Impact of delipidated estrous sheep serum supplementation on in vitro maturation, cryotolerance and endoplasmic reticulum stress gene expression of sheep oocytes
Impact of delipidated estrous sheep serum supplementation on in vitro maturation, cryotolerance and endoplasmic reticulum stress gene expression of sheep oocytes
Natalibeth Barrera 0 1
Pedro C. dos Santos Neto 0 1
Federico Cuadro 0 1
Diego Bosolasco 0 1
Ana P. Mulet 1
Martina Crispo 1
Alejo Menchaca 0 1
0 Instituto de ReproduccioÂn Animal Uruguay , Fundaci oÂn IRAUy, Montevideo , Uruguay , 2 Unidad de Animales Transg eÂnicos y de ExperimentacioÂ n, Institut Pasteur de Montevideo , Montevideo , Uruguay
1 Editor: Yang Yu, Peking University Third Hospital , CHINA
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.
Data Availability Statement: All relevant data are
within the paper.
Funding: This work was supported by Agencia
Nacional de InnovacioÂn e InvestigacioÂn
POSNAC201311217 NB Union Agriculture Group
(UAG) to AM. AM and MC are fellows of Sistema
Nacional de Investigadores (SNI) and PEDECIBA.
The funders had no role in study design, data
collection and analysis, decision to publish, or
preparation of the manuscript.
Within the last decade, there have been significant advances in methods to improve oocyte
cryotolerance. Difficulties associated to oocyte cryopreservation are related with inherent
structural and physiological features. Particularly 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±3]. Furthermore, 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 [
Although the underlying mechanisms behind oocyte competence have not yet been fully
elucidated, there is increasing evidence that oocyte metabolism and the somatic environment play
crucial roles in determining oocyte growth and developmental competence [
metabolism provides a good source of energy during oocyte maturation upon demand. For example,
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 [
]. Therefore, components within the culture medium supplied to the
cumulus-oocytes complexes (COCs) during in vitro maturation have the potential of affecting
oocyte competence [
Studies show that when oocytes are matured in vitro, energy substrates as fatty acids
provided through culture media can lead to increased intracellular lipid droplet (LD)
accumulation [9±11] and alter oocyte metabolism, thereby affecting their quality. A recent report
showed that exposure of mice COCs to high lipid content follicular fluid was associated with
endoplasmic reticulum (ER) stress induction and led to a decrease in oocyte competence [
Moreover, increased accumulation of lipids in oocytes was correlated with reduced
cryopreservation resistance [
]. It is well established that in sheep and other species, in vitro generated
embryos exhibit reduced cryotolerance [
]. The process of cryopreservation leads to
multiple changes, including structural modifications and alterations in gene expression patterns
. For example, increased expression of genes associated with ER stress has been reported
after oocyte cryopreservation. In domestic animals, the high lipid content in oocytes may pose
challenges for cryopreservation [
]. Specifically, bovine, ovine, and porcine oocytes are highly
susceptible to cryoinjuries, with the majority of studies reporting a blastocyst rate of 0 to 20%
[2,17±20] after oocyte vitrification. Additional studies have suggested that tolerance of oocytes
to chilling injuries can be increased when cytoplasmic lipid content is reduced, thereby
improving cleavage and blastocyst rates[
]. Interestingly, the introduction of controlled
stress during in vitro culture led to improved cleavage and blastocyst rates and suggests that
oocytes perform better under specific types of stress signals [
]. Induction of pathways
related to ER stress have been reported in oocytes subjected to vitrification [
]. ER stress is a
mechanism associated with excess intracellular lipid accumulation in COCs [
The addition of animal serum to IVEP medium is a standard practice and helps promote
oocyte maturation and subsequent embryo development [
]. During culture of ovine oocytes,
estrous serum is routinely used to supplement the maturation medium, since it is known that
it contains a range of beneficial components, including hormones, growth factors, amino
acids, binding proteins. Conversely, estrous serum also provides a significant source of lipids.
Therefore, the reduction of lipid content is necessary to improve in vitro embryo cryosurvival
and blastocyst rates in IVP systems. One strategy to reduce lipid exposure involves
incorporation of serum-free media during IVM. [
]. Nutrient restriction in the maturation medium
promotes the use of the oocyte's own endogenous reserves, thereby reducing the amount of
intracellular lipids [
]. However, oocyte competence is impaired under serum-free culture
2 / 17
conditions compared to undefined media in which serum has been added [
restriction of lipid content in the serum may be a more optimal strategy to reduce lipid
droplets in the oocyte and may improve oocyte cryosurvival.
The objective of this study was to determine whether supplementation of IVM medium
with delipidated estrous sheep serum affects the amount of cytoplasmic LD in in vitro matured
sheep oocytes. Furthermore, we investigate whether intracellular lipid content variations in
oocytes is associated with oocyte developmental competencies, cryotolerance, embryo
production, and ER stress.
Materials and methods
Three experiments were conducted by using a total of 2,986 COCs at FundacioÂn IRAUy and
Transgenic and Experimental Animal Unit of Institut Pasteur of Montevideo, Uruguay. The
experimental design is shown in Fig 1. Unless stated otherwise, all media and chemicals were
purchased from Sigma (St. Louis, MO, USA).
Experiment 1. Experiment 1 was conducted to evaluate the effect of the lipid content
(based on the concentrations of Triglycerides, total Cholesterol, and non-esterified fatty acid
(NEFAs)) of IVM medium supplemented with estrous sheep serum on: (a) neutral lipid stored
in LD of in vitro matured oocytes, and (b) oocyte developmental competence. A total of 866
COCs were collected from slaughterhouse ovaries and subjected to IVM in a supplemented
medium with whole estrous sheep serum (Control whole serum group, n = 452) or delipidated
estrous sheep serum (Delipidated serum group, n = 414). Neutral lipid present in LD of
partially denuded COCs, cleavage rate, development rate on Day 6 and Day 8, and hatching rate
on Day 8, were compared between the two experimental groups (Fig 1). Seven replicates of
this experiment were performed for each treatment group.
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).
3 / 17
Experiment 2. Experiment 2 was conducted in order to further investigate the effect of
vitrification on the survival rates of oocytes previously matured under different lipid content
conditions. Viable immature COCs (1280) were randomly assigned to two experimental groups
consisting of IVM medium supplemented with control or delipidated estrous serum sheep.
