The objective of the present experiment was to compare the effects of different maturation media on developmental capacity of in vitro fertilized bovine oocytes when co-cultured with bovine oviduct epithelial cells (BOEC) or cumulus cell monolayer (CCM). Oocytes aspirated from follicles of 2-5mm in diameter were divided into 4 groups and incubated for 22-24 h. Medium for control group was TCM 199 with 10% newborn calf serum (Control group). For the other groups, 30% follicular fluid (FF group), 10% estrus cow serum (ECS group) or 0.02 AU/ml of FSH and 1 μg/ml of estradiol-β (FSH/E2 group) were added to C group. Frozen-thawed semen treated with heparin and caffeine were used for ova insemination. Oocytes were placed on BOEC (n=572) or CCM co-culture (n=1,050) and cleavage rate and numbers of the stage of morula or blastocyst (morula/blastocyst stage) were recorded. The percentage of embryos cleaved were lower in ECS than in other groups (P<0.05)(27.5%, 27.9%, 18.2% and 23.9% for FF, FSH/E2, ECS and control group, respectively). Development to the morula/blastocyst stage was not different among groups (16.1 to 9.3%). Effect of co-culture with BOEC or CCM on cleavage and developmental capacity were not different (P>0.05). The proportion of morula/blastocyst from cleaved ova in FF and ECS groups were significantly higher than FSH/E2 and control groups (P<0.01).Fifteen blastocyst were transferred to 15 heifers and two recipients were confirmed pregant at day 30 and 90, and finally 2 male calves were born. The production of 2 calves from in vitro matured, fertilized and developed embryos was first success in Uruguay. These results indicate that certain biological fluids such as FF and ECS induce successful maturation of bovine follicular oocytes in vitro and that resulting embryos can be successfully cultured on either BOEC or CCM.

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Present experiments were carried out to evaluate positive effects of growth factors and conditioned medium (CM) from bovine granulosa cells (BGC) in a defined medium on bovine embryo development. In Experiment 1, insulin (19%; blastocyst / oocytes examined) and basic fibroblast growth factor (bFGF; 14%) individually stimulated embryonic development to blastocyst stage in a defined medium as effective as serum did (15%; p>0.05), but neither bovine lipoprotein (LP; 3%) nor bovine serum albumin (BSA; 1%) affected the stimulation of embryo development. In Experiment 2, denuded embryos did not traverse beyond 8- to 16-cell block without the co-culture with BGC, regardless of the addition of insulin and bFGF. In Experiment 3, serum-free, CM of BGC in the presence of insulin stimulated blastocyst formation (11%) as well as co-culture system (18%). These results suggest that insulin and bFGF stimulate early bovine embryonic development through the action of a certain embryotrophic factor produced by the activation of the physiological function of BGC.

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We evaluated the effect of two monolayers derived from reproductive cells on in vitro maturation and fertilization of porcine oocytes. Cumulus-oocyte complexes were cultured in medium alone or co-cultured for 48hr on the monolayer of porcine oviductal epithelial cells (pOEC) or granulosa cells (pGC). Assessment of nuclear maturation revealed that oocytes matured in pGC significantly reached metaphase of the second meiotic division compared to those in control (76% vs 55%; p<0.05) and did not differ from the pOEC (69%). Following 18hr of insemination, the proportion of penetrated oocytes in control (82%) was greater (p<0.01) compared with pGC (56%) but did not differ from the pOEC (68%). The proportion of oocytes that underwent polyspermy was lower (p<0.01) in pOEC (38%) and pGC (46%) compared to control (79%). More number of male pronuclei were formed in the oocytes matured on pOEC than pGC (37% vs 19%; p<0.05). There was no difference between pOEC and control (28%). In all groups, irrespective of immature or mature stage, some oocytes at germinal vesicle stage formed a male pronucleus within their cytoplasm. It was suggested that the monolayer of pGC or pOEC was effective for preventing oocytes from polyspermy and that the monolayers from pOEC were superior for promoting male pronuclear formation by increasing monospermy. That immature oocytes may also have an ability to form a male pronucleus in their cytoplasm was indicated.

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Bovine eggs matured in vitro and embryos from the 2-cell to expanded blastocyst stages developed in vitro were studied histochemically for the presence of Δ⁵-3β-hydroxysteroid dehydrogenase (HSD), 17β-HSD, 20α-HSD and 20β-HSD. The activities of Δ⁵-3β-HSD (dehydroepiandrosterone and pregnenolone as the substrates), 17β-HSD (estradiol-17β and testosterone), 20α-HSD (20α-hydroxyprogesterone) and 20β-HSD (20β-hydroxyprogesterone) were always found in the eggs and embryos 1-cell through blastocyst stages. The activity of Δ⁵-3β-HSD (17α-hydroxypregnenolone) was observed in the embryos at the stages of 8-cell to blastocyst, but not in the 1-cell eggs and the 2- to 4-cell embryos. From these results, it seems that progestin, such as progesterone, 20α-hydroxyprogesterone and 20β-hydroxyprogesterone, and androgen and estrogen are always getting synthesized in the cultured bovine eggs and embryos throughout the stages of 1-cell to blastocyst, and that 17α-hydroxyprogesterone, one of progestin, is synthesized in the embryos at the stages of 8-cell, morula and blastocyst.

