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requirements and their subcellular localization. The Disposable 35-mm Petri dishes, 90-mm Petri dishes and
copper/zinc-containing SOD (SOD1) is found in the 0.22-lm syringe lters were obtained from Himedia
cytoplasm, and the manganese-containing SOD (SOD2) Laboratories, Mumbai, India, whereas 15-ml and 50-ml
is mitochondrial. Besides SODs, GPx also plays an centrifuge tubes were obtained from Tarsons Products
important role in antioxidant system and catalyses the Pvt. Ltd., Kolkota, India. Chemicals and kits required for
reduction of H2O2 to the corresponding alcohols at the gene expression study were from Invitrogen, Life Tech-
expense of the reducing equivalent glutathione. Glu- nologies, Carlsbad, CA, USA. Primers for gene expres-
tathione peroxidases depend on the availability of sion study were procured from Sigma-Aldrich Chemical.
reduced glutathione (GSH) for their enzymatic activity.
Glutathione (GSH) is the major cellular non-protein
sulfhydryl compound and plays a major role in regu- In vitro embryo production
lating intracellular ROS concentrations directly as a free Oocyte collection and IVM
radical scavenger and indirectly as a substrate with Sheep ovaries were collected from local slaughter house in
NADPH for detoxifying ROS. Expression pattern of normal saline solution (NSS) fortied with antibiotics
antioxidant enzyme genes is reported in oocytes and and carried to the laboratory within 3 h at 3537C.
embryos of dierent species such as bovine (Takahashi Ovaries were washed in NSS and rinsed in 70% ethyl
et al. 2013), human and mouse (El Mouatassim et al. alcohol for a few minutes to eliminate surface organisms.
1999), but no literature is available on the expression Oocytes were aspirated from follicles (26 mm) with the
pattern of these genes in sheep oocytes and developing help of 20-gauge needle attached to 5-ml syringe contain-
embryos produced in vitro, although qualitative expres- ing oocyte collection medium (OCM) (TCM-199 + BSA
sion of antioxidant enzymes in in vivo sheep oocytes is (3 mg/ml) + 5% FBS + heparin (2 IU/ml). Aspirated
reported (Livingston et al. 2009). The antioxidant oocytes (excellent and good quality) were selected for
enzyme genes are modulated by oxidative stress (Correa IVM. Cumulus oocyte complexes (COCs) (1520
et al. 2008). numbers) were matured in 35-mm Petri dish and incu-
L-carnitine the biologically active form of carnitine (3- bated in CO2 incubator at 5% CO2, 38.5C and 95% RH
hydroxy-4-N-trimethyl amino butyrate, C7H5NO3, in 100 ll of maturation medium (TCM-199 + 10%
M.W.-161.2) is a water-soluble quaternary ammonium FBS + BSA (3 mg/ml) + pyruvate (4 mM) + glutamine
compound and vitamin-like naturally occurring sub- (0.68 mM) + gentamycin (50 lg/ml) + FSH (5 lg/
stance. It is mainly synthesized from amino acids lysine ml) + LH (5 lg/ml) + estradiol (1 lg/ml) with L-carni-
and methionine in liver. It is required to transport fatty tine (0 mM, 2.5 mM, 5 mM, 7.5 mM and 10 mM) under
acids from cytosol to mitochondria during breakdown paran oil for 27 h. Maturation rate was assessed based
of lipids (fats) to generate metabolic energy. Carnitine on the degree of cumulus expansion and extrusion of the
acts as an antioxidant that neutralizes the free radicals rst polar body (oocytes at MII stage) by aceto-orcein
especially superoxide anion and protects cell against staining method. From the result of dierent concentra-
oxidative damage-induced apoptosis (Ye et al. 2010). tion of L-carnitine, nally 10 mM L-carnitine was used for
Eect of L-carnitine on in vitro bovine embryos (Taka- subsequent experiments.
hashi et al. 2013), pig embryos (You et al. 2012) and
mouse embryos (Abdelrazik et al. 2009) has already
been reported, but its eect on sheep blastocyst devel- In vitro fertilization (IVF)
opment rate and its eect on mitochondrial DNA copy In vitro fertilization was performed by collecting fresh
number have been reported recently (Reader et al. semen from the ram with the help of electro-ejaculator.
