Fig 2: AT III attenuates LPS-induced mitochondrial dysfunction in IEC-6 cells via AMPK/SIRT1-mediated deacetylation of PGC-1α. (a) The levels of mtDNA were examined by qPCR. (b) The MMP changes were measured and quantified by the flow cytometry assay using JC-1 probes. (c, d) The complex I and complex IV activities were measured. (e, f) The protein expression levels of PGC-1α, NRF-1, NRF-2, and Tfam were detected and quantified by Western blot. (g, h) The protein expression levels of p-AMPK, AMPK, and SIRT1 were examined and quantified by Western blot. (i, j) The acetylated levels of PGC-1α and quantification results were determined. $p < 0.05, compared to control; &p < 0.05, compared to LPS.

Fig 3: Effect of recombinant IGF1 on bioenergetics markers, ATP production and mitochondrial polarization in FG follicles in vitro. Profiling mRNA (A) and protein (B) expression of nuclear coactivators (PGC-1β) and transcription factors (NRF-1, TFAM) at indicated dose levels of IGF1. FG follicles pooled from separate donor females (n ≥ 5) cultured in vitro either without (−) or with IGF1 (10 nM) (2 h) were subjected to ATP measurement (C) and determination of mitochondrial membrane potential through JC-1 staining (D, E). Representative photomicrographs and the relative fluorescence intensity of J-aggregates and JC-1 monomers from indicated treatment groups are shown. Fluorescence intensity is based on multiple (≥10) fields taking around 30 oocytes in each group (n = 3). Data are representative of at least three separate experiments with identical results and expressed relative to untreated group; (**) denotes p < 0.01. Scale bar ∼200 µm.

Fig 4: Gonadotropin-mediated regulation of igf1 expression vis-à-vis oocyte energy metabolism. Total RNA isolated from FG follicles incubated with hCG (10 IU) for 2 h in vitro was reverse transcribed, followed by qPCR analysis using NRF-1 and TFAM gene-specific primers (left panel, (A)) . Effect of hCG (10 IU) stimulation on ATP levels at 2 h of incubation in vitro (right panel, (A)). Profiling igf1 expression in hCG-treated (1 and 10 IU) follicles at indicated time points through qPCR (B). The impact of hCG stimulation on IGF1Rβ phosphorylation through immunoblot analysis at indicated time intervals (C). Effect of priming (1 h) with anti-IGF1Rβ on hCG-stimulated NRF-1 and TFAM expression (upper panel, (D)). Follicular ATP levels in IGF1Rβ or NRF-1 immunodepleted follicles compared to hCG (10 IU)-treated positive controls (lower panel, (D)). Besides, follicle-enclosed oocytes were primed (1 h) with anti- IGF1Rβ followed by hCG (10 IU) stimulation for 6 h, and % GVBD was determined microscopically (E). Data are representative of at least three separate experiments with identical results. Results are mean ± S.E.M. (n = 3). Data are expressed relative to untreated (A, B, E) and hCG-stimulated (D, E) follicles; (*) denotes p < 0.05 and (**) denotes p < 0.01. Scale bar ∼500 µm.

Fig 5: (A) Western blot for determining the protein levels of p66Shc, SIRT1, PGC-1α, NRF1, TFAM, MFN1, and DRP1 in each group. (B) Quantification of bands in Figure 9A. Mean ± SD. n = 3. ∗p < 0.05 vs. NG group; #p < 0.05 vs. HG group; △p < 0.05 vs. NG + over-p66Shc group.