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.

(1) Background: Polycystic ovarian syndrome (PCOS) is a common and multifactorial disease affecting reproductive-age women. Although PCOS ovarian and metabolic features have received extensive research, uterine dysfunction has been poorly investigated. This research aims to investigate morphological and molecular alterations in the PCOS uterus and search for modulating effects of different carnitine formulations. (2) Methods: CD1 mice were administered or not with dehydroepiandrosterone (DHEA, 6 mg/100 g body weight) for 20 days, alone or with 0.40 mg L-carnitine (LC) and 0.20 mg acetyl-L-carnitine (ALC) in the presence or absence of 0.08 mg propionyl-L-carnitine (PLC). Uterine horns from the four groups were subjected to histology, immunohistochemistry and immunoblotting analyses to evaluate their morphology, collagen deposition, autophagy and steroidogenesis. Oxidative-/methylglyoxal (MG)-dependent damage was investigated along with the effects on the mitochondria, SIRT1, SOD2, RAGE and GLO1 proteins. (3) Results: The PCOS uterus suffers from tissue and oxidative alterations associated with MG-AGE accumulation. LC-ALC administration alleviated PCOS uterine tissue alterations and molecular damage. The presence of PLC prevented fibrosis and maintained mitochondria content. (4) Conclusions: The present results provide evidence for oxidative and glycative damage as the main factors contributing to PCOS uterine alterations and include the uterus in the spectrum of action of carnitines on the PCOS phenotype.

Polycystic ovarian syndrome (PCOS) is a heterogeneous condition characterized by hyperandrogenism (HA), polycystic ovaries, and dysfunctional ovulation, and it is associated with metabolic problems such as insulin resistance (IR) and obesity. After having investigated the morphological and antioxidant/antiglycative alterations on mouse ovaries and uteri, we here focus on PCOS oviducts, a tract of the reproductive system essential for the nourishment and transport of gametes and embryos. The modulating effects of L-carnitine (LC) and acetyl-L-carnitine (ALC) were also assessed. CD1 mice were administered or not with dehydroepiandrosterone (DHEA, 6 mg/100 g body weight) for 20 days, alone or with 0.40 mg of L-carnitine (LC) and 0.20 mg of acetyl-L-carnitine (ALC). Oviducts were then subjected to histology and immunohistochemistry to evaluate their morphology and collagen deposition, and steroidogenesis. Oxidative, mitochondrial, and methylglyoxal (MG)-dependent damage was also investigated. Transmission electron microscopy was used to detect ultrastructural alterations. The PCOS oviducts were affected by hyperfibrosis, hyperplasia, hypertrophy, and altered steroidogenesis, with oxidative alterations associated with MethylGlyoxal-Advanced Glycation End product (MG-AGE) accumulation. A reduced ciliary coverage and numerous dilated intercellular spaces were found in the epithelium. LC-ALC administration mitigated PCOS oviductal alterations. These results provide evidence for the detrimental action of oxidative and glycative stress in PCOS oviducts, confirming a protective role of carnitines on the PCOS phenotype.

Can live real-time determination of mitochondrial bioenergetics represent a reliable assay of the mitochondrial activity and bioenergetic metabolism in mouse oocytes? We demonstrated that real-time determination of mitochondrial oxidative phosphorylation is a feasible method for the characterization of mitochondrial activity and adaptation capacity of mammalian oocytes. Mammalian oocytes possess about 300.000 mitochondria and their alterations represent the main factors underlying reduced oocyte competence. Mitochondrial oxidative phosphorylation, measured as the oxygen consumption rate (OCR) is the largest contributor to cellular ATP demand. Fine-tuning mitochondrial activity is required to sustain bioenergetics homeostasis and maintain physiological levels of ROS production in the female gamete. Despite numerous techniques have been used to measure OCR, the application in oocytes have remained infeasible. Recently the development of a non-invasive, real-time assay has provided a more comprehensive picture of OCR in terms of the components of mitochondrial oxygen flux. Mitochondrial bioenergetic profile was determined in GV oocytes from young unprimed and primed mice; MII oocytes from young hCG-primed mice; oocytes from aged hCG-primed mice; young MII oocytes from IVM; aged MII oocytes from IVM; MII oocytes from IVM following oxidative stress in the presence or absence of the NAD+ booster p7C3 or propionyl-L-carnitine (PLC). Mean values of basal oxygen consumption rate, maximal capacity, reserve capacity and non-mitochondrial sources on oxygen consumption were evaluated. Young (4-8 weeks) and aged (36-40weeks) CD-1 mice were employed. GV oocytes were isolated from PMSG-primed and unprimed mice. MII oocytes were retrieved from hCG-primed mice. Oxidative stress was induced by exposure to 25-100µM H₂O₂ for 10-30’. IVM was performed in M2 medium with/out 1-5 mM P7C3 or 0.2mg/ml PLC. SeahorseXFp96 Agilent was employed to measure OCR (pmol/min/oocytes/wells) in pools of 8 oocytes after establishment of mitochondrial inhibitors concentrations. SigmaStat was used for statistical analysis. From the analysis of the parameters above described, GV oocytes from primed and unprimed mice showed different bioenergetic profiles revealing lower basal OCR in unprimed GV. When exposed to oxidative stress, GV oocytes exhibited of redox state alteration evidenced by the response to the different inhibitors. These effects were prevented by exposure to P7C3 or PLC. A similar profile was present in GV oocytes from aged oocytes. All classes of MII displayed an OCR lower than GV. Ovulated MII oocytes showed an OCR lower than MII from IVM oocytes. Moreover, the latter displayed an increased non-mitochondrial respiration as evidence of oxidative stress. IVM oocytes exposed to oxidative insult revealed increased proton leak associated with reduced ATP levels. The presence of P7C3 in the IVM medium reduced non-mitochondrial respiration and proton leak with positive effects on ATP production and IVM rate. MII oocytes from aged mice when compared with the young group showed a different bioenergetic profile revealing lower OCR in the aged MII along with reduced spare respiratory capacity and increased proton leak. These changes suggest decreased mitochondrial mass and/or altered integrity of ETC components. The presence of P7C3 during IVM allowed a partial recovery of these dysfunctions. Results from animal studies should be extrapolated to humans with caution. Live real-time analysis of mitochondrial bioenergetics can be a reliable approach for the study of mitochondrial dysfunctions linked with reduced oocyte competence associated with aging and other oxidative stress conditions. Thus, it could be employed to set up specific recovery strategies based on metabolic boosters to improve human IVF. not applicable