Antioxidants positively regulate obesity dependent circRNAs - sperm quality - functional axis

Abstract

Obesity is a pathophysiological condition, dependent on body fat accumulation, that progressively induces systemic oxidative stress/inflammation leading to a set of associated clinical manifestations, including male infertility. CircRNAs, covalently closed RNA molecules, are key regulators of sperm quality. Recently, we have characterized a complete profile of high-fat diet (HFD) spermatic circRNA cargo, predicting paternal circRNA dependent networks (ceRNETs), potentially involved in sperm oxidative stress and motility anomalies. In the current work, using HFD C57BL6/J male mice, orally treated with a mix of bioactive molecules (vitamin C; vitamin B12; vitamin E; selenium-L-methionine; glutathione-GSH) for 4 weeks, a reversion of HFD phenotype was observed. In addition, the functional action of the proposed formulations on circRNA biogenesis was evaluated by assessing the endogenous spermatic FUS-dependent backsplicing machinery and related circRNA cargo. After that, spermatic viability and motility were also analyzed. Paternal ceRNETs, potentially involved in oxidative stress regulation and sperm motility defects, were identified and used to suggest that the beneficial action of the food supplements here conveniently formulated on sperm motility was likely due to the recovery of circRNA profile. Such a hypothesis was, then, verified by an in vitro assay.

Keywords: antioxidant agents; backsplicing; circRNAs; male infertility; obesity; oxidative stress; spermatozoa.

Figure 1

Effects of food supplementation (FS) administration on body parameters. (A) Representative image of CTRL, HFD and FS (HFD +FS) male mice. (B–D) Body weight (B), body length (C) and abdominal circumference (D) in CTRL (n=12), HFD (n=12) and FS (n=12) mice. All data are reported as mean ± SEM. Different letters indicate statistical significance (p<0.05 or p<0.01). (E) Hematoxylin and eosin (H&E) staining of Bouin’s fixed CTRL, HFD and FS liver and testis sections (7 μm). Fat vacuole accumulation in hepatic cell was indicated by white arrowheads while in testis, detached germ cells were indicated by black arrowheads. Scale bar: 50 μm. (F) Immunofluorescence analysis of 4-Hydroxy-2-nonenal (4HNE) in cauda SPZ of CTRL, HFD and FS mice. White arrowheads and white asterisk represent 4HNE localization (green) in sperm head and tail, respectively. Nuclei were labeled with DAPI (blue). Scale bar: 5 µm.

Figure 2

Effects of food supplementation (FS) administration on circRNA and FUS expression in SPZ. (A) Expression analysis of 9 circRNAs up-regulated in CTRL, HFD and FS sperm cells. qRT-PCR data are normalized using Cyclophilin expressed as fold expression (nfe) and reported as mean value ± S.E.M. a vs b: p< 0.01. (B) Immunofluorescence analysis of FUS protein in caput and cauda SPZ of CTRL, HFD and FS mice. White arrowheads and white asterisk represent FUS localization (red) in sperm head and tail, respectively. Nuclei were labeled with DAPI (blue). Scale bar: 5 µm. (C) Western blot analysis of FUS protein in caput and cauda SPZ of CTRL, HFD and FS mice (n=6 different samples for each experimental group in triplicate). Signals were quantified by densitometry analysis and normalized to Ponceau Red (Ponceau R). Data were expressed in OD values and reported as mean ± SEM. Different letters indicate statistical significance (p<0.05 or p<0.01).

Figure 3

Effects of food supplementation (FS) administration on sperm backsplicing machinery and functional parameters. (A) Two circRNAs up-regulated in HFD SPZ, circMEMO1 and circMAPT, tether a group of miRNAs and mRNAs as targets, all involved in oxidative stress and motility pathways. Networks were built using Cytoscape. Hexagonal and rectangular symbols represent circRNAs and miRNAs, respectively. The arrow indicates the tethering activity of circRNAs toward miRNAs, while the dotted arrow indicates the pathways downstream of the miRNAs. (B) Hematoxylin and eosin (H&E) staining of cauda SPZ collected from CTRL (n=5), HFD (n=5) and FS (n=5) mice; anomalous sperm heads were indicated by black arrowheads. Scale bar: 50 μm. (C) Percentage of anomalous sperm head in CTRL, HFD and FS cauda SPZ; data were reported as the percentage of anomalous sperm head/total SPZ. Sperm death (D) and motility (E) assay in CTRL, HFD and FS cauda SPZ; data were expressed as the percentage of non-viable/total SPZ and motile/live SPZ, respectively and reported as mean ± SEM. Different letters indicate statistical significance (p<0.01). (F) Sperm motility evaluation in CTRL, HFD and FS cauda SPZ by TLVM analysis. Sperm velocity was calculated in µm/s; different letters indicate statistical significance (p<0.001). (G) The enrichment levels of circMEMO1 and circMAPT in the products of RNA binding protein immunoprecipitation (RIP) assay (FUS-IP compared with IgG-IP) in caput and cauda SPZ of CTRL, HFD and FS mice detected by qRT-PCR. Data were reported as mean ± SEM from three independent experiments. **p<0.01.

Figure 4

Effects of H₂O₂ in vitro treatment on oxidative stress, circRNA expression and sperm functional parameters. (A) Immunofluorescence analysis of 4-Hydroxy-2-nonenal (4HNE) in cauda SPZ in vitro treated with vehicle (Ctrl) or H₂O₂ (Exp). White arrowheads and white asterisk represent 4HNE localization (green) in sperm head and tail, respectively. Nuclei were labeled with DAPI (blue). Scale bar: 5 µm. (B, C) Expression analysis of circMEMO1 (B) and circMAPT (C) in Ctrl and Exp cauda SPZ. qRT-PCR data are normalized using Cyclophilin expressed as fold expression (nfe) and reported as mean value ± S.E.M; **p<0.01. (D, E) The enrichment levels of circMEMO1 and circMAPT in the products of RNA binding protein immunoprecipitation (RIP) assay (FUS-IP compared with IgG-IP) in Ctrl and Exp cauda SPZ by qRT-PCR. Data are reported as mean ± SEM from three independent experiments; **p<0.01. (F, G) Sperm death (F) and motility (G) assay in Ctrl and Exp cauda SPZ; data were expressed as the percentage of non-viable/total SPZ and motile/live SPZ, respectively and reported as mean ± SEM; *p<0.05; **p<0.01.