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Introduction
PKC is initially described as a family of serine/threonine kinases that can be activated by
Ca2+, phospholipid and
diacylglycerol (DAG)[1]. Until now, at least 12 isoforms have been identified and grouped in three subclasses. They are c
protein kinase C (cPKC) including a, b1, bII , g; novel PKCs (nPKC) including
d, e, h, and q; and atypical PKCs (aPKC) including
z, l, and µ. Most PKC research is focused on cPKC.
PKC is a major intracellular kinase that has been implicated in a large number of signal transduction processes that
mediate extracellular signals into intracellular events. In the process of mammalian oocyte fertilization, PKC plays a role in
three major events: (1) a periodic increase in intracellular
Ca2+ (Ca2+ oscillations); (2) the release of CG, which blocks polyspermy;
and (3) release from meiotic arrest.
Sousa et
al[2] reported that fertilization-induced
Ca2+ oscillations showed a periphery-to-centre propagation of
Ca2+ waves and, at the same time, PKC were accumulated in the cell periphery in human preimplantation embryos. However, the
isoforms of PKC were not addressed in the Sousa
et al report. Until now, there has been a lack of information regarding which
members of the PKC family are present in human oocytes. To better understand the role of PKC, it is necessary to clarify this
issue prior to tracking PKC activity in maturation, fertilization and subsequent embryonic
development[3].
The spare oocytes were donated and collected for research over the past 2 years. Due to limited specimens and ethical
reasons, the major objective of this study is to establish the relationship between cPKC and CG exocytosis. We performed
experiments to determine the translocation of cPKCs in human oocytes and tested whether cPKCs were involved in the signal
process of CG exocytosis.
Materials and methods
Collection of oocytes Oocytes used in this study were collected from the Reproduction and Genetic Center of
Peking University First Hospital. These specimens were clinically unsuitable or redundant after manipulations of
intracytoplasmic sperm injection (ICSI). Written consent was obtained from both sides of the couples. This study was approved by
the Ethics Review Committee of Peking University.
Immature oocytes at GV or metaphase I (MI) stage were retrieved using transvaginal retrieval method 36 h after control
oocyte hyperstimulation under the guidance of ultra-sonography. The oocytes of GV-stage were stained with
immunofluorescence. The oocytes of MI-stage were cultured in Hepes medium (Invitrocare, San Diego, CA, USA)
containing 10% serum substitute supplement (Irvine Scientific, Santa Ana, CA, USA) at 37 °C, 5%
CO2 to form MII oocytes. The MII oocytes were used for immunofluorescence or treated with drugs.
Drug treatment of oocytes
To study the relationship between exocytosis of CG and activation of classical PKC
a, β1 and γ, phorbol 12-myristate 13-acetate (PMA, Alexis, San Diego, CA, USA), staurosporine (Alexis) and
4a-phorbol 12,13-didecanoate (4a-PDD; Alexis) were used to treat oocytes. The chemicals were dissolved in dimethylsulfoxide (Sigma, St
Louis, MO, USA), stored at _20 °C, and diluted with Hepes buffer medium prior to use.
It has been reported that 1.62 µmol PMA could induce the translocation of cPKC isoforms and exocytosis of
CG[4]. However, in this experiment, we had to use 3.2 µmol PMA to see translocation. For evaluation of translocation of cPKC and
exocytosis of CG, MII oocytes were treated with 3.2 µmol PMA for 15 min followed by LCSM. As control, oocytes were
treated with 3.2 µmol 4a-PDD, a biologically inactive
phorbol ester. To evaluate exocytosis of CG and distribution of PKC
a, β1 and γ, MII oocytes were treated sequentially with 3.2 µmol PMA for 15 min, 2 µmol staurosporine for 15 min followed by
3.2 µmol PMA plus 2 µmol staurosporine for 15 min.
