Human Reproduction, Vol.29, No.2 pp. 234–241, 2014
Advanced Access publication on November 28, 2013 doi:10.1093/humrep/det427
ORIGINAL ARTICLE Embryology
Addition of lysophosphatidic acid
to mouse oocyte maturation media
can enhance fertilization and
developmental competence
Jun Woo Jo 1,2, Byung Chul Jee 1,2,3,*, Chang Suk Suh 1,2,3
and Seok Hyun Kim 2,3,4
1
Department of Obstetrics and Gynaecology, Seoul National University Bundang Hospital, 300 Gumi, Bundang, Seongnam, Gyeonggi 463-707
Korea 2Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University, Seoul, Korea 3Department of
Obstetrics and Gynaecology, Seoul National University College of Medicine, Seoul, Korea 4Department of Obstetrics and Gynaecology, Seoul
National University Hospital, Seoul, Korea
*Correspondence address. Tel: +82-31-787-7254; Fax: +82-31-787-4054; E-mail: [email protected]
Submitted on May 7, 2013; resubmitted on October 11, 2013; accepted on November 1, 2013
study question: Does exposure to lysophosphatidic acid (LPA) during in vitro maturation (IVM) enhance the maturation and developmental competence of mouse oocytes?
summary answer: Supplementation of IVM medium with 30 mM LPA enhanced the developmental competence of in vitro matured
oocytes and so made them more comparable to in vivo matured control oocytes.
what is known already: LPA is a small phospholipid that acts as an extracellular signaling molecule by binding to and activating at least
five G protein-coupled receptors. LPA has various biological actions, with both developmental and physiological effects.
study design, size, duration: During IVM, LPA at six different doses (0, 1, 10, 30, 50 or 100 mM) was added into the TCM-199
medium. After maturation, the developmental competence and other parameters of the oocytes were assessed.
participants/materials, setting, methods: Immature GV stage oocytes from 5- to 6-week-old female BDF-1 mice were
incubated for 17 –18 h in IVM medium containing 0, 1, 10 or 30 mM LPA and then either fertilized in vitro with epididymal sperm, or assessed for
spindle morphology, mitochondrial membrane potential (DCm) or the mRNA expression of a meiotic checkpoint gene (Mad2), a microtubule
structure gene (Hook1), two maternally derived genes (Mater and Hsf1) and an apoptosis-related gene (Caspase6). The fertilized embryos were
grown in vitro to assess blastocyst-formation rates, differential cell counts and apoptosis.
main results and the role of chance: Rates of maturation, fertilization and blastocyst formation and hatching were significantly
higher in the 30 mM LPA-supplemented group (94.3, 96.3, 79.1 and 51.3%, respectively) than in the unsupplemented control (0 mM) group (80.5,
87.5, 61.3 and 37.8%, respectively) and more comparable to that of the in vivo matured oocytes (100, 96.5, 95.3 and 92.9%, respectively). LPA did
not adversely affect mitochondrial activity, spindle integrity, blastocyst cell number, caspase positivity or Mad2 expression. Oocytes matured in
30 mM LPA had reduced Caspase6 expression, but Hook1, Mater and Hsf1 were up-regulated in all of the LPA-supplemented groups.
limitations, reasons for caution: Chromosomal aneuploidy in the resultant blastocysts and the production of normal pups
were not assessed. Only mouse oocytes were assessed.
wider implications of the findings: Supplementation of IVM medium with 30 mM LPA may enhance the developmental competence of mouse oocytes without affecting apoptosis, spindle normalcy or mitochondrial integrity.
study funding/competing interest(s): This study was supported by a research grant (02-2012-021) from the Seoul National
University Bundang Hospital. The authors declare that they have no competing interests.
Key words: animal model / assisted reproduction / embryo development / fertilization / in vitro maturation
& The Author 2013. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
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235
LPA promotes oocyte developmental competence
Introduction
In vitro maturation (IVM) of oocytes has been introduced as alternative to
traditional stimulated IVF treatment, as it can be safely applied to patients
who are at high risk for ovarian hyperstimulation syndrome (Chian et al.,
2004a,b; Cha et al., 2005). Although more than 2000 births have been
reported after human IVM (Chian et al., 2004a,b; Le Du et al., 2005),
the in vitro culture conditions could improve. In animal studies, typical
supplements added to the basal clinical IVM media include gonadotrophins, serum, EGF and E2.
