Divalent cation influx and calcium homeostasis in germinal vesicle mouse oocytes

Abstract

Prior to maturation, mouse oocytes are arrested at the germinal vesicle (GV) stage during which they experience constitutive calcium (Ca²⁺) influx and spontaneous Ca²⁺ oscillations. The oscillations cease during maturation but Ca²⁺ influx continues, as the oocytes' internal stores attain maximal content at the culmination of maturation, the metaphase II stage. The identity of the channel(s) that underlie this Ca²⁺ influx has not been completely determined. GV and matured oocytes are known to express three Ca²⁺ channels, Ca_V3.2, TRPV3 and TRPM7, but females null for each of these channels are fertile and their oocytes display minor modifications in Ca²⁺ homeostasis, suggesting a complex regulation of Ca²⁺ influx. To define the contribution of these channels at the GV stage, we used different divalent cations, pharmacological inhibitors and genetic models. We found that the three channels are active at this stage. Ca_V3.2 and TRPM7 channels contributed the majority of Ca²⁺ influx, as inhibitors and oocytes from homologous knockout (KO) lines showed severely reduced Ca²⁺ entry. Sr²⁺ influx was promoted by Ca_V3.2 channels, as Sr²⁺ oscillations were negligible in Ca_V3.2-KO oocytes but robust in control and Trpv3-KO GV oocytes. Mn²⁺ entry relied on expression of Ca_V3.2 and TRPM7 channels, but Ni²⁺ entry depended on the latter. Ca_V3.2 and TRPV3 channels combined to fill the Ca²⁺ stores, although Ca_V3.2 was the most impactful. Studies with pharmacological inhibitors effectively blocked the influx of divalent cations, but displayed off-target effects, and occasionally agonist-like properties. In conclusion, GV oocytes express channels mediating Ca²⁺ and other divalent cation influx that are pivotal for fertilization and early development. These channels may serve as targets for intervention to improve the success of assisted reproductive technologies.

Keywords: Calcium; Cav3.2; Influx; Meiosis; Signaling; TRPM7; TRPV3.

Figure 1.. TRPM7 protein is expressed in mouse GV oocytes and MII eggs.

Immunoreactivity of TRPM7 was detected using an anti-TRPM7 + TRPM6 antibody. (A) The immunoreactive band in oocytes and eggs is likely TRPM7 given the ~MW of 210 kDa, and because its functional expression was confirmed by electrophysiology [28]. (B) In all tissues tested here, TRPM6 expression (~MW 240 kDa) appears to be clearly predominant over TRPM7. Red arrows indicate the expected locations of TRPM7 (dark red) and TRPM6 (light red). Representative blots are shown.

Figure 2.. Divalent cations permeate the plasma membrane of GV oocytes.

Divalent cation influx was examined in GV oocytes. (A) Representative traces of Ca²⁺ oscillations caused by enhanced influx after increasing [Ca²⁺]_o from 2 mM to 5 mM. (B) Sr²⁺ induced oscillations. (C) Fluorescence quenching at F360 following Mn²⁺ addition (100 μM). (D) Fluorescence quenching at F380 following Ni²⁺ addition (500 μM) in oocytes loaded with Mag-Fura-2AM in DFM media. Horizontal bars above each panel show the time during which divalent cations were added to the media. All experiments were replicated 4 times. In all panels in this figure and throughout the manuscript, representative traces are shown; darker traces represent the most common response; n: total number of oocytes examined.

Figure 3.. Ca²⁺ influx in GV oocytes is mediated by multiple channels.

Presence of spontaneous oscillations and Ca²⁺ influx examined in WT oocytes and oocytes lacking TRPV3, Ca_V3.2 & V3-Ca_V3.2 channels. (A-four upper panels) Spontaneous Ca²⁺ oscillations in WT and KO oocytes in media containing basal Ca²⁺ levels (2 mM). Fewer oocytes of the Ca_V3.2 and V3-Ca_V3.2 KO lines display oscillations, which are also of decreased frequency (D) (P< 0.05). (B-four medium panels) Ca²⁺ influx triggered by increasing [Ca²⁺]_o from 2 mM to 5 mM induces oscillations in all WT and KO lines, which appear of equal initial frequency (E). (C-four lower panels) After addition of TG (10 μM), Ca²⁺ influx resulted in reduced responses in Ca_V3.2 and V3-Ca_V3.2 KO oocytes, especially in the interval from Ca²⁺addition to the 1^st Ca²⁺ peak (P< 0.05) (F). Filled horizontal bars above each panel show the time of addition of Ca²⁺ (black) or TG (grey) to the media. All experiments were 4 replicated for times. Asterisk(s) above columns in bar graphs denote significant differences from other conditions here and throughout the manuscript.

