Constitutive Expression of TERT Enhances β-Klotho Expression and Improves Age-Related Deterioration in Early Bovine Embryos

Abstract

Age-associated decline in oocyte quality is one of the dominant factors of low fertility. Aging alters several key processes, such as telomere lengthening, cell senescence, and cellular longevity of granulosa cells surrounding oocyte. To investigate the age-dependent molecular changes, we examined the expression, localization, and correlation of telomerase reverse transcriptase (TERT) and β-Klotho (KLB) in bovine granulosa cells, oocytes, and early embryos during the aging process. Herein, cumulus-oocyte complexes (COCs) obtained from aged cows (>120 months) via ovum pick-up (OPU) showed reduced expression of β-Klotho and its co-receptor fibroblast growth factor receptor 1 (FGFR1). TERT plasmid injection into pronuclear zygotes not only markedly enhanced day-8 blastocysts' development competence (39.1 ± 0.8%) compared to the control (31.1 ± 0.5%) and D-galactose (17.9 ± 1.0%) treatment groups but also enhanced KLB and FGFR1 expression. In addition, plasmid-injected zygotes displayed a considerable enhancement in blastocyst quality and implantation potential. Cycloastragenol (CAG), an extract of saponins, stimulates telomerase enzymes and enhances KLB expression and alleviates age-related deterioration in cultured primary bovine granulosa cells. In conclusion, telomerase activation or constitutive expression will increase KLB expression and activate the FGFR1/β-Klotho pathway in bovine granulosa cells and early embryos, inhibiting age-related malfunctioning.

Keywords: FGFR1; TERT; bovine embryo; cycloastragenol; β-Klotho.

Figure 1

β-Klotho and FGFR1 proteins in bovine oocyte and early embryos. (A) The immunofluorescence based FGFR1 and β-Klotho proteins from Pronuclear (PN) stage to day-8 bovine blastocyst 10 per each sample), MII oocyte (20 per each sample), 2-cell embryo (20 per each sample), the 3.5-day embryo (10 per each sample) and day-8 blastocyst stages (5 per each sample and Bar = 100 µm). (B) Immunofluorescence expressions of FGFR1 and β-Klotho in bovine cumulus-oocyte complexes obtained from 5 years, 8 years, and 12 years old Hanwoo (Korea native) cows. The experiments were repeated 3 times, and the data are shown here as a mean ± S.E.M. N.S, not significant. Significance: * = p < 0.05, and ** = p < 0.01 and Bar = 100 µm.

Figure 2

Aging reduces the expression of β-Klotho, and telomerase in cultured cumulus cells. (A) Immunofluorescence images of FOXL2 and SF-1 in bovine cultured granulosa cells treated with control, D-galactose, and D-galactose + cycloastragenol (the experiment was repeated three times independently and Bar = 100 µm). (B) Immunofluorescence expressions of β-Klotho and FGFR1 show significant (p > 0.05) reduction with D-galactose treatment (Bar = 100 µm). (C) Western blot expression of Caspase-3 AND NF-Kb in cultured bovine granulosa cells treated with control, D-galactose, and D-galactose + cycloastragenol. The experiment was repeated three times and the data are shown here as a mean ± S.E.M. N.S, not significant, * = p < 0.05, and ** = p < 0.01.

Figure 3

TERT plasmid injection enhances KLB protein expression and inhibits ROS level. (A) Immunofluorescent expression of KLB in bovine day-8 blastocysts treated with control, D-galactose, and TERT plasmid. (B) Reactive oxygen species (ROS) level was measured via H2DCFA staining in bovine day-8 blastocysts. The result showed a significant reduction in ROS level with hTERT plasmid injection. The data shown here as a mean ± S.E.M. N.S, not significant, * = p < 0.05, and Bar = 100 µm.

Figure 4

Enhanced telomerase expression amplifies mitochondrial genes and improves mitochondrial potential. (A) JC-1 staining in day-8 blastocysts from the control, D-galactose, and hTERT-treated groups (20 per each group) and Bar = 100 µm. (B) TUNEL assay was performed to detect apoptotic cells. The D-galactose group had significantly more TUNEL positive cells than the control and TERT plasmid injected groups (n = 20 per group) and Bar = 100 µm. (C) The relative mRNA expressions of NRF-1 and TFA in bovine day-8 blastocysts. The expression of both the genes were significantly reduced in D-gal group as compared to control and Plasmid groups. The data shown here as a mean ± S.E.M. N.S, not significant, * = p < 0.05, and ** = p < 0.01.

Figure 5

Constitutive expression of TERT enhances embryo implantation potential and inner cell mass (ICM)-related genes. (A) The effects of D-galactose and TERT on blastocyst implantation potential were determined via invasion assay. Bright-field image showing the area of invasion and DAPI for migrant cells in day-8 blastocysts. Image J software (National Institute of Health, Bethesda, MD, USA) was used to quantify the signal intensity of immunofluorescent images. The invaded area and cellular migration were significantly lower in the D-galactose-treated group than the control and TERT-treated groups and Bar = 100 µm.(B) CDX-2 gene related to embryo implantation and invasion of maternal uterine tissue was analyzed via RT-qPCR. (C) Inner Cell Mass (ICM) related genes like OCT-4 and SOX-2 were analyzed to check the embryo quality. The results showed that both the genes were significantly reduced with D-galactose treatment compared to control TERT plasmid injection. The experiments were repeated 3 times, and the data are shown here as the mean ± S.E.M. * = p < 0.05, and ** = p < 0.01.