After IVM, COCs were submitted to IVF and IVC (Control whole serum group, n = 344;
Delipidated serum group, n = 357); or were vitrified using the Cryotop method (Control
whole serum + vitrification group, n = 288; Delipidated serum + vitrification group, n = 291)
before IVF and IVC. Oocyte lipid content was assessed by LD staining before and after
vitrification/warming, and cleavage rate, development rate on Day 6 and Day 8, and hatching rate
on Day 8 were compared among groups (Fig 1). A total of thirteen replicates of this
experiment were performed for each treatment group.
Experiment 3. Experiment 3 was performed in order to determine whether in vitro
matured COCs under different lipid conditions followed by vitrification expresses different
levels of ER stress markers. A total of 840 COCs were used to determine ER stress gene
expression (ATF4, ATF6, GRP78, and CHOP10) by real time PCR in five experimental groups
(Immature, Control whole serum group, Delipidated serum group, Control whole serum +
Vitrification group, and Delipidated serum + Vitrification group). Seven replicates of this
experiment were performed using 30 COCs per replicate for each treatment group.
Estrous sheep serum source, lipid removal and lipid determinations
For serum preparation, blood samples were collected from 15 ewes in estrus previously treated
with a hormonal protocol for estrous synchronization [
]. The procedure was approved by
the Internal Animal Care Committee of FundacioÂn IRAUy that is certified by the National
Council of Animal Care of Uruguay. The blood was allowed to clot at room temperature
during one hour and then centrifuged at 1500 g for 20 min at 4ÊC. Serum was collected, pooled
and heat inactivated at 56ÊC for 30 min. Lipid removal from serum was performed by using
Cleanascite™ (Biotech Support Group, NJ, USA) according to the instructions provided by the
manufacturer. In brief, 1 ml of CleanasciteTM was added to 4 ml of serum (1:4 v/v), samples
were gently mixed for 10 min at room temperature, lipid's agglomeration was improved by
incubation at 4ÊC for 1 h, samples were centrifuged at 1000 g for 15 min at 4ÊC, and then, the
supernatants were pooled and filtered with a 0.22 μm filter.
Aliquots from the whole and the delipidated estrous sheep serum (five samples of each group,
same batch) were analyzed for Triglycerides, total Cholesterol, and NEFAs by using the
commercial kits TG color GPO/PAP AA, Colestat enzimaÂtico (Wiener Lab, Rosario, Argentina), and
NEFA-HR (2) (Wako Chemicals USA, Inc., Richmond, VA, USA), respectively. All enzymatic
colorimetric assays were performed according to the manufacturer's instructions. Measurements
were obtained with a biochemical analyzer (Vitalab Selectra-2 Merck, Darmstadt, Germany). The
lipid removal efficiency was 54.4% for total cholesterol, 21.2% for triglycerides and 30.6% for
NEFAs. For this reason, delipidated serum was in fact partially delipidated. Data presented on
Table 1 show the mean values and inter-assay coefficient of variation (CV) for each metabolite.
Sheep ovaries were collected from the slaughterhouse and transported to the laboratory within
1 h in saline solution with 50 IU/ml of Penicillin and 50 μg/ml of Streptomycin at 35±37ÊC.
The COCs were aspirated from antral follicles (2 to 6 mm) using a 21 gauge needle and a 5 ml
syringe containing 0.5 ml of collection medium containing HEPES-buffered Tissue Cultured
Media 199 (TCM 199) supplemented with 5 IU/ml of Heparin, 50 IU/ml of Penicillin, 50 μg/
ml of Streptomycin, and 0.3% fatty acid-free Bovine Serum Albumin (BSA). Only COCs
4 / 17
surrounded with three or more layers of granulosa cells and with homogeneous cytoplasm
were selected for maturation purposes.
In vitro maturation (IVM)
Embryo production was performed according to the standard operative procedures of our
laboratory using the method described by Menchaca et al. (2016) [
]. Briefly, selected COCs were
washed three times in a washing medium containing TCM 199 + HEPES supplemented with
50 IU/ml of Penicillin, 50 μg/ml of Streptomycin, and 0.3% fatty acid-free BSA. Groups of 25±
30 COCs were placed into 100 μl droplets of maturation medium under mineral oil at 39ÊC in
a humidified atmosphere of 5% CO2 in air for 22±24 hours. For IVM, the medium was
supplemented with either 10% estrous sheep serum (whole or delipidated), 10 μg/ml FSH, 10 μg/ml
LH, 100 μM Cysteamine, 50 IU/ml Penicillin, and 50 μg/ml of Streptomycin.
In vitro fertilization (IVF)
COCs were removed from maturation drops and washed three times in IVF medium
consisting of synthetic oviduct fluid (SOF), 2% estrous sheep serum, 10 μg/ml Heparin, and 10 μg/ml
Hypotaurine. For fertilization (Day 0), frozen semen from a single ram previously frozen and
tested in our lab for IVF was used. Motile spermatozoa were obtained by swim-up method
] with slight modifications. COCs were placed into 100 μl of IVF medium, covered with
mineral oil and inseminated with 1 x 106 spermatozoa/drop. In vitro fertilization was carried
out at 39ÊC in 5% CO2 with humidified atmosphere for 22 hours.
In vitro culture (IVC)
Presumptive zygotes were denuded by gentle pipetting and were washed three times in drops
of culture medium (SOFaaBSA bicarbonate buffered) containing 5% (v/v) Basal Medium
Eagle (BME)-essential amino acids, 2.5% (v/v) Minimum Essential Medium
(MEM)-nonessential amino acids, and 4 mg/ml of BSA. Embryonic development took place in a humidified
atmosphere of 5% CO2, 5% O2, 90% N2 at 39ÊC and the medium was renewed on Day 3 and
Day 6 [
]. The percentage of cleaved embryos on Day 2 was recorded (2±8 cell embryos/total
oocytes). Development rate on Day 6 (number of morulae and blastocysts) and on Day 8
(number of blastocysts) were expressed on the basis of number of presumptive zygotes at the
onset of IVC. Percentage of hatching blastocysts on Day 8 (hatching rate) was determined on
the basis of the total number of blastocysts on the same day.
