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Theriogenology 68 (2007) 223–236

Respiration rates correlate with mRNA expression of G6PD and

GLUT1 genes in individual bovine in vitro-produced blastocysts

A.S. Lopes a,b,*, C. Wrenzycki c, N.B. Ramsing d, D. Herrmann c,H. Niemann c, P. Løvendahl a, T. Greve b, H. Callesen a

a Department of Genetics and Biotechnology, Faculty of Agricultural Sciences, University of Aahrus, DK-8830 Tjele, Denmarkb Department of Large Animal Sciences, Faculty of Life Sciences, University of Copenhagen, DK-1870 Frederiksberg C, Denmark

c Department of Biotechnology, Institute for Animal Breeding, Federal Agricultural Research Centre (FAL), Mariensee, 31535 Neustadt, Germanyd Unisense FertiliTech A/S, DK-8200 Skejby, Denmark

Received 2 February 2007; received in revised form 23 April 2007; accepted 25 April 2007

Abstract

Quantification of embryo respiration is a promising procedure to assess embryonic metabolism and possibly select viable

embryos. At the blastocyst stage, ATP is produced by glycolysis and oxidative phosphorylation, processes that require uptake of

oxygen and glucose, which is regulated by the expression of GLUT1 and G6PD. The purpose of the present study was to investigate

the relationship between respiration rates and relative abundances of G6PD and GLUT1 transcripts in individual bovine blastocysts

produced in vitro. Respiration rates of 104 bovine in vitro-produced blastocysts were measured individually using the

nanorespirometer technology. Real-time RT-PCR was employed to determine the relative abundance of G6PD and GLUT1

mRNA in individual embryos. The mean respiration rates were similar for male and female blastocysts of the same developmental

stage, but the sex ratio was skewed towards males. GLUT1 expression was down-regulated in female versus male embryos. In

contrast, a �1.8-fold increase in the expression of G6PD mRNA was observed in female blastocysts when compared to male

blastocysts, indicating that dosage compensation for this gene had not yet occurred. Both GLUT1 and G6PD expression levels were

affected by morphological quality and stage of development. Expression of GLUT1 and G6PD mRNAs was correlated with

respiration rates, indicating that, in metabolically active blastocysts, uptake of oxygen and glucose are jointly increased. These

findings suggest that expression of genes for oxidative phosphorylation and glycolysis are both involved in oxygen demanding ATP

production.

# 2007 Elsevier Inc. All rights reserved.

Keywords: Respiration; GLUT1; G6PD; Oxygen; Bovine embryo

1. Introduction

Assessment of embryo viability is crucial for the

successful establishment and maintenance of pregnancy

* Corresponding author. Current address: Research Centre for

Reproductive Health, Department of Obstetrics and Gynaecology,

The University of Adelaide, 2nd Floor, Medical School South, Ade-

laide 5005, SA, Australia. Tel.: +61 8 83038188; fax: +61 8 83034099.

E-mail address: ana.sousalopes@adelaide.edu.au (A.S. Lopes).

0093-691X/$ – see front matter # 2007 Elsevier Inc. All rights reserved.

doi:10.1016/j.theriogenology.2007.04.055

following embryo transfer, both in human and livestock

species [1–4]. Usually, single embryos are transferred in

cattle breeding and commercial herds, and elective

single embryo transfer is increasingly applied also in

human IVF clinics [5]. Thus, it is essential to select and

transfer only good quality embryos in order to increase

in vivo development.

Morphological evaluation based on light micro-

scopic observation is still the most commonly used

method to assess embryo quality [2,6–8]. However, this

A.S. Lopes et al. / Theriogenology 68 (2007) 223–236224

type of quality evaluation is subjective and requires

specialized training [2,9,10]. To overcome these limita-

tions, several approaches for embryo quality evaluation

have been proposed in the past few years including

analysis of the kinetics of embryonic development [11–

13], gene expression profiling [14–16] and determination

of embryo metabolism [17,18].

The energy metabolism of the embryo has been

subjected to conspicuous evaluation, and considerable

evidence has accumulated that embryonic metabolic

activity may be used to predict embryonic develop-

mental potential [9,19–27]. At the blastocyst stage, ATP

is generated by glycolysis and in particular oxidative

phosphorylation [28–30], processes that ultimately

require oxygen [31]. The oxygen consumption of

murine [23,25,27,32,33], bovine [28,34–36] and por-

cine [37] embryos have been previously assessed and a

relationship between embryonic oxygen consumption

and viability following transfer has been earlier

described [36,38–40].

Oxygen consumption of individual embryos increases

significantly around compaction and blastulation [25,28,

33,41,42], and is associated with a higher ATP demand to

accommodate increased protein synthesis and activity of

the Na+/K+-dependent ATPase [28,43]. Embryonic

oxygen consumption is significantly affected by culture

conditions, specifically by the glucose [20], serum [44],

amino acids and protein content in culture medium

[45,46] or by oxygen incubation conditions [47]. Oxygen

consumption is higher among embryos of superior

quality [32,36,48–50], but large variations in respiration

rates within each class of morphological quality have

been reported [34,35,50]. Embryonic diameter is some-

what related to respiration as bovine embryos of greater

size have higher respiration rates [35,50]. The stage of the

bovine blastocyst has also been associated with oxygen

consumption, with embryos of more advanced stages

showing higher oxygen consumption [37,51–52]. Over-

all, embryonic sex does not seem to influence respiration

rates [32,35] but the oxygen consumption of good quality

female bovine embryos has been reported to be higher

than that of good quality male embryos. [32].

Glucose is an important energy substrate for the

development of bovine embryos, particularly during

the post compaction period [53]. After entering the

embryonic cells via facilitative glucose transporters

(GLUT), glucose can be metabolized through the

glycolytic pathway or may enter the pentose phosphate

pathway (PPP). The metabolism of glucose through

glycolysis generates pyruvate, which can either be

transformed to lactate or to acetyl CoA that in turn can

be further metabolized by the tricarboxylic acid cycle

and the oxidative phosphorylation pathway. Concomi-

tant with compaction and blastulation, glucose uptake

and metabolism are substantially increased [22,28,

33,41,54]. Despite this increase, the glycolytic pathway

contributes little to ATP production as most of the

glucose is primarely converted to lactate, which is an

ineffective source of ATP production. In fact, glycolysis

only contributes 12, 15 and 28% to the total ATP

produced by mouse, bovine and human blastocysts,

respectively [29]. Glucose uptake tends to be higher

in female as compared to male embryos [19,55].

Furthermore, glucose uptake was shown to be higher in

morphologically normal than in degenerated embryos

[56] and in blastocysts with an increased viability

following transfer to a recipient animal [19,55,57].

Glucose-6-phosphate dehydrogenase gene (G6PD)

maps to the X-chromosome [58] and encodes the rate-

limiting enzyme of the PPP, which is crucially involved

in the metabolism of glucose-6-phosphate. The PPP is

activated during development and generates nicotina-

mide adenine dinucleotide phosphate (NADPH) for

biosynthesis of steroids and fatty acids. It also provides

cells with ribose-5-phosphate for the synthesis of

nucleotides and nucleic acids [59]. Through the

NADPH generation in the PPP, G6PD also participates

in the detoxification of oxygen radicals [60,61] and

therefore has been considered a cytoprotective enzyme

for oxidative stress and DNA damage [61]. The relative

abundance (RA) of G6PD has shown to be nearly two-

fold higher in female pre-implantation bovine embryos

produced in vitro as compared to their male counter-

parts [62–64]. This phenomenon has been associated

with alterations in dosage compensation under in vitro

conditions, likely induced by problems or delay in X-

chromosome inactivation. The high levels of G6PD

were proposed to be responsible for the delayed

development of in vitro female embryos by mediating

NADPH production and thus modulating radical levels

[62,65].

GLUT1 belongs to the family of facilitative glucose

transporters and is expressed after in vitro maturation

of the oocyte [16]. Expression levels of GLUT1 are

significantly higher in the trophectoderm cells than in

the inner cell mass cells of bovine embryos [66], and

within the trophectoderm cells GLUT1 is specifically

found in the lateral membranes. GLUT1 expression

increases with increasing glucose uptake by the embryo

[67,68]. In vitro culture conditions, such as the oxygen

tension [42,69], the presence or absence of serum

[67,70] and the levels of glucose in the culture medium

[71] were shown to affect the expression of GLUT1.

