FBS Group Research Articles

189 EFFECT OF PROGESTERONE AND EPIDERMAL GROWTH FACTOR ON IN VITRO-PRODUCED EIGHT-CELL BOVINE EMBRYOS IN A SERUM-FREE CULTURE MEDIUM

The role of progesterone (P4) and epidermal growth factor (EGF) in early bovine embryo development is still not clear. P4 has been administered at different times of embryo development, and a direct effect on IVF-derived bovine 8-cell embryos has been noted even if there was an interference due to the P4 vehicle (Ferguson et al. 2005 Reprod. Fertil. Dev. 17, 219 abst). EGF has been added to the culture medium from the presumptive zygote stage at different concentrations, resulting in improved blastocyst rates compared to that in control medium (Mtango et al. 2003 Theriogenology 59, 1393–1402; Sirisathien et al. 2003 Anim. Reprod. Sci. 77, 21–32), and gave results similar to those with 5% or 10% FCS (Palasz et al. 2000 Anim. Reprod. Sci. 58, 229–240). The objective if this experiment was to determine the effect of P4 and EGF on development of in vitro-produced bovine embryos when administered alone or in combination at the 8-cell stage in the absence of serum. In vitro-produced bovine 8-cell embryos were randomly allotted to treatments: (1) control, SOFaaBSA medium (BSA, 16 mg mL−1; n = 198); (2) P4, SOFaaBSA + P4 (15 ng mL−1 in ethanol; n = 198); (3) EGF, SOFaaBSA + EGF (25 ng mL−1; n = 200); (4) P4 + EGF, SOFaaBSA + P4 (15 ng mL−1 in ethanol) + EGF (25 ng mL−1; n = 201); and (5) FBS, SOFaaBSA + FBS (5%; n = 197). In order to minimize the toxic effect of ethanol, it was allowed to evaporate from the culture dish and then medium was added. All in vitro procedures were carried out at 38.5°C in a humidified atmosphere of 5% CO2 in air; presumptive zygotes were cultured in SOFaaBSA until 8-cell stage. Embryo development was evaluated on Day 6 and on Day 8 after IVF (Day 0), and rates calculated from 8-cell embryos. The study was done in 4 replicates and chi-square test was used for statistical analysis (Statistica for Windows; Stat Soft Inc., Tulsa, OK, USA); significance was assessed at P < 0.05. Results are reported in Table 1. No differences were found in the number of morulae between P4 and control, between P4 + EGF and FBS, and between P4 + EGF and EGF (P > 0.05), whereas the combination P4 + EGF was better than P4 alone (P < 0.05). Blastocyst rate was not different (P > 0.05) among EGF, P4 + EGF, and FBS groups. P4 achieved an higher (P < 0.05) blastocyst rate than control but it was lower (P < 0.05) than that of P4 + EGF or FBS. In conclusion, P4 alone improves embryo development from the 8-cell embryo to the blastocyst stage in a serum-free culture system, and EGF alone achieves a blastocyst rate not significantly different from that of FBS; furthermore, the combination of P4 and EGF can be considered the most suitable as an alternative to FBS because similar results were obtained in terms of both morulae and blastocysts. Table 1.Eight-cell bovine embryo development in SOFaaBSA medium in presence of P4, EGF, P4+EGF, or FBS

Hematopoietic Differentiation of Embryoid Bodies Derived from the Human Embryonic Stem Cell Line SNUhES3 in Co-culture with Human Bone Marrow Stromal Cells

Human embryonic stem (ES) cells can be induced to differentiate into hematopoietic precursor cells via two methods: the formation of embryoid bodies (EBs) and co-culture with mouse bone marrow (BM) stromal cells. In this study, the above two methods have been combined by co-culture of human ES-cell-derived EBs with human BM stromal cells. The efficacy of this method was compared with that using EB formation alone. The undifferentiated human ES cell line SNUhES3 was allowed to form EBs for two days, then EBs were induced to differentiate in the presence of a different serum concentration (EB and EB/high FBS group), or co-cultured with human BM stromal cells (EB/BM co-culture group). Flow cytometry and hematopoietic colony-forming assays were used to assess hematopoietic differentiation in the three groups. While no significant increase of CD34+/CD45- or CD34+/CD38- cells was noted in the three groups on days 3 and 5, the percentage of CD34+/CD45- cells and CD34+/CD38- cells was significantly higher in the EB/BM co-culture group than in the EB and EB/high FBS groups on day 10. The number of colony-forming cells (CFCs) was increased in the EB/BM co-culture group on days 7 and 10, implying a possible role for human BM stromal cells in supporting hematopoietic differentiation from human ES cell-derived EBs. These results demonstrate that co-culture of human ES-cell-derived EBs with human BM stromal cells might lead to more efficient hematopoietic differentiation from human ES cells cultured alone. Further study is warranted to evaluate the underlying mechanism.

