Expression of Cu/Zn and Mn superoxide dismutases during bovine embryo development: influence of in vitro culture.

peer reviewedTemporal pattern of expression of Cu/Zn and Mn superoxide dismutases (SODs) was investigated in bovine oocytes and embryos produced in vitro in two different culture conditions and in vivo after superovulation. SODs were examined at a transcriptional level in single oocytes and embryos by reverse transcriptase-polymerase chain reaction (RT-PCR) and, at a protein level, by Western blotting on pools of embryos. mRNA encoding Cu/Zn SOD were detected in in vitro bovine embryos throughout preattachment development as well as in in vivo derived morulae and blastocysts. Transcripts for Mn SOD gene were detected in most immature and in vitro matured oocytes as well as in some zygotes and 5- to 8-cell embryos while no transcript was found at the 9-to 16-cell stage in both culture conditions. In vitro embryonic expression of Mn SOD was detected earlier in the presence of serum. Half of the morulae showed the transcript if cultured with 5% serum while none without serum. At the blastocyst stage Mn SOD could be detected independently of culture conditions. For in vivo-derived embryos Mn SOD transcripts were detected both in morulae and blastocysts. Immunoblotting analyses revealed that Cu/Zn SOD and Mn SOD were also present at a protein level in in vitro-derived zygotes and blastocysts. Together these data demonstrate, for the first time, that Mn SOD is transcribed and that Cu/Zn and Mn SOD proteins are expressed in preimplantation bovine embryos. Finally, they suggest that Mn SOD transcription is altered by in vitro culture conditions

In vitro production of cattle embryos: review and Belgian results.

This paper reviews the overall process of in vitro production and cryopreservation of bovine embryos in Belgium. Three laboratories are involved in this field, one at the University of Liège, one at the University of Ghent and ours at the University of Louvain-La-Neuve. Each one uses this technology as a tool to reach different goals. This paper refers mainly to the work done in Louvain-La-Neuve. Oocytes are obtained by punction of 2-4 mm follicles on slaughtered cow ovaries. They are matured in hormone-supplemented TCM199 containing 10% heat-treated fetal calf serum. In vitro fertilization by Percoll-selected spermatozoa is followed by in vitro culture in oviduct-conditioned medium for 7-9 days. Six calves have been born from in vitro produced blastocysts. Recently, full development was obtained in conditioned medium without protein supplementation. This finding will allow further investigations on oviduct/embryo molecular communication and research of oviduct-secreted embryotrophic proteins which were impaired in previous culture systems using serum-supplemented media. In vitro produced blastocysts were frozen-thawed and non-surgically transferred: 7/19 recipients remained pregnant beyond 2 months. Embryo loss was high between day 21 and 35 (31%)

Cinematographic analysis of bovine embryo development in serum-free oviduct-conditioned medium.

Development of bovine embryos produced in vitro from the one-cell to the blastocyst stage in serum-free oviduct-conditioned medium was investigated for 8 days consecutively by time-lapse cinematography. Three movies were analysed (130 embryos). The following observations were made. (1) Development under cine-recording conditions was similar to that in a classical incubator. (2) The highest proportion of embryos at the two-cell, three-four-cell, five-eight-cell, 9-16-cell, morula and blastocyst stages were recorded at 34, 46, 61, 115, 149 and 192 h after insemination, respectively. Cleavage asynchrony between blastomeres within individual embryos started at the two-cell stage. (3) The duration of the first three cell cycles was 35 h, 14 h and 11-62 h, respectively. (4) Detailed analysis of 13 embryos revealed that developmental arrest ('Lag-phase') occurred at the four-cell (1 of 13), five-cell (2 of 13), six-cell (3 of 13), seven-cell (3 of 13) or eight-cell stage (4 of 13); this phase lasted about 59 h. Embryos arrested at the eight-cell stage developed into morula-blastocysts (3 of 4) at a higher rate than did those arrested at earlier stages (2 of 9). (5) The faster the embryos cleaved into early stages (two-cell, three-four-cell and five-eight-cell), the higher the probability that they developed into morula-blastocyst: 70% of the embryos reaching the two-cell stage before 30-31 h after insemination developed into morula-blastocyst.(ABSTRACT TRUNCATED AT 250 WORDS

