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doi: 10.1242/10.1242/dev.00508

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Endoplasmic reticulum reorganizations and Ca²⁺ signaling in maturing and fertilized oocytes of marine protostome worms: the roles of MAPKs and MPF

Department of Biology, MSC03 2020, 1 University Avenue, University of New Mexico, Albuquerque, NM 87131-0001, USA

View larger version (99K): [in a new window]   Fig. 7. (A) Colchicine delays fertilization-induced disassembly of ER clusters (A) and prolongs Ca2+ oscillations (B). Arrows in B indicate timepoints when Ca2+ imaging was discontinued to obtain a z-series of DiI fluorescence. Scale bar: 50 µm. Representative of three doubly injected specimens.

View larger version (29K): [in a new window]   Fig. 1. (A) C. lacteus oocytes treated with 1 µM 5-HT in the presence or absence of 20 µM U0126 and probed for MAPK activity with anti-phospho-ERK1/2 Ab. Note normal levels of GVBD in U0126-treated samples even though MAPK signal is greatly reduced. 'Positive control' was supplied by Cell Signaling as material used to generate antibody. (B) Although 5-HT-treated oocytes mature at high levels without MAPK activity, similar incubations in U0126 or 25 µM PD98059 reduce spontaneous GVBD. Each blot is representative of five replicates. (C) Activity assays of MPF and ERK1/2 MAPK in maturing C. lacteus oocytes. Upper two bands at 66 and 48 kDa represent full-length and truncated Rb fusion protein probed with a phospho-Rb-specific antibody to track MPF activity. Phospho-ELK-1 band at 34 kDa depicts ERK1/2 activity. Note MPF activation continues in the absence of substantial MAPK activity.

View larger version (36K): [in a new window]   Fig. 2. (A,B) Single oocyte of Micrura alaskensis continuously bathed in 20 µM U0126. (A) single confocal plane (left) and compressed z-series (right) before GVBD. (B) Same oocyte in mature state 2 hours after treatment with 5-HT plus U0126, showing ER clusters: single optical plane view (left) and compressed z-series (right). (C) Compressed z-series of a mature Cerebratulus sp. oocyte with ER clusters 2 hours after treatment with 5-HT+U0126. Scale bars: 50 µm. GV, germinal vesicle.

View larger version (62K): [in a new window]   Fig. 3. (A) ER clusters continue to form during 5-HT-induced oocyte maturation in 20 µM curcumin or 20 µM SB-202190 (inset). Large oocyte in upper right of A is from C. lacteus; others are from Cerebratulus sp. (B) SP600125 fails to block 5-HT-induced GVBD in Cerebratulus sp. but does prevent ER clusters formation. Scale bars: 50 µm.

View larger version (76K): [in a new window]   Fig. 4. (A) Post-fertilization loss of ER clusters in a DiI-loaded Cerebratulus sp. oocyte. (B) ER clusters that are present in mature oocytes of Cerebratulus lacteus before fertilization in 25 µM PD98059 (top) continue to be disassembled by 2 hours post-fertilization (bottom). Scale bars: 50 µm.

View larger version (147K): [in a new window]   Fig. 5. (A) Left: two Cerebratulus lacteus oocytes with ER clusters before roscovitine addition. Right: 60 minutes after roscovitine treatment. (B) Roscovitine-induced disassembly of ER clusters and polar body (pb) formation in C. lacteus. Timing from left to right: before roscovitine, and 5 minutes, 10 minutes, 15 minutes and 30 minutes post-treatment. (C, right) A Cerebratulus sp. oocyte that spontaneously progressed through metaphase I arrest and formed polar bodies (pb). Note lack of ER clusters compared with metaphase-I-arrested cohort (left). Scale bars: 50 µm.

View larger version (42K): [in a new window]   Fig. 6. (A) Typical blot of post-fertilization MPF and MAPK declines, as measured by Rb phosphorylation (upper two rows) and ELK-1 phosphorylation (lowest row), respectively. No decrease is observed in uninseminated metaphase-I-arrested controls. (B) MPF activity in 10 fertilization runs subjected to Rb phosphorylation assays. (C) MAPK activity decreases in fertilization runs assayed by phospho-ERK1/2 western blots ('P-MAPK', n=8) or ELK-1 phosphorylations ('P-ELK-1'; n=8). Bars indicate standard errors. pb1, typical onset of polar body 1 formation; pb2, typical onset of polar body 2 formation.

View larger version (12K): [in a new window]   Fig. 8. (A,B) Typical fertilization-induced Ca2+ response of immature, prophase-arrested (A) and mature, metaphase-I-arrested (B) oocyte of C. lacteus. In general, polar body 1 and 2 in C. lacteus form at 45 and 90 minutes post-insemination, respectively.

View larger version (27K): [in a new window]   Fig. 9. Normal fertilization-induced Ca2+ oscillations in Cerebratulus sp. (Inset) two-cell stage that developed at 2.5 hours post-fertilization after generating oscillations shown in graph. In general, polar body 1 and 2 in Cerebratulus sp. form at 20 and 45 minutes post-insemination, respectively.

View larger version (72K): [in a new window]   Fig. 10. (A,B) Essentially normal fertilization-induced Ca2+ response of Cerebratulus lacteus in 25 µM PD98059 (A) or 20 µM U0126 (B). (C) Three consecutive fertilization-induced Ca2+ waves (arrows) in PD98059-treated oocyte (images between waves have been deleted). Scale bar: 50 µm.

View larger version (14K): [in a new window]   Fig. 11. (A,B) Two examples of Ca2+ oscillations of mature Cerebratulus sp. oocytes fertilized in ASW solution of SP600125. (A) Essentially normal oscillations that were characteristic of two out of four specimens showing repetitive Ca2+ elevations after fertilization (compare with Fig. 9 for normal oscillations in a control specimen). (B) Abnormal repetitive spiking that was characteristic of the other two oocytes displaying multiple fertilization-induced Ca2+ rises.

View larger version (13K): [in a new window]   Fig. 12. (A) 50 µM roscovitine pre-treatment prevents normal fertilization-induced Ca2+ oscillations in C. lacteus. (B) When added during the oscillations, 50 µM roscovitine causes premature termination of Ca2+ response.

View larger version (32K): [in a new window]   Fig. 13. General model of the interactions between ER structure, MPF levels and Ca2+ signaling during oocyte maturation and fertilization in nemerteans.