Oocyte-vitrification and warming procedure
For the experimental groups submitted to vitrification (Experiment 2 and 3), cryopreservation
was performed using the Cryotop method first described by Kuwayama et al. (2005)[
method was performed using methodology and media previously reported by our group
5 / 17
]. Following IVM, COCs were mechanically denuded by exposure to 0.1 mg/ml of
hyaluronidase at 37ÊC for 30 seconds through pipetting using a 200 μl pipette tip. Partially
denuded COCs were washed three times in a washing medium. Oocytes were first equilibrated
at room temperature for 15 min in TCM 199 medium supplemented with 20% Fetal Bovine
Serum (FBS), Basic Solution (BS) containing 7.5% (v/v) Ethylene Glycol (EG) and 7.5% (v/v)
Dimethyl sulfoxide (DMSO), referred to as Equilibrium Solution (ES). Four oocytes were
equilibrated at the same time. They were checked for recovery of the initial shape before the
vitrification step. Following equilibration, oocytes were placed in a Vitrification Solution (VS)
containing BS supplemented with 15% (v/v) EG, 15% (v/v) DMSO, and 0.5 M Sucrose. After
90 s in this solution, oocytes were placed on the Cryotop device (Kitazato Biopharma,
Fujinomiya, Japan) in a minimum volume (e.g. <0.1 μl) and immediately submerged in liquid
nitrogen. No more than four oocytes were loaded per Cryotop device. For warming, the Cryotop
was removed from the liquid nitrogen and instantly placed in a solution containing BS plus 1.0
M sucrose at 37ÊC. After 1 minute, the oocytes were transferred to a solution consisting of BS
plus 0.5 M Sucrose for 3 minutes at RT. Finally, a 5minute wash followed by a 1 minute wash
was performed with BS at RT. The oocytes were then placed in IVM medium at 39ÊC with 5%
CO2 humidified atmosphere for 2 h before IVF to allow microtubule repolymerization .
Lipid droplet staining
In Experiment 1 and 2, BODIPY 493/503 dye (Invitrogen, Carlsbad, CA, USA), which stains
intracellular neutral lipids, was used to localize LD. The neutral lipid dye BODIPY493/503 has
been used to demonstrate differences in oocyte lipid content on a variety of species including
mice, cows, sheep, pigs, and humans [
]. The methodology previously described for bovine
oocytes was followed with few modifications [
]. COCs were partially denuded by exposure
to 0.1 mg/ml of Hyaluronidase at 37ÊC for 30 s through pipetting using a 200 μl tip. Oocytes
were washed three times in serum-free Polyvinylpyrrolidone in Phosphate-buffered saline
(PBS-PVP; 0.2% w/v). COCs were fixed in 4% paraformaldehyde at 37ÊC for 1 h and washed
twice in PBS-PVP. Oocytes were allowed to permeate for 30 min in PBS containing 0.1% (w/v)
Saponin, 0.1 M Glycine (PBS-S). The DNA was stained with 10 μg/ml of TO-PRO-3
(Molecular Probes, Eugene, OR) for 20 min and subsequently washed in PBS-S. Next, LDs were stained
with BODIPY 493/503 in PBS (20μg/ml) for 1 h in the dark, and oocytes were washed three
times in PBS-PVP. Oocytes were then placed on a glass slide covered with 80% glycerol (in
PBS) and sealed with a microscope slide.
Lipid droplet determinations
Lipid droplet content was determined in Experiment 1 and 2. Images of oocytes were obtained
using a confocal laser scanning microscope (Model LSM 800; Zeiss, Thornwood, NY, USA)
attached to an inverted microscope (Model AxioObserver Z1; Zeiss, Thornwood, NY, USA) at
25X magnification. BODIPY 493/503 and TO-PRO-3 were subsequently excited with diode
488 nm lasers and diode 640nm laser. Emitted light was selected with emission detection
wavelengths ranges for BODIPY 410±617 nm and for TOPRO-3 645±700 nm. Images were
reconstructed using ZEN 2.1 software (Blue edition). From the in vitro matured groups only
metaphase II stage oocytes were analyzed for lipid determination. Using ImageJ v.1.44g
software, sum slices Z-projection was generated to a stack of images, same number of slices was
used for each projection created (from 1 to 30 endpoint slice). The BODIPY fluorescence
(arbitrary units of fluorescence) in the oocyte was determined after selection of the area
covering entire ooplasm and the background region of each partially denuded oocyte [
6 / 17
background correction Integrated density (Int Den) was calculated using Image J v.144-
software. Finally IntDen mean ± SEM for each experimental group was determined.
RNA isolation and reverse transcription
For RNA isolation, 30±35 COCs from each group of Experiment 3 (Immature, Control whole
serum group, Delipidated serum group, Control whole serum + vitrification group, and
Delipidated serum + vitrification group) were denuded, washed in PBS, and placed into a 1.5 ml
microcentrifuge tube. The tubes were immediately submerged in liquid nitrogen and stored at
-80ÊC until RNA extraction. Total RNA was isolated using the RNeasy Plus Micro Kit
(QIAGEN, Hilden, Germany) according to the manufacturer's instructions. The extracted RNA
concentration and purity was estimated using a ND-1000 spectrophotometer (NanoDrop
Technologies, Delaware USA). Sample purity was assessed using the A260/ A280nm ratio with
expected values between 1.8 and 2.0. Reverse transcription was carried out with 50ng of total
RNA using SuperScript1III First-Strand Synthesis System for RT-PCR (Invitrogen™) and a
random hexamer primer in a final reaction volume of 10 μl according to the manufacturer's
instructions. The cDNA synthesis reactions were carried out at 25ÊC for 5 min for annealing,
50ÊC for 50 min for extension, followed by enzyme heat inactivation at 85ÊC for 5 min.
Real-time polymerase chain reaction
For Experiment 3, gene expression was assessed by quantitative real-time PCR (qPCR).
