BIOLOGY OF REPRODUCTION 73, 737–744 (2005)
Published online before print 15 June 2005.
DOI 10.1095/biolreprod.105.041103
Maturation of Bovine Oocytes in the Presence of Leptin Improves Development
and Reduces Apoptosis of In Vitro-Produced Blastocysts1
Marc Boelhauve,3 Fred Sinowatz,4 Eckhard Wolf,2,3 and Fabı́ola F. Paula-Lopes3
Department of Molecular Animal Breeding and Biotechnology,3 Ludwig-Maximilian University,
85764 Oberschleissheim, Germany
Institute of Veterinary Anatomy, Histology, and Embryology,4 Ludwig-Maximilian University,
80539 Munich, Germany
ABSTRACT
The series of events associated with oocyte growth and mat-
uration determines the oocyte’s ability to undergo successful fer-
tilization, cleavage and embryonic development. Among the
molecules involved in these events, leptin has been identified as
a modulator of oocyte function. Experiments were conducted to
determine whether leptin treatment of oocytes during matura-
tion affects their developmental capacity after fertilization and
whether it has long-lasting effects on apoptosis and gene ex-
pression in the resulting blastocysts. Cumulus-oocyte complexes
(COCs) were matured in serum-free medium containing 0 (con-
trol), 1, 10, or 100 ng/ml leptin or in medium supplemented
with 10% (v/v) estrous cow serum (ECS). Addition of leptin dur-
ing oocyte maturation had no effect on cleavage rate after fer-
tilization. However, an increased proportion of oocytes that ma-
tured in the presence of 1 or 10 ng/ml leptin developed to blas-
tocysts, which exhibited increased cell numbers. The proportion
of apoptotic cells was reduced in blastocysts originating from
leptin- or ECS-treated oocytes. Transcript levels of the genes en-
coding leptin receptor (LEPR), signal transducer and activator of
transcription (STAT3), BCL2 associated X-protein (BAX), and
baculoviral inhibitor of apoptosis protein repeat-containing 4
(BIRC4, also known as XIAP), were determined by reverse tran-
scriptase-quantitative polymerase chain reaction analysis of ex-
panded and hatched blastocysts. Depending on the dose used,
leptin treatment of oocytes resulted in increased LEPR, STAT3,
and BIRC4 mRNA levels and reduced BAX mRNA levels in blas-
tocysts. In conclusion, leptin improved the ability of the oocyte
to sustain embryonic development and had long-term effects on
blastocyst apoptosis and transcript abundance of LEPR, STAT3,
and apoptosis-associated genes.
apoptosis, embryo, leptin, leptin receptor, oocyte development
INTRODUCTION
Leptin, the 16-kDa product of the leptin gene (LEP), is
a pleiotropic peptide secreted primarily by adipocytes. Even
though leptin was initially identified as a signal that regu-
lates food intake and energy expenditure [1, 2], it has also
been implicated in a wider range of physiological functions,
1
Supported by the Deutsche Forschungsgemeinschaft (Research Unit
‘‘Mechanisms of Embryo-Maternal Communication,’’ FOR 478/1).
2
Correspondence: Eckhard Wolf, Department of Molecular Animal Breed-
ing and Biotechnology, Gene Center, Ludwig-Maximilian University, Feo-
dor-Lynen-Strasse 25, 81377 Munich, Germany. FAX: 49 89 2180 76849;
e-mail: ewolf@lmb.uni-muenchen.de
Received: 17 February 2005.
First decision: 14 March 2005.
Accepted: 14 June 2005.
Q 2005 by the Society for the Study of Reproduction, Inc.
ISSN: 0006-3363. http://www.biolreprod.org
737
including reproduction [3–5]. The first evidence that leptin
was required for reproduction came from studies in mice.
Lepob/Lepob mutant mice, which express a truncated leptin
protein, are obese and infertile [2, 6]. Fertility of Lepob/
Lepob mice can be restored by exogenous leptin supple-
mentation. Recently, the leptin system has been localized
in the reproductive tract of several species. Leptin has been
detected in human [7] and mouse [8, 9] oocytes and in
human follicular fluid [7, 10], as well as in granulosa and
cumulus cells [7]. Leptin mRNA expression was found in
human granulosa and cumulus cells [7] and in pig oocytes
at different stages of follicular development and oocyte
maturation [11].
