lncRNA H19 is involved in TGF-β1-induced epithelial to mesenchymal transition in bovine epithelial cells through PI3K/AKT Signaling Pathway

Increased levels of long noncoding RNA H19 (H19) have been observed in many inflammatory and organ fibrosis diseases including ulcerative colitis, osteoarthritis, liver fibrosis, renal fibrosis and pulmonary fibrosis. However, the role of H19 in bovine mastitis and mastitis-caused fibrosis is still unclear. In our study, H19 was characterized as a novel regulator of EMT induced by transforming growth factor-β1 (TGF-β1) in bovine mammary alveolar cell-T (MAC-T) cell line. We found that H19 was highly expressed in bovine mastitis tissues and inflammatory MAC-T cells induced by virulence factors of pathogens. TGF-β1 was also highly expressed in inflammatory MAC-T cells, and exogenous TGF-β1 could induce EMT, enhance extracellular matrix protein expression, and upregulate H19 expression in epithelial cells. Stable expression of H19 significantly promotes EMT progression and expression of ECM protein induced by TGF-β1 in MAC-T cells. Furthermore, by using a specific inhibitor of the PI3K/AKT pathway, we demonstrated that TGF-β1 upregulated H19 expression through PI3K/AKT pathway. All these observations imply that the lncRNA H19 modulated TGF-β1-induced epithelial to mesenchymal transition in bovine epithelial cells through PI3K/AKT signaling pathway, which suggests that mammary epithelial cells might be one source for myofibroblasts in vivo in the mammary glands under an inflammatory condition, thereby contributing to mammary gland fibrosis

Tauroursodeoxycholic acid (TUDCA) alleviates endoplasmic reticulum stress of nuclear donor cells under serum starvation.

Serum starvation is a routine protocol for synchronizing nuclear donor cells to G0/G1 phase during somatic cell nuclear transfer (SCNT). However, abrupt serum deprivation can cause serious stress to the cells cultured in vitro, which might result in endoplasmic reticulum (ER) stress, chromosome damage, and finally reduce the success rate of SCNT. In the present study, the effects of tauroursodeoxycholic acid (TUDCA), an effective ER stress-relieving drug, on the nuclear donor cells under serum deprivation condition as well as following SCNT procedures were first assessed in the bovine. The results showed that TUDCA significantly reduced ER stress and cell apoptosis in those nuclear donor cells. Moreover, it significantly decreased the expression of Hdac1 and Dnmt1, and increased the level of H3K9 acetylation in nuclear donor cells compared with control group. SCNT reconstructed embryos cloned from TUDCA-treated donor cells showed significantly higher fusion, cleavage, blastocyst formation rate, total cell number in day 7 blastocysts, and lower apoptotic index than that from control group. In addition, the expression of Hdac1, Dnmt1 and Bax was significantly lower in blastocysts derived from TUDCA-treated donor cells than that from control group. In conclusion, TUDCA significantly reduced the ER stress of nuclear donor cells under serum starvation condition, and significantly improved the developmental competence of following SCNT reconstructed embryos when these TUDCA-treated cells were used as the nuclear donors

Relative mRNA abundance of <i>Ire1</i>, <i>Bip</i>, <i>Chop</i>, <i>Bax</i>, <i>Bcl-2</i>, <i>Hdac1</i> and <i>Dnmt1</i> genes in SCNT reconstructed blastocysts derived from control or TUDCA-treated donor cells.

<p>a,b: different letters indicate significant difference (P<0.05).</p

Global level of H3K9 acetylation in control and TUDCA-treated donor cells.

<p>(A) H3K9 acetylation was stained as green, nuclei were counterstained with DAPI to visualize as blue. Scale bar: 20 μm. (B) Quantification of H3K9 acetylation/DNA signal intensities. a,b: different letters indicate significant difference (P<0.05).</p

The apoptotic index in SCNT reconstructed blastocysts derived from control or TUDCA-treated donor cells.

<p>Apoptotic nuclei were stained as green by TUNEL assay, DNA was counterstained with DAPI to visualize as blue. Scale bar: 20 μm.</p

Percentages of fibroblasts at different cell cycle phases under control and TUDCA treatment conditions.

<p>Percentages of fibroblasts at different cell cycle phases under control and TUDCA treatment conditions.</p

Apoptosis analysis of donor cells in control and TUDCA treatment groups.

<p>Apoptosis analysis of donor cells in control and TUDCA treatment groups.</p

Effects of embryo-derived exosomes on the development of bovine cloned embryos

<div><p>The developmental competence of <i>in vitro</i> cultured (IVC) embryos is markedly lower than that of their <i>in vivo</i> counterparts, suggesting the need for optimization of IVC protocols. Embryo culture medium is routinely replaced three days after initial culture in bovine, however, whether this protocol is superior to continuous nonrenewal culture method under current conditions remains unclear. Using bovine somatic cell nuclear transfer (SCNT) embryos as the model, our results showed that compared with routine renewal treatment, nonrenewal culture system significantly improved blastocyst formation, blastocyst quality (increased total cell number, decreased stress and apoptosis, enhanced <i>Oct-4</i> expression and ratio of ICM/TE), as well as following development to term. Existence and function of SCNT embryo-derived exosomes were then investigated to reveal the cause of impaired development induced by culture medium replacement. Exosomes were successfully isolated through differential centrifugation and identified by both electron microscopy and immunostaining against exosomal membrane marker CD9. Supplementation of extracted exosomes into freshly renewed medium significantly rescued not only blastocyst formation and quality (<i>in vitro</i> development), but also following growth to term (<i>in vivo</i> development). Notably, ratio of ICM/TE and calving rate were enhanced to a similar level as that in nonrenewal group. In conclusion, our results for the first time indicate that 1: bovine SCNT embryos can secrete exosomes into chemically defined culture medium during IVC; 2: secreted exosomes are essential for SCNT blastocyst formation, blastocyst quality, and following development to term; 3: removal of exosomes induced by culture medium replacement impairs SCNT embryo development, which can be avoided by nonrenewal culture procedure or markedly recovered by exosome supplementation.</p></div