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-Subunit Levels in Granulosa and Thecal Layers of Developing Preovulatory Follicles in the Chicken1
School of Animal and Microbial Sciences, University of Reading (T.M.L., R.T.G., F.J.C., P.G.K.), Whiteknights, Reading, United Kingdom RG6 6AJ; and the School of Biological and Molecular Sciences, Oxford Brookes University (N.P.G.), Oxford, United Kingdom OX3 OBP
Address all correspondence and requests for reprints to: Dr. P. G. Knight, School of Animal and Microbial Sciences, University of Reading, Whiteknights, Reading, United Kingdom RG6 6AJ.
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Abstract |
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and ßA genes are also
expressed in the avian ovary. To investigate the potential involvement
of these proteins in the chicken ovary, thecal and granulosa layers of
the four largest follicles (F1–F4) and the most recent
post-ovulatory follicle were collected from hens (10/group) killed
4, 12, and 20 h before the expected time of F1 ovulation. Inhibin
A and activin A concentrations of tissue extracts (expressed per mg
DNA) were measured using validated two-site enzyme-linked immunosorbent
assays; total immunoreactive inhibin -subunit (ir-) was also
measured by heterologous RIA (Monash assay). Inhibin A and ir- were
largely confined to the granulosa layer, whereas activin A was much
more abundant in the thecal layer. Granulosa inhibin A contents were
similar in F4 and F3, but increased approximately 40-fold from F3–F1
(P < 0.0001). As such, the F1 granulosa layer was
by far the richest source of inhibin A in the chicken ovary, but
contained very little activin A. Total ir- in granulosa was much
more abundant than inhibin A and increased only 3-fold from F4–F1
(P < 0.001). Activin A in both granulosa and theca
showed little variation between F1 and F4 follicles (by ANOVA,
P > 0.05). The inhibin A content of F1 granulosa
was maximal 12 h before ovulation and had fallen approximately
6-fold (P < 0.0001) within 8 h, suggesting an
inhibitory effect of the preovulatory LH surge on the F1 capacity to
synthesize inhibin A. Inhibin A, activin A, and ir- were all less in
the postovulatory follicle compared with F1 before ovulation
(P < 0.0001). In conclusion, application of the
present two-site enzyme-linked immunosorbent assays to the chicken
ovary revealed 1) divergent tissue distribution of inhibin A and
activin A within preovulatory follicles, and 2) differential regulation
of granulosa cell production of inhibin A and activin A dimers during
preovulatory follicular development. These findings of dynamic changes
in inhibin A, activin A, and total ir- support the hypothesis that
these proteins subserve regulatory roles during preovulatory follicular
development in the hen. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Inhibins and activins are dimeric glycoproteins first identified in
gonadal fluids of pigs and cows from their ability to suppress
(inhibin) or stimulate (activin) pituitary FSH secretion (4, 5). Both
molecules have also been implicated as local autocrine/paracrine
regulators of folliculogenesis in several mammalian species (6, 7, 8). Two
isoforms of inhibin (A and B) and three isoforms of activin (A, B, and
AB) are expressed in the ovaries of most mammalian species examined.
Inhibins A and B consist of a common -subunit linked through a
disulfide bond(s) to one of two alternate ß-subunits, termed ßA and
ßB, respectively. Activins A, B, and AB are disulfide-linked dimers
of two inhibin ß-subunits (ßAßA, ßBßB, and ßAßB dimers,
respectively) (4). Recently, the genes for inhibin -subunit (9) and
inhibin/activin ßA-subunit (10) have been cloned in the chicken, and
Northern blot hybridization has revealed differential expression of
- and ßA-subunit messenger RNAs (mRNAs) during preovulatory
follicle development (11).
In the hen, as in mammals, the ovary is the primary source of
immunoreactive (ir) inhibin (12). Experiments in laying hens involving
selective follicle removal have shown that preovulatory follicles are
the major source of ir-inhibin in the ovary (13, 14). Removal of such
follicles leads to a rise in plasma FSH levels, suggesting a possible
endocrine role of inhibin in FSH regulation in the chicken (13).
