Animals and ethics approval

CF-1 female mice (Envigo) aged 6–11 weeks were used in accordance with the Johns Hopkins University Animal Care and Use Committee guidelines. Mice were housed with ad libitum water and food under a 12 h light: dark cycle.

Plasmid constructs and cRNA synthesis

PCR with Phusion High-Fidelity DNA Polymerase (Thermo Fisher, F530S) was used to amplify cDNAs encoding AIDmini-EGFP and osTIR1–9myc (TIR1 from Oryza sativa; both parental plasmids were gifts from Andrew Holland, available from Addgene: AIDmini-EGFP [#101713], TIR1–9myc [#80073] [16]). Following gel purification, PCR products were digested with SalI (New England Biolabs (NEB), R0138S) and XbaI (NEB, R0145S), and cloned into pIVT/KAN2 (a gift from Carmen Williams) using the standard protocol. See Supplementary Table S1 for cloning primers and plasmid constructs. All plasmids were verified using DNA sequencing.

For cRNA synthesis, plasmids were linearized with NdeI (NEB, R0111S) and then purified with QIAquick Gel Extraction Kit (QIAGEN, 28704). In vitro transcription was performed with the mMESSAGE mMACHINE T7 or SP6 Transcription Kit (Thermo Fisher, AM1344 and AM1340) according to the manufacturer's instructions. Complementary RNA was resuspended in nuclease-free water at concentrations of 0.8–4.4 µg/µl.

Oocyte collection, microinjection, and in vitro maturation

Prophase I-arrested oocytes were collected in Whitten's-HEPES medium as previously described [19] (109.5 mM NaCl, 4.7 mM KCl, 1.2 mM KH₂PO₄, 1.2 mM MgSO4, 5.5 mM glucose, 0.23 mM pyruvic acid, 4.8 mM lactic acid hemicalcium salt, with 7 mM NaHCO₃, 15 mM HEPES) supplemented with 0.25 µM milrinone (Sigma-Aldrich, M4659) to hold oocytes at prophase I arrest. For each injection experiment, oocytes were pooled from three to six females. Oocytes were transferred into Whitten's bicarbonate (Whitten's medium without HEPES and with 22 mM NaHCO₃ [19]) supplemented with milrinone. Prophase I oocytes were microinjected with cRNA into the cytoplasm (see the Results section and figure legends for concentrations used; total concentrations of cRNA were 1–3 µg/µl) using a Nikon Eclipse TE 2000–5 microscope equipped with an Eppendorf FemtoJet. An injection pressure of 50–250 hPa and an injection time of 0.1–0.5 s were used. Oocytes were cultured in Whitten's bicarbonate or CZB medium (81.62 mM NaCl, 4.83 mM KCl, 1.18 mM KH₂PO₄, 1.18 mM MgSO4, 25.12 7 mM NaHCO₃, 1.7 mM CaCl₂, 31.3 mM lactic acid hemicalcium salt, 0.27 mM pyruvic acid, 0.11 mM EDTA, 7 mM taurine, and 1 mM L-glutamine) supplemented with 0.25 µM milrinone at 37°C, 5% CO₂ for at least 3 h to allow for protein expression.

Figure 1.

Overview of the auxin-inducible protein degradation system and exogenous expression of AIDm-EGFP and TIR1-myc in oocytes. (A) Schematic illustration of exogenously expressed TIR1 forming a functional SCF E3 ligase with endogenous SKP1 and CUL1 proteins. When cells are not exposed to auxin (Aux, blue circle), auxin-inducible degron (AIDm) tagged proteins are able to function normally. Addition of Aux leads to interaction of TIR1 with the AIDm, polyubiquitination (U, orange circles) of the AID-tagged protein, and ultimately proteasome-mediated degradation. (B) Graphical representation of EGFP fluorescence intensity over time in oocytes injected with 0.4 µg/µl AIDm-EGFP cRNA. EGFP fluorescence normalized to AIDm-EGFP expression at 2 h. (C) TIR1-myc immunofluorescence in prophase I and MII oocytes injected with 2.2 µg/µl TIR1 cRNA 16–18 h post-injection. Line graph shows mean and SEM of EGFP fluorescence every hour.

