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First published online December 14, 2006
Journal of Experimental Biology 210, 138-148 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.02652

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Calcium-regulated fusion of yolk granules is important for yolk degradation during early embryogenesis of Rhodnius prolixus Stahl

I. B. Ramos¹, K. Miranda², W. de Souza², D. M. P. Oliveira¹, A. P. C. A. Lima³, M. H. F. Sorgine⁴ and E. A. Machado^1,*

¹ Laboratório de Entomologia Médica, Instituto de Biofísica Carlos Chagas Filho (IBCCF), Universidade Federal do Rio de Janeiro (UFRJ), Cidade Universitária - Ilha do Fundão, 21941-590 Rio de Janeiro, RJ, Brasil
² Laboratorio de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho (IBCCF), Universidade Federal do Rio de Janeiro (UFRJ), Cidade Universitária - Ilha do Fundão, 21941-590 Rio de Janeiro, RJ, Brasil
³ Laboratório de Bioquímica e Biologia Molecular de Proteases, Instituto de Biofísica Carlos Chagas Filho (IBCCF), Universidade Federal do Rio de Janeiro (UFRJ), Cidade Universitária - Ilha do Fundão, 21941-590 Rio de Janeiro, RJ, Brasil
⁴ Laboratório de Artrópodos Hematófagos, Instituto de Bioquímica Médica (IBQM), Centro de Ciências da Saúde (CCS), Universidade Federal do Rio de Janeiro (UFRJ), Cidade Universitária - Ilha do Fundão, 21941-590 Rio de Janeiro, RJ, Brasil

View larger version (68K): [in this window] [in a new window]   Fig. 1. Internal morphology of R. prolixus eggs. (A,B) DIC micrographs of transverse cryosections of eggs at days 0 and 3 after oviposition, respectively. In B, the chorion (ch) and embryo are visible and white arrows indicate large yolk granules (LYG). (C,D) HistoresinTM sections of day 0 and day 3 eggs, respectively. Black arrows indicate LYGs. Scale bars, 200 µm.

View larger version (4K): [in this window] [in a new window]   Fig. 2. [Ca2+] during R. prolixus early embryogenesis. Ca2+ was measured in eggs using the sensor dye Arsenazo III. The highest [Ca2+] (23±2.3 mmol l-1) was found on the third day of embryogenesis. Values are means ± s.e.m. of 5 different experiments.

View larger version (11K): [in this window] [in a new window]   Fig. 3. Induction of large yolk granule (LYG) formation by in vitro calcium treatment. (A) Formation of LYGs (>40 µm) as a function of calcium concentration. Incubation of day 0 SYGs with increasing concentrations of calcium lead to the formation LYGs (a 13-fold increase of LYGs can be observed in 23 mmol l-1 Ca2+). Values are means ± s.e.m. of 4 different experiments. *Statistically significant difference (P<0.05, one-way ANOVA). (B,C) DIC microscopy showing the general views of YGs incubated with 10 mmol l-1 EGTA (B) and 23 mmol l-1 Ca2+ (C). Note the presence of a higher number of LYGs (arrow) after incubation with calcium. Scale bars, 200 µm.

View larger version (37K): [in this window] [in a new window]   Fig. 4. Fractionation of yolk granules (YGs). (A) DIC light microscopy showing the day 0 total yolk granules before fractionation by differential centrifugation. (B,C) YG fractions after differential centrifugation. Note that the top (B) and pellet (C) fractions are enriched in small YGs (SYGs) and large YGs (LYGs), respectively. Scale bars, 200 µm.

View larger version (39K): [in this window] [in a new window]   Fig. 5. Transfer of the fluorescent membrane dye, PKH67 from labeled small yolk granules (SYGs) to larger YGs after calcium treatment. (A,C,E) DIC images; (B,D,F) corresponding fluorescence images. (A,B) SYGs. Scale bar, 50 µm. (C,D) Dye transfer from SYGs to large ones. Note the appearance of large stained YGs as the result of incubation with Ca2+. Scale bar, 25 µm. (E,F) Incubation of labeled SYGs with day 0 YGs in the presence of 10 mmol l-1 EGTA (calcium-free medium). Note that dye transfer did not occur. Scale bar, 25 µm.

