Takeshi Satoh

Ultrastructure of bovine embryos developed from in vitro–matured and –fertilized oocytes: Comparative morphological evaluation of embryos cultured either in serum-free medium or in serum-supplemented medium

The ultrastructure of bovine embryos developed from in vitro‐matured and ‐fertilized oocytes, cocultured with bovine cumulus/granulosa cells either in a serum‐free medium (IVMD101) or in a serum‐containing medium (TCM199+CS) was compared. Embryos up to the eight‐cell stage had many cellular organelles and cytoplasmic components that were randomly distributed in the cytoplasm. Mitochondria were spherical or ovoid and had only a few peripheral cristae. There were no obvious differences in the ultrastructure between embryos developed in IVMD101 and TCM199+CS up to the eight‐cell stage. However, conspicuous differences in the ultrastructural features between the embryos cultured in IVMD101 and TCM199+CS were observed at the morula and blastocyst stages. At the morula stage, embryos cultured in IVMD101 had cells containing elongated mitochondria, well‐developed Golgi apparatus, lipid droplets, and large vesicles resembling lysosomes. The lysosome‐like vesicles were partially filled with electron‐dense materials and were frequently fused with lipid droplets. The blastomeres of morulae cultured in TCM199+CS contained numerous large lipid droplets and fewer lysosome‐like vesicles than those cultured in IVMD101. In blastocysts cultured in IVMD101, lysosome‐like vesicles were frequently observed in the trophoblast cells and lipid droplets were present in the cytoplasm of trophoblast and inner cell mass (ICM)‐cells, but they were not abundant. On the other hand, the blastocysts developed in TCM199+CS contained fewer lysosome‐like vesicles and large numbers of lipid droplets. This accumulation of lipid droplets was higher in the trophoblast cells than in the ICM‐cells. This study showed major differences in the ultrastructural features between the morulae and blastocysts from serum‐free and serum‐supplemented cultures, suggesting that the ultrastructural differences may reflect physiological characteristics of embryos. Mol. Reprod. Dev. 53:325–335, 1999.

A serum-free culture system for efficient in vitro production of bovine blastocysts with improved viability after freezing and thawing

The aim of this study was to evaluate whether two completely serum-free media (IVMD101 and IVD101) could improve the yield and quality of bovine blastocysts from in vitro matured and fertilized oocytes. The media were evaluated in the presence (IVMD101) or absence (IVD101) of bovine cumulus/granulosa cell (BCGC) cocultures. The proportion of embryos developing to the blastocyst stage in IVMD101 medium with BCGC cocultures (36.5%) and IVD101 medium without BCGC cocultures (37.1%) was significantly higher than in serum-supplemented medium (TCM199 + 5% calf serum) with BCGC cocultures (25.1%). Furthermore, the mean cell numbers per blastocyst on Day 7 developed in IVMD101 medium (179.5 cells) and IVD101 medium (177.1 cells) were greater than in the serum-supplemented medium (145.7 cells). The survival rates of blastocysts derived in IVMD101 medium (73.3%) and IVD101 medium (60.0%) based on hatching after 72 h of post-thaw culture were superior to that of blastocysts derived in the serum-supplemented medium (48.1%). Under microscopic observation, bovine blastocysts derived in the serum-supplemented medium showed abundant lipid droplets, largely into the trophectoderm cells. This morphological difference may partly explain the sensitivity of serum-derived embryos after freezing and thawing. In conclusion, these new serum-free culture media are useful, not only to study the mechanisms of early embryogenesis, but also for mass production of good quality embryos for embryo transfer, cloning and transgenesis.