The leptin receptor (LEPR) has high sequence homology
to the class I cytokine receptor superfamily [12], and alter-
native 39 terminal mRNA splicing generates several recep-
tor isoforms [12–15]. The long transmembrane isoform
containing a 302-residue-long cytoplasmic domain is pre-
sent in the hypothalamus as well as in peripheral tissues
[12, 16]. This isoform is believed to mediate most of the
leptin signaling [12, 15, 17] through the signal transducer
and activator of transcription 3 (STAT3) and mitogen-ac-
tivated protein kinase (MAPK) pathways. The other trans-
membrane receptor isoforms have short cytoplasmic do-
mains and are expressed in many tissues. They have a janus
kinase box and are capable of signaling through the MAPK
pathway. However, there is evidence that this signaling is
weaker than that of the long isoform [16]. Exposure of
oocytes to physiological leptin concentrations increases
STAT3 [18] and MAPK [11] phosphorylation. Moreover,
Lepr/LEPR mRNA is expressed in mouse oocytes [18, 19]
and in human granulosa [7, 14], and cumulus cells [7].
Taken together, these findings suggest that leptin modulates
function of the oocyte and/or its surrounding cells.
Acquisition of oocyte developmental competence is a
limiting step determining the ability of the oocyte to un-
dergo successful fertilization, cleavage. and embryonic de-
velopment. This final differentiation of the oocyte is or-
chestrated by a complex network of growth factors and cy-
tokines leading to proper nuclear and cytoplasmic matura-
tion [20]. There is evidence that leptin stimulates oocyte
maturation. Leptin concentrations of 10–100 ng/ml in-
creased the proportion of pig oocytes reaching metaphase
II stage [11]. In the mouse, culture of follicles in the pres-
ence of 10–1000 ng/ml leptin increased oocyte germinal
vesicle breakdown [9]. Moreover, leptin administration in
rats reduced the incidence of follicular apoptosis [4], im-
plying that leptin can rescue oocytes and follicles from atre-
sia by attenuation of apoptosis. Whereas apoptosis is known
to occur in bovine oocytes [21, 22] and preimplantation
embryos [23–25], there are few studies on the regulation
738 BOELHAUVE ET AL.
of apoptosis and gene expression in bovine blastocysts de-
rived from oocytes matured in different controlled systems.
Therefore, the objectives of the present study were to de-
termine 1) whether addition of leptin (1, 10 or 100 ng/ml)
during maturation of bovine oocytes enhances developmen-
tal capacity of oocytes, as reflected by the ability of the
oocyte to develop to the blastocyst stage, the number of
blastocyst cells, and the incidence of blastocyst apoptosis;
and 2) whether changes in these parameters are associated
with altered expression of genes relevant for leptin signal
transduction or apoptosis-related genes.
MATERIALS AND METHODS
Materials
Recombinant human leptin (purity $ 97% as determined by SDS-
PAGE; endotoxin contamination # 0.1 ng/mg leptin) was purchased from
Sigma Chemical Co. (St. Louis, MO). Bovine pituitary-derived FSH and
LH were purchased from Sioux Biochemicals Inc. (Sioux Center, IA).
Frozen semen from various bulls was donated by Rinderunion Baden-
Württemberg e.V. (Stuttgart, Germany). Tissue culture medium (TCM)-
199 with Earle salts was from Biochrom AG (Berlin, Germany). Unless
otherwise stated, all reagents used for in vitro production of bovine em-
bryos were purchased from Sigma.
In Situ Cell Death Detection Kit (fluorescein) was obtained from Roche
Diagnostics Corporation (Indianapolis, IN). Propidium iodide and poly-
vinylpyrrolidone (PVP) were purchased from Sigma. Prolong Antifade Kit
was obtained from Molecular Probes (Eugene, OR), RQ1 RNA-free DN-
ase was from Promega (Madison, WI), and RNase A was from Qiagen
(Hilden, Germany).
Random hexamer primers, TriZol reagent, DNase, RNaseOut, reverse
transcriptase enzyme Superscript II, TOPO Cloning Kit, TOPO vector,
One Shot chemically competent bacteria, and SOC medium were pur-
chased from Invitrogen (Carlsbad, CA). HotStarTaq polymerase, RNAlater
and QIAprep Spin Mini Kit were obtained from Qiagen. Primer Express
2 software, SybrGreen, uracil N-glycosylase, and ABI PRISM 7000 se-
quence detector system were from Applied Biosystems (Foster City, CA).
EcoRI, dNTPs, and XhoI were purchased from Fermentas Life Sciences
(Hanover, MD), SeeDNA was from Amersham Biosciences Europe GmbH
(Freiburg, Germany), and X-gal was from Carl Roth (Karlsruhe, Germa-
ny).