Lysophosphatidic acid (LPA) is an important member of the phospholipid autacoid family, with growth factor-like and hormone-like activities
(Jalink et al., 1994; Tokumura, 1995). LPA regulates platelet aggregation,
blood pressure and cell proliferation (Tokumura et al., 1978), and acts via
membrane-bound G-protein-coupled receptors (Daub et al., 1996; An
et al., 1998).
Human follicular fluid contains 10 –25 mM LPA (Tokumura et al.,
1999). In mice, LPA has been shown to stimulate phospholipase C
through a G-protein-coupled receptor on the surface of the mouse
cumulus cells and to stimulate both extracellular signal-regulated
kinase and p38 mitogen-activated kinase, resulting in the closure or the
loss of gap junctions between the cumulus cell and the oocyte during
IVM (Komatsu et al., 2006). In that study, treatment with 10 mM LPA significantly enhanced oocyte maturation in vitro and significantly lowered
the intracellular cAMP level of the oocytes (Komatsu et al., 2006).
These findings suggest that lowering intra-oocyte cAMP levels via LPA
treatment may promote the nuclear maturation of mouse oocytes
in vitro.
Although supplementing IVM media with LPA can enhance maturation
(Komatsu et al., 2006), very few reports have looked at the optimal concentration of LPA or the longer term developmental competence of the
oocytes. In the present study, we applied LPA during the IVM of mouse
GV stage oocytes to investigate whether LPA could enhance the developmental competence of immature oocytes. Following IVM with and
without LPA, we examined spindle and mitochondrial integrity, as well
as the expression of several genes related to oocyte function and
development.
cumulus –oocyte complexes (COCs) were released by tearing the ampulla
of the oviducts. The cumulus cells were removed enzymatically using
85 IU/ml hyaluronidase (Cook, Brisbane, Australia) and by mechanical dissociation using a glass pipette. Only morphologically normal mature MII
oocytes, as judged by the presence of a first polar body, were used in our
study.
Retrieval of immature oocytes
Mice were treated with i.p. injections of 7.5 IU PMSG (Sigma) and were killed
by cervical dislocation 48 h later. Both ovaries were excised and placed in
1 ml of washing (¼collection) medium (mMTF) supplemented with 0.4%
(w/v) bovine serum albumin (BSA, Sigma). COCs covered with compact
cumulus cells were collected in washing medium by puncturing the antral
follicles and were then washed three times before culture. All cell culture
experiments were performed under mineral oil (Sigma) and were incubated
at 378C in 5% CO2 in air.
In vitro maturation
COCs were matured for 17 – 18 h in TCM-199 (Invitrogen, Carlsbad, USA)
supplemented with 20% FBS (Invitrogen), recombinant FSH/hCG (75 m and
0.5 IU/ml, respectively) (Serono, Geneva, Switzerland), and recombinant
epidermal growth factor (10 ng/ml, Sigma). For the experiment, LPA
(Oleoyl-L-a-lysophosphatidic acid, L7260, Sigma) at six different doses
(0, 1, 10, 30, 50 or 100 mM) was added into the TCM-199 medium. After
IVM, all COCs were denuded completely by treatment with 85 IU/ml hyaluronidase. The extrusion of the first polar body was used as the maturation
criterion and was scored under an inverted microscope (200×). Only
mature oocytes were fertilized with epididymal sperm.
In vitro fertilization
Materials and Methods
The epididymal spermatozoa were retrieved from the cauda epididymis of
8 – 10-week-old BDF-1 mice and the sperm suspensions were pre-incubated
for 1.5 h in capacitation medium (mMTF supplemented with 0.8% BSA). The
denuded oocytes were washed three times to remove the LPA and then put
in mMTF and inseminated with sperm at a final concentration of 2 × 106/ml.
Inseminated oocytes were incubated at 378C in humidified 5% CO2 in air for
6 h and then washed by pipetting and then placed in embryo maintenance
medium (Global media supplemented with 10% HSA, Life global, USA).
Fertilization was assessed by cleavage to the 2-cell stage on Day 1 after insemination. The cleaved embryos were transferred to new embryo maintenance
medium and development to blastocyst was recorded on Day 5 after
insemination.