For in vitro maturation (IVM), prophase I oocytes were washed through CZB without milrinone. IVM occurred for 16 h at 37°C, 5% CO₂, either in a CO₂ incubator or on a Zeiss Axio Observer Z1 microscope equipped with a Zeiss Heating Unit XL S1, CO₂ Module S1, and Temp Module S1 (Carl Zeiss, Inc.). For experiments in which oocytes were cultured in a CO₂ incubator (i.e. in the dark) or cultured during live-cell imaging (i.e. exposed to light periodically), oocytes were exposed to auxin for the duration of IVM. Data from these experiments on maturation rates reflect the number of MII eggs resulting from the total number of oocytes subjected to IVM. On the other hand, in studies in which auxin was added to oocytes at 180 min into IVM, the reported maturation rates were the percentage of MII eggs resulting from number of oocytes that underwent germinal vesicle breakdown (GVBD) prior to auxin addition. Meiosis I timing was calculated as the difference between GVBD and polar body emission.

Live-cell imaging and image analysis

Oocytes were cultured in CZB medium droplets under mineral oil (Sigma-Aldrich, M3516) in glass bottom culture dishes (Mat-Tek, P35G-0-14-C). For all experiments using prophase I-arrested oocytes, culture media were supplemented with 0.25 µM milrinone. Oocytes were incubated at 37°C, 5% CO₂ with images taken every 20 min or 180 min for the duration of the experiment on a Zeiss Axio Observer Z1 microscope with AxioCam MRm Rev3 camera (Carl Zeiss, Inc.). For degradation studies, auxin, cycloheximide (CHX; Millipore Sigma, 239764; dissolved in ethanol), or auxin with CHX were added to the culture droplets at the indicated time points, to final concentrations of 500, 1000, or 2000 µM auxin and 10 µg/ml CHX. Naturally occurring auxin, 3-indoleacetic acid (IAA; Sigma-Aldrich, I5148; dissolved in H₂O), and synthetic auxin, 1-naphthaleneacetic acid (NAA; Sigma-Aldrich, N0640; dissolved in 1N NaOH) were used, with auxin type indicated for each experiment. Following auxin/CHX addition, imaging continued every 20 min. For experiments using 1000 to 2000 µM IAA, images were only taken four times, prior to IAA addition (t = –20 min) and 180, 360, and 520 min post-IAA addition. For recovery studies, after microinjection oocytes were held arrested in CZB supplemented with 0.25 µM milrinone and vehicle, 500 µM IAA, or 500 µM NAA for 16 h. Oocytes were then washed into CZB with milrinone and vehicle, 500 µM IAA, or 500 µM NAA and imaged every 20 min for 8 h.

ImageJ Fiji distribution (Freeware; National Institutes of Health— https://imagej.nih.gov/ij/[20]) was used to measure the EGFP fluorescence intensity for each oocyte, taking into account background fluorescence and oocyte area as previously described [21]. For degradation experiments, the fluorescence intensity of each oocyte was normalized in order to account for oocyte-to-oocyte variability in starting EGFP fluorescence, by setting a value of 1 for the fluorescence intensity detected at time point t = –180 min or t = –20 min. For examination of GVBD, only oocytes that underwent GVBD within 3 h of milrinone washout (i.e. those that appeared to be meiotically competent) were included in the analysis. For recovery experiments, fluorescence intensity was normalized in two different ways: (1) EGFP fluorescence intensity at t = 20 min was set to 1 for each oocyte; or (2) the average fluorescence intensity of vehicle control group at t = 20 min was set to a value of 1, and this was used to normalize all other groups.

AIDm-EGFP half-life was calculated using GraphPad Prism 7 software (GraphPad Software). The AIDm-EGFP fluorescence intensity over time for oocytes microinjected with 2–2.2 µg/µl TIR1 and exposed to 500 µM auxin was pooled based on stage of meiosis (prophase I, GVBD, and MII) or auxin type (IAA at GVBD, or NAA at GVBD). Data were normalized such that t = –20 min equaled 1, and oocytes that had an EGFP fluorescence intensity greater than 1 at 480 min (prophase I and GVBD) or 180 min (MII) were excluded. Non-linear (one-phase) regression analysis with least squares (ordinary) fit was performed on the average EGFP fluorescence intensity over time.