However, caution is necessary in interpreting published data on
ir-inhibin in both chicken and mammalian species, as it is now
generally accepted that the inhibin RIAs used in the majority of these
studies have limited specificity (15, 16, 17). This is due to the fact that
they cross-react extensively with free -subunit forms that are
synthesized in excess by granulosa cells and have been identified in
follicular fluid and peripheral blood (15, 16, 18). Quantitation of
inhibin/activin subunit mRNAs (11) is a valuable approach but does not
reveal the extent to which the respective gene products are assembled
into inhibin or activin dimers or remain as - and ß-subunit
monomers.
In the last few years, several groups have developed sensitive and
specific two-site immunometric assays for the measurement of different
isoforms of inhibin (19, 20, 21, 22, 23) and activin (24, 25). The inhibin A and
activin A enzyme-linked immunosorbent assays (ELISAs) developed in our
laboratories were designed for use in mammalian species, particularly
human. However, given the considerable homology between chicken and
mammal in the regions of the - and ßA-subunits targeted by the R1
(human C1–26) and E4 (human ßA82–114)
antibodies used in these assays, we considered it likely that they
would also be applicable to the chicken and thereby provide a means of
investigating the potential roles of inhibin A and activin A during
follicular maturation in this species.
In this study we report the validation and application of these
two-site ELISAs for the measurement of inhibin A and activin A in
chicken ovary. To investigate the potential involvement of these
proteins in preovulatory follicular development and to discover their
regional distribution within the ovary, thecal and granulosa layers of
the four largest follicles (F1–F4) and the most recent postovulatory
follicle (POF) were collected at known times during the ovulatory cycle
and assayed for inhibin A and activin A. To facilitate comparison with
previous published data on ir-inhibin in the chicken ovary (12, 13, 14, 26, 27, 28, 29), samples were also assayed using the conventional -subunit
directed inhibin RIA (26) used in these earlier studies.
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Materials and Methods |
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Experimental design
Hens (n = 10/group) were killed by cervical dislocation 4,
12, or 20 h before the expected time of ovulation of a midsequence
egg. The four largest preovulatory follicles (F1–F4) of the ovarian
hierarchy were removed, and thecal and granulosa layers were separated
(30), washed in saline [0.75% (wt/vol) NaCl], and frozen at -20 C.
The most recent POF was also removed and frozen at -20 C. Granulosa
tissue samples were homogenized on ice in 0.5 ml buffer A [PBS
containing 1% (wt/vol) BSA and 0.1% (wt/vol) sodium azide] using an
Ultra-Turrax T8 homogenizer (IKA, Staufen, Germany). Homogenates were
centrifuged at 3500 x g for 15 min, and the
supernatant was stored at -20 C until further analysis. Thecal and POF
tissue samples were homogenized similarly in 2.0 ml buffer A. A 20-µl
aliquot of each granulosa and thecal homogenate was removed before
centrifugation for DNA estimation using the fluorometric assay
described by Labarca and Paigen (31). Extract supernatants were
subsequently assayed for inhibin A, activin A, and total -subunit,
and results were expressed on a per mg DNA basis.
Tissue samples for assay validation
Samples of granulosa and thecal tissue from F1–F4 follicles
were obtained at random times during the ovulatory cycle and prepared
as described above. Pooled tissue extracts were prepared and used for
assay validation tests. These included parallelism tests and recovery
experiments in which known amounts of inhibin A and activin A were
added to aliquots of homogenized samples before the centrifugation
step. In addition, on several occasions tissue pellets remaining after
centrifugation of nonspiked tissue homogenates were resuspended in
fresh extraction buffer and subjected to a secondary extraction. The
amounts of inhibin A and activin A detected in these secondary extracts
represented only 3–8% of the corresponding amounts detected in
primary extracts, indicating that the routine extraction procedure used
was relatively efficient. For assay, extracts were diluted with sample
buffer A as follows: inhibin A assay: 1:2 to 1:128 for granulosa,
undiluted to 1:4 for theca; and activin A assay: undiluted to 1:16 for
theca and undiluted to 1:2 for granulosa.
