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BIOLOGY OF REPRODUCTION 57, 1478-1483 (1997)
Amino Acids in Maturation Medium and Presence of Cumulus Cells at Fertilization
Promote Male Pronuclear Formation in Porcine Oocytes Matured and
Penetrated In Vitro'
Hak-Hyun Ka,3 5 Ken Sawai,s Wei-Hua Wang, 4'5 Kyung-Soon Im,6 and Koji Niwa2,s
Division of Animal Science and Technology,s Faculty of Agriculture, Okayama University, Okayama 700, Japan
Department of Animal Science and Technology,6 College of Agriculture and Life Sciences,
Seoul National University, Suweon, 441-744, Korea
ABSTRACT
The present study was conducted to examine the ability of
porcine oocytes to achieve male pronuclear (MPN) formation
when they are matured and penetrated in vitro under various
culture conditions. When cumulus-enclosed oocytes were cultured for 24-48 h in modified Whitten's medium (pH 7.4) supplemented with 10% porcine follicular fluid, 10 IU eCG/ml, and
10 IU hCG/ml (designated mWM-FG), nuclear maturation of
oocytes reaching metaphase II was completed by 36 h after the
start of culture. However, there were no differences in the proportions (94-95%) of oocytes penetrated in vitro by cryopreserved ejaculated spermatozoa or inthe rates (35-45%) of MPN
formation between oocytes cultured for 36 and 48 h. When cumulus-enclosed oocytes were cultured for 36 h in mWM-FG
supplemented with 2% (v:v) minimal essential medium (MEM)
essential amino acids (EAA) with the addition of 0.1 mM glutamine and/or 1% (v:v) MEM nonessential amino acids (NEAA)
and inseminated in vitro, 93-97% of oocytes were penetrated
regardless of the presence of amino acids during maturation, but
the rates of MPN formation were higher in the presence (7984%) than in the absence (51%) of any amino acids. The addition of EAA+ NEAA and/or 0.57 mM cysteine to mWM-FG also
did not affect sperm penetration in vitro, while it promoted
MPN formation (76-83%) in penetrated oocytes as compared
with those matured in the absence of amino acids and cysteine
(53%). When oocytes were freed from cumulus cells after culture in mWM-FG, sperm penetration rates were not different
between cumulus-enclosed (100%) and cumulus-free (92%) oocytes, but the rate of MPN formation was higher in cumulusenclosed (53%) than in cumulus-free (28%) oocytes. When
EAA+NEAA+cysteine was added to mWM-FG, MPN formation
was not improved in cumulus-free oocytes but was much improved (78%) in cumulus-enclosed oocytes. These results indicate that MPN formation in porcine oocytes is promoted by the
addition of amino acids and/or cysteine in simple maturation
medium and by the presence of cumulus cells at fertilization in
vitro.
INTRODUCTION
Male pronuclear (MPN) formation is generally considered a physiological measure of cytoplasmic maturation of
Accepted August 5, 1997.
Received May 27, 1997.
'This work was supported by Grant-in-Aid for Scientific Research B
(no. 07456123) from the Ministry of Education, Science, Sports and Culture of Japan.
2Correspondence. FAX: (86) 254-0714;
e-mail: [email protected]
3
Current address: Department of Animal Science and Technology, College of Agriculture and Life Sciences, Seoul National University, Suweon,
441-744, Korea.
4
Current address: Department of Animal Sciences, University of Missouri, Columbia, MO 65211.
oocytes cultured in vitro [1, 2]. In the in vitro culture system for porcine oocytes, a low rate of MPN formation is
one of the main problems along with a high incidence of
polyspermy (for review see [3-5]). Improvements in MPN
formation of porcine oocytes penetrated in vitro have been
achieved by supplementing maturation medium with cysteine [6, 7], as a precursor of glutathione whose synthesis
is known to be a prerequisite for sperm chromatin decondensation [8]; by supplementing with porcine follicular fluid (PFF) [9, 10]; by decreasing sodium chloride (NaCI)
concentration in maturation medium [11]; and by coculturing oocytes with follicular shell [12, 13]. However, fully
satisfactory culture systems for maturation and fertilization
of porcine oocytes are yet to be established. Furthermore,
there is little information about the factors affecting MPN
formation with relation to the culture condition. Therefore,
further studies of the effects of individual factors in media
on maturation and fertilization of porcine oocytes in vitro
will be necessary.