Invitro Production of Cattle Embryos - Review and Belgian Results

This paper reviews the overall process of in vitro production and cryopreservation of bovine embryos in Belgium. Three laboratories are involved in this field, one at the University of Liege, one at the University of Ghent and ours at the University of Louvain-La-Neuve. Each one uses this technology as a tool to reach different goals. This paper refers mainly to the work done in Louvain-La-Neuve. Oocytes are obtained by punction of 2-4 mm follicles on slaughtered cow ovaries. They are matured in hormone-supplemented TCM199 containing 10% heat-treated fetal calf serum. In vitro fertilization by Percoll-selected spermatozoa is followed by in vitro culture in oviduct-conditioned medium for 7-9 days. Six calves have been born from in vitro produced blastocysts. Recently, full development was obtained in conditioned medium without protein supplementation. This finding will allow further investigations on oviduct/embryo molecular communication and research of oviduct-secreted embryotrophic proteins which were impaired in previous culture systems using serum supplemented media. In vitro produced blastocysts were frozen-thawed and non-surgically transferred: 7/19 recipients remained pregnant beyond 2 months. Embryo loss was high between day 21 and 35 (31%)

Histologic and autoradiographic study of the in vitro effects of FGF-2 and FSH on isolated bovine preantral follicles: preliminary investigation.

Bovine early preantral follicles (40 to 65 microm diameter) were cultured for 24 or 48 h in the presence of 0, 10, 50 or 100 ng/ml of basic fibroblast growth factor (FGF-2), porcine FSH (pFSH) or both (ratio 1:1); the follicles were also exposed throughout the entire culture period to 2 microCi/ml ((3)H) thymidine. The effects of these factors on oocyte morphology and follicular DNA synthesis were then analyzed. Autoradiography was performed on histological serial sections of follicles after the culture period. Oocyte morphology of each follicle and the rate of follicular DNA synthesis were evaluated at the same time. Oocyte morphology was considerably altered in the presence of exogenous FSH. This effect seemed to be reduced by FGF-2, at least up to 24 h of culture. Analyzable incorporation of ((3)H) thymidine was only detected after 48 h of culture. The FGF-2 significantly increased the number of labeled nuclei per follicle whereas pFSH did not. This responsiveness of granulosa cells to FGF-2 disappeared in the presence of pFSH. No correlation was found between the number of labeled nuclei per follicle and the morphology of its oocyte. These results suggest that in cultured bovine early preantral follicles, pFSH induces oocyte degeneration and that this degeneration seems to be attenuated by FGF-2. In addition, FGF-2 lead to an increase in follicular DNA synthesis that disappeared in the presence of pFSH

The sex ratio of bovine embryos produced in vitro in serum-free oviduct cell-conditioned medium is not altered.

The sex ratio of bovine blastocysts produced in vitro in serum-free oviduct cell-conditioned medium was investigated. Bovine embryos reaching the blastocyst stage were removed from culture medium on Days 6, 7, 8 and 9 and were identified as small, mid-sized or expanded blastocysts. One third (29/91) of the blastocysts appeared on Day 6. Twelve from them were small blastocysts (5 males), 7 were mid-sized blastocysts (4 males) and 10 were expanded blastocysts (5 males). On Day 7, 33 blastocysts were obtained: 8 small (5 males), 9 mid-sized (3 males) and 16 expanded (13 males) blastocysts. Finally, on Days 8 and 9, 29 blastocysts were obtained: 12 small (9 males), 9 mid-sized (6 males) and 8 (3 males) expanded blastocysts. Sexing of the 91 blastocysts was performed by using an original polymerase chain reaction (PCR) protocol generating discreet internal control signals from both female and male samples and Y-specific smears from the male samples. Proportions of male embryos on Days 6, 7 and on Days 8+9 were 48, 64 and 62%, respectively. These values did not differ significantly among days and did not differ from 50%. Fifty-nine percent of small blastocysts, 52% of mid-sized blastocyst and 62% of expanded blastocysts were male. No difference between these values or with respect to 50% could be observed. These results show that bovine blastocysts produced in serum-free oviduct cell-conditioned medium do not have an altered sex ratio

Bovine embryos cultured in serum-poor oviduct-conditioned medium need cooperation to reach the blastocyst stage.