Immunofluorescence

Zona pellucida removal was performed by incubating oocytes briefly in acidic MEM-compatible medium (116.4 mM NaCl, 5.4 mM KCl, 10 mM HEPES, 1 mM NaH₂PO₄, 0.8 mM MgSO4, pH 1.5). Oocytes were then fixed in 4% paraformaldehyde (Sigma-Aldrich, P6148) in PBS at room temperature for 30 min, permeabilized in PBS with 0.1% Triton X-100 (Fisher Scientific, BP151–500) for 15 min at room temperature, and blocked in PBS with 0.1% BSA (Sigma-Aldrich, A9647) and 0.01% Tween-20 (Sigma-Aldrich, P7949) for 1 h at room temperature. Anti-Myc primary antibody (Thermo Fisher, MA1–21316) was used at a final concentration of 4 µg/ml. After overnight incubation with anti-Myc antibody, oocytes were washed three times in PBS with 0.1% BSA and 0.1% Tween-20 before incubation with 7.5 µg/ml goat anti-mouse IgG conjugated to Alexa Fluor 488 (Jackson ImmunoResearch) for 1 h at room temperature. Following three washes in PBS with 0.1% BSA and 0.1% Tween-20, oocytes were mounted in VectaShield containing 0.75 µg/ml 4′-6′-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, D9542). Imaging was performed using a Zeiss Axio Observer Z1 microscope with AxioCam MRm Rev3 camera and ApoTome optical sectioning (Carl Zeiss, Inc.).

Statistics

GraphPad Prism 7.0 software was used for statistical analysis. For categorical data, the Fisher's exact test was used. Numerical data were tested for normal distribution using the D'Agnostino–Pearson omnibus normality test. For data that followed a normal distribution, ANOVA with Tukey's post hoc test was used. For all other data, Kruskal–Wallis test with Dunn's post hoc or Mann–Whitney test was used. A P-value < 0.05 was considered statistically significant. Further information on statistics tests used for each dataset can be found within the figure legends.

Exogenous expression of AIDm-EGFP and TIR1-myc in oocytes

As part of our first evaluation of auxin-inducible degradation in oocytes, we verified expression of our test substrate, AIDm-EGFP, and of a myc-tagged version of the F-box protein TIR1 from the rice O. sativa (TIR1-myc). Prophase I oocytes were microinjected with cRNA encoding AIDm-EGFP (0.4 µg/µl) and live imaged for EGFP fluorescence over a course of 24 h (Figure 1B). Exogenous expression of AIDm-EGFP increased gradually over time, when compared to uninjected control oocytes, and EGFP fluorescence accumulated sufficiently over 3–6 h such that EGFP fluorescence loss could be characterized in further experiments. TIR1-myc cRNA (2.2 µg/µl) was also injected into prophase I oocytes, and then immunofluorescence analysis with an anti-Myc antibody was performed on oocytes that were held arrested at prophase I for 18 h or were in vitro matured to metaphase II for 16 h. This analysis showed that TIR1-myc-cRNAinjected oocytes express TIR1-myc, with TIR1-myc signal observed throughout the cytoplasm (Figure 1C).

Extent of auxin-inducible degradation in prophase I oocytes is dependent on the amount of TIR1

We next sought to determine if auxin-inducible degradation could occur in oocytes, and what concentration TIR1-myc cRNA was needed to induce degradation. Prophase I oocytes were microinjected with cRNA encoding AIDm-EGFP (0.4 µg/µl) and decreasing concentrations of TIR1-myc cRNA (2.2–0.5 µg/µl). Oocytes were cultured for 3 h prior to imaging to allow time for translation of exogenous AIDm-EGFP and TIR1-myc from the injected cRNA. One hundred and eighty minutes after imaging began (6 h post-injection), 500 µM IAA (a naturally occurring auxin) was added to culture. A reduction in AIDm-EGFP fluorescence was observed in all IAA-exposed oocytes injected with TIR1-encoding cRNA (Figure 2A and B). Comparison of fluorescence intensity 20 min prior to IAA addition (noted as “–20 min” hereafter) and 180 min after IAA addition revealed a significant reduction in AIDm-EGFP in all IAA-exposed oocytes, which was not observed in vehicle-only controls (Figure 2C; 2.2 µg/µl TIR1 cRNA, 30.7 ± 2.3% AIDm-EGFP loss, P ≤ 0.0001; 1.1 µg/µl TIR1 cRNA, 23.8 ± 2.0% AIDm-EGFP loss, P ≤ 0.0001; 0.5 µg/µl TIR1 cRNA, 17.4 ± 2.3% EGFP loss, P ≤ 0.0001). This decrease in AIDm-EGFP was found to be dose-dependent, with 2.2 and 1.1 µg/µl TIR1-myc cRNA leading to significantly more loss of AIDm-EGFP fluorescence than 0.5 µg/µl (Figure 2C; 2.2 µg/µl vs. 0.5 µg/µl, P ≤ 0.0001; 1.1 µg/µl vs. 0.5 µg/µl, P = 0.0391). Moving forward, we used 2.2 µg/µl of TIR1-myc cRNA, as it led to the most robust AIDm-EGFP loss in these studies.

