N-acetylcysteine addition after vitrification improves oocyte mitochondrial polarization status and the quality of embryos derived from vitrified murine oocytes

Abstract

Background: Vitrification is the safest method to cryopreserve oocytes, however the process alters mitochondrial function resulting from increased reactive oxygen species (ROS) production. Our aim was to alleviate ROS stress in vitrified mice oocytes using N-acetylcysteine (NAC at 1 mM), to improve the oocyte's developmental competence.

Results: Hence, four experimental groups were compared: fresh oocytes (F-C), vitrified oocytes (V-C), NAC addition prior to oocyte vitrification (V-NAC-Pre) and NAC addition after vitrification (V-NAC-Post). V-NAC-Pre and V-NAC-Post exhibited higher levels of mitochondrial polarization compared to vitrified oocytes (36.5 ± 3.1, 37.7 ± 1.3 and 27.2 ± 2.4 measured as the spatial coefficient of variation/oocyte respectively, mean ± SEM; p < 0.05). However, ROS production increased in vitrified oocytes added with NAC compared to the vitrified control (1124.7 ± 102.1 [V-NAC-Pre] and 1063.2 ± 82.1 [V-NAC-Post] vs. 794.6 ± 164.9 [V-C]; arbitrary fluorescence units/oocyte, mean ± SEM; p < 0.05). ATP significantly decreased in V-NAC-Pre compared to V-NAC-Post oocytes (18.5 ± 6.9 vs. 54.2 ± 4.6 fmol/oocyte respectively, mean ± SEM; p < 0.05), and no differences were observed between V-NAC-Post, F-C and V-C groups. Blastocyst rates derived from F-C oocytes was higher than those derived from V-NAC-Pre (90.7 ± 1.8 vs. 79.1 ± 1.8, respectively, mean % ± SEM,; p < 0.05) but similar to those derived from V-NAC-Post (90.7 ± 1.8, mean % ± SEM, p > 0.05). Total blastomere count of blastocysts derived from V-NAC-Post after in vitro fertilization (IVF) was higher than embryos produced from V-C.

Conclusions: The addition of NAC after vitrification improves the quality of vitrified mature murine oocytes while its addition prior to vitrification is advised against.

Keywords: Mouse; N-acetylcysteine; Oocyte; Vitrification.

Fig. 1

Mitochondrial polarization status of fresh and vitrified murine oocytes in presence or absence of NAC. The groups studied were: F-C: oocytes were cultured in KSOM medium for 2 h prior to IVF; V-C: oocytes were cultured in KSOM medium for 2 h prior to vitrification. After warming, oocytes were allowed to recover in KSOM medium for 2 h and subjected to IVF; V-NAC-Pre: oocytes were cultured in KSOM medium supplemented with 1 mM NAC for 2 h prior to vitrification. Warmed oocytes were allowed to recover for 2 h in KSOM medium before IVF; V-NAC-Post: oocytes were cultured in KSOM medium for two hours prior to vitrification. Warmed oocytes were allowed to recover for 2 h in KSOM medium added with 1 mM NAC before IVF. Representative micrographs of each treatment are provided below each bar and the scale represents 40 μm. Bars bearing different letters differ statistically (p < 0.05); values are expressed as the mean ± SEM

Fig. 2

Assessment of ROS production in fresh and vitrified murine oocytes in presence or absence of NAC. The groups studied were: F-C: oocytes were cultured in KSOM medium for 2 h prior to IVF; V-C: oocytes were cultured in KSOM medium for 2 h prior to vitrification. After warming, oocytes were allowed to recover in KSOM medium for 2 h and subjected to IVF afterwards; V-NAC-Pre: oocytes were cultured in KSOM medium supplemented with 1 mM NAC for 2 h prior to vitrification. Warmed oocytes were allowed to recover for 2 h in KSOM before IVF; V-NAC-Post: oocytes were cultured in KSOM medium for 2 h prior to vitrification. Warmed oocytes were allowed to recover for 2 h in KSOM medium added with 1 mM NAC before IVF; representative micrographs of each treatment are provided below each bar and the scale represents 40 μm. Bars with different letters differ statistically (p < 0.05); values are expressed as mean ± SEM

Fig. 3

ATP content of fresh and vitrified oocytes in presence or absence of NAC. The groups studied were: F-C: oocytes were cultured in KSOM medium for 2 h prior to IVF; V-C: oocytes were cultured in KSOM medium for 2 h prior to vitrification. After warming, oocytes were allowed to recover in KSOM medium for 2 h and subjected to IVF; V-NAC-Pre: oocytes were cultured in KSOM medium supplemented with 1 mM NAC for two hours prior to vitrification. Warmed oocytes were allowed to recover for 2 h in KSOM medium before IVF; V-NAC-Post: oocytes were cultured in KSOM medium for 2 h prior to vitrification. Warmed oocytes were allowed to recover for 2 h in KSOM medium added with 1 mM NAC before IVF. Bars with different letters differ statistically (p < 0.05); values are expressed as mean ± SEM

Fig. 4

Representative micrographs of expanded blastocysts stained with Hoechst 33342 derived from fresh and vitrified murine oocytes after IVF. The letters in each panel represent: a) Fresh oocytes cultured in KSOM medium for 2 h; b) Vitrified oocytes cultured in KSOM medium for 2 h after warming; c) oocytes cultured in KSOM medium supplemented with 1 mM of NAC for 2 h prior to vitrification and cultured in KSOM medium for 2 h after warming; d) Vitrified oocytes cultured in KSOM medium supplemented with 1 mM of NAC for 2 h after warming. The scale represents 100 μm; the micrographs were taken using a 40 × objective

Fig. 5

Diagram explaining the experimental design used in our study. F-C: MII denuded oocytes were cultured in KSOM medium for 2 h prior to IVF; V-C: MII denuded oocytes were cultured in KSOM medium for 2 h prior to vitrification. After warming, oocytes were allowed to recover in KSOM medium for 2 h and subjected to IVF; V-NAC-Pre: MII denuded oocytes were cultured in KSOM medium supplemented with 1 mM NAC for two hours prior to vitrification. Warmed oocytes were allowed to recover for 2 h in KSOM medium before IVF; V-NAC-Post: MII denuded oocytes were cultured in KSOM medium for 2 h prior to vitrification. Warmed oocytes were allowed to recover for 2 h in KSOM medium added with 1 mM NAC before IVF