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Animal Reproduction Science 99 (2007) 306–316

Effects of ovarian cortex cell co-culture during


in vitro maturation on porcine oocytes maturation,
fertilization and embryo development
Xiao-Yu Chen a , Qing-Wang Li a,b,∗ , Shu-Shan Zhang a ,
Zeng-Sheng Han a , Rui Zhao b , Shu-Yun Wu a ,
Jing Huang a
aCollege of Animal Science, Northwest Agriculture & Forestry University, Yangling,
Shannxi Province 712100, People’s Republic of China
b Department of Biological Engineering, College of Enviroment & Chemical Engineering,

Yanshan University, Qinhuangdao, Hebei Province 066004, People’s Republic of China


Received 22 January 2006; received in revised form 17 April 2006; accepted 11 May 2006
Available online 19 June 2006

Abstract
The objective of the experiments was to evaluate the effects of porcine ovarian cortex cells (pOCCs)
during in vitro maturation (IVM) of porcine oocytes on IVM of porcine oocytes, in vitro fertilization (IVF)
parameters and subsequent embryo development. The pOCCs was cultured in the 500 ␮l TCM199 without
hormone until the confluence, and then cultured in 500 ␮l TCM199 supplemented with hormone for 12 h
before the oocytes added. Porcine oocytes were co-cultured with the pOCCs monolayers in the co-culture
system for 44 h, following fertilized in the mTBM for 6 h. Finally, the presumptive zygotes were cultured
for 144 h in the NCSU-23 supplemented with 0.4% BSA. The results showed that matured M II oocytes
in the co-culture group were higher than that in the control group (P < 0.05). Although penetration did not
differ between the co-culture and control groups (P = 0.481), polyspermy declined in the co-culture group
(P < 0.05), whereas male pronucleus (MPN) formation was improved in the co-culture group compared
with the control group (P < 0.05). More blastocysts developed in the co-culture group than that in the control
group (P < 0.05); however, the cleavage rates and the mean number cells per blastocyst showed no significant
difference between the treated group and the control group (P = 0.560 and 0.873, respectively). In conclusion,
the presence of the pOCCs monolayers during IVM enhanced the maturation quality of the porcine oocytes,

∗ Corresponding author at: College of Animal Science, Northwest Agriculture & Forestry University, Yangling, Shannxi

Province 712100, People’s Republic of China.


E-mail address: ysulqw@126.com (Q.-W. Li).

0378-4320/$ – see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.anireprosci.2006.05.007
X.Y. Chen et al. / Animal Reproduction Science 99 (2007) 306–316 307

reduced the polyspermy, increased the percentages of MPN formation and blastocyst, but the blastocyst
quality was not improved.
© 2006 Elsevier B.V. All rights reserved.

