Effect of holding equine oocytes in meiosis inhibitor-free medium before in vitro maturation and of holding temperature on meiotic suppression and mitochondrial energy/redox potential

All chemicals were purchased from Sigma-Aldrich (Milano, Italy) unless otherwise indicated.

Ovaries from mares of unknown reproductive history were obtained at two local abattoirs within 3 hours of slaughter, transported to the laboratory within 30–45 min after collection in a thermal container set at 30°C and processed for recovery of cumulus-oocyte complexes (COCs). A follicle-scraping procedure on follicles 0.5 to 2.5 cm in diameter was used, as previously described [14]. Only COCs classified as having an intact cumulus investment and intact oocyte plasma membrane [14] were selected for culture.

Immature non cultured-oocytes were analyzed either immediately (IMM) or after overnight holding (EH). For IMM oocytes, the time interval between follicle scraping and beginning of COC processing for analysis or onset of IVM culture was less than 2 hours. COCs to be evaluated after IVM were placed into IVM medium either immediately (IMM-IVM) or after overnight holding (EH-IVM). After IVM culture, only oocytes that were at the metaphase II (MII) stage, as visualized after staining, were analyzed for energy/redox status.

COCs assigned to the EH treatment were placed in 5 ml of a mixture of 40% Earle’s and 40% Hank’s salts-buffered M199 with 20% fetal calf serum (FCS) in glass vacuum vials (Venoject – Terumo vt-050sp, Madrid, Spain). Fifteen to 50 COCs were put into each vial and the vial was sealed with a cap and paraffin film. The vial was then laid down on its side and covered with aluminium foil to block exposure to room light, and kept at room temperature for 16–18 h. After this holding period, EH COCs were directly evaluated or were placed in IVM culture (EH-IVM).

In Exp. 1, COCs were held at largely uncontrolled room temperature, estimated ranging from 22 to 27°C.

In Exp. 2, the effects of three holding temperatures (25°C, 30°C, and 38°C) on oocyte initial chromatin configuration were compared with those for immediately-fixed (IMM) oocytes. EH COCs were held either under air conditioning set at 25°C (EH-25 group) or in a water bath set at 30°C (EH-30 group) or in a sealed container in a cell culture CO₂ incubator set at 38.2°C (EH-38 group).

In vitro maturation was performed following the procedure described by Dell’Aquila et al. [14], using TCM-199 with Earle’s salts, buffered with 4.43 mM HEPES and 33.9 mM sodium bicarbonate and supplemented with 0.1 g/L L-glutamine, 2 mM sodium pyruvate, 2.92 mM calcium-L-lactate penthahydrate (Fluka 21175 Serva Feinbiochem GmbH & Co Heidelberg, Germany No. 29760), 50 μg/mL gentamicin, 20% (v/v) FCS, gonadotrophins (10 μg/mL ovine FSH and 20 μg/mL ovine LH) and 1 μg/mL 17β estradiol was used. COCs were cultured for 30 h at 38.2°C under 5% CO₂ in air.

COCs were denuded of cumulus cells by pipetting in 0.05% trypsin–0.02% EDTA (ECM0920D; Euroclone, Milan, Italy; immature COCs) or TCM 199 with 20% FCS containing 80 IU hyaluronidase/mL (mature COCs). Degenerating oocytes (having shrunken, misshapen or fragmented cytoplasm) and oocytes not in MII after IVM, as evaluated after staining, were discarded. Oocytes were washed and then incubated for 30 min in PBS with 3% bovine serum albumin (BSA) containing 280 nM MitoTracker Orange CMTM Ros (Molecular Probes M-7510, Oregon, USA) at 38.5°C under 5% CO₂. Oocytes were then washed and incubated for 15 min in PBS with 0.3% BSA containing 10 μM 2′,7′-dichlorodihydrofluorescein diacetate (DCDHF DA) to detect intracellular ROS [37, 41]. Oocytes were then washed and fixed overnight at 4°C with 2% paraformaldehyde solution in PBS.

After labeling for Mitochondria and ROS determination, oocytes were stained with 2.5 μg/ml Hoechst 33258 in 3:1 (v/v) glycerol/PBS and kept at 4°C in the dark until observation. Nuclear chromatin status was observed under a Nikon Eclipse 600 fluorescent microscope equipped with a B2A (346 nm excitation/460 nm emission) filter and was classified according to the criteria of Dell’Aquila et al. [42] and Hinrichs et al. [9]. Chromatin configurations of germinal vesicle (GV)-stage oocytes were classified as Fluorescent Nucleus (FN), having fluorescence throughout the nucleus; Fibrillar, having chromatin strands throughout the estimated area of the nucleus; Intermediate, having chromatin strands or irregular masses of chromatin over approximately half the estimated nuclear area; or Condensed Chromatin (CC), having chromatin condensed into one small regular or irregular mass. Oocytes having resumed meiosis were characterized as prometaphase I (PI), having chromatids in a circular array, or metaphase I (MI), or MII with first polar body (PB) extruded. Oocytes in anaphase or telophase were rare and were classified as MII. After IVM, only oocytes classified as MII were evaluated further. Oocytes with fragmented or non-detected chromatin were considered to be abnormal and were discarded.