In Vitro Production of Bovine Embryos
Embryos were produced based on procedures described previously
[26]. Ovaries were obtained from slaughtered cows and washed several
times with PBS (1 mM potassium phosphate, 2 mM potassium chloride,
8 mM sodium phosphate, 0.9 mM calcium chloride, 0.49 mM magnesium
chloride and 0.8% sodium chloride) at 25–308C to remove blood and de-
bris. To obtain COCs, 2–10 mm follicles were aspirated with a 20-gauge
needle and a vacuum pressure of approximately 100 mm Hg. COCs that
had at least one layer of compact cumulus cells were washed two times
in serum-free oocyte maturation medium (TCM-199 with Earle salts con-
taining 22 mg/ml FSH and 8 mg/ml LH) supplemented with 1 mg/ml po-
lyvinylalcohol and used for subsequent steps. Groups of 30 COCs were
placed in four-well plates containing 400 ml per well serum-free oocyte
maturation medium supplemented with leptin (1, 10 or 100 ng/ml) or 10%
(v/v) estrous cow serum (ECS) as a positive control. Oocytes were allowed
to mature for 22–24 h at 398C in an atmosphere of 5% [v/v] CO2 in
humidified air. After in vitro maturation, COCs were washed once in IVF-
TALP (modified Tyrode stock solution supplemented with 6 mg/ml essen-
tially fatty-acid-free BSA, 0.022 mg/ml pyruvic acid, and 0.01 mg/ml hep-
arin) and transferred to four-well plates containing 400 ml IVF-TALP per
well. Spermatozoa from a pool of bulls (two to three bulls) were purified
by swim-up procedure in Sperm-TALP (modified Tyrode stock solution
supplemented with 6 mg/ml essentially fatty-acid free BSA and 0.11 mg/
ml pyruvic acid) at 398C in 5% [v/v] CO2 in humidified air for 1 h. Swim-
up was followed by centrifugation of spermatozoa from the upper phase
for 10 min at 700 3 g. The spermatozoa pellet was reconstituted in IVF-
TALP and oocytes were fertilized with 25 ml spermatozoa suspension (;1
3 106 spermatozoa/ml). Approximately 18 h after fertilization, presump-
tive zygotes were denuded of cumulus cells by vortexing in 1 ml synthetic
oviduct fluid (SOF) (SOF stock was modified on the day of use by adding
10% [v/v] ECS, 4% [v/v] essential amino acids, and 1% [v/v] nonessential
amino acids) for 3 min. Groups of 25–30 presumptive zygotes were placed
in 400 ml SOF overlaid with mineral oil and cultured at 398C in a humid-
ified atmosphere of 5% (v/v) CO2, 5% (v/v) O2, and 90% (v/v) N2.
TUNEL and Propidium Iodide Labeling
The TUNEL procedure was used to detect DNA fragmentation ob-
served in late stages of apoptosis. The enzyme terminal deoxynucleotidyl
transferase is a DNA polymerase that catalyzes the transfer of a fluorescein
isothiocyanate-conjugated dUTP nucleotide to a free 39 hydroxyl group
present in DNA strand breaks. Blastocysts were removed from culture
medium (SOF) and washed once in 500 ml PBS (10 mM potassium phos-
phate, 0.9% [w/v] NaCl, pH 7.4) containing 1 mg/ml polyvinylpyrrolidone
(PBS-PVP). Zona pellucida-intact blastocysts were fixed in four-well
plates containing 500 ml per well of 4% (w/v) paraformaldehyde in PBS,
pH 7.4, for 1 h at room temperature, washed once in PBS-PVP, and stored
in 600 ml PBS-PVP at 48C until TUNEL analysis. On the day of TUNEL
staining, blastocysts were permeabilized in four-well plates containing 500
ml permeabilization solution (0.5% [v/v] Triton X-100 containing 0.1%
[w/v] sodium citrate) for 1 h at room temperature. Positive and negative
controls were incubated in 200 ml RQ1 RNase-free DNase (50 U/ml) at
378C for 1 h. Blastocysts were washed in PBS-PVP and incubated in a 25
ml drop of TUNEL reaction mixture (containing fluorescein isothiocya-
nate-conjugated dUTP and the enzyme terminal deoxynucleotidyl trans-
ferase), as prepared by the manufacturer, for 1 h at 378C in the dark.
Negative control was incubated in the absence of terminal deoxynucleo-
tidyl transferase. Blastocysts were then incubated in 500 ml RNase A (50
mg/ml) for 1 h at room temperature, followed by 200 ml propidium iodide
(0.5 mg/ml) for 30 min at room temperature. Blastocysts were washed
three times in PBS-PVP to remove excess propidium iodide, placed on
poly-L-lysine coated slides, and mounted with 16 ml mounting medium
containing Antifade as recommended by the manufacturer. Each blastocyst
was analyzed for total number of nuclei and number of TUNEL-labeled
nuclei using a Zeiss Axiovert 200 M fluorescence microscope coupled
with the Zeiss filter number 9 (FITC) or 15 (Texas red) as well as the
triple band filter 25 (FITC, Texas red and DAPI). Some blastocysts were
also examined for photomicroscopy using a Zeiss laser scanning micro-
scope 510 Meta (LSM 510 Meta). For fluorescein, an argon ion laser
adjusted to below 560 nm was used, and for propidium iodide, a helium-
neon laser adjusted to above 560 nm was used. Digital images were ob-
tained using the LSM 510 Meta system with a 203 objective. Blastocysts
derived from oocytes treated with 10 ng/ml leptin had higher cell numbers,
and therefore these images were acquired using a 103 objective.