Animals
Blastocyst cell counts and caspase staining
Five-to-six-week-old female BDF-1 mice (Orient Co., Seoul, Korea) were
used. Animal care was carried out in accordance with the guidelines established by the Institutional Animal Care and Use Committee (IACUC) of
Seoul National University of Bundang Hospital. IACUC specifically approved
these experiments.
Some of the blastocysts were stained using the CaspaTag Pan-Caspase in situ
assay kit according to the manufacturer’s instructions (Millipore, New
Bedford, USA). Positive controls were incubated in 0.1% H2O2 for 1 min
before staining. Negative controls were incubated in polyvinyl alcohol
(PVA, Sigma)/PBS solution only. The blastocysts were mounted onto
slides and evaluated with fluorescence microscopy (Leica DMIL,
Germany). Caspase-positive cells were stained green and all nuclei were
stained blue. Caspase positivity was expressed as the ratio of caspasepositive blastomeres to the total number of nuclei.
Collection of in vivo matured oocytes
Mice were treated with i.p. injections of 5 IU PMSG (Sigma Chemical,
St. Louis, MO, USA), followed by i.p. 5 IU hCG (Sigma) 48 h later. Mice
were killed by cervical dislocation 13 – 14 h later and the oviducts were collected. The oviducts were dissected and placed in 1 ml of washing (¼
collection) medium [modified mouse tubal fluid (mMTF) supplemented
with 0.4% (w/v) bovine serum albumin (BSA, Sigma) as well as 1% (v/v) nonessential amino acids (11140-050, Gibco, Grand Island, USA), 1% (v/v) essential amino acid (R7131, Sigma), 0.5% (v/v) L-Glutamine (25030-081,
Gibco) and 0.25% (v/v) multi-vitamins (11120-052, Gibco)]. The
Differential staining of blastocysts
Blastocysts were fixed in 4% paraformaldehyde (Sigma) for 20 min at RT and
stored in PBS containing 0.5% BSA at 48C. After overnight incubation in 0.5%
Triton X-100 (Sigma) in PBS at 48C, the blastocysts were washed three times
for 2 min in PBS/BSA and then transferred to blocking solution (10% goat
serum, Invitrogen, in PBS at 48C) overnight. The blastocysts were then
236
washed and incubated in ready-to-use primary Oct3/4 antibody (Invitrogen)
for 1 day at 48C. After another washing step (2 times 15 min), blastocysts
were transferred to goat anti-rabbit secondary antibody (Invitrogen) for
1 day at 48C. After washing in PBS, the samples were mounted onto slides
under a coverslip in Vectorshield mounting medium (Vector laboratories,
Burlingame, CA) containing 0.5 mg of DAPI.
Meiotic spindle and chromosome evaluation
In vitro and in vivo matured oocytes were incubated in embryo maintenance
medium for 2 h at 378C in highly humidified air containing 5% CO2. Spindle
integrity was assessed using previously described methods (Huang et al.,
2007). The localization of tubulin and chromatin revealed by FITC and
DAPI fluorescence was observed under 400× magnification with the use
of a fluorescence microscope with a Hamamatsu digital camera imaging
system. A typical barrel-shaped microtubule structure between both poles
with centrally aligned chromosomes was considered normal. Partially
damaged or incomplete spindles were counted as abnormal (Fig. 1).
Staining of mitochondria
In vitro and in vivo matured oocytes were stained (18 and 13 h after hCG, respectively) by the mitochondrial membrane potential (DCm)-specific probe
5,5′ ,6,6′ -tetrachloro-1,1′ ,3,3′ -tetraethylbenzimidazolyl-carbocyanine
iodide (JC-1, Invitrogen) as described previously (Gualtieri et al., 2009).
Oocytes were placed in JC-1 at a final concentration of 5 mg/ml in mMTF
in a humidified incubator containing 5% CO2 at 378C for 30 min. The
stained oocytes were examined by epifluorescence microscopy with FITC
and rhodamine isothiocyanate (RITC) channels using narrow band path
filter sets. The intensity and distribution pattern of mitochondria was
recorded as weak, moderate or strong (Fig. 2). Strong staining was defined
as intense red fluorescence located around the periphery of oocyte (indicates
higher DCm), with green fluorescence dispersed within the oocyte (indicating lower DCm). Moderate staining was defined as signals with comparable
distribution as above, but with less intense brightness. Weak staining was
defined as both signals sparse and dull.
