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Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556
Address all correspondence and requests for reprints to: A. L. Johnson, Ph.D., Department of Biological Sciences, P.O. Box 369, University of Notre Dame, Notre Dame, Indiana 46556.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has previously been reported that during follicle development, cells
from the hen granulosa layer first become competent to produce
significant amounts of progesterone only at or shortly after a
prehierarchal follicle (defined as a follicle 
8 mm) has been selected
into the preovulatory hierarchy (at the 9–12 mm stage of development)
(8). Prehierarchal follicles are highly susceptible to
undergoing atresia, and granulosa cells from such follicles are
considered undifferentiated. By comparison, preovulatory follicles
rarely become atretic under normal physiological conditions. Follicle
recruitment into the preovulatory hierarchy is accompanied by the first
evidence of FSH-induced cAMP accumulation (8) and
increased basal levels of LH receptor (LH-R) mRNA (9)
within the rapidly differentiating granulosa cell layer. Of additional
significance is that although granulosa from prehierarchal follicles
express low, but detectable, levels of P450scc mRNA and immunoreactive
protein, cells fail to actively convert cell-permeable
25-hydroxycholesterol to pregnenolone in vitro
(3). On the other hand, preculture of 6- to 8-mm follicle
granulosa cells with forskolin or FSH for 24 h renders cells
competent to synthesize progesterone in response to a challenge with LH
or 8-bromo-cAMP during a subsequent 3-h incubation. This induction of
steroidogenesis can be blocked by coculture with the growth factors,
transforming growth factor-
(TGF) and epidermal growth factor
(EGF) (10). The latter finding suggests that the
differentiation of granulosa cells from prehierarchal follicles is
tonically suppressed by paracrine and/or autocrine factors up to the
time at which the follicle is selected into the preovulatory follicle
hierarchy. In fact, it has been hypothesized that the removal of this
inhibitory influence may be a prerequisite for follicle selection and
the subsequent progression of granulosa cell differentiation
(11).
We recently reported expression of very low levels of StAR mRNA (by Northern blot analysis) in undifferentiated granulosa cells from prehierarchal follicles. By comparison, readily detectable levels of the transcript are present in granulosa from the third (F3) and second (F2) largest follicles, and the highest basal levels expressed within the granulosa layer during follicle development are consistently found in the largest (F1) preovulatory follicle (7).
Taken together, the above observations led to the working hypothesis, tested herein, that gonadotropin-induced StAR expression is linked to the initial differentiation of granulosa from prehierarchal follicles and to the full potentiation of progesterone production in preovulatory follicles. The results demonstrate that StAR expression in granulosa cells from prehierarchal follicles is promoted initially by FSH and subsequently by LH over a 20-h culture period, and preexisting elevated levels of StAR expression in preovulatory follicle granulosa are enhanced by LH treatment. Furthermore, inhibition of mitogen-activated protein (MAP) kinase signaling (using the MAP kinase kinase inhibitors U0126 and PD98059) potentiates gonadotropin-induced StAR and LH-R expression and progesterone synthesis in granulosa cells from prehierarchal follicles. These findings further support a balance between positive and negative regulators of StAR expression in hen granulosa cells and are consistent with the proposal that follicle selection and the subsequent progression of granulosa cell differentiation are initiated only after the removal of differentiation-inhibiting signals targeted at the granulosa layer.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recombinant human TGF and insulin-like growth factor I (IGF-I) were
obtained from PeproTech, Inc. (Rocky Hill, NJ), and 8-bromo-cAMP
(8-br-cAMP) was acquired from Sigma (St. Louis, MO). Ovine
LH (lot 26) and recombinant human FSH (lot AP8468A) were provided by
the National Hormone and Pituitary Program. The MAP kinase kinase
inhibitors U0126 and PD98059 were obtained from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA). These agents, at the
doses used, are reported to be selective for the MAP kinase pathway and
do not attenuate signaling via protein kinase A or B in hen granulosa
cells (12).
