Generation of live offspring from vitrified mouse oocytes of C57BL/6J strain.

In mammals, unfertilized oocytes are one of the most available stages for cryopreservation because the cryopreserved oocytes can be used for assisted reproductive technologies, including in vitro fertilization (IVF) and intracytoplasmic sperm injection. However, it has generally been reported that the fertility and developmental ability of the oocytes are reduced by cryopreservation. C57BL/6J mice, an inbred strain, are used extensively for the production of transgenic and knockout mice. If the oocytes from C57BL/6J mice can be successfully cryopreserved, the cryopreservation protocol used will contribute to the high-speed production of not only gene-modified mice but also hybrid mice. Very recently, we succeeded in the vitrification of mouse oocytes derived from ICR (outbred) mice. However, our protocol can be applied to the vitrification of oocytes from an inbred strain. The aim of the present study was to establish the vitrification of oocytes from C57BL/6J mice. First, the effect of cumulus cells on the ability of C57BL/6J mouse oocytes to fertilize and develop in vitro was examined. The fertility and developmental ability of oocyte-removed cumulus cells (i.e., denuded oocytes, or DOs) after IVF were reduced compared to cumulus oocyte complexes (COCs) in both fresh and cryopreserved groups. Vitrified COCs showed significantly (P<0.05) higher fertility and ability to develop into the 2-cell and blastocyst stages compared to the vitrified DOs with cumulus cells and vitrified DOs alone. The vitrified COCs developed to term at a high success rate, equivalent to the rate obtained with IVF using fresh COCs. Taken together, our results demonstrate that we succeeded for the first time in the vitrification of mouse oocytes from C57BL/6J mice. Our findings will also contribute to the improvement of oocyte vitrification not only in animals but also in clinical applications for human infertility

The effects of cumulus cells on the fertility and developmental ability of vitrified C57BL/6J mouse oocytes after IVF.

<p>IVF was carried out using frozen-thawed BDF1 mouse sperm. Data are means±S.E.M. ^a-b,c-d,e-f Different superscripts denote significant difference (<i>P</i><0.05). In each treatment group, more than 100 oocytes were examined.</p

In vivo development of vitrified COCs from C57BL/6J mouse after in vitro fertilization.

<p>In vivo development of vitrified COCs from C57BL/6J mouse after in vitro fertilization.</p

The effects of cumulus cells on the fertility and developmental ability of C57BL/6J mouse oocytes after IVF.

<p>Data are means±S.E.M. ^a-b,c-d,e-f Different superscripts denote a significant difference (<i>P</i><0.05). In each treatment group, more than 100 oocytes were examined. PN, pronuclear stage; COC, cumulus oocyte complexes; DO, denuded oocytes.</p

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

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 ε-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

In vivo development of mouse oocytes vitrified with COOH-PLL.

<p>In vivo development of mouse oocytes vitrified with COOH-PLL.</p

Cryoprotectants in equilibration and vitrification medium for mouse oocytes.

<p>*These cryoprotectants were added into calcium free PB1 supplemented with 20% FCS.</p><p>^# These cryoprotectants were added into calcium free PB1 supplemented with 20% FCS and 0.5 M sucrose.</p

The kinetics of COOH-PLL in mouse oocytes.

<p>Mouse oocytes were exposed to PB1(-) supplement with FITC-labeled COOH-PLL (5% (w/v)). Green: FITC-labeled COOH-PLL.</p

The effects of COOH-PLL on survival, fertility and developmental ability of vitrified mouse oocytes after IVF.

<p>Data are shown as means ± S.E.M. Different superscripts denote a significant difference (P<0.05). Numbers of oocytes used in each group were described under the treatment group.</p