Immunoassays
Inhibin A and activin A were determined using recently developed
two-site ELISAs that employ monoclonal antibodies raised against
synthetic peptide fragments of the human - and ßA-subunits (21, 24). Both assays were validated for use in the domestic fowl as
described in Results. Total -subunit levels were measured
using a heterologous RIA employing a rabbit polyclonal antiserum
against purified bovine inhibin (32) that has been validated previously
for use in the domestic fowl (26). Recombinant human activin A and
32-kDa bovine inhibin A were used as assay standards. Activin A was
generously provided by Genentech (San Francisco, CA), and 32-kDa bovine
inhibin was isolated from bovine follicular fluid in this laboratory as
reported previously (33). The detection limit of the activin A ELISA
was 10 pg/well. The same 32-kDa bovine inhibin preparation was used as
the standard in the inhibin A ELISA and total RIA, and respective
detection limits were 3 pg/well and 100 pg/tube.
Fast protein liquid chromatography (FPLC) gel permeation
chromatography
Granulosa layers from the F1 follicle were obtained at random
times during the hen ovulatory cycle, and a tissue extract was prepared
as described above (n = 20 pooled granulosa layers). A sample (100
µl) was applied to a FPLC column (Superose 12, Pharmacia, Milton
Keynes, UK) that was equilibrated and eluted with PBS (pH 7.4)
containing 0.1% (wt/vol) Polypep (Sigma Chemical Co., Poole, Dorset,
UK) and 0.05% (wt/vol) sodium azide at a flow rate of 0.5 ml/min.
Eluant was collected in 0.5-ml fractions for inhibin A ELISA, and total
RIA. To calibrate the column the retention times of the following
marker proteins were determined: 2-macroglobulin (void
volume), 750 kDa; apoferritin, 450 kDa; alcohol dehydrogenase, 150 kDa;
BSA, 66 kDa; and recombinant human activin, 25 kDa.
Statistical analysis
Confirmation of parallelism between assay standard and test
sample dilution curves was made using linear regression analysis of
transformed data. The log-log transformation was used to linearize the
ELISA dose-response curves. Comparison of the slopes (±95% confidence
intervals) of the regression lines for standards and test samples
indicated no significant departure from parallelism. This analysis was
not possible in the case of activin A levels in the granulosa because
only the first two dilutions of each gave responses above the detection
limit. Linear regression analysis was used to evaluate the recovery
experiment data. One-way ANOVA of log-transformed data was used in
conjunction with post-hoc Fisher’s protected least
significant difference (PLSD) test to determine whether concentrations
of inhibin A, activin A and total subunit differed between
different follicle groups. Post-hoc tests were only
performed when the ANOVA yielded a significant F ratio.
P < 0.05 was considered to be significant. Unless
stated otherwise, values are the mean ± SEM
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, a and b). The recovery
(y) of known amounts of exogenous inhibin A standard
(x) added to representative tissue homogenates before
centrifugation was linear and was described by the following regression
equations: y = 0.93x - 0.15 (r =
0.999; n = 6) for granulosa homogenates and y =
0.93x + 1.26 (r = 0.987; n = 6) for thecal
homogenates. Similarly, the recovery (x) of known amounts of
activin A standard (y) was linear and was described by the
following regression equations: y = 1.02x +
1.07 (r = 0.999; n = 6) for granulosa homogenates and
y = 0.94x + 3.65 (r = 0.994; n =
6) for thecal homogenates. Additional recovery tests were performed in
which known amounts of inhibin A and activin A standard were spiked
into randomly selected extracts after centrifugation. The mean recovery
of inhibin A was 98.3 ± 1.7% for granulosa (n = 8) and
100.7 ± 1.9% for theca (n = 8). Activin A recovery values
were 100.3 ± 2.0 for granulosa (n = 8) and 101.8 ±
2.0% for theca (n = 8). The cross-reactivities of a range of
related substances in the inhibin A and activin A ELISA have been
reported previously (21, 24) and were shown to be acceptably low. In
particular, bovine free -subunit showed minimal (<0.3%)
cross-reaction in the inhibin A ELISA. Mean intra- and interassay
coefficients of variation for the inhibin A ELISA determined using
pooled laying hen plasma were 5.0% and 7.0%, respectively. Intra- and
interassay coefficients of variation for the activin A ELISA determined
using a pooled bovine follicular fluid were 3.9% and 6.9%,
respectively.
