64 USING PORCINE GRANULOSA CELLS AS FEEDERS FOR PORCINE AND BOVINE TROPHECTODERM CELL CULTURE

Abstract

The trophectoderm cells, arising from the outer side of the blastomere in the blastocyst stage, are the first differentiated embryonic cells with specific potential as stem cells. The physiology of trophectoderm cells has been studied; however, their functions still remain unclear, because the lack of definitive information of cell lineages. Here, we aimed to establish in culture different feeder-dependent trophectoderm cell lines from 9-day, preimplantation, in vitro-produced porcine and bovine embryos. We used 2 different feeders: porcine granulosa cells (PGC) and mouse embryonic fibroblasts (MEF). Both cells were mitotically inactivated by mitomycin-C and then cultured with a density of 5 × 10⁴ mL^–1 on 0.1% (wt/vol) gelatin coated 4-well dishes in DMEM-199 medium supplemented with 10% (vol/vol) fetal bovine serum (FBS), nonessential amino acids (NEAA), β-mercaptoethanol and nucleosides (Talbot et al. 2000 Biol. Reprod. 62, 235–247). Trophectoderm cells were observed by light microscopy and characterised by reverse transcription-PCR using specific primers for both species. Different feeders and trophectoderm cells growth rates were compared after trypsinization using a hemocytometer. Data were analysed using 1-way ANOVA. In results, trophectoderm cells display epithelial characteristics, cuboidal morphology and express mRNA of homebox protein CDX2, cytokeratin 8 (KRT8) and interferon (IFN) gamma or tau for porcine or bovine cells, respectively. Moreover, oestrogen receptor (ESR1) and progesterone receptor (PGR) were expressed in trophectoderm cells of both species. Porcine granulosa cells were highly proliferative with doubling time of 24 h when compared to MEF (P ≤ 0.5), easy to recover and provided a reasonable source of steroids, 17β-oestradiol (E2; 31.21 ± 3.1 ng mL^–1) and progesterone (P4; 6.36 ± 0.4 ng mL^–1). Moreover, trophectoderm cell colonies of both species that cultured on PGC grew faster, with a doubling time of 48 h when compared to those cultured on MEF (P ≤ 0.5). We speculate that the continuous supplement of steroids and other cytokines during the co-culture of trophoblasts with granulosa cells might help the trophectoderm cells growth more than that of MEF. Further investigations are required in this regard. In conclusion, porcine granulosa cells can be good alternative feeders to culture porcine and bovine trophectoderm.

This research was supported by MKE (Grant # 10033839-2011-13) and IPET.