Katsuhiko Narimoto

Oxidative stress on mouse embryo development in vitro

Oxygen radicals are involved in the in vitro block phenomenon of embryo development, because a low oxygen tension and superoxide dismutase (SOD) have been shown to promote the in vitro development of mouse embryos. One of the target molecules damaged by oxygen radicals may be the thiol (SH) group of proteins because it is readily oxidized. In this study, we evaluated the effects of thioredoxin, which is a powerful protein disulfide reductase, on mouse (Institute of Cancer Research, ICR) preimplantation embryo development. Culture of mouse pronuclear embryos recovered 17 h after human chorionic gonadotrophin (hCG) administration in the presence of thioredoxin (200 micrograms/mL) significantly increased the blastulation rate (75.3%) when compared to the control culture system (8.9%). The effects of thioredoxin were observed only from the pronuclear stage to the two-cell stage (17-48 h after hCG administration). An additive effect of thioredoxin and SOD, or thioredoxin and a low oxygen tension, was observed. These results suggest that the oxidation of the SH group of proteins is one of the causes of developmental blockage of embryos in vitro. The target protein for reduction by thioredoxin has not been identified yet, but thioredoxin will be a new clue for clarifying the mechanism of blocking development in vitro.

EFFECTS OF SUPEROXIDE DISMUTASE ON MOUSE IN VITRO FERTILIZATION AND EMBRYO CULTURE SYSTEM

PurposeWe recently found that, for mouse embryos fertilized in vivo, the two-cell block could be attenuated by adding superoxide dismutase (SOD), a scavenger of superoxide radicals, to the culture medium. In this study, we evaluated the effects of SOD on the process of fertilization and on the further development of the embryos fertilized in vitro.MethodsWe performed incubation of mouse epididymal spermatozoa, in vitro fertilization, and further cultivation in Biggers—Whitten—Whittinghams medium supplemented with various concentrations of Cu · Zn—SOD.ResultsHigh concentrations (2000 µg/ml or more) of SOD prevented loss of motility in mouse sperm over time. The addition of SOD (less than 2000 µg/ml) to the basic medium showed no significant difference in the fertilization rate. Also, no significant difference was observed in the rate of polyspermy or parthenogenesis between the basic and the SOD-supplemented media. However, 18% of the two-cell-stage embryos developed to the expanded blastocyst stage in the 500 µg/ml SOD-supplemented medium, while no blastocysts were found in the basic medium. Furthermore, the addition of SOD 7 hr after insemination increased the expanded blastocyst rate (28%).ConclusionsThese results indicate that the addition of SOD exerts a protecting effect from oxidative stress both on sperm viability and on the development of embryos fertilized in vitro as well as in vivo, while its addition showed no effect on the process of fertilization.

Effect of visual light on in vitro embryonic development in the hamster.

The effect of light exposure during collection and culture of hamster embryos on their subsequent development in vitro was examined. When embryos were collected under dark conditions (70 lux) within 10 minutes and then cultured in a HECM-1 medium in 5% CO2 in air, the developmental rates of 1-cell embryos to the 4- and 8-cell stages were 88.6% (93/105) and 66.7% (70/105), respectively. These rates were significantly higher than those under light conditions (1600 lux): 51.9% (56/108) and 34.3% (37/108). Light irradiation during the culture of 1-cell embryos suppressed subsequent development. The degree of suppression correlated inversely with duration of light irradiation, and light irradiation of 30 minutes or more completely blocked development to the 2-cell stage. When 1-cell embryos were irradiated through a yellow filter, cutting the light wavelengths to less than 500 nm, embryonic development was still suppressed. However, the degree of the suppression varied and 45.7% (53/116), 6.0% (7/116), and 0.9% (1/116) of the embryos developed to the 2-, 4-, and 8-cell stages, respectively, under 30 minute light irradiation. Inhibitory effects of light irradiation on the development of 2- and 8-cell embryos were also observed, showing an inverse correlation with duration; the developmental rates of 2-cell embryos to the 8-cell stage under 0, 10, and 30 minutes of irradiation were 85.6% (107/125), 1.6% (2/122), and 0% (0/129), respectively, and those of 8-cell embryos to the blastocyst stage were 79.8% (91/114), 74.8% (86/115), and 0% (0/110), respectively. These findings indicate that early-stage embryos are sensitive to light exposure; however, severe light exposure adversely affects the development of embryos at any stage. Thus, the protection of embryos from light exposure at all stages of embryo manipulation, from collection to culture, is essential.