2015). There is no report available to nd out Semen after collection was washed twice with washing
L-carnitine-mediated alteration in expression of antiox- medium [Fert-TALP + heparin (10 lg/ml) + pyruvate
idant enzyme genes in sheep oocytes and embryos. (1 mM)] by centrifuging at 400 x g for 5 min. After washing,
Therefore, this experiment was designed in sheep model supernatant was removed, pellet was reconstituted in
with the objective to nd out the eect of L-carnitine fertilization medium [Fert-TALP + fatty acid free BSA
supplementation on in vitro oocyte maturation and (4 mg/ml) + heparin (10 lg/ml) + pyruvate (1 mM) +
subsequent embryo development with L-carnitine- BME (1009) (1%) + MEM (509) (1%)], and nal sperm
mediated reduction in oxidative stress and alteration concentration was adjusted to 239 106 sperms/ml which
any in transcript level of antioxidant enzymes [glu- was assessed through Neubauers chamber. The sperm
tathione peroxidase (GPx), Cu/Zn-super oxide dismu- suspension after process was kept in 5% CO2, 38.5C and
tase (SOD1) and Mn-super oxide dismutase (SOD2)]. 95% RH till matured oocytes were washed 45 times in
fertilization medium. Finally, in vitro matured oocytes (15
20 numbers) were inseminated with 100 ll of processed
Materials and Methods
spermatozoa, and fertilization was carried out by co-
Reagents and media incubation of sperm and oocytes for 18 h in fertilization
All the chemicals used in this study were obtained medium in the same temperature and gaseous condition
from Sigma-Aldrich Chemical, St. Louis, MO, USA. described for maturation.
In vitro culture (IVC) 0.5, 1, 2.5, 5, 10, 20 lM) of hydrogen peroxide (H2O2) an
Following 18 h co-incubation, presumptive zygotes were oxidant supplemented to maturation medium to nd out
cultured in 100 ll of culture medium [TCM-199 + 20% the concentration of H2O2 which arrests completely the
FBS + BSA (3 mg/ml) + pyruvate (4 mM) + glutamine developmental potential of oocytes to embryos after
(0.68 mM) + gentamycin (50 lg/ml) + BME (1009) fertilization. From these concentrations of H2O2, it was
(1%) + MEM (509) (1%)] in the same temperature observed that at concentration of 10 lM of H2O2 in
and gaseous condition described for maturation maturation medium, there was signicant (p < 0.05)
and fertilization to get embryos of dierent develop- decrease in cleavage and further development, whereas
mental stages from two cells to blastocysts stage. at 20 lM of H2O2 there was no cleavage at all.
Cleavage rates were recorded on day 2 (48 hpi) of Therefore, to assess the antioxidant eect of L-carnitine,
culture, and stages of embryonic development were there were two studies conducted: in rst study, oocytes
evaluated every 24 h. Blastocyst development was were matured (27 h) with 20 lM H2O2 with and without
recorded on day 7 (day 0 = day of IVF). Every 48 h 10 mM L-carnitine, and in second study, embryos (2
medium was replaced with 50% of freshly prepared IVC 4 cells) (obtained from oocytes matured without H2O2
medium. and L-carnitine) were cultured with 20 lM H2O2 for 48 h
post-fertilization followed by replacing 50% fresh
culture medium without H2O2 with and without
Intracellular ROS and GSH levels in oocytes and 10 mM L-carnitine to nd out developmental potential
embryos of embryos.
Intracellular ROS levels in oocytes and embryos were
quantied using 20 ,70 -dichlorodihydrouorescein diac-
Commet assay to assess integrity of oocytes DNA
etate (DCHFDA) and detected as green uorescence at
excitation wave length of 495 nm and emission wave- Integrity of oocytes DNA due to H2O2-induced oxida-
length of 520 nm. ROS level was quantied by mea- tive damage was assessed by commet assay as described
suring 20 ,70 -dichlorouorescein (DCF) uorescence by Men et al. (2003) with modication for sheep
generated from the reaction of the 20 ,70 -dichlorodihy- oocytes. There were three groups of oocytes to be
drouorescein diacetate (DCHFDA) with intracellular assessed such as (i) oocytes cultured neither with H2O2
H2O2 (Nasr-Esfahani et al. 1990). GSH levels was (20 lM) nor L-carnitine (10 mM) (ii) oocytes cultured
quantied by using 4-chloromethy L-6.8-diuoro-7- with H2O2 (20 lM) and (iii) oocytes cultured with both
hydroxycoumarin (CMF2HC) and detected as blue H2O2 (20 lM) and L-carnitine (10 mM). Zona pellucidae
uorescence at excitation wave length of 350 nm and of the oocytes were removed by 1% pronase digestion.