Cloning of Selected Genes
Amplification primers (Table 1) were designed according to sequences
found in GenBank and using the software Primer Express 2. Selected
primers were submitted to BLAST analysis to check for sequence speci-
ficity. Complementary DNA was produced by reverse transcription of bo-
vine ovary and brain total RNA (20 ng/ml). A polymerase chain reaction
(PCR) was performed during the primer optimization procedure to deter-
mine the presence of specific PCR products and the absence of primer-
dimer formation. For each of the selected primers, the PCR reaction in-
cluded 0.25 ml HotStarTaq (5 IU/ml), 1 ml primer (5 mM), 1 ml cDNA, 2
ml dNTPs (1 mM), 1.25 ml magnesium chloride (25 mM) and 11.5 ml
water. The thermal cycler was programmed to run an initial incubation at
958C for 15 min to activate HotStarTaq. This was followed by 35 PCR
cycles of DNA denaturation (958C for 15 sec), primer annealing (608C for
30 sec), and elongation (728C for 30 sec). A sample corresponding to 10%
of the PCR products was subjected to 2.5% agarose gel electrophoresis
containing ethidium bromide (10 mg/ml). PCR products were visualized
by exposure to ultraviolet light.
Plasmid standards were established for every gene of interest with the
TOPO Cloning Kit as recommended by the manufacturer. The cloned bac-
teria suspension (One Shot chemically competent bacteria) was spread on
an agarose plate containing antibiotic (50 ng/ml ampicillin) and X-gal (50
mg/ml) selection and incubated overnight at 378C. Selected positive col-
onies were cultured in Luria-Bertani medium (10 mg/ml tryptone, 5 mg/
ml yeast extract, and 10 mg/ml sodium chloride) containing 50 ng/ml
ampicillin overnight at 378C. The cloned plasmids were isolated using the
QIAprep Spin Mini Kit following the manufacturer’s instructions. A sam-
ple from the isolated plasmids was digested with the restriction endonu-
clease EcoRI (10 IU/ml) to determine the length of the cloned fragments.
Isolated plasmids were linearized using XhoI (10 IU/ml) digestion. Plas-
mids were subjected to 1% agarose gel electrophoresis as described before.
Linearized plasmids with the sequence of interest were diluted to 1 mil-
lion/ml. To confirm plasmid identity, the PCR products were subjected to
 LONG-TERM EFFECTS OF LEPTIN IN BLASTOCYSTS 739

TABLE 1. Amplification primers.

Product size GenBank

Gene Oligo Primer sequence Tm (8C) (bp) accession No.
H2AFZ Sense 59 TTC GAA ATG GCT GGC GG 39 60 91 NMp174809
Antisense 59 GGA ACT GCA AAC CGG CTC T 39 59
LEPR Sense 59 GGA CGT TAT GAG GCA GTT GTT G 39 58 150 U83512
Antisense 59 TGC CTT CCC TTC AAT GTC ATC 39 59
BAX Sense 59 GCA GAG GAT GAT CGC AGC TG 39 61 148 U92569
Antisense 59 CAG GGC CTT GAC CAC CAG 39 59
STAT3 Sense 59 CTG CAG CAG AAG GTT AGC TAC AAA 39 59 85 AJ620655
Antisense 59 TTC TAA ACA GCT CCA CGA TTC TCT C 39 59
BIRC4 Sense 59 GAA GCA CGG ATC ATT ACA TTT GG 39 59 89 AP458770
Antisense 59 CCT TCA CCT AAA GCA TAA AAT CCA G 39 58

sequence analysis (Laboratory for Functional Genome Analysis—LAFU-
GA, Munich, Germany).
RNA Isolation and Reverse Transcription
Total RNA was isolated from pools of blastocysts (4 blastocysts/tube).
TriZol reagent (500 ml) was added to the tube followed by a 15-sec vor-
texing to homogenize the samples. Samples were centrifuged (10 min,
11 900 3 g, 48C) and the supernatant (450 ml) was mixed with 100 ml
chloroform in a new tube, vortexed, and incubated for 10 min at room
temperature. Following a second centrifugation step, the top phase (250
ml) was transferred to a new tube and gently mixed with 2 ml of SeeDNA,
0.1 volume of sodium acetate (3 M), and 2 volumes 100% ethanol and
incubated for 10 min at room temperature. Samples were centrifuged as
described before, and the pellet was washed in 500 ml 75% ethanol and
centrifuged again for 5 min. The pellet was dried on air, resuspended in
16.75 ml nuclease-free water, heated for 10 min at 558C, and chilled on
ice. A DNase digestion step was performed using 2 ml DNase digestion
buffer and 0.25 ml DNase (1 IU/ml) at 258C for 15 min. DNase was
inactivated with 1 ml EDTA (25 mM) and denaturation at 658C for 10
min.