Immature bovine oocytes were matured and fertilized in vitro, and the resulting zygotes were cultured to the blastocyst stage in droplets of tissue culture medium 199 (TCM 199) conditioned by oviduct cells in the absence of serum. In Experiment 1, the effect of the number of zygotes in a constant culture volume was investigated by culturing 1, 4 or 40 zygotes in 40 mul of culture medium. The cleavage rate was low with a single embryo (36%) but increased with the number of embryos, to reach 50% with 4 embryos/40 mul and 59% with 40 embryos/40 mul. Blastocyst formation was nil with 1 embryo per 40 mul, reaching 2.5% with 4 embryos/40 mul and 18% with 40 embryos/40 mul. The effect of the size of the drop was assessed in Experiment 2, the concentration of embryos remaining constant (1 embryo/1 mul). The volumes tested were 10, 20, 30 and 40 mul. Development into blastocysts increased gradually from 12% in the 10 10 group to 20% in the 40 40 group. Experiment 3 was designed to find a minimal droplet volume able to support the development of a single embryo to the blastocyst stage. The minimum tested volume was 5 mul and was not successful. These results show that bovine embryos cultured in oviduct-conditioned TCM 199 need to cooperate to reach the blastocyst stage. The mechanism of this cooperation is not known, but some autocrine/paracrine factors, probably growth factors, could promote embryo development as was demonstrated in mice. From Experiment 2 we can hypothesize that the surface volume ratio of the droplets could play a role in the culture conditions by interfering with the exchanges between the culture medium and the surrounding environment

Characterization of in vitro growth of bovine preantral ovarian follicles: A preliminary study.

Preantral follicles were mechanically extracted from bovine ovaries collected at slaughter. On average 90 early growing follicles were collected per ovary. The follicles were cultured for 7 days in Dulbecco's modified Eagle medium F-12 nutrient mixture + 10% fetal calf serum + 10% newborn calf serum. The individual follicular diameter and the follicular DNA content were recorded at the start and the end of culture. The follicular DNA content was estimated by a microfluorometric method using the fluorochrome, 4'-6 diamidino-2-phenylindol-2HCl (DAPI). After culture, 86% of the follicles looked morphologically normal. Of the surviving follicles, 73% showed an increase in diameter varying between 5 and 30 mum, and the follicular DNA content increased from 1 to 72% under the same culture conditions. These results indicate that bovine preantral follicles can survive in vitro and even grow in a single medium

Establishment of an embryonic stem cell line from 8-cell stage mouse embryos.

The aim of this study was to try establishing mouse ES cell lines from the early developmental stage. Fifty-two uncompacted 8-cell stage embryos were dissociated and single blastomeres were seeded on primary embryonic fibroblasts in DMEM/F12 completed with 10% foetal calf serum, 10% new born calf serum. 10(-4) M beta-mercaptoethanol. After approximately 5 days of culture, multiple cell clones exhibiting stem cell morphology grew out and were dissociated. One cell line was established (MSB1) and characterised. The karyotype and the G-banding revealed a male diploid cell line. MSB1 cells were injected into syngenic mice and produced teratocarcinomas. Detailed histological examination of the tumours showed a great variety of cell types including representatives of all three primary germ layers. Several nests of undifferentiated stem cells were also present. Microinjections of MSB1 cells into 52 blastocysts produced 2 chimeras, 1 male and 1 female. These results demonstrate that a highly pluripotents ES cell line can be derived from 8-cell stage mouse embryos. However, the male chimera appeared sterile. More experiments would thus be necessary to prove that the cell line obtained is capable to colonise the germ line

Effects of dilution procedure and culture conditions after thawing on survival of frozen bovine blastocysts produced in vitro.

Blastocysts derived from bovine zygotes fertilized and matured in vitro and cultured for 7 days in conditioned medium were frozen in 1.36 mol glycerol l-1 and 0.25 mol sucrose l-1. In vitro survival after thawing was unaffected by dilution rate in 0.25 mol sucrose l-1. The proportion of blastocysts that re-expanded after 24 h was 81% (70 of 86) and 47% (33 of 70) hatched. Seven pregnancies beyond 2 months resulted from transfer of 21 blastocysts to 19 recipients. Total embryonic loss was 46.2%, of which 31% occurred between days 21 and 35. In vitro survival after thawing was influenced by culture conditions, the best being culture with oviduct epithelial cells, where 55-82% of blastocysts re-expanded, of which 41-54% hatched. Conditioned medium also supported re-expansion, but low hatching (6%), whereas M199 plus fetal calf serum allowed only limited re-expansion (19-40%). This behaviour was not a consequence of freezing. It is suggested that blastocysts produced in vitro have reduced metabolic activity leading to high embryonic loss before or just at the time of implantation and that oviduct cells create a favourable environment after thawing, allowing hatching in vitro