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Promote Zebrafish Oocyte Maturation in Vitro: Potential Role of the Ovarian Activin Regulatory System
Department of Biology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China
Address all correspondence and requests for reprints to: Dr. Wei Ge, Department of Biology, Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China. E-mail: weige{at}cuhk.edu.hk
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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are well known for their
activities in the ovary. Both factors initiate signal transduction by
binding to the common EGF receptor that has been demonstrated in the
ovary across vertebrates from fish to humans. Using zebrafish
as the model, we demonstrated in the present study that recombinant
human EGF and TGF significantly enhanced final maturation of the
fully grown, follicle-enclosed oocytes (0.58–0.65 mm) in
vitro in a clear time- and dose-dependent manner. The effect of
EGF/TGF was additive to that of hCG at low concentrations, but the
additivity diminished when the concentration increased. Both
actinomycin D and cycloheximide completely blocked the effect of
EGF/TGF, indicating that the promotion of oocyte maturation by
EGF/TGF requires de novo mRNA transcription and
protein synthesis. Interestingly, the effect of EGF/TGF could be
blocked by cotreatment with follistatin, a potent binding protein for
activin, an ovarian growth factor belonging to the TGFß superfamily.
Semiquantitative RT-PCR assays showed that both EGF and TGF
significantly stimulated the expression of activin ßA and activin
type II receptor in the cultured zebrafish ovarian follicle cells in a
clear time- and dose-dependent manner. This together with our
previous report that activin had a potent stimulatory effect on
zebrafish oocyte maturation strongly suggests that the intrinsic
ovarian activin system is probably a downstream mediator of EGF/TGF
actions in the zebrafish ovary. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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belong to the same family of growth factors. EGF and TGF are
structurally related by sharing about 40% overall amino acid sequence
homology with six cysteines fully conserved (1). All
members of EGF family are characterized by the presence of EGF-like
structural unit, and therefore share some degree of amino acid
homology, including the six conserved cysteines over a sequence of
35–40 amino acids and three disulfide bonds formed by these cysteines
(1, 2, 3). EGF and TGF exert their functions through
binding to the same EGF receptor with similar affinity in the same
species (4, 5).
EGF and TGF have mitogenic effects and are involved in a variety of
physiological processes, including reproduction (6, 7, 8, 9).
The roles played by EGF and its related proteins in the function of
vertebrate gonads have received increasing attention during the past
decade (10, 11, 12, 13). Both EGF and its receptors are expressed
in the ovary of birds (14), mammals (15, 16, 17),
and humans (18, 19), suggesting their regulatory roles in
ovarian functions. Similarly, TGF is also expressed in the rat
ovary, and its expression is stimulated by FSH (20). Both
follicle thecal cells (20) and granulosa cells
(21) have been reported to be the production sites of
TGF using immunocytochemical staining and RT-PCR, respectively.
EGF/TGF exhibits a variety of biological activities in the ovary,
including stimulation of progesterone secretion (17, 22),
suppression of FSH receptor (23, 24) and FSH-induced LH
receptor expression in the granulosa cells (25), and
inhibition of the biosynthesis of E2 (25) and androgen
(17) as well as inhibin secretion
(26). EGF is also well known for its potent stimulatory
effect on the final oocyte maturation in a variety of mammalian species
and humans (10, 27, 28, 29, 30, 31, 32).
In the lower vertebrates, such as teleosts, the studies on EGF and its
related peptides are limited. EGF was found to mediate the action of
hCG on DNA synthesis in goldfish vitellogenic follicles
(33) and suppress apoptosis in rainbow trout preovulatory
ovarian follicles (34). EGF/TGF may play a role in the
regulation of ovarian PG synthesis during ovarian follicular
development in the goldfish (35). In addition, both EGF
and TGF increase final oocyte maturation in the goldfish
(36). These findings suggest that EGF and its related
peptides may exist in the ovary of teleost fish and serve as local
paracrine factors to regulate fish ovarian functions.
