Hiroshi Kanagawa

Evidence for the presence of macrophage migration inhibitory factor in murine reproductive organs and early embryos

Cytokines appear to play a critical role in the establishment of early pregnancy. In this study, we examined the mRNA expression of murine macrophage migration inhibitory factor (MIF), one of the cytokines activating macrophages, in the murine reproductive system by Northern blot analysis. It revealed that MIF mRNA was expressed in the ovary, the oviduct and the uterus during the pre-implantation period and all stages of the estrus cycle. The histological localization of MIF in the uterus was examined by an immunohistochemical method, which revealed that MIF was mostly present in the tunica muscularis. It was, moreover, demonstrated by reverse transcription-polymerase chain reaction (RT-PCR) that MIF mRNA was expressed in the ovulated oocytes, zygotes, 2-cell embryos, 8-cell embryos and blastocysts. MIF is known to be present in T-lymphocytes and monocytes/macrophages; however, it was revealed by the present study that MIF mRNA is expressed in the reproductive organs and the early stages of embryos. Considering these results, it s suggested that MIF plays an important role in the establishment of pregnancy.

Stimulation of the development of bovine embryos by insulin and insulin-like growth factor-I (IGF-I) is mediated through the IGF-I receptor

To study the effects of insulin and insulin-like growth factor-I (IGF-I) on the development of bovine embryos, fertilized bovine embryos in vitro were cultured in a chemically defined, protein-free medium: modified synthetic oviduct fluid (mSOF) supplemented with 1 mg/ml polyvinyl alcohol. Dose-response studies showed that insulin (0.5 to 10 microg/ml) and IGF-I (2 to 200 ng/ml) stimulated the development of bovine embryos to the morula stage 5 d after in vitro fertilization. The addition of 0.5 microg/ml insulin or 2 ng/ml IGF-I to the mSOF had beneficial effects on embryonic development to the morula stage in the presence of amino acids, but insulin and IGF-I did not affect the development of bovine embryos to the morula stage in the absence of amino acids. The antiIGF-I receptor antibody (alphaIR-3) completely blocked the stimulation of development to the morula stage by insulin and IGF-I. These findings suggest that the stimulation of embryonic development by insulin and IGF-I is mediated through the IGF-I receptor.

Effects of water quality on in vitro fertilization and development of bovine oocytes in protein-free medium

Examination was made of the effects of water quality in medium preparation on fertilization and early development of bovine in vitro matured (IVM) oocytes in a protein-free medium. The IVM oocytes were inseminated and cultured for 7 d in protein-free media prepared with 4 different types of water preparations: tap, deionized, twice-distilled, and purified water using the Milli-Q system (Milli-Q water). High frequencies (70 to 83%) of normal fertilization were obtained in media prepared with all types of water. However, the frequency of development to the blastocyst stage in media prepared with Milli-Q water (31 +/- 3%) was significantly higher than with the 3 other types of water (11 to 13%). Moreover, the effects of storage period of Milli-Q water on early development of bovine embryos was also examined. The frequency of development to the blastocyst stage in media prepared with Milli-Q water immediately after preparation (fresh Milli-Q water; 35 +/- 4%) was significantly higher than for Milli-Q water stored for 1 wk (18 +/- 4%) or 2 wk (18 +/- 3%). Effects of commercially available purified water on early development of bovine embryos were also examined. The frequency of development to the blastocyst stage in media prepared with Milli-Q water (33 +/- 5%) was significantly higher than for purified water purchased from 3 different suppliers (Brand A; 21 +/- 6%, Brand B; 21 +/- 2%, Brand C; 21 +/- 4%). Each water sample was analyzed by the measurement of electrical conductivity, organic compounds and/or inorganic ion and endotoxin concentrations to evaluate purity. Fresh Milli-Q water showed the lowest level of electrical conductivity and contained the lowest concentration of organic compounds. These results indicate that in vitro fertilization of bovine oocytes is not affected by the water quality in the preparation of medium; however, early development of bovine embryos is seriously affected by the purification method and the storage period of water used for medium preparation.