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shows the distribution of
inhibin A and total -subunit in pooled chicken granulosa tissue
extract fractionated by FPLC gel permeation chromatography under
nondissociating conditions. The FPLC profile for inhibin A differed
markedly from that of total ir-, confirming that chicken free
forms detected by the RIA do not cross-react in the inhibin A ELISA.
Inhibin A eluted as a single major peak with an apparent molecular mass
of approximately 100 kDa, in contrast to total -subunit, where two
major peaks of immunoreactivity were found in fractions corresponding
to molecular masses of about 30 and 60 kDa. Activin A levels in
granulosa extract were too low to permit chromatographic analysis.
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-subunit, and activin A in F1–F4 follicles-subunit, and activin A in the granulosa and thecal layers of F1–F4
follicles recovered at different times during the ovulatory cycle are
shown in Figs. 3 and 4. Concentrations of inhibin A and total
-subunit were substantially higher in granulosa than in thecal
tissue at all times during the cycle and at all positions within the
follicular hierarchy. In contrast, concentrations of activin A were
much higher in thecal than in granulosa tissue in each follicle
analyzed. Total -subunit in granulosa tissue was present in marked
excess over inhibin A, being approximately 570-fold higher in F4
granulosa, and falling to about 30-fold in F1 granulosa. Total
-subunit in thecal tissue was present in an approximately 15-fold
excess over inhibin A.
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). The mean concentration
increased only about 2-fold from F4–F2, but showed a further
approximately 20-fold increase from F2–F1. When the follicle was in
the F1 position, the level of inhibin A in the granulosa was maximal
12 h before the expected time of ovulation (43.2 ng/mg DNA),
declining significantly over the following 8-h period (7.3 ng/mg DNA).
Inhibin A levels in the thecal layer increased about 3-fold from F4–F1
(71.4–195.9 pg/mg DNA), although values were much lower than those in
the granulosa layer in all cases (Fig. 4).
Mean concentrations of total ir- in granulosa layers increased about
3-fold from F4–F1 (Fig. 3). In each case maximal levels were observed
in hens killed 12 h before the expected time of ovulation, peaking
in the F1 follicle (1070 ng/mg DNA). In contrast to the approximately
6-fold decline in inhibin A, total ir- level in the F1 granulosa did
not fall significantly between -12 and -4 h relative to the expected
time of ovulation. Total ir- levels in the theca were at least
100-fold lower than those in the corresponding granulosa layer. In each
case (F1–F4) levels were maximal 4 h after the expected time of
ovulation.
Mean concentrations of activin A in granulosa and thecal layers are
presented in Figs. 3c and 4c, respectively. Overall, activin A
concentrations were about 35-fold higher in theca (
780 pg/mg DNA)
than in granulosa (22 pg/mg DNA) tissue. There were no significant
differences between F1–F4 follicles in the activin A content of either
thecal or granulosa layers.
F1 follicle/POF transition
After ovulation of the F1 follicle, the remaining tissue becomes
the POF, and the F2 moves up the hierarchy to become the new F1
follicle. Figure 5 shows data for this
transition period plotted relative to the predicted time of ovulation.
The most recent POF contained significantly lower inhibin A, activin A,
and total ir- levels compared with the preovulatory F1 follicle.
There were no significant changes in inhibin A, activin A, and total
ir- levels in the POF at 4, 12, and 20 h after the expected
time of F1 follicle ovulation.
