a-LIPOIC ACID: AN ANTIOXIDANT THAT IMPROVES MAMMALIAN
Donald W. Linck
B.A., University of Washington, 1998
M.B.A, University of Colorado Denver, 2008
A thesis submitted to the
University of Colorado at Denver
in partial fulfillment
of the requirements for the degree of
Masters of Chemistry
This thesis for the Master of Science
Donald W Linck
has been approved
Linck, Donald W. (M.S., Chemistry)
a-LIPOIC ACID: AN ANTIOXIDANT THAT IMPROVES MAMMALIAN
Thesis research directed by Professor Douglas F. Dyckes.
Oxidative damage caused by reactive oxygen species (ROS) has major
implications in cell culture. Interestingly, a-lipoic acid (LA) has been referred to as the
antioxidant of antioxidants due to its unique chemical properties, reductive power,
and involvement in aerobic metabolism. Therefore, the aim of this study was to
investigate the potential benefits of LA in culture media.
In this experimental study zygotes from CF1 outbred female mice were cultured
in G1/G2 (Series III) media in the presence or absence of LA (1, 10, 100 pM), under
high (20%) and low (5%) O2 tension. Blastocyst development was assessed after 72 h
and 96 h of culture. Blastocyst cell number and allocation were counted following
differential nuclear staining. Compared to control embryos, culture in 10 pM LA
significantly improved the percentage of embryos reaching the blastocyst stage after 72
h of culture at high O2. Blastocyst cell number and allocation were significantly
increased following culture in 10 pM LA at both high and low O2. Pyruvate uptake,
measured through a microfluorimetric technique, suggests that LA has no significant
effect on embryo metabolism. Using a fluorescent ROS indicator (H2DFFDA)
demonstrated that LA significantly reduces ROS production (in response to UV
exposure) in media and embryos.
The data presented here support the hypothesis that addition of LA, a potent
antioxidant, provides protection against oxidative stress and ROS production,
significantly improving embryo culture.
This abstract accurately represents the content of the candidates thesis. I recommend
I dedicate this work to my loving wife, Ashlee, and express my heartfelt thanks and
appreciation for her continued support, understanding, and motivation through and
I would like to express sincere gratitude and appreciation to Dr. David K. Gardner for
his guidance, direction, and supervision; both professionally and personally.
I would like to give endless thanks to Dr. Mark G. Larman for his scientific insight and
trouble-shooting expertise throughout the research.
TABLE OF CONTENTS
List of Figures..................................................................viii
List of Tables......................................................................x
2. Experimental Procedure..........................................................9
2.1 Lipoic Acid Preparation........................................................9
2.2 Embryo Collection and Cell Culture.............................................9
2.3 Cell Allocation...............................................................10
2.4 Intracellular and Extracellular ROS detection.................................12
2.5 Pyruvate Utilization..........................................................15
3.1 Cell Culture .................................................................19
3.2 Intracellular and Extracellular ROS detection.................................23
3.3 Pyruvate Utilization..........................................................26
5. Suggestions for Future Experiments.............................................34
LIST OF FIGURES
1. Structure and Enantiomeric Forms of Lipoic Acid.................................4
2. Example of chelate complex with R-(+)-aLA and divalent metal ion................6
3. Photo image of differentially stained hatching blastocyst......................12
4. Sample pyruvate calibration curve..............................................16
5. Calibrated constriction pipet..................................................17
6. Percentage Blastocysts Developed After 78 h of Culture in Different Concentrations
of Lipoic Acid in G1/G2 Media in 6% CO2 and two O2 Concentrations (5% and
7. Percentage Blastocysts Developed After 96 h of Culture in Different Concentrations
of Lipoic Acid in G1/G2 Media in 6% CO2 and two O2 Concentrations (5% and
8. Cell Allocation of Mouse Blastocysts After Culture for 96 h in Different
Concentrations of Lipoic Acid in G1/G2 Media in 6% C02 and Ambient Atmosphere22
9. Cell Allocation of Mouse Blastocysts After Culture for 92 Hours in Different
Concentrations of Lipoic Acid in G1/G2 Media in 6% CO2 and 5% O2.................23
10. Extracellular ROS Levels After 10 Minutes Exposure to UV Light for G-MOPS
Media Drops with Different Concentrations of Lipoic Acid in Ambient Atmosphere.. 24
11. Intracellular ROS Levels After 10 Minutes Exposure to UV Light for Embryos in
G-MOPS Media Drops with Different Concentrations of Lipoic Acid in Ambient
12. Fluorescent Readings for the Pyruvate Uptake of Embryos After 24 Hours of
Culture in G1 Media With and Without the Presence of 10 pM Lipoic Acid in Ambient
LIST OF TABLES
1. Standard reduction potentials at 298 K and pH = 7.0...............................5
2. Radical scavenging properties of lipoate-dihydrolipoate couple...................5
3. Metal chelation properties of lipoate-dihydrolipoate couple......................7
4. Statistical interpretation of Figure 11...........................................29
5. Embryo transfer results for blastocysts cultured for 96 h in G-series medium with and
without the presence of 10 pM Lipoic Acid in 6% C02 and Ambient Atmosphere...........32
6. Metal chelation properties comparison of lipoate-dihydrolipoate couple and EDTA34
It is widely recognized that oxidative damage caused by free radicals and
reactive oxygen species (ROS) has major implications in cell culture due to the
damaging effects on lipids, proteins, and DNA (Halliwell and Gutteridge, 1990). More
recently, the role of ROS during in vitro embryo culture has been investigated due to the
potential oxidative damage on spermatozoa, oocytes, and embryos, which may occur
during their manipulation, culture and cryopreservation (Nasr-Esfahani et al., 1990a;
Noda et al., 1991; Goto et al., 1992; Johnson and Nasr-Esfahani, 1994; Sharma and
Argawal, 1996; Blondin et al., 1997; Fujitani et al., 1997; Yang et al., 1998; Armstrong
et al., 1999; Takahashi et al., 2000; Guerin et al., 2001; Argawal et al., 2003, 2004,
2005, 2006; Sikka, 2004; Kitigawa et al., 2005; Bedaiwy et al., 2006; Choi et al., 2007).
