Introduction
Mammalian oocytes are usually fertilized soon after ovulation. If fertilization does not occur in time, unfertilized oocytes that remained in the oviduct (aging in vivo) or cultured in a suitable medium (aging in vitro) overall undergo time-dependent aging (Yanagimachi & Chang, Reference Yanagimachi and Chang1961; Whittingham & Siracusa, Reference Whittingham and Siracusa1978). It is well known that fertilization or artificial activation of aged oocytes could result in abnormal development (Juetten, Reference Juetten1983; Tarin et al., Reference Tarin, Perez-Albala, Aguilar, Minarro, Hermenegildo and Cano1999, Reference Tarin, Perez-Albala, Perez-Hoyos and Cano2002; Gordo et al., Reference Gordo, Rodrigues, Kurokawa, Jellerette, Exley, Warner and Fissore2002). Despite inherent difficulties, gamete cryopreservation has revolutionized mammalian husbandry and reproductive medicine. It has been reported that frozen–thawed gametes can often regain their viability and be used for fertilization (Watson, Reference Watson and 1990; Wakayama et al., Reference Wakayama, Whittingham and Yanagimachi1998; Nakagata, Reference Nakagata2000). Conventional storage of gametes in liquid nitrogen (−196 °C) is complicated. It requires the constant supply of liquid nitrogen and, during transportation, dry ice might sometimes be insufficient for maintaining suitable temperatures. Thus, it would be ideal if we could store gametes for 1 day, or even several days, at room temperature. Previous investigations have already shown that mouse spermatozoa can be freeze-dried and stored at room temperature for up to 1 month without losing their genetic and reproductive potential (Wakayama & Yanagimachi, Reference Wakayama and Yanagimachi1998) and mouse cumulus–oocyte complexes (COCs) stored at room temperature for 24 h can still retain full developmental potential (Wakayama et al., Reference Wakayama, Thuan, Kishigami, Ohta, Mizutani, Hikichi, Miyake and Wakayama2004).
Oocytes matured both in vivo and in vitro are enclosed with cumulus cells (CCs). The CCs stay with the oocyte matured in vivo for a variable period after ovulation, depending upon the species (Yanagimachi & Chang, Reference Yanagimachi and Chang1961; Longo, Reference Longo1974, Reference Miao, Liu, Qiao, Miao, Luo and Tan1980; Tan, Reference Tan1985, Reference Tan1988), but they will always stay with oocytes matured in vitro until artificially removed. The roles of the surrounding CCs in maturation, ovulation and fertilization of oocytes have been extensively studied (Eppig, Reference Eppig1982, Reference Eppig1991; Buccione et al., Reference Buccione, Schroeder and Eppig1990; Tanghe et al., Reference Tanghe, Van Soom, Nauwynck, Coryn and de Kruif2002), yet little is known about their roles in oocyte aging. Early studies have shown that ovulated oocytes with CCs aged in vitro displayed similar morphological alterations as those aged in vivo (Longo, Reference Longo1980; Webb et al., Reference Webb, Howlett and Maro1986; Tan, Reference Tan1988) and spontaneous reduction in MPF activity (Kikuchi et al., Reference Kikuchi, Izaike, Noguchi, Furukawa, Daen, Naito and Toyoda1995, Reference Kikuchi, Naito, Noguchi, Shimada, Kaneko, Yamashita, Aoki, Tojo and Toyoda2000; Wu et al., Reference Wu, Ignotz, Currie and Yang1997) and cytoskeletal alteration (Kim et al., Reference Kim, Moon, Prather and Day1996) have been reported in the aging process of oocytes with CCs matured in vitro. Abbott et al. (Reference Abbott, Xu, Kopf, Ducibella and Schultz1998) indicated that in vitro culture of mouse oocytes free of CCs could retard the spontaneous activation of cell-cycle progression that normally occurred in unfertilized eggs in vivo (Xu et al., Reference Xu, Abbott, Kopf, Schultz and Ducibella1997; Abbott et al., Reference Abbott, Xu, Kopf, Ducibella and Schultz1998). Recently, Miao et al. (Reference Miao, Liu, Qiao, Miao, Luo and Tan2005) also demonstrated that CCs could accelerate the progression of in vitro aging of mouse oocytes.
