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M. Kuwayama, S. Hamano, and T. Nagai

Summary. Two experiments were conducted to assess the viability of bovine blastocysts obtained by in vitro fertilization of oocytes matured in vitro (IVM–IVF) and cryopreserved by vitrification. In Expt 1, the optimal concentrations of glycerol and 1,2-propanediol in the basic medium (modified TCM199) for cooling and warming without formation of ice crystals were determined by plunging the solution into liquid nitrogen and then warming it in a water bath at 15°C; when both glycerol and 1,2-propanediol were present in the solution (>45% v/v), vitrification of the medium was observed. In Expt 2, IVM–IVF blastocysts were equilibrated to the mixture of glycerol and 1,2-propanediol (0% to 45%) at 15°C in a stepwise manner as follows: (i) in one step, for 18 min to the final vitrification solution; (ii) in two steps, for 8 min in the first step and 10 min in the second step; (iii) in four steps, for 4 min in the first three steps and 6 min in the last step; (iv) in eight steps, for 2 min in each step, but 4 min in the last step; and (v) in 16 steps, for 1 min in each step, but 3 min in the last step. After removal of cryoprotectants, the blastocysts were cultured for 24 h in vitro. The survival rates for the embryos equilibrated in 1, 2, 4, 8 and 16 step(s) were 56, 89, 100, 100 and 100%, respectively. The blastocysts equilibrated in 1, 2, 4, 8 and 16 steps were vitrified by plunging the straws containing them into liquid N2, thawed and cultured in vitro. Higher survival rates were obtained for blastocysts equilibrated in 4, 8 and 16 steps (79, 82 and 87%, respectively) than for those in one (0%) or two (10%) steps. Ten blastocysts that survived after vitrification were transferred to ten recipients and six of these became pregnant. These results indicate that vitrification can be used for cryopreservation of blastocysts obtained by in vitro culture of IVM–IVF bovine follicular oocytes.

Keywords: in vitro; vitrification; fertilization; embryo transfer; cow

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T. Nagai, K. Niwa, and A. Iritani

Summary. Pig follicular oocytes cultured in a defined medium for 28–29 h were inseminated in vitro by epididymal or ejaculated boar spermatozoa that were preincubated in a modified KRB solution at various sperm concentrations for 4 h at 37°C. Sperm concentration at insemination was 2 × 106 cells/ml. When epididymal spermatozoa were preincubated at concentrations of 4–16 x 108 cells/ml, 71–75% of oocytes were penetrated. In contrast, preincubation at a low concentration (0·8 × 108 cells/ml) resulted in a low penetration rate (11%). Epididymal spermatozoa preincubated at a concentration of 4 × 108 cells/ml could also penetrate denuded oocytes. None of the oocytes were penetrated by epididymal spermatozoa that were exposed to seminal plasma before preincubation or by ejaculated spermatozoa. After preincubation, whiplash motility was observed in the epididymal spermatozoa, but not in the ejaculated spermatozoa.

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Y. Hatanaka, T. Nagai, T. Tobita, and M. Nakano

Summary. Several hundred fertilized pig eggs were prepared by an in vitro fertilization (IVF) technique in which follicular phase ovarian eggs were matured in vitro to metaphase II before incubation with capacitated epididymal spermatozoa for 12 h at 39°C. Parthenogenetic eggs were also prepared by stimulation of the mature eggs with an electric pulse. The zonae were solubilized with 0·2% pronase/phosphate-buffered saline (PBS) or lactic acid/PBS. The time taken for solubilization was 30–40% shorter than for unfertilized eggs, indicating that zona hardening was induced during fertilization. At the same time, the sperm receptor activity of the zona was reduced. Electrophoretic analyses of zona glycoproteins from the ovarian, mature and fertilized eggs revealed that the amount of 90 kDa proteins decreased substantially during fertilization. This fraction could barely be detected in the zonae from parthenogenetic eggs. However, modification with a fluorescent probe showed that the general architecture of the zona remained unchanged during fertilization. These results suggest that the minor 90 kDa proteins are specifically degraded by the protease(s) released from the oocyte at fertilization, thereby leading to the block to polyspermy.