Oocytes and pre-implantation embryos are exposed to numerous sources of
ROS. ROS can be generated as a by-product of normal oxidative phosphorylation as
well as other metabolic pathways that are active during embryo development such as
NADPH oxidase and xanthine oxidase (Nasr-Esfahani and Johnson, 1991; Lehninger,
2004). The production of ROS during mammalian embryo development increases in
ambient atmosphere culture conditions (Goto et al., 1993; Nagao et al., 1994). The
presence of reactive, metallic cations, such as Fe2+ and Cu2+, have also been shown to
accelerate the generation of ROS within cells through the Fenton and Haber-Weiss
reactions (Nasr-Esfahani, 1990b; Guerin et al., 2001; Argawal el al., 2006).
Spermatozoa produce high levels of ROS and initiate lipid peroxidation (de Lamirande
and Gagnon, 1995; Sharma and Agarwal, 1996). Additionally, cryopreservation
produces large amounts of ROS and decreases the levels of protective antioxidants
during the lengthy exposure of oocytes and embryos to high levels of cryoprotectants
(Bilodeau et al., 2000; Lane et al., 2002). Other potential sources of ROS include
elevated glucose levels (Karja et al., 2006) and exposure to light (Fischer et al., 1988;
Mauthe et al., 1995; Takahashi et al., 1999).
In situ, embryos are protected from ROS by various components. The
reproductive system of mammalian species is under low O2 tension (1-10%)
(Mastroianni and Jones, 1965; Ross and Graves, 1974; Mass et al., 1967; Fischer,
1993), which limits the number of oxygen molecules available for reactivity.
Furthermore, multiple in vitro research studies indicate that reduced oxygen levels are
beneficial for embryo development to the blastocyst stage (Nasr-Esfahani et al., 1992;
Evers et al., 1999; Orsi and Leese, 2001; Bavister, 2004; Bacheci et al., 2004; Choe et
al., 2007; Gardner, 2008; Nilsson et al., 2008). Additionally, it is now common practice
to supplement culture media with antioxidant compounds found in follicular and
oviduct fluids (Vermeiden and Bast, 1994; Tarin, 1995; Menezo and Guerin, 1995) such
as ascorbate (Alcain and Buron, 1994; Bonilla-Musoles et al., 1994; Lane et al., 2002;
Wang et al., 2002), EDTA (Orsi and Leese, 1991), glutathione (Gardiner and Reed,
1994; Gardiner et al, 1998), pyruvate (Orsi and Leese, 1991), superoxide dismutase
(SOD) (Alvarez et al., 1987; Mori et al., 1991; Nonogaki et al., 1992) and tocopherol
(Varda et al., 2001; Wang et al., 2002). However, despite major improvements in
culture media performance in the past decade, there is still much debate over the most
suitable antioxidant strategy to implement within a preimplantation embryo culture
There are multiple criteria a compound may possess to be an effective
antioxidant; radical scavenging, ability to accept electrons (reduction potential),
metabolic regeneration and metal ion chelation. Some of the most biologically
important antioxidants, vitamin E and ascorbate, are limited in their capabilities. For
example, it would seem that vitamin E is limited to scavenging peroxyl radicals only
within cell membranes due to its lipid-soluble characteristics. Ascorbate has questions
regarding its ability to re-generate once it has been oxidized to dehydroascorbate
(Halliwell, 1996b). Although glutathione is a well studied antioxidant and most likely
deserves its claim as the most important cellular antioxidant, the stores of reduced
glutathione are depleted rapidly when in the presence of oxidative stress and have an
impaired ability to regenerate its antioxidant defense system (Mesiter, 1992; Gardiner
and Reed, 1994; Hansen et al., 1999; Bilodeau et al., 2000). However, a-lipoic acid
(LA) has been referred to as the antioxidant of antioxidants due to its unique chemical
properties, reductive power, and involvement in aerobic metabolism (Wtodek and
LA is a disulfide, amphiphilic compound which contains a chiral center creating
two enantiomeric forms (Figure 1), with the most biologically active form being the R-
enantiomer (Hermann et al., 1996, Kramer and Packer, 2001). Further research (Smith
Main physiological form
Figure 1. Structure and enantiomeric forms of lipoic acid.
et al., 2005) has shown the S-enantiomer to be pharmacologically inactive in three
different cell culture systems. However, it has been proposed that the S-enantiomer
works in a synergistic fashion to aid in the reduction of the R-enantiomer when a
racemic mixture is present (Biewenga et. ai, 1997). Additionally, research has shown
the disulfide component provides the molecule with the majority of its ability to react
with the free radical electron within ROS that causes much of the damage to cells. The
dihydrolipoic acid/lipoic acid (DHLA/LA) couple has a reported redox potential of
-0.29 V (Clark, 1960) relative to a standard hydrogen electrode. This standard
reduction potential value gives the pair a greater affinity to acquire electrons in
comparison to some of the other known biological antioxidants (Table 1). From this
Table 1. Standard reduction potentials at 298 K and pH = 7.0. (Lehninger, p. 511, Table
Half-reaction E (mV)
V2 02 + 2H+ + 2e' H20 +816
FeJ+ + e ^ Fei+ +771
aTO + H+ + e' - aTOH +480
Dehydroascorbic acid + 2H+ + 2e' -> ascorbic acid +54
Pyruvate' + 2H+ + 2e~ -> lactate' -185
GSSG + 2H+ + 2e -> 2 GSH -230
aLA + 2H+ + 2e' ^ DHLA -290
NAD+ + H+ + 2e~ -> NADH -320
NADP+ + H+ + 2e - NADPH -324
table it is apparent that LA has the potential to recycle other reduced compounds.