Considering that final oocyte maturation is a complex event that
involves regulation by both endocrine hormones and intraovarian
paracrine factors, we carried out the present study with the aim of
examining the effects of EGF and TGF on final oocyte maturation in
the zebrafish in vitro. Moreover, as our previous study
suggested that the zebrafish ovarian activin system plays important
roles in both the spontaneous and gonadotropin-induced oocyte
maturation (37), the present study also aimed at
investigating the relationship between the ovarian activin system and
EGF activity. The results showed that both EGF and TGF promoted
final oocyte maturation in the zebrafish, and their effects could be
suppressed by follistatin, an activin-binding protein, suggesting that
the ovarian activin system could be a downstream mediator of EGF and
TGF action in the ovary. This hypothesis was further substantiated
by evidence that the expression of both activin ßA and activin type
II receptor was up-regulated by these two growth factors in cultured
zebrafish ovarian follicle cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,20ß-dihydroxy-4-pregnen-3-one
(DHP) were obtained from Sigma (St. Louis, MO), and
recombinant human EGF and TGF were obtained from Promega Corp. (Madison, WI) unless otherwise stated. hCG, EGF, and
TGF were dissolved in the medium directly, whereas DHP was first
dissolved in ethanol and then diluted to the desired concentration with
the medium before use. Recombinant goldfish activin B was produced by a
Chinese hamster ovary cell line established in our laboratory and
purified according to Schmelzer et al. (38).
One unit of activin B is defined as the amount per ml that induces a
half-maximal differentiation of F5-5 cells (ED50)
in the erythroid differentiation factor assay according to Eto
et al. (39) and Schmelzer et al.
(38). Recombinant human follistatin (rhFS-288) was
supplied by Dr. A. F. Parlow (National Hormone and Pituitary
Program, NIDDK, Torrance, CA).
Animals
Young zebrafish (Danio rerio) were purchased from
local pet stores and maintained without separation of males and females
in flow-through aquaria (36 liter) at 25 C on a 14-h light, 10-h dark
photoperiod. The fish were fed twice daily with commercial tropical
fish food with supplement of live brine shrimp once or twice a
week.
Isolation and incubation of follicles
Gravid female zebrafish were anesthetized with 0.01% tricaine
methanesulfonate solution for 2 min. The ovaries were removed after
decapitation and placed in a 35-mm culture dish containing 60% medium
Leibovitz L-15 (Life Technologies, Inc., Gaithersburg, MD)
(40). The follicles from 8–10 females were carefully
separated with the aid of fine forceps and blades. The diameter of
follicles was measured with an ocular micrometer in a dissecting
microscope, and healthy follicles, 0.58–0.65 mm in diameter, were
selected, pooled, and randomly distributed in wells of 24-well plates
(Falcon, Becton Dickinson and Co., Franklin Lakes, NJ).
The incubation of follicles was modified from that reported by Selman
et al. (40). Briefly, the follicles
(30–40/well) were cultured at 28 C for up to 24 h in 1 ml 60%
Leibovitz L-15 in 24-well plates. The follicles were scored at
different times of incubation for those that had turned translucent due
to germinal vesicle breakdown (GVBD), an easily identifiable marker for
oocyte maturation (40). All experiments were repeated two
or three times to confirm the results.
Culture of the follicle cells and drug treatment
The dispersed follicles were washed three times with medium L-15
and three times with medium 199. The follicles were then cultured in
medium 199 and 10% FBS in a culture dish under the condition of 28 C
and 5% CO2 for 6 d. The medium was changed
once on the third day of incubation. During the 6-d incubation, the
follicle cells proliferated significantly. On the sixth day of
incubation, the culture medium was discarded, and the proliferated
cells in the dish were washed once with medium 199 and 10% FBS. The
cells were then suspended by treatment with 0.25% trypsin in 2.68
mM KCl, 137 mM NaCl, 0.68 mM
KH2PO4, and 7.85
mM Na2HPO4 at
28 C for 15 min. After three washes with medium 199 and twice with
medium 199 and 10% FBS, the cell suspension was filtered with a
40-µm pore size cell strainer (Falcon, Becton Dickinson and Co.) to remove the follicle debris. The cells were then
subcultured in medium 199 and 10% FBS in 24-well plates at a density
of 100,000 cells/ml·well for 24 h before treatment. The medium
was changed before the drug treatment was performed. For the
time-course study, EGF or TGF was applied to each well to reach a
final concentration of 10 nM, and the treatment lasted for
1, 2, and 4 h before RNA extraction; concentrations of 5, 10, and
20 nM EGF or TGF were used to treat the cells for 2
h for the dose-response assay.