RT–PCR analysis
Total mRNA was extracted from five in vitro matured oocytes in each experimental group using a Dynabeads mRNA DIRECT micro kit (Dynal Asa, Oslo,
Norway) according to the manufacturer’s instructions. cDNA was synthesized from mRNA using 50 ng/ml random hexamer primers, according to
the SuperScript Preamplification System protocol (Gibco-BRL, Grand
Island, USA). PCR was carried out according to the Nova Taq amplification
protocol (Nova Clean-Taq, Genenmed, Korea). Five oocyte equivalents of
cDNA were used as the template for PCR analysis. Primer sequences for
RT– PCR and their amplification conditions are listed in Table I. The
mRNA expression for meiotic checkpoint gene (Mad2), microtubule structure gene (Hook1), maternally derived genes (Mater and Hsf1), and
apoptosis-related gene (Caspase6) were analyzed in cDNA prepared from
oocytes in four different experimental groups (Fig. 3). All experiments
were repeated three times. GAPDH and oocyte-specific Gdf9 were used as
internal controls.
Acquisition of gel images and
semi-quantitative RT– PCR analysis
Images of ethidium bromide-stained agarose gels following electrophoresis of
the RT– PCR products were obtained under UV light at 300 nm with
Bio-Capt software (Vilber Lourmat, Cedex, France). Band intensity was
expressed as relative absorbance units. The ratio between the target
cDNA and GAPDH cDNA was calculated to normalize for initial variations
in sample concentration and as a control for amplification efficiency. The
Jo et al.
mean and standard deviation of all target genes were calculated after normalization to GAPDH.
Statistical analysis
The experiments of developmental competence erratum. Deleted and
spindle staining were repeated at least four times with different pools of
samples. The mitochondrial membrane potential tests were repeated at
least three times. Means and standard deviations were compared using the
Student’s t-test. The proportions were compared using the x 2 tests. Differences were considered statistically significant when P , 0.05. All data were
analyzed using the Statistical Package for the Social Sciences for Windows
(Version 18.0, SPSS, Inc., Chicago, IL, USA).
Results
In our preliminary tests to confirm the proper concentration of LPA in
IVM medium, we evaluated six different doses (0, 1, 10, 30, 50 or
100 mM) of LPA, respectively. Maturation rates gradually increased as
the concentration of LPA increased up to 30 mM, and then the rates
decreased at concentrations of 50 and 100 mM (80.5, 83.5, 91.6, 94.3,
86.4 and 78.9%, for 0, 1, 10, 30, 50, and 100 mM, respectively). After
IVF, the cleavage rate was significantly higher in the 10 and 30 mM LPA
supplemented groups (93.4 and 96.3%) than in the control (0 mM)
group (87.5%), or groups supplemented with 50 and 100 mM LPA
(75.4 and 68.2% cleavage, respectively). The blastocyst formation
rates increased dose dependently up to 30 mM, and then the rates
decreased at concentrations of 50 and 100 mM (61.3, 67.2, 71.8, 79.1,
62.6 and 57.4%, for 0, 1, 10, 30, 50 and 100 mM, respectively).
Because the 50 and 100 mM LPA groups showed lower maturation
and fertilization rates compared with the 30 mM-LPA supplemented
group, we did not perform the further experiments with concentrations
of 50 or 100 mM LPA.
The maturation rate of the IVM oocytes was significantly higher in the
10 and 30 mM LPA-supplemented groups than in the control (0 mM)
group (91.6 and 94.3% versus 80.5%) (Table II). The fertilization and
blastocyst-formation rates were significantly higher in the 30 mM LPAsupplemented group than in the control group (0 mM) (96.3 and
79.1%, respectively versus 87.5 and 61.3%, respectively). The fertilization rate was similar in the 10 and 30 mM LPA groups compared with
in vivo control group (93.4 and 96.3% versus 96.5%). In addition, only
the 30 mM LPA-supplemented group had a significantly higher number
of hatching blastocysts than the control (0 mM) group (51.3 versus
37.8%). However, the in vitro control group and all LPA-supplemented
groups showed lower blastocyst formation and hatching rates than the
in vivo control group (61.3, 67.2, 71.8, 79.1 versus 95.3%, respectively,
37.8, 35.3, 44.4, 51.3 versus 92.9%, respectively). The total or differential
blastomere counts among the five groups were similar. Caspase positivity in the blastocysts was also similar.