This transcript is down-regulated in in vitro-produced

A.S. Lopes et al. / Theriogenology 68 (2007) 223–236 225

bovine embryos as compared to their in vivo counter-

parts [70,72,73].

The present study investigated the relationship

between individual respiration rates of day 7 bovine

in vitro-produced blastocysts and the relative abundance

of GLUT1 and G6PD mRNA transcripts. To gain a

better understanding on whether genetic patterns can

be reflected at the phenotypic level, we evaluated the

effects of morphological quality, stage of embryonic

development, sex and embryo diameter on the GLUT1

and G6PD transcriptional activity. Preliminary results

of this work have been presented earlier [74].

2. Materials and methods

2.1. Experimental design

A total of 750 bovine oocytes were used in this study

(50 oocytes per replicate with 15 independent repli-

cates). Oocytes were submitted to in vitro production

(IVP) and from the resulting embryos, 5 or 10

blastocysts were randomly selected for subsequent

assessment of oxygen consumption. On each day of

measurement, the respiration rate of 5 (one rosette/day)

or 10 (two rosettes/day) day 7 embryos produced in

vitro were measured in duplicate. In total, the

respiratory activity of 104 embryos was assessed

during 15 experimental days. The measured embryos

were classified in six classes, according to the stage of

embryonic development: not expanded (n = 16), early

expanded (n = 20), expanded (n = 35), late expanded

(n = 22), collapsed (n = 5) or hatching (n = 6) blas-

tocysts. Sex diagnosis and gene expression analysis

were performed on 81 embryos. The RA of GLUT1 and

G6PD transcripts was not considered for 3 embryos due

to insufficient amount of DNA of the samples. Oxygen

measurements on 4 embryos were regarded as non-

consistent and these results were not included in the

analysis. Furthermore, 3 embryos with extreme values

for RA of G6PD or respiration rate were considered as

outliers and thus not included in the analysis. Thus, only

71 embryos were included in the full statistical analysis

to evaluate the relationship between respiration rate,

embryo morphology, sex, stage of development and

abundance of GLUT1 and G6PD transcripts.

2.2. In vitro embryo production (IVP)

The method used for IVP has been reported

previously [75]. Briefly, bovine immature cumulus-

oocyte complexes (COCs) were aspirated from slaugh-

terhouse ovaries, selected (i.e. excluding denuded

COCs and COCs with clear degenerative signs) and

matured for 24 h in four-well dishes (Nunc, Roskilde,

Denmark). Each well contained 400 ml of bicarbonate

buffered TCM-199 medium (Gibco BRL, Paisley, UK)

supplemented with 15% cattle serum (CS; Danish Food

and Veterinary Institute, Frederiksberg, Denmark),

10 IU/ml eCG and 5 IU/ml hCG (Suigonan Vet; Intervet

Scandinavia, Skovlunde, Denmark). The oocytes were

matured under mineral oil at 38.5 8C in 5% CO2 in

humidified air. Fertilization was performed in modified

Tyrode’s medium [76] using frozen–thawed, Percoll-

selected sperm. After 22 h, cumulus cells were removed

by vortexing and presumptive zygotes were transferred

to 400 ml of culture medium composed of synthetic

oviduct fluid (SOF) medium containing amino acids,

citrate and inositol (SOFaaci [75]) supplemented with

antibiotics (gentamycin sulphate, 50 mg/ml) and 5%

CS, and subsequently incubated at 38.5 8C in 5% CO2,

5% O2, 90% N2 atmosphere with maximum humidity.

2.3. In vitro embryo selection and evaluation

On each day of experiment, 5–10 embryos at the

blastocyst stage (day 7) were randomly selected to

represent all classes of morphological quality, and

individually transferred into one well of a four-well dish

containing culture medium. Blastocysts were evaluated

according to the stage of embryonic development (see

above) and classified as quality I, II, III or IV based on

microscopic observation of the morphological quality

(excellent, good, fair or poor blastocysts, respectively).

In a previous work using the same IVP system the mean

cell numbers of day 7 expanded or haching blastocysts,

early expanded blastocysts and not expanded blasto-

cysts were 134 � 33, 95 � 26 and 76 � 22, respectively

[75]. The diameter of each blastocyst (i.e. from the outer

periphery of the zona pellucida) was measured under a

stereomicroscope (SMZ800; Nikon, Tokyo, Japan)

using an ocular micrometer eyepiece. Digital images

of each embryo were acquired using a digital still

camera (GC-X3E; JVL, Yokohama, Japan) mounted on

an inverted optical microscope (TDM, Nikon) with a

thermal control microscope stage (CO 102; Linkam

Scientific Instruments Ltd., Tadworth, Surrey, UK).

2.4. Measurement of oxygen consumption

The nanorespirometer system (Unisense A/S, Aar-

hus, Denmark) used to measure the oxygen consump-

tion of single embryos has recently been described [35].

Briefly, each of the embryos was transferred from the

four-well dish into the bottom of each glass capillary in

A.S. Lopes et al. / Theriogenology 68 (2007) 223–236226

the rosette (measuring unit composed of seven fused

glass capillaries). A total of five blastocysts were

loaded into each rosette while two glass capillaries

were left empty to serve as negative references

without respiratory activity (=control). The rosette

was subsequently placed into the rosette disc holder

and submerged into the culture medium in the beaker

(80 ml), which was maintained in a semi-closed

system at 38.5 8C and under constant flow of

humidified air (21% oxygen) with 5% CO2. The

system was left undisturbed for one h, until a steady-

state linear oxygen gradient was established inside the

glass capillaries of the rosette. Subsequently, oxygen

concentration gradients generated by the respiration

of the embryo were determined by measuring the

oxygen concentration at consecutive measurement

points inside the capillary with an oxygen micro-

sensor. Both embryo-containing capillaries and empty

capillaries were measured in duplicate. Calculation of

the mean respiration rate was based on the two oxygen

concentration profiles obtained from each embryo,

after subtraction of the background noise measured in

the empty reference capillaries.

2.5. Embryo processing after the measurements and

freezing procedure

With the purpose of preserving the genetic material

for sexing and gene expression analysis, day 7

embryos produced in vitro were individually trans-

ferred from the rosette into a well of a four-well dish

containing culture medium. Subsequently, individual

embryos were transferred to a four-well dish contain-

ing 500 ml Ca2+-free and Mg2+-free phosphate-

buffered saline (Biowhittaker, Walkersville, MD)

supplemented with 4 mg/ml of polyvinylpyrrolidone

and washed four times using a glass pipette. Finally,

embryos were individually transferred under the

stereomicroscope in a minimum volume of medium

(5 ml or less) into 1.5 ml eppendorf tubes, which were

immediately snap-frozen in liquid nitrogen and stored

at �80 8C.

2.6. Sex determination

DNA from each individual embryo was prepared

using the Microcon YM-100 (42412; Millipore,

Eschborn, Germany) columns according to the instruc-

tions of the manufacturer, except for minor modifica-

tions. The supernatant from the RNA extraction was

diluted with 200 ml H2O and centrifuged for 10 min.

Another 200 ml were then added to the column followed

by centrifugation for 8 min. Subsequently, the column

was inverted and centrifuged at 350 � g for 2 min. One

half of the supernatant (�10 ml) was used for PCR.

PCR analysis was performed using bovine-specific

(50-AGGTCGCGAGATTGGTCGCT-AGGTC ATGC

A-30 and 30-AAGACCTCGAGAGACCCTCTTCAA-

CACGT-50; accession number PCT WO 86/07095) and

Y-chromosome-specific primers (50-CCTCCCCTTGT-

TCAAACGCCCGGAATCATT-30 and 30-TGCTTGA-

CTGCAGGGACCGAGAGGTTTGGG-50; accession

number PCT WO 86/07095) as previously described

[61]. The PCR products were subjected to electrophor-

esis on a 2% agarose gel in 1� TBE buffer (90 mM Tris,

90 mM borate, 2 mM EDTA, pH 8.3) containing

0.2 mg/ml ethidium bromide. The image of each gel

was recorded using a CCD camera (Quantix; Photo-

metrics, Munchen, Germany). Sex determination was

performed in duplicate for each individual embryo.

