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

Abstract

Abstract. Cryopreservation of matured oocytes is a useful technique because the oocytes can be used for some assisted reproductive technologies after warming. Even though rats, like mice, have been used in various research fields including reproductive technology, information about cryopreservation of rat oocytes is limited. The objective of the present study was to improve the vitrification protocol for matured rat oocytes. To determine the optimal equilibration time, oocytes were equilibrated in 7.5% ethylene glycol (EG) + 7.5% dimethyl sulfoxide (DMSO) + 20% fetal calf serum (FCS) for 1, 4, 7 or 10 min at 24 C and then 15% EG + 15% DMSO + 0.5 M sucrose + 20% FCS for 1 min at 24 C before being plunged into liquid nitrogen on Cryotops. Oocytes exposed to equilibration medium for 4 min showed higher survival and cleavage rates after artificial activation than those of oocytes exposed for 1, 7 or 10 min. The survival and cleavage rates of vitrified oocytes after activation were 98.3 and 78.4%, respectively. However, the perivitelline spaces of most of the vitrified/warmed oocytes (6/168, 3.6%) could not be penetrated by sperm after in vitro fertilization, and cortical granule exocytosis (CGE) was observed in the oocytes. Therefore, the inhibitory effect of calcium and cryoprotectants in vitrification medium on CGE was examined. In most of the oocytes vitrified in calcium-free media, CGE was strongly suppressed independent of cryoprotectants. Oocytes vitrified in EG-supplemented calcium-free media showed high survival rates after warming (79.4%). After artificial activation, the cleavage and blastocyst formation rates of the oocytes were also high (72.8 and 23.1%, respectively). The zona penetration rate of vitrified/ warmed oocytes was dramatically improved by using EG-supplemented calcium-free media after in vitro fertilization (111/155, 63.9%). Thus, our data suggest that EG-supplemented calcium-free media improve zona penetration of vitrified rat oocytes by sperm cells. Key words: Cryopreservation, Oocytes, Rat, Vitrification (J. Reprod. Dev. 56: [169][170][171][172][173][174][175] 2010) ryopreservation of germ cells and reproductive organs is a useful and important technology for efficient production and maintenance of transgenic, mutant, knock-out and other animals. In particular, embryo and sperm cryopreservation has been routinely used for not only efficient production of experimental animals but also clinical medicine. On the other hand, the most desired germ cells for freezing or vitrification are matured oocytes because successfully cryopreserved oocytes can be used for in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI) or somatic-cell nuclear transfer (SCNT) [1,2]. Although successful freezing or vitrification of oocytes has been reported in several mammalian species [3][4][5][6], the survival is generally low. Especially in rats, a high rate of successful vitrification of early-stage embryos has not been obtained until recent years, possibly because rat germ cells seem to be sens...

Alterations in calcium oscillatory activity in vitrified mouse eggs impact on egg quality and subsequent embryonic development

“…In that case, performing vitrification in calcium-free medium reduced zona pellucida hardening and enhanced the fertilization rate and development [7,17]. In the present study, we produced vitrified eggs using EG only or EG+ DMSO, which recovered from cryodamage after a period of incubation and then, we investigated the changes of longlasting [Ca 2+ ] i oscillation [30].…”

Current progress in oocyte and embryo cryopreservation by slow freezing and vitrification

“…Its effect seems to be beyond being a permeable CP because some interaction between EG and calcium seems to take place. When rat oocytes were vitrified in vitrification solution of 15% EG, 15% DMSO, and 0.5 M sucrose and 20% FCS, survival and cleavage rate after activation of vitrified warmed oocytes was 98.3 and 78.4% respectively, but zona pellucida sperm penetration rate was very low (3.6%, 6/168) and a high level of cortical granule exocytosis was noted (Fujiwara et al 2010). When the oocytes were vitrified in EG-supplemented calcium-free media without DMSO, they had 79.4% survival, 72.8% cleavage after activation, 63.9% zona pellucida penetration, and 23.1% of the oocytes developed to blastocyst.…”

Generation of rats from vitrified oocytes with surrounding cumulus cells via in vitro fertilization with cryopreserved sperm

“…The females were superovulated by intraperitoneal injections of equine chorionic gonadotropin (eCG: PMS‐A 1000 for animals, Nippon Zenyaku Kogyo Co., Ltd., Fukushima, Japan) 300 IU/kg of body weight and of human chorionic gonadotropin (hCG: Gonatropin, ASKA Pharmaceutical Co., Ltd. Tokyo, Japan) 300 IU/kg of body weight at 48 h intervals, as previously reported by Fujiwara et al . (). During the first 12 to 14 h after the hCG injection, oocytes composed of masses of surrounding cumulus cells were collected from the oviductal ampullae of the donor females with modified Dulbecco's phosphate buffered medium (PB1: Whittingham & Wales ) calcium free supplemented with 1% (v/v) fetal calf serum (FCS: Life Technologies, Carlsbad, CA, USA), and then COCs were individually separated from the mass of cumulus‐matrix including several COCs by pipetting.…”

“…Although matured rat oocytes vitrified with ethylene glycol (EG) survived after warming, the oocytes were not fertilized after IVF with fresh sperm (Fujiwara et al . ). In that study, sperm have been observed in the perivitelline space of the vitrified/warmed oocytes after IVF.…”