Biol. Bull. 202: 100-103. (April 2002)
© 2002 Marine Biological Laboratory
Twin Meiosis 2 Spindles Form After Suppression of Polar Body 1 Formation in Oocytes of the Marine Shrimp Sicyonia ingentis
Philip L. Hertzler
Department of Biology, Central Michigan University, 100 W. Preston Rd., Mt. Pleasant, Michigan 48859
E-mail: Philip.L.Hertzler{at}cmich.edu
Abbreviations: CD, cytochalasin D • DMSO, dimethyl sulfoxide • PB1, polar body 1 • PB2, polar body 2 • PS, post-spawning
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 post-spawning 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 µM or greater, and could be completely inhibited at 0.5–0.8 µM (Fig. 1). Inhibition of polar body formation required at least 1 µM 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.
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Figure 1. Dose response of inhibition of cleavage to treatment with cytochalasin D (CD). Gravid specimens of Sicyonia ingentis were collected by otter trawl one mile south of Palos Verdes, California, from the R/V Vantuna, operated by Occidental College. Animals were spawned as described previously (16). Cytochalasin D was obtained from Sigma (Cat. #C8273) and dissolved in dimethyl sulfoxide (DMSO) to make a stock solution of 1 mM. In three separate experiments, eggs were spawned into artificial seawater (ASW) then transferred to ASW containing 0–1 µM CD at 15 min post-spawning (PS). In each group, 300 eggs were scored for % cleavage. Cleavage was completely inhibited with 0.5–0.8 µM CD, whereas evidence of polar body suppression required at least 1 µM CD.
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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 µM 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 microtubule-based 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 DMSO had no effect, whereas CD treatment completely inhibited embryonic cleavage (Table 1).
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Figure 2. Effects of cytochalasin D (CD) treatments. For inhibition of polar body 2 (PB2) formation, eggs were transferred at 40 min post-spawning (PS) to 1 µM (0.5 mg/l) CD in artificial seawater containing 50 µM 3-amino-1,2,4-triazole (ATA-ASW), to facilitate removal of the hatching envelope for staining (17). At 55 min PS, eggs were washed 4 times with 0.1% DMSO in ATA-ASW. For suppression of polar body 1 (PB1) formation, eggs were transferred at 15 min PS to 1 µM CD in ATA-ASW. At 35 min PS, CD-treated eggs were washed 4 times with 0.1% DMSO in ATA-ASW. Control groups (0.1% DMSO carrier control and No Treatment control) were subjected to identical physical manipulations as CD-treated eggs. CD-treated eggs and control groups were fixed with 4% paraformaldehyde in ASW and examined by phase-contrast microscopy. Other samples were fixed in 90% methanol/50 mM EGTA, pH 8.0, and stained with ß-tubulin monoclonal antibody E7 as described previously (14). Nuclei and chromosomes were stained with Sytox green (Molecular Probes, Inc.) for confocal microscopy. (A) DMSO control from 40–55 min PS, dark-field image of embryos at the 16-cell stage, 3-h PS. (B) CD treatment from 40–55 min PS, dark-field image of embryos at 3-h PS. Arrows indicate cleared regions of cytoplasm. (C) DMSO control from 40–55 min PS, embryo at 3-h PS, stained with Sytox green. The image is a composite of optical slices through one-half of the embryo. (D) CD treatment from 40–55 min PS, embryo at 3-h PS, stained with Sytox green. The image is a composite of optical slices through the entire embryo. (E) Phase-contrast image of DMSO control from 15–40 min PS, egg at 60 min PS. Note the positions of PB1 and PB2 (arrowheads) relative to the hatching envelope. (F) Phase-contrast image of CD treatment from 15–40 min PS, egg at 60 min PS. Two "second" polar bodies (arrowheads) have formed beneath the hatching envelope. (G) Confocal fluorescence image of "twin" meiosis 2 spindles in PB1-suppressed egg, revealed by anti-ß-tubulin immunofluorescence. (H) Composite of optical sections of PB1-suppressed egg, stained with Sytox green. A single male pronucleus (m) and two female pronuclei (f) are present. Scale bars = 100 µm in A–F and H, and 20 µm in G.
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PB1 formation was inhibited by treating oocytes with 1 µM CD from 15–35 min PS, then washing out the CD with artificial seawater. In No Treatment and DMSO controls, polar bodies formed normally, with PB1 outside the hatching envelope and PB2 inside of it (Fig. 2E). In the 15–35 min CD treatment, two "second" polar bodies formed simultaneously in some eggs, both positioned inside the hatching envelope (Fig. 2F). Oocytes treated from 15–35 min with CD and stained with anti-tubulin antibodies revealed that "twin" meiosis 2 spindles were present in the cortex (Fig. 2G). Two maternal chromosome sets were expelled in the two polar bodies, while two maternal chromosome sets were retained in the egg and decondensed into two female pronuclei (Fig. 2H). Based on the data from two experiments, percent cleavage in DMSO controls showed no significant difference from No Treatment controls, and the CD treatment from 15–35 min showed no significant difference from No Treatment controls (Table 1). Normal cleavage divisions continued in the CD-treatment group.
