DrosophilaTrpm 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, germline specific RNAi knockdown, and germline 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 reduced egg hatchability in trpm germline knockout mutant females, although eggs were able to complete some egg activation events including cell cycle resumption. Since the mouse Trpm ortholog 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. SignificanceA rise in intracellular free calcium is a conserved feature of the egg-to-embryo transition in almost all animals. In Drosophila, as in vertebrates, the rise starts at one end of the egg, and then travels across the egg in a wave. The Drosophila calcium rise is mediated by an influx of calcium, due to the action of mechanically-gated ion channels. Here we identify the ion channel that is critical for the calcium entry as TRPM. TRPM is the ortholog of the channel recently

RETRACTED: AgNPs reduce reproductive capability of female mouse for their toxic effects on mouse early embryo development

“…[36][37][38] In mammalian reproduction, it had been well known that Ca 2+ signaling in fertilization plays a vital role in egg activation and normal development of embryo, 24,39 and the inhibitor of ion channel can effectively affect early embryo development. 40,41 Therefore, our evidence of AgNPs depolarizing CMP of zygotes strongly suggests that it should not be ignored to explore effects of extracellular AgNPs on CMP of eggs, zygotes, and embryos, and this effect has been partly confirmed by other previous studies including those of carbon nanotubes being ion channel blockers 42 and AgNPs inhibiting action potential in pyramidal neurons. 17 Although our data provided primary evidence of AgNPs influencing CMP of zygotes, further studies should explore why AgNPs can depolarize CMP of embryonic cells, and what the key compound is in the process of AgNPs changing CMP.…”

Osmolarity-regulated swelling initiates egg activation in Drosophila

“…Together with previous work, our data support the following model of Drosophila egg activation (figure 5): (i) at ovulation, the meiotically arrested mature oocyte passes into the lateral and then common oviduct; (ii) the mature oocyte then takes up fluid due to the difference in osmolarity between the oviduct fluid and the ooplasm; (iii) the increase in volume results in tension at the plasma membrane and dispersion of the cortical actin; (iv) volume increase is mediated by association of RPK with cortical actin; (v) decreased density of cortical actin at the poles, prior to dispersion, primes these regions for calcium entry; (vi) calcium likely enters the egg from the perivitelline space through the mechanosensitive Trpm channels in the plasma membrane; (vii) starting at the posterior pole, further increase in intracellular calcium may be relayed across the oocyte by the opening of the neighbouring Trpm channels via the dispersion of the cortical actin cytoskeleton at the lateral sides, in addition to IP3- and PLC-mediated calcium propagation [6,24]; (viii) the calcium wave is then followed by an F-actin wavefront, which ensures the reorganization of the actin cytoskeleton; (ix) intracellular calcium returns to basal levels, likely through channels that transport calcium back into the perivitelline space or intracellular stores. Collectively, the single calcium wave prepares the oocyte for pronuclear apposition and embryogenesis [44].…”

“…Rehydration at egg activation can be recapitulated ex vivo through the addition of a hypotonic solution, known as activation buffer (AB), which when added to an isolated mature egg results in swelling and a single calcium wave [5,6,15]. This influx of calcium requires the transient receptor potential M (Trpm) mechanosensitive channel in the plasma membrane and results in the activation of Plc21C that sustains the wave [8,24,25]. Regulation of calcium entry was hypothesized to be related to the distribution of the Trpm protein in the membrane, as calcium entry is often seen first at the poles.…”

Spindle F-actin coordinates the first metaphase-anaphase transition in Drosophila meiosis

“…Unlike mammals and most vertebrates, the final arrest in Drosophila oocytes is at metaphase I, and the bipolar spindle structure that forms is more defined and has more focused spindle poles when compared to the “barrel” shaped spindles of mammals. Activation in Drosophila occurs prior to fertilization as the oocyte passes into the oviduct, resulting in calcium influx through transient receptor potential melastatin (TrpM) ion channels in the plasma membrane of the oocyte 14,15 . This calcium event enables the resumption of meiosis from its arrested state, and can be observed using a variety of microtubule labelling tools 16 .…”