emission wavelength of 450 nm as described by You Microscopic slides pre-coated with 1% L-poly lysin were
et al. (2012). Briey, DCHFDA (Sigma-Aldrich Che- coated with 1% normal melting agarose and dried at
mical) and CMF2HC (Sigma-Aldrich Chemical) stock room temperature. A drop of 10 ll 1% low melting
was prepared by dissolving with dimethyl sulfoxide agarose was quickly mixed with the oocytes, put onto
(DMSO) at 1 mM concentration. Further stock solution microscope slide pre-coated with 1% agarose and
was diluted in phosphate-buered saline (PBS) to a quickly placed on ice to solidify the agarose. The
working concentration of 10 lM. Oocytes and embryos oocytes were then lysed by immersing the slides into a
were washed twice in PBS + polyvinyl pyrrolidone pre-cooled (4C) lysis buer (1% sodium sarcosinate,
(PVP) (0.5%) (wt/vol) and xed with 4% 2.5 mM NaCl, 100 mM Na2-EDTA, 1% Triton X-100 in
paraformaldehyde (PFA) and then placed in 50 ll of 10 mM Tris buer, pH 10) for 1 h. After 1 h, the slides
10 lM DCHFDA and 10 lM of CMF2HC for 15 min were removed from lysing buer and placed in a
at 5% CO2, 38.5C and 95% RH to detect ROS and horizontal gel electrophoresis unit lled with pre-chilled
GSH, respectively. Finally, the oocytes and embryos (4C) alkali electrophoresis buer (1 mM Na2-EDTA,
were washed three times by PBS + PVP (0.5%), care- 300 mM NaOH, pH > 13) and electrophoresis was
fully mounted on glass slide and covered with coverslip. conducted for 30 min at 25V, 300 mA. After elec-
The uorescence intensity of oocytes and embryos in trophoresis, slides were neutralized by in 0.4 M Tris-HCl
each group was observed under an epiuorescence (pH 7.5) for 5 min at room temperature. The slides were
microscope (Euromex, Arnhem, Holland) equipped stained with ethidium bromide (20 lg/ml) for 10 min.
with a digital camera. Fluorescence intensities of The integrity of oocyte DNA was examined under
oocytes and embryos were analysed by grey pixel uorescent microscopy (Euromax), and images were
intensity using ImageJ software (NIH, USA) normal- captured. A lter combination of 510- to 560-nm
izing untreated control oocytes and embryos as 1. excitation lter and 590-nm barrier lter was used for
the study. The integrity of oocyte DNA was determined
by the migration of DNA. If no DNA migrated outside
Antioxidant eect of L-carnitine the oocyte, then DNA was considered as intact but
To proof the antioxidant eect of L-carnitine, rst DNA was considered as damaged if DNA was migrated
oocytes were matured with dierent concentrations (0, outside the oocyte.
Expression prole of antioxidant enzyme genes 75% ethanol by centrifuging at 7500 9 g for 5 min at
The transcript abundance of antioxidant enzyme genes 4C and supernatant was discarded. The pellet was dried
[glutathione peroxidase (GPx), Cu/Zn superoxide dis- in an incubator for 10 min at 37C and was dissolved in
mutase (SOD1) and Mn superoxide dismutase (SOD2)] 10 ll of DEPC water. The dissolved pellet was incu-
was analysed by rea L-time quantitative PCR (qPCR). bated for 10 min at 5560C with little shaking in
For gene expression study, oocytes were matured with between incubation. The genomic DNA contamination
and without 10 mM L-carnitine for treatment and was removed by using TURBO DNA-freeTM kit
control groups, respectively, but during post-fertiliza- (Ambion, Life Technologies, Carlsbad, CA, USA).