2.7. Determination of the relative abundance of

GLUT1 and G6PD transcripts in individual

embryos by real-time reverse transcription-

polymerase chain reaction (RT-PCR)

Poly(A)+ RNA was isolated from single blastocysts

as previously described [67] and immediately used for

reverse transcription (RT), which was carried out in a

total volume of 20 ml using 2.5 mM random hexamers

(Applied Biosystems, Vaterstetten, Germany). Prior to

RNA isolation, 1 pg of rabbit globin RNA (Invitrogen,

Karlsruhe, Germany) was added as an internal standard

and was analyzed simultaneously. The rabbit globin

mRNA represents a mixture of the a- and b-chains

derived from polyribosomes of reticulocytes and was

purified via oligo-dT cellulose chromatography. The

reaction mixture consisted of 1� RT buffer (50 mM

KCl, 10 mM Tris–HCl, pH 8.3, Invitrogen), 5 mM

MgCl2, 1 mM of each dNTP (Amersham, Brunswick,

Germany), 2.5 mM random hexamers (Applied Biosys-

tems), 20 IU RNase inhibitor (Applied Biosystems) and

50 IU MuLV reverse transcriptase (Applied Biosys-

tems). The RT reaction was carried out in a PTC-200

thermocycler (MJ Research, Watertown, MA, USA) for

10 min at 25 8C, 60 min at 42 8C, followed by a

denaturation step for 5 min at 99 8C and flash cooling on

ice.

Immediately after RT, the samples were subjected to

real-time RT-PCR, which was performed with 0.05

embryo equivalents (percentage of the volume from

the RT reaction employing one embryo in a defined

volume). The PCR primers for each gene were designed

using the OLIGOTM program (Plymouth, MN, USA).

A.S. Lopes et al. / Theriogenology 68 (2007) 223–236 227

The sequences of the primer pairs to detect globin,

GLUT1 and G6PD transcripts were respectively:

50-GCAGCCACGGTGGCGAGTAT-30 (position 241–

260) and 30-GTGGGACAGGAGCTT-GAAAT-50 (posi-

tion 555–657), 50-CAGGAGATGAAGGAGGAGAGC-

30 (position 894–915) and 30-CACAAATAGCGACAC-

GACAGT-50 (position 1131–1152), 50-GTTCTTCAA-

CCC-CGAGGAGTC-30 (position 748–769) and 30-ATGTGGTGGAGCAGTGGAGTG-50 (position 910–

931). The corresponding accession numbers were

X04751, M60448.1 and XM_583628.1, respectively.

A standard curve consisting of dilutions of globin

cDNA originating from 1 pg globin RNA was included

as reference in each run. The standard curve consisted

of five standards of the globin cDNA diluted from 50 to

0.08 fg. This standard curve was used for calculation of

the concentration of each target gene as the amplifica-

tion efficiency of the target genes (GLUT1, G6PD)

and the reference gene (globin) is comparable. The

amplification efficiency of GLUT1, G6PD and globin

was compared by preparing serial dilutions for all the

genes from cDNA samples. After real-time amplifica-

tion, the threshold cycle (CT) values obtained were

used for the standard curve construction. The ampli-

fication efficiency (E) was calculated according to the

following equation: E = 10(�1/S) � 1 (S: slope of the

standard curve). Differences in the CT values between

the target genes and the reference gene were then

plotted against the logarithm of the template amount

(slope of the resulting straight line <0.1; data not

shown).

Real-time RT-PCR was carried out in a Lightcycler

apparatus using the LightCyclerFastStart DNA Mas-

ter + SYBR Green I Kit (Roche Diagnostik, Man-

nheim, Germany) according to the instructions of

the manufacturer. The PCR reaction consisted of a

cDNA equivalent to 0.05 blastocysts, 0.5 mM of each

primer, 4 ml of the mix containing dNTP’s, MgCl2,

FastStart Taq DNA polymerase and SYBR Green I,

in a total volume of 20 ml. The cycling profile

included the denaturation of the sample and activation

of the FastStart Taq DNA polymerase at 95 8Cfor 15 min, and 44 PCR cycles (denaturation at

96 8C for 10 s, annealing at 60 8C for 10 s and

elongation at 72 8C for 10 s) followed by a melting

curve. The LightCycler Software Version 3.5 (Roche

Diagnostik) was used to quantify and analyze the

results. Purity and specificity of both GLUT1 and

G6PD amplicons were verified by analysis of the

melting curve and the correct size of the products

was confirmed by electrophoresis on a 2% agarose

gel.

2.8. Statistical analysis

Sex ratios of the embryos were compared with

an expected 50:50 ratio by a corrected chi square

procedure (Freq Procedure [77]; Yate’s adjustment

[78]).

The background noise of the nanorespirometer,

measured by assessing the apparent oxygen consump-

tion within the empty reference capillaries (zero-value),

was evaluated by a two-tailed Student’s t-test. Linear

regression (Reg Procedure, [77]) was used to determine

the association between the first and second oxygen

measurements of each embryo. Normalization of the

results obtained for the individual genes of each

blastocyst by Real-Time RT-PCR was performed by

calculating the ratio of abundance of mRNA for the

target genes to globin mRNA.

Respiration rates and the RA of GLUT1 and G6PD in

in vitro embryos were tested for the distributional

properties and deviation from normal distribution was

significant (Univariate procedure [77]). Data was

subsequently square root transformed, as this was

effective in stabilizing variance and obtaining approx-

imate normal distribution of data and residuals.

Simple relationships between respiration rates and

RA of GLUT1 and of G6PD were firstly investigated as

correlations and through linear regression of respiration

rate on RA of GLUT1 and G6PD.

The mRNA expression of GLUT1 and G6PD were

analyzed independently using linear mixed models

(Mixed Procedure, [77]). For GLUT1, the model

included sex, diameter, time point of the measurement

in relation to in vitro fertilization, square root of

the RA of G6PD as well as interaction between

morphological quality and stage of embryonic

development as fixed effects. For G6PD, the model

included sex, diameter, time point of the measurement

in relation to in vitro fertilization, square root of the

RA of GLUT1 expression as well as interaction

between morphological quality and stage of embryo-

nic development as fixed effects. Random effects

were not included in these models as the effects of

rosette and date of measurement were non-significant

in relation to the variable in question. Least square

means (LSM) were produced for the factors in the

final model.

Factors affecting embryo diameter were assessed

using a linear mixed model (Mixed Procedure [77]).

The model included sex, time point of the measurement

in relation to in vitro fertilization, morphological

quality, stage of embryonic development, RA of GLUT1

and G6PD and the interaction of morphological quality

A.S. Lopes et al. / Theriogenology 68 (2007) 223–236228

and stage of development as fixed effects. Date of

measurement was tested as random effect but it was

regarded as non-significant and consequently excluded

from the model. LSM were produced by the final model

after removal of both interactions, which were shown to

be non-significant.

Respiration rates of day 7 embryos were subse-

quently analyzed using a linear mixed model (Mixed

Procedure [77]), including time points of the mea-

surement in relation to in vitro fertilization, diameter,

stage of embryonic development, morphological

quality, sex, square root of the RA of GLUT1 and

G6PD transcripts and interaction between morpho-

logical quality and stage of embryonic development

as fixed effects. The random part of the model

contained an interaction between rosette and date of

measurement. LSM were produced by the previous

model after removal of non-significant effects. The

interaction of date of measurement and rosette was

tested by a linear mixed model (Asycov, Covtest [77])

before being introduced in the random part of the

model.

All statistical procedures were performed using the

computational software of SAS [77]. Data was

presented as LSM � S.E. and the level of significance

was P < 0.05, unless otherwise indicated.

3. Results

3.1. Sex ratio of embryos selected for oxygen

measurements

Among the 81 blastocysts evaluated for sex and

morphology (Table 1), a total of 51 were male and thus

the average ratio of male to female blastocysts was

63:37, which deviated significantly from the expected

50:50 ratio in all morphological quality categories

(P < 0.05).