Furthermore, the couples affinity to gain or lose electrons makes the molecule a
versatile radical scavenger unit (Jocelyn, 1967) (Table 2).
Table 2. Radical scavenging properties of lipoate-dihydrolipoate couple. (Noda et al,
Oxidant LA DHLA
H202 V V
02 1 -
OH V V
o2" - V
OOH - V
NO V V
HCIO V V
ONOO V V
The sulfide components also provide the molecule with divalent metal ion
chelating properties (Figure 2). This is an important property of the compound because
''' M '
Figure 2. Example of chelate complex with R-(+)-aLA and divalent metal ion.
chelators of divalent metallic ions have been shown to support the preimplantation
embryo in overcoming in vitro developmental arrest (Nasr-Esfahani, 1990b; Nasr-
Esfahani and Johnson, 1992; Nasr-Esfahani et al., 1992). Both LA and DHLA can
chelate heavy metals, but R-(+)-aLA is most effective in chelating Cu2+, Zn2+ and Pb2+.
LA can also form complexes with Mn2+ and Cd2+, but is unable to chelate Fe3+ (Sigel et
al., 1978; Sigel, 1997; Suh et al., 2004). In contrast, DHLA is able to chelate Fe3+
(Bonomi et al., 1985; Kawabata et al., 1995) as well as complex with Cu2+, Zn2+, Pb2+,
Co2+, Ni2+ (Sigel, 1997) and Hg2+ (Brown and Edwards, 1969; Suh et al., 2004).
However, the DHLA complexes are reportedly poorly soluble in water and difficult to
analyze at biological pH (Biewenga et al., 1997). Table 3 summarizes the metal
Table 3. Metal chelation properties of lipoate-dihydrolipoate couple. (Ou et al, 1995)
Metal LA DHLA Kf
Cd2+ V - 2.6 x 105
Co2+ - V 5.0 x 104
Cu2+ V V 1.1 x 10u
Fe3+ - V 3.3 x 102U
Hgz+ - V 1.8 x 10IV
Mn21 V - 6.5 x 105
Ni2+ - V 2.0 x 108
Pb2* V V -
Zni+ V V 7.8 x 10s
chelation affinity for the LA/DHLA couple where Kf is the formation constant and
represents the equilibrium constant for the formation of the metal-ligand complex in
aqueous solution. This constant is pH dependent and provides a measure of the ability
of the ligand (eg, lipoate) to bind a metal ion in comparison to water.
Lipoates amphiphilic properties allow the relatively small molecule
(MW=206.34 g/mol) to diffuse across both the mitochondrial and cell membranes
(Booker, 2004). Once in the cytosol or the media surrounding the cell, lipoate can be
converted to its dithiol, reduced form (DHLA) (Jones et. al., 2002, Booker, 2004)
giving the molecule its potential for both intra- and extra-cellular ROS protection.
However, lipoate pre-dominantly appears as the primary co-factor in the pyruvate
dehydrogenase complex (PDC); a vital aerobic step in energy metabolism (Lehninger,
2004). Additionally, previous research has shown that the developing pre-implantation
embryo shows a preference for pyruvate and lactate as its energy substrates during its
early-cleavage stages (Biggers et al., 1967; Gardner and Leese, 1986; Leese, 1991;
Gardner et al., 1996). Therefore, it would be reasonable to believe that introducing a
culture agent that may increase the activity of enzyme complexes involved in the
aerobic metabolism of pyruvate would improve early embryo development.
Despite LAs well-documented chemical versatility, there is little evidence, as
well as differing reports, about the effect of LA when added directly to in vitro embryo
culture media. The levels of ROS have been shown to increase during in vitro embryo
development, particuarly under atmospheric culture conditions (Goto et al., 1993;
Nagao et al., 1994). It was therefore the aim of this study to investigate the potential
benefits of adding LA to the embryo culture system. A defined concentration range of
LA was found to increase embryo development, including cell numbers. Further
investigation revealed that the beneficial affects caused by the addition of 10 pM LA
are likely due to a reduction of ROS within the embryo and its surrounding medium.
2. Experimental Procedure
2.1 Lipoic Acid Preparation
Lipoic acid (T1395; Sigma, St. Louis, MO, USA) was prepared as a 100 mM
stock solution in 95% ethanol. Final working equivalents (1, 10, 100 pM) of lipoate
were then added to pre-made culture media (G Series; Vitrolife Inc., Englewood, CO,
USA) using dilutions from this stock. A vehicle control (95% ethanol), equivalent to
the dilution of the highest concentration solution, was used in all culture experiments.
2.2 Embryo Collection and Cell Culture
All media were supplemented with 5 mg/mL human serum albumin (HSA)
unless otherwise stated. Zygotes were collected from 4-week-old CF1 outbred female
mice (Charles River Laboratories, Wilmington, MA, USA). Superovulation was
induced by an intraperitoneal injection of 5 iu pregnant mares serum gonadotropin
(PMSG; Sigma, St. Louis, MO, USA) followed 48 h later by 5 iu human chorionic
gonadotropin (hCG; Sigma, St. Louis, MO, USA). Following the administration of the
hCG hormone, females were placed with males of the same strain and mating was
confirmed by the presence of a vaginal plug the following morning. Cumulus-enclosed
zygotes were collected at 21 h post-hCG and denuded with 1 mg/mL hyaluronidase in
G-MOPS medium (Lane and Gardner, 2004). Collected embryos were then cultured for
a total of 96 h in groups of 10 in 20 pL drops of medium at 37 C, 6% CO2 and either
ambient air (20% O2) or low oxygen tension (5% O2) under oil overlay (Ovoil;
Vitrolife, Englewood, CO, USA). Embryos were placed into G1.2 medium (Gardner
and Lane, 2004) for the initial 48 h of culture and then moved to fresh G2.2 (Gardner
and Lane, 2004) medium for the remaining 48 h. All cultures were performed in 35x10
mm Petri dishes (Falcon; BD, Franklin Lakes, NJ, USA). To avoid variation between
females, embryos from each donor were pooled together and evenly distributed between
treatments. To monitor the rate of embryo development, embryo scoring was
performed at 78 h and 96 h post-zygote collection.