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Naturally ovulated eggs from mature rats were inseminated with spermatozoa recovered from the cauda epididymidis of mature males in modified Krebs-Ringer bicarbonate solution. High proportions (77-100%) of eggs were penetrated with no statistical difference among different sperm concentrations (0.05-1.0 × 10⁶ spermatozoa/ml) and 25-61% of penetrated eggs showed polyspermy. When penetrated eggs were transferred into modified rat 1-cell embryo culture medium (mR1ECM) 10 h after insemination, 58% of them developed to the blastocyst stage 130 h after insemination. These results indicate that rat eggs collected from naturally ovulated females can be penetrated in vitro by epididymal spermatozoa and the eggs penetrated in vitro can develop to the blastocyst stage in a chemically defined medium.

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Glucose incorporation and utilization in mouse embryos increases during preimplantation development, which may depend at least in part on the hexokinase activity in the embryos. Microdetermination methods including NADP cycling were used to quantitatively examine the enzymatic activity of hexokinase. Hexokinase activity in 1-cell embryos was low (0.035 ± 0.010 pmol of NADPH formed/embryo/min), but progressively increased during preimplantation development. Although there is a significant delay, this increase is also observed when 2-cell embryos are developed in vitro. This increase in hexokinase activity was inhibited by the administration of actinomycin-D in the medium. A reverse transcription-polymerase chain reaction was used to study the expression of hexokinase mRNA in preimplantation mouse embryos. Messenger RNA was obtained from 100 of 2-cell embryos and blastocysts using the Micro-Fast Track mRNA isolation kit. Hexokinase mRNA is detectable after the 2-cell embryo stage. The levels of mRNA increased during embryonic development. These results suggest that hexokinase may be a key enzyme synthesized as the expression of zygotic genome in preimplantation embryos, and help in assessing the quality of embryos developed in vitro.

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The objective of this experiment was to examine the effect of co-culture with epithelial cells on development of porcine IVF embryos. Monolayers of epithelial cells for the co-culture were prepared from porcine oviducts or uteri. The developmental rates to the blastocysts stage were significantly higher (P<0.05) in the embryos co-cultured with epithelial cells from oviducts (16.9%) than in the embryos cultured without epithelial cells (control; 6.6%). However, the developmental rates to the blastocysts stage were not significantly higher (P<0.05) in the embryos co-cultured with epithelial cells from uteri (9.2%) than the control. The effects of ampulla, isthmus and utero-tubal junction on development of porcine IVF embryos were examined. The developmental rates to the blastocysts stage were significantly higher (P<0.05) in the embryos co-cultured with epithelial cells from ampulla (15.3%) and isthmus (15.3%) than in the embryos co-cultured with epithelial cells from utero-tubal junction (9.9%) and control (6.7%). These results suggest that the co-culture system with oviductal epithelial cells supports the development of porcine IVF embryos to blastocysts in vitro.

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Rat preimplantation embryos were subjected to analysis of programmed SDS-electrophoresis with silver staining using Phast system (Pharmacia Co.Ltd.). Embryos in early development with ovulated ova, one cells, 2 cells, 4 cells, 8 cells, morulas, and early blastocysts were aspirated into 1 μ l capillary tube and kept at -60 °C until used. The embryos of each developmental stage were collapsed by repeating a temperature difference with dry ice and warm water (50 °C) several times and SDS-solution (1.5%) including 2-mercapotoethanol were aspirated into the tube, kept overnight, and applied to the electro-phoresis. By examining a minimum number of embryos per a lane (a capillary) for Phast gel to apply silver staining , five embryos were known to be the minimum number for the visual recognition of protein patterns and the number was standerized for each stage later on. The pattern of protein bands of preimplantation embryos development were scanned and calculated for molecular weight, relative percentage and protein content with Image Master (Pharmacia Co.Ltd). Typical bands of 50-52KD and 15-16kD were recognized in ovulated ova,1-cells, 2-cells, 4-cells, 8-cells, and morulas but the relative percent of the 2 bands was remarkably decreased in early blastocysts. When the protein patterns of serum, uterine fluid and granulosa cells of rat origin were compared with those of embryos, the band 48-51KD and 15-16KD was found to be specific for embryos. In addition, albumin,which is one of the main constituents of protein in serum, uterine fluid, and granulosa cells, was found to be remarkably less in the rat embryos. When zona pellucida originated from 6-11 ova were subjected to Phast system, no band was visible. Therefore, the pattern of protein band was considered to exist in substantial region of embryos.

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To investigate protein synthesis associating with in vitro meiotic maturation in porcine oocytes, follicular oocytes were treated with cycloheximide (10 μg/ml) at 6 h intervals in the culture up to 48 h. Germinal vesicle breakdown(GVBD) but not chromatin condensation was blocked in the oocytes when they were treated at either 0, 6 or 12 h after culture. Also, the transition from metaphase-1(M1) to metaphase-2(M2) was prevented. Pronuclear formation was observed in the oocytes when treated at 24 to 30 h after culture. Using [35S]methionine, the profile of protein synthesis revealed that the proteins of 27, 47, 49 and 70kDa were detected only at 0 to 18 h and that new proteins of 25 and 63kDa were appeared from 24 to 48 h with presence of proteins of 39 and 92kDa throughout culture. These results suggest that in porcine oocytes GVBD and the transition from M1 to M2 require protein synthesis and that the remodelling of protein synthesis occurrs at 24 h after in vitro culture.

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