Granulosa cell cultures
Granulosa cells from the F1 follicle and from the combined F2
plus F3 follicle layers were studied separately because it had
previously been established that levels of StAR mRNA were dramatically
higher in F1 compared with F2 or F3 follicles (7). In
addition, granulosa layers from 8–12 prehierarchal (6–8 mm) follicles
were pooled and prepared for culture as previously described (8, 13). Portions of granulosa cell layers from each follicle group
were immediately frozen at -70 C (T0 controls) or were dispersed with
0.3% collagenase (type 2; Worthington Biochemical Corp.,
Freehold, NJ) and plated in 6-well polystyrene culture plates (Falcon
3046, Fisher Scientific, Pittsburgh, PA) at a density of
approximately 106/well in 1 ml medium 199-HEPES
supplemented with Hanks’ salts (Life Technologies, Inc.,
Gaithersburg, MD) plus 1 ml DMEM containing 5% FBS (Life Technologies, Inc.).
In the first set of experiments, granulosa cells from 6- to 8-mm follicles were cultured for 0 (T0), 1, 3, 6, or 20 h in the absence or presence of 8-br-cAMP (1 mM), LH (100 ng/ml), FSH (100 ng/ml), IGF-I (50 ng/ml), or FSH plus IGF-I. The agents and doses chosen for study were based upon previous experiments that evaluated steroidogenesis in hen granulosa cells (8, 10). After the appropriate culture time, only the plated cells were collected and frozen at -70 C until prepared for total cellular mRNA.
In addition, 6- to 8-mm follicle granulosa cells were prepared and
immediately cultured in the absence or presence of FSH (100 ng/ml), LH
(100 ng/ml), U0126 (50 µM), FSH plus U0126, LH plus
U0126, PD98059 (50 µM), or FSH plus PD98059 for 20
h. After culture, plated cells were collected and frozen for analysis
of StAR mRNA and protein, and LH-R mRNA, and media were assayed for
progesterone. In a related experiment the effectiveness of these
inhibitors to block MAP kinase signaling through Erk1/2 was tested by
pretreating freshly cultured granulosa cells from prehierarchal
follicles for 1 h in the absence or presence of U0126 (50
µM) or PD98059 (50 µM), then culturing
cells for an additional 20 min in the absence or presence of 50 ng
TGF/ml. Samples were processed for the analysis of phosphorylated
Erk (Erk-P). Treatment with this dose of TGF has previously been
demonstrated to be an effective inducer of Erk-P in hen granulosa cells
(12). Freshly plated cells were also pretreated without or
with TGF (50 ng/ml) for 30 min, then cultured for 20 h in the
absence or presence of FSH (100 ng/ml). Media were removed and assayed
for progesterone, whereas the plated cells were collected and analyzed
for levels of StAR and LH-R mRNA.
In a third set of experiments, granulosa cells from F1 or F2 plus F3 follicles were plated in the absence and presence of 1 mM 8-br-cAMP or LH (100 ng/ml) and cultured for 1, 3, or 6 h. At the end of each culture period, cells were collected and rapidly frozen at -70 C until prepared for total cellular RNA and analysis of StAR mRNA and protein and LH-R mRNA.
In the final experiments, granulosa cells from F2 plus F3 preovulatory
follicles were precultured for 1 h in the absence or presence of
U0126 (50 µM) or PD98059 (50 µM), then
treated for an additional 1 h in the absence (Con) or presence of
100 ng/ml LH. Cells were collected and rapidly frozen at -70 for
analysis of StAR and LH-R mRNA, and in some instances the media were
analyzed for progesterone. In addition, F2 and F3 follicle granulosa
cells were preplated for 6 h, then pretreated for 1 h in the
absence or presence of U0126 or PD98059 and cultured for an additional
20 min in the absence or presence of TGF (50 ng/ml). Cells were
collected and frozen at -70 for the analysis of Erk-P.
Northern blot analysis
The chicken StAR cDNA used for probing Northern blots was
recently described by Bauer et al. (7), and the
chicken LH-R cDNA was described by Johnson et al.
(9). Each blot was probed separately for StAR mRNA and
LH-R mRNA and was finally probed with a chicken 18S ribosomal RNA cDNA
to standardize for equal loading of RNA samples. Conditions for
conducting Northern blot analysis have been previously detailed
(13), and all blots were visualized on phosphor screens
using a Storm 840 PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA). Analysis of blots was conducted using the
ImageQuant data reduction system (Molecular Dynamics, Inc.).
