Two steps required for male pronucleus formation in the sea urchin egg

We have developed a system for microinjection of sea urchin sperm nuclei into live eggs and oocytes in order to study conditions which are required for pronuclear development. The system bypasses sperm-initiated surface events that result in egg activation. By comparison to male nuclei of normally fertilized eggs or immature oocytes, the injected nuclei faithfully reflect the ability of the egg cytoplasm to promote male pronuclear development. Isolated nuclei injected into fertilized eggs swell and transform into male pronuclei like the endogenous male pronuclei. Nuclei injected into immature oocytes remain conical like fertilizing sperm nuclei. If sperm nuclei are injected into unfertilized, unactivated eggs, they undergo incomplete decondensation and fail to form pronuclei. This partial decondensation can be inhibited with 6-dimethylaminopurine (6-DMAP). If unfertilized eggs are first incompletely activated by raising internal pH with ammonia, injected sperm nuclei will complete pronuclear development. Increasing pH or Ca2+ does not cause sperm nuclear swelling or chromatin decondensation in immature oocytes. These experiments reveal two conditions necessary for pronuclear formation. The first condition, resulting in partial decondensation, develops during meiotic maturation and can be blocked by 6-DMAP. The second requires cytoplasmic alkalinization, but not the fertilization-induced internal Ca2+ increase or inositol triphosphate pathway activation.