For reverse transcription, 2 ml random hexamer primers (3 mg/ml), 2
ml dNTPs (10 mM) and 2.5 ml water were added to the samples, incubated
for 5 min at 658C, and chilled on ice. Samples were subjected to a short
centrifugation step and incubated with 8 ml 53 transcription buffer, 4 ml
dithiothreitol (0.1 M), and 1 ml RNaseOut (40 IU/ml) for 10 min at 258C.
This was followed by a 2-min incubation step at 428C and addition of 0.5
ml reverse transcriptase enzyme Superscript II (200 IU/ml). The reverse
transcription reaction was carried out for 50 min at 428C and terminated
by incubating the samples for 15 min at 708C.
Quantitative PCR
Quantitative PCR (qPCR) was performed using the real-time PCR ABI
PRISM 7000 sequence detector system and SybrGreen as a double-strand-
ed DNA-specific fluorescent dye. Amplification mixes contained 2 ml
cDNA, 12.5 ml SybrGreen PCR Mix, 0.25 ml uracil N-glycosylase (1 IU/
ml), 1.5 ml primer (5 mM), and 7.25 ml water. The ABI PRISM 7000
sequence detector system was programmed to start with uracil N-glyco-
sylase activation for 2 min at 508C. The program continued with 45 cycles
of DNA denaturation (15 sec at 958C), primer annealing (30 sec at 608C),
and elongation (30 sec at 728C). Serial dilutions of plasmids were used to
establish a standard curve for each gene of interest. Each qPCR was run
in triplicate. Transcript copy numbers for LEPR, STAT3, BCL2 associated
X-protein (BAX), and baculoviral inhibitor of apoptosis protein repeat-
containing 4 (BIRC4, also known as XIAP) were calculated using the stan-
dard curve method with determination of PCR amplification efficiency and
normalized for histone 2A (H2AFZ) transcript levels [27]. Transcript levels
in the control group (0 ng/ml leptin) were set to 1 and data in the treatment
groups were calibrated accordingly.
Experiments
Effect of leptin on developmental capacity of bovine oocytes and blas-
tocyst apoptosis. COCs were matured in serum-free maturation medium
containing 0, 1, 10 or 100 ng/ml recombinant human leptin or maturation
medium containing 10% (v/v) ECS as positive control. After 22–24 h
maturation, oocytes were subjected to in vitro fertilization and culture.
Cleavage rate was assessed at Day 3 and development to the blastocyst
stage at Days 7 and 8 postinsemination. This experiment was replicated
eight times, using 312–374 oocytes per treatment. Blastocysts from five
in vitro production (IVP) replicates (46–72 blastocysts per treatment) were
harvested at Day 8 and fixed in 4% paraformaldehyde for TUNEL analysis
and determination of total cell number.
Effect of leptin on transcript levels in blastocysts. COCs were matured
in serum-free maturation medium containing 0, 1, 10 or 100 ng/ml recom-
binant human leptin or maturation medium containing 10% (v/v) ECS as
positive control. After 22–24 h maturation, oocytes were subjected to in
vitro fertilization and culture. Expanded and hatched blastocysts were har-
vested from six IVP replicates (three replicates in May–June 2004 and
three replicates in March–April 2005). Expanded blastocysts were har-
vested at Day 7 and hatched blastocysts at Day 8 postinsemination. For
each treatment group, six pools of four expanded and six pools of four
hatched blastocysts were analyzed.
Statistical Analysis
Data were analyzed by least-squares analysis of variance. For experi-
ments on the effect of leptin on developmental capacity of bovine oocytes
and blastocyst apoptosis, independent variables were treatment and repli-
cate. For experiments on blastocyst gene expression, independent variables
were treatment, experimental period, and replicate. Dependent variables
were percentage of oocytes cleaved, percentage of blastocyst development,
percentage of apoptotic cells, total cell number, mRNA copy number nor-
malized to the housekeeping gene H2AFZ, and the arcsine of the per-
centage variables. Two analyses were performed: one by the General Lin-
ear Models (GLM) procedure of SAS [28], in which replicate (i.e., day of
IVP procedure) was considered a fixed variable, and one by the Mixed
Models procedure of SAS, where replicate was considered a random var-
iable. Probability values were similar for both methods, and probability
values reported herein are derived from the analysis by GLM. Percentage
data were analyzed without transformation and after being subjected to
the arcsine transformation to correct for problems of nonnormality asso-
ciated with analysis of percentage data. Probability values were similar for
both analyses. Orthogonal contrasts and the PDIFF procedure of SAS were
performed when appropriate to determine differences between various lev-
els of a treatment. Data are presented as least squares means 6 SEM.