Immunofluorescence Oocytes were briefly treated with acidic Tyrode's solution (pH
2.1-2.5; Sigma, St Louis, MO, USA) to remove zona pellucida
[4]. Zona-free cells were fixed for 15 min in 4% paraformaldehyde (2% paraformaldehyde for MII
oocytes). Double staining of PKC isoforms and CG was performed as described in a previous
study[5]. Briefly, oocytes were permeabilized with incubation buffer (0.5% Triton-100 in 20 mmol/L Hepes, pH 7.4, 3 mmol/L
MgCl2, 50 mmol/L NaCl, 300 mmol/L sucrose and 0.02%
NaN3) for 30 min at 37 °C in a incubation chamber, followed by blocking in 1% BSA for 1 h and
incubation overnight with Lens Culinaris
Agglutinin-biotin (LCA-biotin; Vector Labs, Burlingame, CA, USA) for CG
staining or the specific antibodies (mouse anti-human) for PKC
a, β1 and γ (1:80; Alexis) at 4 °C. After extensive washes, oocytes
were incubated with Fluorescein isothiocynate (FITC)-labeled streptavidin for CG stain or rhodamine-labeled
rabbit-anti-mouse IgG antibody (KPL, Gaithersburg, MD, USA) for
PKCα, b1 or g (Southern Biotech, Birmingham, AL, USA) for 1 h.
Finally, cells were stained with 0.02 g/L Hoechst 33258 (Molecular Probes, Eugene, OR, USA) 10 min for displaying chromatin,
mounted on slides using GVA-mount (Zymed, San Francisco, CA, USA) and examined by using LCSM (MRC-2100, Bio-Rad,
Richmond, USA). Nonspecific staining was determined by incubation with isotype control antibodies.
Data analysis The SPSS version 11.0 for Windows (SPSS Inc, Chicago, IL, USA) software was used for the statistical
analysis including the Student-Neumann-Keul's test and the paired
t-test. The values were presented as the mean±SEM.
P<0.05 was considered to be statistically significant.
Oocytes displaying signs of degeneration as indicated by dark cytoplasm or fragmented cytoplasm were excluded from
analysis.
Results
Changes of cPKCs in the maturation processes of
oocytes from GV to MII stage In this study, human oocytes at GV and MI stage retrieved from patients were the redundant
cells after artificial fertilization manipulations, and those at MII stage were obtained by
in vitro culture of the MI oocytes. Immunofluorescence using antibodies against PKC
a, β1 and γ showed that in GV oocytes, PKC a, b1 and
g were localized to the germinal vesicles, with a weak expression in the ooplasm
(PKCα=100%, n=9; PKCβ1=88.89%, n=9;
PKCγ=83.33%, n=12). In MII oocytes, PKC a,
β1 and γ were distributed evenly in ooplasm (PKCα=88.89%,
n=9; PKCβ1=100%, n=10; PKCγ=90%,
n=10), and the expression of PKCα in the membrane was stronger than in the ooplasm. However, the expression of PKC
g in MII oocytes was weaker than that in GV
(P<0.05). No staining was seen in the isotype control (Figure 1).
Changes of classical PKCs in oocytes treated with PKC activator, PKC inhibitor or
4a-PDD After exposure of MII oocytes to PKC activator PMA for 15 min, cPKC were transferred to the peripheral cytoplasm and cell membrane, with a
marginal expression in ooplasm (PKCα=75%, n=8;
PKCβ1=
87.5%, n=8; PKCγ=60%, n=10) (Figure 2A_2C). Incubation with the PKC inhibitor staurosporine followed by PMA plus
staurosporine blocked translocation of cPKC induced by PMA. The three PKCs (ie,
a, β1 and γ) were evenly distributed in ooplasm under this condition
(PKCα=100%, n=8; PKCβ1=100%, n=9;
PKCγ=85.71%, n=7) (Figure 2D_2F). The biologically
inactive phorbol ester, 4a-PDD could not
induce translocation of PKC (PKCα=90%,
n=10; PKCβ1=100%, n=10; PKCγ=85.71%,
n=7) (Figure 2G_I). The difference was significant
(P<0.05).
Exocytosis of CG in oocytes
Exocytosis of CG was not found in mature oocytes (0%,
n=29) (Figure 3A). Treatment with PKC activator PMA resulted in considerable exocytosis of CG in MII oocytes (80.77%,
n=26) (Figure 3B). No significant exocytosis of CG was induced by treatment with PKC inhibitor and
4a-PDD (3.92%, n=51) (Figure 2D-I). The difference was
significant (P<0.05).