Extraction of total RNA from the cultured follicle cells
After discarding the medium, 500 µl Tri-Reagent
(Molecular Research Center, Inc., Cincinnati, OH) were
added to each well. The plate was then shaken for 10 min at 700 rpm on
the Thermomixer comfort (Eppendorf, Hamburg, Germany) to
extract the RNA. The extract from each well was transferred to a new
microtube containing 100 µl chloroform. After vortexing on the
thermomixer for 1 min at 1,400 rpm and incubating at room temperature
for 10–15 min, the samples were spun at 15,000 rpm for 15 min. The
aqueous phase from each sample was transferred to a new tube containing
250 µl isopropanol, vortexed for 15 sec, left at -20 C for 10–30
min, and centrifuged for 20 min at 15,000 rpm. After rinsing with 400
µl 75% ethanol and brief air-drying, the RNA pellet from each well
was dissolved in 10 µl diethylpyrocarbonate-H2O
and stored in a -80 C freezer.
RT-PCR
Total RNA from each well was reverse transcribed into cDNA at 42
C for 3 h in a total volume of 10 µl consisting of 1 x
First Strand Buffer (Life Technologies, Inc.), 10
mM dithiothreitol, 0.5 mM of each dNTP, 0.5
µg oligo(deoxythymidine), and 100 U SuperScript II (Life Technologies, Inc.). PCR was carried out in a volume of 50 µl
consisting of 1 x PCR buffer, 0.2 mM of each dNTP,
2.5 mM MgCl2, 0.2 µM of
each primer, and 1 U Taq polymerase. One half microliter of
RT reaction was used in each PCR reaction. The primers for activin
ßA-subunit (sense, 5'-TGCTGCAAGCGACAATTTTA-3';
antisense, 5'-CGATTTTCTGCTCCTCGTTG-3'), activin type II receptor
(sense, 5'-AAGAGTGTTGGACCATGAAG-3'; antisense,
5'-TTATACTGAAATGACACGAG-3'), and ß-actin (sense, 5'-CCCCTTGTTCACAAT
AACCT-3'; antisense, 5'-TCTGTTGGCTTTGGGATTC-3') were designed according
to the sequences from GenBank (accession no. AJ238980, AA497191, and
AF057040).
The semiquantitative RT-PCR assays were validated by running PCR reaction on the Thermal Controller PTC-100 (MJ Research, Inc., Watertown, MA) for different cycles to determine the cycle number that generates half-maximal PCR reaction, and these cycle numbers are 17, 27, and 28 for ß-actin, activin ßA, and activin type II receptor, respectively. After an initial denaturation for 4 min at 94 C, the reactions were performed for the cycle numbers determined above with the cycling profile of 30 sec at 94 C, 30 sec at 58 C, and 1 min at 72 C, followed by a 5-min extension at 72 C. PCR (5 µl) was electrophoresed on agarose gel containing ethidium bromide, the products were visualized with the Gel Doc 1000 System (Bio-Rad Laboratories, Inc., Hercules, CA), and the yield of the products was quantitated with Molecular Analyst software (Bio-Rad Laboratories, Inc.). The specificity of the reactions was confirmed by cloning the PCR products into pBluescript II KS+ (Stratagene, La Jolla, CA) followed by sequencing on an ABI 310 genetic analyzer (PE Applied Biosystems, Foster City, CA). The PCR for ß-actin was performed to control the variation in mRNA concentration in the RT reaction.
Data analysis
The data for percent oocyte maturation were analyzed by one-way
ANOVA after arcsine transformation, followed by Fisher’s least
significance difference comparison. The mRNA levels of activin ßA and
activin type II receptor were expressed as a ratio to those of
ß-actin, which was amplified simultaneously as an internal control,
and statistically analyzed by one-way ANOVA, followed by Fisher’s
least significance difference comparison. All experiments were
performed two or three times to confirm the results.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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on zebrafish oocyte maturation on the final oocyte maturation, the full-grown follicles
were incubated in the presence or absence of recombinant human EGF or
TGF at different concentrations, and the GVBD of the oocytes was
scored at different times of incubation. Both EGF and TGF
significantly enhanced zebrafish oocyte maturation in a clear dose- and
time-dependent manner. The effects of EGF and TGF were observed as
early as 2 h after treatment, and the maximal effect was achieved
at 10–20 nM after 16-h incubation (Fig. 1
). When the follicles were cotreated
with gonadotropin (hCG) and EGF or TGF, the effects of hCG and EGF
or TGF were additive over the low concentration range of EGF/TGF,
and the additivity diminished at high concentrations of EGF/TGF
(Fig. 2).
