Novel Insights into circRNA Saga Coming from Spermatozoa and Epididymis of HFD Mice

Abstract

Obesity is a pathophysiological disorder associated with adiposity accumulation, oxidative stress, and chronic inflammation state that is progressively increasing in younger population worldwide, negatively affecting male reproductive skills. An emerging topic in the field of male reproduction is circRNAs, covalently closed RNA molecules produced by backsplicing, actively involved in a successful spermatogenesis and in establishing high-quality sperm parameters. However, a direct correlation between obesity and impaired circRNA cargo in spermatozoa (SPZ) remains unclear. In the current work, using C57BL6/J male mice fed with a high-fat diet (HFD, 60% fat) as experimental model of oxidative stress, we investigated the impact of HFD on sperm morphology and motility as well as on spermatic circRNAs. We performed a complete dataset of spermatic circRNA content by a microarray strategy, and differentially expressed (DE)-circRNAs were identified. Using a circRNA/miRNA/target network (ceRNET) analysis, we identified circRNAs potentially involved in oxidative stress and sperm motility pathways. Interestingly, we demonstrated an enhanced skill of HFD sperm in backsplicing activity together with an inefficient epididymal circRNA biogenesis. Fused protein in sarcoma (FUS) and its ability to recruit quaking (QKI) could be involved in orchestrating such mechanism.

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

“…Considering that in our previous work we have demonstrated an impaired testicular morphology and several spermatic anomalies, oxidative stress dependent in HFD male mice ( 35 ), we assessed a potential beneficial effect of FS on HFD reproductive phenotype. In detail, we analyzed testis morphology and sperm oxidative state by H&E staining and 4HNE immunofluorescence analysis, respectively.…”

“…We have previously characterized a spermatic circRNA cargo in HFD SPZ, focusing on a set of differentially expressed circRNAs (DE-circRNAs). An enhanced endogenous backsplicing in sperm cells was the driving force for circRNAs up-regulated in HFD SPZ and potentially involved in the oxidative stress pathway ( 35 ). With this wealth of knowledge, in the current work, we aimed to assess the potential FS ability in restoring a physiological spermatic circRNA cargo.…”

“…With this wealth of knowledge, in the current work, we aimed to assess the potential FS ability in restoring a physiological spermatic circRNA cargo. In detail, epididymal SPZ collected from CTRL, HFD and FS mice were used to analyze the expression levels of 9 selected circRNAs up-regulated in HFD SPZ, as previously identified ( 35 ). In agreement with our data, all the analyzed circRNAs were significantly (p<0.01) up-regulated in HFD SPZ ( Figure 2A ).…”

“…In order to clarify the molecular mechanism underlying the resetting of circRNA profile in HFD SPZ following FS treatment, we pointed on FUS protein, the main RBP orchestrating spermatic circRNA biogenesis ( 34 ) and previously reported as key player in the enhanced endogenous backsplicing in HFD SPZ ( 35 ). We carried out a qualitative and quantitative FUS characterization in SPZ collected from caput and cauda epididymis of CTRL, HFD and FS mice, by immunofluorescence and western blot analyses, respectively.…”

“…In particular, the endogenous skill to circularize mRNAs in circRNA counterpart, highlighted in both murine and human SPZ, seems to be tightly correlated with the intrinsic sperm quality state, thus electing circRNAs as potential biomarkers of sperm quality ( 33 , 34 ). We have previously profiled a spermatic circRNA cargo in HFD murine SPZ and demonstrated a dysregulation in the endogenous backsplicing machinery dependent on Fused protein in Sarcoma (FUS), the main RNA binding protein (RBP) orchestrating circRNA biogenesis ( 35 ). In addition, we have predicted circRNA dependent networks (ceRNETs) potentially involved in sperm oxidative stress and motility anomalies occurring in HFD model.…”