Impact of delipidated estrous sheep serum supplementation on in vitro maturation, cryotolerance and endoplasmic reticulum stress gene expression of sheep oocytes

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.

Fig 1. Experimental design.

Schematic representation for determination of the effect of control whole estrous sheep serum vs. delipidated serum used during in vitro maturation of cumulus oocytes complexes (COCs) on oocyte lipid content and: embryo development (Experiment 1), cryotolerance after vitrification (Experiment 2), and expression of endoplasmic reticulum (ER) stress genes (Experiment 3).

Fig 2. Lipid quantification of partially denuded COCs in vitro matured with control whole serum or delipidated serum (Experiment 1).

a) Nuclei of cells were stained with ToPro-3 (red) and merged with bright field image (BF). Neutral lipid staining with BODIPY 493/503 (green) show an increase in lipid droplets of oocytes after exposure to IVM media supplemented with control whole serum. b) Comparison of the lipid content of immature (Immature, n = 23) and COCs in vitro matured in a medium supplemented with control whole (n = 24) or delipidated (n = 24) estrous sheep serum. Values are expressed as average of BODIPY fluorescence intensity in the ooplasma per area ± SEM. a vs b indicates significant differences (P<0.05).

Fig 3. Effect of vitrification on lipid droplet content of partially denuded COCs after IVM under different lipid content conditions (Experiment 2).

a) Nuclei of cells were stained with ToPro-3 (red) and merged with bright field image (BF). Neutral Lipid staining with BODIPY 493/503 (green) shows a decrease in lipid droplets of oocytes after vitrification. b) Comparison of the lipid content of immature (Immature, n = 12) versus IVM COCs in a medium supplemented with control whole (n = 16) or delipidated estrous sheep serum (n = 19) before or after vitrification (control whole serum+ vitrification group, n = 12; Delipidated serum+ vitrification n = 11). Values are expressed as average of BODIPY fluorescence intensity in the ooplasma per area ± SEM. Different superscripts indicate significant differences (P< 0.05).

Fig 4. Expression of endoplasmic reticulum (ER) stress genes induced in cumulus oocytes complexes (COCs) submitted to in vitro maturation (IVM) in whole or delipidated serum with subsequent vitrification.

Total RNA was extracted from denuded COCs, and expression of ER stress marker genes (ATF4, GRP7878, ATF6 and CHOP10) was determined by qPCR. Gene expression of 5 experimental groups, interaction was found when COCs were matured in delipidated serum and subsequently vitrified. Within the same gene, different letters indicates significant differences (P< 0.05). Mean ± SEM is expressed as fold change compared with calibrator sample (control whole serum).