RESULTS
Effect of Leptin on Developmental Capacity of
Bovine Oocytes
The purpose of this experiment was to determine wheth-
er leptin enhances developmental capacity of bovine oo-
cytes. There was no effect of leptin supplementation during
oocyte maturation on cleavage rate after fertilization (81.7
6 2.1, 85.6 6 2.0, 85.0 6 2.0, 81.4 6 2.0, and 82.0 6
2.3% cleavage rate for 0, 1, 10 and 100 ng/ml leptin and
ECS, respectively). However, an increased (P , 0.05) pro-
portion of oocytes developed to blastocysts in the 10 ng/
ml leptin treatment group at days 7 and 8 and in the 1 ng/
ml leptin treatment group at Day 8 (Fig. 1, A and B). Treat-
ment of oocytes with 100 ng/ml leptin or ECS did not affect
their capacity to develop to blastocysts as compared to the
serum-free control (Fig. 1).
740
FIG. 1. Effect of leptin on developmental capacity of bovine oocytes.
The proportion of oocytes that developed to the blastocyst stage at Days
7 (A) and 8 (B) postinsemination are shown. Results are presented as least
squares means 6 SEM of 8 replicates, using 312–374 oocytes per treat-
ment. Significant (P, 0.05) differences from the control group (0 ng/ml
leptin) are indicated by an asterisk.
Effect of Leptin on Blastocyst Apoptosis
Figure 2, A–D, displays representative confocal images
of bovine blastocysts subjected to TUNEL analysis. A blas-
tocyst derived from a control oocyte (0 ng/ml leptin) is
shown in Figure 2A. Figure 2, B and C, shows blastocysts
derived from oocytes matured in the presence of 1 and 10
ng/ml leptin, respectively. Note that these blastocysts have
reduced incidence of TUNEL-positive blastomeres (yellow
in color). Indeed, the percentage of blastomeres undergoing
apoptosis was reduced in blastocysts originating from oo-
cytes matured in the presence of leptin (1, 10 or 100 ng/
ml, P , 0.001) or ECS (P , 0.001, Fig. 3A). Propidium
iodide staining demonstrated that blastocyst cell number
was increased by 1 (P , 0.05) and 10 (borderline signifi-
cance, P 5 0.06) ng/ml leptin (Fig. 3B). There was no
effect of ECS on blastocyst cell number (Fig. 3B).
Effect of Leptin on Blastocyst Gene Expression
Messenger RNA levels for H2AFZ, LEPR, STAT3, BAX,
and BIRC4 were determined by reverse transcriptase-quan-
titative PCR (RT-qPCR) to evaluate whether the effect of
leptin during oocyte maturation has consequences for the
expression of these selected genes at the expanded and
hatched blastocyst stage. There was no effect of leptin on
expression of the housekeeping gene H2AFZ in expanded
and hatched blastocysts. Expanded but not hatched blasto-
cysts originating from ECS-treated oocytes exhibited in-
creased (P , 0.001) H2AFZ mRNA levels. Therefore, the
ECS treatment group was not included for the final analysis
of transcript levels in expanded blastocysts.
BOELHAUVE ET AL.
FIG. 2. Representative confocal images illustrating the frequency of ap-
optotic nuclei in bovine blastocysts subjected to TUNEL analysis. Blas-
tocysts were labeled with fluorescein isothiocyanate-conjugated dUTP
(green channel) and propidium iodide (red channel). A) A blastocyst de-
rived from control oocytes matured in the absence of leptin (0 ng/ml
leptin). Arrows point to TUNEL-positive nuclei. Note the higher frequency
of TUNEL-positive blastomeres in the control blastocyst. B and C) Blas-
tocysts derived from oocytes matured in the presence of 1 and 10 ng/ml
leptin, respectively. D) Representative picture of a positive control blas-
tocyst pretreated with DNase. Digital images were obtained using the
LSM 510 Meta system with a 203 objective. Note that the blastocyst
derived from oocytes treated with 10 ng/ml leptin (C) had higher cell
numbers, and that therefore the image was acquired using a 103 objec-
tive.
Maturation of bovine oocytes in the presence of physi-
ological doses of leptin increased LEPR mRNA abundance
in expanded (1 and 10 ng/ml, P , 0.05) and hatched blas-
tocysts (1 ng/ml, P , 0.05), whereas blastocysts originating
from oocytes treated with a higher dose of leptin (100 ng/
ml) tended to have reduced levels of LEPR mRNA (Fig.
4A). ECS had no effect regarding this parameter.