2.3 Cell Allocation
Differential nuclear staining was used to determine cell allocation to the Inner
Cellular Mass (ICM) or the Trophectoderm (TE) of expanded and hatching blastocysts
after 96 h in culture. The technique used was previously described (Gardner et al.,
2004), but is a modified protocol based upon methods used by Handyside and Hunter
(1984). Initially, blastocysts were incubated in 0.5% Protease (P5147; Sigma) solution
in G-MOPS (Gardner and Lane, 2004; MOPS buffered version of G1.2 medium used
for handling embryos outside of the incubator in ambient air) at 37C for up to 5
minutes in order to dissolve the zona pellucida. Secondly, blastocysts were incubated
for 10 min at 4C in 10 mM 2,4,6-trinitrobenzenesulfonic acid (P2297; Sigma) in 4
mg/ml polyvinyl alcohol in PBS (PBS-PVA). Thirdly, blastocysts were incubated in 0.1
mg/mL anti-dinitrophenyl-BSA conjugate (D9656; Sigma) in protein-free G-MOPS for
10 min at 37C. Next, a complement mediated lysis was induced by incubating the
blastocysts in guinea pig complement (SI639; Sigma) diluted 1:4 in protein-free G-
MOPS with 10 pg/mL propidium iodide (P4170; Sigma) for 5-10 min at 37C.
Blastocysts were then placed into a 25 pg/mL bisbenzimide (B2883; Sigma) solution in
ethanol overnight at 4C. The following morning, embryos were mounted in 100%
glycerol (G2025; Sigma) on a glass slide and overlaid with a coverslip. Finally, the
differential color of the nuclei was examined under ultraviolet light using an Olympus
AH-3 fluorescent microscope (Olympus Optical) with a 100W mercury lamp.
Excitation filter UG 1 (365 nm) was used in combination with barrier filter 41 (410 nm)
resulting in bisbenzimide-labelled ICM nuclei fluorescing blue and TE-nuclei stained
with both fluorochromes fluorescing pink (Figure 3). Classification of Pi-labelled TE
nuclei can be confirmed by viewing with interference green excitation filters (546 nm)
and barrier filter 59 (590 nm) which excludes blue fluorescence. Once the ICM and TE
cells were obtained for each embryo, and ratios of ICM to total number of cells were
calculated for individual embryos. These cell number parameters provide an indirect
measurement of embryo viability.
2.4 Intracellular and Extracellular ROS Detection
For this assay, the fluorinated analogue of 2,7-dichlorodihydrofluorescein
diacetate (H2DFCDA), was used to determine the concentration of generalized ROS
level within the embryo and medium (C13293; Molecular Probes, Eugene, OR, USA).
5-(and-6)-carboxy-2',7'-difluorodihydrofluorescein diacetate (carboxy-FFDFFDA)
exhibits improved photostability and carries additional negative charges that improve
retention over its parent molecule. Once inside the cell, the diacetate is deacetylated by
intracellular esterases producing the cell impermeable, non-fluorescent
difluorodihydrofluorescein (H2DFF). The non-fluorescent product will then react with
reactive oxygen species intracellularly to produce DFF; visualized by fluorescence at
525 nm when excited at 488 nm. Stocks of 20pM H2DFFDA were made with DMSO
and stored frozen (in the dark) under nitrogen until the day of the experiment.
Pronuclear oocytes were pooled and incubated in 20pM FI2DFFDA (in G-MOPS with
5mg/ml HSA under oil) for 45 mins at 37C in the dark. Pronuclear oocytes were
washed in G-MOPS and then divided into G-MOPS +/- lipoic acid (1, 10, 100 or
lOOOpM) for 30 mins. Pronuclear oocytes were then washed through G-MOPS (without
pyruvate or HSA) +/- lipoic acid. Pyruvate is a known antioxidant and was therefore
removed to reduce any masking of the antioxidant effects of lipoic acid. Protein was
removed since it is autofluorescent at the wavelengths used for imaging of H2DFFDA.
Pronuclear oocytes were imaged in a glass bottom dish (Fluorodish; WPI, Sarasota,
Florida). Groups of 10-12 pronuclear oocytes were placed in 5 pi droplet of G-MOPS
(without pyruvate or HSA) overlaid with oil and imaged using a 20x plan fluor (NA
0.45) objective on a Nikon TE2000-S microscope with a heated stage. Images were
captured and analyzed using MetaMorph software (Molecular Devices, Downingtown,
Pennsylvania, USA). Excitation light was provided by a high-pressure 100W mercury
lamp through a 480/30 (excitation/bandwidth) nm filter. Each image was obtained
through a 100ms exposures controlled by a mechanical shutter (Sutter Instruments,
Novato, California, USA). Fluorescence emission was collected by a Cascade 512B
CCD camera (Roper Scientific, Tucson, Arizona, USA) through a dichroic mirror
(505nm) and a 535/40 nm filter. An image of the pronuclear oocytes before and after
exposure to UV light was recorded. UV exposure for 10 minutes was carried out using a
350/50 nm excitation filter and 400 nm dichroic mirror. Regions of interest were drawn
around each of the pronuclear oocytes together with three background measurements.
