Rat one-cell embryos recovered from naturally mated females were cultured in modified hamster embryo culture medium 1 without amino acids. In the presence of 0.4 mmol phosphate l−1 (NaH2PO4), no embryos developed beyond the two-cell stage, regardless of the presence of 5.0 mmol glucose l−1. This inhibition was dose dependent at very low concentrations of phosphate in the medium supplemented with 7.5 mmol glucose l−1 and osmolarity adjusted to 244 mosmol; development to the blastocyst stage was not inhibited at 0.001–0.01 μmol phosphate l−1, but development to the morula and four-cell stages was markedly inhibited at 0.1 and 1.0 μmol phosphate l−1. In the medium without phosphate, glucose did not inhibit or promote development to the morula stage, but adequate concentrations of glucose were necessary for the development of morulae to the blastocyst stage; the percentage of one-cell embryos that developed to the blastocyst stage at 7.5 mmol glucose l−1 (67%) and 10.0 mmol glucose l−1 (60%) were not statistically different from the percentage at 5.0 mmol glucose l−1 (46%), but was significantly greater than the percentage at 2.5 mmol l−1 (33%). When osmolarity of the medium with 5.0 mmol glucose l−1 was varied by adjusting the amount of NaCl added, more (82–98%) of the one-cell embryos developed to the four-cell stage at 212–276 mosmol, but development was greatly inhibited at 304 mosmol. Development to the blastocyst stage was largely dependent on osmolarities; at 244 mosmol, 61% of embryos developed to the blastocyst stage, although this percentage was not significantly different from the percentage (43%) at 264 mosmol.
K. Miyoshi, H. Funahashi, K. Okuda and K. Niwa
K. Miyoshi, L. R. Abeydeera, K. Okuda and K. Niwa
Rat one-cell embryos, recovered from naturally mated females, were cultured in a chemically defined medium (R1ECM) under different experimental conditions. When the osmolarity of the medium with a reduced concentration (63.8 mmol l−1) of NaCl was varied by adding different amounts of d-sorbitol, more (79–91%) of the one-cell embryos developed to the four-cell stage at 212–278 mosmol than at 306 mosmol (13%). The greatest proportions of morulae (74%) and blastocysts (60%) were obtained at 246 mosmol. When the medium was supplemented with amino acids in various combinations and the osmolarity adjusted to about 246 mosmol, more (80–98%) of the embryos developed to the morula stage. More blastocysts were obtained in medium supplemented with glutamine (Gln: 80%), minimal essential medium (MEM) essential amino acids (EAA) (90%), Gln + EAA (83%), EAA + MEM nonessential amino acids (NEAA) (83%) or EAA + Gln + NEAA (90%) than in medium without amino acids (59%). Few (3–10%) hatching or hatched blastocysts were observed 120 h after the start of culture in the medium with EAA plus Gin or NEAA. The mean number of cells in blastocysts developed in the medium with EAA + Gin + NEAA was 46.7 ± 7.2. When a total of 82 morulae or early blastocysts that had developed in culture were transferred to eight pseudopregnant rats on day 4, six recipients into which 62 embryos were transferred maintained their pregnancies beyond day 23, although no deliveries had occurred by day 25 or 26. When the rats were killed, 42 (68%) implantation sites and eight (13%) full-term fetuses with no gross abnormality were observed in the uterine horns.
K. Niwa, K. Ōhara, Y. Hosoi and A. Iritani
Summary. Newly ovulated eggs from mature queens treated with PMSG and hCG were inseminated in modified KRB solution with spermatozoa recovered from the cauda epididymidis of male cats. When 5 eggs were examined 15 min after insemination, no signs of sperm penetration into the vitellus were observed. However, in an egg examined before fixation 20 min after insemination, a spermatozoon whose head had passed through the zona pellucida was observed. Very high proportions (90– 100%) of the eggs were penetrated when they were examined 0·5–5 h after insemination. Male and female pronuclei were first observed in eggs examined 4 h after insemination.
K. Niwa, H. Imai, C. I. Kim and A. Iritani
Summary. Hamster and mouse eggs with follicular cells were penetrated by epididymal spermatozoa in a modified Krebs—Ringer—bicarbonate solution known to be suitable for fertilization of rat eggs in vitro. The highest proportion (84–88%) of hamster eggs penetrated was observed when 6–30 eggs with follicular cells were introduced into 10 μl sperm suspension. Greater volumes of sperm suspension reduced the proportions of eggs fertilized, while increased numbers of eggs in the same volume gave greater penetration rates. No fertilization was observed when 21– 30 denuded hamster eggs were introduced into 10–100 μl sperm suspension, indicating that capacitation and/or acrosome reaction of hamster epididymal spermatozoa was induced only by the post-ovulatory oviduct contents in this medium. However, when small numbers (10–20) of mouse eggs with or without follicular cells were incubated in a large volume (400 μl) of sperm suspension, 84–96% and 91% were penetrated, respectively, suggesting a lack of importance of the products of ovulation on mouse sperm capacitation. The incidence of polyspermy was very low for the hamster (0–13%) and mouse eggs (0–10%).
