A combined treatment with ethanol and 6-dimethylaminopurine is effective for the activation and further embryonic development of oocytes from Sprague-Dawley and Wistar rats

Abstract

In nuclear-transferred or round spermatid-injected oocytes, artificial activation is required for further development in mammals. Although strontium chloride is widely used as the reagent for inducing oocyte activation in mice, the optimal method for oocyte activation remains controversial in rats because ovulated rat oocytes are spontaneously activated in vitro before artificial activation is applied. In our previous study, we found that cytostatic factor activity, which is indispensable for arrest at the MII stage, is potentially low in rats and that this activity differs greatly between two outbred rats (Slc: Sprague-Dawley (SD) and Crj: Wistar). Therefore, it is necessary to establish an optimal protocol for oocyte activation independent of strains. Given that comparative studies of the in vitro development of oocytes activated by different activation protocols are very limited, we compared four different protocols for oocyte activation (ethanol, ionomycin, strontium and electrical pulses) in two different SD and Wistar rats. Our results show that oocytes derived from SD rats have significantly higher cleavage and blastocyst formation than those from Wistar rats independent of activation regimes. In both types of rat, ethanol treatment provided significantly higher developmental ability at cleavage and blastocyst formation compared to the other activation protocols. However, the initial culture in a fertilization medium (high osmolarity mR1ECM) for 24 h showed a detrimental effect on the further in vitro development of parthenogenetic rat oocytes. Taken together, our results show that ethanol treatment is the optimal protocol for the activation of rat oocytes in SD and Wistar outbred rats. Our data also suggest that high-osmolarity media are inadequate for the in vitro development of parthenogenetically activated oocytes compared with fertilized oocytes.

Role of AMPK throughout meiotic maturation in the mouse oocyte: Evidence for promotion of polar body formation and suppression of premature activation

“…This is not unprecedented, since other protein kinase inhibitors have been shown to promote this developmental response. For example, a wide range of studies have shown that the general kinase inhibitor 6-dimethylaminopurine is an effective inducer of parthenogenesis in oocytes of many mammalian species, including mouse (Szllosi et al, 1993;Liu et al, 1997;Nakasaka et al, 2000), rat (Jiang et al, 2002;Sano et al, 2009), cat (Yin et al, 2007), rabbit (Liu et al, 2002), pig (Leal and Liu, 1998;Jilek et al, 2001), bovine (Winger et al, 1997;Liu et al, 1998), sheep (Loi et al, 1998;Choi et al, 2004;Alexander et al, 2006), goat (Ongeri and Krisher, 2001), camel (Wani, 2008), rhesus monkey (Mitalipov et al, 2001), and human (Nakagawa et al, 2001). This is due in large measure to interruption of maturation promoting factor (MPF) activity, which is required to maintain MII arrest and is regulated by mitogen-activated protein kinase (Fan and Sun, 2004).…”

Ethylene Glycol-supplemented Calcium-free Media Improve Zona Penetration of Vitrified Rat Oocytes by Sperm Cells

“…The oocytes were activated by the following protocol, and the survival and cleavage rates were evaluated at 0 and 24 h after artificial activation. Artificial activation was carried out by a combined treatment with ethanol and 6-dimethylaminopurine (6-DMAP) according to our previous report [32]. In brief, oocytes were put in a fertilization medium (modified Rat 1-cell Embryo Culture Medium (mR1ECM) containing 110 mM NaCl and 4 mg/ml bovine serum albumin (BSA) and omitting polyvinyl alcohol (PVA) [33]) supplemented with 7% (v/v) ethanol (Kanto Chemical, Tokyo, Japan) for 3 min.…”