Mitochondrial distribution pattern was evaluated at 630× magnification under oil immersion with a Nikon C1/TE2000-U laser scanning confocal microscope. A helium/neon laser ray at 543 nm and the G-2 A filter (551 nm exposure/576 nm emission) was used to evaluate MitoTracker Orange CMTM Ros fluorescence. An argon ion laser ray at 488 nm and B-2 A filter (495 nm exposure/519 nm emission) was used to evaluate DCF fluorescence. Scanning to allow three-dimensional distribution analysis was conducted utilizing 25 optical series from the top to the bottom of the oocyte, with a step size of 0.45 μm. Criteria for mitochondrial pattern definition were based on previous studies in equine oocytes [39‐41]: oocytes were separated into two groups as having a homogeneous/fine pattern of fluorescence due to small mitochondrial aggregates (SA) diffused throughout the cytoplasm, which was considered an indication of cytoplasmic immaturity, or an heterogeneous perinuclear/pericortical (P/P) distribution pattern of larger mitochondrial aggregates, with accumulation around the nucleus and in the periphery, considered to be characteristic of cytoplasmic maturation. Oocytes showing irregular distribution of large mitochondrial clusters were classified as abnormal and were not analyzed further.

Fluorescence intensity was measured in each oocyte, at the equatorial plane, with the aid of the EZ-C1 Gold Version 3.70 software platform for Nikon C1 confocal microscope, as described previously [37, 41]. Fluorescence intensity within the programmed scan area was recorded and plotted against the conventional pixel unit scale (0–255). Images were taken under fixed conditions with respect to laser energy, signal detection (gain) and pinhole size.

Colocalization analysis of Mitochondria and ROS was performed with the EZ-C1 Gold Version 3.70 software. Degree of colocalization was reported as a Pearson’s correlation coefficient quantifying the overlap degree between Mitotracker and DCF fluorescence signals [37, 43].

The proportions of oocytes showing the different chromatin configurations and mitochondrial distribution patterns were compared among groups by Chi-square test with the Yates correction for continuity. The Fisher’s Exact Test was used when a value of <5 was expected in any cell. For confocal quantification analysis of mitochondrial activity, intracellular ROS levels and Mitochondria/ROS colocalization, mean values of MitoTracker CMTM Ros and DCF fluorescence intensity and Pearson’s correlation coefficient were compared by one-way ANOVA followed by Multiple Comparison methods (Dunn’s or Holm-Sidak; Sigma Plot software version 11.0). All comparisons were performed between treated and control groups and among meiotic stages. A level of P <0.05 was considered significant.

A total of 95 non-cultured oocytes was evaluated for chromatin configuration, 45 in the IMM group and 50 in the EH group, comprising four replicates. EH holding at uncontrolled room temperature significantly affected the chromatin configuration of non-cultured oocytes (Figure 1). EH treatment was associated with a significant decrease in the more juvenile FN (P <0.001) and Fibrillar/Intermediate chromatin configurations (P <0.05), and with a significant increase in oocytes exhibiting the more meiotically competent CC configuration (P <0.01). Notably, 22% of oocytes in the EH treatment had resumed meiosis (prometaphase I and metaphase I), vs 0 in the IMM treatment (P <0.01).

Of these 95 oocytes, 11 were not evaluated further due to degenerated chromatin (n =2) or breakage during processing (n =9). The mitochondrial distribution pattern in the remaining 84 oocytes is presented in Table 1. Thirteen oocytes (9 in the IMM treatment and 4 in the EH treatment) exhibited abnormal mitochondrial distribution; this was highest in IMM oocytes having the FN configuration (44%). There were no significant differences in mitochondrial distribution within chromatin configurations between EH and IMM oocytes (P >0.1), thus, results for oocytes within the IMM and EH treatments were pooled to determine the association of chromatin configuration with mitochondrial distribution. Oocytes in the CC configuration had a significantly higher proportion of the more mature P/P mitochondrial distribution (16/32, 50%) than did oocytes having Fibrillar/Intermediate chromatin (2/15, 13%; P <0.05).

Quantitative data of fluorescence intensities for Mitotracker Orange CMTM Ros and DCF for 71 non-cultured oocytes are reported in Table 2; 13 oocytes which had an abnormal mitochondrial distribution pattern were excluded from quantitative analysis. There was no effect of EH treatment on mitochondrial activity, intracellular ROS levels, or Mitochondria/ROS colocalization either when grouped as total non-cultured oocytes (including oocytes at the PI/MI stage) or when grouped as total GV-stage oocytes. In oocytes having the CC chromatin configuration, EH treatment did not affect quantitative energy/redox parameters. For oocytes having the FN chromatin configuration, EH treatment was associated with significantly lower mitochondrial activity (P <0.05) and Mitochondria/ROS colocalization (P <0.05) and a tendency for lower DCF fluorescence (P =0.08).