For each experiment, regions for the pronuclear oocytes and background (before and
after UV exposure) were averaged and then subtracted. The background subtracted
values for the oocytes before and after UV exposure could then be divided to determine
the fold increase in fluorescent signal and therefore ROS level in the pronuclear oocyte.
Due to the photo-lability and sensitivity to oxidation of the dye, freshly diluted
H2DFFDA was used for each experiment and only one concentration of lipoic acid was
compared per experiment.
In order to quantitate the levels of generalized oxidative stress in culture
medium alone, the same imaging process was used as outlined above in the absence of
an embryo. Images of the same area as an embryo and referenced against the
background were taken before and after 10 minutes of UV exposure to determine the
fold increase in fluorescence and therefore ROS level in the medium.
2.5 Pyruvate Utilization
The microfluorimetric technique used to measure pyruvate uptake has been
previously described (Leese and Barton, 1984; Gardner and Leese, 1986; Garnder and
Leese, 1990; Gardner and Leese, 1999; Gardner et al., 2004). The fluorometric assay is
based upon the fluorescent capabilities of the pyridine nucleotides, NADH and
NADPH, which are known to fluoresce when excited with 340 nm light in contrast to
their respective oxidized products, NAD+ and NADP+, which do not fluoresce. Sample
measurements are calculated from a five-point, pyruvate standard linear curve based
upon the fluorescence range for those standards. Under experimentally controlled
conditions, the levels of decrease in fluorescence are proportional to the amount of
pyruvate consumed by the embryo in the following reaction:
Pyruvate + NADH + H
Lactate + NAD
Reagents: 0.075 mM NADH, 28 U lactate dehydrogenase/mL (EC 126.96.36.199), in EPPS
buffer (4-(2-hydroxyethyl)-l-piperazine propane-sulphonic acid), pH 8.0.
Initially, CF1 zygotes were co-cultured overnight (24 h) in G1.2 at 37 C, 6%
CO2 and air (20% O2) under oil overlay in the presence or absence of 10 pM LA.
Individual, 2-cell embryos were then transferred to 15 nL drop of a metabolic analysis
medium, buffered with MOPS in the presence or absence of 10 pM LA and incubated
for 3 hours at 37 C, 6% CO2 and air (20% O2) under oil overlay. The metabolic
analysis medium was formulated to contain 0.32 mM pyruvate, 0 mM lactate, and 0
mM glucose. This medium ensured the embryo would metabolise pyruvate as its sole
energy substrate for the allotted time; allowing for an indirect measurement of the
embryos ability to convert pyruvate to Acetyl-CoA in the absence or presence of
lipoate. Serial 1-nL samples were taken from each drop at 60-min intervals and
pyruvate concentrations were determined based upon results from 5-point standard
curve (Figure 4). Linear rates of pyruvate uptake were then determined for individual
embryos and expressed as pmol/embryo per hour.
0 ___ r ___________ ___________ , ___ ___________ ________
0.06 Q 12 0.25 0.50 1 00
Pyruvate Concentration (mM)
Figure 4. Sample pyruvate calibration curve.
The submicroliter volumes necessary for this assay are created using specially
constructed constriction micropipettes (Figure 5) pulled from borosilicate glass
capillaries (GC100T-15, Harvard Apparatus Ltd., UK). The micropipettes are
calibrated using tritiated water in a triplicate measurement of radioactivity that falls
within a 95% confidence interval (Gardner, 2007).
Figure 5. Calibrated constriction pipet.
Cell number, ROS, and embryo transfer data were subjected to Least Squares
analysis of variance (ANOVA) and Student Mest. Blastocyst development data were
compared relative to the control group. When comparing each treatment to the control
group, an F-test was performed initially to test for equality of variances. Statistical
comparisons were then made using the Student Mest. Statistical comparisons were
done with InStat (GraphPad, San Diego, CA). No between-replicate differences were
detected and data from all experiments were subsequently pooled for analysis. Pyruvate
uptake data was assessed by analysis of variance, followed by the Bonferroni procedure
for multiple comparisons. Differences were detected by least squares means. A
probability value of P < 0.05 was considered to be significant.
3.1 Cell Culture
Mouse embryos were grown in sequential G-media from the pronuclear oocyte
to blastocyst stage in the absence or presence of LA (1,10 and lOOpM) at high (20%)
(control, n=202; vehicle, n=216; 1 pM, n=198; 10 pM, n=231; 100 pM, n=201) and low
(5%) O2 (control, n=205; vehicle, n=209; 1 pM, n=209; 10 pM, n=215; 100 pM,
n=206). There was no difference noticed between the control and vehicle (ethanol)
groups in any treatments throughout the study, indicating that the percentage of lipoic
acid solvent used for these experiments did not impact on embryo development. The
dose-dependent effect of LA on embryo development after 78 h and 96 h post-zygote
collection is shown in Figures 6 and 7, respectively.
High (20%) 02
Low (5%) 02
0 1.0 10 100
Lipoic acid concentration (pM)
Figure 6. Percentage Blastocysts Developed After 78 h of Culture in Different
Concentrations of Lipoic Acid in G1/G2 Media in 6% CO2 and two O2 Concentrations
(5% and Ambient Atmosphere).
Lipoic acid concentration (pM)
High (20%) 02
E3 Low (5%) 02
Figure 7. Percentage Blastocysts Developed After 96 h of Culture in Different
Concentrations of Lipoic Acid in G1/G2 Media in 6% CO2 and two O2 Concentrations
(5% and Ambient Atmosphere).