The percentage of oocytes with normal spindles (Fig. 1) was not significantly different between the in vivo control group and the 30 mM LPAsupplemented group (Table III). The spindle integrity in the latter
group was higher than in the other groups of in vitro matured occytes,
but the difference was not significant. The percentage of oocytes with
strong mitochondrial staining was highest in the in vivo developed
oocytes and in the oocytes matured in vitro in medium supplemented
with 30 mM LPA, but this latter group was not significantly different
from the other in vitro matured groups (Table IV).
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LPA promotes oocyte developmental competence
Figure 1 Microphotographs showing representative normal and abnormal meiotic spindle organization in in vivo and in vitro matured mouse oocytes
(400X). LPA, lysophosphatidic acid.
The mRNA expression of Hook1, Mater and Hsf1 was higher in all
three LPA-supplemented groups than in the control (0 mM) group. In
contrast, caspase6 mRNA expression was markedly lower in the
30 mM LPA-supplemented group than in the control (0 mM) group,
while the mRNA expression of Mad2 was not altered. The mRNA expression of the oocyte-specific marker Gdf9 was similar in all of the
four groups (Fig. 3).
Discussion
Our study demonstrated that supplementation of the IVM medium with
30 mM LPA significantly enhanced the maturation, fertilization and development of oocytes up to the blastocyst stage when compared with the
unsupplemented control. Moreover, 30 mM LPA did not have a detrimental effect on apoptosis, blastocyst cell number, spindle normalcy,
238
Jo et al.
Figure 2 Microphotographs showing representative mitochondrial images of in vivo and in vitro matured mouse oocytes. The upper panel shows strong,
moderate and weak signal stained by fluorescent JC-1. The middle and lower panels show images of experimental groups.
mitochondrial integrity or the mRNA expression of a meiotic checkpoint
gene (Mad2), a microtubule structural gene (Hook1) and two maternally
derived genes (Mater and Hsf1).
According to a previous report by Komatsu et al. (2006), treatment
with 10 mM LPA significantly improves oocyte maturation in vitro.
Based on that article, we attempted to decide the most appropriate concentration of LPA in the IVM medium. When we determined the LPA
concentration to add to the IVM medium, we found that LPA could
improve the rates of maturation, as well as fertilization and blastocystformation rate of in vitro matured mouse oocytes. We supplemented
the IVM medium with 0–30 mM LPA. These LPA concentrations were
based on our preliminary experiment; we tested the dose-dependent
effects of LPA concentrations from 0 to 100 mM (0, 1, 10, 30, 50 and
100 mM). The survival and cleavage rates gradually increased as the concentration of LPA increased up to 30 mM, but then the rates decreased at
concentrations of 50 and 100 mM. The blastocyst development rate (per
cleaved oocyte) was remarkably higher in the 30 mM LPA-supplemented
group than in the control group (0 mM) (79.1 versus 61.3%) yet still not
comparable to that of the in vivo matured oocytes.
Mitochondria play an important role in cell metabolism because they
provide ATP for use as an energy source. Oocytes need ATP for proper
fertilization and preimplantation embryo development (Dumollard et al.,
2007; Van Blerkom, 2011). We did not measure intra-oocyte ATP levels,
instead, we evaluated the mitochondrial distribution within the oocytes
239
LPA promotes oocyte developmental competence
Table I Primer sequences and the conditions for RT–PCR analysis.
Gene
Primer forward (5′ –3′ ) Primer reverse (5′ – 3′ )
Accession no.
Size (bp)
Annealing temp (88 C)
Cycles
.............................................................................................................................................................................................
