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A single and rapid calcium wave at egg activation in Drosophila.

York-Andersen AH, Parton RM, Bi CJ, Bromley CL, Davis I, Weil TT - Biol Open (2015)

Bottom Line: Here, we utilise ratiometric imaging of Ca(2+) indicator dyes and genetically encoded Ca(2+) indicator proteins to identify and characterise a single, rapid, transient wave of Ca(2+) in the Drosophila egg at activation.We further show that mechanical pressure alone is not sufficient to initiate a Ca(2+) wave.We also find that processing bodies, sites of mRNA decay and translational regulation, become dispersed following the Ca(2+) transient.

View Article: PubMed Central - PubMed

Affiliation: Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK.

No MeSH data available.


Related in: MedlinePlus

Suction alone is not sufficient to cause a Ca2+ wave.(A,B) Time series of a mature oocyte expressing UAS-myrGCamp-5 dissected into Schneider's Insect medium with suction applied to the posterior half of the mature oocyte with a 135 µm diameter plastic tube for 2 minutes (A). Subsequent time series show no morphological or intracellular Ca2+ changes (B) (n = 7). (C) Whole female reproductive machinery dissection, white box highlights the region of interest for experiment in panels D and E. (D,E) Higher magnification of whole female reproductive machinery expressing UAS-myrGCamp-5 cultured in Schneider's Insect medium imaged with fluorescence (D) and corresponding bright-field image (E). A mature oocyte preparing to enter or slightly entered the lateral oviduct (white star) shows an increase in intracellular Ca2+ at the posterior pole (n = 3 due to technical challenges in imaging live at this magnification). Scale bars A,B,D,E = 100 µm, C = 1000 µm. Max projection B = 40 µm.
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f05: Suction alone is not sufficient to cause a Ca2+ wave.(A,B) Time series of a mature oocyte expressing UAS-myrGCamp-5 dissected into Schneider's Insect medium with suction applied to the posterior half of the mature oocyte with a 135 µm diameter plastic tube for 2 minutes (A). Subsequent time series show no morphological or intracellular Ca2+ changes (B) (n = 7). (C) Whole female reproductive machinery dissection, white box highlights the region of interest for experiment in panels D and E. (D,E) Higher magnification of whole female reproductive machinery expressing UAS-myrGCamp-5 cultured in Schneider's Insect medium imaged with fluorescence (D) and corresponding bright-field image (E). A mature oocyte preparing to enter or slightly entered the lateral oviduct (white star) shows an increase in intracellular Ca2+ at the posterior pole (n = 3 due to technical challenges in imaging live at this magnification). Scale bars A,B,D,E = 100 µm, C = 1000 µm. Max projection B = 40 µm.

Mentions: It has long been postulated that both mechanical stimulation and external cues contribute to Drosophila egg activation during deposition (Heifetz et al., 2001; Horner and Wolfner, 2008; Krauchunas and Wolfner, 2013). Various methods have previously been used to study this question, for example, physical pulling on the dorsal appendages of mature oocytes has been shown to cause the resumption of meiosis (Endow and Komma, 1997) and placing mature oocytes into a hypotonic buffer causes them to swell and activate (Mahowald et al., 1983; Page and Orr-Weaver, 1997). To test whether mechanical pressure is able to prompt and propagate a wave of Ca2+, we applied pressure with a glass rod to the outside of an oocyte mounted in halocarbon oil (Fig. 4A,B). Despite a local increase in Ca2+ where pressure was applied, no propagating wave or physical hallmarks of activation were observed (Fig. 4C). To better represent the physiological process of the passage of an egg chamber through the oviduct, we applied pressure on the ex vivo mature oocyte with plastic tubes, normally used for cell isolation prior to IVF treatments (Fig. 5A,B). We show that cylindrical pressure for 1–3 minutes on the posterior half of the mature oocyte did not result in a Ca2+ or physical change (Fig. 5A,B). These results further support the model that the external environment, likely the fluid in the oviduct (Heifetz et al., 2001) is required for initiation of a Ca2+ wave.