Oligonucleotide primers were designed for GRP78, CHOP10, ATF4, and ATF6 using NCBI Primer
Blast. Genes were selected as markers of activation of UPR, signaling branch activated under
]. The primer sequences for PPIA and TUBB were taken from a published report
where those genes were found most stable for normalization when random hexamers were
used for cDNA priming in ovine oocytes [
]. Expected fragment size and GenBank accession
numbers are listed on Table 2. All primers were synthesized by IDT (Integrated DNA
Technologies, Coralville, IA, USA). The PCR mix in each include 5 μl of Power SYBR Green PCR Mix
(2X) (Applied Biosystems, UK), 2μl of nuclease-free water, 1 μl of each forward and reverse
primer pair (10 μM), and 1 μl of cDNA in a final volume of 10 μl. The PCR was carried out on
an Eco™ Real-time PCR System (Illumina, San Diego, USA). The program used for the
Product Size (bp)
7 / 17
amplification of the genes consisted of an enzyme activation step of 10 min at 95ÊC followed
by 45 cycles of PCR of a 15s denaturation at 95 ÊC, 60 s annealing/extension at 60 ÊC and a
dissociation step consisting of 95ÊC for 15 s, 60ÊC for 15 s and finally 95ÊC for 15 s. At the end of
the PCR reactions, melt curve analyses were performed for all genes to confirm the integrity of
PCR products and specificity by the presence of a single peak. All samples were run in
duplicate and mean value was used for calculations. Standard curves were created for each gene
using a 3-fold dilution of cDNA and used to calculate individual real time PCR efficiencies (E)
according to the formula %E = (10−1/slope± 1) × 100 [
]. The data generated by Eco Real-Time
PCR System Software v5.0 (IIlumina, CA, USA) were transferred to Microsoft Excel for
All target gene transcriptions were expressed as an n-fold difference relative to the
calibrator (Control serum group). Different amplification efficiencies for individual genes were
]. The geometrical mean of two internal reference genes (TUBB and PPIA) was
used to correct the raw values for the genes of interest.
Statistical analysis was performed using Infostat software (Cordoba, Argentina). The criterion
of data normality was evaluated by the Shapiro-Wilk test and percentage data was subjected to
arcsine transformation and expressed as Mean ± SEM. Significance differences in Experiment
1 were tested by a two-way analysis of variance (ANOVA) followed by Tukey's test when
variables were normally distributed. A non-parametric Kruskal-Wallis test was conducted for
comparison of variables that did not follow normal distribution. For Experiment 2 and 3, in
which 2x2 factorial design was performed, mixed models were used with fixed effect for
cryopreservation (vitrification or not), serum (whole or delipidated) and its interaction, and the
replicate as random effect (13 replicates for Experiment 2 and seven replicates for Experiment
3). Values of p less than 0.05 were considered statistically significant.
The lipid content of immature oocytes measured soon after follicular aspiration was 9.9 ± 2.9
(expressed as the mean fluorescence intensity). After maturation, the lipid droplet content in
oocytes cultured in medium containing whole estrous serum increased more than twofold
compared to immature oocytes (22.8 ± 5.0; P< 0.05). In contrast, oocytes that were matured in
medium containing delipidated estrous serum showed a significantly lower content of lipid
droplets (7.6 ± 1.7; P< 0.05), which was similar to the levels measured in immature oocytes
(P = NS). Fig 2 shows the results of lipid droplet localization and quantification of immature,
control whole, and delipidated in vitro matured COCs after BODIPY 493/503 staining.
Differences found in the oocyte neutral lipid content did not influence subsequent embryo
development (Table 3). No significant differences (P = NS) were observed in the cleavage rate,
developmental rates on Day 6 and Day 8, or hatching rate between groups.
Oocyte lipid content assessed by LD staining before and after vitrification are shown in Fig 3.
Lipid droplets in oocytes exhibited reduced fluorescence after vitrification when matured in
vitro with either medium supplemented with control whole serum or delipidated serum.
Despite IVM treatment, oocytes subjected to vitrification exhibited reduced competence after
in vitro fertilization and culture, with lower cleavage rate, embryo development rates on Day 6
8 / 17
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).
and Day 8, and hatching rate when compared with the non-vitrified oocytes (P<0.05). No
significant differences (P = NS) were found among non-vitrified groups, which is in agreement
with the results obtained in Experiment 1 (serum effect: whole vs. delipidated). No interaction
between vitrification and the type of serum (whole or delipidated) were found for cleavage,
embryo development, and hatching rates (P = NS) (Table 4).
69.1 ± 7.3
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).
The expression of ER stress genes was affected by the serum type used during IVM of COCs as
well as by the vitrification/warming process, with interaction between both main effects
(P<0.05). The expression of classic ER stress markers (ATF4, ATF6, GRP78, and CHOP10)
For the same column within main or simple effects, a vs. b differ (P < 0.05)
53.3 ± 6.3a
54.6.2 ± 6.2a
80.3 ± 2.9a
27.2 ± 3.6b
24.6 ±6 a
48.68 ± 4.9a
5.1 ± 4.0b
PLOS ONE | https://doi.org/10.1371/journal.pone.0198742
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).
examined in COCs exposed to IVM media supplemented with control whole or delipidated
serum and either submitted to vitrification or not is shown in Fig 4. The COCs matured in
delipidated serum and subjected to vitrification exhibited increased expression of ATF4 (4.4
fold), ATF6 (4.0 fold), GRP78 (3.6 fold), and CHOP10 (2.5 fold) compared with COCs matured
in IVM media supplemented with control whole serum (calibrator sample). No significant
differences (P = NS) were found in the expression levels of those ER stress markers in COCs
matured in control whole serum and subject to vitrification (control whole serum +
vitrification group). COCs matured in delipidated serum (non-vitrified group) exhibited similar
expression levels of the four ER stress markers respect to the control whole serum group.
These results demonstrate that vitrification induces ER stress in COCs when they are
previously matured in vitro in medium deprived of lipids.
The current study demonstrates that 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. However, reduced numbers of
cytoplasmic lipid droplets did not improve oocyte competence and embryo production in fresh
and vitrified oocytes. While the vitrification process impairs oocyte developmental
competence, the serum type (delipidated or whole) used during IVM did not appear to have any
adverse effects. Notably, mRNA expression levels of ER stress genes increased significantly
after vitrification of oocytes but only when maturation was performed with medium
supplemented with delipidated serum.
