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Increase of cAMP upon release from prophase arrest in surf clam oocytes

¹ Département d’Obstétrique-Gynécologie, Université de Montréal, Centre de Recherche du CHUM, Hôpital Saint-Luc, 264 René-Lévesque Est, Montréal, Québec, Canada H2X 1P1
² Département des Sciences Biologiques, Université de Montréal, C.P. 6128, Succ. Centre-Ville, Montréal, Québec, Canada H3C 3J7
³ Urology Research Laboratory, Royal Victoria Hospital, McGill University, 687, Pine Avenue W., Montréal, Québec, Canada H3A 1A1

View larger version (30K): [in a new window]   Fig. 1. Effect of forskolin and IBMX pretreatments on GVBD and cAMP levels in Spisula oocytes. In (A) and (B), oocytes were pretreated with various combinations of forskolin and IBMX at indicated concentrations for 15 minutes before adding 5-HT (5 µM, A) or KCl (45 mM, B). DMSO, DMSO vehicle alone; Forsk/IBMX: Forskolin and IBMX (65/60 µM) without any activating agent. GVBD was scored after 20 minutes. Mean results (±s.e.m.) of four experiments are shown. In (C) and (D), the effect of incubating oocytes for one hour in the presence of forskolin (120 µM), IBMX (115 µM) or a combination of both (60/65 µM) on oocyte cAMP concentration (A) and on subsequent 5-HT-induced GVBD (B) is shown. Unt, oocytes left untreated; DMSO, oocytes treated with the DMSO vehicle alone (Panel C) and to which 5-HT was added (Panel D). Asterisk (*), significantly different from untreated oocytes. Mean results (±s.e.m.) of three experiments are shown.

View larger version (18K): [in a new window]   Fig. 2. Effect of dbcAMP, 8-bromo-cAMP or Sp-cAMPs pretreatment on 5-HT-induced GVBD. Oocytes were pretreated using various indicated concentrations of dbcAMP (A), 8-bromo-cAMP (B) or Sp-cAMPs (C) for 15 minutes prior to the addition of 5-HT (5 µM). dbcAMP, dbcAMP alone; 8-Br-cAMP, 8-bromo-cAMP alone; Sp-cAMPs, Sp-cAMPs alone. Mean results (±s.e.m.) of four experiments are shown.

View larger version (15K): [in a new window]   Fig. 3. Effect of IBMX and forskolin on sperm-induced GVBD. Oocytes were either left untreated (, Control) or pre-incubated in presence of 65 µM IBMX and 60 µM forskolin (, Forsk/IBMX) or an equivalent amount of DMSO (, DMSO), for 15 minutes prior to insemination at time t=0. Aliquots were sampled at indicated times for determination of percentage GVBD. Mean results (±s.e.m.) of two separate experiments are shown.

View larger version (36K): [in a new window]   Fig. 4. Effect of IBMX and forskolin on sperm incorporation into the oocytes. (A) Control fertilized oocyte after 30 minutes (metaphase I of meiosis) showing alignment of maternal chromosomes (mc) and a decondensed male pronuleus (mp). (B) Control fertilized oocyte 60 minutes (metaphase II of meiosis) after the addition of sperm, showing a polar body (pb), maternal chromosomes (mc) and a decondensed male pronucleus (mp). (C) Oocytes 30 minutes after insemination in the presence of IBMX (65 µM) and forskolin (60 µM), showing an intact germinal vesicle (gv) and undecondensed sperm heads (sh) at the periphery of the oocyte. (D) Similarly treated oocytes 60 minutes after insemination, still showing intact germinal vesicles and the absence of any male pronucleus in their cytoplasm.

View larger version (34K): [in a new window]   Fig. 5. Effect of 5-HT, NH4Cl or KCl on the cAMP concentration of Spisula oocytes. (A) Oocytes were either treated with 5-HT (5 µM, ) or left untreated (). Aliquots were removed at different times and processed for cAMP determinations, and the results are expressed as a percentage of the initial cAMP concentration of untreated oocytes. (B) A similar experiment using oocytes pre-incubated in the presence of forskolin and IBMX (60/65 µM) for 15 minutes prior to the addition of 5-HT. The insets depict the corresponding time course of GVBD (±s.e.m.) for sampled oocytes. Mean results of three experiments are shown. (C) shows the effect of adding 5-HT (5 µM), KCl (10% v/v) or NH4Cl (10 mM) on oocyte cAMP concentration at indicated times, as compared with untreated oocytes. Percentages of GVBD scored 15 minutes after adding 5-HT, KCl or NH4Cl were 100%, 100% and 0% percent, respectively. Mean results (±s.e.m.) of three experiments are shown. (D) shows the effect of adding K+ on oocyte cAMP concentration. Various amounts of isotonic KCl (0.52 M) were added to oocytes to obtain the final concentrations shown (% v/v). Oocytes were sampled 10 minutes later for determination of cAMP concentration, as described in the Materials and Methods. GVBD, as scored after 15 minutes, had occurred only at KCl concentrations of 5 and 10% (v/v), in 86% and 100% of the oocytes, respectively, with all lower KCl concentrations resulting in less than 2% GVBD. Mean results (±s.e.m.) of three experiments.

View larger version (16K): [in a new window]   Fig. 6. Effect of fertilization on cAMP concentration in Spisula oocytes. Oocytes (0.2% v/v) were divided in two lots, which were either inseminated at time t=0 (, Fertilized) or not (, Unfertilized). At indicated times, oocytes were sampled for determinations of cAMP concentration, as described in Materials and Methods. GVBD was less than 2% in unfertilized oocytes. Mean results (±s.e.m.) of two experiments are shown.

View larger version (18K): [in a new window]   Fig. 7. Effect of pretreatment of oocytes with SQ 22,536 on cAMP concentration and the time course of GVBD. (A) Oocytes were either pretreated with 1 mM SQ 22,536 (open symbols, ,) or left untreated (filled symbols, , ) for one hour prior to the addition of 5-HT (5 µM, , ) or KCl (45 mM, , ), at time 0. At indicated times, oocytes were fixed for determination of the percentage of GVBD. Mean results (±s.e.m.) of two experiments are shown. (B) Oocytes were divided in two lots, one to which SQ 22,536 (1 mM) was added () while the other was left untreated (). One hour later, 5-HT (5 µM) was added to an aliquot of both SQ 22,536-treated () and -untreated oocytes (). At indicated times, samples of oocytes were frozen and later processed for determinations of cAMP concentration expressed as a percentage of the initial cAMP concentration of oocytes at the beginning of the experiment. The inset depicts the corresponding time course of GVBD after addition of 5-HT, in both groups of oocytes. Mean results of two experiments are shown.