Table 1

Morphological quality and sex ratio of day 7 bovine in vitro-produced

blastocysts

Morphological

quality

Number of

embryos

Male:female

number/(ratio)

I 19 13:6/(68:32a)

II 27 17:10/(63:37a)

III 27 16:11/(59:41a)

IV 8 5:3/(62:38a)

Total 81 51:30/(63:37a)

a Significantly different from anticipated 50:50 ratio of males to

females (P < 0.05).

3.2. Consistency and accuracy of the oxygen

measurements

The background noise of the nanorespirometer

system was very small, although significantly different

from zero, with the empty reference capillaries showing

an apparent oxygen consumption of 0.016 � 0.0047 nl/

h (n = 35; P < 0.01). The results of the first and second

oxygen measurements of the same embryos were highly

correlated (R2 = 0.985; n = 85; P < 0.0001) and the

slope of the regression line was not significantly

different from one (y2 = 0.981 (�0.014) y1 + 0.025

(�0.023); P > 0.05).

3.3. Relative abundance of GLUT1 and G6PD

transcripts in in vitro-produced embryos

Significantly lower levels of RA of GLUT1 transcript

were observed among female embryos compared to

male embryos (0.023 � 0.0029 versus 0.040 � 0.0031,

for female and male embryos respectively; P < 0.0001;

Fig. 1). G6DP expression correlated closely with

GLUT1 expression (n = 77, r2 = 0.54, P < 0.0001), as

confirmed by the positive and highly significant

marginal regression of G6PD on GLUT1 (b = 0.82 +

0.010, P < 0.0001; Fig. 2).

The interaction of morphological quality and stage

of embryonic development influenced the RA of

GLUT1 (the mean RA of the GLUT1 transcript accord-

ing to morphological quality and stage of embryonic

development are separately shown in Fig. 3a and b).

However, the time point of the measurement in relation

to in vitro fertilization and diameter did not affect the

expression of this gene.

Likewise, the RA of G6PD was influenced by the sex

of the embryos, with female embryos showing a 1.8-

fold higher mean RA compared to male embryos

(P < 0.0001; Fig. 1). Similarly to GLUT1, the interaction

Fig. 1. Relative abundance of GLUT1 and G6PD transcripts (left axis)

and respiration rate (right axis) of individual male and female blas-

tocysts (LSM � S.E.). Numbers between parentheses inside columns

represent the number of blastocysts measured. a,bP < 0.05. Columns

with different superscripts vary significantly.

A.S. Lopes et al. / Theriogenology 68 (2007) 223–236 229

Fig. 2. Relationship between relative abundance of GLUT1 and

G6PD transcripts in day 7 bovine in vitro-produced blastocysts.

of morphological quality and stage of development

affected the RA of G6PD (P < 0.05; the mean RA of the

G6DP transcript according to morphological quality and

stage of embryonic development are separately shown in

Fig. 3c and d), which is possibly influenced by the very

low expression of this gene in quality IV expanded

blastocysts.

3.4. Embryo diameter

The diameter of the embryos was directly affected by

embryonic stage (P < 0.001), as embryos of more

Fig. 3. Relative abundance of (a and b) GLUT1 transcript and (c and d) G6P

blastocysts; EXB, early expanded blastocysts; XB, expanded blastocysts; LX

blastocysts) and stage of embryonic development. Superscripts were not incl

and only the joint effect of morphological quality and stage of developme

Numbers between parentheses below columns represent the number of bla

advanced stages were associated with larger diameter.

Furthermore, morphology had an effect on diameter,

such that embryos of better morphological quality had

larger diameters (P < 0.05).

3.5. Respiration rates of in vitro-produced embryos

The mean respiration rate of day 7 in vitro-produced

embryos was 1.375 � 0.081 nl/h (n = 100). Respiration

rates were closely related with the RA of GLUT1

(n = 77, r = 0.73, P < 0.0001; Fig. 4a) and G6PD

(n = 77, r = 0.78, P < 0.005; Fig. 4b), regardless of the

sex of the embryos.

Respiration rates were affected by the interaction

between morphological quality and stage of embryonic

development (P < 0.001; Fig. 5), with embryos of

better morphological qualities showing slightly higher

respiration rates. The time point of the measurement in

relation to in vitro fertilization and sex of the embryo

did not affect respiration rates (1.332 � 0.090 nl/h for

male (n = 45) versus 1.218 � 0.089 nl/h for female

(n = 29) blastocysts (P > 0.05; Fig. 1). The diameter

did not affect the respiration rates of individual

embryos.

The mean embryonic respiration rate was signifi-

cantly affected (P = 0.01) by the interaction of rosette

used in the measurements (rosette 1 versus 2) and date

D transcript, according to morphological quality (NXB, not expanded

, late expanded blastocysts; CB, collapsed blastocysts; HB, hatching

uded because the values shown are mean � S.E. and not LSM � S.E.,

nt significantly affected the relative abundance of these transcripts.

stocysts measured.

A.S. Lopes et al. / Theriogenology 68 (2007) 223–236230

Fig. 4. Relationship between respiration rate (nl/h) and mRNA

expression of (a) GLUT1 transcript and (b) G6PD transcript in day

7 bovine in vitro-produced blastocysts. Trendlines and equations of

regression are not shown as regressions included joint effects of

GLUT1 and G6PD, where units of respiration rate, GLUT1 and

G6PD were square root transformed, but the correlation coefficients

are reported in the text.

of measurement (15 experimental days), which was

considered as a random effect.

4. Discussion

The present study is the first to demonstrate a

correlation between the RA of GLUT1 and G6PD

transcripts and the respiration rate of individual day 7

bovine in vitro-produced blastocysts. Real-time RT-

PCR was combined with sex determination, careful

Fig. 5. (a) Mean respiration rates (LSM � S.E.; back transformed from sq

number and stage of development of the blastocysts measured in each morpho

blastocysts; XB, expanded blastocysts; LXB, late expanded blastocysts; CB,

included, as only the joint effect of morphological quality and stage of dev

morphological evaluation and measurement of embryo-

nic metabolic activity, which allowed insight into

the biochemistry of the early bovine embryo. The

nanorespirometer technology is sensitive, rapid, accu-

rate and consistent enough to measure individual

embryonic respiration rates as demonstrated by the

high correlation between first and second measurement

of the same embryo as well as by the low background

noise, which is likely caused by a slight oxygen

consumption of the electrochemical microsensor during

the measurements.

Here we demonstrate that respiration rates of in

vitro-produced embryos correlate with the mRNA

levels of both G6PD and GLUT1 genes (Fig. 1a and

b). The observed differences in mRNA copy number

should reflect the transcription of the respective genes

assuming that the stability of both gene transcripts is

constant. The mRNA abundance is a likely indicator of

the synthesis of the particular enzymes through

translation of the particular mRNAs, so the relative

abundance of each mRNA most likely reflects level of

gene expression for each gene. At the blastocyst stage,

glucose uptake and oxygen consumption are augmented

to support the high ATP demand for protein synthesis

and blastocoel formation [25,28,33,43,52,54]. A direct

correlation between oxygen consumption and glucose

uptake in blastocysts has been previously described

[28,52]. Increased glucose uptake is associated with

higher activity of glycolytic enzymes [17] and increased

glucose transport across the membrane via glucose

transporters such as GLUT1 [67,79]. Although glyco-

lysis only contributes to 15% of the total ATP produced

by the bovine blastocyst [29], both oxidative phosphor-

ylation, accounting for 70% of the oxygen consumed at

this stage [33], and glycolysis seem to be increased for

the maximization of ATP production. This is supported

by findings from Khurana and Niemann [80], who

suggested that bovine embryos increase glycolysis and

energy production in parallel when other pathways are

uare root) of blastocysts according to morphological quality and (b)

logical quality (NXB, not expanded blastocysts; EXB, early expanded

collapsed blastocysts; HB, hatching blastocysts). Superscripts were not

elopment significantly affected embryonic respiration rates.

A.S. Lopes et al. / Theriogenology 68 (2007) 223–236 231

already operating at a maximum rate. Furthermore,

glycolysis is affected by oxygen availability in each

blastomere via the hypoxia-inducing factor 1 (HIF-1),

which is regulated by the oxygen sensor, that itself

accounts for part of the oxidative phosphorylation-

independent oxygen consumed by the blastocyst [41].