We found that the standard method used for in vitro maturation of COCs with a medium
supplemented with whole estrous sheep serum increased oocyte neutral lipid content in
comparison with immature oocytes. This increase was prevented by serum delipidation, since
oocytes subjected to IVM medium with partially delipidated serum had less lipid droplets than
COCs matured in control whole serum. Lipid droplets are intracellular sites of neutral lipid
storage, which have been shown to play an important role in the metabolism of lipids and
cellular energy homeostasis [
]. During in vitro maturation, serum lipids are incorporated into
the oocyte cytoplasm [
], with the presence of lipids in culture media causing an increase in
11 / 17
the number of lipid droplets in the produced embryos [
]. Although lipid droplets play a
pivotal role during oocyte maturation, since oxidative phosphorylation is the main pathway to
produce ATP [
], high accumulation of lipid droplets content has been correlated with poorer
cryosurvival rates and reduced development competence of oocytes.[
]. In this study, we
demonstrate that in vitro maturation of COCs in medium containing whole estrous serum
increase lipid accumulation in matured oocytes, a finding that could be important for oocyte
cryotolerance and embryo development.
Studies show that high lipid content in IVM oocytes correlates with reduced cryotolerance,
therefore different strategies have been used to reduce numbers of lipid droplets in oocytes.
One way to regulate the lipid content is the use of serum free media, which contains restricted
nutrients, during IVM [
]. However, efforts to reduce lipid content in the oocytes and
embryos, have been met with limited success and resulted in lower oocyte competence [
]. Unlike other approaches, the protocol described herein for delipidation of estrous sheep
serum was effective in decreasing levels of Triglycerides, total Cholesterol, and NEFAs. To our
knowledge this is the first study to use the Cleanascite HC method to generate estrous sheep
serum yielding significantly reduced lipid levels. Subsequent use of the partially delipidated
serum as supplemented in IVM media resulted in effective reduction of oocyte lipid content.
The advantage of this method over other traditional methods (i.e. chloroform) includes
increased feasibility and reduced toxicity and biosafety concerns [
]. Similar results have
been found when oocyte delipidation is achieved by stimulating lipid metabolism (i.e.
]. The approach of encouraging embryos and oocytes to deplete intracellular lipids to
increase cryosurvival rates has been shown to be a more benign alternative to mechanical
delipidation and safer for oocytes and embryos [
]. Furthermore, our protocol enables
simultaneous delipidation of large numbers of oocytes and does not require micromanipulation,
thereby increasing efficiency and improving viability of oocytes.
The current study shows that oocyte competence was affected by cryopreservation
independently of the lipid environment in which the COCs were matured. Cryopreservation protocols
are not well established for oocytes, especially in domestic animals due to their high lipid
]. Ultra-rapid vitrification methods have made it possible to overcome some challenges
associated with oocyte cryopreservation. Specifically, the use of Cryotop with minimum
volume of vitrification (<0.1 μl) and high cooling rates (22.800ÊC min-1) has allowed human
oocytes to obtain post-warming survival rates of >90% and blastocyst rates of roughly 50%
]. Compared to human oocytes, oocytes from some species of domestic animals have high
lipid content which increases their sensitivity to cooling processes and exacerbates outcomes
after IVM [
]. Overall, species that have higher lipid content in their ooplasma have lower
survival rates following cryopreservation [
]. As expected, we found that vitrified oocytes that
had been in IVM medium containing whole serum showed reduced cleavage rates and embryo
development compared to fresh control oocytes that were not subjected to vitrification.
Interestingly, although incorporation of partially delipidated serum in the IVM medium resulted in
lower lipid content in the matured oocytes, it did not improve outcomes after vitrification
including cleavage rates and embryo development rates in comparison to IVM oocytes with
control whole serum medium. Collectively, our results show that reducing lipid content during
the COC maturation process prior to cryopreservation is not sufficient to improve oocyte
competences following, suggesting that lipid content is one factor amongst many
variables/factors that can lead to reduced tolerance to cryopreservation. Some studies attribute the lower
cryotolerance of in vitro produced embryos to an imbalance of oxidation-reduction
metabolism leading to greater accumulation of reactive oxygen species in the culture medium and
reduced survival rates of embryos [
]. Our current results suggest that a negative correlation
exists between lipid content and oocyte cryotolerance. It is well known that embryos derived
12 / 17
from live animals have fewer lipid droplets compared to IVP embryos [
]. This difference in
lipid neutral content may explain, in part, the differences in cryotolerance between in vivo
versus in vitro embryos [
]. Some studies have reported similar differences in bovine and porcine
oocytes matured in vivo vs. in vitro [
]. These observations support the idea that if IVM
oocytes in delipidated systems could resemble in vivo matured oocytes in terms of lipid
droplets content and that cryotolerance could be improved. However, despite the lower lipid
droplet content found in those COCs subjected to IVM with partially delipidated serum, no
differences in oocyte competence were found. It is likely that either the variations observed are
insufficient to alter oocytes' cryotolerance to vitrification, or that a reduction of lipid droplets
using our experimental conditions is not a key factor to overcome damages associated with
Finally, we demonstrate that vitrification induces greater expression of ER stress genes in
COCs matured in a medium with reduced lipid content. The ER folding capacity can be
disturbed by biological stimuli, resulting in an accumulation of misfolded and unfolded proteins
in the ER lumen and ER stress [
]. ER stress triggers a homeostasis response, referred to as
the unfolded protein response (UPR) [
], which involves the activation of ER transmembrane
signaling molecules (PERK, IRE1 and ATF6). Activation of these three master regulators of
UPR influence the transcription of several genes involved in UPR. IRE1α regulates the splicing
of XBP1 promoting components of ER-associated protein degradation (ERAD) as Bip/GRP78.
Activation of PERK favor translation of ATF4, which regulates genes involved in protein
folding, degradation, and apoptosis, including CHOP. In this study, we have selected downstream
regulators ATF4, CHOP, and GRP78 as representative genes of the PERK/IRE1 pathway to
determine UPR activation [
]. Differences in the expression levels of UPR genes has been
related with lipid droplet content in mice and expression of GRP78 is greater when oocytes are
matured in vivo [
]. Lipid peroxidation has also been linked to induction of UPR in
endothelial cells [
] and augmentation in the expression levels of XBP1 has been reported in
vitrification-warmed mouse oocytes [
]. Our data revealed that GRP78 is expressed highly in
immature sheep oocytes compared to COCs that were matured in vitro. No significant
differences were found in the expression levels of ATF4, ATF6, GRP78, and CHOP10 in COCs that
were matured in vitroÐeither in whole serum or in delipidated serum. Attenuation in the
expression levels of GRP78 in in vitro matured COCs could be a consequence of FSH
supplementation in the IVM media as reported previously for mice [
]. COCs that were matured in
vitro in IVM media supplemented with delipidated serum and vitrification upregulated the
expression of ATF4, ATF6, CHOP10, and GRP78. It has been previously suggested that lipid
droplets may serve as a site of storage to sequester unfolded or excessive proteins, thereby
alleviating ER stress [
]. Therefore, the amount of lipid droplets in COCs matured in whole
serum and vitrified reduced their sensitivity to ER stress induction post-warming. Another
possible explanation for our results, is that oocyte competence is regulated by adaptive
machineries governed by ER stress. Various genes associated with ER stress were found in
oocytes and preimplantation embryos of mice and pigs, as a normal part of preimplantation
embryos adaptive machineries [
]. However, some studies oppose the notion that the UPR
response promotes oocyte competence. The inhibition of the UPR response by different
inhibitors (TUDCA or Salubrinal) was shown to enhance maturation of pig and mouse oocytes by
preventing ER stress mediated apoptosis in vitro [
], suggesting that ER stress may
negatively impact oocyte developmental competence. To our knowledge, this is the first time that
expression of the ATF4, ATF6, CHOP10, and GRP78 genes has been evaluated in ovine oocytes.