Overall, 218 in vitro-cultured oocytes (six replicates for each treatment) were evaluated. Oocyte maturation rate in the IMM-IVM group was significantly higher than that for the EH-IVM group (48/85, 56.5% vs 47/133, 35.3%, respectively; P <0.01, Figure 1). Forty-six IMM MII and 34 EH MII oocytes underwent confocal analysis of energy/oxidative status; the remaining 2 IMM MII and 13 EH MII oocytes were not analysed, as they were damaged during handling. The proportion of oocytes showing the heterogeneous P/P distribution pattern was significantly higher (P <0.05) in the EH-IVM than in the IMM-IVM treatments (Table 1). Fluorescence intensities of Mitotracker Orange CMTM Ros and DCF evaluated in MII oocytes after IVM are reported in Table 2; 5 oocytes which had an abnormal mitochondrial distribution pattern were excluded from quantification analysis. Measured energy status, intracellular ROS levels and Mitochondria/ROS colocalization did not differ between EH-IVM and IMM-IVM MII oocytes. In comparing energy/redox data between non-cultured and MII oocytes, MII oocytes, independent of treatment, showed significantly higher values for all energy/redox parameters than did total non-cultured oocytes (P <0.05; Table 2). In addition, EH-IVM MII oocytes showed significantly higher rates of P/P mitochondrial pattern than did EH (non-cultured immature) oocytes (23/34, 67.6% vs 15/41, 45.3%; P <0.05, Table 1).

Because the results obtained in Exp. 1, conducted under variable temperature, indicated that EH treatment failed to completely suppress meiosis, in contrast to previous reports [1], a second experiment was conducted to determine whether the holding temperature could affect the ability of EH treatment to suppress meiosis.

One hundred and nineteen oocytes were evaluated in five replicates. Holding temperature significantly affected oocyte nuclear chromatin configuration (Figure 2). When oocytes were held in EH at 25°C, no differences in chromatin configuration were observed in comparison with the IMM group; however, holding at higher temperatures (30°C and 38°C) was associated with significant increases in oocytes resuming meiosis (metaphase I and metaphase II) compared with oocytes in the IMM or 25°C EH held groups.

Subsequently, in order to determine whether holding at 25°C maintained Mitochondria/ROS equivalent to that for IMM oocytes, oocytes held in EH at controlled room temperature (25°C) were analysed for initial chromatin configuration, meiotic competence and mitochondrial energy/redox potential. A total of 72 non-cultured oocytes was evaluated in two replicates for chromatin configuration, 39 in the IMM group and 33 in the EH group. EH treatment performed at 25°C did not affect oocyte initial chromatin configuration (Figure 3), as no differences were observed in the rates of oocytes of any chromatin configuration between EH and IMM groups. Of these 72 oocytes, 7 were not evaluated further due to degenerated chromatin (n =4 in the EH group and n =3 in the IMM group). The mitochondrial distribution pattern in the remaining 65 oocytes is presented in Table 3. Two oocytes in the EH treatment exhibited abnormal mitochondrial distribution and were excluded from further analysis. There were no significant differences in mitochondrial distribution within chromatin configurations between EH and IMM oocytes (P >0.1). In the IMM group, three oocytes were found at the metaphase I stage and two oocytes were in metaphase II with the second polar body extruded. All these oocytes exhibited the P/P mitochondrial distribution pattern.

Quantitative data of fluorescence intensities for Mitotracker Orange CMTM Ros and DCF for 62 non-cultured oocytes are reported in Table 4. Two EH oocytes which had an abnormal mitochondrial distribution pattern, and an IMM CC oocyte with an abnormal shape were excluded from quantitative analysis. In non-cultured oocytes held in EH at controlled room temperature, there was no effect of EH treatment on mitochondrial activity, intracellular ROS levels, or Mitochondria/ROS colocalization at any meiotic stage. Within EH-treated oocytes, no differences were observed among chromatin configurations for any energy/redox parameter, however mitochondrial activity and intracellular ROS levels of oocytes showing FN chromatin configuration tended to be lower than those for the other configurations, as observed in Exp. 1. Within IMM oocytes, significantly higher mitochondrial activity and ROS levels were found in oocytes resuming meiosis compared with GV oocytes (F/I vs PI/MI and CC vs PI/MI; P <0.05). Photomicrographs representative of oocyte morphology, nuclear chromatin configuration, mitochondrial and ROS distribution patterns and Mitochondria/ROS colocalization of EH-25 and IMM oocytes are presented in Figure 4. The merged image shows the simultaneous distribution of overlapping fluorescence signals (corresponding to the Mitochondria- and ROS-specific probes) within the oocyte cytoplasmic area; an increasingly yellow image denotes increasing colocalization. The scatter plot displays how the two variables (MitoTracker and DCF fluorescent intensities) are correlated. Increasing width of the scatter denotes increasing variability, increasing length denotes increasing signal intensity. The chromatin categories with the highest prevalence are shown. Oocytes in lines A and B show homogeneous mitochondrial distribution and lower fluorescence intensity; oocytes in lines C-F show heterogeneous P/P mitochondrial pattern and higher fluorescence intensity, as also expressed by their longer scatter plot graphs. It should be noted that, in this figure, mitochondrial distribution are shown on only one confocal plane, whereas recording of distribution was performed using all 25 planes obtained.