Keywords: Porcine; Ovarian cortex cells; Monolayers; IVM; IVF

1. Introduction

In vitro production (IVP) technology in domestic animals has been considered as an important
technology in agriculture and the biomedical field. However, there are still many inadequacies in
IVP of the pig compared with other technologies currently used in domestic animals. Incomplete
cytoplasmic maturation, polyspermy, low percentage of MPN formation and week development
capability of the blastocyst have not been overcome completely until today (Day, 2000; Abeydeera,
2002). In order to improve the quality of matured porcine oocytes and embryos in vitro, all kinds
of methods were attempted in IVP of pig. Supplement of IVM medium with follicular cell-
conditioned medium (Mattioli et al., 1988; Ding and Foxcroft, 1994) or co-culture with follicular
shell pieces (Abeydeera et al., 1998) have positive effects on nuclear maturation, subsequent
fertilization and porcine embryo development in vitro. Addition of porcine oviductal epithelial
cells (pOECs) during the IVM resulted in more blastocysts formation as well as the blastocyst
quality is improved (Bureau, 2000; Kidson et al., 2003; Qian et al., 2005). These studies indicated
the IVM system at present is still suboptimal; therefore, the co-culture with the homologous cell
during IVM is an efficient method which can improve the quality of porcine matured oocytes and
embryos in vitro.
Most mammalian oocytes enter meiosis during the fetal stage, arrest at the dictyotene stage
of the first meiotic division until re-initiation of meiosis under the surge of gonadotropins or
degradation because of follicles atresia. During the follicles growth, oocytes gradually gain more
development capacity until arriving at Prophase II (M II) stage after undergoing germinal vesicle
breakdown (GVBD) and Prophase I (M I). Synchronization of nuclear and cytoplasmic mat-
uration of oocytes is regarded as pivotal precondition in the following development. How to
perfect the maturation condition in vitro and improve the synchronization of nuclear and cyto-
plasmic maturation has become a focus during porcine embryos IVP (Day, 2000; Niemann and
Rath, 2001; Abeydeera, 2002). Clearly, ovarian cortex performs important function during the
reproductive life span of the female mammalian as the best optimal environment of oocytes mat-
uration or follicles growth in vivo. In order to find out the positive effect of ovarian cortex on the
follicles growth in vitro, the ovarian cortex was co-cultured successfully with follicles in vitro
(Fortune et al., 1998; Parrot and Skinner, 1999; Oktay et al., 2000; Cushman et al., 2002; Silva
et al., 2004). Moreover, the effects of oviductal epithelial, granulosa and cumulus monolayers
co-cultured with oocytes on the meiotic resumption competence were also studied during IVM
(Teotia et al., 2001; Abeydeera, 2002; Kidson et al., 2003; Romar et al., 2003). Now, we won-
der whether the maturation and subsequent development capacity of porcine oocytes could be
improved when porcine ovarian cortex cells (pOCCs) are directly co-culture with oocytes during
whole IVM.
In the present study, we investigated the effects of pOCCs co-cultured during IVM on the
maturation, fertilization parameters, embryo development capacity and resultant blastocyst quality
of the pig in vitro.
308 X.Y. Chen et al. / Animal Reproduction Science 99 (2007) 306–316

2. Materials and methods

2.1. Culture medium

Unless otherwise stated, all chemicals used in this study were purchased from Sigma Chem-
ical Co. (St. Louis, MO, USA). The maturation medium was modified TCM-199 medium
(TCM199; Gibco, Grand Island, NY; Cat. No. 31100-035) supplemented with 0.1% PVA (Cat.
No. P8136), 3.05 mM glucose (Ameresco, Cat. No. 0188), 0.91 mM sodium pyuvate (Cat. No.
P5280), 0.57 mM/ml l-cysteine (Cat. No. C7352), 10 IU/ml PMSG (Cat. No. G4877), 10 IU/ml
hCG (Cat. No. C1063), 50 IU/ml penicillin G and 50 IU/ml streptomycin sulfate. The fertilization
medium was a modified Tris-buffered medium (mTBM, pH 7.2) (Abeydeera and Day, 1997b)
containing 113.1 mM NaCl, 3 mM KCl, 7.5 mM CaCl2 ·2H2 O, 20 mM Tris, 11 mM d(+)-glucose,
5 mM sodium pyuvate, 1 mg/ml BSA (Ameresco, Cat. No. 0332, pH 7.0) and 2 mM caffeine
(Cat. No. C0750). The embryo culture medium was NCSU-23 (Petters and Wells, 1993) supple-
mented with 0.4% BSA. The cell culture medium was TCM-199 supplemented with 2.2 mg/ml
NaHCO3 , 20% fetal calf serum (FCS, Hyclone, Cat. No. SH30406.02), 1 mg/ml glutamine (Cat.
No. G8540), 1% (v/v) non-essential amino acids (Cat. No. M7145), 150 IU/ml penicillin, and
100 ␮g/ml streptomycin.

2.2. Ovaries selection, oocytes collection and in vitro maturation

Porcine ovaries were obtained from prepubertal swine within 5 min after slaughter. The selected
ovaries were in the follicular phase with no apparent corpus luteum as previously described (Parrott
and Skinner, 1998a,b) and were transported to the laboratory in d-PBS containing 150 IU/ml
penicillin, and 100 ␮g/ml streptomycin at 35 ◦ C within 2 h. In the laboratory, cumulus–oocyte
complexes (COCS ) were collected from non-atretic follicles (2–6 mm in diameter) by aspiration
with an 18 G needle fixed to a 10 ml disposable syringe. Only oocytes with compact cumulus cells
showing a homogeneous and granulated cytoplasm were selected and rinsed twice in TL-HEPES-
PVA (Funahashi et al., 1997), at last the COCS was washed three times with maturation medium
that previously equilibrated in incubator for 3 h under 5% CO2 in air at 38.5 ◦ C. About 40–50
washed COCS were placed into each well of the 4-well multidish (Nunclon, Roskilde, Denmark,
Cat. No. 176740) containing 500 ␮l of IVM medium which had previously been covered with
mineral oil (Cat. No. M8410) and equilibrated for 12 h with the pOCCs monolayers at 38.5 ◦ C
under 5% CO2 in air prior use. The COCS were co-cultured at the same medium with pOCCs
monolayers for 44 h.