The observed increase in GLUT1 expression among

embryos with higher respiration rates, and hence with

higher metabolic activity, suggests an important role of

this glucose transporter in the metabolism of the pre-

implantation bovine embryo. However, it has been

suggested that GLUT3 instead of GLUT1 is the main

transporter of glucose from the external environment

and that GLUT1 is primarily responsible for mediating

the glucose efflux into the blastocoel cavity and the

uptake by the inner cell mass cells [79,81]. It would thus

be interesting to investigate if the expression of GLUT3

is equally increased in embryos with higher respiration

rates.

Similar to GLUT1, mRNA expression of G6PD was

up-regulated in embryos with a higher level of

respiratory activity (Fig. 4b). The finding that G6PD

expression was correlated with GLUT1 expression

(Fig. 2) suggests that higher levels of GLUT1 increase

the uptake of glucose by the cells, which in turn leads to

higher amounts of glucose metabolized through the PPP

in the form of glucose-6-phosphate. The high influx of

glucose is likely to be associated with an allosteric

increase of the activity of G6PD, which in turn

stimulates glucose cycling through the PPP. It thus

seems that higher ATP demands are responsible for

higher respiration rates and glucose uptake by the

embryos, leading to enhanced glycolytic and pentose

phosphate pathways and consequently to increased

GLUT1 and G6PD expression. Alternatively, the

increased G6PD expression associated with the higher

oxygen uptake by the embryos could be caused by the

presence of reactive oxygen species (ROS). A higher

oxygen consumption causes an enhanced ATP produc-

tion by mitochondrial oxidative phosphorylation, but

also produces ROS [41]. G6PD is critically involved in

the production of NADPH, which detoxifies ROS.

Furthermore, G6PD is the only NADPH-producing

enzyme activated in response to oxidative stress [82]. It

is therefore possible that the presence of higher levels of

ROS generated by the increased oxygen consumption

triggers G6PD activation. To further unravel this

intriguing hypothesis, the expression pattern of genes

related to stress response, such as the CU/ZN-SOD and

in particular the HSP70.1, should be investigated. This

would help clarifying whether the higher levels of

respiratory activity and G6PD are indicative of embryo

viability or are merely a reaction to an increased stress

response. Moreover, assessment of cell number in each

individual blastocyst would be useful for understanding

if the increased expression of GLUT1 and G6PD in

more advances stages is or not merely a reflection of a

higher cell number.

Individual respiration rates were correlated with the

RA of GLUT1 and G6PD transcripts. Furthermore, the

interaction between morphological quality and stage of

embryonic development also influenced respiration

rates (Fig. 5), but the limited number of observations

within each stage of development and morphological

quality made results interpretation difficult. The fact

that collapsing embryos might not necessarily have

been at a more advanced stage than expanding embryos

could have also been a confounding factor. Differences

in respiration rates between embryos of different

morphological qualities have shown that oxygen

consumption is higher among embryos of superior

quality [32,35,38,49,50]. However the partial correla-

tion between morphological quality and respiration rate

of individual embryos observed in the present and

recent studies [35,41] also supports the concept that

morphological differences are to a certain extent

reflected at the physiological level. Thus, a combination

of oxygen consumption measurements and morpholo-

gical assessment of quality will be instrumental to

improve embryo selection. However, it should be noted

that high respiration rates could reflect an overproduc-

tion of ROS, and thus very high respiration rates could

represent a manifestation of metabolic stress instead of

superior quality.

Changes in morphology at the blastocyst stage

require the expression of key genes from the embryonic

genome. To our knowledge, differences in GLUT1 and

G6PD expression in relation to the morphological

quality of the embryos have not been reported

previously. This is likely explained by the fact that

transcriptional profiles are normally analyzed solely in

good quality embryos. Patterns of gene expression in

relation to morphological quality have been analyzed by

comparing gene expression of in vitro-produced

embryos with that of their in vivo counterparts

[16,42,70], which are considered to be of superior

quality. Blastocysts of the best morphological quality

and at the hatching/hatched stage expressed the highest

amount of GLUT1 mRNA among all analyzed embryos

(Fig. 3a and b). In contrast, GLUT1 expression was

relatively low in embryos of inferior morphological

quality (IV) (Fig. 3a). In agreement with the present

study, Wrenzycki et al. [70] and Harvey et al. [42]

reported that GLUT1 expression was reduced in in

A.S. Lopes et al. / Theriogenology 68 (2007) 223–236232

vitro-produced embryos as compared to embryos

produced in vivo and suggested that reduced amounts

of GLUT1 were indicative of compromised develop-

mental potential (poor bovine embryo quality). Based

on these results, it is plausible to speculate that a higher

expression of the GLUT1 transcript is observed in better

quality embryos, but a larger study including a higher

number of embryos representative of all morphological

qualities and stages of development is required to fully

clarify this. Furthermore, quantification of the total cell

number of each blastocyst will help elucidating if the

abundance of transcripts represents increased quality or

is merely a result of the number of cells in each

blastocyst, which is not related to embryo quality as

evaluated following embryo transfer [3].

Quality I embryos seemed to show the highest G6PD

mRNA levels (Fig. 3c). Similar to GLUT1 expression,

mRNA expression of G6PD was relatively low in

quality IV embryos. Previous reports have shown that

G6PD expression was significantly higher in in vitro-

produced blastocysts than in their in vivo counterparts

[64]. However, these differences probably reflect

incomplete X-chromosome inactivation of in vitro-

produced blastomeres rather than differences in embryo

quality.

Embryonic diameter was not correlated with

respiration rates or expression of GLUT1 and G6PD.

However, diameter was affected by the embryonic

stage, which might explain the lack of influence of

diameter on G6PD and GLUT1 expression levels as

well as on respiration, when both parameters were

simultaneously analyzed. Embryonic diameter had

previously been related to respiration, with significantly

larger embryos associated with higher respiration rates

[35,50], but not with glucose metabolism [80]. In the

present study, the number of cells of each blastocyst

was not determined. The number of cells of individual

embryos appears to be correlated with oxygen consum-

ption [83] but effects of cell number on the mRNA

expression of GLUT1 and G6PD have only been

indirectly described [42,66].

We observed that the majority of the analyzed

blastocysts were males. Similar skewed sex ratios have

been reported in previous studies [35,64,84–88]. The

higher number of male blastocysts in culture at defined

points has been attributed to their faster development in

glucose-containing culture media [86–89]. According

to Gutierrez-Adan et al. [90], the presence of serum in

the culture media also affects the sex and kinetics of the

blastocysts, favoring male development. The culture

media (SOFaaci) employed in this study were not

supplemented with glucose but included 5% bovine

serum, which contains non-defined levels of amino

acids, proteins, vitamins, growth factors and energy

substrates such as glucose [91,92]. Thus, the differential

response of the embryos to serum components might

have been responsible for the observed sex ratio.

Irrespectively of the skewed sex ratio observed for all

morphological qualities, the respiration rates were

similar for male and female blastocysts (Fig. 1). Inte-

restingly, Agung et al. [32] also found no differences in

the oxygen consumption between male and female

embryos of excellent quality, but observed that the

oxygen consumption of good quality female embryos

was higher than that of male embryos. Thus, it seems

that sexual dimorphism does not exist at the level of

oxidative phosphorylation, and oxygen consumption is

mostly associated with quality rather than with the sex

of the embryos.

On the contrary, expression of GLUT1 and G6PD

was influenced by the sex of embryos (Fig. 1). In the

present study, the DNA used for the sexing procedure

was extracted from the supernatant of the RNA isolation

buffer and no biopsy had to be performed, ruling out the

possibility of alterations in gene expression patterns

caused by the manipulation procedure. The lower levels

of GLUT1 in day 7 female blastocysts (Fig. 1) have not

previously been reported. This observation conflicts

with other reports, in which female embryos showed

higher glucose uptake [19,55]. Later in development,

Bertolini et al. [72] also observed that day 16 male

bovine in vivo-produced embryos had higher levels of

GLUT1 and GLUT3 expression than female in vivo-

produced embryos. Moreover, Morton et al. [93] found

that the levels of GLUT3 were lower in female embryos

as compared to male embryos. Potentially, the increased

level of expression of the GLUT1 gene in males is

associated with the faster development of male embryos

under in vitro conditions [84,85]. A faster development

could lead to an increase in glucose uptake, mediated by

GLUT1 expression. However, the increased metabolism

of faster growing embryos (males) was not significantly

reflected at the respiratory level, even though the

respiration rate of male embryos was 9% higher than

that of female embryos. In fact, if GLUT1 contributes to

glucose uptake and the mRNA level corresponds to

GLUT1 activity then we would only expect a 15%

difference in respiration rate between the sexes

(considering that glycolysis only contributes in 15%

for the overall oxygen consumption), even though the

GLUT1 transcript level is twice as large for males. Even

so, the contribution of GLUT1 to the glucose transport

from the external environment requires further inves-

tigation.

