Reproductive nanotechnology: tretinoin-loaded lipid-core nanocapsulesand in vitro embryos production

Background

The improvement of in vitro maturation (IVM) protocols through the supplementation with different molecules has become an alternative to increase the culture medium efficiency. Tretinoin (TTN, all-trans retinoic acid, ATRA), is an active metabolite of vitamin A [1], that mediates cell proliferation, cell differentiation, and embryonic development process. In in vitro production embryos systems, TTN acts improving cytoplasmic maturation process in oocytes, developmental competence in early embryos, and quality in blastocysts [2]. Studies have been demonstrated the presence of α, β and γ subtypes of retinoic acid receptors (RARα, RARβ, RAR_γ) for TTN in oocyte, hatched blastocysts, and cumulus cells [3]. This molecule can also be used for treatment of skin disorders and for anti-tumor treatment, so researchers have been associated TTN with polymeric nanoparticles to protect it from degradation and to improve its chemical stability and efficacy [4]. The aim of present study was to test the concentration-dependent effect of supplementation of free tretinoin (TTN) and tretinoin-loaded lipid-core nanocapsules (TTN-LNC) in bovine in vitro maturation media, and its influence in reactive oxygen species (ROS) production in two-to four-cell stage embryos. In conclusion, tretinoin- loaded lipid-core nanocapsules added in in vitro maturation media highly protects embryos at early stage of development against oxidative stress.

Methods

The experimental groups were established, and cumulus-oocyte complexes (COCs) were matured in oocyte in vitro maturation medium supplemented with 0.25, 0.5 and 1 μM of TTN-LNC or TTN. Control groups of COCs matured without treatment and treated only with blank lipid-core-nanocapsules (LNC) were also examined. The oocytes were in vitro fertilized in order to evaluate the ROS levels in embryos produced by the different treatments. The ROS formation was evaluated in two-to four-cell stage embryos as previously described [5] with some modifications.

Results and conclusions

ROS production was lower in embryos derived from oocytes matured in the presence of TTN or TTN-LNC. Both treatments TTN and TTN-LCNC protect the cell more effectively against oxidative damage reducing the ROS production. A significant reduction (p <0.05) in ROS production was detected in the presence of TTN-LCNC compared with controls. There was no difference between the concentrations in TTN and TTN-LNC groups. Tretinoin-loaded lipid-core nanocapsules offers an increased protection against oxidative stress in embryos produced in vitro. The studies at the molecular level using oocyte competence markers are alternatives to clarify the function of lipid-core nanocapsules in in vitro maturation and in embryonic development.