Extended culture time and inhibition of translation does not enhance loss of exogenously expressed AIDm-EGFP with auxin-induced degradation

Having established that the extent of auxin-inducible degradation was dependent on the dose of TIR1-myc cRNA injected, we next examined if time of exogenous expression of AIDm-EGFP and TIR1 following cRNA injection would influence AIDm-EGFP loss. In these experiments, oocytes were allowed 16 h prior to live-cell imaging and IAA addition to translate AIDm-EGFP and TIR1-myc from the injected cRNA, increasing the post-injection culture time from the 6 h used in the experiments noted above. Over a period of 3 h of live-cell imaging, during which oocytes were treated with 500 µM IAA or vehicle, a reduction in exogenous AIDm-EGFP fluorescence was observed over time in both groups, but was more pronounced in the IAA group (Figure 3A). Analysis of fluorescence intensity just prior to (t = –20 min) IAA addition and at 180 min after IAA addition revealed that this reduction was statistically significant for both IAA-treated and vehicle-only control oocytes (Figure 3B; IAA, 29.5 ± 0.9% AIDm-EGFP loss, P ≤ 0.0001; vehicle, 5.2 ± 0.8%, P ≤ 0.0001). Addition of auxin at 0 min led to an increase in AIDm-EGFP loss when compared to vehicle-only oocytes (IAA vs. vehicle, P ≤ 0.0001). Taken together, these results showed that allowing 16 h for protein expression after cRNA injection led to comparable auxin-inducible AIDm-EGFP degradation as observed with 6 h (30.7% loss with 6 h culture after cRNA injection (Figure 2C) vs. 29.5% with 16 h culture after cRNA injection (Figure 3B)).

Figure 2.

Range of TIR1-myc cRNA concentrations allows for depletion of exogenously expressed AIDm-EGFP. Prophase I oocytes were microinjected with 0.4 µg/µl AIDm-EGFP and 2.2, 1.1, or 0.5 µg/µl TIR1 cRNA 6 h prior to 500 µM IAA treatment. (A) Live-cell EGFP fluorescence (gray) in prophase I oocytes 20 min prior to (–20 min) and 180 min after IAA addition. (B) Graphical representation of EGFP fluorescence over time. 500 µM IAA was added at 0 min (arrow). EGFP fluorescence normalized to expression at –180 min. (C) Graphical comparison of EGFP fluorescence before (–20 min) and after (180 min) IAA addition. EGFP fluorescence was normalized to expression at –20 min (ANOVA with Tukey's post hoc). n = number of oocytes analyzed (15–29 over 2–3 technical replicates), P ≤ 0.0391. Line graph shows mean and SEM of EGFP fluorescence every 20 min. Bar graph shows mean, SEM, and individual oocyte scatter plot.

We observed that EGFP fluorescence intensity never dropped below 0.7 arbitrary units (AU) by 180 min post-IAA treatment. To determine what effect continued translation of exogenous AIDm-EGFP cRNA had on EGFP loss, we treated prophase I oocytes with the translation inhibitor cycloheximide (CHX), either alone or in combination with 500 µM IAA; oocytes treated with 500 µM IAA only were another experimental group. As expected, addition of CHX prevented further EGFP accumulation (Figure 3C). The combination of IAA + CHX resulted in a further decrease in EGFP fluorescence as compared to IAA, but only at 480 min post-IAA exposure (Figure 3C; 0.66 ± 0.03 (IAA) vs. 0.50 ± 0.04 (IAA + CHX) AU) and not 180 min post-IAA exposure. Analysis of EGFP fluorescence intensity at 180 min following IAA/IAA + CHX addition revealed no significant decrease between oocytes treated with IAA and oocytes treated with IAA + CHX (Figure 3D; 0.75 ± 0.02 [IAA] vs. 0.64 ± 0.02 [IAA + CHX], P > 0.9999). Data analysis for these experiments was performed in two different ways: (a) measurement of oocyte fluorescence intensity adjusted to field background (the area outside the oocyte) and oocyte area prior to normalization of –180 min (Figure 3C) or –20 min (Figure 3D) to 1; or (b) subtraction of fluorescence intensity of uninjected oocytes prior to normalization. This second method of normalization decreased final EGFP fluorescence at 480 min by a further 20% in IAA-exposed oocytes (Supplementary Figure S1; IAA, 0.39 ± 0.02 AU; IAA + CHX, 0.20 ± 0.03 AU). This suggests that oocyte autofluorescence and continued AIDm-EGFP cRNA translation contribute to the fluorescence signal detected. As such, an EGFP fluorescence intensity value of ∼0.6 AU equates to almost complete EGFP loss.

