The role of microtubules during blastodisc formation of the squid, Loligo pealei.

After fertilization, cytoplasm streams from the vegetal region of the squid egg towards the animal cap to form a blastodisc where meroblastic cleavage will occur (1, 2). This process begins at fertilization, accelerates after second polar body formation (90 min, at 20°C), and continues through third cleavage (6.5–7.0 h). A blastodisc cap is formed, although at a slower rate, in eggs that have been artificially activated with 10 mg/ml A23187 (Molecular Probes) (3). To explore the role of the cytoskeleton in this process, in vitro fertilized (4) or activated embryos were placed in small petri dishes lined with 0.2% agarose (Sigma, Type II) and filled with 20°C Millipore-filtered seawater (MFSW). The dishes were placed on ice and cooled to 4°C. Exposure to cold was chosen to perturb cytoplasmic movements targeting microtubules (5), so that the effect on the embryos could be easily reversed. Cold treatment periods were selected to include the first and second polar body meiotic divisions (20 min and 1.5 h respectively), and the first (3.5 h), second (4.0 h) and third (6.5 h) cleavage events. Treatment periods were 20 min to 3 h, 3 to 4 h, 4 to 5 h, 5 to 6 h and 6 to 7 h of development. After treatment, dishes of embryos were removed from the ice and allowed to return to room temperature (20°C). Embryos were compared to control embryos for blastodisc formation, the presence of polar bodies, and cleavage pattern. Cleavage in squid is bilateral (Fig. 1a). First cleavage occurs along the line between the polar bodies and the apex of the embryo where the male pronucleus enters the egg. Second cleavage occurs perpendicular to this, and third cleavage is unequal and distinguishes the future right and left sides of the developing embryo. Exposure to cold inhibited blastodisc cap formation in all embryos treated prior to cytoplasmic streaming; it also arrested streaming in embryos treated after second polar body formation. Twenty minutes after removal from cold exposure, precleavage stage embryos develop a blister-like swelling of clear cytoplasm surrounding the male pronucleus. Activated eggs do not form blisters of cytoplasm when removed from cold treatment, although a small crescent of cytoplasm may form over the female pronucleus after 50 minutes. Over this same period of time, the polar bodies that are present swell to more than 4 times their normal diameter of 10 mm and then slowly return to normal size. Over the next 20 min the blister of cytoplasm around the male pronucleus relaxes into a small but growing blastodisc cap that resembles a normal cap in most (95%) cases. Abnormal cap formation was observed in about 5% of the embryos examined and included displacement of the cytoplasm to one side of the animal pole or splitting of the cap at the apex into two regions. Normal cleavage did not occur in these cases. In contrast to control squid embryos, which form two polar bodies, in vitro fertilized embryos treated during polar body formation possessed one (59/73, 37%) or two (3/73, 4%) and more frequently no (43/73 or 59%) polar bodies. Similar results were observed in activated eggs treated with cold during polar body formation. Fertilized embryos that failed to complete their meiotic divisions often possessed three nuclei at the apex of the blastodisc cap prior to cleavage, indicating that cold shock at this early stage induces polyploidy. These embryos seldom underwent normal cleavage. Interestingly, in contrast to the 2%–10% of control-activated eggs that underwent a cleavage event, 60% (79/132) of activated eggs treated with cold during their meiotic divisions possessed cleavage furrows. Embryos treated with cold from 3 to 4 h, the time when control embryos undergo first cleavage, possessed two polar bodies (as did all other embryos treated at later times), formed normal blastodisc caps, and cleaved normally. In contrast, even though first cleavage begins at 3.5 h, embryos treated from 4 to 5 h of development and returned