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, and activin A in
individual follicles (r = 0.45; P < 0.0001) and
activin A (r = 0.26; P < 0.01) in the granulosa
layer. However, there was no significant relationship between activin A
and total ir- in granulosa tissue (r = 0.17; P
= 0.07). In the thecal layer, inhibin A was significantly correlated
with total ir- (r = 0.33; P < 0.001), but not
with activin A (r = 0.09; P = 0.32). Likewise,
thecal activin A was not correlated with thecal total ir- (r =
0.06; P = 0.55). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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was present in a large (15- to 900-fold) excess
over inhibin A; and 5) inhibin A, activin A, and total ir- levels in
the POF were appreciably lower than those in the F1 follicle.
To facilitate comparison of our inhibin A ELISA data with results from
previous studies of ir-inhibin in the chicken ovary (12, 13, 14), we also
assayed each sample using the same heterologous RIA (Monash assay) as
that used by these investigators. In common with most other inhibin
RIAs, the Monash assay cross-reacts extensively with free -subunit
forms (17, 33) and is no longer considered satisfactory for
quantitating dimeric inhibins because -subunit is synthesized in
excess by mammalian granulosa cells but lacks inhibin-like
biological activity (15, 16, 18). Our results indicate that total
ir- is indeed much more abundant than inhibin A in both granulosa
(570-fold in F4 falling to 30-fold in F1) and thecal (15-fold
higher) layers of chicken follicles. These findings accord well with
the recent data of Chen and Johnson (11), who estimated that the ratio
of - to ßA-subunit mRNA expressed in chicken granulosa layers
ranged from 70:1 in F5 follicles to 7:1 in F1 follicles. Recent
preliminary studies (our unpublished data) have shown that total ir-
is more abundant than inhibin A in chicken granulosa cell-conditioned
culture medium (F1, 21-fold; F2, 37-fold) and in peripheral hen
plasma (5-fold), consistent with these observations and confirming
that both free -subunit and inhibin A dimer are secreted by the
chicken ovarian follicle. Moreover, granulosa cells from F1 follicles
secreted 2-fold more inhibin A than cells from F2 follicles during a
24-h incubation period in preliminary culture experiments. Analysis of
data for all 120 follicles examined in the present study showed only a
moderate (although statistically significant) degree of correlation
between inhibin A measured by ELISA and total ir- levels measured by
RIA in granulosa (r = 0.45) and thecal layers (r = 0.33).
Taken together with our chromatographic evidence that inhibin A
immunoreactivity and total ir- in F1 granulosa extract are distinct
molecular entities, these observations firmly indicate that, as in
mammals, the ovary of the chicken produces a marked excess of free
-subunit. These results also highlight the need to reappraise
earlier studies of ir-inhibin secretion in vivo (13, 14, 26, 28, 29) and in vitro (12, 27), as the Monash assay used
would grossly overestimate the amount of inhibin dimer present and
would be relatively insensitive to changes in (much lower) levels of
"authentic" inhibin dimer. The finding of two major chromatographic
peaks of total ir- in granulosa extracts is consistent with
differential posttranslational processing of the full-length
-subunit precursor molecule, as is known to occur in mammalian
granulosa cells (4, 5, 8). However, the apparent molecular masses of
these two major peaks (60 and 30 kDa) are substantially greater
than the predicted values for the chicken inhibin precursor (31
kDa) and mature (C) polypeptide (13 kDa) reported by Wang and
Johnson (9), and posttranslational glycosylation would not fully
account for this size discrepancy. The apparent molecular mass of the
single peak of inhibin A identified (100 kDa) is similar to a major
peak observed previously in human follicular fluid and serum (21) and
could either represent unprocessed -ßA dimer or mature, fully
processed -ßA dimer (30 kDa) associated with binding protein(s)
(e.g. follistatin). Further detailed fractionation studies
will be required to establish the molecular identity of these
immunoreactive inhibin forms in the chicken. No correlation was found
between activin A and total ir- levels in either the thecal (r
= 0.06; P = 0.55) or granulosa (r = 0.17;
P = 0.07) layers of preovulatory follicles, suggesting
independent regulation of - and ßA-subunit production. In support
of this, it was recently reported that expression of mRNA for inhibin
-subunit in chicken granulosa decreases during follicular
development, whereas that of inhibin/activin ßA-subunit mRNA
increases dramatically (11).