Figure 3.

Increasing cRNA expression time or inhibiting translation with cycloheximide has no impact on loss of exogenously expressed AIDm-EGFP. (A, B) Oocytes were injected with 0.4 µg/µl AIDm-EGFP cRNA and 2.0–2.2 µg/µl TIR1-myc cRNA, and allowed to express cRNA for 16 h prior to 500 µM IAA treatment. (A) Graphical representation of EGFP fluorescence over time. 500 µM IAA was added at 0 min (arrow). EGFP fluorescence normalized to expression at –20 min. (B) Graphical comparison of EGFP fluorescence before (–20 min) and after (180 min) IAA addition. EGFP fluorescence was normalized to expression at –20 min (ANOVA with Tukey's post hoc). n = number of oocytes analyzed (61–71 over two technical replicates), P < 0.0001. (C, D) Oocytes were injected with 0.4 µg/µl AIDm-EGFP cRNA and 2.0 µg/µl TIR1-myc cRNA, and allowed to translate protein from the injected cRNA for 6 h prior to 500 µM IAA and/or CHX treatment. (C) Graphical representation of EGFP fluorescence over time. IAA and/or CHX were added at 0 min (arrow). EGFP fluorescence normalized to expression at –180 min. (D) Graphical comparison of EGFP fluorescence before (–20 min) and after (180 min) IAA and/or CHX addition. EGFP fluorescence normalized to expression at –20 min (Kruskal–Wallis test with Dunn's post hoc). n = number of oocytes analyzed (25–29 over two technical replicates), P ≤ 0.0072. Line graphs show mean and SEM of EGFP fluorescence every 20 min. Bar graphs show mean, SEM, and individual oocyte scatter plots.

Auxin-inducible degradation occurs in oocytes during meiosis I and at metaphase II

The ability to perform precise and temporally controlled depletion of proteins at distinct points of meiosis is a key motivation for establishing use of auxin-inducible degradation in oocytes. Having optimized conditions for auxin-inducible degradation in prophase I oocytes, we next ascertained if auxin-inducible protein degradation could be utilized at other stages of oocyte meiosis, namely after oocytes had resumed meiosis (GVBD), and at metaphase II (MII) arrest. In these experiments, prophase I-arrested oocytes were microinjected with AIDm-EGFP and TIR1-myc cRNAs. For analyses of GVBD oocytes, oocytes were washed out of milrinone-supplemented medium 180 min prior to IAA treatment; only oocytes that underwent GVBD before IAA exposure were included in analyses. For MII oocytes, oocytes injected at prophase I were subjected to IVM for 16 h, and treated with IAA at metaphase II arrest. A significant reduction in EGFP signal was observed between vehicle-only control and 500 µM IAA-treated oocytes at each of these stages of meiosis (Figure 4A–D; prophase I, 20.3 ± 3.1% AIDm-EGFP loss, P ≤ 0.0001; GVBD, 28.3 ± 3.8% AIDm-EGFP loss, P ≤ 0.0001; MII, 49.8 ± 2.3% AIDm-EGFP loss, P ≤ 0.0001). Comparison of exogenous AIDm-EGFP fluorescence before (t = –20 min) and after (t = 180 min) IAA addition showed similar extents of EGFP loss in oocytes at these three stages of meiosis. However, no IAA-treated GVBD oocytes progressed through meiosis to produce first polar bodies during these live-cell imaging experiments. This failure to complete meiosis I was not observed in vehicle-only control oocytes (data not shown), suggesting a deleterious effect of IAA and/or auxin-inducible degradation on completion of meiotic maturation.