Western blot analysis
The phospho-specific Erk1/2 monoclonal antiserum was
obtained from Upstate Biotechnology, Inc. (Lake Placid,
NY), and the -tubulin monoclonal antibody used for standardization
was purchased from Sigma. The StAR polyclonal antibody was
provided by Dr. D. B. Hales (University of Illinois, Chicago, IL)
(14). Western blot analyses were conducted essentially as
previously described (12, 13). Briefly, tissues were
homogenized in a protein lysis buffer containing an enzyme and
phosphatase inhibitor cocktail (Sigma). Proteins were
separated on a 10% polyacrylamide gel under denaturing conditions,
then transferred to a polyvinylidene difluoride membrane (Immobilon-P,
Millipore Corp., Fisher Scientific, Hanover
Park, IL). Incubations with primary antibodies (phospho-Erk and
phospho-Akt at 1:1000 dilution; StAR at 1:5000 dilution) were conducted
overnight at 4 C, whereas those for the horseradish
peroxidase-conjugated antimouse IgG secondary antibody (for
phospho-Erk; Pierce Chemical Co., Rockford, IL) or
horseradish peroxidase -conjugated antirabbit IgG secondary antibody
(for StAR; Pierce Chemical Co.) were performed for 1
h at room temperature. Blots were incubated with ECL Western blotting
agent (Amersham Pharmacia Biotech, Arlington Heights, IL)
for 1 min and exposed to x-ray film for 1–15 min. The extent of
antibody binding was quantitated by densitometry (UltraScan XL laser
densitometer, Pharmacia LKB, Piscataway, NJ).
Progesterone assays
Progesterone levels in medium samples were quantitated using an
enzyme immunoassay (Diagnostics Systems Laboratories, Inc., Webster TX) according to the manufacturer’s protocol.
Data analysis
All experiments were repeated a minimum total of three times
unless otherwise stated. Levels of StAR and LH-R mRNA, Erk-P, and
progesterone were expressed as the fold difference (mean ±
SEM) vs. a designated reference treatment such
as freshly collected (T0) or cultured control cells (value for
reference treatment arbitrarily set at 1). Data were analyzed by
one-way ANOVA (analysis of fold difference data did not include the
reference treatment) and the Fisher protected least significant
difference multiple range test. Post-hoc analysis of
selected data was conducted by paired t test using original
(untransformed) data (e.g. LH-R mRNA, T20 control
vs. T0; Fig. 1
).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, top panel). Within 6 h of culture, FSH, but not LH,
treatment increased StAR mRNA levels (10.4 ± 3.5-fold increase
vs. T0), and by 20 h of culture, treatment with both LH
and FSH stimulated increased StAR mRNA levels (by 5.0 ± 1.2- and
4.3 ± 0.9-fold, respectively). By comparison, there was a small,
but significant, increase in LH-R mRNA levels within control cultured
cells after 20 h of culture (P < 0.05
vs. T0, by paired t test; Fig. 1, bottom
panel). Treatment with 8-br-cAMP, LH, or FSH further increased
LH-R mRNA levels above those detected in T20 control cultured cells
(P < 0.05). On the other hand, IGF-I treatment for
20 h failed to increase levels of StAR or LH-R mRNA compared with
those in T20 control cells, and IGF-I had no additive effect on StAR or
LH-R expression when combined with FSH compared with the FSH treatment
alone (P > 0.10; Fig. 2). As a positive control for biological
activity, IGF-I was determined to induce a greater than 10-fold
increase in Akt phosphorylation after a 20-min treatment of granulosa
cells from prehierarchal follicles (data not shown).
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). Whereas a 20-min
treatment with TGF induced a 2.6 ± 0.6-fold increase in levels
of Erk-P within prehierarchal follicle granulosa cells
(P < 0.05, by paired t test), pretreatment
with the MAP kinase kinase inhibitors U0126 and PD98059 effectively
blocked this increase.