In vitro fertilization (IVF) has been used frequently for the treatment of sterility in humans; to improve the quality and increase production in cattle (Suzuki et al., Reference Suzuki, Yorozu, Watanabe, Nakura and Adachi1996). In laboratory animals, IVF has been used for analysis of fertilization events and to ameliorate defects in the reproductive ability of mutant mice (Suzuki et al., Reference Suzuki, Shingai, Togashi, Miyai and Okamoto1987, Reference Suzuki, Shingai, Togashi, Miyai, Motohide and Okamoto1988, Reference Suzuki, Togashi, Moriguchi and Adachi1996; Yokoyama et al., Reference Yokoyama, Katsuki and Nomura1995) or aged mice (Suzuki et al., Reference Suzuki, Togashi, Moriguchi and Adachi1994). Early studies have shown that the addition of E2 to oocyte maturation medium could increase the fertilization and cleavage rates of the in vitro matured human oocytes (Tesarik & Mendoza Reference Tesarik and Mendoza1995). Although 1 μg/ml E2 is often used routinely in in vitro maturation (IVM) and in in vitro fertilization (IVF) (Beker et al., Reference Beker, Colenbrander and Bevers2002), yet little is known about its roles in embryos development.
In this study, mouse oocytes without CCs were stored at room temperature (25 °C) for 24 h and were used for in vitro fertilization with fresh spermatozoa. We also tested the effect of E2 in CZB medium on subsequent embryo development.
Materials and Methods
Animals
Kunming female (white) and C57Bl/6 male (black) mice, 2–4 months old, were used in this study. Kunming mice were originally derived from ICR mice (CD-1) (Yu et al., Reference Yu, Ma, Miao, Tan, Liu, Lu and Tan2006). They were kept in an air-conditioned room (23 °C, 50% relative humidity) under 14-h light and 10-h dark cycles.
Oocyte collection and storage
Kunming female mice were superovulated by injection of 7.5 IU pregnant mare serum gonadotropin (PMSG, Tianjin Animal Hormone Factory), followed by 7.5 IU human chorionic gonadotropin (hCG, Ningbo Animal Hormone Factory) 48 h later. Approximately 13–14 h after hCG injection, COCs were collected from oviducts. COCs were placed in modified HEPES-buffered CZB (H-CZB) medium and treated with 0.1% hyaluronidase in order to disperse cumulus cells. Then 10 oocytes were stored in a 20 μl H-CZB medium drop for 24 h at room temperature (25 °C). Medium droplets were covered with mineral oil to prevent evaporation.
Sperm collection and capacitation
Spermatozoa were obtained from C57Bl/6 male mice. After the male mice were killed, one caudae epididymis was removed and the spermatozoa were gently squeezed out. Then the spermatozoa were placed into a 400 μl HTF drop in the incubator (5% CO2 in air, 37 °C) for 1.5 h for capacitation.
Parthenogenetic activation
Fresh oocytes and categories of survived oocytes after 24 h storage were cultured in Ca2+-free CZB medium supplemented with 5.56 mM d-glucose containing 10 mM SrCl2 for 5 h at 37 °C in a humidified atmosphere containing 5% CO2, then washed and cultured in modified CZB medium (Cummins et al., Reference Cummins, Wakayama and Yanagimachi1998; Kishikawa et al., Reference Kishikawa, Wakayama and Yanagimachi1999; Takeda et al., Reference Takeda, Tasai, Iwamoto, Onishi, Tagami, Nirasawa, Hanada and Pinkert2005) with or without 1 μg/ml E2 (Beker et al., Reference Beker, Colenbrander and Bevers2002; Zhang, Reference Zhang2004) at 37 °C in a humidified atmosphere containing 5% CO2 for further culture.
In vitro fertilization
Both fresh and stored oocytes were inseminated in 400 μl microdroplets of sperm suspension in HTF medium (2 × 106 motile sperm/ml) overlaid with mineral oil for 5 h at 37 °C in a humidified atmosphere containing 5% CO2. They were then washed and trans-ferred into fresh CZB medium with or without 1 μg/ml E2 (Beker et al., Reference Beker, Colenbrander and Bevers2002; Zhang, Reference Zhang2004) for further culture.