Figure 4.. Pharmacological inhibitors disrupt Ca²⁺ influx/oscillations in GV oocytes.

Ca²⁺ influx in GV oocytes was tested by increasing [Ca²⁺]_o from 2- to 5 mM (white/black horizontal bars above each panel) in the presence of pharmacological inhibitors (dashed bars). Increasing [Ca²⁺]_o in (A) control, untreated oocytes, or oocytes treated with (B) Ca_V3.2 inhibitors: MBF (1 μM) or Ni²⁺ (100 μM) induced oscillations in the majority of oocytes. Conversely, Increasing [Ca²⁺]_o in the presence of the (C) general TRP channel inhibitors 2-APB (50 μM) or MgCl₂ (10 mM), or (D) in the presence of the TRPM7 inhibitors NS8593 (10 μM) or waixenicin-A (5 μM) failed to induce oscillations (P <0.05). All experiments were performed in 4 replicates.

Figure 5.. Sr²⁺-induced oscillations are diminished in Ca_V3.2- and V3-Ca_V3.2-KO oocytes.

Sr²⁺ induced oscillations were tested in WT and in oocytes lacking TRPV3, or Ca_V3.2 or V3-Ca_V3.2 channels. Experiments were performed in nominal Ca²⁺-free media containing 10 mM Sr²⁺. (A) Sr²⁺ induced the expected oscillations in WT oocytes, and largely similar responses in (B) TRPV3-KO oocytes. Sr²⁺ induced oscillations were reduced in (C) Ca_V3.2-KO and in (D) V3-Ca_V3.2-KO oocytes both in the (E) number of cells showing oscillations, and in the (F) number of rises during the first 1h of imaging (P<0.05). Horizontal bars above each panel denote the time during which Sr²⁺ was present in the media. All experiments were performed in 3–4 replicates.

Figure 6.. Ca_V3.2 channels inhibitors rapidly terminate Sr²⁺ induced oscillations.

Experiments were performed in nominal Ca²⁺-free media containing 10mM Sr²⁺ (black bar) and pharmacological inhibitors (dashed horizontal bars) were added 25 min following initiation of imaging. (A) Control, untreated oocytes showed normal oscillations, which were immediately terminated (B) by the addition of the Ca_V3.2 inhibitors MBF (1 μM) and Ni²⁺ (100 μM). (C) The general TRP channels inhibitors 2-APB (50 μM) & MgCl₂ (5 mM) and (D) the TRPM7 inhibitor NS8593 (10 μM) also terminated oscillations but more protractedly especially by the latter; waixenicin-A (5 μM) was without effect. All experiments were replicated 4 times.

Figure 7.. Multiple channels enable Mn²⁺ entry.

Mn²⁺ influx was tested in WT and in oocytes lacking TRPV3, or Ca_V3.2 or V3-Ca_V3.2 channels. Experiments were performed in media containing 2 mM Ca²⁺. (A) Mn²⁺ influx in WT oocytes, (B) TRPV3-KO oocytes, (C) Ca_V3.2-KO oocytes, (D) V3-Ca_V3.2-KO oocytes. (E) Fluorescence quenching assessed by the rate of F360 nm fluorescence decrease per minute relative to baseline fluorescence (P<0.05). (F) Time to inflection measured from the time of Mn²⁺ addition (5 min) to the time of first significant change in fluorescence values, which shortened in V3-Ca_V3.2-KO oocytes (P<0.05). Black horizontal bars denote addition of 100 μM Mn²⁺. Experiments were replicated 4 times.

Figure 8.. Inhibitors of Ca_V3.2 & TRPM7 channels suppress Mn²⁺ influx.

Mn²⁺ influx monitored using traces depicting quenching of F360 nm fluorescence. Experiments were performed in media containing 2 mM Ca²⁺ in the absence/presence of specific pharmacological inhibitors (dashed bars) after addition of Mn²⁺ (black bars): (A) control untreated oocytes, (B) oocytes treated with the Ca_V3.2 inhibitors MBF (1 μM) or Ni²⁺ (100 μM), (C) or with the general TRP inhibitors 2-APB (50 μM) and MgCl₂ (10 mM), or (D) with the TRPM7 inhibitors NS8593 (10 μM) or waixenicin-A (1 μM). (E) Rate of fluorescence quenching per minute at F360 nm relative to baseline fluorescence was reduced by all inhibitors examined (P<0.05), and (F) time to inflection measured from the time of addition (5 min) to the time fluorescence quenching begins was prolonged by all inhibitors examined(P<0.05). Experiments were replicated 3 times.