A single and rapid calcium wave at egg activation in Drosophila.

York-Andersen AH, Parton RM, Bi CJ, Bromley CL, Davis I, Weil TT - Biol Open (2015)

Suction alone is not sufficient to cause a Ca2+ wave.(A,B) Time series of a mature oocyte expressing UAS-myrGCamp-5 dissected into Schneider's Insect medium with suction applied to the posterior half of the mature oocyte with a 135 µm diameter plastic tube for 2 minutes (A). Subsequent time series show no morphological or intracellular Ca2+ changes (B) (n = 7). (C) Whole female reproductive machinery dissection, white box highlights the region of interest for experiment in panels D and E. (D,E) Higher magnification of whole female reproductive machinery expressing UAS-myrGCamp-5 cultured in Schneider's Insect medium imaged with fluorescence (D) and corresponding bright-field image (E). A mature oocyte preparing to enter or slightly entered the lateral oviduct (white star) shows an increase in intracellular Ca2+ at the posterior pole (n = 3 due to technical challenges in imaging live at this magnification). Scale bars A,B,D,E = 100 µm, C = 1000 µm. Max projection B = 40 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4400597&req=5

f05: Suction alone is not sufficient to cause a Ca2+ wave.(A,B) Time series of a mature oocyte expressing UAS-myrGCamp-5 dissected into Schneider's Insect medium with suction applied to the posterior half of the mature oocyte with a 135 µm diameter plastic tube for 2 minutes (A). Subsequent time series show no morphological or intracellular Ca2+ changes (B) (n = 7). (C) Whole female reproductive machinery dissection, white box highlights the region of interest for experiment in panels D and E. (D,E) Higher magnification of whole female reproductive machinery expressing UAS-myrGCamp-5 cultured in Schneider's Insect medium imaged with fluorescence (D) and corresponding bright-field image (E). A mature oocyte preparing to enter or slightly entered the lateral oviduct (white star) shows an increase in intracellular Ca2+ at the posterior pole (n = 3 due to technical challenges in imaging live at this magnification). Scale bars A,B,D,E = 100 µm, C = 1000 µm. Max projection B = 40 µm.
Mentions: It has long been postulated that both mechanical stimulation and external cues contribute to Drosophila egg activation during deposition (Heifetz et al., 2001; Horner and Wolfner, 2008; Krauchunas and Wolfner, 2013). Various methods have previously been used to study this question, for example, physical pulling on the dorsal appendages of mature oocytes has been shown to cause the resumption of meiosis (Endow and Komma, 1997) and placing mature oocytes into a hypotonic buffer causes them to swell and activate (Mahowald et al., 1983; Page and Orr-Weaver, 1997). To test whether mechanical pressure is able to prompt and propagate a wave of Ca2+, we applied pressure with a glass rod to the outside of an oocyte mounted in halocarbon oil (Fig. 4A,B). Despite a local increase in Ca2+ where pressure was applied, no propagating wave or physical hallmarks of activation were observed (Fig. 4C). To better represent the physiological process of the passage of an egg chamber through the oviduct, we applied pressure on the ex vivo mature oocyte with plastic tubes, normally used for cell isolation prior to IVF treatments (Fig. 5A,B). We show that cylindrical pressure for 1–3 minutes on the posterior half of the mature oocyte did not result in a Ca2+ or physical change (Fig. 5A,B). These results further support the model that the external environment, likely the fluid in the oviduct (Heifetz et al., 2001) is required for initiation of a Ca2+ wave.

Bottom Line: Here, we utilise ratiometric imaging of Ca(2+) indicator dyes and genetically encoded Ca(2+) indicator proteins to identify and characterise a single, rapid, transient wave of Ca(2+) in the Drosophila egg at activation.We further show that mechanical pressure alone is not sufficient to initiate a Ca(2+) wave.We also find that processing bodies, sites of mRNA decay and translational regulation, become dispersed following the Ca(2+) transient.

View Article: PubMed Central - PubMed

Affiliation: Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK.

No MeSH data available.


Related in: MedlinePlus