2.3. Porcine ovarian cortex cells (pOCCs) monolayers preparation

The pOCCs monolayers were prepared beforehand for IVM. The same ovaries as above
described were prepared. After aspiration, the fat tissue and ligaments of ovaries were trimmed off
carefully. The ovaries were chipped into small tissues, then the medulla and visible antral follicles
were removed. Following this, the ovarian cortex was micro-dissected in fragments approxi-
mately 0.5 mm × 0.5 mm (1 mm thick) for next digestion as previously described (Silva et al.,
2004). The pieces of ovarian cortex were digested with 1 mg/ml collagenase (Cat. No. C2674),
2.5 mg/ml trypsinase (Ameresco, Cat. No. 0458) and 1 mg/ml hyaluronidase (Cat. No. H2126)
for 1 h at 37 ◦ C. After terminated the digestion, the digested cells clusters were dissociated by
gentle, repeated pipetting followed by centrifugation at 750 × g for 4 min. The supernatant was
X.Y. Chen et al. / Animal Reproduction Science 99 (2007) 306–316 309

removed before the pellet was re-suspended with D-Hank’s medium. The procedure above was
repeated thrice.
After the final centrifugation, discarded the resultant supernatant, re-suspended the pellet
in fresh cell culture medium, then seeded in the 4-well multidish at a density of 0.5 × 106 to
1 × 106 cells/ml, the cell was maintained in cell culture medium at 38.5 ◦ C under 5% CO2 in air
for 48 h and the medium was changed after 48 h. Once the pOCCs monolayers arrived to 80% of
confluence stage, changed the cell culture medium with the maturation medium and incubated at
38.5 ◦ C under 5% CO2 at least 12 h before oocytes added for IVM.

2.4. Glutathione (GSH) assay

The concentration of intracellular GSH was determined as described previously (Brian and
James, 2004). Basically, denuded matured oocytes in each group were washed three times in
stock buffer (0.2 M sodium phosphate containing 10 mM EDTA, pH 7.2). Groups of 30 oocytes
from each group were transferred with 5 ␮l stock buffer to a 1.5 ml Eppendorf tube and stored at
−80 ◦ C until the day of the assay. On the day of the assay, 5 ␮l of 1.25 M H3 PO4 was added to
every tube and the oocytes were ruptured using a blunt glass rod. The intracellular concentration
of GSH in oocytes was determined using the dithionitrobenzonic acid-glutathione disulphide
(DTNB-GSSH) reductase recycling assay using spectrophotometer.

2.5. In vitro fertilization and embryo culture

When maturation was completed, the expanded cumulus cells were removed by pipetting
in NCSU-23 containing 0.1% hyaluronidase. The denuded oocytes with first polar (Pb I) were
washed three times in 100 ␮l drops of mTBM equilibrated for 24 h in an incubator at 38.5 ◦ C
under 5% CO2 in air. About 40–50 oocytes were stored in 50 ␮l drops of mTBM under mineral
oil then equilibrated in a 35 mm × 10 mm Petri dish (Nunc, Roskilde, Denmark, Cat. No. 153066)
at 38.5 ◦ C under 5% CO2 in air at least 0.5 h. A frozen semen straw was prepared for IVF as
described previously (Brian and James, 2004). Briefly, the frozen–thawed semen were thawed
in 8 ml of d-PBS supplemented with 0.1% BSA, 0.10 g/l CaCl2 ·2H2 O, 150 IU/ml penicillin, and
100 ␮g/ml streptomycin at 40 ◦ C in 50 ml polypropylene conical tube. The washed semen was
centrifuged at 73 × g for 5 min to remove dead spermatozoa in the subnatant. The supernatant
was transferred to a new tube and the d-PBS was added to 15 ml. The semen was centrifuged at
1052 × g for 5 min to collect viable spermatozoa in the pellet. The pellet was re-suspended with
mTBM and the spermatozoa density was regulated to 1.5 × 105 spermatozoa/ml. Then 50 ␮l of
the spermatozoa was added to each drop, mixed, and the oocytes and spermatozoa co-incubated
at 38.5 ◦ C in an atmosphere of 5% CO2 for 6 h. Then the fertilized oocytes were washed three
times with embryos culture medium and cultured for another 18 h in the same medium.
After IVF, zygotes were washed three times in 100 ␮l drops of embryo culture medium and
transferred to the 4-well multidish which containing 500 ␮l embryo culture medium under warm
mineral oil. The zygotes were cultured at 38.5 ◦ C in a mixture of 5% CO2 in air. After 48 h post-
IVF, embryos were placed in fresh embryo culture medium in the same way as described above.