Influence of combined activation treatments on the success of bovine nuclear transfer using young or aged oocytes

This study was determined if the combined activation of young or aged oocytes influence their development. The 16-cell stage in vitro maturation/fertilization embryos were exposed to 10 microL nocodazole for 18-20 h, blastomeres that divided within 3 h after the release from nocodazole were used as synchronized donor blastomeres. Metaphase II oocytes were enucleated at 20-22 h post onset of maturation. Enucleated oocytes were divided into 2 groups: oocytes activated at 24 h (young) and oocytes activated at 38 h (aged). In both groups (young and aged), one group of oocytes was activated in 7% ethanol alone for 5 min (alone) and the other group (combination) was activated in ethanol and subsequently incubated in 5 micrograms/ml cycloheximide in TCM199 for 6 h (combination). Electrofusion was carried out at 30 h (young) and 44 h (aged). The nuclear morphology of the blastomere-oocyte complexes at 1 h post-fusion and their development to the blastocyst stage after 6 days of culture in modified synthetic oviduct fluid were examined. Interphase and swollen nuclei were observed at 1 h post-fusion following nuclear transfer to the cytoplasm from young oocytes of combined activation and aged oocytes of combined and ethanol alone activation. When young oocytes were treated with the combined activation method, the reconstituted embryos had a significantly higher developmental rate to the blastocyst stage than the aged oocyte groups (P < 0.05). We conclude that the combined activation of young oocytes leads to a more efficient development of bovine nuclear transfer embryos.

Factors affecting survival of mouse blastocysts vitrified by a mixture of ethylene glycol and dimethyl sulfoxide

The present study was conducted to determine suitable conditions for mouse blastocysts vitrified by a solution containing 25% v/v (4.5M) ethylene glycol and 25% v/v (3.4M) dimethyl sulfoxide (VSi). In Experiment 1, blastocysts were exposed to 50% diluted VSi (50% VSi) for 10 minutes then to VSi for 0.5 minutes at room temperature (22 approximately 24 degrees C) or at 4 degrees C, followed by vitrification. The survival rates of these embryos exposed at each temperature were not significantly different. In Experiment 2, embryos were exposed directly to VSi for various time periods at room temperature and diluted in mPBS with 0.5 M sucrose without vitrification. The viability of embryos decreased after more than a 3 minute exposure. When the embryos were exposed to VSi for 0.5, 1, 1.5 and 2 minutes followed by vitrification, the survival rates were 78, 80, 76 and 50%, respectively. In Experiment 3, embryos were vitrified after exposure to 50% VSi for various time periods and then to VSi for 0.5 minutes at room temperature. One minute exposure to 50% VSi resulted in the highest survival rate. In Experiment 4 and 5, the cooling rate (from approximately 70 to approximately 2500 degrees C/minute), sucrose concentration (0, 0.5 and 1 M) of dilution solution, and dilution time (1 or 5 minutes) did not affect the viability of the vitrified embryos. Following exposure to 50% VSi for 1 minute and to VSi for 0.5 minutes at room temperature, embryos were cooled 1) at approximately 2500 degrees C/minute and diluted in 0.5 M sucrose in mPBS after warming or 2) at approximately 200 degrees C/minute and diluted in mPBS. In vivo development rates after the transfer to recipients were 38 and 42%, respectively. These values were similar to that of fresh control embryos.

Effects of equilibration time, precooling and developmental stage on the survival of mouse embryos cryopreserved by vitrification

Factorial experiments were carried out to examine the effects of equilibration time, precooling and developmental stage on the postthaw in vitro survival of vitrified mouse embryos. Eight-cell embryos, compacted morulae, or blastocysts were cryopreserved using vitrification Solution 1 (VS1; 10% glycerol + 20% propylene glycol), and vitrification Solution 2 (VS2; 25% glycerol + 25% propylene glycol) in phosphate buffered saline + 10% calf serum. Each embryo stage group was first equilibrated in VS1 for 5, 10 or 20 min and then exposed to either a precooled ( approximately 4 degrees C) or nonprecooled ( approximately 20 degrees C) VS2 in a 0.25-ml straw before they were plunged directly into liquid nitrogen. Results of this study showed an interaction between precooling, equilibration time and developmental stage which affect significantly post-thaw embryo survival (P< 0.05). High survival rates were obtained after 10 min equilibration in VS1 irrespective of the embryo developmental stage. Precooling of the VS2 significantly improved the survival mainly of blastocysts. However, eight-cell and morula-stage embryos also showed high survival rates when they were exposed to precooled VS2 after 5 min equilibration in VS1. It was further observed that morulae usually exhibit high survival rates, and vitrification conditions are more critical for early and advanced stage embryo development.