Comparison of IAA and NAA for live-cell imaging experiments

We next sought to determine the cause of the meiotic failure observed during live-cell imaging in IAA-treated oocytes exogenously expressing AIDm-EGFP and TIR1-myc. In yeast, exposure to IAA during live-cell imaging suppressed cell proliferation, and this impaired cell proliferation was not observed with the synthetic auxin NAA [22]. This growth defect was speculated to be caused by free radicals and nucleophiles accumulating as a result of IAA photodegradation, which occurs at UV to blue wavelengths [22–24]. To test whether the oocytes' failure to complete meiotic maturation could be associated with IAA photodegradation, oocytes were subjected to IVM, in the presence of 500 µM IAA or NAA, in the dark (i.e. in a CO₂ incubator) or during live-cell imaging with white light and 470 nm wavelength light, as performed in studies noted above (Figures 2–4). Vehicle-only control and NAA-treated oocytes progressed through first polar body emission in both culture conditions (Figure 5A and B; percentages of oocytes that emitted first polar bodies, vehicle-treated, 85.7% in dark, 62.9% during imaging, P = 0.0539; NAA-treated, 80.0% in dark, 84.1% during imaging, P = 0.7769). In contrast, IAA-treated oocytes only completed meiosis I in the dark. The extent of meiosis I completion of IAA-treated oocytes in the dark was comparable to vehicle-treated and NAA-treated oocytes, whereas exposure of IAA-treated oocytes to live-cell imaging conditions completely prevented meiotic maturation, as measured by first polar body emission (Figure 5A and B; IAA, 73.8% in dark vs. 0.0% during imaging; P ≤ 0.0001). Of note, in plant cell culture medium this IAA photodegradation can be eliminated with yellow long-pass filters that remove UV, violet, and blue wavelengths [23]. Therefore, we next sought to determine if IAA-treated oocytes could complete meiosis when exposed to 556 nm (green wavelength) or 640 nm (red wavelength) excitation every 20 min during IVM. Excitation of IAA-treated oocytes at 556 nm significantly impacted their ability to complete meiosis I and emit the first polar body (Figure 5C; 91.8% (Veh) vs. 51.1% (IAA); P < 0.0001). In contrast, excitation at 640 nm had no effect on maturation of IAA-exposed oocytes (Figure 5C; 90.0% (Veh) vs. 80.5% (IAA); P = 0.3493).

Given that NAA did not appear toxic to completion of meiotic maturation during live-cell imaging with white light and wavelength 470 nm, we evaluated if NAA could induce exogenous AIDm-EGFP loss. Prophase I oocytes were microinjected with cRNAs encoding exogenous AIDm-EGFP and TIR1-myc as described above. After 180 min of expression, oocytes were subjected to IVM and imaged for 180 min prior to auxin addition (500 µM IAA or NAA). IAA and NAA led to comparable extents of EGFP loss, and EGFP loss was not observed in vehicle-only controls (Figure 6A and B; 34.1 ± 1.4% [IAA] vs. 30.1 ± 1.6% [NAA] EGFP loss; P > 0.9999). Importantly, NAA-treated oocytes were able to complete meiosis I, with extents of first polar body emission comparable to vehicle-only control oocytes (Figure 6D; 76.8% (vehicle) vs. 73.9% (NAA) MII eggs; P = 0.8186). Meiosis I timing, measured as the time from GVBD to first polar body emission, was also comparable between NAA-treated oocytes and vehicle-only control oocytes (Figure 6E; 646.4 ± 10.0 min (vehicle) vs. 643.5 ± 12.3 min (NAA); P = 0.6655).

Half-life of exogenously expressed AIDm-EGFP varies by meiotic stage but not auxin type

A key feature of auxin-inducible degradation is the ability to rapidly deplete proteins, with complete protein loss in mammalian cell lines occurring in 30–90 min, with a half-life (t_1/2) of AID-tagged proteins of 10–50 min [13, 15, 25–28]. The t_1/2 times of exogenous AIDm-EGFP in 500 µM IAA-treated prophase I, GVBD, and MII oocytes injected with 2–2.2 µg/µl TIR1-myc cRNA were 106.9, 82.4, and 41.0 min, respectively. The differences in t_1/2 time between stage of meiosis were statistically significant (P < 0.0001). Treatment of GVBD oocytes with 500 µM NAA resulted in an AIDm-EGFP t_1/2 of 65.1 min, which was not different from the t_1/2 of AIDm-EGFP in IAA-treated GVBD oocytes (P = 0.4275).