Each method of polar body suppression should theoretically produce triploid zygotes. In Sicyonia, normal embryonic cleavage follows from suppression of PB1 but not PB2. The responses to polar body suppression in marine invertebrates have been best studied in oysters (2,3,6). In Crassostrea gigas, inhibition of PB1 with cytochalasin B resulted in a number of possible chromosome segregation patterns, including united bipolar, tripolar, and separated bipolar (3). Tripolar spindles have been demonstrated in such treatments by anti-tubulin staining (4). The "twin" meiosis 2 spindles described here correspond to the "separated bipolar" spindle pattern inferred from observations of chromosome staining in C. gigas. This report describes the first direct observation of the "separated bipolar" pattern by tubulin immunofluorescence localization. Tripolar spindles or other unusual patterns were not observed as a result of ploidy manipulation in S. ingentis, so the occurrence of aneuploidy following PB1 suppression may be less of a problem than it is for oysters (2,3). However, the studies in oysters used cytochalasin B instead of cytochalasin D, so they may not be directly comparable. The separated bipolar meiosis 2 spindles in PB1-blocked oocytes appear to be of the same size and orientation as normal meiosis 2 spindles, and they progress through the cell cycle at the same time. Therefore, following meiosis 1, both centrosomes retain the ability to duplicate and form meiosis 2 spindle poles.
In response to PB1 suppression, only one "second" polar body forms in C. gigas (2), but in S. ingentis two polar bodies formed simultaneously. The simultaneous formation of two polar bodies occurs naturally in the androgenetic clam Corbicula leana following meiosis 1, which eliminates all maternal chromosomes; development is then directed by the incorporated diploid sperm nucleus (15). A specialized microtubular structure causes this process in C. leana. In contrast, the two polar bodies formed after PB1 suppression in S. ingentis apparently utilize the same spindle mechanics as does normal meiosis 2. Maternal centrosome function, pronuclear formation, pronuclear migration, and embryonic mitoses and cytokinesis seem to occur normally following PB1 suppression. Further work is needed to determine if these embryos develop to the nauplius larval stage and if they are indeed triploid. The diploid number of S. ingentis is known to be 64 (10), so ploidy analysis could be performed to answer this question.
Why does PB1 suppression in S. ingentis allow embryonic cleavage while PB2 suppression does not? The time of exposure to CD was less for PB2 suppression than for PB1 suppression, and the number of washes was the same, yet cleavage was blocked. The difference may be due to the presence of the hatching envelope during the treatment time for PB2 suppression. The hatching envelope formed more slowly in CD-treated eggs, suggesting that the vesicle secretion needed for this process requires microfilaments. Perhaps the newly formed hatching envelope also retards the loss of CD during the washing steps, creating a higher concentration of CD within the microenvironment of the perivitelline space and thereby inhibiting embryonic cleavage.
The results show that paired meiosis 2 spindles form following PB1 suppression in penaeoidean shrimp, and normal meiotic spindle function follows, so that triploid induction may be possible by this method. Polar body extrusion occurs rapidly in tropical shrimp species; for example, in L. vannamei, the polar bodies form at 8 and 15 min PS. Triploidy induction may be more feasible in cold-water penaeoidean species such as S. ingentis, where the events of meiotic maturation occur more slowly (at 35 and 45 min), allowing time for CD treatment and wash steps. The technique may also be possible in warm-water species if performed at the minimum temperature for successful development.
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Acknowledgments
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I am grateful to Dr. Gary Martin, Occidental College, for his generosity in sharing laboratory space and time, to the crew of the R/V Vantuna for assistance in animal collection, and to Jhonatan Sepulveda Villet and Dan Kiernan, Central Michigan University, for technical assistance. The ß-tubulin monoclonal antibody E7 developed by Michael Klymkowsky was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242. Thanks to Felix Famoye, Dept. of Mathematics, Central Michigan University, for help with statistical analysis and to two anonymous reviewers for suggestions on improving the manuscript. This work was supported by a State of Michigan/Central Michigan University Research Excellence Funds award and funds from the Department of Biology and the College of Science and Technology at Central Michigan University.
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Footnotes
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Received 8 November 2001; accepted 5 February 2002.
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