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, top panel). However,
although FSH and LH induced 3.4- and 6.4-fold increases, respectively,
in StAR mRNA levels compared with control cultured cells
(P < 0.05), 20 h of culture with these
gonadotropins in the presence of U0126 or PD98059 induced a further
5.1- to 6.9-fold increase in StAR mRNA compared with FSH or LH
treatment alone. By comparison, FSH, LH, U0126, and PD98059 each
induced a significant increase in LH-R mRNA compared with levels in
control cultured cells (P < 0.05; Fig. 4, bottom
panel). Furthermore, cultures containing FSH or LH combined with
U0126 or PD98059 expressed 2.2- to 5.4-fold higher levels of LH-R mRNA
than cultures with either gonadotropin alone. The synergistic effects
of U0126 and PD98059 on FSH-induced StAR mRNA levels were paralleled by
increases in the mature form of the 30-kDa StAR protein (Fig. 5).
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).
Neither U0126 nor PD98059 alone had any discernable effect on
progesterone levels after 20 h of culture; however, each inhibitor
in combination with FSH or LH produced a synergistic effect on
progesterone synthesis (a 2.2- to 9.9-fold increase) compared with
gonadotropin treatment alone.
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did not alter levels of StAR or
LH-R mRNA or progesterone production compared with those in control
cultures; however, TGF effectively blocked FSH-induced StAR and LH-R
mRNA as well as the initiation of progesterone synthesis (Fig. 7).
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, inset).
Levels of StAR mRNA were increased in F2/F3 follicle granulosa cells
after treatment with 8-br-cAMP or LH for 1 h, whereas in F1
follicle granulosa the first significant increase in response to each
treatment was not observed until 3 h (Fig. 8, top
panel). Both 8-br-cAMP and LH stimulated a greater fold increase
in F2 plus F3 granulosa cells compared with F1 granulosa at each
culture interval investigated. The rapid increase in LH-induced StAR
mRNA levels was subsequently reflected by increased StAR protein (Fig. 8, bottom panel). By contrast, neither 8-br-cAMP nor LH
treatment altered levels of LH-R mRNA after any of these relatively
short culture periods compared with the respective control value
(P > 0.10; data not shown).
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induced a significant (4.4 ± 0.9-fold)
increase in Erk-P within granulosa from F2 plus F3 preovulatory
follicles compared with the control, whereas pretreatment with U0126 or
PD98509 effectively prevented (U0126) or significantly reduced
(PD98059) this response (Fig. 9).
However, unlike granulosa from prehierarchal follicles, neither U0126
nor PD98059 further increased levels of LH-induced StAR mRNA after
1 h of culture beyond those found in LH-treated cells (Fig. 10). Furthermore, treatment with
PD98059 plus LH did not stimulate progesterone secretion above levels
found in cells treated with LH alone (a 9.0 ± 1.6- vs.
11.1 ± 2.5-fold increase, respectively, compared with control
cultured cells; P > 0.05). Not unexpectedly, levels of
LH-R mRNA were not affected by any treatment after only a 1-h culture
period (P > 0.10; data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Expression of both StAR and LH-R mRNA in freshly collected granulosa from prehierarchal follicles is extremely low, and this reflects the undifferentiated state of the follicles used. The finding that after 6 h of culture, FSH, but not LH, induces StAR mRNA expression reflects the fact that granulosa cells from prehierarchal follicles predominantly express the FSH receptor and considerably lesser amounts of the LH receptor (9, 16, 17). Significantly, LH-R mRNA levels are increased by 2- to 3-fold in adhering, viable control cells cultured for 20 h, whereas LH treatment induced a further doubling of LH-R mRNA expression. These results indicate that isolated granulosa cells from undifferentiated follicles begin the process of differentiation during culture, possibly due to the release from inhibitory influences that exist in vivo, and in addition that this initial increase in LH-R expression is sufficient to mediate StAR mRNA transcription in the presence of LH. Bypassing gonadotropin receptors with the relatively stable cAMP analog, 8-br-cAMP, results in the greatest induction of StAR mRNA and protein (data not shown) after 20 h of culture, and this provides direct evidence for the involvement of protein kinase A signaling.
