Three-dimensional in vitro maturation of rabbit oocytes enriched with sheep decellularized greater omentum

Abstract

Background: To prevent ovarian hyperstimulation syndrome, in vitro maturation (IVM) allows the oocytes for infertility treatment without hormone therapy. Although many oocytes matured during IVM, some deficiencies in the culture conditions lead to inhibition of the growth and development of the cumulus cells and the oocyte nuclear and cytoplasmic maturation. Objectives: The challenge of improving the oocyte culture conditions prompted us to use greater omentum (GOM), full of growth factors and proteins, as a rich supplement to the base culture medium. Methods: Cumulus-oocyte complexes were recovered from rabbits and divided into 3D and 2D conditions cultured for 12 and 24 h. In 3D cultures, the oocytes embedded in alginate containing FBS decellularized GOM. Corresponding supplements were also added in 2D conditions—maturation of the oocytes evaluated by Aceto-Orcein, TEM, and RT-PCR for MAP2K1 and Cdk2. Results: DNA quantification, Hoechst, and H&E staining confirmed cell depletion from GOM, and SEM showed the preservation of ultra-architecture after decellularization. Histochemical staining methods showed appropriate extracellular matrix preservation. ELISA assessment showed retention of VEGF content. MTT assessment indicated decellularized GOM was non-toxic. Both Aceto-Orcein assessment and ultra-structure study of the oocytes showed that supplementation of 2D or 3D cultures with decellularized omentum promoted oocyte maturation. Expression of MAP2K1 and Cdk2 also increased in the presence of GOM. Conclusions: GOM supplementation has a beneficial impact on oocyte maturation, probably due to the presence of growth factors and proteins.