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. 2019 Oct 22;14(10):e0224087.
doi: 10.1371/journal.pone.0224087. eCollection 2019.

N-acetyl cysteine restores the fertility of vitrified-warmed mouse oocytes derived through ultrasuperovulation

Ayumi Mukunoki  1 , Toru Takeo  1 , Naomi Nakagata  1
Affiliations

N-acetyl cysteine restores the fertility of vitrified-warmed mouse oocytes derived through ultrasuperovulation

Ayumi Mukunoki et al. PLoS One. .

Abstract

Oocyte cryopreservation is useful for preserving fertility and storing genetic resources. However, the small number of oocytes acquired using conventional treatment to induce superovulation and the reduction of fertility due to cryopreservation represent significant problems. Herein, we vitrified the oocytes derived through high-yield superovulation using inhibin antiserum and equine chorionic gonadotropin (IAS + eCG: IASe) and examined the yield of cryopreserved oocytes and survival rates relative to those of vitrified-warmed mouse oocytes derived through conventional superovulation using equine chorionic gonadotropin (eCG). Furthermore, we investigated the effects of N-acetyl cysteine on the fertility and developmental potential of vitrified-warmed oocytes derived using IASe. Compared with eCG, IASe increased the yield of cryopreserved oocytes and achieved equivalent survival rates. N-acetyl cysteine (0.5 mM) increased the fertilization rate of vitrified-warmed oocytes derived using IASe. Vitrification decreased thiol levels in the zona pellucida (ZP), while warming followed by N-acetyl cysteine treatment increased free thiol levels in ZP. Moreover, N-acetyl cysteine treatment recovered zona hardening by cleaving disulfide bonds and promoting the expansion of ZP. Two-cell embryos derived via in vitro fertilization using N-acetyl cysteine developed into normal pups through embryo transfer. Therefore, we developed an efficient technique for the production of cryopreserved oocytes using IASe through superovulation and found that N-acetyl cysteine improves the fertility of vitrified-warmed oocytes by cleaving the disulfide bonds and promoting the expansion of ZP.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Effect of superovulation induced by eCG or IASe treatment on the viability and yield of vitrified–warmed oocytes.
A) The number of ovulated oocytes per oocyte donor was counted after removing the cumulus cells. B) The number of surviving oocytes per oocyte donor was counted after vitrifying and warming. C) The rate of survival of oocytes (%) was calculated as the number of morphologically normal oocytes divided by the sum of morphologically normal and dead oocytes × 100. Morphologically normal oocyte (D) and dead oocyte (E) after vitrifying and warming are shown. The results are expressed as mean ± SD (n = 5). *P < 0.05 compared with eCG.
Fig 2
Fig 2. Effect of NAC on the fertilization rate of vitrified–warmed oocytes.
The fertilization rate was calculated as follows: total number of two-cell embryos divided / (the total number two-cell embryos + unfertilized oocytes) × 100. Results are expressed as mean ± SD (n = 6). *P < 0.05 compared with control (0 mM NAC).
Fig 3
Fig 3. Thiol levels following treatment with cryoprotectants.
Relative fluorescence intensity was calculated as follows: fluorescence intensity of vitrified–warmed oocytes precultured with NAC / fluorescence intensity of fresh oocytes. Results are expressed as mean ± SD (fresh: n = 85, DMSO: n = 85, DAP213: n = 52, thawed: n = 86). *P < 0.05 compared with fresh oocytes [DMSO (−), DAP213 (−), vitrified–warmed (−)].
Fig 4
Fig 4. Effect of NAC on thiol levels in vitrified–warmed oocytes.
The level of thiol group of vitrified warmed oocytes treated with or without NAC was measured by fluorescence intensity of the thiol-selective fluorescent compound AFM (31–34 oocytes/experiment). The oocytes were shown (A: 0 mM, C: 0.25 mM, E: 0.5 mM: bright field, B: 0 mM, D: 0.25 mM, F: 0.5 mM: fluorescence, scale bar shows 50 μm). G) Thiol levels in the ZP were calculated as follows: Fluorescence intensity of each group / Fluorescence intensity of 0 mM NAC) × 100. Results are expressed as mean ± SD (n = 4). *P < 0.05 compared with control (0 mM NAC).
Fig 5
Fig 5. Effect of NAC on zona pellucida expansion in vitrified–warmed oocytes.
A) Cross-sectional area of the zona pellucida was measured (31–34 oocytes/experiment). B) The extent of expansion of the zona pellucida was calculated as follows: (cross-sectional area of each group / cross-sectional area in 0 mM NAC) × 100. Results are expressed as mean ± SD (n = 4). *P < 0.05 compared with control (0 mM NAC).
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Grants and funding

TT received a grant for Research on Development of New Drugs from the Japan Agency for Medical Research and Development (ID: 16769865), URL: https://www.amed.go.jp/en/index.html. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.