We have found that modified Whitten's medium containing PFF and gonadotropins could support nuclear maturation and penetration of porcine oocytes in vitro by frozen-thawed ejaculated spermatozoa, but that the rate of
MPN formation was very low (unpublished results). Therefore, the present study was undertaken to determine whether various factors-including maturation time, the presence
of amino acids in maturation medium, and the presence of
cumulus cells during fertilization in vitro-affect MPN formation of penetrated porcine follicular oocytes.
MATERIALS AND METHODS
Media
The basic medium used for the maturation of oocytes
was BSA-free Whitten's medium [14] composed of 87.95
mM NaCl, 4.83 mM KC1, 1.18 mM KH2PO 4, 1.18 mM
MgSO 4, 22.62 mM NaHCO 3, 5.56 mM glucose, 21.58 mM
sodium lactate, 0.31 mM sodium pyruvate, 2.66 mM hemicalcium lactate, 80 fIg potassium penicillin G/ml, 50 jug
streptomycin sulfate/ml, and 0.01 mg phenol red/ml and
supplemented with 10% (v:v) PFF, 10 IU eCG/ml (Serotropin; Teikoku-Zoki Co., Tokyo, Japan) and 10 IU hCG/ml
(Puberogen; Sankyo Co., Tokyo, Japan). This medium was
designated mWM-FG. The PFF was withdrawn using an
18-gauge needle fixed to a 10-ml disposable syringe from
superficial follicles (2-5 mm in diameter) on maturing porcine ovaries. The drawn PFF was centrifuged at 1500 x g
for 15 min at room temperature to remove any cells or
debris, and the supernatant was stored at -20°C until used
[9]. The basic medium, designated TCM-199B (pH 7.8) and
used for the treatment of spermatozoa and the fertilization
in vitro of oocytes, was modified tissue culture medium
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MALE PRONUCLEAR FORMATION IN PIG OOCYTES
(TCM) 199 (with Earle's salts; Gibco Lab., Grand Island,
NY) supplemented with 3.05 mM glucose, 0.91 mM sodium pyruvate, 75 jig potassium penicillin G/ml, and 50 jLg
streptomycin sulfate/ml [15].
Preparation of Oocytes
Ovaries were collected from maturing gilts at a local
abattoir and transported to the laboratory within 1-1.5 h in
0.9% (w:v) NaCI containing 75 g potassium penicillin
G/ml and 50 Ig streptomycin sulfate/ml at 37-39°C. Oocytes were aspirated from antral follicles (2-5 mm in diameter) with an 18-gauge needle fixed to a 10-ml disposable syringe and washed four times with mWM-FG with
various supplements according to the experiment. Ten oocytes surrounded by compact cumulus were transferred to
a 100-,ul drop of the same medium, which had previously
been covered with warm paraffin oil in a polystyrene culture dish (35 x 10 mm; Becton Dickinson Labware, Lincoln Park, NJ) and equilibrated in an atmosphere of 5%
CO 2 in air for a minimum of 3 h, and were cultured for
24-48 h (experiment 1) or 36 h (experiments 2-4) at 39°C
under the same atmospheric conditions. For in vitro fertilization, cumulus-enclosed oocytes cultured for 36 h (experiments 1-4) or 48 h (experiment 1) were washed twice
in TCM-199B supplemented with 10 mM caffeine-sodium
benzoate (Sigma Chemical Co., St. Louis, MO), placed into
50 jil of the same medium, and kept in a CO2 incubator
(5% CO2 in air at 39 0C) for 30 min until spermatozoa were
added for fertilization.
