygote spindle formation in mice. Speriolin is a binding partner of Cdc20, a substrate-specific activator of the anaphase-promoting complex (APC). It is present in centrosomes during meiosis and spermiogenesis and in the centrosomal region of sperm. This region is devoid of both proximal and distal centrioles, but is surrounded by a proteinaceous collar containing speriolin. Speriolin is carried into the egg during fertilization. To determine the contributions of speriolin to the production of functional sperm, fertilization, and the onset of the first mitotic division, we produced speriolin-null mice (Spatc1-/-). Fertility often was reduced in male, but not female Spatc1-/- mice. Sperm lacking speriolin had ultrastructural defects in the centrosomal region and were easily decapitated by treatment with low concentrations of detergents. After mating wild-type females with Spatc1+/+ or Spatc1-/- males, eggs were collected 15 hr after fertilization and then incubated in M16 medium for 21 hr (36 hr after fertilization). 87% of eggs fertilized by Spatc1+ sperm developed into two-cell embryos, 5% degenerated, and 8% were unfertilized. In contrast, 26% of eggs fertilized by Spatc1- sperm reached the two-cell stage, 44% degenerated, and 30% were unfertilized. Most of the abnormal zygotes underwent nuclear fragmentation or abnormal cytokinesis without chromosome segregation. To characterize the defects in early embryogenesis, zygotes were fixed 21 hr after fertilization and stained with monoclonal antibodies to tubulin and pericentrin, and with DAPI. Abnormal zygotes often had two nuclei, failed to extrude the secondary polar body, and lacked bipolar spindles. These results indicate that speriolin is an essential component of the connecting piece of the sperm flagellum. In addition, speriolin may be required for secondary polar body extrusion and the onset of the first mitotic division after fertilization, possibly by interacting with APCCdc20 in the zygote. Supported by NIH grant HD045655.
KEY WORDS: spindle, centrosome, sperm, cdc20