Keywords: zona pellucida; fertilization; pig; in vitro fertilization

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M Sakaguchi, T Dominko, N Yamauchi, ML Leibfried-Rutledge, T Nagai, and NL First

The mechanism for the accelerating effects of epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) on the meiotic cell cycle of bovine oocytes cultured in vitro was investigated. Cumulus-oocyte complexes (COCs) were obtained from small (< or = 3 mm in diameter), medium (4-6 mm in diameter) or large (7-10 mm in diameter) ovarian follicles and cultured with or without a combination of EGF and IGF-I (growth factors). Growth factors significantly increased the frequency of first polar body extrusion of oocytes derived from small follicles at 16 h of culture (PB16 oocytes; with growth factors: 75%; without growth factors: 55%), but did not increase the frequency in oocytes from medium or large follicles. COCs from small follicles were cultured with individual growth factors and sampled for kinase activity. The frequencies of polar body extrusion in EGF only (67%) and EGF + IGF-I (75%) treatment groups were significantly higher than those in the control (no growth factor) group (49%), but not significantly higher than in the IGF-I only group (63%). The H1 kinase activity at 6-8 h of culture in each group increased significantly from the baseline value at 0 h of culture, and the H1 kinase activities in the EGF only, IGF-I only and EGF + IGF-I treatment groups were significantly higher than those in the control group at 8 h of culture. MAP kinase activity was significantly higher than the baseline value and significantly higher than that in the control group at 6 h of culture in the EGF treatment group only. In conclusion, EGF and IGF-I act on COCs from small follicles to accelerate the meiotic cell cycle of the oocytes. This accelerating effect may be related to increased H1 and MAP kinase activities during the early stages of maturation.

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Y. Hirao, T. Nagai, M. Kubo, T. Miyano, M. Miyake, and S. Kato

Preantral follicles containing oocytes of 70–89.5 μm in diameter were isolated from pig ovaries and cultured in collagen gel for up to 16 days, in the presence of serum, FSH and oestradiol. Formation of follicular antra occurred as the culture proceeded. The oocytes had been enclosed by granulosa cells and contacts between the oocytes and processes of the enclosing cumulus cells were maintained over the culture period. After 16 days of culture, 30–40% of the oocytes were of normal appearance, and the diameters of about half of these oocytes were larger than 100 μm. When the oocytes grown in vitro were liberated from the follicles and cultured for a further 48 h in modified Krebs–Ringer bicarbonate solution, 6, 30 and 60% of the oocytes larger than 90, 100 and 110 μm underwent germinal vesicle breakdown, respectively. Progression to metaphase II was observed in 40% of oocytes that were over 110 μm in diameter, whereas no oocyte less than 90 μm in diameter resumed meiosis. The relationship between the size and meiotic competence of oocytes was similar for oocytes grown in vitro or in vivo. Oocytes grown and matured in vitro were penetrated by spermatozoa and formed a female pronucleus, but decondensation of the sperm head was incomplete. The results demonstrate for the first time that pig oocytes from preantral follicles can grow up to their final size, acquire meiotic competence, and be penetrated by spermatozoa in vitro.

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T. Nagai, T. Takahashi, H. Masuda, Y. Shioya, M. Kuwayama, M. Fukushima, S. Iwasaki, and A. Hanada

Summary. In Exp. 1 pig oocytes matured in vitro were used to evaluate the fertilizability in vitro of frozen epididymal (4 boars) and ejaculated (3 boars) spermatozoa that were preincubated in modified TCM-199 for 4 h at 37°C. The percentages of penetrated oocytes with the frozen epididymal spermatozoa were 0–40%. In contrast, none of the occytes were penetrated with the frozen ejaculated spermatozoa. In Exp. 2, oocytes matured in vivo were inseminated in vitro with the frozen epididymal spermatozoa that were known to penetrate oocytes matured in vitro. The penetration rate was 79% and the percentage of polyspermic oocytes was 57%. Culture for 30 h of oocytes matured in vivo and fertilized in vitro resulted in 51% (34/67) developing to the 2-cell stage. These embryos were transferred to 2 recipient gilts. One gilt became pregnant and a litter of 3 (1 live and 2 dead) was born. These results indicate that frozen epididymal spermatozoa can be used for in-vitro fertilization in the pig.