Sperm Preparation and In Vitro Fertilization
Spermatozoa were prepared as described by Wang et al.
[15]. Briefly, three 0.1-ml pellets of frozen ejaculated spermatozoa obtained from boars of proven fertility were
thawed in 2 ml of TCM-199B at 37°C for 1 min. The medium had previously been equilibrated in an atmosphere of
5% CO 2 in air for a minimum of 3 h. After thawing, spermatozoa were washed three times in the same medium by
centrifugation at 550 x g for 5 min at room temperature.
The sperm pellet was then resuspended to give a sperm
concentration of 2-3 X 107 cells/mil. A 50-pIl aliquot of
final sperm suspension was introduced into 50 ptl of TCM199B containing 10 mM caffeine that included the oocytes,
and the mixture was cultured at 39C in an atmosphere of
5% CO2 in air. The mixture had final concentrations of 11.5 x 107 spermatozoa/ml and 5 mM caffeine.
Assessment of Nuclear Maturation
After 24-48 h of maturation culture (experiment 1), the
oocytes were freed from cumulus cells by treatment with
Dulbecco's PBS (D-PBS; Sigma) containing 0.1% (w:v)
hyaluronidase from bovine testis (Sigma), followed by repeated passage through a fine pipette. The denuded oocytes
were mounted, fixed for 48-72 h in 25% (v:v) acetic acid
in ethanol at room temperature, stained with 1% (w:v) orcein in 45% (v:v) acetic acid, and examined under a phasecontrast microscope at magnifications of x200 or x400 for
assessment of nuclear maturation. Oocytes at the stage of
metaphase II (M-II1) were regarded as matured.
TABLE 1. Composition of amino acids added to the medium.
Amino acids
mM
EAA (2%, v:v)a
L-Arginine HCI
L-Cystine
L-Glutamine
L-Histidine HCIlH,O
L-Isoleucine
L-Leucine
L-Lysine-HCI
L-Methionine
L-Phenylalanine
L-Threonine
L-Tryptophan
L-Tyrosine
L-Valine
L-Glutamine
0.6
0.1
0.1
0.2
0.4
0.4
0.4
0.1
0.2
0.4
0.05
0.2
0.4
0.1
NEAA (1%, v:v)b
L-Alanine
L-AsparagineHO
L-Aspartic acid
L-Glutamic acid
Glycine
L-Proline
L-Serine
0.1
0.1
0.1
0.1
0.1
0.1
0.1
Minimal Essential Medium (MEM) essential amino acid solution (no.
320-1130; Gibco).
b MEM nonessential amino acid solution (no. 320-1140; Gibco).
a
of sperm penetration as described above for the assessment
of nuclear maturation. Oocytes were considered penetrated
when they had one or more swollen sperm heads and/or
male pronuclei and their corresponding sperm tails.
Experimental Studies
In experiment 1, to determine the optimal time for maturation of oocytes in mWM-FG, oocytes were cultured for
periods ranging from 24 to 48 h. Furthermore, to determine
the effect of maturation time on sperm penetration in vitro,
cumulus-enclosed oocytes cultured for 36 and 48 h for maturation in mWM-FG were inseminated in vitro. In experiment 2, to study the effects of amino acids in maturation
medium on sperm penetration in vitro, cumulus-enclosed
oocytes were cultured for 36 h in mWM-FG supplemented
with 2% (v:v) MEM essential amino acid solution (EAA;
no. 320-1130; Gibco) with the addition of 0.1 mM glutamine (no. G-5763; Sigma) and/or 1% (v:v) MEM nonessential amino acid solution (NEAA; no. 320-1140; Gibco)
and inseminated in vitro. The composition of amino acids
added to the medium was exactly the same as that used by
Miyoshi et al. [16] for the development of rat one-cell embryos as indicated in Table 1. In experiment 3, to examine
the effects of amino acids and/or cysteine on sperm penetration in vitro, cumulus-enclosed oocytes were cultured for
36 h in mWM-FG supplemented with EAA+NEAA and/or
0.57 mM cysteine (Sigma) and inseminated in vitro. In experiment 4, to study the effects of the presence of cumulus
cells during fertilization on sperm penetration in vitro, some
of cumulus-enclosed oocytes that had been cultured for 36
h in mWM-FG with or without EAA+NEAA+cysteine
were freed from cumulus cells. Then cumulus-enclosed and
cumulus-free oocytes were inseminated in vitro.
