Appendix 11: Production of Transparent Mini-Cells Lacking Yolk

Egg fragmentation by centrifugation has been a common technique for a hundred years. Procedures used for Chaetopterus are similar to those for other marine invertebrates. With Chaetopterus, the opacity of the egg makes this especially attractive, because use of such mini-cells facilitates the microscopic observation of processes that otherwise would be obscured by the yolk. It has been reported that the cortical cytoskeletal domain (Jeffery, 1985), which contains most of the stored maternal mRNA, enters the centrifugal (yolk) half of the egg upon centrifugation (Jeffery, 1985; Swalla et al., 1985) despite early reports that centrifuged eggs and egg fragments develop normally (Lillie, 1906; Wilson, 1929, 1930; Harvey, 1939)! Either of the methods described below will yield a clean population of transparent mini-cells containing the meiotic spindle.

Method I (Goode and Sarma, 1986)

This method is simpler than Method II.

  1. Wash eggs several times in natural seawater to induce germinal vesicle breakdown and spindle formation.
  2. Layer over 1 M sucrose in a centrifuge tube for a swinging bucket rotor.
  3. Centrifuge 24,000 x g x 10 min at 25°C
  4. Collect yolk-free mini-cells at the seawater/sucrose interface.

Method II (Harvey, 1939; Jeffery, 1985)

This method allows the collection of relatively pure populations of both the yolk-free mini-cells and non-nucleated mini-cells.

  1. Make a discontinuous sucrose gradient consisting of 3 ml 60% sucrose in seawater and 8 ml of a 2.2:1 ratio of 1 M sucrose:seawater.
  2. Wash eggs as in Method I.
  3. Layer eggs in seawater over sucrose.
  4. Centrifuge 13,000 x g for 13 min (8 min for zygotes).
  5. Collect light halves (yolk-free mini-cells) at upper gradient interface; collect yolky halves (non-nucleated mini-cells) at lower gradient interface.