tion period, presumptive zygotes were cultured without
L-carnitine. The gene-specic primers used in this study
RNA integrity and cDNA synthesis
were designed from NCBI, PRIMER BLAST
(www.ncbi.nlm.nih.gov/BLAST) (Table 1). The speci- The integrity of total RNA was checked on 1% agarose
city of the primers was tested using BLAST analysis gel electrophoresis using 19 TAE buer. The bands of
against the genomic NCBI database. 28sRNA and 18sRNA reected the quality of extracted
total RNA. The purity of total RNA (free from protein
and genomic DNA contamination) was checked using
RNA isolation nanodrop by OD 260:OD 280 values which was more
Immature oocytes, in vitro matured oocytes and devel- than 1.8. 150 ng RNA was used in the Reverse
oping embryos (zygote, 24 cells, 816 cells, morula and Transcription (RT) as the template for rst-strand
blastocysts) generated in vitro were used for RNA synthesis by using SuperScript III First-Strand Synthesis
isolation. Before RNA isolation immature and in vitro kit (Invitrogen, Life Technologies) as per manufac-
matured oocytes were treated with 0.25% Trypsin turers guide line using oligo dT (50 lm), dNTP
EDTA solution, vortexed and washed 56 times in the (10 mM), RT buer, mgCl2 (2.5 mM), reverse transcrip-
handling medium (TCM-199 +5% FBS) to remove tase (RT) (200 U), RNAse inhibitor (40 U), DTT
attached cumulus cells. Total RNA was isolated from (0.1 M) in volume of 20 ll. The synthesized cDNA
pools of oocytes, immature (n = 20), in vitro matured was stored in 20C until used for rea L-time quanti-
(n = 20) and embryos of zygote (n = 20), 24 cell tative PCR.
(n = 20), 816 cell (n = 20), morula (n = 10) and blas-
tocyst (n = 10) by Trizol (Invitrogen, Life Technologies)
Real time quantitative PCR
method as per manufacturers guide line with some
modications. Briey, 200 ll of Trizol was added to the The expression levels of specic genes in oocytes and
oocytes and embryos, mixed thoroughly by pipetting up embryos were quantied by qPCR using step one plus
and down and the mixture was incubated at room qPCR system (Applied Biosystem, Carlsbad, CA, USA).
temperature for 10 min. 50 ll of chloroform was added Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
to the tubes, mixed and incubated again at room was used as the reference gene in this study. The qPCR
temperature for 10 min. Sample mixture was cen- reactions were performed using Maxima SYBR Green/
trifuged at 12 000 9 g for 15 min at 4C, upper Rox qPCR master mix (29) (Fermentas, Waltham, MA,
aqueous phase was collected without touching inter- USA). Each run was performed in duplicate in a 10 ll
phase and transferred to a new RNAse free tube. 2 ll reaction containing 5 ll qPCR master mix, 5 pM of gene-
(20 lg) of acrylamide (20 mg/ml stock) and 100 ll of specic forward and reverse primers, 2 ll of cDNA as
isopropanol were added to the aqueous phase collected, template, and nal volume of 10 ll was made up with
mixed them well by inversion and incubated on ice for nuclease free water. The PCR condition used to amplify
30 min. The tubes were centrifuged at 12 000 9 g for all genes was initial denaturation at 95C for 10 min with
10 min at 4C after incubation and supernatant was 40 cycles of denaturation at 95C for 15 s followed by
discarded. The pellet was washed twice with 150 ll of annealing and extension at 60C for 1 min. The melting
curve analysis was carried out to conrm the qPCR
specicity. Ct (threshold cycle for target amplication)
Table 1. Primers used for gene expression study values were analysed using 2Ct (normalized expres-
sion ratio) method to determine the relative level of
Product expression of each mRNA. Ct = Ct (target gene)-Ct (-
Sl No Genes Primer sequence size (bp) housekeeping gene) and Ct = Ct (target gene sam-
ple) Ct (calibrator). qPCR was conducted for three
1 GAPDH F-ATGGGCGTGAACCACGAGAA 146
R-ATGGCGTGGACAGTGGTCAT
times for three dierent sets of embryos.
6 GPx F-CGTGCAACCAGTTTGGGCAT 141
R-GATGCGCCTTCTCGCCATTC
7 SOD1 F-CCACTTCGAGGCAAAGGGAGA 167 Conrmation of qPCR amplicons
R-CCTTTGGCCCACCGTGTTTT The qPCR amplicons of antioxidant enzymes were
8 SOD2 F-CCGTCAGCCTTACACCAAGT 112
R-CAAGCCACGCTCAGAAACAC
conrmed by ethidium bromide (0.5 lg/ml) stained 2%
agarose gel electrophoresis.