The data in Figure 5 show the percentage of embryos reaching the blastocyst
stage by 78 h of culture at high and low O2. The addition of 1 pM LA did not improve
embryo development to the blastocyst stage in comparison to the control (high O2:
control 27.2%; lpM LA 37.4%, low O2: control 61.4%; lpM LA 64.7%). Culture in
the presence of 10 pM LA significantly improved blastocyst development in
comparison to the control in a high oxygen culture environment only (high O2: control
27.2%; lOpM LA 41.7%, low O2: control 61.4%; lOpM LA 68.2%). In contrast, the
addition of 100 pM had a negative impact on blastocyst development, significantly
reducing the number of blastocysts compared to the control group (high O2: control
27.2%; lOOpM LA 9.1%, low 02: control 61.4%; lOOpM LA low 02: 38.8%).
No significant increase in the percentage of embryos reaching the blastocyst
stage was observed at high or low O2 following 96 h of culture (Figure 6), although
there was a trend for lOpM to support development of more blastocysts (high O2:
control 64.4%; lOpM LA 70.8%, low 02: control 73.7%; lOpM LA 82.8%). The
negative impact of 100 pM on blastocyst development continued through 96 h of
culture, significantly reducing the number of blastocysts compared to the control group
(high O2: control 64.4%; lOOpM LA 42.8%, low O2: control 73.7%; lOOpM LA
As an indicator of embryo viability, differential cell staining was performed to
determine total cell numbers and allocation to the trophectoderm (TE) and inner cell
mass (ICM). The data shown in Figure 8 represent the cell number and allocation for
all treatments after culture for 96 h at high O2 in an oil overlay.
Figure 8. Cell Allocation of Mouse Blastocysts After Culture for 96 h in Different
Concentrations of Lipoic Acid in G1/G2 Media in 6% CO2 and Ambient Atmosphere.
The addition of 1 pM LA had no impact on cell number or allocation in
comparison to the control (control TE 51.2 2.5, ICM 13.3 0.7; lpM LA TE 55.3
4.0, ICM 13.1 1.2). The presence of 10 pM LA significantly increased total cell
numbers with increased cells within the TE and ICM compared to the control (lOpM
LA TE 61.1 2.3, ICM 16.5 0.7). In contrast, total cell number and cell allocation
were significantly decreased during culture in the presence 100 pM LA (lOOpM LA TE
43.7 3.2, ICM 10.3 1.0). A similar trend was observed at low O2 (Fig. 9). The
I I Trophectoderm
0 1 10 100
Lipoic acid concentration (pM)
Figure 9. Cell Allocation of Mouse Blastocysts After Culture for 96 h in Different
Concentrations of Lipoic Acid in G1/G2 Media in 6% CO2 and 5% O2.
presence of 10 pM LA significantly increased total cell numbers with increased cells
within the TE and ICM compared to the control (control: TE 71.9 2.5 ICM 20.2 0.8;
lOpM LA: TE 79.3 2.1, ICM 22.5 0.8).
3.2 Intracellular and Extracellular ROS Detection
The data presented in Figure 10 represent changes in extracellular (i.e. media
drops containing H2DFFDA) ROS levels in response to 10 minutes of UV light
exposure in the presence of increasing concentrations of LA. There was no significant
0 1 10 100 1000
Lipoic acid concentration (pM)
Figure 10. Extracellular ROS Levels After 10 mins Exposure to UV Light for G-MOPS
Media Drops with Different Concentrations of Lipoic Acid in Ambient Atmosphere.
difference measured between the control and vehicle, indicating that the percentage of
ethanol used for these experiments did not impact on the measurements (control 7.5
0.4; vehicle 7.4 0.5). The presence of LA significantly reduced the ROS level in the
media drops compared to the control (lpM 5.8 0.4; lOpM 4.7 0.4; lOOpM 3.5 0.3;
ImM 2.4 0.2).
Figure 11 shows the levels of generalized ROS when embryos placed in
different concentrations of LA are exposed to 10 minutes of UV light exposure.
Embryos that had been loaded with H2DFFDA to report intracellular ROS levels were
incubated for 30 minutes in the presence or absence of LA before being exposed to UV
0 1.0 10 100 1000
Lipoic acid concentration (pM)
Figure 11. Intracellular ROS Levels After 10 mins Exposure to UV Light for Embryos
in G-MOPS Media Drops with Different Concentrations of Lipoic Acid in Ambient
light. There was no difference measured between the control and vehicle, indicating that
the percentage of ethanol used for these experiments did not impact on the
measurements (Control 13.5 0.7; Vehicle 13.2 0.8). The presence of lpM LA did not
significantly reduce the ROS level in the embryos compared to the control (lpM 13.3
0.6). However, the presence of 10pM, lOOpM, and ImM significantly reduced the
levels of ROS in the embryos relative to the control (lOpM 8.6 1.6; lOOpM 8.3 1.3;
ImM 8.6 0.3).
3.3 Pyruvate Utilization
The results shown in Figure 12 represent the uptake of pyruvate by CF1 2-cell
embryos cultured in the absence or presence of lOpM LA. After 3 h of exposure to the
0.28 0.30 0.32
Pyruvate Concentration (mM)
Figure 12. Fluorescent Readings for the Pyruvate Uptake of Embryos After 24 h of
Culture in G1 Media With and Without the Presence of 10 juM Lipoic Acid in Ambient
metabolic assessment medium containing 0.32 mM pyruvate, CF1 2-cell embryos
cultured in the presence of lOpM LA showed no significant changes in their linear rate
of pyruvate uptake compared to the control embryos (Control 1.3 0.3; 1 OpM LA 0.8
Previous research has shown that the levels of ROS, both intra- and
extracellularly, increase in the mammalian preimplantation embryo through various
mechanisms. It is thought that increased levels of glucose will increase the levels of
ROS within the embryo through increased activity of oxidative phosphorylation (Iwata
et al., 1998; Machaty et al., 2000; Thompson et al., 2000) and purine metabolism
(Nureddin et al., 1990). Additionally, intracellular sources of ROS may occur from
various metabolic enzymes (Guerin et al., 2001), such as NADPH oxidase (Manes and
Lai, 1995) and xanthine oxidase (Alexiou, 1982; Nasr-Esfahani and Johnson, 1991).
