In ruminants, interferon-τ (IFN-τ) is a major pregnancy factor, secreted by the embryonic trophoblast cells during the pre-implantation period, being important for the maternal-fetal recognition. The co-transfer of bovine trophoblastic vesicles (bTVs) derived from in vivo recovered conceptuses is known to promote the successful implantation of embryos with expected lower viability, such as in vitro handled embryos, through the effects of IFN-τ secreted by bTVs. We have also reported that the pregnancy rate was improved using this technique in early pregnancy phase (Hashiyada et al. 2005 J. Reprod. Dev. 51, 749-756). However, the IFN-τ secretion level from bTVs has not been well known. Therefore, the objective of the present study was to measure concentration of IFN-τ released from individually cultured bTVs in vitro. Furthermore, we also investigated the transition of IFN-τ level in continuous culture of bTVs. Blastocysts were produced by artificial insemination of Japanese black cows following superstimulatory treatment and were recovered on 16 or 18 days post-estrus. Sixty-eight bTVs were prepared from 23 elongating blastocysts, 3 to 20 mm in length, by dissection using a surgical blade. Each trophoblastic fragment, 1 to 1.5 mm in width, was cultured in a well of 96-well plates using TCM-199 supplemented with 20% (v/v) fetal bovine serum and 0.1 mM β-mercaptoethanol at 38.5°C in a humidified atmosphere of 5% CO2 in air. After 24 h of culture, fragments of unformed vesicles were re-cultured for an additional 24 h; 10 bTVs from this group were continuously cultured until Day 24 (the day of insemination was defined as Day 0). The volume of culture medium was 100 μL/well/day until Day 2 and thereafter changed to 200 μL/well/2 days to terminate. The viability of bTVs was assessed based on maintained spherical shape of vesicle, morphologically. Exchange and collection of culture media, morphological observation of bTVs were performed on Days 1, 2, 4, 8, 10, 12, 14, 16, 18, 20, 22, and 24. Culture fluids were stored at -30°C. IFN-τ was measured by RIA (Takahashi H et al. 2005 Theriogenology 63, 1050-1060). Data were analyzed by Student’s τ-test Initial IFN-τ secretion did not differ between groups that had formed and unformed vesicles on Day 1, 89.8 ± 7.1 (mean ± SEM, n = 41) and 76.6 ± 7.2 ng mL-1 (n = 27), respectively. On Day 2, in the unformed group, all of the fragments had made vesicles and the IFN-τ increased to 99.4 ± 11.8 ng mL-1. In the extended culture group (n = 10), IFN-τ secretion tended to increase from Day 2 (66.9 ± 14.2 ng mL-1) to Day 8 (166.0 ± 46.7 ng mL-1) (P = 0.06). However, this large amount of IFN-τ on Day 8 significantly decreased from Day 10 (32 ± 4.9 ng mL-1, P < 0.05) to Day 24 (9.2 ± 1.0 ng mL-1, P < 0.05) gradually. The survival rate of these bTVs decreased to 90% (9/10) on Day 10 and then to 60% (6/10) during Days 18 to 22. These results indicate that bTVs cultured for a long term in vitro might decrease IFN-τ secretion.
Improving In Vitro Culture of Human Male Fetal Germ Cells