Experimental design were taken for RNA isolation and subsequent study of
Experiment 1 antioxidant enzyme genes expression. Objective of this
experiment was to nd out the L-carnitine-mediated
Immature oocytes were randomly divided into ve alterations in antioxidant enzymes gene expression in
groups and matured in vitro with dierent concentra-
developmental stages of embryos.
tions of L-carnitine (0 mM, 2.5 mM, 5 mM, 7.5 mM and
10 mM) supplemented to maturation medium (described
in method). The objective of this experiment was to nd Statistical analysis
out the eect of L-carnitine on maturation and subse-
The results are expressed in mean SEM. Statistical
quent embryo development as well as to select a suitable
analysis was carried out using GraphPad Prism 5, San
concentration of L-carnitine for subsequent experiment.
Diego, CA, USA. The mean between groups for
embryonic development, ROS and GSH level and gene
Experiment 2 expression level was compared by analysis of variance
From the result of experiment 1, nally 10 mM (ANOVA). Embryo development data have been presented
L-carnitine was used in subsequent experiments. in the percentage in relation with total oocytes cultured.
Matured oocytes and embryos produced from 10 mM Percentage values were arcsine-transformed before anal-
L-carnitine were taken to measure the intracellular ROS ysis. p < 0.05 values were considered as signicant.
and GSH levels in oocytes and embryos. The objective
of this experiment was to nd out the eect of
L-carnitine on intracellular ROS and GSH levels.
Results
Experiment 1
Experiment 3 Eect of L-carnitine supplementation on IVM and
subsequent embryo development
Immature oocytes were matured in vitro with dierent
concentrations (0, 0.5, 1, 2.5, 5, 10, 20 lM) of H2O2 Supplementation of dierent concentrations (0 mM,
supplemented to maturation medium. The objective of 2.5 mM, 5 mM, 7.5 mM and 10 mM) of L-carnitine in
this experiment was to nd out the concentration of the maturation medium did not inuence the matura-
H2O2 which arrests completely the developmental tion rate (80.2083.83%) (Fig. 1). The result of in vitro
potential of oocytes to embryos after fertilization. embryo development in the presence and absence of
L-carnitine in maturation medium is detailed in Fig. 2.
10 mM L-carnitine during IVM resulted signicantly
Experiment 4 (p < 0.05) higher percentage of cleavage (66.80% vs
From the result of experiment 3, 20 lM of H2O2 was used 39.66, 41.76, 44.64, 64.31%) followed by morula
for subsequent study. Oocytes were randomly divided into (48.50% vs 20.88, 26.01, 26.99, 44.72%) and blastocyst
three groups and matured for 27 h supplemented with (33.22% vs 7.66, 9.19, 10.71, 28.57%) as compared
20 lM H2O2 with and without 10 mM L-carnitine. Control other groups of lower concentration (0 mM, 2.5 mM,
group oocytes were matured without H2O2 and L-carnitine. 5 mM and 7.5 mM). Cleavage percentage between
Group 1 oocytes were matured with H2O2, and Group 2 10 mM and 7.5 mM L-carnitine groups were not
oocytes were matured with H2O2 and L-carnitine. Ran- signicantly dierent. For subsequent studies, 10 mM
domly oocytes were taken from the three groups, and L-carnitine was used in maturation medium. In this
commet assay was conducted. Objective of this experiment study, cleavage was observed on day 2 (48 hpi) and
was to assess the antioxidant eect of L-carnitine on oocyte blastocyst was observed on day 7 (day 0 = day of IVF).
maturation and subsequent development.
Experiment 5
Embryos (24 cells) were cultured with 20 lM H2O2 with
and without 10 mM L-carnitine for 48 h followed by
replacing 50% fresh culture medium without H2O2 up to
blastocyst development. Control group embryos were
cultured without H2O2 and L-carnitine. Group 1
embryos were cultured with H2O2, and Group 2 embryos
were cultured with H2O2 and L-carnitine. Objective of
this experiment was to assess the antioxidant eect of
L-carnitine on embryo development.