Developmental potentiality of embryos cultured under low oxygen tension with superoxide dismutase

A low-oxygen culture of preimplantation embryos with superoxide dismutase (SOD) has been demonstrated to produce a marked increase in the blastocyst formation. In this study, we examined the effects of low oxygen-SOD cultures of embryos on the potentiality of embryonic development after intrauterine transplantation and the reproductive ability of the offspring. Mouse pronuclear embryos were cultured in Biggers-Whitten-Whittinghams solution containing 0.3% bovine serum albumin and 500 μg/ml SOD under a low-oxygen condition (5% O2, 5% CO2, 90% N2), and 37.2% (110/296) of the embryos developed to blastocysts, with a significant difference from the 1.3% (3/231) under an atmospheric oxygen condition without SOD (P<0.01). The blastocysts obtained under a low oxygen-SOD culture condition were transplanted by direct insertion into the uterus of pseudopregnant females 3 days after infertile coitus. The implantation rate was 65.0% (65/100), and the embryo viability rate was 46.0% (46/100). In the control group, in which in vivo developed blastocysts were transplanted into 3-day pseudopregnant females, the implantation rate was 72.5% (81/112), and the embryo viability rate was 51.8% (58/112). The differences between the two groups were not significant. Significant differences also were not observed in either the body weight of pups at birth and 35 days after birth or the mean number of pups delivered by successive mating of the offspring between the two groups. These results suggest that the low oxygen-SOD culture conditions in this study are closer to the in vivo intratubal environment than the conventional culture conditions and that the protection of embryos from superoxide anion radicals is a prerequisite for the acquisition of embryos in vitro showing a developmental potentiality equal to that of embryos in vivo. Furthermore, this low oxygen-SOD culture system may be applicable for human IVF-ET.

Role of protein supplements in the culture of mouse embryos

The effect of various types of proteins used in single protein supplements for Bigger-Whitten-Whittingham (BWW) medium on the in vitro development of mouse preimplantation embryos was evaluated. Thioredoxin, superoxide dismutase (SOD), and apotransferrin showed prominent growth-promoting activity, whereas bovine serum albumin (BSA), fatty acid-free BSA, and catalase showed moderate promoting effects. beta-lipoprotein, ovalbumin and hemoglobin were ineffective, and holo-type transferrin and ceruloplasmin were actually toxic to the embryos. These results suggest that the growth-promotive effect of proteins on mouse pronuclear stage embryos is due to their antioxidative action, or to the removal of some free metal ion(s) such as Fe(3+). The mild growth promoting effect of both BSA and fatty acid free BSA suggest that the effect mediated by BSA is not dependent on bound fatty acids, but more likely is due to their antioxidative effect or chelating effect.

Development of hamster one-cell embryos recovered under different conditions to the blastocyst stage

One-cell stage embryos, recovered from superovulated golden hamsters (8 to 12 weeks of age) 12 hours after egg activation, were cultured in HECM-1 medium at 37 degrees C and 5% CO(2) in air. The culture conditions investigated were the time and temperature required for embro recovery, the pH shift of the washing medium, and the oxygen concentration of the gas phase during and after embryo recovery. Each condition was assessed by the developmental efficiency of the embryo as determined by morphological criteria. As the time required for embryo recovery was reduced, the developmental rates of the embryos were improved: 2.3% (3 128 ) 26.9% (35 130 ) at 5 and 3 minutes, respectively, as determined by the number of embryos developed to the blastocyst stage. No blastocysts were obtained when more than 10 minutes were required for embryo recovery. As the oxygen concentration was reduced from 40 to 20% or to 5%, rather high developmental rates were obtained even when the time required for embryo recovery was prolonged: 6.9% (9 130 ) and 21.7% (28 129 ) of the embryos developed to the blastocyst stage when they were recovered under 5% oxygen within 10 and 5 minutes, respectively. Neither the temperature during embryo recovery (37 degrees C and 25 degrees C) nor the pH shift (pH 7.22 to 7.52) of the washing medium used in embryo recovery procedures influenced the development of the embryos. These findings suggest that the developmental block in hamster embryos may involve oxidative stress, which may result from exposure to high oxygen concentration and light during the manipulation of oocytes and embryos.

Development to the four-cell stage of hamster embryos fertilized in vitro

Abstract In an attempt to overcome the 2-cell block phenomenon in hamster embryos fertilized in vitro, the time for ovum recovery was shortened, and culture was performed under low oxygen conditions (5% O2). The effects of culture media and observation procedures during culture were also investigated. Fertilization rates were significantly increased by recovering the ova at 3 minutes, under standard and low oxygen conditions (82 and 69%, respectively) rather than at 10 minutes (59 and 54%, respectively). Thirty percent of the embryos developed to the 2-cell stage when ova were recovered at 3 or 10 minutes, and culture was performed in a TALP-PVA or HECM-1 medium under standard or low oxygen conditions, with no significant difference between groups. Four-cell stage embryos (8%) were obtained only when ova were recovered at 3 minutes and culture was performed in a HECM-1 medium under low oxygen conditions. When observation procedures were performed 4 hours (at the 1-cell stage) and 24 hours (at the 2-cell stage) after insemination, the development to the 2- and 4-cell stages decreased significantly. Four-cell stage embryos were obtained only when culture was continued without observation. In this study, the 2-cell block in hamster embryos fertilized in vitro was successfully overcome for the first time. Evidence suggests that the developmental block may be related to oxygen toxicity, which results from exposure to high oxygen concentrations and light exposure during the manipulation of oocytes and embryos.

Human Decidual Function in Trophoblast and Uterine Interaction

The decidua constitutes an anatomical compartment of the placenta in deciduate mammals including humans. By virtue of its anatomical location at the interface between maternal and fetal tissues, it has long been recognized that the decidua possesses a particular function which leads to the formation of an immunologically privileged site to accommodate the semi-allograft implant and its subsequent development.