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Reproductive Biology Unit (M.L., J.L., B.K.T.), Department of Obstetrics and Gynaecology and Department of Physiology, University of Ottawa, Ottawa Civic Hospital Loeb Research Institute, Ottawa, Ontario, Canada K1Y 4E9; Program in Developmental Biology and Division of Gastroenterology (P.A.H., T.T., J.L.W.), Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8; and Department of Anatomy and Cell Biology (L.A.), University of Toronto, Toronto, Ontario, Canada M5S 1A8
Address all correspondence and requests for reprints to: Dr. Benjamin K. Tsang, Department of Obstetrics and Gynaecology, Ottawa Civic Hospital, 1053 Carling Avenue, Ottawa, Ontario, Canada K1Y 4E9. E-mail: ben{at}civich.ottawa.on.ca
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ovarian follicular development is the culmination of proliferation and
differentiation of granulosa and theca cells, the nature and extent of
which are dependent on the actions and interactions of gonadotropins
and intraovarian regulators (13). TGFß, a secretory product of hen
granulosa and theca cells throughout follicular development (14), seems
to act antagonistically with TGF
in the regulation of granulosa cell
function (15, 16, 17, 18). Previous studies from our laboratory have
demonstrated that PGs play an important role in the mitogenic response
of granulosa cells to TGF and that the production of PGs by
granulosa cells is modulated by TGFß in a follicular stage-dependent
manner (19). Although cyclooxygenase II seems to be a site of
regulation by TGFß (20), more recent evidence points to the fact that
this growth factor also suppresses the expression of cytosolic
phospholipase A2 (cPLA2), a key enzyme in the biosynthesis
of PGs in granulosa cells (21). The decrease in granulosa cell
cPLA2 messenger RNA (mRNA) abundance in the presence of
TGFß seemed to be the consequence of suppressed gene transcription
and, interestingly, was more pronounced in cells at early stages of
follicular development, when they were less differentiated and
proliferatively more active (21). The reason(s) for this developmental
dependency of TGFß action is not clear, nor is it known if the
follicular stage-specific regulation of MADR2 expression by this growth
factor is a determinant of the relative responsiveness of this
signaling pathway to TGFß. To date, the presence and regulation of
MADR2 in the ovary by TGFß have not been examined.
The overall objective of the present studies was to demonstrate the presence of MADR2 in the hen granulosa cells and to study the regulation of its expression by TGFß in vitro during ovarian follicular development, particularly with reference to its possible association with the follicular stage-dependent control of granulosa cell cPLA2 gene transcription by the growth factor.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-32 P deoxycytidine triphosphate
and the enhanced chemiluminescence Western blotting detection kit were
purchased from Amersham (Oakville, ON, Canada). 
-probe blotting
membrane, trans-blot supported nitrocellulose membrane, and the Bio-Rad
protein assay kit were from Bio-Rad Laboratories (Hercules, CA). X-ray
films were from Eastman Kodak Company (Rochester, NY). Recombinant
human TGFß was purchased from Collaborative Research (Bedford, MA).
The 2.8-kb hen cPLA2 complementary DNA (cDNA) probe was a
gift from Dr. Lih-Ling Lin (Genetics Institute, Cambridge, MA). The
1.4-kb human MADR2 and 1.2-kb human MADR1 probes have been described
previously (3, 22).
Isolation and culture of granulosa cells
White Leghorn hens in their first year of lay were obtained from
a local egg farm and were individually caged in a windowless,
air-conditioned room with a 14-h light, 10-h dark cycle. The birds had
free access to feed and water. The time of ovulation was predicted from
the time of the previous oviposition, on the basis that the former
occurs 15–75 min from the latter. Approximately 10–14 h before the
expected time of ovulation, hens were killed by cervical dislocation,
and the ovaries were excised and placed in ice-cold Medium 199
supplemented with HEPES (25 mM, pH 7.4). Follicles from two
to three hens were grouped together according to the stage of
development, as follows: the largest (F1), third largest (F3), and
fifth and sixth largest (F5–6) preovulatory follicles and large white
follicles (LWF). Granulosa cells from each group of follicles were
isolated, as described previously (Asem et al., 1984) and
dissociated by incubation (15 min at 37 C) in 2 ml Medium 199-HEPES
containing collagenase (540 U) and trypsin inhibitor (0.2 mg/follicle).