It is of interest to note that the increase in StAR mRNA levels after 3–6 h of culture in prehierarchal follicle granulosa cells in response to 8-br-cAMP or FSH occurs more rapidly than the 8- to 16-h culture period required for the increased expression of P450scc mRNA (10). Although this finding emphasizes the acute nature of StAR induction via the protein kinase A signaling pathway in hen granulosa, it is interesting to note that P450scc expression appears to precede that of StAR during follicle development (3). By comparison, in PMSG-primed prepubertal rats the first detectable expression of StAR within granulosa was found almost 2 days after an increase in P450scc (18). Given this observed relationship, it is likely that the low levels of P450scc protein previously determined to be expressed within mitochondria from prehierarchal follicle granulosa cells (3) are sufficient to initiate low, but physiologically relevant, amounts of steroid production immediately after the initial induction of StAR expression. The potential for a rapid initiation of steroid production is probably a critical component of the follicle selection process. This proposal is supported by the fact that FSH induced a small, but significant, increase in progesterone production after 20 h of culture.
In contrast to prehierarchal follicles, granulosa cells from F3 through F1 follicles express similar and comparatively high levels of LH receptor mRNA, and LH represents the primary gonadotropin responsible for promoting granulosa cell steroidogenesis (predominantly progesterone) during the final stages of follicle development (17). Thus, it is not surprising that LH or 8-br-cAMP treatment rapidly induces StAR mRNA expression (within 1–3 h of culture), as a rapid up-regulation of StAR by gonadotropins via the protein kinase A signaling pathway has been well documented in mammalian species (2, 5, 19). It is also noteworthy that inhibition of MAP kinase signaling failed to enhance LH- induced StAR mRNA levels after short-term culture, suggesting that MAP kinase signaling is not involved in acute regulation of steroidogenesis (e.g. as would occur during the rapid and transient preovulatory surge of serum progesterone).
It was recently established that constitutive expression of StAR mRNA is dramatically increased in granulosa during the transition from the F2 stage to the F1 stage of development (7). Thus, it is reasonable to speculate that the increased potential for F1 follicle granulosa to synthesize progesterone compared with those from the remaining hierarchal follicles (previously reviewed in Ref. 20) may be linked to this preexisting elevation in StAR expression. In general, high levels of preexisting StAR mRNA expression within preovulatory follicle granulosa ensure rapid protein translation to facilitate the 8- to 10-fold increase in serum progesterone levels that occurs after initiation of the LH surge (21). Furthermore, the present data suggest that an additional mechanism to provide maximal transport of steroid precursor to the inner mitochondrial membrane during the LH surge occurs via new transcription of StAR. It is reasonable to conclude that the comparatively smaller fold increase in StAR mRNA levels after short-term culture of granulosa from F1 (compared with F2 plus F3 follicles) with 8-br-cAMP or LH probably reflects an already near-maximal rate of StAR transcription by F1 follicle granulosa.
One additional point worthy of discussion is the inability to reliably detect basal (T0) levels of StAR protein in F1 follicle granulosa by Western blot analysis despite the relatively high preexisting levels of the StAR transcript. One possible explanation is a lack of sensitivity to basal levels of StAR protein due to the use of a heterologous antibody. However, alternative explanations include the possibility that StAR protein expression is controlled at the level of translation, and/or that StAR protein may be rapidly turned over on a continuing basis such that elevated levels of LH (such as those that occur during the preovulatory LH surge) are obligatory for the production of sufficient StAR protein to enable the generation of the preovulatory progesterone surge. Further experiments to evaluate these possibilities are currently underway. In any event, the rapid translational response, and subsequent elimination, of StAR would allow for the precise regulation of steroidogenesis required for a species, such as the hen, that contains a hierarchy of ovarian follicles differing in steroidogenic potential. The proposed requirement for newly synthesized StAR protein during the 4- to 6-h preovulatory LH surge is supported by the finding that in the absence of continued LH stimulation in vitro, protein levels in whole cell extracts rapidly decline by 95% after 3 h of culture (data not shown).
Furthermore, it is significant to note that despite the high level of LH receptor mRNA expression previously reported in F2 and F3 follicle granulosa in vivo (9), and the ability of LH treatment to markedly increase StAR expression in cultured F2 plus F3 granulosa in vitro, basal levels of StAR mRNA in F2 and F3 follicle granulosa are markedly lower than those found within granulosa from the F1 follicle (7). This relationship suggests that gonadotropin-mediated StAR mRNA transcription in F2 and F3 follicles may be tonically suppressed in vivo, and that a putative inhibitory signal (via a paracrine and/or autocrine factor) is lost during the transition to the F1 stage of development. Of relevance is a previous report of a suppressive effect of the F2, but not F1, thecal layer on granulosa cell progesterone production in vitro (22). Accordingly, it is reasonable to speculate that factors produced by the thecal layer from hierarchal follicles other than the F1 follicle may be responsible for tonic suppression of StAR transcription, and thus the attenuation of steroidogenesis. The subsequent loss of or acquired insensitivity to such putative factors within F1 follicle granulosa could explain both the increase in StAR expression (7) and the heightened potential for progesterone production from the F1 follicle (20).
