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Efficient production of live offspring from mouse oocytes vitrified with a novel cryoprotective agent, carboxylated ?-poly-L-lysine.

ABSTRACT: In cryopreservation of mammalian germ cells, unfertilized oocytes are one of the most available stages because these cryopreserved oocytes can be used for assisted reproductive technologies, including in vitro fertilization (IVF) and intracytoplasmic sperm injection. However, it has been generally reported that the fertility and developmental ability of the oocytes are reduced by cryopreservation. Therefore further improvement will be required. Very recently, a new cryoprotective agent (CPA), called as carboxylated ?-poly-L-lysine (COOH-PLL), has been developed to reduce physical and physiological damage by cryopreservation in mammalian stem cells. However, it is unclear the effect of COOH-PLL on fertility and developmental ability of vitrified oocytes. In this study, we used COOH-PLL as a CPA with ethylene glycol (EG) for vitrification of mouse oocytes. Cumulus-oocyte complexes (COCs) were collected from ICR mice and then vitrified with Cryotop using different concentration of COOH-PLL and EG. A combined treatment with COOH-PLL and EG showed high survival rate (more than 90%) of vitrified-warmed COCs after in vitro fertilization. In addition, the fertility and developmental ability of COCs vitrified with E20P10 [EG 20% (v/v) and COOH-PLL 10% (w/v)] or E15P15 group (EG 15% and COOH-PLL 15%) were significantly higher than those with E10P20 (EG10% and COOH-PLL 20%) or P30 group (PLL30%). The vitrified COCs in E20P10 group developed to term at a high success rate (46.2%) and it was significantly higher than that in control (E30) group (34.8%). Our present study demonstrated for the first time that COOH-PLL is effective for vitrification of mouse oocytes.

SUBMITTER: Watanabe H

PROVIDER: S-EPMC3871522 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Efficient production of live offspring from mouse oocytes vitrified with a novel cryoprotective agent, carboxylated ε-poly-L-lysine.

Watanabe Hitomi H Kohaya Natsuki N Kamoshita Maki M Fujiwara Katsuyoshi K Matsumura Kazuaki K Hyon Suong-Hyu SH Ito Junya J Kashiwazaki Naomi N

PloS one 20131223 12

In cryopreservation of mammalian germ cells, unfertilized oocytes are one of the most available stages because these cryopreserved oocytes can be used for assisted reproductive technologies, including in vitro fertilization (IVF) and intracytoplasmic sperm injection. However, it has been generally reported that the fertility and developmental ability of the oocytes are reduced by cryopreservation. Therefore further improvement will be required. Very recently, a new cryoprotective agent (CPA), ca ...[more]

PMID: 24376724

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Project description:BackgroundWe had previously demonstrated that vitrification reduces the levels of certain phospholipid classes, and that oocytes from aged mice show a similar lipidome alteration, even without vitrification. In the current investigation, we examined if vitrification-warming of mouse oocytes from young and aged mice causes any changes in molecular aspects of lipid-associated features.MethodsMetaphase II (MII) stage oocytes were harvested from young (10-14-week-old) and aged (45-54-week-old) mice by a superovulation regime with PMSG followed by hCG. We examined the status of the intracellular lipid pool and the integrity of the plasma membrane by staining oocytes with BODIPY 500/510 and CellMask live dyes. Expression of lipid uptake- and necroptosis-associated genes was assessed by quantitative PCR analyses, in oocytes from young and old mice, before and after vitrification. Localization patterns of two crucial necroptosis proteins, phosphorylated MLKL (pMLKL) and phosphorylated RIPK1 (pRIPK1) were examined in mouse oocytes by immunofluorescence staining. Necrostain-1 (Nec1), an inhibitor of RIPK1, was used to examine if RIPK1 activity is required to maintain oocyte quality during vitrification.ResultsWe confirmed that vitrified-warmed oocytes from aged mice showed noticeable decrease in both CellMask and BODIPY 500/510 dyes. Among the lipid uptake-associated genes, Cd36 expression was higher in oocytes from aged mice. Necroptosis is a type of programmed cell death that involves damage to the plasma membrane, eventually resulting in cell rupture. The expression of necroptosis-associated genes did not significantly differ among groups. We observed that localization patterns of pMLKL and pRIPK1 were unique in mouse oocytes, showing association with microtubule organizing centers (MTOCs) and spindle poles. pMLKL was also localized on kinetochores of MII chromosomes. Oocytes treated with Nec1 during vitrification showed a decreased survival rate, indicating the importance of RIPK1 activity in oocyte vitrification.ConclusionsWe report that oocytes from aged mice show differential expression of CD36, which suggests that CD36-mediated lipid uptake may be influenced by age. We also show for the first time that pMLKL and pRIPK1 exhibit unique localization pattern in mouse oocytes and this may suggest role(s) for these factors in non-necroptosis-associated cellular processes.

Dataset Information

Efficient production of live offspring from mouse oocytes vitrified with a novel cryoprotective agent, carboxylated ?-poly-L-lysine.

Publications

Efficient production of live offspring from mouse oocytes vitrified with a novel cryoprotective agent, carboxylated ε-poly-L-lysine.

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