Dispersed granulosa cells (2 x 105) were incubated in
16-mm tissue culture wells (Falcon, Becton Dickinson Labware, Lincoln
Park, NJ) in 0.5 ml MEM, without phenol red but supplemented with
L-glutamine (0.29 mg/ml), nonessential amino acids (0.1
mM), penicillin (100 U/ml), and streptomycin (100 µg/ml),
at 39 C under an atmosphere of 5% CO2 and 95% air. After
an incubation period of 3 h to allow cell plating, the medium was
changed to one containing different test agents, and cells were
cultured for up to 24 h. At the end of the culture period, the
media were removed, and protein and RNA extracts were prepared. Cell
viability, determined by the trypan blue dye exclusion test in both
experimental groups (F1 and F5–6), was greater than 90% before and
after culture.
Solubilized cell extracts and immunoblot analysis
Because MADR proteins translocate from the cytosol to the
nucleus upon receptor activation (3, 23), whole-cell lysates, rather
than cytosolic fractions from granulosa cells, were used for Western
blot analysis. Total cell protein extracts were prepared as follow:
granulosa cells were sonicated (8 sec/cycle, 3 cycles) on ice in 10
mM HEPES buffer (pH7.4) containing 1 mM EGTA
and 2 mM phenylmethylsulfonyl fluoride. The sonicates were
stored at -20 C until electrophoretic analyses were performed. Protein
concentration was determined using the Bio-Rad Protein Assay
Method.
Equal amounts of proteins (60–100 µg per lane, depending on specific experiment) present in cell extracts were resolved by one-dimensional SDS-PAGE and electrophoretically transferred to nitrocellulose membrane. Membranes were blocked with 5% nonfat milk and subsequently incubated with polyclonal human MADR1 or MADR2 antibody (or preimmune sera for negative controls) diluted in 10 mM Tris-buffered saline (pH7.5) containing 5% milk. An enhanced chemiluminescence kit was used to visualize immunopositive proteins. For preparation of anti-MADR1 and MADR2 antibodies, the nonconserved domain (3) of each protein was expressed as a glutathione-S-transferase fusion protein in pGEX4T1 (Pharmacia). Rabbits were immunized with 1 mg of bacterially expressed protein, purified by glutathione-sepharose chromatography, using standard protocols (24).
RNA isolation and Northern analysis
Total RNA from granulosa cells was isolated by using an RNeasy
Kit (Qiagen) and quantified spectrophotometrically at 260 nm. RNA
samples (6–12 µg) were denatured at 55 C for 15 min in 45%
formamide and 5.4% formaldehyde and electrophoresed at room
temperature in 1% formaldehyde-agarose gel. Gels were stained with
ethidium bromide to confirm equal loading of RNA samples (by comparing
staining of 28S and 18S ribosomal bands). After transfer to a membrane,
blots were UV-cross-linked and hybridized to 32P-labeled
hen cPLA2, human MADR1, and MADR2 cDNA probes. The probes
were labeled using a Random Primed [ -32 P] DNA
Labeling Kit. Blots were washed twice (5 min/wash) at room temperature,
4 times (15 min/wash) at 65 C in 0.3 x saline sodium citrate,
0.5% SDS, and subsequently exposed to x-ray film at -80 C.
Densitometric analysis of mRNA and 28S ribosomal ribonucleic acid
(rRNA) band was performed using an Image Analysis System from Bio-Rad
Laboratories. Data were normalized by the respective 28S RNA and
expressed as a percentage of the control (defined as 100%).
Statistical analysis
Follicles from two or three birds were used for each experiment.
Results were expressed as the mean ± SEM of three to
five experiments. Statistical analysis were carried out by ANOVA, and
significant differences between treatment groups were determined by the
Tukey test. Where required, data were transformed logarithmically,
before statistical analysis, to remove heterogeneity of variance, as
determined by Barlett’s test. Statistical significance was inferred at
P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, MADR2 appeared as a 60-kDa protein in
granulosa cells at all stages of follicular development examined.
