The oocytes of C. elegans, like the oocytes of many animals, are capable of an extended arrest during meiotic prophase. This arrest, which can last for several days, only occurs in the absence of sperm (McCarter et al., 1999). When sperm are present, oocytes sequentially undergo nuclear envelope breakdown, cortical rearrangement (collectively termed oocyte meiotic maturation), and ovulation every ~23 min in an assembly linelike fashion starting with the most proximal oocyte (McCarter et al., 1999). An increase in the basal contraction rate of the proximal gonadal sheath cells surrounding the oocytes also occurs in the presence of sperm (McCarter et al., 1999). These observations suggest that sperm-derived factors promote both oocyte meiotic maturation and sheath cell contraction. In order to identify these putative factors, we have developed an in vivo bioassay using
fog-2(
q71) hermaphrodites, which lack sperm (Schedl and Kimble, 1988). Oocyte meiotic maturation in
fog-2 mutants occurs at a slow rate causing them to "stack-up" in the proximal gonad arm. Microinjection of sperm-conditioned medium (SCM), which was prepared as described below, into the uterus of
fog-2 mutants causes a dramatic increase in the rate of oocyte meiotic maturation and sheath cell contraction. SCM was prepared by purifying sperm from enriched male cultures using pressure and filtration (Klass and Hirsch, 1981). After incubating these sperm for a period of several hours in M9 buffer, all sperm were subsequently removed from the medium using centrifugation (10,000 x g) and filtration through a 0.2 micron filter. The maturation and contraction-inducing factors present in SCM are heat resistant, protease sensitive, and larger than 3000 daltons. Fractionation of SCM is being performed using reversed phase high pressure liquid chromatography (HPLC) and the identities of molecules from biologically active fractions are being determined using MALDI mass spectrometry. Currently, we have isolated a single active fraction that promotes oocyte meiotic maturation and sheath cell contraction. This fraction contains one major protein species as well as one minor species, which we believe is a contaminant. The fact that both biological activities are copurifying during HPLC fractionation suggests that sperm secrete a single protein species that promotes both oocyte meiotic maturation and sheath cell contraction. Progress in the identification of the active species will be revealed and its evolutionary implications will be discussed. Klass, M. R., and Hirsh, D. 1981. Sperm isolation and biochemical analysis of the major sperm protein from Caenorhabditis elegans. Dev. Biol. 84, 299-312. McCarter, J., Bartlett, B., Dand, T., and Schedl, T. 1999. On the control of oocyte meiotic maturation and ovulation in Caenorhabditis elegans. Dev. Biol. 205, 111-128. Schedl, T., and Kimble, J. 1988.
fog-2, a germ-line specific sex determination gene required for hermaphrodite spermatogenesis in Caenorhabditis elegans. Genetics 119, 43-61.