Extracellular ROS levels are thought to increase due to, but not limited to: elevated
oxygen tension (Goto et al., 1993; Nagao et al., 1994), exposure to light (Goto et al.,
1993; Nakayama et al., 1994; Takenaka et al., 2007), presence of metallic cations
(Nasr-Esfahani et al, 1990), and the presence of spermatozoa during insemination
(Aitken, R. et al., 2001; Saleh, A., et al., 2002; Aziz, A., et al., 2004).
Since experiments within this study looked at increased ROS levels due to
increased oxygen tension and exposure to UV light, then it is presumed that LAs
beneficial effects were due to the compounds ability to decrease the levels of
exogenous ROS. LAs effectiveness at reducing the levels of ROS, both intra- and
extracellularly, is thought to be attributed to the compounds strong reducing qualities.
Both LA and its reduced form, DHLA, have been shown previously to be effective ROS
scavengers (Smith et al., 2004). Although the reduction of LA is presumed to take
place within cells, the reduced form of the compound that is generated leaks from the
cells into the surrounding medium implying both intracellular and extracellular
antioxidant capabilities (Handelman, 1994). A theoretical Nernst equation calculation
under physiological equilibrium conditions and 37 C suggests that the addition of 10
pM LA produces ~4 pmol DHLA (Appendix A). Additionally, the data from Table 4
Table 4, Statistical interpretation of Figure 11.
Pyruvate Uptake n = Pyruvate Utilization Rate SEM p-value
Control 15 1.263 0.238 -
+10 pM Lipoate 15 0.794 0.542 0.451
suggest that the addition of LA to the medium does not have an effect on the PDH
complex as manifest by no increase in pyruvate utilization by the embryos. However,
LA has been shown to inhibit xanthine oxidase activity within cells (Packer, 1995), so
may be involved with intracellular metabolic enzyme activity.
Previous reports have shown conflicting results on the addition of lipoic acid to
culture media (Bavister, 2000; Kane, 1988; Volubueza, 2005). In initial experiments,
lipoate was added to Hams F-10 in concentrations an order of magnitude lower than this
study. In contrast, Volubueza and colleagues (2005) using epithelial cells required 20-
50 times more lipoate to observe an effect. Thus, indicating the higher degree of
sensitivity associated with preimplantation embryos compared to somatic cells.
However, recent research shows that the addition of the antioxidant L-carnitine in
millimolar ranges improves murine preimplantation embryo development (Abderlazik
et al., 2008). Unfortunately, all anti-oxidants are a biological double-edged sword
(Argawal, 2003) because they will exhibit pro-oxidant behavior when present at
inappropriately high concentrations. This is partly the reason why determining the most
effective range of an anti-oxidant can be difficult. Having determined a lipoate
concentration that improves embryo development and increases cell numbers, three
potential mechanisms of action were investigated; intracellular and extracellular
protection against ROS and an effect on carbohydrate metabolism. The results from
this study demonstrate that the addition of lOpM LA improves murine pre-implantation
embryo development in ambient air through increased blastocyst development and
increased cell allocation. The results presented here suggest that this may be attributed
to effective ROS scavenging within extracellular and intracellular domains and not from
a metabolic rate increase in pyruvate uptake.
Furthermore, the data in these experiments suggest that LAs pro-oxidant
properties are detected at a concentration of lOOpM within the pre-implantation embryo
model. Research has shown that other antioxidants, such as Vitamin C, Vitamin E, and
glutathione (GSH), may also demonstrate pro-oxidant behavior (Halliwell, B., 1996b;
Weinberg, R.B. et al., 2001; Joshi, S. et al., 2005; Horsley, E.T. et al., 2007).
Theoretically, since the chemical product of any oxidized antioxidant is a radical, it has
been proposed that these three anti-oxidants exhibit pro-oxidant behavior due to their
inability to re-cycle once they have been oxidized (Halliwell, B., 1996b).
Consequently, since glutathione (GSH) has been labeled the bodys most important
antioxidant and plays a crucial role during gamete development, then introducing a
strong thiol reducing agent such as lipoate, which stimulates GSH synthesis (Han, D. et
al., 1997), into the culture environment may increase intracellular GSH concentrations
and improve embryo development (Luberda, 2005; Gardiner and Reed, 1994).
Attempts were made to measure the changes in intracellular GSH through the use of
monochlorobimane (data not shown); however, due to the probes non-selectivity for
GSH as opposed to other thiol compounds, it became apparent that lipoates thiol-
reactivity was hindering the probes success.
LA is universally present in prokaryotes and eukaryotes (Carreau, 1979).
