Abstract
High lipid content of oocytes and embryos in domestic animals is one of the well-known factors associated with poor cryosurvival. Herein, we wanted to determine whether the use of delipidated estrous sheep serum during in vitro maturation (IVM) of ovine oocytes reduces the cytoplasmic lipid droplets content and improves embryo development and cryotolerance after vitrification. Cumulus oocytes complexes (COCs) were matured in vitro for 24 h in medium supplemented with whole or delipidated estrous sheep serum prior to vitrification. Neutral lipid present in lipid droplets of COCs, cleavage rate, embryo development rate on Day 6 and Day 8, and hatching rate on Day 8, were compared among experimental groups. Endoplasmic reticulum stress genes were evaluated in in vitro matured COCs under different lipid conditions prior to vitrification. The lipid droplets’ content (mean fluorescence intensity) of oocytes cultured with IVM media supplemented with delipidated serum was lower than COCs matured with whole serum (7.6 ± 1.7 vs. 22.8 ± 5.0 arbitrary units, respectively; P< 0.05). Despite IVM treatment, oocytes subjected to vitrification showed impaired competence compared with the non-vitrified groups (P<0.05). No significant differences in embryo production were observed in non-vitrified COCs after maturation in delipidated or whole serum (33.4±4.9 vs 31.9 ±4.2). COCs matured in delipidated serum and subjected to vitrification showed increased expression of ATF4, ATF6, GRP78, and CHOP10 genes (ER stress markers). Collectively, our results demonstrate that although supplementation of IVM medium with delipidated estrous sheep serum reduces the presence of cytoplasmic lipid droplets in oocytes after maturation, oocyte cryotolerance is not improved. Notably, the expression of genes associated with the unfolded protein response (UPR) was increased in COCs, with fewer lipid droplets subjected to vitrification, suggesting that oocyte cryopreservation is associated with ER stress and activation of adaptive responses.
Highlights
IntroductionThere have been significant advances in methods to improve oocyte cryotolerance
Within the last decade, there have been significant advances in methods to improve oocyte cryotolerance
The current study demonstrates that cumulus-oocytes complexes (COCs) matured in vitro in medium supplemented with delipidated estrous sheep serum contain fewer cytoplasmic lipid droplets than those matured in medium supplemented with whole estrous serum
Summary
There have been significant advances in methods to improve oocyte cryotolerance. Difficulties associated to oocyte cryopreservation are related with inherent structural and physiological features. In domestic animals, the high intracellular lipid content of oocytes adds greater complexity to cryopreservation. Different strategies have been developed to reduce lipid droplets in oocytes including mechanical removal and pharmacological options[1,2,3]. It is generally accepted that in vitro embryo production (IVEP) systems are not as efficient as in vivo embryo production, mainly due to lower oocyte competence acquisition when maturation is induced under in vitro conditions [1,4,5]. Lipid metabolism provides a good source of energy during oocyte maturation upon demand. Fatty acids stored within lipid droplets provide adenosine triphosphate (ATP) molecules through β-oxidation, largely serving as an energy source during oocyte maturation and early embryo development [7]. Components within the culture medium supplied to the cumulus-oocytes complexes (COCs) during in vitro maturation have the potential of affecting oocyte competence [8]
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