Assessment of Sperm Penetration
Fourteen hours after insemination, oocytes were freed
from cumulus cells by repeated passage through a fine pipette, mounted, fixed, stained, and examined for evidence
Statistical Analysis
The percentages of oocytes reaching M-II, penetration,
MPN formation, and polyspermy were subjected to an arc-
1480
KA ET AL.
(4 39
(42d
100-
amino acids (Table 3). When the oocytes were matured in
medium supplemented with amino acids (EAA and/or
NEAA), the incidence (79-84%) of MPN formation was
significantly (p < 0.01) greater than in the absence of amino acids (51%), while the incidence (97-99%) of polyspermy and the mean number (4.7-5.7) of spermatozoa in
penetrated oocytes were not affected.
4(46)
d
80c)q 0
(49
Experiment 3
U2 -
CD
V 20
(45)
20-
(45)
a
0-
*y
24
-
I
I
I
1
I
28 32
36 40 44
Maturation time (h)
FIG. 1. Time course of nuclear maturation (metaphase II)of porcine oocytes cultured in mWM-FG containing 10% (v:v) PFF, 10 IU eCG/ml, and
10 IU hCG/ml. The numbers of oocytes examined at each time of culture
are shown in parentheses. The values with different letters are significantly
different (p < 0.01). Experiments were repeated three times.
sine transformation, and the transformed values were analyzed using one-way (experiments 1 and 2) or two-way
(experiments 3 and 4) ANOVA. When ANOVA revealed a
significant effect, the treatments were compared by Duncan's Multiple Range test. Tukey's studentized range tests
were applied to compare mean numbers of spermatozoa in
penetrated oocytes. All analyses were carried out using
GraphPad-PRISM (GraphPad Software, San Diego, CA).
RESULTS
Experiment 1
The proportion of oocytes completing nuclear maturation was significantly (p < 0.05) higher when they were
cultured in mWM-FG for 36-48 h (90-98%) as compared
to 24-32 h (0-43%; Fig. 1). There were no differences in
the rates of nuclear maturation among oocytes cultured for
36-48 h. When oocytes cultured for 36 or 48 h were inseminated in vitro, very high penetration rates (94-95%)
were obtained, and there were no differences in the proportions of MPN formation (35-45%) and polyspermy (8790%), or in the mean numbers (3.6-4.0) of spermatozoa in
penetrated oocytes, between the two different culture periods (Table 2). Therefore, in all subsequent experiments, oocytes cultured for 36 h were used.
Experiment 2
Very high penetration rates (93-97%) were obtained in
oocytes matured both in the presence and in the absence of
TABLE 2.
When cysteine was added to the maturation medium
without amino acids, the incidence (83%) of MPN formation was increased significantly (p < 0.05) as compared
with the value (53%) in the absence of cysteine (Table 4).
However, the beneficial effects of amino acids
(EAA+NEAA) on MPN formation was not increased by
supplementation with cysteine. Penetration rates (89100%), the incidence (85-98%) of polyspermy, and the
mean number (4.3-6.4) of spermatozoa in penetrated oocytes were not affected by supplementation with amino acids and/or cysteine.