View larger version (43K): [in this window] [in a new window]   Fig. 6. H+-PPase specific activity levels in different yolk granule (YG) fractions at days 0 and 3 of development. Membrane fractions were separated from LYGs and SYGs and assayed for H+-PPase activity. (A) H+-PPase activity levels during days 0 and 3 of embryogenesis. White bars, day 0 activity; gray bars day 3 activity. EH, total egg homogenate activity. Values are means ± s.e.m. of 3 different experiments. (B) H+-PPase specific activity levels in day 0 LYGs with and without Ca2+. *Statistically significant difference (P<0.05, t-test). (C-F) To determine the location of the H+-PPase, YGs were also incubated with Acridine Orange in the presence of PPi during day 0 (C,D) and day 3 (E,F) of embryogenesis. Arrows indicate acidic SYGs and arrowheads indicate acidic LYGs. (C,E) DIC images; (D,F) corresponding fluorescence images. Scale bars, 25 µm.

View larger version (50K): [in this window] [in a new window]   Fig. 7. Vacuolar H+-ATPase specific activity levels in different yolk granule (YG) fractions during days 0 and 3 of development. LYGs and SYGs were submitted to membrane fraction separation and assayed for H+-ATPase activity. (A) Vacuolar H+-ATPase activity levels during days 0 and 3 of embryogenesis. White bars represent day 0 activities and gray bars represent day 3 activities. Values are means ± s.e.m. of 3 different experiments. To investigate the location of the H+-ATPase, YGs were incubated with AO in the presence of ATP during day 0 (B,C) and day 3 (D,E) of embryogenesis. Arrows indicate acidic SYGs and arrowheads indicate acidic LYGs. (B,D) DIC images; (C,E) corresponding fluorescence images. Scale bars, 25 µm.

View larger version (14K): [in this window] [in a new window]   Fig. 8. Specific activity levels of hydrolases in different yolk granule (YG) fractions during days 0 and 3 of development. (A) Acid phosphatase activity levels. (B) Cathepsin D activity levels. White bars, activity on day 0; gray bars, activity on day 3. Values are means ± s.e.m. of 3 different experiments. EH, total egg homogenate. *Statistically significant difference (P<0.05, t-test).

View larger version (37K): [in this window] [in a new window]   Fig. 9. SDS-PAGE of yolk granule (YG) fractions and vitellin (VT) immunolocalization. (A) SDS-PAGE of the different YG fractions. Arrows indicate the four apoproteins in SYGs, LYGs and total egg homogenate (EH); molecular mass is given on the left. (B-E) Vitellin immunolocalization in R. prolixus eggs. Vitellins were present in both LYGs and SYGs during day 0 (B,C) and day 3 (D,E) of embryogenesis. The chorion (ch) was shown to be auto-fluorescent in control groups (data not shown). (B,D) DIC images; (C,E) corresponding fluorescence images. Scale bars, 50 µm.

View larger version (17K): [in this window] [in a new window]   Fig. 10. Vitellin (VT) proteolysis after calcium incubation. (A) SDS-PAGE showing VT proteolysis after calcium incubation. Yolk granules (YGs) were extracted from day 0 and day 3 eggs and incubated in Ringer saline containing 1 mmol l-1 ATP and 1 mmol l-1 PPi. For experimental groups 23 mmol l-1 Ca2+ was added. (Lane 1) Day-0 YGs after incubation with EGTA; (lane 2) day-0 YGs after incubation with calcium; (lane 3) day-3 YGs after incubation with EGTA; (lane 4) day-3 YGs after incubation with calcium. (B) Cathepsin D activities tested in all samples used in A. AU, arbitrary units.