Development of early bovine embryos to the blastocyst stage in serum-free conditioned medium from bovine granulosa cells

SummaryBovine granulosa cell — conditioned medium (BGC-CM) was prepared in a serum-free medium consisting of TCM 199, 5µg/ml insulin, and 0.5µg/ml aprotinin (TCM 199 IAP). Granulosa cells surrounded with embryos were denuded 24 to 30 h after in vitro fertilization. The proportion of denuded granulosa cell-free embryos that developed to the blastocyst stage in BGC-CM (43/219; 20%) as well as in the co-culture system (43/178; 24%) was significantly greater (P<0.001) than in fresh TCM 199 IAP medium (FM: 10/191; 5%), whereas the proportion of embryos that developed to the eight-cell stage was similar (P>0.05) in all three culture systems (95/178; 53% in co-culture, 111/219; 51% in BGC-CM, and 86/191; 45% in FM, respectively). Higher rates of hatching and hatched blastocysts 8.5 days after in vitro fertilization were observed in co-culture (13/44; 29.5%) and in conditioned medium (8/39; 20.5%). On the other hand, no hatching or hatched blastocysts were obtained in the fresh medium (0.7; 0%). Cell numbers per blastocyst in BGC-CM (178.3 cells/blastocyst) were approximately two-fold higher than those in FM (97.1 cells/blastocyst). However, higher cell numbers (249.3 cells/blastocyst) were observed in co-culture with BGC than in BGC-CM. The embryotrophic activity in BGC-CM was stable upon freezing and thawing, lyophilization, and heating at 56° C whereas activity was reduced by dilution in fresh medium, dialysis, pronase digestion, and heating at 80° C. These results suggest that BGC cultured in a serum-free medium can synthesize and secrete an embryotrophic factor(s) that supports blastocyst formation in vitro beyond the 8- to 16-cell stage.

Ultrastructural features of goat oviductal secretory cells at follicular and luteal phases of the oestrous cycle

The aim of the present study was to investigate the ultrastructure of secretory cells in the various regions of the goat oviduct during the follicular and luteal phases of the oestrous cycle. During the follicular phase in the fimbriae, the secretory cells contained small secretory granules with electron‐dense matrices. In the luteal phase, the secretory granules disappeared and cytoplasmic protrusions, extending beyond the luminal border of the ciliated cells and often containing the nucleus, were predominant. During the follicular phase in ampullary secretory cells, numerous secretory granules with moderately electron‐dense matrices were present in the supranuclear cytoplasm and exocytosis of secretory granules was observed. The number of secretory granules was dramatically reduced in the ampullary secretory cells at the luteal phase. Conspicuous cytoplasmic protrusions of secretory cells were observed similar to those of the fimbrial epithelium. Isthmic cells were almost free of secretory granules and lysosome‐like bodies were found both at the follicular and luteal phases. In conclusion, our ultrastructural observations of goat oviduct revealed marked cyclic changes in the ultrastructural features of secretory cells and the ultrastructural features and the numbers of secretory granules were distinctive for each particular segment.

Fibroblast growth factor stimulates the gene expression and production of tissue inhibitor of metalloproteinase-1 in bovine granulosa cells.

SummaryThe hormonal control of tissue inhibitor of metalloproteinase-1 (TIMP-1) gene expression and production by growth factors, gonadotrophins, and serum factors in cultured bovine granulosa cells (BGC) were investigated. Confluent cultures of BGC were exposed to various factors in a defined medium and levels of TIMP-1 in the conditioned medium were determined by enzyme immunoassay. Basic fibroblast growth factor (bFGF) and acidic fibroblast growth factor (aFGF) showed potent stimulation of cell proliferation and TIMP-1 production by BGC, while insulin stimulated growth but not TIMP-1 production. Basic FGF stimulated TIMP-1 production and BGC cell proliferation in a dose-dependent manner. A time course of TIMP-1 production showed substantially increased levels between 18 and 24 h in both control and bFGF-stimulated BGC cultures with bFGF-stimulated cultures having markedly higher TIMP-1 production at all time points. Consistent with the TIMP-1 production data, bFGF and aFGF increased the expression of TIMP-1 mRNA as determined by northern blot analysis, while insulin, inhibited TIMP-1 mRNA levels. These results indicate that FGF-induced TIMP-1 production by BGC may support bovine embryo development in vitro.