In vitro embryo culture
In vitro culture (IVC) of embryos was carried out in 20 μl microdroplets of CZB medium with or without 1 μg/ml E2 (Beker et al., Reference Beker, Colenbrander and Bevers2002; Zhang, Reference Zhang2004) under mineral oil (10 embryos per drop) at 37 °C in a humidified atmosphere containing 5% CO2. Embryos were cultured in CZB medium with or without 1 μg/ml E2 (without glucose) for 48 h. On day 2 of culture, the embryos were checked for further development. Cleaved embryos were transferred to fresh culture medium supplemented with glucose (5 mg/ml) and incubated for another 2 days to the morula/blastocyst stage.
Embryo transfer to foster mothers
After 96 h in culture, morulae/blastocysts embryos were transferred into the uteri of day 3 pseudopregnant females. The uteri of pseudopregnant females were examined for fetuses on day 19 or 20.
Statistical analysis
The data were analysed using the chi-squared test (SPSS 13.0). A value of p < 0.05 was considered significant.
Results
Comparison of oocyte activation and fertilization rates
The parthenogenetic activation rates of fresh and storage oocytes were 90.35% and 84.31% respectively (p > 0.05) (Table 1). Furthermore, as shown in Table 2, the fertilization rates of fresh and storage oocytes were significantly different (90.40% and 47.12%, respectively, p < 0.05).
Table 1 Activation rates of fresh oocytes and storage oocytes
aValues with same superscripts within a column do not differ significantly (p > 0.05).
Table 2 Fertilization rates of fresh oocytes and storage oocytes
a ,b Values with different superscripts within a column differ significantly (p < 0.05).
Parthenogenetic development of storage and fresh oocytes
Most parthenogenetic embryos from storage oocytes (93.58% and 87.70%, p > 0.05) developed to the 2-cell stage after cultured in CZB medium or CZB medium + E2 for 24 h. There was significant difference between the development rates of 4–8-cell embryos in the two media (59.63% and 68.85%, p < 0.05). In addition, the development rates of morula/blastocyst were significantly different (41.18% and 48.36%, respectively, p < 0.05) (Table 3). There was also significant difference between the development rates of blastocysts derived from activated fresh oocytes cultured in the two media (6.33% and 18.60%, p < 0.05) (Table 4).
Table 3 The effects of E2 on development of parthenogenetic oocytes stored at room temperature for 1 day
a ,b Values with different superscripts within a column differ significantly (p < 0.05).
Table 4 The effects of E2 on development of parthenogenetic fresh oocytes
a ,b Values with different superscripts within a column differ significantly (p < 0.05).
Development of IVF embryos from storage oocytes
As shown in Fig. 1a, 47.12% of IVF eggs formed two pronuclei (2PN) after IVF. The shape and size of 2PN in storage oocytes were similar to those in fresh oocytes after IVF. These results suggest that a large proportion of mouse oocytes stored at room temperature for 24 h can be fertilized by IVF. Most of these embryos (90.35% vs. 94.92%, p > 0.05) developed to the 2-cell stage (Table 5, Fig. 1b) after being cultured in the two media for 24 h, while the rates of embryos developed to the 4–8-cell stage (Table 5, Fig. 1c,d) in the two media were significantly different (60.53% vs. 67.97%, p < 0.05). In addition, the rates of morula/blastocyst (Table 5, Fig. 1e,f) (48.83% and 55.08%, respectively) were also significantly different (p < 0.05).
Table 5 The effects of E2 on development of fertilized embryos from storage oocytes
a ,b Values with different superscripts within a column differ significantly (p < 0.05).
Figure 1 In vitro development of mouse embryos derived from IVF of oocytes stored at room temperature for 1 day (×400). (a) zygote with a male and a female pronuclei; (b) 2-cell embryo; (c) 4-cell embryo; (d) 8-cell embryo; (e) morula; (f) blastocyst.
Birth of pups after embryo transfer
One out of seven foster females transferred with embryos cultured in CZB medium gave birth to a female pup (agouti) (Table 5, Fig. 2a). One out of six foster females with embryos cultured in CZB with E2 medium gave birth to six pups, two males (agouti) and four females (agouti) on day 20 (Table 5, Fig. 2b). All pups had black eyes and a pigmented coat. The donors of spermatozoa were C57Bl/6 (black coat) and foster mothers were Kunming mice (white coat), who had never been exposed to pigmented males, therefore, the offspring were derived from the IVF eggs. All the pups developed normally to date (134 days and 147 days at the time of submission).
