entering the egg. This prompts rapid modification of the vitelline coat, through processes such as the egg's release of glycosidase into the seawater, so no more sperm can bind and polyspermy is avoided. After fertilization, free calcium ions are released in the egg cytoplasm in waves, mostly from internal stores. The temporary large increase in calcium concentration prompts the physiological and structural changes of development. The dramatic rearrangement of egg cytoplasm following fertilization, called ooplasmic segregation, determines the dorsoventral and anteroposterior axes of the embryo. There are at least three types of sea squirt egg cytoplasm: ectoplasm containing vesicles and fine particles, endoderm containing yolk platelets, and myoplasm containing pigment granules, mitochondria, and endoplasmic reticulum. In the first phase of ooplasmic segregation, the myoplasmic actin-filament network contracts to rapidly move the peripheral cytoplasm (including the myoplasm) to the vegetal pole, which marks the dorsal side of the embryo. In the second phase, the myoplasm moves to the subequatorial zone and extends into a crescent, which marks the future posterior of the embryo. The ectoplasm with the zygote nucleus ends up at the animal hemisphere while the endoplasm ends up in the vegetal hemisphere. Promotion of out-crossing Ciona intestinalis is a hermaphrodite that releases sperm and eggs into the surrounding seawater almost simultaneously. It is self-sterile, and thus has been used for studies on the mechanism of self-incompatibility. Self/non-self-recognition molecules play a key role in the process of interaction between sperm and the vitelline coat of the egg. It appears that self/non-self recognition in ascidians such as C. intestinalis is mechanistically similar to self-incompatibility systems in flowering plants. Self-incompatibility promotes out-crossing, and thus provides the adaptive advantage at each generation of masking deleterious recessive mutations (i.e. genetic complementation). Ciona savignyi is highly self-fertile. However, non-self sperm out-compete self-sperm in fertilization competition assays. Gamete recognition is not absolute allowing some self-fertilization. It was speculated that self-incompatibility evolved to avoid inbreeding depression, but that selfing ability was retained to allow reproduction at low population density. Botryllus schlosseri is a colonial tunicate able to reproduce both sexually and asexually. B. schlosseri is a sequential (protogynous) hermaphrodite, and in a colony, eggs are ovulated about two days before the peak of sperm emission. Thus self-fertilization is avoided, and cross-fertilization is favored. Although avoided, self-fertilization is still possible in B. schlosseri. Self-fertilized eggs develop with a substantially higher frequency of anomalies during cleavage than cross-fertilized eggs