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effects by actinomycin D (AD) and
cycloheximide (CH)-enhanced
zebrafish oocyte maturation, we examined the effects of AD (an
inhibitor of transcription) and CH (an inhibitor of protein
translation) on EGF/TGF-enhanced oocyte maturation. For these
experiments we chose animals 1 or 2 months older than those used in
other experiments to achieve higher spontaneous maturation rates so
that the extent of inhibition by the two drugs could be better
demonstrated. Despite the high spontaneous maturation rates, both EGF
and TGF still exhibited a significant promoting effect on final
oocyte maturation (Figs. 3 and 4). AD or
CH was added to the medium 2 h
before treatment with EGF/TGF. The effect of EGF/TGF on oocyte
maturation after 16-h incubation was significantly suppressed by AD in
a dose-dependent manner and was completely abolished at 100 ng/ml. AD
also significantly, but not completely, inhibited spontaneous oocyte
maturation (Fig. 3). Compared with AD, CH had an even more potent
inhibitory effect on oocyte maturation. It significantly reduced both
spontaneous and EGF/TGF-enhanced oocyte maturation dose dependently.
In contrast to AD, CH completely abolished spontaneous and
EGF/TGF-enhanced oocyte maturation at a concentration of 1000 ng/ml
(Fig. 4). To demonstrate that the complete suppression of oocyte
maturation by CH is not due to a nonspecific toxic effect of the drug
on the follicles, we further examined the ability of the follicles to
recover from suppression after removal of the chemical. After 16-h
treatment with CH, the follicles were washed, the medium was changed,
and culture was continued without CH for 20 h in the absence or
presence of TGF (20 nM). The results showed
that the oocytes could resume spontaneous maturation and responsiveness
to TGF enhancement after removal of the drug (Fig. 5).
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effects on oocyte maturation by recombinant
human follistatin action during the process of oocyte
maturation, we performed experiments by treating the follicles with EGF
or TGF in the presence of recombinant human follistatin, a specific
binding protein of activin. Follistatin (350 ng/ml) was applied 2
h before the addition of EGF/TGF and activin B. As expected,
follistatin treatment significantly reduced spontaneous and
activin-stimulated oocyte maturation (Fig. 6). Interestingly, cotreatment with
follistatin significantly suppressed the effects of EGF and TGF on
oocyte maturation (Fig. 6). As a negative control, follistatin had no
effect on DHP-stimulated oocyte maturation (Fig. 6).
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regulation of the expression of activin ßA and activin
type II receptor in the zebrafish follicle cells on oocyte maturation
involves activin in the signaling pathway, we further investigated the
effect of EGF/TGF on the expression of activin ßA and activin type
II receptor using cultured zebrafish ovarian follicle cells.
Semiquantitative RT-PCR assays were used to assess the expression
levels of the genes. The results showed that both EGF (10
nM) and TGF (10 nM) significantly stimulated
the expression of activin ßA and activin type II receptor, with
maximal effects achieved after 1–2 h of treatment, and the effect
tended to diminish after further treatment (Fig. 7). When assayed at 2 h of
treatment, EGF and TGF both exhibited clear dose-dependent
stimulation of activin ßA and activin type II receptor (Fig. 8).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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have significant biological activities
in fish ovary. EGF and TGF stimulate the production of goldfish
ovarian PGs (35), but suppressed gonadotropin-induced T
secretion (36). In agreement with reports in mammalian
species, both EGF and TGF significantly enhance final oocyte
maturation in the goldfish (36).
