Establishment and characterization of immortalized bovine endometrial epithelial cells

Abstract

Bovine primary uterine endometrial epithelial cells (EECs) are not ideal for long-term studies, because primary EECs lose hormone responsiveness quickly, and/or they tend to have a short life span. The aims of this study were to establish immortalized bovine EECs and to characterize these cells following long-term cultures. Immortalized bovine EECs were established by transfecting retroviral vectors encoding human papillomavirus (HPV) E6 and E7, and human telomerase reverse transcriptase (hTERT) genes. Established bovine immortalized EECs (imEECs) showed the same morphology as primary EECs, and could be grown without any apparent changes for over 60 passages. In addition, imEECs have maintained the features as EECs, exhibiting oxytocin (OT) and interferon tau (IFNT) responsiveness. Therefore, these imEECs, even after numbers of passages, could be used as an in vitro model to investigate cellular and molecular mechanisms, by which the uterine epithelium responds to IFNT stimulation, the event required for the maternal recognition of pregnancy in the bovine species.

Keywords: bovine; endometrial epithelial cell; immortalization.

Figure 1

The morphology and characterization of immortalized bovine uterine endometrial epithelial cells (EECs). (A) Immortalized bovine uterine EECs were obtained by transfection of retroviral vectors, containing HPV-16 E6, E7 and hTERT genes to extend their life span. The representative photographs of EECs are shown (left: primary, right: immortalized). Bar = 200 μm. (B) The expression of HPV-16 E6, E7 and hTERT mRNAs in transfected EECs. Transcripts were examined by RT-PCR in primary and immortalized EECs. ACTB was used as an internal control. (C) The expression of steroid and interferon A (IFNA) receptor gene transcripts in primary and immortalized EECs. RNAs, extracted from primary or immortalized EECs at 60^th passage, were subjected to PCR analysis. ACTB was used as an internal control.

Figure 2

Proliferation of primary and immortalized endometrial epithelial cells (EECs). Primary (Pri) and immortalized at 60th passage (Im, broken line) EECs were cultured on collagen type IA-coated (+, triangle) or non-coated (−, circle) six-well plates for up to 48 h. The values of CCK-8 (MTT absorbance at 450 nm) were measured at 450 nm at 0, 24, or 48 h (n = 3). Duplicate samples were examined for each treatment, and three independent experiments were performed. Values represent means ± SEM. Statistical significance: **P –; 0.01 vs. Pri (−) within a time period.

Figure 3

Effect of OT on PGF2α accumulation and the expression of interferon (IFN) stimulated genes following recombinant ovine IFNT treatment in immortalized endometrial epithelial cells (imEECs). (A) The primary (white bar) and immortalized at 60^th passage (black bar) EECs were cultured on 48-well plates coated with collagen type IA. The cells were treated with increasing concentrations of OT (0, 1, 10, 100 or 1000 ng/mL) and PGF2α accumulation in conditioned media after 24 h incubation were measured using an enzyme immunoassay. The concentrations were corrected for cell numbers as determined by Cell Counting Kit-8 (CCK-8) (see Materials and Methods). The mean concentration of prostaglandin F2α (PGF2α) was derived from an average of three wells within a single plate experiment. Values represent means ± SEM. (B) The expression of IFN-stimulated gene transcripts in primary and immortalized EECs. The primary (Pri) and immortalized (Im) EECs were seeded on six-well plates coated with collagen type IA. The cells were treated with IFNT (1 μg/mL) for 24 h and the expression of IFN stimulated gene transcripts was analyzed by RT-PCR. IFNT stimulated genes include interferon-inducible Mx proteins (Mx1 and Mx2), interferon regulatory factors (IRF1, 2 and 9) and signal transducers and activator of transcriptions (STAT1 and 2). ACTB was used as an internal control.

Figure 4

Immunofluorescence characterization of the immortalized endometrial epithelial cell (imEECs). Immunofluorescence staining analysis on primary (left panels) or immortalized at 60th passage (right panels) EECs was carried out using antibodies against cytokeratin (upper panels) as epithelial marker, or vimentin (lower panels) as stromal marker. Bar = 200 μm.