We demonstrate that vitrification can cause activation of UPR when oocytes are diminished in
lipid droplets content.
13 / 17
In conclusion, this study demonstrates that partial delipidation of estrous sheep serum used
for supplementation of IVM medium reduces the neutral lipid content and the presence of
cytoplasmic lipid droplet in oocytes. However, the culture of oocytes in delipidated serum did
not result in improved cryotolerance when in vitro matured oocytes were subjected to
vitrification. COCs with reduced amounts of lipid droplets subjected to vitrification were observed to
have higher expression of UPR genes. Overall, this study provides a feasible method to reduce
lipid droplets in oocytes that are in vitro matured, and suggests the need of a revision of the
idea that oocyte cryotolerance may be improved by lipid content depletion.
The authors thank Victoria de Brun and Marcela DÂõaz for their technical support. The authors
would also like to thank Dr. Stephan du Plessis and Dr. Sumantha Bhatt for their help in
preparing this manuscript. This study received financial support of Union Agriculture Group
(UAG) and Agencia Nacional de InvestigacioÂn e InnovacioÂn (ANII; POSNAC201311217).
AM and MC are fellows of Sistema Nacional de Investigadores (SNI) and PEDECIBA.
Conceptualization: Natalibeth Barrera, Ana P. Mulet, Alejo Menchaca.
Data curation: Natalibeth Barrera.
Formal analysis: Natalibeth Barrera.
Investigation: Pedro C. dos Santos Neto, Federico Cuadro, Ana P. Mulet.
Methodology: Natalibeth Barrera, Federico Cuadro, Diego Bosolasco, Ana P. Mulet.
Project administration: Natalibeth Barrera.
Supervision: Martina Crispo, Alejo Menchaca.
Validation: Martina Crispo, Alejo Menchaca.
Writing ± original draft: Natalibeth Barrera, Alejo Menchaca.
Writing ± review & editing: Martina Crispo.
14 / 17
15 / 17
16 / 17
1. Wirtu G , Mcgill J , Crawford L , Reddy G , Bergen WG , Simon L . Targeting Lipid Metabolism to Improve Oocyte Cryopreservation (OCP ) in Domestic Animals . 2013 ; 1 : 15 ± 20 .
2. Vajta G. Vitrification of the oocytes and embryos of domestic animals . Anim. Reprod. Sci . 2000 ; 60 ± 61 : 357 ± 64 .
3. Menchaca A , Barrera N , Santos PC dos, Cuadro F , Crispo M. Advances and limitations of in vitro embryo production in sheep and goats . Anim. Reprod. [Internet]. 2016 ; 13 : 273 ± 8 . Available from: http:// www.cbra.org.br/pages/publicacoes/animalreproduction/issues/download/v13/v13n3/p273- 278 ( AR871 ).pdf
4. Lonergan P , Fair T . Maturation of Oocytes in Vitro. Annu. Rev. Anim. Biosci . United States; 2016 ; 4 : 255 ± 68 . https://doi.org/10.1146/annurev-animal- 022114 -110822 PMID: 26566159
5. Wrenzycki C , Stinshoff H . Maturation environment and impact on subsequent developmental competence of bovine oocytes . Reprod . Domest. Anim. Germany; 2013 ; 48 Suppl 1 : 38 ± 43 .
6. Collado-Fernandez E , Picton HM , Dumollard ReÂ . Metabolism throughout follicle and oocyte development in mammals . Int. J. Dev. Biol . 2012 ; 56 : 799 ± 808 . https://doi.org/10.1387/ijdb.120140ec PMID: 23417402
7. Sturmey RG , Leese HJ . Energy metabolism in pig oocytes and early embryos . Reproduction . 2003 ; 126 : 197 ± 204 . PMID: 12887276
8. Dunning KR , Russell DL , Robker RL . Lipids and oocyte developmental competence: The role of fatty acids andβ-oxidation . Reproduction . 2014 ; 148 .
9. Listenberger LL , Brown D a. Fluorescent detection of lipid droplets and associated proteins . Curr. Protoc. Cell Biol . 2007;Chapter 24:Unit 24.2.