Experiment 4
The proportions (92-100%) of oocytes penetrated were
not different among different experimental groups, but the
incidence of MPN formation was significantly (p < 0.01)
higher in cumulus-enclosed (53-78%) than in cumulus-free
(28%) oocytes matured in both the presence and absence
of amino acids + cysteine (Table 5). In cumulus-enclosed
oocytes, MPN formation was promoted when the oocytes
were matured in the presence (78%) as compared to the
absence (53%) of amino acids + cysteine (p < 0.05). The
incidence (69-96%) of polyspermy and mean number (3.14.7) of spermatozoa in penetrated oocytes were not affected
by the different conditions for maturation and fertilization
in vitro.
DISCUSSION
The results of the present study indicate that 1) nuclear
and cytoplasmic maturation of porcine oocytes can be completed by 36 h of culture in chemically simple medium
(mWM-FG) containing 10% (v:v) PFF and gonadotropins
(10 IU eCG/ml + 10 IU hCG/ml); 2) supplementation of
the medium with amino acids (EAA and/or NEAA) or cysteine (0.57 mM) promotes MPN formation in penetrated
oocytes; 3) the presence of cumulus cells at insemination
also promotes MPN formation in penetrated oocytes; and
4) the efficacy of cumulus cells to promote MPN formation
is higher when the oocytes are cultured for maturation in
the presence as compared to the absence of amino acids
and cysteine.
Porcine oocytes usually require a long time for maturation, and various times in different culture conditions have
been reported (36 h [15, 17, 18]; 40 h [11]; 44-46 h [1];
Effects of the time of maturation on sperm penetration in vitro of porcine oocytes.a
Number of oocytes penetrated
Time (h) of
maturation
36
48
Number of
oocytes examined
81
87
Total (%)
With swollen
sperm head (%)b
With male
pronucleus (%)b
77 (95)
82 (94)
42 (55)
53 (65)
35 (45)
29 (35)
Number of
polyspermic
oocytes
(%)
b
Mean number of
spermatozoa in
penetrated oocytes
69 (90)
71 (87)
4.0
3.6
a Cumulus-enclosed oocytes were cultured in modified Whitten's medium containing 10% (v:v) PFF, 10 IU eCG/ml, and 10 IU hCG/ml; experiments
were repeated four times.
b Percentage of oocytes penetrated.
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MALE PRONUCLEAR FORMATION IN PIG OOCYTES
TABLE 3.
Effects of addition of amino acids to the maturation medium (mWM-FG) on sperm penetration in vitro of porcine oocytes.a
Number of oocytes penetrated
Amino acidsb
Number of oocytes
inseminated
None
EAA
NEAA
EAA + NEAA
73
75
79
76
With swollen
sperm head (%)c
Total (%)
70
73
77
71
(96)
(97)
(97)
(93)
Number of
With male
pronucleus (%)'
34 (49)d
15 (21)e
12 (16)e
14 (20)e
(51) d
(79)e
(84)e
(80) e
36
58
65
57
Mean number of
polyspermic oocytes spermatozoa in
(%)c
penetrated oocytes
68
72
75
69
(97)
(99)
(97)
(97)
5.0
5.7
4.8
4.7
a Experiments were repeated four times.
b EAA: minimal essential medium (MEM) essential amino acid solution (2%, v:v) + 0.1 mM glutamine; NEAA: MEM nonessential amino acid solution
(1%, v:v).
' Percentage of oocytes penetrated.
d. Values with different superscripts within each column differ significantly (p < 0.01).
TABLE 4.
oocytes.a
Effects of addition of amino acids and/or cysteine (0.57 mM) to the maturation medium (mWM-FG) on sperm penetration in vitro of porcine
Addition of
EAA + NEAAb
Cysteine
_
+
+
+
Number of oocytes
inseminated
Number of
Mean number of
With swollen
sperm head (%)c
polyspermic
oocytes (%)c
spermatozoa in
penetrated oocytes
Total (%)
44
48
46
45
+
Number of oocytes penetrated
43
46
41
45
(98)
(96),
(89)
(100)
20
8
10
10
With male
pronucleus (%)c
(47)d
(17)e
(24)0
(22)e
23
38
31
35
(53) d
(83)e
(76)e
(78)0
41
42
39
44
(95)
(91)
(85)
(98)
5.7
6.4
4.3
5.1
Experiments were repeated four times.