Experiment 6
Immature oocytes, matured oocytes and embryos pro- Fig. 1. Eect of L-carnitine on in vitro maturation of sheep oocytes
duced from 10 mM L-carnitine in maturation medium (p < 0.05)
Experiment 3
Eect of H2O2 exposure during oocyte maturation for
subsequent embryo development
The result (Fig. 4) of the experiment showed that up to
2.5 lM H2O2 in maturation medium, there was no
signicant (p < 0.05) change in cleavage (37.5639.66%)
percentage followed by further development to morula
(16.6620.88%), but the percentage of blastocysts (5.13
7.66%) was signicantly decreased at 2.5 lM H2O2. At
5 lM H2O2, there was signicant (p < 0.05) decrease in
Fig. 2. Eect of L-carnitine during in vitro maturation on embryo
development. Percentage results are presented as mean + SEM cleavage (29.43%), morula (6.93%) and blastocyst
(p < 0.05). Dierent superscripts in the same group dier signicantly (2.15%) percentage. At 10 lM H2O2, there was further
at p < 0.05. Six experiments were performed for each group signicant (p < 0.05) decrease in cleavage (18.12%) and
morula (2.13%) but blastocyst was nil, whereas at 20 lM
of H2O2 there was no cleavage at all.
Experiment 2
Eect of L-carnitine on intracellular ROS and GSH levels
in oocytes and embryos Experiment 4
L-carnitine-treated oocytes with subsequent embryos Antioxidant eect of L-carnitine on oocyte maturation
showed signicantly (p < 0.05) lower intensities for and subsequent development
ROS indicating decreased intracellular ROS, and To assess the antioxidant eect of L-carnitine on
signicantly (p < 0.05), higher intensities for GSH oocyte maturation and subsequent development,
indicating increased intracellular GSH as compare to oocytes were matured with 20 lM H2O2 with and
oocytes and embryos not treated with L-carnitine without 10 mM L-carnitine. There was no cleavage at
(Fig. 3A,B). concentration of 20 lM H2O2 but when 10 mM
(A)
a b c d
e f g h
(B)
Fig. 3. (A) Fluorescent photograph of in vitro matured sheep oocytes and embryo for ROS and GSH level. (a) ROS in oocyte mature with 10 mM
L-carnitine.(b) ROS in oocyte mature without 10 mM L-carnitine. (c) ROS in embryo developed from oocyte mature with 10 mM L-carnitine. (d)
ROS in embryo developed from oocyte mature without 10 mM L-carnitine. (e) GSH in oocyte mature with 10 mM L-carnitine. (f) GSH in oocyte
mature without 10 mM L-carnitine. (g) GSH in embryo developed from oocyte mature with 10 mM L-carnitine. (h) GSH in embryo developed
from mature without 10 mM L-carnitine. (B) Eect of L-carnitine on intracellular ROS and GSH level in oocytes and embryos. A: Control
oocytes/embryos; B: L-carnitine-treated oocytes; C: Embryos from L-carnitine-treated oocytes. Dierent superscripts in the same group dier
signicantly at p < 0.05. Three experiments were performed for each group
Discussion
The micromilieu during in vitro culture of preimplanta-
tion embryos is a critical determinant to the develop-
mental competence of the embryos. During embryo
development, ROS generated from embryos and their
surrounding cause defective embryo development
(Guerin et al. 2001). The present study was undertaken
to ameliorate OS in IVMFC protocol by L-carnitine
supplementation for better embryo development. Sub-
sequent studies were conducted to nd out the antiox-
idant eect of L-carnitine followed by intracellular GSH
and ROS level in oocytes and embryos due to
L-carnitine supplementation. L-carnitine-mediated alter-
Fig. 4. Embryo development in the presence and absence of H2O2
during maturation with and without L-carnitine (10 mM). Percentage
ations any in transcript level of antioxidant enzymes
results are presented as mean + SEM. Dierent superscripts in the (GPx, SOD1 and SOD2) were also analysed. To our
same group dier signicantly at p < 0.05. Six experiments were knowledge, this is the rst report describing L-carnitine-
performed for each group mediated alteration any in transcript level of antioxidant
SOD1 was expressed signicantly highest at 816 cells, considered as an indicator of oxidative stress in cells
and there were no signicant dierences in expression and embryos (He et al. 2004). Downregulated expres-
of SOD1 in rest of the developmental stages. Relative sion of SOD2 due to L-carnitine supplementation
expression level of SOD2 in both control and treatment might be due to the fact that L-carnitine helps in
groups was signicantly low among all the antioxidant transportation of long-chain fatty acid to mitochon-
enzymes studied in this experiment in a pattern of dria and synthesize adenosine triphosphate (ATP).