10. Abe H , Yamashita S , Satoh T , Hoshi H . Accumulation of cytoplasmic lipid droplets in bovine embryos and cryotolerance of embryos developed in different culture systems using serum-free or serum-containing media . Mol. Reprod . Dev. [Internet]. John Wiley & Sons, Inc.; 2002 ; 61 : 57 ± 66 . Available from: http://dx.doi.org/10.1002/mrd.1131 PMID: 11774376
11. Ferguson EM , Leese HJ . Triglyceride content of bovine oocytes and early embryos . J. Reprod. Fertil . 1999 ; 116 : 373 ± 8 . PMID: 10615263
12. Zhao N , Liu XJ , Li JT , Zhang L , Fu Y , Zhang YJ , et al. Endoplasmic reticulum stress inhibition is a valid therapeutic strategy in vitrifying oocytes . Cryobiology [Internet]. Elsevier Inc.; 2015 ; 70 : 48 ± 52 . Available from: http://dx.doi.org/10.1016/j.cryobiol. 2014 . 12 .001 PMID: 25499542
13. Zhou G Bin , Li N. Cryopreservation of porcine oocytes: Recent advances . Mol. Hum. Reprod . 2009 . p. 279 ± 85 . https://doi.org/10.1093/molehr/gap016 PMID: 19251762
14. dos Santos Neto PC , Vilariño M , Barrera N , Cuadro F , Crispo M , Menchaca A . Cryotolerance of Day 2 or Day 6 in vitro produced ovine embryos after vitrification by Cryotop or Spatula methods . Cryobiology . 2015 ;
15. dos Santos-Neto PC , Cuadro F , Barrera N , Crispo M , Menchaca A . Embryo survival and birth rate after minimum volume vitrification or slow freezing of in vivo and in vitro produced ovine embryos . Cryobiology . 2017 ; 78 :8± 14 . https://doi.org/10.1016/j.cryobiol. 2017 . 08 .002 PMID: 28803846
16. Shirazi A , Naderi MM , Hassanpour H , Heidari M , Borjian S , Sarvari A , et al. The effect of ovine oocyte vitrification on expression of subset of genes involved in epigenetic modifications during oocyte maturation and early embryo development . Theriogenology [Internet]. Elsevier Inc; 2016 ; 86 : 2136 ± 46 . Available from: http://dx.doi.org/10.1016/j.theriogenology. 2016 . 07 .005 PMID: 27501872
17. Bhat MH , Yaqoob SH , Khan FA , Waheed SM , Sharma V , Vajta G , et al. Open pulled straw vitrification of in vitro matured sheep oocytes using different cryoprotectants . Small Rumin. Res . 2013 ; 112 : 136 ± 40 .
18. Bhat MH , Sharma V , Khan FA , Naykoo NA , Yaqoob SH , Vajta G , et al. Open pulled straw vitrification and slow freezing of sheep IVF embryos using different cryoprotectants . Reprod. Fertil. Dev . 2015 ; 27 : 1175 ± 80 . https://doi.org/10.1071/RD14024 PMID: 24871337
19. Mullen SF , Fahy GM . A chronologic review of mature oocyte vitrification research in cattle, pigs, and sheep . Theriogenology . 2012 . p. 1709 ± 19 .
20. Hwang IS , Hochi S. Recent progress in cryopreservation of bovine oocytes . Biomed Res. Int . 2014 ; 2014 .
21. Shabankareh HK , Zandi M. Developmental potential of sheep oocytes cultured in different maturation media: effects of epidermal growth factor, insulin-like growth factor I, and cysteamine . Fertil. Steril. [Internet]. Elsevier Ltd; 2010 ; 94 : 335 ± 40 . Available from: http://dx.doi.org/10.1016/j.fertnstert. 2009 . 01 . 160 PMID: 19324348
22. Du Y , Pribenszky CS , MolnaÂr M , Zhang X , Yang H , Kuwayama M , et al. High hydrostatic pressure: A new way to improve in vitro developmental competence of porcine matured oocytes after vitrification . Reproduction . 2008 ; 135 : 13 ±7. https://doi.org/10.1530/REP-07-0362 PMID: 18159079
23. Pribenszky C , Lin L , Du Y , Losonczi E , Dinnyes A , Vajta G . Controlled stress improves oocyte performanceÐcell preconditioning in assisted reproduction . Reprod . Domest. Anim. Germany; 2012 ; 47 Suppl 4 : 197 ± 206 .
24. Wu LL , Russell DL , Norman RJ , Robker RL . Endoplasmic reticulum (ER) stress in cumulus-oocyte complexes impairs pentraxin-3 secretion, mitochondrial membrane potential (DeltaPsi m), and embryo development . Mol. Endocrinol. [Internet] . 2012 ; 26 : 562 ± 73 . Available from: http://www.ncbi.nlm.nih. gov/pubmed/22383462 PMID: 22383462
25. Gordon I. Laboratory production of cattle embryos 2nd edition . CAB Int . Univ. Press. 2003 .
26. Leroy J , Genicot G , Donnay I , Van Soom A. Evaluation of the lipid content in bovine oocytes and embryos with nile red: a practical approach . Reprod.Domest.Anim . 2005 ; 40 : 76 ±8. https://doi.org/10. 1111/j.1439- 0531 . 2004 . 00556 . x PMID : 15655006
27. Menchaca A , Rubianes E . New treatments associated with timed artificial insemination in small ruminants . Reprod. Fertil. Dev . 2004 ; 16 : 403 ± 13 . https://doi.org/10.10371/RD04037 PMID: 15315739
28. Parrish JJ , Susko-Parrish JL , Leibfried-Rutledge ML , Critser ES , Eyestone WH , First NL . Bovine in vitro fertilization with frozen-thawed semen . Theriogenology . United States; 1986 ; 25 : 591 ± 600 . PMID: 16726150
29. Kuwayama M , Kato O , Leibo SP , Genetics P. Article Highly effi cient vitrifi cation method for cryopreservation of human oocytes . Biomedicine . 2005 ; 11 : 300 ± 8 .
30. Tamura AN , Huang TTF , Marikawa Y. Impact of vitrification on the meiotic spindle and components of the microtubule-organizing center in mouse mature oocytes . Biol. Reprod. [Internet] . 2013 ; 89 : 112 . Available from: http://www.ncbi.nlm.nih.gov/pubmed/24025740 PMID: 24025740
31. Aardema H , Vos PL a M , Lolicato F , Roelen B a J , Knijn HM , Vaandrager AB , et al. Oleic acid prevents detrimental effects of saturated fatty acids on bovine oocyte developmental competence . Biol. Reprod. [Internet] . 2011 ; 85 : 62 ± 9 . Available from: http://www.ncbi.nlm.nih.gov/pubmed/21311036 PMID: 21311036
32. Oslowski CM , Urano F . Measuring ER stress and the unfolded protein response using mammalian tissue culture system . Methods Enzymol . [Internet]. 2013 ; 490 : 71 ± 92 . Available from: http://www. sciencedirect.com/science/article/pii/B9780123851147000040
33. O 'Connor T , Wilmut I , Taylor J. Quantitative evaluation of reference genes for real-time pcr during in vitro maturation of ovine oocytes . Reprod. Domest. Anim . 2013 ; 48 : 477 ± 83 . https://doi.org/10.1111/ rda.12112 PMID: 23066791
34. Rutledge RG , CoÃteÂ C. Mathematics of quantitative kinetic PCR and the application of standard curves . Nucleic Acids Res . 2003 ; 31 :e93. PMID: 12907745
35. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR . Nucleic Acids Res . [Internet]. 2001 ; 29 : e45 . Available from: http://www.ncbi.nlm.nih.gov/pubmed/11328886 PMID: 11328886
36. Welte MA . Expanding roles for lipid droplets . Curr. Biol. [Internet]. Elsevier Ltd ; 2015 ; 25: R470 ± 81 . Available from: http://dx.doi.org/10.1016/j.cub. 2015 . 04 .004 PMID: 26035793
37. Kim JY , Kinoshita M , Ohnishi M , Fukui Y. Lipid and fatty acid analysis of fresh and frozen-thawed immature and in vitro matured bovine oocytes . Reproduction . 2001 ; 122 : 131 ± 8 . PMID: 11425337
38. Leroy J , Genicot G , Donnay I , Soom A Van. Short Communication Evaluation of the Lipid Content in Bovine Oocytes and Embryos with Nile Red: a Practical Approach . 2005 ; 78 : 76 ± 8 .
