Acyl-Carnitines Exert Positive Effects on Mitochondrial Activity under Oxidative Stress in Mouse Oocytes: A Potential Mechanism Underlying Carnitine Efficacy on PCOS

Abstract

Carnitines play a key physiological role in oocyte metabolism and redox homeostasis. In clinical and animal studies, carnitine administration alleviated metabolic and reproductive dysfunction associated with polycystic ovarian syndrome (PCOS). Oxidative stress (OS) at systemic, intraovarian, and intrafollicular levels is one of the main factors involved in the pathogenesis of PCOS. We investigated the ability of different acyl-carnitines to act at the oocyte level by counteracting the effects of OS on carnitine shuttle system and mitochondrial activity in mouse oocytes. Germinal vesicle (GV) oocytes were exposed to hydrogen peroxide and propionyl-l-carnitine (PLC) alone or in association with l-carnitine (LC) and acetyl-l-carnitine (ALC) under different conditions. Expression of carnitine palmitoyltransferase-1 (Cpt1) was monitored by RT-PCR. In in vitro matured oocytes, metaphase II (MII) apparatus was assessed by immunofluorescence. Oocyte mitochondrial respiration was evaluated by Seahorse Cell Mito Stress Test. We found that Cpt1a and Cpt1c isoforms increased under prooxidant conditions. PLC alone significantly improved meiosis completion and oocyte quality with a synergistic effect when combined with LC + ALC. Acyl-carnitines prevented Cpt1c increased expression, modifications of oocyte respiration, and ATP production observed upon OS. Specific effects of PLC on spare respiratory capacity were observed. Therefore, carnitine supplementation modulated the intramitochondrial transfer of fatty acids with positive effects on mitochondrial activity under OS. This knowledge contributes to defining molecular mechanism underlying carnitine efficacy on PCOS.

Keywords: acetyl-l-carnitine (ALC); carnitine palmitoyltransferase-1 (CPT1); fatty acid beta-oxidation; l-carnitine (LC); mitochondria; oocyte; oxidative stress; oxygen consumption rate (OCR); polycystic ovarian syndrome (PCOS); propionyl-l-carnitine (PLC).

Figure 1

Representative images of MII plate in MII oocytes. Chromosomes were classified as normal (a), slightly aberrant (b), or aberrant (c). Spindle was classified as normal (d), slightly aberrant (e), or aberrant (f). Merged images of DNA and spindle staining are presented (g–i). MII oocytes were labeled by mouse anti α-tubulin primary antibody and secondary antibody conjugated with DyLight^® 594, and chromatin staining was performed by Hoechst 33342. Scale bars: 10 μm.

Figure 2

Changes in gene expression of Cpt1a in GV and MII oocytes stressed by H₂O₂. Values are means ± SEM of three determinations. The fold change of each gene is shown as 2^−ΔΔCt. Statistical comparison was performed by t-test. * p < 0.05 vs. control; n.d.: not detectable.

Figure 3

Changes in gene expression of Cpt1c in GV and MII oocytes stressed by H₂O₂. Values are means ± SEM of three determinations. The fold change of each gene is shown as 2^−ΔΔCt. Statistical comparison was performed by t-test. * p < 0.05 vs. control; n.d.: not detectable.

Figure 4

Changes in gene expression of Cpt1a in GV oocytes exposed to PLC or to LC–ALC–PLC for 2 h prior to stress by H₂O₂. Values are means ± SEM of three determinations. The fold change of each gene is shown as 2^−ΔΔCt. Statistical comparison was performed by t-test. * p < 0.05 vs. control.

Figure 5

Changes in gene expression of Cpt1c in GV oocytes exposed to PLC or to LC–ALC–PLC for 2 h prior to H₂O₂. Values are means ± SEM of three determinations. The fold change of each gene is shown as 2^−ΔΔCt. Statistical comparison was performed by t-test. * p < 0.05 with respect to the control; ^# p < 0.05 compared to H₂O₂.

Figure 6

Optimization of drug concentrations used for dissecting the components of oxygen consumption in mouse GV oocytes. Data show mean ± SEM. Statistical comparison was performed by t-test. * p < 0.05.

Figure 7

Comparison of bioenergetics profile of GV oocytes exposed to plain medium (CTRL), H₂O₂, or incubated with PLC or LC–ALC–PLC prior to H₂O₂ analyzed by extracellular flux analysis. Data show mean ± SEM. Statistical comparison was performed by t-test. * p < 0.05 vs. CTRL; # p < 0.5 vs. H₂O₂.

Figure 8

Analysis of ATP production of GV oocytes exposed to PLC or to LC–ALC–PLC for 2 h prior to H₂O₂. Data show mean ± SEM.