STAT3 mRNA levels were increased in expanded blas-
tocysts derived from the 100 ng/ml leptin treatment group
(P , 0.001) and in hatched blastocysts derived from all
leptin-treated oocytes (1 and 10 ng/ml, P , 0.001; 100 ng/
ml, P , 0.05). STAT3 transcript levels were increased with
borderline significance (P 5 0.06; Fig. 4B) in blastocysts
originating from ECS-treated oocytes.
BAX transcript levels were reduced in blastocysts de-
rived from 10 ng/ml leptin-treated oocytes (P , 0.001 in
expanded and P , 0.05 in hatched blastocysts) and in ex-
panded blastocysts originating from the 100 ng/ml leptin
treatment group (P , 0.05, Fig. 4C). There was no effect
of 1 ng/ml leptin regarding this parameter at the expanded
or hatched blastocyst stage. Moreover, treatment of oocytes
with 100 ng/ml leptin or ECS did not affect the level of
BAX transcripts in hatched blastocysts (Fig. 4C).
In contrast, transcript levels of the antiapoptotic gene
BIRC4 were increased in blastocysts originating from 1 ng/
ml leptin-treated oocytes (P , 0.05 in expanded and P ,
0.001 in hatched blastocysts) and the abundance of BIRC4
mRNA was—with borderline significance—also increased
 LONG-TERM EFFECTS OF LEPTIN IN BLASTOCYSTS 741

in expanded blastocysts from the 100 ng/ml leptin treatment

group (P 5 0.06, Fig. 4D). Hatched blastocysts derived
from other leptin- and ECS-treated oocytes did not exhibit
altered BIRC4 transcript levels as compared to the control
(Fig. 4D).
DISCUSSION
The present study demonstrated that leptin at physiolog-
ical concentrations exerted a beneficial effect during mat-
uration of bovine oocytes. Leptin concentrations (1 and 10
ng/ml) used in the present study represent physiological
serum leptin levels found in periparturient [29, 30] and cy-
clic [31] cows. Leptin improved oocyte developmental
competence in a dose-dependent manner. Addition of 1 and
10 ng/ml leptin to serum-free maturation medium did not
affect cleavage rate but increased development to the blas-
tocyst stage and blastocyst cell number. It is well known
that oocyte developmental potential is a reflection of proper
cytoplasmic maturation. Even though most bovine oocytes
resume meiosis and progress to metaphase II following in
vitro maturation [32], cytoplasmic maturation in vitro is
generally compromised, leading to low rates of develop-
ment. Thus, the positive effect of leptin on the develop-
mental potential of oocytes may be related to their cyto-
plasmic maturation. Potential modes of action include di-
rect or indirect (cumulus cell-mediated) effects of leptin
restructuring oocyte cytoskeleton [33], reprogramming pro-
tein synthesis [33], or inhibiting apoptosis [4]. FIG. 3. Effect of leptin during maturation of bovine oocytes on the pro-
This study shows for the first time that leptin supple- portion of apoptotic cells and total cell number of in vitro derived blas-
mentation during bovine oocyte maturation has long-term tocysts. The proportion of TUNEL-positive blastomeres (A) and blastocyst
effects on in vitro-produced blastocysts, i.e., reduction in cell numbers (B) are shown. Results are least squares means 6 SEM of
the percentage of apoptotic cells. Moreover, the reduction five replicates using 46–72 blastocysts per treatment. Significant differ-
ences from the control group (0 ng/ml leptin) are indicated by * (P ,
in TUNEL-positive cells in blastocysts derived from leptin- 0.05) and ** (P , 0.001). Borderline significance is indicated by † (P 5
treated oocytes was associated with increased BIRC4 and 0.06).
reduced BAX expression. As a member of the inhibitor of
apoptosis protein family, BIRC4 acts as an antiapoptotic
molecule and blocks apoptosis by inhibiting caspase 3, 7,
and 9 [34]. Inhibition of caspases prevents the oligonucleo- supplementation but not by ECS during oocyte maturation.
somal DNA fragmentation typical of apoptosis. In contrast, Thus, it is likely that leptin regulated the apoptotic ma-
BAX is a major proapoptotic molecule from the BCL2 pro- chinery in a specific manner.
tein family [35]. The relative ratio between proapoptotic Exposure of oocytes to physiological leptin concentra-
and antiapoptotic proteins determines the fate of the cell in tions resulted in a dose-dependent increase in LEPR and
many cases. Leptin acted in the oocyte and/or cumulus cells STAT3 mRNA levels in bovine blastocysts. In contrast,
causing a long-term shift in the expression of proapoptotic LEPR mRNA levels in blastocysts originating from oocytes
and antiapoptotic genes. It possibly reduced intracellular matured with a high dose of leptin (100 ng/ml) had a ten-
BAX available to form BAX-BAX homodimers and dency to be reduced. Moreover, this high leptin concentra-
blocked caspase activity through BIRC4 leading to cell sur- tion had only a limited effect: it reduced blastocyst apopto-
vival. sis but had no influence on blastocyst development or blas-
Apoptosis plays an important role in mammalian devel- tocyst cell number. A potential reason for the limited effect
opment as a quality control mechanism to eliminate cells under this condition might be a dose-dependent downreg-
that are damaged, nonfunctional, abnormal, or misplaced ulation of LEPR mRNA and protein at the oocyte stage.
