Research in the Singson lab

Fertilization is a biological process that has important medical, economic and social implications. Our primary research interest is the mechanisms of sperm-egg interactions. This includes understanding the molecular events that mediate gamete recognition, adhesion, signaling and fusion. The genetic and molecular dissection of these events will also provide insights relevant to other important cell-cell interactions during the life and development of multicellular organisms.

Above: A sterile C. elegans hermaphrodite with a lawn of E. coli (worm food) to itself.

We are helping to pioneer the use of the nematode worm Caenorhabditis elegans for addressing the mechanisms of sperm-egg interactions. The powerful tools of classical and molecular genetics developed for the worm are not available or are very difficult to utilize in the other organisms traditionally used for studying fertilization. The amoeboid sperm of C. elegans, despite lacking an acrosome and flagellum, carry out the same basic functions common to all spermatozoa.

Singson, A. (2001) Figure 1

C. elegans spermatozoa

  • (A) Transmission electron micrograph. P = pseudopod, N = nucleus, M = mitochondria, MO = membranous organelles, LM = laminar membranes.
  • (B) Scanning electron micrograph.
  • (C) Light micrograph.

From Singson (2001) Developmental Biology 230, 101-109.

The reproductive biology of C. elegans facilitates the identification of mutations that affect gametes and no other cells. The worm exists as a hermaphrodite that makes both sperm and oocytes or as a male that makes only sperm. Sterile hermaphrodites can be identified because they produce no progeny and the uterus fills with unfertilized oocytes instead of developing embryos.

wild-type worm from Singson, A. (2001) Figure 2

Wild-type worm

Wild type worms are fertile. Note the dividing embryos in the uterus on the right side of the image. From Singson (2001) Developmental Biology 230, 101-109.

sterile worm

Mutant worm with defective fertilization

Sperm-sterile (spe or fer) and egg-sterile (egg or ooc) worms display this phenotype. Note the oocytes (with single nuclei) in the uterus on the right side of the image.

Mutants that affect sperm function at fertilization

Mutant hermaphrodites that are spermatogenesis-defective (spe) or fertilization defective (fer) are self-sterile and lay unfertilized oocytes. However, when these otherwise healthy worms are crossed to wild type males (a source of sperm) they can produce outcrossed progeny. We have focused on a set of spe and fer genes that produce sperm with normal morphology and motility that cannot fertilize eggs even after contact. From this phenotype we infer that these mutants disrupt sperm-egg recognition, adhesion, signaling or fusion. The characterization of these genes is a critical step in formulating a model concerning their role in wild-type fertilization.

Mutants that affect egg function at fertilization

Mutant hermaphrodites that are oocyte formation abnormal (ooc) or egg sterile (egg) produce oocytes that cannot be fertilized by wild type sperm. Two approaches have been used to identify worms with this phenotype. In a forward genetic screen, we have identified temperature sensitive mutants that are fertile when raised at low culture temperature but sterile when raised at high culture temperature. In an RNAi screen, we have knocked down the function of oocyte enriched genes. Any genes that lead to an egg sterile RNAi phenotype are then further analyzed to fully determine their function at fertilization.