Lipoate, the unprotonated base, is the most prevalent form at physiological conditions
and is thought to be synthesized within the cell by lipoic acid synthetase from the
precursors octanoic acid and a sulfur source; most likely a cysteine residue (Carreau,
1979; Miller et al., 2000; Zhao et al., 2003; Cicchillo et al., 2004, 2005). This makes
the molecule a non-essential nutrient, but LA has been reported to be a valuable dietary
supplement in diseases associated with excessive oxidative stress, including arthritis,
diabetes, atherosclerosis and Alzheimers (Evans and Goldfine, 2000; Bilska and
Wlodek, 2005). Endogenous lipoate production has been reported to be essential for
early embryonic development (Yi, 2005). Furthermore, lipoates safety evaluation by
Cremer and colleagues (2006) suggest the potential dangers associated with the addition
of alpha-lipoic acid are non-existent. Preliminary embryo transfer data during these
experiments support Cremers findings as there were no fetal abnormalities observed
Table 5. Embryo transfer results for blastocysts cultured for 96 h in G-series medium
with and without the presence of 10 pM Lipoic Acid in 6% CO2 and Ambient
Treatment n = Implantation Rate Fetal Development Rate Fetal Weight (mg) SEM (mg) p-value
Control 30 36.7% 50.0% 284.3 22.3 -
+10 pM Lipoate 24 50.0% 33.3% 273.1 34.1 0.642
Since oxidative stress is pre-eminent throughout in vitro embryo development,
continued research must focus on limiting the embryos exposure to ROS. Simply
adding necessary ROS scavengers is a multipart problem due to difficulties in
determining useful concentrations of different compounds and their potential for
deleterious effects as pro-oxidants. The data from this study suggests that the addition
of LA may help to create a more suitable antioxidant defense system within
preimplantation mammalian embryo culture systems through its effective intracellular
and extracellular ROS scavenging abilities.
5. Suggestions for Future Studies
Although the data presented in these studies suggest that the presence of 10 pM
LA is advantageous for the murine preimplantation embryo, further studies are
necessary to ascertain the molecules full potential or otherwise. Early research from
the 1990s shows that certain strains of mice are susceptible to the 2-cell block
without the presence of chelating agents like EDTA, etc. Since LA is reported to have
metal chelating properties similar to EDTA for certain metal ions like Cu2+, Fe3+, and
Hg (Table 6), then a simple hypothesis would be to substitute LA for EDTA in a
simple, salt solution media devoid of amino acids to see if it may alleviate the 2-cell
block in an outbred strain of mice.
Table 6. Metal chelation properties comparison of lipoate-dihydrolipoate couple and
DTA. (Martel! and Smith, 1974; Ou et al, 1995)
Metal LA DHLA Kf EDTA Kf
OP V - 2.6 x ltE V 1.6 x 1011
Co2+ - V 5.0 x 104 V 1.0 x 10Jb
Cu2+ V 1.1 x 10IJ V 5.0 x 1018
FeJ+ - V 3.3 x 102u V 1.7 x 1024
- V 1.8 x 10ly V 6.3 x 1021
MrP V - 6.5 x 105 V -
nP - V 2.0 x 10* V 3.6 x 1018
Pb2+ V V - V 2.0 x 10IS
Zn2+ V V 7.8 x 10s V 3.0 x 10'6
Studies to gain additional quantitative insight into lipoates anti-oxidant
properties on the embryo are also advised. The first of such supplemental studies
should involve lipoates strong reduction potential and theoretical ability to regenerate
other oxidized molecules, notably ascorbate radical, tocopherol radical, and glutathione.
Additionally, advances in incubator technology have created real-time videography of
cultured embryos, with internal laboratory data showing delays in cell division timings
of 20% oxygen vs. 5% oxygen environments. This proposes the hypothesis that the
addition of lipoic acid to culture media may reduce the cell division delays seen in 20%
oxygen cultures. Furthermore, if there are specific house-keeping genes involved
with oxidative metabolism of the murine pre-implantation embryo, then perhaps the
addition of lipoate to the medium may regulate the expression of these genes.
Although the metabolism study done in this research shows non-significant
results, the trend in the data suggests that further research is necessary. The replicate
sample size is small, so if more embryos were analyzed, perhaps significance would
appear. Additionally, one potential enhancement to the metabolic assay would be to use
radio-labelled pyruvate. This would be a more precise alternative because exact
quantities and endpoints of the carbon molecules could be tracked. Further
collaborations could allow for the proteomic profile of individual embryos (Katz-Jaffe
et al 2005) to be studied as well, which could aid in determining lipoates effect on
Finally, more recent research suggests the damaging effects of cryopreservation
techniques on embryo development due to oxidative stress and the generation of ROS.
Since cryopreservation solutions typically contain strong solvent molecules such as
dimethyl sulfoxide (DMSO) and propanediol (PrOFI), then the addition to lipoate to
these solutions would not require an ethanol carrier in solution like the culture media.
This could potentially increase lipoates bioactivity and aid in the embryos ability to
combat the oxidative stress associated with cryopreservation.
In summary, lipoates unique chemical structure and properties make the
molecule an intriguing antioxidant supplement. The early findings from the studies in
this paper demonstrate the compounds ability as an effective combatant of oxidative
stress in the preimplantation murine embryo model, both intracellularly and
extracellulary. However, further studies are necessary in order to discover the
molecules full potential or otherwise.
Nemst equation calculation for 10 pM LA at 37C and pH = 7.2.
Half-reaction for LA: aLA + 2H+ + 2e -> DHLA where E = -0.29 V
E = E + (RT/nF)*ln([electron acceptor]/[electron donor])
Universal Gas Constant (R) = 8.314 J.K'VmoF1
Faradays Constant (F) = 96485 C.moF1
Temperature (T) = K = C + 273.15 = 37 + 273.15 = 310.15
n = # of electrons = 2
Reaction is at equilibrium, so E = 0.
Racemic mixture of LA is equivalent to HALF starting concentration, so
[LA] = 5 pM.
Therefore, the equation now looks like:
0 = -0.29 V + ((8.314 J.K-'.mol'1 310.15 K)/(2 96485 C.moF1)) ln([LA]/[DHLA])
0 = -0.29 V + (0.0134 V) ln([DHLA]/[5 pM])
21.64 = ln([5 pM]/[DHLA])
[DHLA] = -1.90 pM
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