Effect of encapsulation on development of mouse pronuclear stage embryos in vitro.

Pronuclear stage embryos with intact (ZI), slit (ZS) or completely removed (ZF) zona pellucida were encapsulated with an artificial zona pellucida (AZP) made of 1.5% sodium alginate. Embryos were cultured in KSOM medium with or without protein and their development in vitro to the blastocyst stage was recorded. AZP significantly (P < 0.05) improved the development of embryos to the blastocyst stage regardless of the presence of the natural zona pellucida. The encapsulated embryos developed at a higher rate (P < 0.05) in the absence of protein as compared with non-encapsulated embryos. Furthermore, the cell contacts at the 4-cell stage were significantly improved (P < 0.05) with encapsulation. AZP improved (P < 0.05) the development of pronuclear stage embryos with a slit zona pellucida to morula and blastocyst stages as compared with ZS embryos. It is concluded that AZP improves the in vitro development of pronuclear stage embryos with intact or completely removed zona pellucida as well as micromanipulated embryos to the blastocyst stage.

Quick freezing of mouse embryos using ethylene glycol with lactose or sucrose

Abstract Mouse compacted morulae were quickly frozen in liquid nitrogen (LN2) vapor at approximately −170°C after 5 min equilibration in 2 M or 3 M ethylene glycol or glycerol in the presence of either 0.25 M lactose or sucrose to examine their post-thaw viability. After thawing in a water bath at 37°C and a one-step dilution of the cryoprotectant, the embryos were cultured for 48 h. Significantly higher in vitro survival rates were obtained with 3 M ethylene glycol than with 2 M ethylene glycol or 2 or 3 M glycerol irrespective of the sugar used (P

In vitro maturation and fertilization of swamp buffalo (Bubalus bubalis) oocytes

Abstract Cumulus-oocyte complexes (COC) collected at slaughter from adult (n=4) and gonadotrophin-treated prepuberal (n=4) swamp buffaloes were matured in TCM199 with fetal calf serum (FCS), antibiotics and hormones (follicle stimulating hormone (FSH) and E2), at 39°C in 5% CO2 and 95% air for 24 h. Freshly collected buffalo semen was diluted with defined medium (DM) containing 5 mg ml−1 bovine calf serum (BSA), and washed by centrifugation. COC were co-incubated with sperm for 24 h in DM supplemented with heparin and theophylline. Oocytes were then examined for fertilization after fixation and staining with orcein. The numbers of COC harvested were 29 35 and 29 39 for adults and prepuberal ovaries, respectively. The maturation rates were 41% ( 12 29 ) and 52% ( 15 29 ), and fertilization rates were 21% ( 6 29 ) and 21% ( 6 29 ) for adult and prepuberal buffaloes, respectively. Two out of nine inseminated oocytes co-cultured with oviductal cells developed into morulae 6 days following in vitro insemination. It is concluded that swamp buffalo oocytes can be fertilized in vitro and the prepuberal buffalo could serve as a potential oocyte donor.

Production of bovine identical twins via transfer of demi-embryos without zonae pellucidae

Embryos were collected nonsurgically from nåturally-cycling or superovulated donors 7 d after estrus. Forty-four morulae, early blastocysts and blastocysts classified as good to excellent were bisected using a fine glass needle to produce forty-four identical demi-embryos. The bisected demi-embryos, without zonae pellucidae, were nonsurgically transferred, either by twin or single transfer. An additional forty-eight embryos were collected from the same donors and transferred as a control. Among the twin transfers, 8 of the 13 recipients became pregnant (61.5%). Seven of them conceived twin fetuses (87%) and one a single fetus. However, only two sets of normal identical twin calves were obtained. Among the single transfers, 72.6% (45/62) of bisected embryos without zonae pellucidae resulted in pregnancy, of which 48.4% (30/62) were identical twins, and 24.2% (15/62) were singletons. Another 27.4% (17/62) of the recipients did not became pregnant. The pregnancy rate for whole embryos with zonae pellucidae was 72.9% (35/48). These data show that there was no significant difference between the pregnancy rates of bisected embryos without zonae pellucidae and whole embryos with zonae pellucidae transferred 7 d after estrus. Bisection of bovine embryos was simplified and even morula stage embryos were transferred without zonae pellucidae.