Increasing auxin concentration has no impact on auxin-inducible degradation

We observed that exogenously expressed AIDm-EGFP in prophase I and GVBD oocytes had a half-life of ∼2- to 11-fold greater than the protein half-life observed in mammalian mitotic cell lines [13, 15, 25–28]. As mouse oocytes are ∼4 times larger than mitotic cells, we wondered if this could be a consequence of a decreased auxin gradient within the cell. Therefore, we examined the effects of higher auxin concentrations in the culture medium on auxin-inducible degradation. Complementary RNAs encoding AIDm-EGFP and TIR1-myc were microinjected into prophase I oocytes. Live-cell imaging began 180 min post-injection. Addition of NAA (500 and 1000 µM) at t = 0 min led to a significant reduction in EGFP fluorescence at t = 180 min when compared to vehicle-only treated oocytes (Figure 7A and B; P < 0.0001). Increasing NAA concentration to 1000 µM had no impact on EGFP loss at t = 180 min (Figure 7B; 40.0 ± 1.5% (500 µM NAA) vs. 33.1 ± 1.8% (1000 µM NAA) EGFP loss; P > 0.9999). NAA at concentrations higher than 1000 µM were not tested to minimize the amount of solvent, 1N NaOH, in culture medium. Similar studies were performed with IAA. Due to the toxicity of light-induced degradation of IAA, we tested increased IAA concentrations under altered imaging conditions, reducing light exposure by imaging every 180 min for 540 min (Figure 7C). Addition of IAA led to a reduction in EGFP fluorescence over time when compared to vehicle-treated oocytes (Figure 7D; P < 0.0001). However, as with NAA, increasing the concentration of IAA had no impact on the extent of EGFP loss at t = 180 min (Figure 7D; 40.1 ± 2.5% (500 µM IAA) vs. 28.4 ± 4.4% (1000 µM IAA) vs. 22.9 ± 4.4% (2000 µM IAA) EGFP loss; P > 0.9999 (500 µM vs. 1000 µM) and 0.3581 (500 µM vs. 2000 µM)).

Figure 4.

Auxin-inducible degradation works during meiosis I and at metaphase II (MII) arrest. Oocytes were injected with 0.4 µg/µl AIDm-EGFP cRNA and 2.0–2.2 µg/µl TIR1-myc cRNA, and allowed to express cRNA for 3 h prior to imaging and initiation of meiotic maturation (GVBD oocytes). (A) Live-cell EGFP fluorescence (gray) in oocytes 20 min prior to (–20 min) and 180 min after IAA addition. (B, C) Graphical representation of EGFP fluorescence over time in prophase I, GVBD (B), or MII (C) oocytes. IAA was added at a concentration of 500 µM at t = 0 min (arrow). EGFP fluorescence normalized to expression at –180 min (prophase I and GVBD) or –20 min (MII). (D) Graphical comparison of EGFP fluorescence before (–20 min) and after (180 min) IAA addition. EGFP fluorescence normalized to expression at –20 min (Kruskal–Wallis test with Dunn's post hoc). n = number of oocytes analyzed (23–44 over three technical replicates), P < 0.0001. Line graphs show mean and SEM of EGFP fluorescence every 20 min. Bar graph shows mean, SEM, and individual oocyte scatter plot.

Figure 5.

Auxin type impacts meiotic maturation with live-cell imaging. Impact of exposure to light and/or auxin (IAA or NAA) on oocyte maturation rate. (A) DIC images of oocytes at 540 min post-milrinone washout. Oocytes were exposed to white light + 470 nm wavelength light every 20 min, and 500 µM IAA or NAA throughout in vitro maturation (IVM). (B) Graphical representation of percentage of oocytes at MII following 16 h of IVM in vehicle, 500 µM IAA, or 500 µM NAA with (+) or without (–) exposure every 20 min to white light and 470 nm wavelength light. n = number of oocytes analyzed (35–79 over three technical replicates), ^***P ≤ 0.0001. (C) Graphical representation of percentage of oocytes at MII following 16 h IVM in vehicle or 500 µM IAA with exposure every 20 min at 556 or 640 nm wavelength light. n = number of oocytes analyzed (40–49 over three technical replicates), ^***P ≤ 0.0001. Bar graphs show percentage of oocytes that emitted a first polar body (PB).