Low oxygen and glucose improves the development of fertilized bovine oocytes in defined medium without somatic cells

Dear Editor: Bovine embryos in culture often fail to develop beyond the eightcell stage in serum-free or serum-containing medium under standard culture conditions of 5% CO2 and 95% humidified air (reviewed by Wright and Bondioli, 1981). This commonly used culture system provides 20% O2 in the gas phase. The gaseous environment of cultured mammalian embryos is important in their developmental processes. When embryos are grown in defined medium without potential macromolecular scavengers such as serum or bovine serum albumin, embryos are more sensitive to suboptimal culture conditions (Ellis, 1990a,b). To overcome the developmental block associated with a high oxygen atmosphere, bovine embryos are cocultured with bovine oviduct epithelial cells (Eyestone and First, 1989; Ellington, et al. 1990a), bovine cumulus/granulosa cells (Fukui and Ono, 1989; Kobayashi et al. 1992), and other types of cells (Camous et al. 1984; Voelkle et al. 1985). Alternatively, it has been reported that significantly improved development of domestic animals in vitro can be achieved under 5-10% 02 low oxygen cultures (Tervit et al. 1972; Tervit and Rowson, 1974; Thompson et at. 1990). Recent studies have suggested that the presence of glucose in early preimplantation embryos up to the eight-cell stage is harmful to further embryo development of mouse (Chatot et at. 1989), hamster (Sheshagiri and Bavister, 1989), and cow (Ellington, et al. 1990b; Pinyopummintr and Bavister, 1991). On the other hand, embryos of mouse (Brinster, 1968), cow (Rieger et at. 1992), and sheep (Thompson et al. 1992) utilize glucose as a major energy source during preimplantation development, particularly from the eight-cell stage to the blastocyst stage. It is possible that the optimal requirement of glucose on animal embryo development might be modified by other medium components, the presence or absence of somatic cells, and/or oxygen concentration in culture. This study was designed to establish a simple culture system that can support the better development of bovine embryos matured and fertilized in vitro. We found that a combination of low oxygen atmosphere (5% 02) and low glucose medium (0.4 mg/ml) greatly improved bovine embryo development in vitro in a chemically defined medium without somatic cells. Collection of bovine oocytes and the maturation and fertilization of oocytes were carried out by the methods of Kobayashi et al (1992). Briefly, isolated cumulus-oocyte complexes were cultured in a maturation medium (TCM 199 containing 10% fetal bovine serum) in a humidified atmosphere of 5% CO2/95% air at 38.5 ° C. For in vitro fertilization, the cumulus-oocyte complexes were transferred into a Japanese Black bull spermatozoa suspension and incubated for 6 h in a humidified atmosphere of 5% CO2/95% air at 38.5 ° C. After in vitro fertilization, the effect of oxygen concentration and the presence or absence of bovine cumulus/granulosa cells (BGC) on the development of bovine in vitro matured (1VM) and in vitro fertilized (IVF) embryos in high glucose medium (1 mg/ml) was investigated (Table 1). Glucose-free TCM199 was a gift from Kyokuto Pharmaceutical Co., Tokyo, Japan. Inseminated oocytes were incubated for 24 h in a defined medium for embryo culture (TCM199 supplemented with 4.13 mM Na-lactate, 0.27 mM Na-pyruvate, 5 #g/ml insulin, and 10 ~g/ml gentamycin) under mineral oil. This original TCM199 contains 1 mg/ml (5.56 mM) glucose. Then, fertilized ooeytes were denuded by careful pipetting with capillary pipets to remove the BGC. The denuded oocytes were transferred with or without BGC and incubated under two different oxygen environments (20% 02 and 5% 02). The medium was replaced 3 days after IVF and embryos were euhured for up to 9 days. Cultured embryos were examined daily under a fight microscope. In a gas atmosphere of 5% CO2/95% air (20% O2), the proportion of embryos developing to the blastoeyst stage was