2.6. Assessment of maturation, fertilization results and development of oocytes

After IVM culture, about 50–60 denuded oocytes from every treatment were mounted on slides
with a cover slip, secured by two lines of vaseline, and then fixed with acetic acid:ethanol (1:3,
310 X.Y. Chen et al. / Animal Reproduction Science 99 (2007) 306–316

v/v) for 48–72 h. The fixed oocytes were stained with acetic–orcein (Cat. No. O7380) (1% orcein
in 45% acetic acid) then sealed with nail polish. Maturation degree of oocytes was evaluated under
a phase-contrast microscope.
At the end of IVF, about 50–60 fertilized oocytes from each treatment were stained with
acetic–orcein as above method. Oocytes were considered to be polyspermic that had more than one
swollen sperm head or male pronuclei with corresponding sperm trail. Oocytes were considered
to be monospermic that had only one swollen sperm head. MPN formation was considered with
a visual identification of a MPN. Oocytes were considered to be cleaved that the embryos with
two to eight blastomers 48 h after IVF.
When the embryos culture period (144 h post-IVF) was completed, the percentages of the
blastocyst in every group were calculated. The blastocystes were placed in 110 ␮l PBS containing
10 ␮l of 1 mg/ml bis-benzimide H33342 (Hoechst 33342, Cat. No. B2261) staining for 10 min,
then the embryos were de-stained in PBS three times for 5 min and the cell number of the blastocyst
was counted under a fluorescent microscope.

2.7. Experimental design

Experiment 1 investigated the effect of pOCCs monolayers co-culture during IVM on mat-
uration of porcine oocytes. Before IVM, all of the selected COCS were assigned to two
treatment groups randomly and equally. One group consisted of COCS that maturated in
vitro with the pOCCs monolayers for 44 h, the other group, as the control group, in which
the COCS were cultured in vitro for 44 h alone. At termination of IVM, one-quarter of
denuded oocytes from every group were stained with orcein to evaluate the meiotic compe-
tence of the oocytes, one-quarter of denuded oocytes from every group were prepared for
GSH assay, the other oocytes were prepared for IVF. This experiment consisted of six repli-
cates.
Experiment 2 investigated the effect of pOCCs monolayers co-culture during IVM on the
penetration, polyspermy and MPN formation rates of porcine oocytes after IVF. After IVF,
half of the fertilized oocytes from both of the treatment groups were removed and stained
with orcein respectively, and then the percentages of the penetration, the polyspermy and
the MPN formation were counted. This experiment consisted of six replicates. The other
fertilized oocytes were cultured in NCSU-23 supplemented with 0.4% BSA for continuous
development.
Experiment 3 investigated the effect of pOCCs monolayers co-culture during IVM on the
cleavage and embryo development rates of fertilized porcine oocytes. The rates of the cleav-
age and the blastocyst of the embryos were counted respectively at 48 and 144 h after IVF.
Finally, the cell number in each blastocyst was recorded. This experiment consisted of six
replicates.

