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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 mm, at 20[degrees]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 [micro]g/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[degrees]C Millipore-filtered seawater (MFSW). The dishes were placed on ice and cooled to 4[degrees]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 divis ions (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[degrees]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 [micro]m 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 embry os 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 to room temperature failed to retain their first cleavage furrow (Fig. 1b) in 90% of the cases examined (36/40). In these embryos, because the polar bodies mark the region through which first cleavage will form, it is possible to determine that second cleavage occurred normally, while third cleavage--which is normally unequal--was equalized to mirror the cells in the future dorsal region of the embryo. Most embryos treated between 5 and 6 h of development retained a reduced first cleavage furrow at the center of the blastodisc and formed normal second and third cleavage furrows (76/89 or 85%), while other embryos from this group developed without a second cleavage furrow (Fig. 1c). This pattern was also observed in embryos treated between 6 and 7 h. Surprisingly, most of the embryos from all treatment groups continue to develop and at 48 h appeared fairly normal, although they often possessed clumps of large cells, uneven blastoderm yolk boarders, and regions where cell layers appeared thicker than controls.

These results suggest that the ordered movement of cytoplasm, which forms the blastodisc in the squid, is disturbed by cold treatment. Cold exposure also induced polyploidy and perturbed cleavage furrows. The failure to retain or create specific cleavage furrows may be due to the direct action of cold on the microfilaments responsible for furrow formation, cell membranes, or specific factors that regulate mitosis. However, the formation of the blister-like swellings of cytoplasm around the male pronucleus, likely initiated by the sperm centriole to form microtubule arrays, suggests that cytoplasmic movements are rapidly resuming and may disturb the previously formed or forming microfilaments responsible for cleavage. That the polar bodies, which are little more than unwanted chromosomes and microtubules, swell rapidly during this same period further suggests that microtubules may be partially responsible for these events, although this does not rule out the possibility that cold exposure results in destabili zation of membranes in these cells. Microtubules originating from the sperm pronucleus are crucial for the reorganization of cytoplasm after fertilization in frog eggs [6]. The result that cold exposure can equalize third cleavage in squid embryos is nearly identical to what was reported when squid embryos were treated with the microfilament inhibitor cytochalasin B, although first cleavage furrows were still present in some of those embryos [7]. To address the importance of microtubules and microfilaments alone and in concert to blastodisc formation and cleavage in the squid, it will be necessary to selectively challenge each element with specific inhibitors and characterize their appearance over time with immunohistochemistry.

This work was supported by a Research Opportunity Award from the National Science Foundation to Karen Crawford.

Literature Cited

(1.) Brooks, W. K. 1880. Anniv. Mem. Boston Soc. N.H. 1-22.

(2.) Arnold, J. M. 1968. Dev. Biol. 18: 180-197.

(3.) Crawford, K. 1985. Biol. Bull. 169: 540.

(4.) Klein, K. C., and L. A. Jaffe. 1984. Biol. Bull. 167: 518.

(5.) Yahara, I., and F. Kakimoto-Sameshima. 1978. Cell 15: 251-259.

(6.) Elinson, R. P., and B. Rowning. 1988. Dev. Biol. 128: 185-197.

(7.) Arnold, J. M., and L. D. Williams-Arnold. 1974. J. Embryol. Exp. Morphol. 31: 1-25.
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Author:Crawford, Karen
Publication:The Biological Bulletin
Geographic Code:1USA
Date:Oct 1, 2000
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