immature oocytes > morula > blastocyst > matured During ATP synthesis, a large quantity of oxygen is
oocytes >24 cells > zygote >816 cells. Highest utilized causing decrease in concentration of oxygen
upregulated expression of SOD2 was observed in and reduction in ROS formation that requires less
immature oocytes, and signicant downregulation SOD2 to neutralize free radicals causing downregu-
was at 816 cells as compared to all the rest of the lation of SOD2 gene (Gulcin 2006). L-carnitine might
developmental stages which were non-signicantly be acting as a buer for excessive acetyl groups in
dierent from each other. The antioxidant enzyme mitochondria, so there may be decrease in mitochon-
genes GPx, SOD1 and SOD2 in this experiment were drial superoxide production causing downregulation
expressed in all stages of developing embryos including of SOD2 gene expression (Lee et al. 2014). Our
immature and in vitro matured oocytes. Culture- nding on SOD2 expression due to L-carnitine
induced developmental arrest of sheep embryos is supplementation is in contrast to the previous report
normally observed at 816-cell (Gandol and Moor that L-carnitine supplementation showed no alter-
1987). At this stage, sheep embryos undergo transition ation in expression of SOD2 (Takahashi et al. 2013),
from maternal to embryonic genome control (Telford but Correa et al. (2008) has reported that high
et al. 1990). So maternal SOD2 mRNA might have oxygen concentration during culture upregulated the
degraded progressively during embryo development expression of SOD2 which is indirectly in accordance
because of which the level of transcript of SOD2 was to our nding because L-carnitine reduces oxidative
signicantly downregulated at 816 cells stage in our stress in micromilieu causing decrease in expression
study. of SOD2. As discussed earlier, expression of SOD2
To our knowledge, there is no such report available (mitochondrial SOD) is culture condition dependant
to nd out the eect of L-carnitine on expression (Lequarre et al. 2001) and oxidative stress aects
pattern of antioxidant enzyme genes in sheep oocytes mainly to mitochondria, so expression of SOD2 is
and embryos. The antioxidant enzyme genes expres- inuenced. In contrast, the relative expression of
sion is modulated by oxidative stress (Correa et al. SOD1 is not aected by L-carnitine supplementation
2008). Expression of these antioxidant enzyme genes which is might be due to the fact that oxidative stress
throughout the developmental stages indicates their during our in vitro culture condition might have not
inheritance from maternal pool mRNA. The intra- inuenced so much to alter SOD1 (cytoplasmic
cellular redox potential can modulate the activity of SOD).
some transcription factors, and ROS may activate the In conclusion, the results of the present study
antioxidant defence genes (Schultz 1993). L-carnitine demonstrated that L-carnitine supplementation during
supplementation in maturation medium signicantly in vitro sheep oocyte maturation improved develop-
upregulated the expression of GPx and downregu- mental potential of embryos by reducing ROS and
lated the expression of SOD2 in all stages of increasing GSH and alter the expression of antioxi-
developing embryos including matured oocytes, dant enzyme genes throughout the embryo develop-
whereas expression of SOD1 and GAPDH (reference ment.
gene) was not altered in any stage. GPx is considered
the major antioxidant enzyme within the glutathione
peroxidase family and its deciency renders cells Acknowledgements
more sensitive to stress that results in elevated The authors are thankful to the Director, National Institute of Animal
induction of apoptosis (Flentjar et al. 2002). GPx Nutrition and Physiology, Bangalore, India, to provide the necessary
depends on availability of reduced glutathione (GSH) facilities.
and acts in conjunction with GSH a tripeptide
cofactor for their enzymatic activity. GSH constitutes
a vital component of the cellular antioxidant system Conict of interest
(Mari et al. 2009). In our study, L-carnitine increased None of the authors have any conict of interest to declare.
the GSH level in oocytes and embryos. Therefore, L-
carnitine-mediated upregulation of GPx might be
also due to increase in cofactor GSH concentration. Author contributions
There is no in vitro study to support this nding, but
AM contributed towards experimental design, embryo culture work
an in vivo study reported that L-carnitine supplemen- and statistical analysis. IJR was involved in manuscript preparation
tation increased glutathione and glutathione peroxi- and gene expression study. PSP was involved in embryo culture work.
dase activity (Fatouros et al. 2010). SOD2 is SM carried out the gene expression study.
roundings. Hum Reprod Update 72, Reader KL, Cox NR, Staton JA, Juengel JL,
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Guerin P, El Mouatassim S, Menezo Y, the mouse preimplantation embryos: ish Mishra, NIANP, Bangalore 560030,
2001: Oxidative stress and protection BAT6 a new medium for improved cul- India. E-mail- ashishvet1@gmail.com
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