39. Prates EG , Nunes JT , Pereira RM . A role of lipid metabolism during cumulus-oocyte complex maturation: Impact of lipid modulators to improve embryo production . Mediators Inflamm . Hindawi Publishing Corporation; 2014 ; 2014 .
40. Accorsi MF , Leão BC da S , Rocha-Frigoni NA de S , Perri SHV , Mingoti GZ . Reduction in cytoplasmic lipid content in bovine embryos cultured in vitro with linoleic acid in semi-defined medium is correlated with increases in cryotolerance . Zygote . 2015 ; 24 : 485 ± 94 . https://doi.org/10.1017/ S0967199415000428 PMID: 26350684
41. Mishra A , Gupta PSP , Sejian V , Reddy IJ , Ravindra JP . Maturation timing and fetal bovine serum concentration for developmental potential of sheep oocytes in vitro . 2016 ; 54 : 630 ± 3 .
42. Castro AR , Morrill WE , Pope V . Lipid removal from human serum samples . Clin. Diagn. Lab. Immunol . 2000 ; 7 : 197 ± 9 . PMID: 10702492
43. Dunning KR , Robker RL . Promoting lipid utilization with l-carnitine to improve oocyte quality . Anim . Reprod. Sci. [Internet]. Elsevier B.V. ; 2012 ; 134 : 69 ± 75 . Available from: http://dx.doi.org/10.1016/j. anireprosci. 2012 . 08 .013 PMID: 22917873
44. Woods EJ , Benson JD , Agca Y , Critser JK . Fundamental cryobiology of reproductive cells and tissues . Cryobiology . 2004 ; 48 : 146 ± 56 . https://doi.org/10.1016/j.cryobiol. 2004 . 03 .002 PMID: 15094091
45. Sudano MJ , Paschoal DM , da Silva Rascado T , Magalhães LCO , Crocomo LF , de Lima-Neto JF , et al. Lipid content and apoptosis of in vitro-produced bovine embryos as determinants of susceptibility to vitrification . Theriogenology [Internet]. Elsevier Inc.; 2011 ; 75 : 1211 ± 20 . Available from: http://dx.doi.org/ 10.1016/j.theriogenology. 2010 . 11 .033 PMID: 21247620
46. Seidel GE . Modifying oocytes and embryos to improve their cryopreservation . Theriogenology . 2006 . p. 228 ± 35 .
47. Del Collado M , Saraiva NZ , Lopes FL , Gaspar RC , Padilha LC , Costa RR , et al. Influence of bovine serum albumin and fetal bovine serum supplementation during in vitro maturation on lipid and mitochondrial behaviour in oocytes and lipid accumulation in bovine embryos . Reprod. Fertil. Dev . 2016 ; 28 : 1721 ± 32 .
48. Kikuchi K , Ekwall H , Tienthai P , Kawai Y , Noguchi J , Kaneko H , et al. Morphological features of lipid droplet transition during porcine oocyte fertilisation and early embryonic development to blastocyst in vivo and in vitro . Zygote . 2002 ; 10 : 355 ± 66 . PMID: 12463532
49. Volmer R , Ron D . Lipid-dependent regulation of the unfolded protein response . Curr. Opin. Cell Biol . [Internet]. Elsevier Ltd; 2015 ; 33 : 67 ± 73 . Available from: http://dx.doi.org/10.1016/j.ceb. 2014 . 12 .002 PMID: 25543896
50. Yang X , Wu LL , Chura LR , Liang X , Lane M , Norman RJ , et al. Exposure to lipid-rich follicular fluid is associated with endoplasmic reticulum stress and impaired oocyte maturation in cumulus-oocyte complexes . Fertil . Steril. [Internet]. Elsevier Inc.; 2012 ; 97 : 1438 ± 43 . Available from: http://dx.doi.org/10. 1016/j.fertnstert. 2012 . 02 .034 PMID: 22440252
51. Vladykovskaya E , Sithu SD , Haberzettl P , Wickramasinghe NS , Merchant ML , Hill BG , et al. Lipid peroxidation product 4-hydroxy-trans-2-nonenal causes endothelial activation by inducing endoplasmic reticulum stress . J. Biol. Chem . 2012 ; 287 : 11398 ± 409 . https://doi.org/10.1074/jbc. M111.320416 PMID: 22228760
52. Babayev E , Lalioti M D , Favero F , Seli E . Cross-talk between FSH and endoplasmic reticulum stress: A mutually suppressive relationship . Reprod. Sci . 2016 ; 23 : 352 ± 64 . https://doi.org/10.1177/ 1933719115602770 PMID: 26342052
53. Zhang X , Zhang K. Endoplasmic reticulum stress-associated lipid droplet formation and type II diabetes . Biochem. Res. Int . 2012 ; 2012 .
54. Michalak M , Gye MC . Endoplasmic reticulum stress in periimplantation embryos . Clin. Exp. Reprod. Med . 2015 ; 42 : 1±7 . https://doi.org/10.5653/cerm. 2015 . 42 . 1 .1 PMID: 25874167
55. Zhang JY , Diao YF , Oqani RK , Han RX , Jin DI . Effect of Endoplasmic Reticulum Stress on Porcine Oocyte Maturation and Parthenogenetic Embryonic Development In Vitro. Biol. Reprod . 2012 ; 86 : 128 ± 128 . https://doi.org/10.1095/biolreprod.111.095059 PMID: 22190710