2.8. Statistical analysis

Each experiment was repeated six times. Data are presented as means ± S.E.M. All rates were
modeled according to binomial model of parameters and were analyzed by one-way ANOVA. The
percentages of oocytes reaching each stage of meiosis, polyspermy, MPN, as well as the cleavage
and blastocyst formation rates, and the mean number of cells per blastocyst were analyzed by
Tukey test in SPSS 11.0 soft. Differences among treatments were considered significant when the
P-value < 0.05.
X.Y. Chen et al. / Animal Reproduction Science 99 (2007) 306–316 311

Table 1
Effect of pOCCs mono layers co-culture during IVM on in vitro matured oocytes after IVM
Treatment No. of total oocytesa Percentage of oocytes at each meiotic stage (mean ± S. E. M.)

GVb M Ic AI/TId M IIe

Co-culture 463 2.8 ± 0.7 a 3.3 ± 1.6 a 8.7 ± 1.l a 85.2 ± 3.6 a
Control 452 3.3 ± 0.3 a 6.4 ± 1.9 b 9.7 ± 1.4 a 80.5 ± 4.1 b

Values with different letters (a and b) within columns are significantly different (P < 0.05).
a The experiment was replicated six times.
b GV: germinal vesicle.
c M I: Metaphase I.
d AI/TI: Anaphase I or Telophase I.
e M II: Metaphase II.

3. Results

3.1. Experiment 1

Effect of pOCCs monolayers co-culture during IVM on maturation of porcine oocytes is shown
in Table 1. The presence of the pOCCs monolayers during IVM significantly increased percentage
of oocytes in M II stage compared to the control group (P = 0.023, and P < 0.05, respectively).
Although the percentages of oocytes in GV and Anaphase I/Telophase I (AI/TI) stage were
lower than that in the co-culture group, the percentages of oocytes in the stages above showed
no significant decrease in the co-culture group after IVM (P = 0.116 and 0.705, respectively).
More oocytes in M I stage were found in the control group than co-culture group (P = 0.036,
and P < 0.05, respectively). After maturation (Table 2), it was found that GSH concentration per
oocyte was higher than the oocyte before IVM (P = 0.024 and 0.004, and P < 0.05, respectively)
and GSH concentration per oocyte in co-culture group was higher than that in the control group
(P = 0.031, and P < 0.05, respectively).

3.2. Experiment 2

Effect of the pOCCs monolayers cell co-culture during IVM on penetration, polyspermy and
MPN formation rates of porcine oocytes after IVF are presented in Table 3. The total penetration
rates were similar in both groups (P = 0.481), with the proportion of total penetration ranging
from 88.01 to 90.45%. It was found that the polyspermy rate showed significant decrease in the
co-culture group compared with that in the control group (P = 0.039 and P < 0.05, respectively),
but the mean number of spermatozoa per penetrated oocyte showed no significant decrease in the

Table 2
Effect of pOCCs mono layers co-culture during IVM on intracellular GSH concentration of porcine oocytes
IVM group No. of oocytes examineda GSH concentration (pmol/oocyte; mean ± S. E.M.)

Before maturation in vitro 96 4.1 ± 0.3 a


Co-culture group 96 8.1 ± 1.3 b
Control group 96 6.3 ± 1.1 c

Values with different letters (a–c) within columns are significantly different (P < 0.05).
a The experiment was replicated six times.
312 X.Y. Chen et al. / Animal Reproduction Science 99 (2007) 306–316

Table 3
Effect of pOCCs mono layers co-culture during IVM on fertilization parameters 6 h post-IVF of porcine oocytes
Treatment No. of total fertilized oocytesa Percentage of fertilized oocytes (% ± S.E.M.)

Total penetration Polyspermyb MPNc S/Od

Co-culture 354 88.0 ± 2.4 a 39.4 ± 1.5 a 97.5 ± 3.2 a 1.9 ± 0.1 a
Control 341 90.4 ± 3.6 a 45.0 ± 2.8 b 90.0 ± 3.9 b 1.6 ± 0.5 a

Values with different letters (a and b) within columns are significantly different (P < 0.05).
a The experiment was replicated six times.
b Percentage of the number of penetrated oocyte.
c MPN: male pronucleus.
d S/O: spermatozoa/penetrated oocyte.