Keywords: in vitro; oocyte; fertilization; pig; embryo transfer

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K. Kikuchi, T. Nagai, J. Ding, N. Yamauchi, J. Noguchi, and Y. Izaike

A large population (62–90% of pig follicular oocytes can mature to metaphase II after culture for 48 h. However, a proportion (6–22%) remain in an immature stage at metaphase I (metaphase I-arrested). The main objective of this study was to determine whether the cytoplasm of metaphase I-arrested pig oocytes is capable of being activated by sperm penetration or parthenogenetic stimulation. After culture for 48 h, oocytes without a polar body (73% were shown to be at metaphase I after staining) and those with a polar body (94% were at metaphase II) were fertilized in vitro. A total of 69% and 62% of the oocytes were activated to form a female pronucleus, respectively, and the rate of polar body extrusion induced by fertilization in the activated oocytes was 90% (the first polar body) and 95% (the second polar body), respectively. When oocytes without and with a polar body were stimulated with an electric pulse, 53% and 81% of the oocytes were activated, respectively. The rate of polar body extrusion in the activated oocytes was 73% (the first polar body) and 79% (the second polar body), respectively. In contrast, young metaphase I oocytes cultured for 24 h had low (6%) or zero activation rate after in vitro fertilization or electric pulse stimulation. However, about one-third of the young metaphase I oocytes penetrated by spermatozoa after in vitro fertilization responded to electric pulse 12 h after insemination, and almost all (93%) were activated when they were stimulated 24 h after insemination. Patterns of polypeptide synthesis and histone H1 kinase activity were similar in metaphase I-arrested and metaphase II oocytes, and were characterized by increase in a 25 kDa polypeptide and by decrease in kinase activity. Although the first step of meiotic division is impaired, these results indicate that metaphase I-arrested oocytes are mature cytoplasmically.

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R. Procházka, E. Nagyová, Z. Rimkevičová, T. Nagai, K. Kikuchi, and J. Motlík

Summary. Oocyte–cumulus cell complexes (OCC) and complexes with an attached piece of membrana granulosa (C + P), isolated from prepubertal or cyclic gilts stimulated with pregnant mares' serum gonadotrophin, were cultured in media supplemented with follicle-stimulating hormone (FSH; 0·01–1·0 μg/ml) or forskolin (50–100 μmol/l) for 24 and 32 h. FSH and forskolin each induced dose-dependent cumulus and membrana granulosa expansion. After 2 h of culture, FSH (0·1 μg/ml) or forskolin (100 μmol/l) increased the contents of intracellular adenosine 3′,5′-phosphate (cAMP) in OCC from prepubertal gilts to almost 10 times that in unstimulated complexes. After 24 h of culture in media supplemented with FSH (0·1 μg/ml) or forskolin (100 μmol/l), the oocytectomized OCC and C + P showed similar expansion to that of the control groups. The intracellular cAMP contents in intact and oocytectomized OCCs were similar in all groups except those treated with FSH, in which the intact OCCs had significantly higher contents than their oocytectomized counterparts (P < 0·01). After hyaluronidase treatment, cumulus and membrana granulosa cells of intact and oocytectomized OCC and C + P were suspended, except for those of the innermost layers of the corona radiata. The results suggest that increases in cAMP contents and synthesis of an extracellular, hyaluronidase-sensitive mucus by pig OCC and C + P induced by FSH or forskolin are not dependent on the oocyte.

Keywords: FSH; cumulus expansion; hyaluronidase; oocytectomy; pig