Similarly, it is of interest to speculate regarding mechanisms that
contribute to the relative absence of constitutively expressed StAR
mRNA in granulosa cells from prehierarchal follicles (7).
As noted above, such cells predominantly express FSH-R and are exposed
to fluctuating levels of serum FSH on a continual basis. We previously
reported that the increase in FSH-promoted P450scc mRNA levels and the
initiation of steroidogenesis in undifferentiated granulosa cells
in vitro are blocked by coculture with TGF
(10). Furthermore, inhibition of MAP kinase kinase
signaling (with U0126 or PD98059) enhanced gonadotropin-induced StAR
and LH-R mRNA levels, StAR protein levels, and progesterone synthesis
in cultured granulosa cells from prehierarchal follicles. The
comparatively lower net production of progesterone in response to LH
plus U0126 (compared with FSH plus U0126) probably reflects the delay
in expression of the LH-R after the initiation of culture.
Results presented in Fig. 7 further demonstrate that the inhibition of
FSH-induced initiation of steroidogenesis by TGF is associated with
the complete inhibition of StAR expression. TGF treatment has
previously been reported to stimulate DNA synthesis and promote cell
proliferation in cultured granulosa cells (23, 24). Taken
together, these results implicate MAP kinase signaling via TGF (and
possibly additional growth factors) as a mechanism to promote granulosa
cell proliferation in prehierarchal follicles while at the same time
preventing premature differentiation. Significantly, both
immunoreactive TGF and the TGF/EGF receptor have been localized
to cells within the granulosa and thecal layers in follicles throughout
development, yet levels of both are reported to decrease with
follicular maturation (25). It remains to be unequivocally
demonstrated whether the decline in the expression of either the ligand
or receptor is directly related to the maintenance of undifferentiated
prehierarchal follicles and subsequently follicle selection and/or the
progression of differentiation in vivo. Finally, it is noted
that the structurally related growth factor, EGF, has previously been
reported to dramatically decrease steady state expression of StAR in
the porcine corpus luteum (26).
It has been proposed that the nuclear transcription factor SF-1 is an essential regulator of StAR expression and that activation of SF-1-dependent transcription requires MAP kinase-dependent phosphorylation (27). Nevertheless, the results presented herein clearly demonstrate that inhibition of MAP kinase signaling potentiates gonadotropin-induced StAR expression and steroidogenesis in granulosa cells from prehierarchal follicles. This finding argues against a role for phosphorylated SF-1 in mediating gonadotropin-induced StAR expression in hen granulosa cells. Although this is consistent with results derived from site-directed mutagenesis studies in mice, which demonstrate that activation of protein kinase A signaling (using 8-br-cAMP) can fully potentiate StAR transcription after deletion of one or both SF-1 promoter elements (19), the requirement for SF-1 appears to be species specific (5).
Inhibition of MAP kinase signaling with U0126 or PD98059 alone was found to increase LH-R (but not StAR) mRNA levels in granulosa cells from prehierarchal follicles, and the combination of FSH or LH plus MAP kinase kinase inhibitor further enhanced this increase in LH-R transcript. Although the activation of MAP kinase signaling via gonadotropins has previously been described (28), the present results are among the first to implicate MAP kinase signaling in the regulation of a gonadotropin receptor. Moreover, this result further supports a role for MAP kinase signaling in the regulation of granulosa cell differentiation.