302 PROTEIN SUPPLEMENTATION TO IVM MEDIUM IN RELATION TO THE INCIDENCE OF APOPTOSIS IN BOVINE OOCYTES MATURED IN VITRO

Mammalian embryos derived from in vitro fertilization display lower developmental competence and quality when compared to their in vivo counterparts. The composition of culture media significantly contributes to this phenomenon. Media supplemented with FBS or the serum derivate BSA are described as biochemically undefined. Those macromolecules were shown to exert a wide range of effects on cultured embryos, dependent on batch-to-batch variability. Therefore, replacement of these protein sources with a synthetic macromolecule such as polyvinyl pyrrolidone (PVP) or polyvinyl alcohol (PVA) provides a possibility to use a chemically defined culture medium (Ali et al. 2002 Biol. Reprod. 66, 901–905). Apoptosis as programmed cell death naturally occurs in mammalian oocytes and embryos; however, its incidence is significantly higher in vitro. The aim of this study was to investigate whether protein supplementation (FBS, fatty acid-free (faf)-BSA, PVP40) of IVM medium affects the incidence of apoptotic oocytes. In the present study, the IVM system previously described by Makarevich et al. (2002 Biol. Reprod. 66, 386–392) was used. Briefly, follicular oocytes aspirated from slaughterhouse ovaries were matured in vitro in one of three maturation media supplemented with FBS (10%), faf-BSA (6 mg mL−1) or PVP40 (4 mg mL−1). The terminal TUNEL assay kit was used to detect the DNA fragmentation in apoptotic cells (DeadEndTM Fluorometric TUNEL system, Promega, Madison, WI, USA). The data were analyzed by chi-square test of independence. Altogether, 630 oocytes collected during 12 IVM experiments were subjected to the Tunel test, and 563 (89.4%) of them were successfully investigated: 426 after maturation in vitro and 137 follicular, non-matured. The remaining 67 cells were lost during manipulation. The rate of Tunel-positive cells differed (P < 0.001) between matured (11.8%) and follicular oocytes (1.5%). Protein supplementation of IVM media did not significantly affect the rate of apoptotic oocyte occurrence, which was 9% in the faf-BSA group, 11.5% in the FBS group, and 15% in the PVP group. No differences were observed in the rate of Tunel-positive cells between oocytes at MII and MI stages. In conclusion, protein supplementation of IVM medium used in the present study did not affect the incidence of apoptotic oocytes after maturation in vitro. This research was supported by the State Committee for Scientific Research as a Solicited Project PBZ-KBN-084 from 2003 to 2005 year.