Table 4
Effect of pOCCs mono layers co-culture during IVM on embryo development and mean number cells per blastocyst
Treatment No. of total Percentage of embryo at different Mean number of cells/
fertilized oocytesa development stage of fertilized blastocyst (% ± S.E.M.)
oocytes (% ± S.E.M.)
Cleavage Blastocystb

Co-culture 373 78.5 ± 2.3 a 25.6 ± 2.7 a 35.7 ± 2.3 a


Control 374 80.1 ± 2.1 a 21.9 ± 2.2 b 33.6 ± 2.0 a

Values with different letters (a and b) within columns are significantly different (P < 0.05).
a The experiment was replicated six times.
b Percentage of the number of cleavage oocytes.

co-culture group (P = 0.766). Co-culture of the pOCCs monolayers during IVM showed positive
effect on the MPN formation of oocytes after IVF, the proportion of the fertilized oocytes with
MPN was significantly increased in the co-culture group compared with that in the control group
(P = 0.013, and P < 0.05, respectively).

3.3. Experiment 3

Effect of the pOCCs monolayers co-culture during IVM on the cleavage rate and embryo
development rate of fertilized porcine oocytes are presented in Table 4. The pOCCs monolayers
co-cultured during IVM had a positive effect on the blastocyst rate (P = 0.024, and P < 0.05,
respectively), although the cleavage rate showed no significant increase in the co-culture group
(P = 0.560). At the end of 144 h after IVF, it was found that the mean number of cells per blastocyst
showed no significant increase in the co-culture groups (P = 0.861).

4. Discussion

The results indicated that we first used pOCCs monolayers during IVM and were successful
in developing a co-culture system. Porcine oocytes nuclear and cytoplasm maturation degree,
fertilization parameters and blastocyst development were promoted significantly in the present
co-culture system.
The ovary cortex is poorly vascularized in vivo (Van Wezel and Rodgers, 1996; Herrman and
Spanel-Borowski, 1998), in which atresia follicles constantly occur. When oocytes co-cultured
X.Y. Chen et al. / Animal Reproduction Science 99 (2007) 306–316 313

with pOCCs monolayers, there will be a new environment that is richer in hormones, nutri-
ents and/or oxygen during IVM because the distribution of those will not be influenced by
microcirculation. Ovarian cell cultured in vitro could secret keratinocyte growth factor (KGF)
which is a 28-kilodalton (kDa) protein and belongs to the fibrobkast growth factor family (FGF-
7) (Parrott et al., 2000). It has been reported that KGF is primarily produced by stromal- or
mesenchymal-derived cells in many tissues and acts as an epithelial cell-specific mitogen to
proliferate granulose/cumulus cell growth in vitro (Jewgenow, 1996). Furthermore, the feasible
progesterone concentration produced by cumulus cells is responsible for an acceleration of GVBD
in porcine oocytes (Shimada et al., 2002; Yamashita et al., 2003). It seemed that the presence of
pOCCs monolayers during IVM resulted a higher percentage of oocytes completing M I syn-
chronally to reach the M II stage after IVM in the co-culture group compared with the control
group. Oocytes matured in vivo keep cytoplasmic contact with cumulus cells by gap junctions
until the M I stage and then lose the contact during progression towards the M II stage. But, in
vitro, oocytes progressively lose contact with surrounding cumulus cells immediately from the
onset of maturation (Motlik et al., 1986). Lower rate of oocytes at GV stage and higher GSH
content in the co-culture group also shown that the pOCCs monolayers co-culture system could
provide a better microenvironment for cumulus cells keeping the gap junction from GV stage to
M I stage. It has been reported that gap junctions between oocytes and cumulus cells are related
to GSH content of porcine oocytes cultured in vitro through the synthesis of GSH in cumulus
cells or oocytes (Mori et al., 1998).
When porcine oocytes were cultured in the co-culture system of the present study, secretions
by the pOCCs monolayers probably participated in the regulation mechanism and facilitated
maturation progress of oocytes by modulating the intercellular cooperation during IVM. In mam-
mals, sperm penetration triggers oocyte activation, premature migration and partial exocytosis of
cortical granule (CGs). The lower polyspermy in the co-culture group showed that the pOCCs
monolayers may have contributed to the normal distribution of intracellular organelles (mitochon-
dria and CGs) during IVM. It has been reported that normal distribution of CGs during IVM may
play an important role in preventing polyspermy (Day, 2000), and the occurrence of polyspermy
of porcine matured oocytes from in vitro may be due to a delay in CGs exocytosis (Wang et al.,
1997). The ability to form a male pronucleus after IVF is correlated with cytoplasmic maturation
(Funahashi and Day, 1993) and intracellular GSH concentration in pigs (Yoshida et al., 1993).
GSH participates in sperm decondensation in parallel with oocyte activation after fertilization, as
well as in the transformation of the penetrated sperm head into the MPN. In the present study, the
high concentration of GSH in the co-culture group were mainly attributable to the two reasons
below: (1) the microenvironments containing the pOCCs monolayers was propitious to synthesis
GSH utilizing the cysteine by reduced the oxygen stress; (2) the presence of pOCCs monolayers
during IVM maintained availably the gap junctions from the GV stage to the M I stage, which
promoted intracellular cumulation of the extracellular GSH. Intracellular GSH was beneficial to
normal male MPN formation presumably by reduction of oxidative stress (Yoshida et al., 1992,
1993). It seems probable that the presence of pOCCs monolayers induces a lower oxygen ten-
sion in vitro culture environment, as reported for oviduct cell in culture (Bavister, 1988), so
that oocytes may effectively utilized cysteine to synthesis GSH by preventing the oxidation of
cysteine to cystine. The rates of total penetration showed no significant difference between the
both of two groups, which due to suboptimal fertilization time in vitro and suboptimal semen
concentration. Generally, porcine sperm–oocytes incubation time changes during 3–12 h, and the
number of polyspermic oocytes increases with incubation time when oocytes were fertilized in
vitro (Abeydeera and Day, 1997a). In our study, although the percentages of cleavage in the two
314 X.Y. Chen et al. / Animal Reproduction Science 99 (2007) 306–316