Discussion
The expression of PKC a, b1 and
g has been reported in animals but not in
humans[6-9]. In our study, cPKC isoforms were
found to be concentrated in germinal vesicles at the GV stage and dispersed in MII oocytes. In mouse GV oocytes,
PKCα and PKCbII uniformly distribute in the cytoplasm, while
PKCbI localizes in the cytoplasm and plasma membrane. In mouse MII
eggs, although distribution of PKCα and PKCbI is the same as GV stage,
PKCbII can distribute in the cytoplasm and plasma
membrane[8]. In pig eggs, the three PKC isoforms (PKC
a, β1 and γ) concentrate in the GV but evenly distribute in the
cytoplasm of MII eggs[4]. The different location of cPKC in different species might correlate with their function. The
functions of PKC in GV are unknown at present, but their compartmentalization during oocyte development may be
physiologically important. Germinal vesicle breakdown (GVBD) was one of the most important steps during the maturation of
oocytes. PKC elicits GVBD by activating maturation-promoting
factor[10]. When activated by FSH and PKC,
mitogen-activated protein kinases (MAPK) may stimulate the synthesis of specific proteins in cumulus cells followed by the secretion
of an unknown positive factor that is capable of inducing GVBD in
oocytes[11]. Direct activation of PKC in oocyte suppresses
maturation, while stimulation in cumulus cells generates a positive trigger that leads to meiotic
resumption[12-14]. PKC activity could disrupt the MI-to-MII transition, leading to a precocious exit from meiosis
too[15].
There was further evidence suggesting that an increase in PKC activity occurred at fertilization as the activation of PKC
was often associated with a translocation to the plasma membrane from cytoplasm. This is similar to our observations.
Treatment with PMA, a PKC activator, PKC a, b1 and
g moved to the periphery and cell membrane, and CG exocytosis was
induced at the same time. These effects were blocked in the presence of staurosporine, a potent inhibitor of PKC, suggesting
that the activation of PKC is necessary for PKC translocation and CG exocytosis. As a control,
4a-PDD, a biologically inactive phorbol ester, could not induce translocation and CG exocytosis. Such a transition has also been observed in
hamster oocytes after treatment with Ca2+[16,
17] and spermatozoa[18].
Following the fusion of sperm and oocyte, the contents of CG release into the perivitelline space (cortical reaction),
causing the zona pellucida to become refractory to sperm binding and penetration (zona reaction). The sperm-oocyte fusion,
mediated by GTP-binding protein (G-protein), might elicit the generation of two second messengers, inositol 1,4,5
triphosphate (IP3)[19] and DAG. The former induce
Ca2+ exocytosis from intracellular stores and the latter activate PKC, leading to CG
exocytosis. The CG exocytosis cause zona sperm receptor modification and zona hardening, and thus blocks polyspermic
penetration. Oolemma modification after sperm-oocyte fusion and formation of CG envelope following cortical reaction may
also contribute to polyspermy
block[20-22].
Pharmacological agents including phorbol esters and synthetic diacylglycerols have been used to imitate the events of
egg activation, such as CG exocytosis and the second polar body
initiation[23-25]. However, contradictory results have been
obtained from the pharmacological studies using PKC activators or inhibitors on mammalian eggs. It has been found that
phorbol esters induce the initiation of second polar body-like structures in hamster eggs; while in mouse eggs such
structures have not been detected[26]. In mouse denuded oocytes, meiotic resumption was inhibited by phorbol
esters[13].
Because a parthenogenetic agent (phorbol esters) does not fully mimic the egg activation, PKC activation following the
sperm-induced egg activation should be assessed before drawing conclusions. Ducibella and
LeFevre[27] addressed this issue and demonstrated the failure of PKC inhibitors to block fertilization-induced CG exocytosis. However, their results did
not exclude the possibility of PKC involvement in exocytosis as either a very small amount of PKC was sufficient for cortical
reaction or that other active pathways could compensate for PKC inhibition.
A key phenomena associated with PKC function
in vivo is likely to be subcellular distribution of both the enzyme
and substrate[28]. PKC isoforms have been found to be distributed differentially among different cell types together with a
number of targeting proteins[29]. An aim in future study would be to identify the specific roles of each of these isoforms in
human oocyte fertilization.
In conclusion, we revealed for the first time that PKC
a, β1 and γ existed in human oocytes. Their subcellular distribution
is differently regulated during oocyte maturation. cPKC isoforms participate in CG exocytosis in human oocytes and the
activation of these subunits can induce CG exocytosis. However, the function of each of the isoforms needs to be investigated.
Acknowledgements
Thanks to Prof Lin LIU from Marine University and Prof Qing-yuan SUN from the Institute of Zoology of the Chinese
Academy of Sciences who helped with the immunofluo-rescence.
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