Twin meiosis 2 spindles form after suppression of polar body 1 formation in oocytes of the marine shrimp Sicyonia ingentis.

The production of triploid shellfish has proven beneficial to the aquaculture industry since triploid organisms have reduced gonadal development, resulting in improved product quality and growth characteristics (1). Triploidization has been achieved for several molluscan species (2, 3, 4, 5, 6). In contrast, little is known about the effects of ploidy manipulation in decapod crustaceans. Triploidy has been induced in Penaeus chinensis and other penaeid shrimp using heat shock or cold shock, cytochalasin B, and 6-dimethylaminopurine (7, 8, 9, 10, 11). Triploidy was induced by suppression of polar body 2 in the shrimp Litopenaeus vannemei using cold shock, but resulted in poor survival for unknown reasons (11). One difficulty of ploidy manipulation in L. vannemei is the rapid sequence of meiotic events, since the polar bodies are produced at 8 and 15 min postspawning at 28°C (12). In addition, the actual behavior of chromosomes and spindles was not addressed. Here I report that in the penaeoidean shrimp Sicyonia ingentis, inhibition of polar body 2 resulted in embryos that failed to undergo embryonic cleavage, while inhibition of polar body 1 produced oocytes with “twin” meiosis 2 spindles followed by the simultaneous formation of two “second” polar bodies. I performed a dose response study to determine the minimum level of cytochalasin D (CD) needed to inhibit cytokinesis in zygotes of Sicyonia ingentis. Embryonic cleavage was sensitive to CD concentrations of 0.1 M or greater, and could be completely inhibited at 0.5–0.8 M (Fig. 1). Inhibition of polar body formation required at least 1 M CD, but percent inhibition of polar bodies was difficult to judge, since they could only be seen if present along the edge of the egg. One hundred percent suppression of polar bodies did not occur, so a qualitative judgement of inhibition was made if few were observed relative to controls. In later experiments, qualitative evidence of first polar body (PB1) suppression was provided if eggs were observed with two second polar bodies (PB2). No inhibition of cleavage or polar body formation was seen in controls that received no treatment or treatment with the carrier dimethyl sulfoxide (DMSO) only. Oocytes of S. ingentis are spawned at metaphase of meiosis 1. At 21°C, PB1 forms at 30–35 min post-spawning (PS), the hatching envelope forms at 40–45 min PS, PB2 formation occurs at 50–55 min PS, and first cleavage occurs about 90 min PS (13, 14). Thus, after meiotic maturation, PB1 lies outside of the hatching envelope while PB2 lies within it (see Fig. 2E). PB2 formation was inhibited by treatment of oocytes with 1 M CD from 40–55 min PS, after formation of PB1. In No Treatment and DMSO controls, eggs extruded both polar bodies, formed hatching envelopes, and went through normal cleavage (Fig. 2A). In the 40–55 min CD treatment, PB1 formed but not PB2, hatching envelopes elevated about 15 min later than the control groups, and cleavage was completely inhibited (Fig. 2B). Cleared regions of cytoplasm were observed within these eggs. When control-group embryos were stained using Sytox green, nuclei were present in the blastomeres (Fig. 2C). In the CD-treatment group, the uncleaved embryo contained many nuclei (Fig. 2D), indicating that the CD treatment from 40–55 min PS did not prevent microtubulebased chromosome segregation. Based on the data from two trials, a comparison of the No Treatment controls with the DMSO controls during PB2 suppression showed no significant difference in percent cleavage, indicating that the Received 8 November 2001; accepted 5 February 2002. E-mail: [email protected] Abbreviations: CD, cytochalasin D; DMSO, dimethyl sulfoxide; PB1, polar body 1; PB2, polar body 2; PS, post-spawning. Reference: Biol. Bull. 202: 100–103. (April 2002)