Mad2
TAC GCG TGG CAT TTA TCC
ATT GCG GTC CCG ATT CTT
NM_019499
422
54
35
Hook1
TGG AAG AAG AGC TGA AGA AGG
TGT ATT CCA CGG GCA TGA TCT
NM_030014
320
56
35
Mater
GAG CAT CAT GGA GGT GAA GAG
CTT CTG GTT AAT CAG CAG CCA
NM_011860
300
56
35
Hsf1
ACA ACA ACA TGG CTA GCT TCG
GGA GTC CAT ACA CTC CTG TTT
AF082485
280
56
35
Caspase6
GGC AAC CAC GTT TAC GCA TAC
GGC GCT GAG AGA CCT TTC TGT
NM_009811
406
56
35
GAPDH
ACC ACA GTC CAT GCC ATC AC
TCC ACC ACC CTG TTG CTG TA
BC092294
451
60
35
Gdf9
GGT TCT ATC TGA TAG GCG AGG
GGG GCT GAA GGA GGG AGG
NM_008110
446
64
35
Figure 3 The mRNA expression of meiotic checkpoint gene (Mad2),
microtubule structure gene (Hook1), maternally derived genes (Mater
and Hsf1) and apoptosis-related gene (Caspase6) in in vitro matured
mouse oocytes. LPA, lysophosphatidic acid; control, 0 mM LPA.
using the JC-1 staining method, which can detect mitochondrial membrane potential (DCm). A previous report showed that DCm is a sensitive index of oocyte damage because it is influenced by cryopreservation
(Gualtieri et al., 2009). At relatively low potentials, JC-1 usually exists as a
monomer that can be detected by the green fluorescence FITC channel.
As the potential increases, JC-1 monomers assemble to form metastable
stacks or arrays termed J-aggregates that can be detected by the red
fluorescence RITC channel (Reers et al., 1995). JC-1 is an suitable
probe for detecting DCm, whose red and green fluorescence emission
only relies on DCm and is not altered by oocyte size or mitochondria
number (Reers et al., 1995).
In the present study, a higher percentage of oocytes with strong staining was observed in the 30 mM LPA-supplemented group compared with
the in vitro control, although the difference was not significant. This finding
indicates that IVM with 30 mM LPA supplementation does not adversely
affect, and possibly protects mitochondrial integrity within the oocyte,
thus allowing the production of the energy needed by the oocyte
during fertilization and subsequent embryo development.
In the present study, supplementation of IVM medium with 30 mM
LPA did not affect spindle normalcy in the oocytes. This finding may be
partly explained by the preservation of Mad2 and Hook1 mRNA levels.
Mad2 is a meiotic checkpoint protein that regulates anaphase onset
and genome integrity in the oocyte (Musacchio and Hardwick, 2002;
Wang and Sun, 2006). Hook1 plays a role in configuring the microtubule
cytoskeleton and regulating chromosome segregation. This protein is necessary for the correct positioning of microtubule structures within
haploid germ cells (Hamatani et al., 2004; Simpson et al., 2005). Insufficient Hook1 expression can lead to chromosomal abnormalities
(Hamatani et al., 2004). In the present study, Mad2 expression was
not different in the 30 mM LPA-supplemented group compared with
the control group (0 mM). Hook1 expression was relatively higher in
the 30 mM LPA-supplemented group than in the control group
(0 mM). Further studies are needed to determine the frequency of
chromosomal aneuploidy in these oocytes matured in vivo and in vitro
under LPA supplementation and in the resultant blastocysts.
During folliculogenesis and oogenesis, oocytes accumulate maternal
gene products, which are essential for early embryonic development.
The maternally derived genes Mater and Hsf1 have been well studied
(Christians et al., 2000; Tong et al., 2000; Wu et al., 2003). These
genes are conserved during oocyte maturation and embryonic progression. In mice, disruption of Mater in embryos results in arrest at the 2-cell
stage (Tong et al., 2000). In Hsf1 mutants, embryos are arrested at the
1-cell stage (Christians et al., 2000; Wu et al., 2003). In the present
study, Hsf1 expression levels were well conserved in the oocytes that
were matured in vitro under LPA supplementation. In fact, Hsf1 and
Mater expression levels were higher in the LPA-supplemented groups.
Further studies are needed to determine the link between LPA supplementation and fertilization rate.
Caspases are the final effectors during the apoptosis cascade (Izawa
et al., 1998; Exley et al., 1999). In the present study, we measured
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Jo et al.
Table II Developmental competence of in vivo and in vitro matured mouse oocytes according to the concentration
of LPA in the maturation medium.
0 mm
In vivo control
1 mM
10 mM
30 mM
.............................................................................................................................................................................................