EAA: minimal essential medium (MEM) essential amino acid solution (2%, v:v) + 0.1 mM glutamine; NEAA: MEM nonessential amino acid solution
(1%, v:v).
c Percentage of oocytes penetrated.
d,eValues with different superscripts within each column differ significantly (p < 0.01).
b
48 h [9, 12, 13]). The factors that regulate the timing of
nuclear maturation in porcine oocytes in vitro have not been
strictly identified. However, it has been reported that the
proportion of porcine oocytes reaching M-II in vivo increased markedly 36 h after hCG injection [19]. This timing
of oocyte maturation is comparable to that observed in vitro
in complex medium, modified TCM-199 supplemented
with fetal calf serum (FCS) [15, 18]. In the present study
using simple medium supplemented with PFF and gonadotropins, most oocytes reached M-II also by 36 h of culture.
These results may indicate that FCS, PFF, and gonadotropins contain the factors that regulate oocyte maturation. The
maturation rate and/or the time for reaching M-II in the
presence of EAA and/or NEAA were not examined in the
present study. However, since in our previous study [15], a
large proportion (73%) of porcine oocytes reaching M-II
was not obtained until 36 h after culture in modified TCM-
199, which contains some amino acids, it is postulated that
the addition of EAA and/or NEAA to mWH-FG may not
alter the timing of meiotic progression of porcine oocytes.
Although some degree of decondensation of the sperm
nucleus can occur independently of oocyte maturation and
activation [15], MPN formation is clearly dependent not
only upon germinal vesicle breakdown but also on oocyte
activation [20]. In the present study, oocytes had already
been matured after 36 h of culture in mWM-FG and there
were no differences in the rates of penetration in vitro and
MPN formation between oocytes cultured for 36 h and 48
h. However, in a study involving hCG-regulated ovulation,
Hunter [21] reported that aging in porcine oocytes was revealed within 6-8 h of ovulation and that oocytes penetrated by spermatozoa after these intervals showed an increased risk of polyspermic penetration and subsequent
fragmentation. It has been also reported that as a result of
TABLE 5. Effects of the presence of cumulus cells during fertilization on sperm penetration in vitro in porcine oocytes matured in the presence or
absence of amino acids and cysteine (0.57 mM).
Presence (+) or absence (-) of
EAA + NEAA +
cysteine during
maturationb
Number of oocytes penetrated
Cumulus cells during
fertilization
+
+
+
+
Number of oocytes
inseminated
50
43
45
46
Total (%)
46
43
43
45
(92)
(100)
(96)
(98)
With swollen
sperm head
(%)c
33 (72) d
20 (47)e
31 (72) d
10 (22 )f
Mean number of
With male
pronucleus
(%)
13
23
12
35
(28) d
(53)e
(28)d
(78) f
Number of
polyspermic
oocytes (%)
37
38
30
43
(80)
(88)
(69)
(96)
spermatozoa in
penetrated
oocytes
3.6
4.7
3.1
4.0
aExperiments were repeated four times.
b EAA: minimal essential medium (MEM) essential amino acid solution (2%, v:v) + 0.1 mM glutamine; NEAA: MEM nonessential amino acid solution
(1 %, v:v).
Percentage of oocytes penetrated.
d,-'fValues with different superscripts within each column differ significantly (d-e, d-f, p < 0.01; e-f, p < 0.05).
1482
KA ET AL.
aging the spindle of the second meiotic metaphase might
break down, which results in the loss of chromosomes [22].
Therefore, in the present experimental conditions, it seems
that oocytes cultured for 36 h are more suitable for use for
in vitro fertilization than those cultured for 48 h.