groups showed no significantly different, the rate of blastocyst showed higher in the co-culture
group compared with the control group. The pOCCs monolayers co-culture of oocytes enhanced
the subsequent percentage of oocytes developed to blastocysts, which showed the homologous
cells co-culture with porcine oocytes during IVM may become a potential method to increase the
maturation (nuclear and cytoplasm) degree in porcine embryos IVP. It has been evaluated that the
blastocyst development and the hatchability of the blastocyst were enhanced, when the pOECs
was co-cultured with porcine oocytes during IVM (Bureau et al., 2000; Kidson et al., 2003).
When the in vivo-derived porcine embryos cultured in vitro, distinct ultrastructural differences
was observed from the 2- and 4-cell stage to the blastocyst stage compared with their in vivo
counterparts. These deviations were related to the quality of porcine embryos cultured in vitro
(Hyttel and Niemann, 1990). It has been evaluated that abnormal cleavage and low cell numbers
in blastocyst produced in vitro were due to apparent deficiencies in actin filament distribution
within the cytoplasm and inadequate cytoplasmic maturation of oocytes (Wang et al., 1999). Low
rate of blastocyst was correlated with the cytoplasmic maturation degree of porcine oocytes in the
control group compared with the co-culture group. In the present study, the presence of pOCCs
monolayers had no influence on the mean number of cells per blastocyst, which was different from
the effect of the pOEC co-culture during the IVM (Kidson et al., 2003). Perhaps, the diversity
between the both was caused by the secretion of the two types of cells. Although the NCSU-
23 supplemented BSA was considered the best medium at present of porcine embryos culture in
vitro (Wang and Day, 2002), suboptimal embryo culture medium here may be responsible for poor
mean number of cells per blastocyst cultured in vitro when compare with the embryos derived in
vivo (Machat et al., 1998; Wang and Day, 2002).
In summary, the present study demonstrated that the pOCCs monolayers co-culture during IVM
enhanced the quality of nuclear and cytoplasmic maturation degree of the oocytes. Furthermore,
the co-culture of the pOCCs monolayers with oocytes during IVM reduced the percentages of
polyspermy, but improved rate of MPN after fertilization. Although the co-culture seemed have
no positive effect on both the total penetration and mean number sperm per oocyte here, the rate of
the blastocyst increased at the presence of the pOCCs monolayers during IVM. Finally, it seemed
that the presence of pOCCs monolayers during the IVM had no effect on the mean cell number
of the blastocyst at 144 h after IVF.

Acknowledgement

This research was supported by the Institute of Science and Technology of Qin Huangdao City
of Hebei Province of China (No. D08).

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