Inducible Nitric Oxide Synthase and Heme Oxygenase 1 Are Expressed in Human Cumulus Cells and May Be Used as Biomarkers of Oocyte Competence

Abstract

The interplay between oocyte and surrounding cumulus cells (CCs) during follicular growth influences oocyte competence to undergo fertilization and sustain embryo development. The expression of many genes and proteins in CCs has been suggested as potential biomarker of oocyte competence in human in vitro fertilization (IVF). In the present study, we analyzed 90 human cumulus-oocyte complexes obtained during IVF procedure: 30 CCs were analyzed using quantitative real-time polymerase chain reaction and 60 CCs using Western blotting analysis to detect gene and protein expression of some enzymes related to oxidative stress, that is, the 3 nitric oxide synthase (NOS) isoforms and heme oxygenase 1 (HO-1). In the group of 60 CCs, we also investigated the expression and phosphorylation of IkBα, a known inhibitor of the nuclear factor κB (NF-κB) pathway, which controls several redox-sensitive genes. The expression of the messenger RNAs (mRNAs) was related to the oocyte morphological analysis performed by polarized light microscopy and to the occurrence of normal fertilization after intracytoplasmic sperm injection. We observed that the amount of iNOS and HO-1 mRNAs and proteins is significantly higher, and that in the meanwhile the NF-κB pathway is activated, in CCs corresponding to oocytes that were not fertilized in comparison to CCs whose corresponding oocyte showed normal fertilization. Instead, no correlation between the fertilization and the oocytes' morphological data was observed. These results suggest that the increase in iNOS and HO-1 mRNAs expression in CCs is a negative index of oocyte fertilizability and might be an useful tool for oocyte selection.

Oocyte polarized light microscopy, assay of specific follicular fluid metabolites, and gene expression in cumulus cells as different approaches to predict fertilization efficiency after ICSI

“…Our finding that the level of 4-hydroxynonenal-protein conjugates was significantly higher in the FF of nF oocytes confirms that oxidative stress, and lipo-peroxidation in particular [42], acts as an oocyte damaging condition, predisposing to fertilization failure. Noticeably, these observations agree with a previous study in which we observed that in cumulus cells the mRNA and protein expression of inducible nitric oxide synthase, heme-oxygenase-1 and IkBα (inhibitor of the redox-sensitive transcription factor NF-kB) phosphorylation - all pathways controlling redox-sensitive genes - was significantly related to fertilization failure [43]. …”

“…As AMH induces the redox-sensitive transcription factor NF-kB binding activity in breast and prostate cancer cells [45], it is possible that an induction of NF-kB in CCs, significantly related to fertilization failure [43], could be linked to the increase of AMH signal transduction in nF oocytes, in which the mRNA expression of AMH-receptor II gene and the oxidative status are greater than in normally fertilized oocytes.…”

Oocyte environment: follicular fluid and cumulus cells are critical for oocyte health

“…Increased oxidative stress-related genes in human cumulus cells, including iNOS, HO-1, and those regulating the NF-lB pathway, are associated with reduced oocyte fertilization by intracytoplasmic sperm injection (107). Other cumulus cell genes as markers of oocyte quality include COX2, GREM1, HAS2, and PTX3 (108,109).…”

A simple method for assessing hyaluronic acid production by cumulus-oocyte complexes

“…Based on our knowledge, the quality of cumulus expansion can determine oocyte meiotic maturation and its developmental potency (Qian et al 2003;Feuerstein et al 2012;Bergandi et al 2014). Conversely, cumulus expansion physiologically reflects the meiotic stage of oocyte secreting CEEFs (Prochazka et al 1998;Dragovic et al 2005).…”