TheDrosophilaTrpm channel mediates calcium influx during egg activation

Abstract

Egg activation is the process in which mature oocytes are released from developmental arrest and gain competency for embryonic development. In Drosophila and other arthropods, eggs are activated by mechanical pressure in the female reproductive tract, whereas in most other species, eggs are activated by fertilization. Despite the difference in the trigger, Drosophila shares many conserved features with higher vertebrates in egg activation, including a rise of intracellular calcium in response to the trigger. In Drosophila, this calcium rise is initiated by entry of extracellular calcium due to opening of mechanosensitive ion channels and initiates a wave that passes across the egg prior to initiation of downstream activation events. Here, we combined inhibitor tests, germ-line–specific RNAi knockdown, and germ-line–specific CRISPR/Cas9 knockout to identify the Transient Receptor Potential (TRP) channel subfamily M (Trpm) as a critical channel that mediates the calcium influx and initiates the calcium wave during Drosophila egg activation. We observed a reduction in the proportion of eggs that hatched from trpm germ-line knockout mutant females, although eggs were able to complete some egg activation events including cell cycle resumption. Since a mouse ortholog of Trpm was recently reported also to be involved in calcium influx during egg activation and in further embryonic development, our results suggest that calcium uptake from the environment via TRPM channels is a deeply conserved aspect of egg activation.

A calcium‐mediated actin redistribution at egg activation in Drosophila

“…Using AB to activate the mature oocyte, we observed and analyzed a calcium wave and a wavefront of actin ( Figure 3a legend for details). We confirmed this result using modified Robb's Buffer (RB, Hu & Wolfner, 2019) to initiate egg activation and show that the speed of the F-tractin wavefront is similar to that of the calcium wave (0.56 μm/s; Figure S3, Movie 8). The average completion times of the calcium wave and the F-actin wavefront highlights the similarities between the dynamics of these waves.…”

“…IB contains 55 mM NaOAc, 40 mM KOAc, 1.2 mM MgCl 2 , 1 mM CaCl 2 , 110 mM sucrose, 100 mM HEPES in ddH 2 O. IB is adjusted to pH 7.4 with NaOH and filter sterilized. RB contains 55 mM NaOAc, 8 mM KOAc, 20 mM sucrose, 0.5 mM MgCl 2 , 2 mM CaCl 2 , 20 mM HEPES in ddH 2 O. RB was adjusted to pH 6.4 with NaOH and filter sterilized (Hu & Wolfner, ); For the high‐resolution 3D live imaging oocytes were mounted in a glass‐bottomed culture dish (MatTek) in Schneider's insect culture medium (GIBCO‐BRL) with a 1 mm 2 coverslip on the oocyte. For activation, Schneider's medium was removed and replaced with AB (Weil et al, ).…”

“…IB contains 55 mM NaOAc, 40 mM KOAc, 1.2 mM MgCl 2 , 1 mM CaCl 2 , 110 mM sucrose, 100 mM HEPES in ddH 2 O. IB is adjusted to pH 7.4 with NaOH and filter sterilized. RB contains 55 mM NaOAc, 8 mM KOAc, 20 mM sucrose, 0.5 mM MgCl 2 , 2 mM CaCl 2 , 20 mM HEPES in ddH 2 O. RB was adjusted to pH 6.4 with NaOH and filter sterilized (Hu & Wolfner, );…”

“…2) Oocytes were dissected in IB (Page & Orr-Weaver, 1997) RB was adjusted to pH 6.4 with NaOH and filter sterilized (Hu & Wolfner, 2019);…”

“…When a mature oocyte is ovulated and moves from the ovary to the lateral oviduct, it is thought to experience pressure and fluid uptake. This results in its swelling and a transient wave of increased cytoplasmic calcium that initiates from oocyte poles, predominantly the posterior, due to calcium influx through Trpm channels (Horner & Wolfner, 2008;Hu & Wolfner, 2019;Kaneuchi et al, 2015). This calcium influx and wave can be recapitulated ex vivo by submerging dissected mature oocytes in certain hypotonic buffers (Kaneuchi et al, 2015;York-Andersen et al, 2015).…”