Initiated germinal vesicle
—
169
164
166
174
Matured (%, per initiated)
198
136 (80.5)
137 (83.5)
152 (91.6)*
164 (94.3)*
Cleaved (%, per matured)
191 (96.5)
119 (87.5)**
119 (86.9)**
142 (93.4)
158 (96.3)*
Blastocyst (%, per cleaved)
182 (95.3)
73 (61.3)**
80 (67.2)**
102 (71.8)**
125 (79.1)*,**
Hatching blastocyst (%, per cleaved)
169 (92.9)
45 (37.8)**
42 (35.3)**
63 (44.4)**
81 (51.3)*,**
Total blastomere count
69.4 + 12.6
61.1 + 12.8
59.6 + 17.2
62.7 + 16.2
65.9 + 16.1
Trophectoderm count
57.5 + 12.2
49.8 + 9.1
51.4 + 13.2
53.1 + 9.3
56.3 + 9.8
Inner cell mass count
11.9 + 1.8
10.5 + 1.9
11.7 + 2.2
11.5 + 2.1
12.3 + 1.6
Caspase positive blastomeres
4.5 + 0.9
5.6 + 1.8
5.7 + 2.1
6.0 + 2.0
6.0 + 2.4
Each experiment involved four replicates. Mean + SD.
*P , 0.05 versus control (0 mM),
**P , 0.05 versus in vivo control.
Table III Meiotic spindle normalcy of in vivo and in vitro matured mouse oocytes according to the concentration of LPA in the
maturation medium.
0 mM
In vivo control
1 mM
10 mM
30 mM
.............................................................................................................................................................................................
Oocytes examined
187
138
116
144
125
Oocyte with normal spindle
181 (96.8%)
123 (89.1%)*
102 (87.9%)*
132 (91.7%)*
118 (94.4%)
Each experiment involved four replicates.
*P , 0.05 versus in vivo control.
Table IV Mitochondrial grading of in vivo and in vitro matured mouse oocytes according to the concentration of LPA in the
maturation medium.
In vivo control
0 mM
1 mM
10 mM
30 mM
.............................................................................................................................................................................................
Oocytes examined
68
59
66
60
61
Strong
62 (91.2%)
40 (67.8%)*
47 (71.2%)*
47 (78.3%)*
51 (83.6%)
Moderate
4 (5.9%)
12 (20.3%)*
15 (22.7%)*
11 (18.3%)*
7 (11.5%)
Weak
2 (2.9%)
7 (11.9%)
4 (6.1%)
2 (3.3%)
3 (4.9%)
Each experiment involved three replicates.
*P , 0.05 versus in vivo control.
Caspase6 mRNA expression as a representative apoptosis-related gene.
Its expression was much lower in the 30 mM LPA-supplemented group
than in the control (0 mM) and the other LPA-supplemented groups.
Although apoptosis within an oocyte is generally a rare event (Gualtieri
et al., 2009), the near absence of Caspase6 mRNA expression possibly
reflects the healthiness of the oocytes matured in vitro under 30 mM
LPA supplementation.
In conclusion, we demonstrated that LPA exerts positive effects on the
IVM process in a mouse model. The addition of LPA to the IVM media,
especially at 30 mM, improved oocyte maturation, fertilization and subsequent blastocyst development. In the 30 mM LPA-supplemented
group, these developmental rates were brought closer to the values
for in vivo matured oocytes. This positive effect on developmental potential may be partly explained by the increased levels of maternally derived
gene products. Treatment with LPA did not affect the rate of apoptosis of
the resultant blastocysts, nor did it have a detrimental effect on spindle
normalcy or the reservation of intact mitochondria in the in vitro
matured occytes.
Authors’ roles
J.W.J. was responsible for the study conception and design, the data acquisition, analysis and interpretation, and the drafting and revision of the
article. B.C.J. was responsible for the study conception and design, the
LPA promotes oocyte developmental competence
data analysis and interpretation, and the revision and final approval of the
article. C.S.S. and S.H.K. were responsible for the study conception and
design, the data analysis and interpretation and the revision of the article.
Funding
This study was supported by a research grant (02-2012-021) from the
Seoul National University Bundang Hospital.
Conflict of interest
The authors declare that they have no competing interests.
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