*2.2. Ovine Trophoblast Cells Generated from Day 16 Trophectoderm Had a Significant Reduction in LIN28*

To further investigate the regulation of ovine trophoblast cell proliferation by LIN28 in vitro, we used day 16 TE to generate ovine trophoblast cells. The day 16 TE was minced and plated in

collagen-coated plates and was passaged to obtain a cell line. The cells used for further experiments were collected at passage 4–6, so we called these cells non-immortalized ovine trophoblast (OTR) cells. Interestingly, real-time PCR data showed that OTR cells had a significant reduction in *LIN28A* and *LIN28B* mRNAs compared to day 16 TE (Figure 5A). Densitometric analysis of Western blots showed that LIN28A and LIN28B proteins were also significantly reduced in OTR cells compared to day 16 TE (Figure 5B). Furthermore, real-time PCR data showed significant increase in *let-7* miRNAs (*let-7a, let-7b, let-7c, let-7d, let-7e, let-7f, let-7g, let-7i*) in OTR cells compared to day 16 TE (Figure 5C). The significantly reduced LIN28 and high *let-7* miRNAs in OTR cells after only 4–6 passages suggest that these cells differentiated to a different phenotype compared to trophoblast cells in day 16 TE.

**Figure 5.** LIN28A, LIN28B, and *let-7* miRNAs in non-immortalized ovine trophoblast (OTR) cells. (**A**) *LIN28A* and *LIN28B* mRNA in OTR cells compared to day 16 sheep TE. (**B**) Representative immunoblots for LIN28A, LIN28B, and β-actin, and densitometric analysis of immunoblotting results in OTR cells compared to day 16 TE. (**C**) *Let-7* miRNAs in OTR cells compared to day 16 TE (*n* = 3), \* *p* < 0.05 vs. TE.

The effect of low LIN28 and high *let-7* miRNAs on proliferation-associated genes in OTR cells was determined by measuring IGF2BP1, IGF2BP2, IGF2BP3, HMGA1, ARID3B, and c-MYC mRNAs and proteins. Real-time PCR showed that mRNA levels of *IGF2BP1, IGF2BP2, IGF2BP3, HMGA1, ARID3B,* and *c-MYC* were significantly reduced in OTR cells compared to day 16 TE (Figure 6). Densitometric analysis of Western blots revealed a significant reduction in protein levels of IGF2BP1, IGF2BP2, IGF2BP3, HMGA1, ARID3B, and c-MYC in OTR cells compared to day 16 TE (Figure 7). These results suggest that reduced LIN28 and high *let-7* miRNAs led to reduced expression of proliferation-associated genes in OTR cells.

**Figure 6.** *IGF2BP1, IGF2BP2, IGF2BP3, HMGA1, ARID3B,* and *c-MYC* mRNA in OTR cells compared to day 16 TE (*n* = 3), \* *p* < 0.05 vs. TE.

**Figure 7.** Representative immunoblots for IGF2BP1, IGF2BP2, IGF2BP3, HMGA1, ARID3B, c-MYC, and β-actin, and densitometric analysis of immunoblotting results in OTR cells compared to day 16 TE (*n* = 3), \* *p* < 0.05 vs. TE.

The OTR cells originated from day 16 TE undergo senescence after only a few passages. Therefore, the OTR cells were immortalized by overexpressing human telomerase reverse transcriptase (hTERT)

to keep them growing for further in vitro experiments. The newly generated immortalized cells were referred to as immortalized ovine trophoblast (iOTR) cells.