Figure 2 The foster mothers (white) and pups (agouti). (a) One pup from embryos cultured in CZB medium without E2; (b) Six pups from embryos cultured in CZB with E2.
Discussion
Cryopreservation of gametes has been very successful in humans and some mammalian species (Polge, Reference Polge1952; Wakayama et al., Reference Wakayama, Whittingham and Yanagimachi1998; Nakagata Reference Nakagata2000), but it needs a constant supply of liquid nitrogen, which is inconvenient and also expensive. New inexpensive methods for gamete preservation are therefore of interest at the current time. Wakayama et al. (Reference Wakayama, Thuan, Kishigami, Ohta, Mizutani, Hikichi, Miyake and Wakayama2004) found that mouse COCs stored at room temperature for 24 h can retain full developmental potential. In this experiment, storage of denuded oocytes at room temperature for 1 day decreased fertilization rate when compared with that of fresh oocytes (90.40% and 47.12%, respectively, p < 0.05). On the other hand, there was no significant difference in the activation percentages of fresh oocytes and storage oocytes when they were activated by Sr2+ (90.35% and 84.31%, respectively, p > 0.05). Miao et al. (Reference Miao, Liu, Qiao, Miao, Luo and Tan2005) showed that although oocytes aged in vitro without CCs showed a less degree of cortical granule (CG) exocytosis, they displayed an unusually long t50 for zona pellucida (ZP) dissolution. Downs et al. (Reference Downs, Schroeder and Eppig1986) found that serum maintained the ability to fertilize mouse oocytes matured in vitro by preventing the hardening of the ZP. Our oocytes were stored in H-CZB medium without serum. The results of fertilization and activation suggested that, although most DOs stored at room temperature for 1 day were still normal, the ZP of stored DOs became hard during storage at room temperature, so their fertilization rate became low. Serum might prevent the hardening of the ZP during DOs that were stored at room temperature in H-CZB medium.
Tesarik et al. (1995) reported that the addition of E2 to oocyte maturation medium did not produce any apparent effects on either germinal vesicle breakdown or further progression of meiosis, but it did increase the fertilization and cleavage rates of the in vitro matured oocytes (Tesarik et al., 1995). Guler et al. (Reference Guler, Poulin, Mermillod, Terqui and Cognie2000) found that E2 played an important role in the nuclear and cytoplasmic maturation of sheep oocytes in vitro in defined conditions. Zhang et al. (2004) found that the 2-cell block of reconstructed oocytes of hamster could be overcome by adding E2 to M199. In this experiment, some mouse oocytes stored at room temperature for 24 h can be fertilized normally by IVF. Most of these embryos (90.35% vs. 94.92%, p > 0.05) developed to 2-cell stage after being cultured in the two media for 24 h, while the rates of embryos developed to the 4–8-cell stage in the two media were significantly different (60.53% vs. 67.97%, p < 0.05). In addition, the rates of morula/blastocyst formation (48.83% and 55.08%, respectively) were significantly different (p < 0.05). There was also significant difference between the rates of blastocysts derived from activated fresh oocytes cultured in the two media (6.33% and 18.60%, p < 0.05). E2 is the most potent steroidal estrogen (female sex hormone) and is produced endogenously by all mammalian species. It is produced in the ovary, placenta and possibly the adrenal cortex. It can diffuse through the plasma membrane and bind with the receptor. The receptor with bound ligand is then translocated into the nucleus, where its DNA-binding domain binds to response elements, allowing E2 to stimulate transcription of target genes. We therefore proposed a hypothesis that E2 would improve mouse embryos development in vitro through stimulating transcription of some important genes.
In conclusion, we have demonstrated that some DOs stored at room temperature for 1 day can be fertilized normally by IVF and that E2 might improve mouse embryo development in vitro. The birth of seven agouti pups in this study and their healthy growth indicate that the storage of DOs at room temperature for 1 day may become a practical procedure in mammalian reproduction.
Grant sponsor: This work was supported by the ‘973’ Project (grant A1902164) from the Ministry of Science and Technology of China.
Acknowledgments
We are grateful to Ms Xiang-Fen Song, Ms Yi Hou, Drs Yong-Zhong Zhang, Zhen-Jun Zhao, Li-Ying Yan, Jun-Shu Ai and Shen Yin for their technical assistance.