Expression of this protein was minimal in granulosa cells from LWF,
highest in F5–6 granulosa cells, and decreased with follicular
development (Fig. 1).
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), whereas the abundance of MADR2
transcript (
3.6 kb) was increased six-fold by the growth factor
(P < 0.05; Fig. 3).
MADR1 (55 kDa), an MADR specifically phosphorylated in bone
morphogenetic protein (BMP) signaling pathways (3), also was present in
granulosa cells. It has a transcript size of 3.6 kb but was not
significantly affected by the presence of TGFß at concentrations as
high as 20 ng/ml (P > 0.05; see Figs. 3 and 5). In
addition, the presence of MADR1 and MADR2 in the hen granulosa cell
seems authentic, because no detectable signals were evident in
immunoblots when the anti-MADR1 and anti-MADR2 antibodies were replaced
with their respective preimmune antisera (data not shown).
Interestingly, the basal MADR2 transcript level was highest early in
the culture period (0.5 h) but significantly decreased with the
duration of culture (Fig. 4). Addition of
TGFß (20 ng/ml) to the cell cultures had no significant influence on
granulosa cell MADR2 mRNA abundance in both F1 and F5–6 until the last
time point examined (6 h), when the transcript levels were
significantly elevated by 250% (Fig. 4a) and 500% (Fig. 4b),
respectively. Thus, granulosa cell MADR2 is selectively regulated by
TGFß at both the transcriptional and translational levels.
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) and protein content at 12 h (Fig. 5) in granulosa cells in both F1 and
F5–6 follicles (P < 0.05), MADR2 expression in the
presence of TGFß (20 ng/ml) seemed to be more pronounced in the less
differentiated cells (F5–6; Figs. 4 and 5). This is supported by the
outcome of statistical analysis by ANOVA, which shows a significant
interaction between follicular stage and TGFß effects
(P < 0.05), presumably brought about by the greater
stimulatory action of the growth factor at the F5–6 stage (Fig. 4).
Similarly, MADR2 protein content in F5–6 granulosa cells in the
presence of TGFß (20 ng/ml) was two times higher than in the F1 cells
(Fig. 5). In contrast, TGFß had no significant influence on granulosa
cell MADR1 protein content, irrespective of the stage of follicular
development (P > 0.05; Fig. 5).
Reciprocal expression of MADR2 and cPLA2 in granulosa
cells cultured in the absence and presence of TGFß
To investigate whether the TGFß-induced changes in
cPLA2 expression in hen granulosa cells possibly could be
associated with the up-regulation of MADR2, experiments were performed
to compare concentration-dependent and temporal responses to TGFß. In
the absence of TGFß, cPLA2 mRNA abundance increased,
whereas MADR2 mRNA levels decreased, with the duration of culture. The
earliest significant changes were observed at the same time point (6 h;
Fig. 4). Addition of TGFß to the cultures increased MADR2 mRNA
abundance and decreased that of cPLA2, after 6 h of
culture, when compared with their respective controls (Fig. 4). The
divergent effects of TGFß on the expression of MADR2 and
cPLA2 both were concentration-dependent, with significant
responses evident at the same concentration of the growth factor (20
ng/ml; Fig. 3). Moreover, both MADR2 and cPLA2 responses to
TGFß were follicular stage-dependent and were greater in the
granulosa cells from the early stage of follicular development (Figs. 4 and 5). Thus, a reciprocal relationship seems to exist between
cPLA2 and MADR2 expression in granulosa cells that are
responsive to TGFß. Furthermore, this relationship is follicular
stage-dependent.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, A and B), suggesting that the
growth factor may be necessary for the maintenance of physiologically
important cellular levels of MADR2 in vivo. Previous studies from our laboratory have demonstrated that TGFß suppresses granulosa cell cPLA2 and cyclooxygenase II mRNA abundance and PG production and increases urokinase plasminogen activator activity in vitro. These responses were more pronounced at early stages of follicular development (19, 20, 27). In the present study, we have demonstrated that MADR2 is up-regulated by TGFß in vitro, with maximum response observed in granulosa cells from F5–6 follicles. Whereas TGFß is expressed in hen granulosa and theca cells throughout follicular development (14), our present in vivo studies indicate that MADR2 abundance was minimal in granulosa cells from LWF, highest in F5–6 granulosa cells, and decreased with follicular maturation. These findings raise the interesting possibility that the follicular stage-specific high abundance of MADR2 may be an important determinant for the relative responsiveness of the signaling pathway to the growth factor and that homologous up-regulation of MADR2 may be physiologically important. The physiological significance for the low MADR2 abundance in LWF granulosa cells observed in the present studies is not clear. It has been shown that TGFß is capable of inducing apoptosis in other systems (28). It is possible that the suppression of MADR2 expression in vivo at this follicular stage may be one of the mechanisms in place to prevent these cells from undergoing cell death until the fate of these follicles (i.e. development vs. atresia) has been determined.