286 THE EFFECT OF IN VITRO MATURATION MEDIUM ON CRYOSURVIVAL, CELL NUMBERS AND APOPTOTIC INDEXES OF BOVINE EMBRYOS

The success of in vitro maturation (IVM) has a significant impact on the oocytes ability to develop to blastocyst stage. The quality of the produced blastocysts can be evaluated using staining techniques. The aims of this study were (a) to compare the effect of different IVM media on embryo production rates, and (b) to utilize differential (DF) and TUNEL staining to evaluate the quality and cryosurvival of the produced blastocysts. Abattoir-derived oocytes were randomly divided into the IVM groups: (1) M199 IVM (n = 2305): TCM-199 with glutamax-I (GIBCO, Paisley, UK), 0.25 mM Na-pyruvate, 100 IU mL−1 penicillin, and 100 μg mL−1 streptomycin; (2) FBS IVM (n = 2484): M199 IVM medium with hormones (10 μg mL−1 LH, 2 μg mL−1 FSH, and 100 μg mL−1 β-estradiol), and 10% FBS (GIBCO, New Zealand); and (3) FAFBSA IVM (n = 2411): as group (2), but FBS was replaced with 4 mg mL−1 fatty acid free albumin. Fertilized oocytes were denuded and cultured in modified SOFaaci + 4 mg mL−1 FAFBSA in 5% O2 (Holm P et al. 1999 Theriogenology 52, 683–700). Fresh, Grade I Day 7 blastocysts were stained with TUNEL (n = 114) or with DF (n = 149). In addition, 184 Grade I Day 7 blastocysts were frozen in AG Freeze (AB Technology, Pullman, WA, USA), thawed, and cultured for 24 h. The re-expanded embryos were stained with TUNEL (n = 96) or with DF (n = 88). Modified TUNEL protocol (Upstate, Lake Placid, NY, USA; Makarevich A and Markkula M 2002 Biol. Reprod. 66, 386–392) and DF staining protocol (Thouas G.A. et al. 2001 Reprod Biomed Online 3, 25–29) were used. The results of embryo cleavage and D7 embryo development are based on logistic regression models, and the results of proportion of ICM and apoptotic index on general linear mixed models. After FBS IVM, 83.6% of the fertilized oocytes were estimated to cleave and 25.5% to develop to the blastocyst stage by Day 7. The estimations for embryo cleavage and Day 7 development rates were significantly lower in FAFBSA IVM and M199 IVM groups (P < 0.0001): 74.0% and 15.0% for the FAFBSA, and 76.1% and 8.8% for the M199, respectively. The re-expansion rates (%) after thawing were 86.5, 90.6, and 73.3 for the FBS, FAFBSA, and M199 IVM groups, respectively. Freezing reduced the ICM proportions and elevated the apoptotic indexes (P < 0.001). The rate of ICM reduction after freezing was not influenced by the IVM medium. There was a significant interaction between the apoptotic index and the IVM group (P = 0.04). The increase of the apoptotic index was smallest in FAFBSA IVM and greatest in M199 IVM. The results indicate that exclusion of serum from IVM medium results in lower embryo cleavage and development rates. Freezing reduced ICM and increased apoptotic index of Day 7 embryos in every IVM group studied. FAFBSA IVM seemed to produce embryos of better quality as evidenced by the smallest increase in the apoptotic index after freezing.

In vitro maturation of bitch oocytes from advanced preantral follicles in synthetic oviduct fluid medium: serum is not essential

The ability of oocytes from preantral follicles to mature in vitro was assessed using a synthetic oviduct fluid (SOF) medium. Advanced preantral follicles (approximately 210μm diameter) were isolated from the ovaries of domestic bitches and assigned to one of four treatment groups: (1) SOF (n=230); (2) SOF+3mg/ml bovine serum albumin (+BSA, n=220); (3) SOF+20% fetal bovine serum (+FBS, n=227); or (4) SOF+3mg/ml BSA+20% FBS (+BSA+FBS, n=232), then cultured for up to 72h. A group of control follicles was not cultured (n=103). The percentages of oocytes reaching metaphase I to metaphase II stages (MI to MII) did not differ between treatments at each culture period. Within treatments, the percentages of oocytes at MI to MII stages did not differ with duration of culture. However, when compared to the control group (0.97%) the percentages of oocytes at MI to MII increased (P<0.05) in the SOF group after 48h (10.0%) and 72h (12.2%) of culture. In the +BSA (10.1%) and +FBS (9.7%) groups, the percentages of oocytes at MI to MII increased (P<0.05) above control values only after 72h of culture. The percentage of oocytes at MI to MII did not significantly increase in the +BSA+FBS group (3.9, 6.6 and 7.6% at 24, 48 and 72h of culture, respectively) compared to the control group. These results indicate that under the described conditions supplementation of culture medium with BSA or FBS is not essential, and the simple medium SOF can support nuclear maturation of a small proportion of bitch oocytes in vitro.

Immunocytochemical expression and localization of protein kinase C in bovine aortic endothelial cells

Total PKC activity in BAEC incubated for 24 hrs in either 10% serum (FBS) or serum-deprived media (SDM) was similar. However, most of the activity (69%) in the FBS group was detected in the particulate fraction, while it was mainly in the cystosolic fraction (66%) in the SDM group. By confocal microscopy, there was diffuse cytoplasmic localization of the antibodies to the α and β PKC isoforms. γ PKC was not detected. Treatment of FBS or SDM cells with a phorbol ester resulted in an increase in PKC activity with translocation to the particulate fraction. PKC α immunofluorescence redistributed to the perinuclear region whereas PKC β staining remained mostly cytosolic. Calphostin C, a PKC inhibitor, prevented the phorbol ester-induced increase in PKC activity and translocation.