Figure 9.. Enhanced Ni²⁺ influx in V3-Ca_V3.2-KO oocytes.

Ni²⁺ influx was tested in WT and oocytes lacking TRPV3, Ca_V3.2 or V3-Ca_V3.2 channels. Experiments were performed using Mag-Fura-2AM loaded oocytes in divalent free medium (DFM). Ni²⁺ influx in (A) WT oocytes, (B) TRPV3-KO oocytes, (C) Ca_V3.2-KO oocytes, (D) V3-Ca_V3.2-KO oocytes. (E) Fluorescence quenching at F380 assessed by the rate of fluorescence decrease per minute relative to baseline fluorescence (*P<0.05). (F) Time to inflection measured from the time of addition (5 min) to the time fluorescence quenching begins (*P<0.05). Black bars show the time at which Ni²⁺ was added. Experiments were replicated three times.

Figure 10.. TRPM7 channel inhibitors block Ni²⁺ influx.

Inhibition of Ni²⁺ influx (black bars) was examined in the absence/presence of pharmacological inhibitors (dashed bars) and assessed by the rate of fluorescence quenching per minute at F380 relative to baseline fluorescence. Experiments were performed using Mag-Fura-2AM loaded oocytes in divalent free medium. (A) Control, untreated group, (B) Ca_V3.2 inhibitor MBF (1 μM), (C) general inhibitors of TRP channels 2-APB (100 μM) or MgCl₂ (10 mM), (D) TRPM7 inhibitors NS8593 (10 μM) or waixenicin-A (5 μM). (E) Maximal inhibition of fluorescence quenching of Mag-Fura by general TRP inhibitors, and more specifically by the TRPM7 inhibitor NS8593 (P<0.05). (F) Time to inflection measured from the time of addition (5 min) to the time of fluorescence quenching begins, was also maximally inhibited by TRP inhibitors and more specifically by NS8593 (P<0.05). Experiments were replicated 3 times.

Figure 11.. The absence of TRPV3- and V3-Ca_V3.2 channels reduces internal Ca²⁺ stores.

Ca²⁺ release from ER and total stores was measured using TG (10 μM) and IO (2.5 μM) in WT and in oocytes lacking TRPV3, Ca_V3.2, or V3-Ca_V3.2 channels. Experiments were performed in nominal Ca²⁺-free medium. (A) TG responses in WT, TRPV3-, Ca_V3.2- and V3-Ca_V3.2-KO oocytes. (B) Ionomycin responses in WT, TRPV3-, Ca_V3.2- and V3-Ca_V3.2-KO oocytes. (C) Peak amplitude of TG-induced Ca²⁺ release was reduced in Ca_V3.2 (P<0.05). (D) Peak amplitude of ionomycin-induced Ca²⁺ release was reduced V3-Ca_V3.2 (P<0.05), whereas (E) AUC response induced by IO was reduced in all KO lines (P<0.05). Open bars indicate nominal Ca²⁺ free media; filled gray bars show the time at which TG/IO was added. Experiments were replicated 3 times.

Figure 12.. Internal Ca²⁺ stores are reduced in the presence of Ca_V3.2 and general TRP channels inhibitors.

Oocytes were incubated with pharmacological inhibitors for 1 h after which internal Ca²⁺ store content was tested following addition of TG and IO-induced. Peak amplitude of intracellular Ca²⁺ release was graphed and quantified. (A and D) the Ca_V3.2 inhibitors MBF (1 μM) and Ni²⁺ (100 μM) reduced responses to both treatments (P<0.05), and similar effects were observed after the addition of (B and E) the TRP channels general inhibitors 2-APB (100 μM) and MgCl₂ (5 mM) (P<0.05). (C & F) The TRPM7 inhibitors NS8593 (10 μM) & waixenicin-A (1 μM) were without effect (P>0.05) regarding TG responses, while NS8593 affected the amplitude of the IO response (P>0.05). Dashed bars indicate the presence of pharmacological inhibitors; black and white bars indicate the presence/absence of Ca²⁺; gray bars show the time at which IO/TG was added.