Removal of auxin allows for recovery of exogenous AIDm-EGFP expression

Having examined the time frame of degradation of an exogenous protein (AIDm-EGFP) in an auxin-inducible-dependent manner, we wanted to determine if levels of this protein would recover after auxin removal. Prophase I oocytes were injected with cRNAs encoding TIR1-myc and AIDm-EGFP, and then subjected to 16 h of incubation in medium with vehicle, 500 µM NAA, or 500 µM IAA (treatment 1). Following this 16 h incubation, oocytes were washed into medium containing vehicle, 500 µM NAA, or 500 µM IAA (treatment 2), and live-cell imaged. Due to oocyte-to-oocyte variability in starting EGFP fluorescence, statistical analysis was performed with EGFP fluorescence at t = 20 min set to a value 1 for all oocytes (Figure 8A). The trends observed from this normalization are in agreement with those observed when oocyte EGFP fluorescence intensity was normalized to the average of vehicle-only exposed oocytes at t = 20 min (Figures 8B, Supplementary Figure S2A). There was no change in EGFP fluorescence in oocytes treated with vehicle throughout the duration of the experiment, i.e. in treatments 1 and 2 (Figure 8A; P > 0.9999). In oocytes incubated in vehicle for treatment 1 then exposed to IAA in treatment 2, there was a significant reduction in EGFP fluorescence when comparing t = 20 min to t = 500 min, indicating a continued sensitivity of the system to auxin (Figure 8A; 46.6 ± 2.4% EGFP loss; P < 0.0001). With regard to the ability of levels of AIDm-EGFP to recover, this was different between oocytes initially treated with NAA vs. IAA. After removal of NAA (i.e. exposure to NAA in treatment 1, then transfer to vehicle for treatment 2), EGFP fluorescence showed an 83.2 ± 1.1% increase by 500 min (Figure 8A; P < 0.0001), which was not observed with continued NAA exposure (46.1 ± 4.2% EGFP loss with continuous NAA exposure; P < 0.0001). Although this increase in EGFP fluorescence was significant, AIDm-EGFP expression did not recover to the level observed in vehicle-only oocytes (Figures 8B, and Supplementary Figure S2A). Oocytes exposed to IAA, whether for the duration of the experiment (treatment 1 + 2) or for treatment 1 only (then vehicle for treatment 2), showed a small increase in EGFP fluorescence over the 500 min imaging (21.0 ± 6.1% EGFP recovery (IAA + Veh), P = 0.0005; 31.6 ± 8.8% EGFP recovery (IAA + IAA, i.e. continuous IAA), P = 0.0077). Of note, EGFP signal was distinctly different in oocytes continually exposed to IAA, with the fluorescence pattern being reminiscent of signals observed in oocytes just prior to death (Supplementary Figure 2B). This suggests that an increase in autofluorescence detected in the GFP channel could be occurring in these oocytes exposed to IAA for 24 h, and that the increase in fluorescence does not represent a true increase in AIDm-EGFP fluorescence. Importantly, oocytes treated with IAA continuously (IAA + IAA) had low survival compared to all other groups (12/33 oocytes survived (IAA + IAA) vs. 27/29 oocytes survived (IAA + Veh) vs. 31/32 oocytes survived (NAA + NAA) vs. 18/22 oocytes survived (NAA + Veh) vs. 19/26 oocytes survived (Veh + Veh) vs. 23/32 oocytes survived (Veh + IAA) oocytes survived). Note that only oocytes that survived the full 500 min of live-cell imaging were included in analysis.

Figure 6.

NAA induces auxin-inducible degradation to the same extent as IAA. Oocytes were injected with 0.4 µg/µl AIDm-EGFP cRNA and 2.0 µg/µl TIR1-myc cRNA, and allowed to express cRNA for 6 h prior to 500 µM IAA or NAA treatment. (A) Live-cell EGFP fluorescence (gray) in prophase I oocytes 20 min prior to (–20 min) and 180 min post IAA or NAA addition. (B) Graphical representation of EGFP fluorescence over time in vehicle, IAA, or NAA-exposed oocytes. Auxin was added at a concentration of 500 µM at t = 0 min (arrow). EGFP fluorescence normalized to expression at –180 min. (C) Graphical comparison of EGFP 180 min after auxin addition. EGFP fluorescence normalized to expression at –20 min (Kruskal–Wallis test with Dunn's post hoc). n = number of oocytes analyzed (39–46 over four technical replicates), P < 0.0001. (D, E) Graphical representation of percentage of oocytes at MII following 16 h of IVM and their time spent in meiosis I (D: Fisher's exact test; E: Mann-Whitney test). Bar graphs show mean, SEM, and individual oocyte scatter plot (C) or percentage of oocytes that emitted a first polar body (D). Line graph shows mean and SEM of EGFP fluorescence every 20 min. Scatter plot shows mean and individual oocyte meiosis I timing.