[36, 37]. The occurrence of apoptosis has been demonstrat- This point needs to be clarified in future studies. Inhibition
ed in many cell types, including bovine oocytes [22], cu- of LEPR expression by high doses of leptin has previously
mulus cells [21, 38], and preimplantation embryos [23, 25, been observed in other tissues, including hypothalamus
39–41]. The beneficial effect of leptin during oocyte mat- [42] and adrenal gland [43]. The fact that STAT3 mRNA
uration suggests a role for leptin as a survival factor min- levels in blastocysts were also affected by leptin supple-
imizing cellular damage to oocyte and/or cumulus cells. It mentation in the oocyte maturation medium suggests com-
is possible that leptin rescued oocytes that would otherwise plex and long-lasting regulatory effects of leptin on its sig-
have generated arrested embryos or embryos with poor de- naling machinery.
velopmental potential because of a high incidence of apo- On the basis of our findings, it is tempting to speculate
ptosis. The beneficial effect of leptin during oocyte matu- how leptin might function in the bovine ovary to exert a
ration resulted in blastocysts with reduced apoptosis. positive effect on oocytes. Perhaps leptin produced by oo-
Whereas ECS mimicked leptin’s effect of minimizing blas- cytes and cumulus cells acts in an autocrine/paracrine man-
tocyst apoptosis, it had no effect on the expression of ap- ner to 1) upregulate LEPR expression; 2) activate STAT3
optosis-associated genes. The levels of BIRC4 and BAX and MAPK pathways enhancing nuclear and cytoplasmic
transcripts in blastocysts were strongly affected by leptin oocyte maturation; and 3) alter the ratio between antiapop-
742 BOELHAUVE ET AL.

FIG. 4. Effect of leptin during maturation

of bovine oocytes on the abundance of
LEPR (A), STAT3 (B), BAX (C) and BIRC4
(D) mRNAs in expanded (white bars) and
hatched (black bars) blastocysts. Results
are least squares means 6 SEM of six
pools of four expanded and six pools of
four hatched blastocysts per treatment
group. Significant differences from the
control group (0 ng/ml leptin) are indicat-
ed by * (P , 0.05) and ** (P , 0.001).
Borderline significance is indicated by † (P
5 0.06).
 LONG-TERM EFFECTS OF LEPTIN IN BLASTOCYSTS
totic and proapoptotic molecules, reducing cumulus cell
and oocyte apoptosis. There is evidence that the long LEPR
isoform mediates most of the leptin signaling [12, 15, 17]
through the STAT3 and MAPK pathways. Indeed, leptin
has been shown to induce STAT3 phosphorylation in
mouse oocytes [18] and MAPK phosphorylation in porcine
oocytes [11]. In porcine oocytes, leptin improved oocyte
maturation through activation of MAPK signaling cascade
[11]. It has been suggested that this effect is mediated by
the short receptor isoform [11], which has not yet been
detected in bovine ovarian tissue [44].
The developmental potential of oocytes matured in vitro
is reduced as compared to in vivo oocytes [45]. To char-
acterize the specific requirements for bovine oocyte matu-
ration, it is necessary to establish a serum-free oocyte mat-
uration medium. In the current study, development to the
blastocyst stage and blastocyst cell number were not dif-
ferent between oocytes matured in serum-free medium or
medium supplemented with 10% serum. Other studies have
also reported successful development to the blastocyst stage
following serum-free oocyte maturation [46–48]. However,
blastocysts derived from oocytes matured under serum-free
conditions had increased incidence of apoptosis. This neg-
ative effect was prohibited by the addition of leptin to se-
rum-free maturation medium.
In conclusion, leptin treatment during oocyte maturation
improved developmental potential, resulting in increased
development to the blastocyst stage with reduced numbers
of apoptotic cells. Further, increased BIRC4 and reduced
BAX mRNA levels as well as effects on transcript levels of
LEPR and STAT3 mRNA were detected in blastocysts orig-
inating from oocytes of some of the leptin-treatment
groups. Thus, physiological doses of leptin during oocyte
maturation may have long-term effects on the expression
of developmentally important genes in early embryos.
ACKNOWLEDGMENTS
The authors thank Tuna Guengoer for collecting ovaries and blasto-
cysts for RT-qPCR analyses, Myriam Weppert for collecting ovaries, and
the Rinderunion Baden-Württemberg e.V. (Stuttgart, Germany) for donat-
ing the semen.
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