A.S. Lopes et al. / Theriogenology 68 (2007) 223–236 233

The nearly 1.8-fold higher levels of G6PD observed

among female embryos produced in vitro (Fig. 1) was

expected and confirmed the delayed dosage com-

pensation of G6PD reported by others [62–64,94].

By inhibiting G6PD with dehydroepiandrosterone

(DHEA), Perez-Crespo et al. [65] observed similar

mRNA expression of G6PD between the two sexes and

did not detect a skewed sex ratio for in vitro-produced

embryos. Thus, the sexual dimorphism regarding the

G6PD transcript could also be responsible for the

skewed sex ratio observed in our study. It is likely that

the higher expression of G6PD leads to higher PPP

activity in females [95] and thus higher production of

NADPH and higher antioxidative capacity by female

embryos. This leads to a reduction in the amount of free

radical levels, which have a growth stimulating effect

[59] and thus conditions the subsequent development of

female embryos. Even so, it seems that the delayed

dosage compensation of G6PD and the higher activity

of the PPP in female embryos did not affect oxygen

consumption and thus oxidative phosphorylation.

In conclusion, the present study showed sexual

dimorphism in regards to the expression of both GLUT1

and G6PD transcripts, which was not observed in the

levels of oxygen consumed by individual bovine

embryos produced in vitro. Furthermore, the RA of

GLUT1 and G6PD transcripts correlate with each other

and with individual respiration rates, suggesting that

more metabolically active embryos may have higher

developmental competence. However, the present study

only investigated two gene transcripts, and thus only

provides a limited view of the involved complex

metabolic pathways. It is important to stress that

changes in transcriptional levels do not necessarily

reflect cellular and physiological alterations and there-

fore caution should be applied when interpreting

transcriptional profiles. Combining assessment of

embryonic respiration with genetic investigations using

microarray technology, which is capable of analyzing

the expression patterns of thousands of genes simulta-

neously, and with quantification of protein abundance

may allow for a better understanding of the involvement

of key genes in specific metabolic processes.

Acknowledgements

The authors thank Anette Pedersen, Klaus Villemoes

and Ruth Kristensen for their technical assistance. Novo

Nordisk Foundation financially supported the acquisi-

tion of the nanorespirometer technology and the Danish

Directorate for Food, Fisheries and Agri Business

(TFH03-8) the embryo work. Ana Sofia Lopes is

supported by a grant from Foundation for Science and

Technology (FCT), Ministry of Science and Technol-

ogy, Portugal (SFRH/BD/8486/2002). Dr. Per Horn is

thanked for critically revising the manuscript.

References

[1] Ziebe S, Petersen K, Lindenberg S, Andersen AG, Gabrielsen A,

Nyboe Andersen A. Embryo morphology or cleavage stage: how

to select the best embryos for transfer after in-vitro fertilization.

Hum Reprod 1997;12:1545–9.

[2] Merton S. Morphological evaluation of embryos in domestic

animals. In: Van Soom A, Boerjan M, editors. Assessment of

mammalian embryo quality: invasive and non-invasive techni-

ques. 1st ed, Dordrecht, The Netherlands: Kluwer Academic

Publishers; 2002. p. 31–55.

[3] Donnay I. Metabolic markers of embryo viability. In: Van Soom

A, Boerjan M, editors. Assessment of mammalian embryo

quality: invasive and non-invasive techniques. 1st ed, Dordrecht,

The Netherlands: Kluwer Academic Publishers; 2002. p. 57–94.

[4] Scott L. The biological basis of non-invasive strategies for

selection of human oocytes and embryos. Hum Reprod Update

2003;9:237–49.

[5] Bergh C, Aittomaki K, Loft A, Hazekamp J, Nygren KG.

Attitudes to single embryo transfer among Nordic IVF doctors.

Hum Reprod 2005;20:54. Abstract.

[6] Van Royen E, Mangelschots K, De Neubourg D, Laureys I,

Ryckaert G, Gerris J. Calculating the implantation potential of

day 3 embryos in women younger than 38 years of age: a new

model. Hum Reprod 2001;16:326–32.

[7] Boiso I, Veiga A, Edwards RG. Fundamentals of human embryo-

nic growth in vitro and the selection of high-quality embryos for

transfer. Reprod Biomed Online 2002;5:328–50.

[8] Corcoran D, Fair T, Lonergan P. Predicting embryo quality:

mRNA expression and the preimplantation embryo. Reprod

Biomed Online 2005;11:340–8.

[9] Overstrom EW. In vitro assessment of embryo viability. Ther-

iogenology 1996;45:3–16.

[10] Farin PW, Slenning BD, Britt JH. Estimates of pregnancy out-

comes based on selection of bovine embryos produced in vivo or

in vitro. Theriogenology 1999;52:659–70.

[11] Holm P, Shukri NN, Vajta G, Booth P, Bendixen C, Callesen H.

Developmental kinetics of the first cell cycles of bovine in vitro

produced embryos in relation to their in vitro viability and sex.

Theriogenology 1998;50:1285–99.

[12] Sakkas D, Shoukir Y, Chardonnens D, Bianchi PG, Campana A.

Early cleavage of human embryos to the two-cell stage after

intracytoplasmic sperm injection as an indicator of embryo

viability. Hum Reprod 1998;13:182–7.

[13] Lonergan P, Khatir H, Piumi F, Rieger D, Humblot P, Boland MP.

Effect of time interval from insemination to first cleavage on the

developmental characteristics, sex ratio and pregnancy rate after

transfer of bovine embryos. J Reprod Fertil 1999;117:159–67.

[14] Munne S, Wells D. Preimplantation genetic diagnosis. Curr Opin

Obstet Gynecol 2002;14:239–44.

[15] Gianaroli L, Magli M, Ferraretti A, Munne S. Preimplantation

diagnosis for aneuploidies in patients undergoing in vitro ferti-

lization with a poor prognosis: identification of the categories for

which it should be proposed. Fertil Steril 1999;72:837–44.

[16] Wrenzycki C, Herrmann D, Lucas-Hahn A, Korsawe K, Lemme

E, Niemann H. Messenger RNA expression patterns in bovine

A.S. Lopes et al. / Theriogenology 68 (2007) 223–236234

embryos derived from in vitro procedures and their implications

for development. Reprod Fertil Dev 2005;17:23–35.

[17] Houghton FD, Hawkhead JA, Humpherson PG, Hogg JE, Balen

AH, Rutherford AJ, et al. Non-invasive amino acid turnover

predicts human embryo developmental capacity. Hum Reprod

2002;17:999–1005.

[18] Brison DR, Houghton F, Falconer D, Roberts SA, Hawkhead J,

Humpherson PG, et al. Identification of viable embryos in IVF

by non-invasive measurement of amino acid turnover. Hum

Reprod 2004;19:2319–24.

[19] Lane M, Gardner DK. Selection of viable mouse blastocysts

prior to transfer using a metabolic criterion. Hum Reprod

1996;11:1975–8.

[20] Donnay I, Feugang JM, Bernard S, Marchandise J, Pampfer S,

Moens A, et al. Impact of adding 5.5 mM glucose to SOF

medium on the development, metabolism and quality of in vitro

produced bovine embryos from the morula to the blastocyst

stage. Zygote 2002;10:189–99.

[21] Leese HJ. Non-invasive amino acid turnover predicts human

embryo developmental capacity. Hum Reprod 2002;17:999–

1005.

[22] Thompson JG. In vitro culture and embryo metabolism of cattle

and sheep embryos—a decade of achievement. Anim Reprod Sci

2000;60/61:263–75.