Using zebrafish as the model, we demonstrated in the present study that
both EGF and TGF significantly enhanced the rate of final oocyte
maturation, consistent with the effects of these two growth factors
reported in the goldfish (36) and mammals (11, 27, 28, 29, 30, 31, 32, 41, 42). When administered together with hCG, EGF and
TGF had additive interaction with hCG, but only over the low
concentration range, and the addition of effects diminished when the
concentrations of EGF and TGF increased. This suggests that
pituitary gonadotropin and intraovarian EGF/TGF may share similar
signaling pathways in regulating oocyte maturation. The mechanism of
EGF induction of oocyte maturation in mammals and nonmammalian species
is still unknown. In mammals, although EGF receptor has been
demonstrated in both cumulus cells and oocytes, EGF internalization
studies showed that cumulus cells are probably the major action site of
EGF (43). However, the possibility also exists that EGF
may act directly on the oocytes to promote oocyte maturation, because
EGF exhibited similar effects on both denuded bovine oocytes and
cumulus-oocyte complexes (41). Both cAMP-dependent
(44) and PKC (29) pathways have been
implicated in EGF signaling, but these mechanisms remain controversial,
and more studies are needed to confirm these findings. The effects of
EGF and TGF on zebrafish oocyte maturation could be significantly
suppressed by AD and CH, suggesting the involvement of de
novo transcription and protein synthesis in the action of EGF and
TGF. Interestingly, when applied together with follistatin, the
activin-binding protein, EGF and TGF had little effect on oocyte
maturation, strongly suggesting that the effects of EGF and TGF on
oocyte maturation are probably mediated at least partially by promoting
the activity of the intrinsic ovarian activin, which has a potent
stimulatory effect on final oocyte maturation in the zebrafish
(37, 45). This idea has not been reported in any
vertebrate. The observation that follistatin had no effect on
DHP-induced final oocyte maturation is in agreement with the fact that
DHP is the most potent oocyte maturation-inducing hormone in most
teleosts and acts directly on the surface of oocytes to initiate the
process of maturation (46). The mechanism by which activin
stimulates final oocyte maturation is still unknown. Our recent
evidence showed that activin had a powerful effect in enhancing the
maturational competence of oocytes, and it is likely that activin
exerts this effect by acting directly on the oocytes (Pang, Y., and W.
Ge, unpublished data). The expression of activin receptors by oocytes
has been reported in a variety of vertebrate species, including
zebrafish (47, 48, 49, 50).
To provide direct evidence for involvement of the ovarian activin
system in the action of EGF and TGF in zebrafish ovary, we further
investigated the regulation of expression of activin ßA and activin
type II receptor by EGF and TGF in cultured zebrafish ovarian
follicle cells. Our results clearly showed that treatment of follicle
cells with EGF or TGF significantly increased the expression levels
of both activin ßA and activin type II receptor in a clear dose- and
time-dependent manner, strongly suggesting that EGF and TGF may act
in the ovary by stimulating the ovarian activin system, including
activin subunits and activin receptors. The role of ovarian activin in
oocyte maturation and its involvement in the action of pituitary
gonadotropin in the event have been demonstrated in our previous study
(37) and a similar study from others (45).
The present study provides further evidence that the zebrafish ovarian
activin system is important in mediating not only the endocrine
regulation of ovarian functions by hormones such as gonadotropin(s),
but also the regulation by intraovarian paracrine factors, including
EGF/TGF.
In summary, the present study demonstrated that EGF and TGF, two
peptide growth factors that play important regulatory roles in the
mammalian ovary, increased the rate of final oocyte maturation in the
zebrafish. The effect of EGF/TGF involved de novo
transcription and protein translation. Follistatin, an activin-binding
protein, could significantly suppress the effects of EGF and TGF,
suggesting the involvement of the intraovarian activin system in the
action of EGF and TGF. This hypothesis was further supported by our
evidence that EGF and TGF significantly stimulated the expression of
activin ßA and activin type II receptor in cultured zebrafish ovarian
follicle cells. The present study not only provides consistent evidence
for the involvement of the ovarian EGF/TGF system in the regulation
of vertebrate oocyte maturation, but also demonstrates, for the first
time, the relationship between the ovarian activin system and the
EGF/TGF system in the event, which undoubtedly contributes to our
understanding of the complex regulatory network involved in controlling
the oocyte development of vertebrates.
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Acknowledgments |
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Footnotes |
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Abbreviations: AD, Actinomycin D; CH, cycloheximide; DHP,
17,20ß-dihydroxy-4-pregnen-3-one; EGF, epidermal growth factor;
GVBD, germinal vesicle breakdown.
Received May 7, 2001.
Accepted for publication September 12, 2001.
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