It has been reported that amino acids improve the development of mouse [23], rat [16], rabbit [24], hamster [25,
26], bovine [27], and ovine [28] embryos in vitro and also
that they support rabbit [29] and hamster [30] oocyte maturation in vitro. The results of the present study show that
the presence of EAA and NEAA, either alone or together
in mWM-FG during maturation culture, is beneficial for
MPN formation in porcine oocytes after penetration in
vitro. The mechanism(s) by which amino acids during oocyte maturation improve MPN formation and the required
amount or composition of amino acids are not known.
However, it seems that amino acids in PFF may not be
sufficient in their amounts or composition for porcine oocytes during maturation to acquire the ability to form male
pronuclei after sperm penetration.
In the present study, when mWM-FG was supplemented
with 0.57 mM cysteine, MPN formation rate was increased
significantly. This finding is consistent with the reports of
Yoshida et al. [6] and Sawai et al. [7], who found that
supplementing the maturation medium with cysteine as a
substrate for glutathione synthesis is important for MPN
formation in porcine oocytes after sperm penetration. However, when the medium was supplemented with amino acids
(EAA+NEAA), additional beneficial effects of the addition
of cysteine on MPN formation were not observed. These
results might indicate that cysteine is not the only amino
acid that can be utilized for glutathione synthesis or augment MPN formation and that other amino acids are also
important for cytoplasmic maturation of oocytes, although
it is unknown whether the effects of EAA, NEAA, and
cysteine are brought about through the same mechanism or
different mechanisms.
Research in the pig and in other species showed that
cumulus cells have a beneficial effect on maturation and
fertilization of oocytes in vitro [1, 2, 31-33]. However, reports concerning the effect of cumulus cells during fertilization on MPN formation are very few. Recently it was
reported that porcine oocytes surrounded by expanded cumulus cells exhibited higher frequency of MPN formation
than did denuded oocytes [3, 15, 34]. Zhang et al. [35] also
reported a significant decrease in fertilization rate when cumulus cells were removed from bovine oocytes 7 h after
insemination. In agreement with these observations, the
present study showed that the presence of cumulus cells
during fertilization increased the incidence of MPN formation in porcine oocytes. Moreover, amino acids in maturation medium improved MPN formation only when cumulus cells were attached to the oocytes during fertilization
in vitro. The fact that amino acids are transferred to the
oocytes by the action of gap junctions between cumulus
cells and an oocyte is well known [36-38], but the mechanisms by which amino acids and cumulus cells regulate
MPN formation are unknown at present. Although no systematic assessment of the degree of cumulus expansion was
done in the present study, full expansion of the cumulus
mass was observed at 36 h of culture in mWM-FG, and no
morphological changes in cumulus expansion were observed after the EAA and/or NEAA supplementation. However, one possibility is that amino acid supplementation alters the composition of extracellular matrix and subsequently changes the timing of sperm penetration, which influ-
ences MPN formation. On the other hand, since
intracellular uncoupling of porcine cumulus-oocyte complexes initiated during the first meiotic division is nearly
completed at M-II [39], and since the ability of denuded
oocytes to form male pronuclei is not increased when they
are cocultured with cumulus-intact oocytes, Ball et al. [40]
suggested that damage of oocytes by exposure to hyaluronidase and/or mechanical pipetting for the removal of cumulus cells may result in a low rate of MPN formation.
Furthermore, Funahashi et al. [11] reported that decreasing
the NaCl concentration of maturation medium can produce
a high rate of MPN formation despite fertilization in vitro
without cumulus cells. Since the requirement of the presence of cumulus cells could be reduced under conditions
that are more favorable for oocyte maturation, additional
studies will be necessary to elucidate the relationships between cumulus cells, culture conditions, and MPN formation.
ACKNOWLEDGMENTS
We thank Dr. A. Bartke, Southern Illinois University, for critical reading and valuable suggestions for the manuscript and Dr. H. Funahashi,
Okayama University, for useful comments on the manuscript.
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