MADR2 is believed to be a potential transcriptional regulator, based on its ability to accumulate in the nucleus in response to TGFß and the observation that MADR1 can act as transcriptional activator (4). Furthermore, MAD3, another member of MAD family, seems to be able to potentiate a transcriptional response to TGFß after its overexpression (29). However, the physiological target gene for MADR2 in the granulosa cell is not known, although it is tempting to speculate that the down-regulation of cPLA2 by TGFß may be related to the follicular stage-dependent expression of MADR2. This notion is consistent with our earlier observation that the suppression of granulosa cell cPLA2 level by the growth factor resides at the level of gene transcription and is not a result of decrease in mRNA stability (Li et al., unpublished data). In addition, it is of interest to note, from the present studies, that alterations in MADR2 expression seem tightly coupled to reciprocal changes in cPLA2 mRNA abundance, as evidenced by increased basal MADR2 mRNA and protein levels, whereas cPLA2 transcript abundance decreased with follicular development. Furthermore, basal MADR2 expression decreased, whereas that of cPLA2 increased, with the duration of culture. Although the precise mechanism for the increase in cPLA2 mRNA and protein content is not known, one possibility is that granulosa cell cPLA2 expression is suppressed by endogenous inhibitory factors (e.g. TGFß) and their signaling machinery to maintain appropriate levels in vivo. When these inhibitory factors were removed by culturing cells in serum-free medium, cPLA2 expression spontaneously increased. This notion is consistent with our present observation that the increase in basal cPLA2 expression, with increased duration of culture, is accompanied by reciprocal decreases in mRNA abundance and protein content of MADR2. Finally, the reciprocal MADR2 and cPLA2 response to TGFß followed a similar time course and concentration- and follicular stage-dependency. Further experiments, including MADR2 knock-out with antisense, are required to determine directly whether and what precise relationship exists between MADR2 signaling and cPLA2 expression in the granulosa cell.
It has been demonstrated that MADR1 shares considerable sequence homology with MADR2 but is not involved in the TGFß signaling pathway (3). Although significant levels of MADR1 mRNA and protein were detected in granulosa cells in the present studies, they were not significantly affected by the presence of TGFß, indicating that the response in MADR2 expression to the growth factor was specific. It is known also that MADR4 (DPC4), another MADR protein, is involved in TGFß signaling (30). Whether MADR4 also is upregulated by TGFß and can act as a transcriptional coregulator in the regulation of gene expression remains to be determined.
In summary, we have demonstrated, for the first time, the presence of MADR1 and MADR2 in hen granulosa cells. The expression of MADR2, but not of MADR1, was up-regulated by TGFß in vitro in a concentration- and time-dependent manner. Granulosa cell MADR2 expression was maximal during early stages of follicular development, when the cells are proliferatively most active and the cPLA2 system most responsive to TGFß. These findings are consistent with the hypothesis that MADR2 expression is autoregulated and that this regulation may be an important determinant in the follicular stage-specific responsiveness of the cells to TGFß.
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Footnotes |
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2 Recipient of the Genesis Research Foundation Graduate Studentship
and the Ontario Graduate Scholarship.
Received March 18, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ß and does not control plasminogen activator
activity during follicular development. Biol Reprod 51:787–794[Abstract]
in hen granulosa cells during ovarian follicular
development. Biol Reprod 52:1050–1058[Abstract]
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