[23] Trimarchi JR, Liu L, Porterfield DM, Smith PJ, Keefe DL. A

non-invasive method for measuring preimplantation embryo

physiology. Zygote 2000;8:15–24.

[24] Rieger D, Loskutoff NM. Changes in the metabolism of glucose,

pyruvate, glutamine and glycine during maturation of cattle

oocytes in vitro. J Reprod Fertil 1994;100:257–62.

[25] Houghton FD, Thompson JG, Kennedy CJ, Leese HJ. Oxygen

consumption and energy metabolism of the early mouse embryo.

Mol Reprod Dev 1996;44:476–85.

[26] Houghton FD, Humpherson PG, Hawkhead JA, Hall CJ, Leese

HJ. Na+, K+, ATPase activity in the human and bovine pre-

implantation embryo. Dev Biol 2003;263:360–6.

[27] Houghton FD, Leese HJ. Metabolism and developmental com-

petence of the preimplantation embryo. Eur J Obstet Gynecol

Reprod Biol 2004;115:92–6.

[28] Thompson JG, Partridge RJ, Houghton FD, Cox CI, Leese HJ.

Oxygen uptake and carbohydrate metabolism by in vitro derived

bovine embryos. J Reprod Fertil 1996;106:299–306.

[29] Leese H. What does an embryo need? Hum Fertil 2003;6:180–5.

[30] Houghton FD. Energy metabolism of the inner cell mass and

trophectoderm of the mouse blastocyst. Differentiation 2006;74:

11–8.

[31] Benos DJ, Baladan RS. Energy metabolism of preimplantation

mammalian blastocysts. Am J Physol 1983;245:40–5.

[32] Agung B, Otoi T, Abe H, Hoshi H, Murakami M, Karja NW,

et al. Relationship between oxygen consumption and sex of

bovine in vitro fertilized embryos. Reprod Domest Anim

2005;40:51–6.

[33] Trimarchi JR, Liu L, Porterfield DM, Smith PJ, Keefe DL.

Oxidative phosphorylation-dependent and -independent oxygen

consumption by individual preimplantation mouse embryos.

Biol Reprod 2000;62:1866–74.

[34] Overstrom EW. Manipulation of early embryonic development.

Anim Reprod Sci 1992;28:277–85.

[35] Lopes AS, Larsen LH, Ramsing N, Løvendahl P, Raty M, Peippo

J, et al. Respiration rates of individual bovine in vitro-produced

embryos measured with a novel, non-invasive and rapid micro-

sensor system. Reproduction 2005;130:669–79.

[36] Lopes AS, Madsen SE, Ramsing N, Løvendahl P, Greve T,

Callesen H. Respiration of individual bovine in vivo and in

vitro-produced embryos: correlation with viability following

transfer. Hum Reprod 2006;22:558–66.

[37] Sturmey RG, Leese HJ. Energy metabolism in pig oocytes and

early embryos. Reproduction 2003;126:197–204.

[38] Abe H, Shiku H, Yokoo M, Aoyagi S, Moriyasu S, Minamihashi

A, et al. Evaluating the quality of individual embryos with a non-

invasive and highly sensitive measurement of oxygen consump-

tion by scanning electrochemical microscopy. J Reprod Dev

2006;52:55–64.

[39] Moriyasu S, Hirayama H, Sawai K, Kageyama S, Aoyagi S,

Shiku H, et al. Relationship between respiratory activity and the

pregnancy rate of bisected bovine embryos in vivo. Reprod Fertil

Dev 2007;19:219. Abstract.

[40] Sakagami N, Akiyama K, Nakazawa Y. The relationship

between oxygen consumption rate and pregnancy rate of bovine

embryos. Reprod Fertil Dev 2007;19:225. Abstract.

[41] Harvey AJ, Kind KL, Thompson JG. REDOX regulation of early

embryo development. Reproduction 2002;123:479–86.

[42] Harvey AJ, Kind KL, Pantaleon M, Armstrong DT, Thompson

JG. Oxygen-regulated gene expression in bovine blastocysts.

Biol Reprod 2004;71:1108–19.

[43] Leese HJ. Metabolism of the preimplantation mammalian

embryo. Oxf Rev Reprod Biol 1991;13:35–72.

[44] Abe H, Shiku H, Aoyagi S, Matsue T, Hoshi H. Respiration

activity of bovine embryos cultured in serum-free and serum-

containing media. Reprod Fertil Dev 2005;17:215. Abstract.

[45] Eckert J, Pugh PA, Thompson JG, Niemann H, Tervit HR.

Exogenous protein affects development competence and meta-

bolic activity of bovine pre-implantation embryos in vitro.

Reprod Fertil Dev 1998;10:327–32.

[46] Manser RC, Leese HJ, Houghton FD. Effect of inhibiting nitric

oxide production on mouse preimplantation embryo develop-

ment and metabolism. Biol Reprod 2004;71:528–33.

[47] Hooper K, Lane M, Gardner DK. Reduced oxygen concentration

increases mouse embryo development and oxidative metabo-

lism. Theriogenology 2001;55:334. Abstract.

[48] Abe H, Hoshi H. Evaluation of bovine embryos produced in high

performance serum-free media. J Reprod Dev 2003;49:193–202.

[49] Magnusson C, Hillensjo T, Hamberger L, Nilsson L. Oxygen

consumption by human oocytes and blastocysts grown in vitro.

Hum Reprod 1986;1:183–4.

[50] Shiku H, Shiraishi T, Ohya H, Matsue T, Abe H, Hoshi H, et al.

Oxygen consumption of single bovine embryos probed by

scanning electrochemical microscopy. Anal Chem 2001;73:

3751–8.

[51] Thompson JG, Partridge RJ, Houghton FD, Kennedy CJ, Pullar

D, Wrathall AE, et al. Preliminary observations of the uptake of

oxygen by day 7 bovine blastocysts. Theriogenology 1995;43:

337. Abstract.

[52] Donnay I, Leese HJ. Embryo metabolism during the expansion

of the bovine blastocyst. Mol Reprod Dev 1999;53:171–8.

[53] Leese HJ. Metabolic control during preimplantation mammalian

development. Hum Reprod Update 1995;1:63–72.

[54] Rieger D, Loskutoff NM, Betteridge KJ. Developmentally

related changes in the metabolism of glucose and glutamine

by cattle embryos produced and co-cultured in vitro. J Reprod

Fertil 1992;95:585–95.

[55] Gardner DK, Leese HJ. Assessment of embryo viability prior to

transfer by the noninvasive measurement of glucose uptake. J

Exp Zool 1987;242:103–5.

A.S. Lopes et al. / Theriogenology 68 (2007) 223–236 235

[56] Rieger D. The measurement of metabolic activity as an approach

to evaluating viability and diagnosing sex in early embryos.

Theriogenology 1984;21:138–49.

[57] Renard JP, Philippon A, Menezo Y. J In-vitro uptake of glucose

by bovine blastocysts. J Reprod Fertil 1980;58:161–4.

[58] Shimizu N, Shimizu Y, Kondo I, Woods C, Wegner T. The bovine

genes for phosphoglycerate kinase, glucose-6-phosphate dehy-

drogenase, alpha-galactosidase, and hypoxanthine phosphoribo-

syltransferase are linked to the X chromosome in cattle-mouse

cell hybrids. Cytogenet Cell Genet 1981;29:26–31.

[59] Rieger D. Relationships between energy metabolism and devel-

opment of early mammalian embryos. Theriogenology 1992;37:

75–93.

[60] Peippo J, Bredbacka P. Sex-related growth rate differences in

mouse preimplantation embryos in vivo and in vitro. Mol Reprod

Dev 1995;40:56–61.

[61] Nicol CJ, Zielenski J, Tsui LC, Wells PG. An embryoprotective

role for glucose-6-phosphate dehydrogenase in developmental

oxidative stress and chemical teratogenesis. FASEB J 2000;14:

111–27.

[62] Gutierrez-Adan A, Oter M, Martinez-Madrid B, Pintado B, De

La Fuente J. Differential expression of two genes located on the

X chromosome between male and female in vitro-produced

bovine embryos at the blastocyst stage. Mol Reprod Dev

2000;55:146–51.