K. Niwa, M. Miyake, A. Iritani and Y. Nishikawa
Department of Animal Science, College of Agriculture, Kyoto University, Kyoto, Japan 606
In a previous study, it was shown that nuclear maturation from the germinal vesicle stage to division of the first polar body in rat oocytes could be completed in a defined medium, but that the proportion of oocytes penetrated and cleaved was low (Niwa & Chang, 1975), perhaps because of some fault in the maturation process. The present study was to determine the number of oocytes penetrated and cleaved in vitro when they were cultured at various stages during maturation in vivo.
White rats of the Wistar strain were used. Immature females (21-25 days old) were induced to superovulate by a s.c. injection of 10 i.u. PMSG (Serotropin: Teikoku-Zoki Co.) 48 hr before an i.p. injection of 10 i.u. HCG (Puberogen: Sankyo Co.). Oocytes were recovered at various times after the HCG injection according to the procedures described
A. Iritani, M. Kasai, K. Niwa and H. B. Song
Summary. Cattle follicular oocytes were collected from the ovarian follicles and cultured for 28 h in m-KRB solution in a CO2 incubator (5% CO2 in air at 37°C, 95% humidity). After further culture for 15–18 h with spermatozoa, 78·6% of the oocytes had matured to the second metaphase. The highest fertilization rates (36–58%) were obtained when spermatozoa were collected 1 day before insemination, kept for 14–18 h at 20°C in a test tube (10 × 108/ml), preincubated in m-KRB solution (1–1·2 × 108/ml) in a CO2 incubator for 0–12 h, and then inseminated at a concentration of 1·5–2·0 × 106/ml. The optimum period of preincubation in the CO2 incubator was 8 h: 6/15 denuded oocytes (40%) were fertilized with spermatozoa preincubated for 8 h. No polyspermic fertilization was observed in any of the 139 penetrated oocytes. When spermatozoa were preincubated in the uteri of oestrous cows and gilts for 4–4.5 h instead of the m-KRB solution, the fertilization rates were 80 and 86%, respectively. However, in this system, 13 and 24% of the penetrated oocytes were polyspermic. These results indicate that ejaculated bull spermatozoa can be capacitated in a chemically defined isotonic medium, and about half of the oocytes matured in culture are normally fertilized in vitro.
W. H. Wang, L. R. Abeydeera, L. R. Fraser and K. Niwa
Cumulus-enclosed pig oocytes were matured in vitro, freed from cumulus cells, and inseminated with frozen–thawed ejaculated spermatozoa in a chemically defined protein-free medium containing 37.0 mmol NaHCO3 l−1 and 5 mmol caffeine l−1. When the medium was supplemented with 1 mg polyvinylalcohol (PVA) ml−1, more penetrated oocytes were observed 14 h after insemination with 7–12 × 106 cells ml−1 than with 4–5 × 106 cells ml−1 and the incidence of polyspermy reflected the sperm concentration used. Varying the NaHCO3 concentration but maintaining the sperm concentration at 8 × 106 cells ml−1 resulted in significantly more oocytes being penetrated in media containing 45.83–50.25 than 37.0–41.42 mmol NaHCO3 l−1; there were no significant differences in the incidence of either male pronuclear formation or polyspermy. In medium containing 45.83 mmol NaHCO3 l−1, the inclusion of PVA at 0–5 mg ml− had no effect on proportions of penetrated oocytes, male pronuclear formation or polyspermy. However, when spermatozoa from three different boars were evaluated, the penetration and male pronuclear formation rates were highly variable, unlike the incidence of polyspermy. Penetration of cumulus-free oocytes was first detected at 6 h. When spermatozoa were incubated for 6 h in the absence of oocytes, motility, but not vitality, decreased whether or not PVA was included in the medium. Chlortetracycline (CTC) fluorescence analysis of the capacitation state indicated a rapid decline in the proportion of live uncapacitated, acrosome-intact cells and a rapid rise in the proportion of live capacitated, acrosome-reacted cells during the first hour. Smaller changes in the distribution of CTC patterns occurred during the later stages, suggesting that the rapidly responding cells were non-fertilizing, owing to damage by freeze–thawing, and that the fertilizing spermatozoa were drawn from the remaining pool of cells which underwent capacitation more slowly. This is the first report indicating that capacitation of frozen–thawed ejaculated boar spermatozoa and penetration of oocytes matured in culture are possible in a chemically defined, protein-free medium.
T. Nishimoto, I. Yamada, K. Niwa, T. Mori, T. Nishimura and A. Iritani
Summary. Human oocytes were obtained randomly from the ovaries excised from 28 patients at different stages of the menstrual cycle. When 114 oocytes were cultured for 40–40·5 h in a chemically defined medium, 80 showed degeneration. When 16 of the remaining 64 oocytes were examined for their maturation, germinal vesicle breakdown (GVBD) had occurred in 11 oocytes. Another 48 oocytes were inseminated by ejaculated spermatozoa which were washed twice and preincubated for 3 h. When examined 10 h later, 19 (39·6%) were penetrated with an enlarged sperm head or male pronucleus(ei) and its corresponding sperm tail(s).