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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.
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Summary. Bovine immature oocytes cultured for various times in TC-199 medium were inseminated with frozen–thawed spermatozoa in Medium BO with caffeine (5 mm) and heparin (10 μg/ml). Very high penetration rates (95–100%) were obtained in all oocytes which had been cultured for 0–20 h. When oocytes cultured for 0 and 4 h were inseminated, 100% of them were penetrated and had a decondensing sperm head and most of the oocytes remained at the stage of condensed germinal vesicle (GV) to telophase-I 20–22 h after insemination. The formation of male and female pronuclei was first observed in oocytes inseminated 8 h after culture. The proportions of polyspermy and average number of spermatozoa in penetrated oocytes gradually decreased as oocyte maturation proceeded. Penetration of at least one spermatozoon with a decondensing head into oocytes at the GV stage (without culture) was almost completed up to 8 h after insemination and at that time most of the penetrated oocytes were still at the stage of GV or condensed GV. These results indicate that maturation of bovine oocytes is not required for sperm penetration into the vitellus or for sperm nuclear decondensation under the in-vitro conditions used.
Keywords: in-vitro fertilization; immature oocytes; maturation; bovine; sperm nuclear decondensation
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Summary. Pig oocytes matured in culture were inseminated with frozen–thawed ejaculated spermatozoa without preincubation in modified tissue culture medium (TCM) 199. High penetration rates (85–89%) and increased incidence of polyspermy were obtained at 25–100 × 106 spermatozoa/ml. Wide variation in penetration rates (16–89%) was observed in oocytes inseminated in medium containing 5mm caffeine and at 25–50 × 106 spermatozoa/ml obtained from 6 boars, regardless of sperm motility. At 25–50 × 106 spermatozoa/ml, penetration rates of oocytes were dependent upon the concentration of caffeine in the medium: there was no penetration without caffeine, but penetration was highest (89%) with 5mm caffeine. None of the oocytes was penetrated in the medium supplemented with heparin at 5–40 μg/ml. When heparin was included in the medium with 5mm caffeine, it inhibited the efficacy of caffeine to promote sperm penetration of oocytes.
Keywords: in-vitro fertilization; oocyte; pig; frozen ejaculated sperm
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Bovine oocytes at the germinal vesicle stage were inseminated in Brackett and Oliphant's medium in the presence of BSA (10 mg ml−1), caffeine (5 mmol l−1) and heparin (10 μg ml−1). When oocytes were transferred into tissue culture medium (TCM)-199 containing 10% fetal calf serum (FCS) with or without 6-dimethylaminopurine (6-DMAP; 2 mmol l−1) 8 h after insemination and cultured for 15–40 h at 39°C in 5% CO2 in air, 74–83% of oocytes were penetrated and polyspermy (67–80%) was common. At 40 h after culture in 6-DMAP-free medium, 65% and 63% of unpenetrated and penetrated oocytes, respectively, reached metaphase II or beyond. A few (6%) oocytes were activated and contained both male and female pronuclei. Sperm metaphase chromosomes were observed in 90% of the penetrated oocytes. Penetration by more than four spermatozoa greatly retarded the meiotic maturation of the oocyte. However, sperm chromosomes were never observed in oocytes cultured in 6-DMAP supplemented medium and oocyte maturation did not proceed beyond the stage of prometaphase I. These results demonstrate the possible participation of maturation-promoting factor in metaphase chromosome formation in spermatozoa.
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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.
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The abundance of mRNA encoding the PGF2α receptor in bovine corpora lutea at different phases of the oestrous cycle and pregnancy was examined in relation to the number of [3H]PGF2α binding sites. Corpora lutea were removed from cyclic (early: 3–5 days after ovulation; mid-cycle: 8–12 days after ovulation; late: 15–18 days after ovulation; and regressed: 20–21 days after ovulation) and pregnant (early: fetal size 9–13 cm (2–3 months); mid-cycle: fetal size 42–43 cm (5–6 months); and late: fetal size 78–80 cm (8 months)) cows and subjected to total RNA preparation, in situ hybridization and membrane preparation for [3H]PGF2α binding assay. Northern blot analysis demonstrated that expression of PGF2α receptor mRNA progressively increased from the early phase to the late phase of the oestrous cycle, and was markedly reduced at the regressed phase; while constant amounts of mRNA were observed in early and middle pregnant corpora lutea, and there was a significant reduction at late pregnancy. Specific high affinity [3H]PGF2α binding sites with K d values of 18.3–31.1 nmol−1 were observed in the luteal membrane during the oestrous cycle and pregnancy; this is consistent with the expression of PGF2α receptor mRNA. The number of receptors decreased at the regressed phase and in early pregnancy. These results strongly suggest that PGF2α is involved in not only luteolysis but also luteal functions in both pregnant and nonpregnant cows.
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Cytokines are thought to regulate prostaglandin (PG) secretion in the bovine endometrium. However, there is no consensus about the role of interleukin-1α (IL1A) on PG secretion. The objective of this study was to examine the influence of IL1A on basal and interferon-τ (IFNT)-regulated PG in vitro secretion, as well its effects on PG secretion, progesterone (P4) output, and corpus luteum (CL) in vivo lifespan. Explants of bovine endometrium (days 16–17 of the estrous cycle or early pregnancy) were stimulated with IL1A (10 ng/ml), IFNT (30 ng/ml), or IL1A combined with IFN. IL1A alone strongly stimulated luteotrophic PGE2 secretion by endometrial tissues of both pregnant and nonpregnant cows. IL1A also stimulated luteolytic PGF2α output in the late luteal phase. IFNT augmented the stimulatory effect of IL1A on PGE2 secretion. In an in vivo experiment, saline or IL1A at different doses (0.001–10 μg/per animal) was applied to the uterine lumen on day 16 of the cycle. Only the highest dose of IL1A caused a temporal increase in PGF2α secretion, while it had no effect on P4 secretion or CL lifespan. Application of 0.1 and 1 μg IL1A stimulated P4 and PGE2 output and prolonged the CL lifespan. Although IL1A may stimulate in vitro luteolytic PGF2α secretion during the estrous cycle, it only acts as a luteotrophic factor in vivo. IL1A increased luteotrophic PGE2 and P4 output, inhibiting spontaneous luteolysis. These luteotrophic effects may result in appropriate luteal development and function in cows during the estrous cycle and early pregnancy.