The absence of an additive effect of IGF-I on FSH-induced StAR and LH-R mRNA after a 20-h culture is consistent with previous findings regarding a lack of synergism by the combination of these same factors on P450scc enzyme mRNA levels or steroid production in granulosa cells from prehierarchal follicles (10). This inactivity cannot be attributed to an absence of bioactivity, as recombinant human IGF-I has been found here and previously to rapidly activate Akt/protein kinase B signaling in this model system (12). By contrast, IGF-I has been demonstrated to promote a direct effect on StAR expression and a synergistic effect on FSH-induced StAR expression in porcine granulosa cells after 24–48 h of culture (29). Although cultures in the present studies were not maintained beyond a duration of 20 h, it is possible that longer cultures in the presence of IGF-I would facilitate gonadotropin-induced StAR expression. If so, the mechanisms mediating this long-term effect are proposed to be more complex than those mediated directly by FSH or LH (within 1–6 h) or those promoted by MAP kinase inhibition (<20 h).
In summary, gonadotropin-induced expression of StAR is linked to the earliest stages of granulosa cell differentiation and the initiation of steroidogenesis in prehierarchal follicles as well as the potentiation of progesterone production in preovulatory follicles. Moreover, results suggest that cell signaling via the MAP kinase pathway may act to tonically suppress gonadotropin-mediated StAR and LH-R expression in undifferentiated granulosa cells; thus, the removal of such inhibition may be critical for follicle recruitment into the preovulatory hierarchy as well as for the progression of granulosa cell differentiation. On the other hand, LH- induced StAR expression in preovulatory follicle granulosa cells, although not potentiated by inhibition of MAP kinase signaling, is probably required for full potentiation of progesterone synthesis such as occurs in response to the preovulatory LH surge in vivo. The dramatic increase in StAR protein in response to LH treatment is predicted to result from the initiation of translation from preexisting mRNA and is supported by the induction of new transcription.
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Acknowledgments |
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Footnotes |
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Received November 21, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and LH on proliferation of chicken granulosa cells and
production of progesterone in culture. J Reprod Fertil 104:259–265[Abstract]
-like peptides in chicken ovarian
follicles. J Reprod Fertil 102:147–153[Abstract]
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R. Rusovici, Y. Y. Hui, and H. A. LaVoie Epidermal Growth Factor-Mediated Inhibition of Follicle-Stimulating Hormone-Stimulated StAR Gene Expression in Porcine Granulosa Cells Is Associated with Reduced Histone H3 Acetylation Biol Reprod, April 1, 2005; 72(4): 862 - 871. [Abstract] [Full Text] [PDF]
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D. C. Woods and A.L. Johnson Regulation of Follicle-Stimulating Hormone-Receptor Messenger RNA in Hen Granulosa Cells Relative to Follicle Selection Biol Reprod, March 1, 2005; 72(3): 643 - 650. [Abstract] [Full Text] [PDF]
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A.L. Johnson, J.T. Bridgham, and D.C. Woods Cellular Mechanisms and Modulation of Activin A- and Transforming Growth Factor {beta}-Mediated Differentiation in Cultured Hen Granulosa Cells Biol Reprod, December 1, 2004; 71(6): 1844 - 1851. [Abstract] [Full Text] [PDF]
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J.T. Bridgham and A.L. Johnson Alternatively Spliced Variants of Gallus gallus TNFRSF23 Are Expressed in the Ovary and Differentially Regulated by Cell Signaling Pathways Biol Reprod, April 1, 2004; 70(4): 972 - 979. [Abstract] [Full Text] [PDF]
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B. Schmierer, M. K. Schuster, A. Shkumatava, and K. Kuchler Activin and Follicle-Stimulating Hormone Signaling Are Required for Long-Term Culture of Functionally Differentiated Primary Granulosa Cells from the Chicken Ovary Biol Reprod, February 1, 2003; 68(2): 620 - 627. [Abstract] [Full Text] [PDF]
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A.L. Johnson, E.V. Solovieva, and J.T. Bridgham Relationship Between Steroidogenic Acute Regulatory Protein Expression and Progesterone Production in Hen Granulosa Cells During Follicle Development Biol Reprod, October 1, 2002; 67(4): 1313 - 1320. [Abstract] [Full Text] [PDF]
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M. Kusakabe, T. Todo, H. J. McQuillan, F. W. Goetz, and G. Young Characterization and Expression of Steroidogenic Acute Regulatory Protein and MLN64 cDNAs in Trout Endocrinology, June 1, 2002; 143(6): 2062 - 2070. [Abstract] [Full Text] [PDF]
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