[63] Peippo J, Farazmand A, Kurkilahti M, Markkula M, Basrur PK,

King WA. Sex-chromosome linked gene expression in in-vitro

produced bovine embryos. Mol Reprod Dev 2002;8:923–9.

[64] Wrenzycki C, Lucas-Hahn A, Herrmann D, Lemme E, Korsawe

K, Niemann H. In vitro production and nuclear transfer affect

dosage compensation of the X-linked gene transcripts G6PD,

PGK, and Xist in preimplantation bovine embryos. Biol Reprod

2002;66:127–34.

[65] Perez-Crespo M, Ramirez MA, Fernandez-Gonzalez R, Rizos D,

Lonergan P, Pintado B, et al. Differential sensitivity of male and

female mouse embryos to oxidative induced heat-stress is

mediated by glucose-6-phosphate dehydrogenase gene expres-

sion. Mol Reprod Dev 2005;72:502–10.

[66] Wrenzycki C, Herrmann D, Niemann H. Timing of blastocyst

expansion affects spatial messenger RNA expression patterns of

genes in bovine blastocysts produced in vitro. Biol Reprod

2003;68:2073–80.

[67] Wrenzycki C, Herrmann D, Carnwath JW, Niemann H. Altera-

tions in the relative abundance of gene transcripts in preimplan-

tation bovine embryos cultured in medium supplemented with

either serum or PVA. Mol Reprod Dev 1999;53:8–18.

[68] Leppens-Luisier G, Urner F, Sakkas D. Facilitated glucose

transporters play a crucial role throughout mouse preimplanta-

tion embryo development. Hum Reprod 2001;16:1229–36.

[69] Kind KL, Collett RA, Harvey AJ, Thompson JG. Oxygen-

regulated expression of GLUT-1, GLUT-3, and VEGF in the

mouse blastocyst. Mol Reprod Dev 2004;70:37–44.

[70] Wrenzycki C, Herrmann D, Keskintepe L, Martins Jr A, Sir-

isathien S, Brackett B, et al. Effects of culture system and protein

supplementation on mRNA expression in pre-implantation

bovine embryos. Hum Reprod 2001;16:893–901.

[71] Kimura K, Spate LD, Green MP, Roberts RM. Effects of D-

glucose concentration, D-fructose, and inhibitors of enzymes of

the pentose phosphate pathway on the development and sex ratio

of bovine blastocysts. Mol Reprod Dev 2005;72:201–7.

[72] Bertolini M, Beam SW, Shim H, Bertolini LR, Moyer AL,

Famula TR, et al. Growth, development, and gene expression

by in vivo- and in vitro-produced day 7 and 16 bovine embryos.

Mol Reprod Dev 2002;63:318–28.

[73] Knijn HM, Wrenzycki C, Hendriksen PJ, Vos PL, Herrmann D,

van der Weijden GC, et al. Effects of oocyte maturation regimen

on the relative abundance of gene transcripts in bovine blas-

tocysts derived in vitro or in vivo. Reproduction 2002;124:365–

75.

[74] Lopes AS, Wrenzycki C, Ramsing NB, Herrmann D, Niemann

H, Greve T, et al. Relationship between respiration rates and

mRNA expression of G6PD and Glut-1 genes in individual

blastocysts produced in vitro. Hum Reprod 2006;21:48–9.

Abstract.

[75] Holm P, Booth PJ, Schmidt MH, Greve T, Callesen H. High

bovine blastocyst development in a static in vitro production

system using SOFaa medium supplemented with sodium citrate

and myo-inositol with or without serum-proteins. Theriogenol-

ogy 1999;52:683–700.

[76] Parrish JJ, Susko-Parrish JL, Liebfried-Rutledge ML, Critser ES,

Eyestn WH, First NL. Bovine in vitro fertilization with frozen–

thawed semen. Theriogenology 1986;25:591–600.

[77] SAS Institute. SAS/STAT user’s guide (release 8.2). Cary, NC:

Statistical Analysis System Institute Inc.; 1999.

[78] Box G, Hunter WG, Hunter JS. Statistics for experimenters, 1st

ed, New York: John Wiley & Sons; 1978. p. 150.

[79] Pantaleon M, Kaye PL. Glucose transporters in preimplantation

development. Rev Reprod 1998;3:77–81.

[80] Khurana NK, Niemann H. Energy metabolism in preimplanta-

tion bovine embryos derived in vitro or in vivo. Biol Reprod

2000;62:847–56.

[81] Augustin R, Pocar P, Navarrete-Santos A, Wrenzycki C, Gan-

dolfi F, Niemann H, et al. Glucose transporter expression is

developmentally regulated in in vitro derived bovine preimplan-

tation embryos. Mol Reprod Dev 2001;60:370–6.

[82] Filosa S, Fico A, Paglialunga F, Balestrieri M, Crooke A, Verde

P, et al. Failure to increase glucose consumption through the

pentose-phosphate pathway results in the death of glucose-6-

phosphate dehydrogenase gene-deleted mouse embryonic stem

cells subjected to oxidative stress. Biochem J 2003;370:935–43.

[83] Kaidi S, Bernard S, Lambert P, Massip A, Dessy F, Donnay I.

Effect of conventional controlled-rate freezing and vitrification

on morphology and metabolism of bovine blastocysts produced

in vitro. Biol Reprod 2001;65:1127–34.

[84] Avery B, Madison V, Greve T. Sex and development in bovine in-

vitro fertilized embryos. Theriogenology 1991;35:953–63.

[85] Xu KP, Yadav BR, King WA, Betteridge KJ. Sex-related differ-

ences in developmental rates of bovine embryos produced and

cultured in vitro. Mol Reprod Dev 1992;31:249–52.

[86] Bredbacka K, Bredbacka P. Glucose controls sex-related growth

rate differences of bovine embryos produced in vitro. J Reprod

Fertil 1996;106:169–72.

[87] Gutierrez-Adan A, Granados J, Pintado B, De La Fuente J.

Influence of glucose on the sex ratio of bovine IVM-IVF

embryos cultured in vitro. In: Proceedings of the 14th scientific

meeting, AETE; 1998. p. 166. Abstract.

[88] Larson MA, Kimura K, Kubisch HM, Roberts RM. Sexual

dimorphism among bovine embryos in their ability to make

the transition to expanded blastocyst and in the expression of the

signaling molecule IFN-tau. Proc Natl Acad Sci USA

2001;98:9677–82.

[89] Gutierrez-Adan A, Granados J, Pintado B, De La Fuente J.

Influence of glucose on the sex ratio of bovine IVM/IVF

embryos cultured in vitro. Reprod Fertil Dev 2001;13:361–5.

A.S. Lopes et al. / Theriogenology 68 (2007) 223–236236

[90] Gutierrez-Adan A, Lonergan P, Rizos D, Ward FA, Boland MP,

Pintado B, et al. Effect of the in vitro culture system on the

kinetics of blastocyst development and sex ratio of bovine

embryos. Theriogenology 2001;55:1117–26.

[91] Kane MT. A low molecular weight extract of bovine serum

albumin stimulates rabbit blastocyst cell division and expansion

in vitro. J Reprod Fertil 1985;73:147–50.

[92] Maurer HR. Towards serum-free, chemically defined media for

mammalian cell culture. In: Freshney RI, editor. Animal cell

culture: a practical approach. 2nd ed, Oxford, UK: Oxford

University Press; 1992. p. 15–46.

[93] Morton KM, Herrmann D, Sieg B, Struckmann C, Maxwell

WMC, Rath D, et al. Altered mRNA expression patterns in

bovine blastocysts after fertilization in vitro using flow-cyto-

metrically sex-sorted sperm. Mol Reprod Dev 2007;74:931–

40.

[94] Lonergan P, Gutierrez-Adan A, Pintado B, Fair T, Ward F,

Fuente JD, et al. Relationship between time of first cleavage

and the expression of IGF-I growth factor, its receptor, and

two housekeeping genes in bovine two-cell embryos and

blastocysts produced in vitro. Mol Reprod Dev 2000;57:

146–52.

[95] Tiffin GJ, Rieger D, Betteridge KJ, Yadav BR, King WA.

Glucose and glutamine metabolism in pre-attachment cattle

embryos in relation to sex and stage of development. J Reprod

Fertil 1991;93:125–32.