One of our most striking findings was that the F1 follicle displayed a
greatly enhanced capacity to produce dimeric inhibin A over other
members of the hierarchy in the absence of any appreciable change in
activin A. The concentration of inhibin A in the granulosa layer
increased slightly (2-fold) from F4–F2, but showed an additional
approximately 20-fold increase from F2–F1. This observation
contradicts an earlier report (12) that the concentration of ir-inhibin
in granulosa layers decreases 2- to 3-fold from F4–F1, but is
consistent with other evidence (34, 35) that the F1 follicle secretes
more bioactive inhibin than smaller follicles. This discrepancy can
largely be accounted for by cross-reactivity of the Monash RIA used by
Vanmontfort et al. (12) with free inhibin -subunit, as
discussed above. However, the fact that we observed a modest (2- to
3-fold) increase from F4–F1 in total ir- measured using the same
RIA is more difficult to account for. One possible explanation could be
that Vanmontfort et al. (12) expressed their granulosa layer
inhibin concentrations on a per mg protein basis, whereas we expressed
our data on a per mg DNA basis because we were concerned about possible
contamination of granulosa tissue with residual yolk that could lead to
overestimation of tissue protein.
Our finding that the F1 granulosa layer is by far the richest source of
inhibin A in the chicken ovary is in excellent accordance with the
recent report of Chen and Johnson (11), who observed a dramatic
enhancement of ßA mRNA expression in the F1 granulosa and predicted
that this tissue would prove to be the primary source of inhibin A and
activin A dimers in the hen. Surprisingly, however, the activin A
concentration in the F1 granulosa layer remains low (900-fold lower
than inhibin A) despite this markedly increased expression of ßA
mRNA. Given that information is currently lacking on the intracellular
regulatory signals that allow differential posttranslational processing
of inhibin/activin subunits leading to assembly of their respective
dimers, the F2–F1 granulosa cell transition in the hen may prove to be
a valuable in vivo model for cell biologists addressing this
issue.
After ovulation, the remaining F1 tissue (comprising both thecal and
granulosa layers) is referred to as the POF. The early POF appears to
be endocrinologically active, producing steroids (36) and high levels
of PGF (37) and a relaxin-like peptide (38). The POF has been
implicated in the control of oviposition, as its removal delays
oviposition of the egg derived from it, whereas administration of POF
extract can promote premature oviposition (39). Inhibin A and total
ir- levels in the POF fell dramatically within 4 h of ovulation
of the F1 follicle, indicating an abrupt cessation of biosynthesis,
which contrasts with the greatly increased production of PGF (37) and
relaxin-like peptide (38) in the early POF. Activin A only fell by
about 50%, raising the possibility that the POF continues to express
ßA-subunit to permit de novo activin A synthesis.
Alternatively, this may simply reflect a longer half-life of activin A
compared with that of inhibin A or total ir-. Further studies are
required to determine whether the maintained activin A content of the
POF has any physiological importance.
Hens were killed at three different times during the 24- to 26-h
ovulatory cycle to enable a comparison of successive follicles of the
hierarchy at an early, mid, and late stage of their development. For
each position in the follicular hierarchy (i.e. F1–F4),
total ir- levels in granulosa increased from the early to midstage,
whereas total ir- levels in theca showed the reverse trend. Whether
this divergent pattern has any physiological relevance remains to be
investigated, but it should be emphasized that total ir- levels in
the thecal layer were always substantially (60- to 300-fold) lower than
total ir- levels in the corresponding granulosa layer. These fairly
rapid (<8 h) fluctuations in follicular total ir- levels presumably
reflect changes in -subunit mRNA expression in response to cyclic
changes in systemic and/or intraovarian regulatory factors. Regulatory
factors known to modulate inhibin -subunit mRNA expression in the
mammalian gonad include FSH, LH, activin, transforming growth
factor-ß, epidermal growth factor, insulin-like growth factor I,
estrogen, and androgen (40), and it is likely that at least some of
these also inhibin -subunit expression in the chicken ovary. Inhibin
A levels in the F1 granulosa were maximal 12 h before the expected
time of ovulation, showing an abrupt 6-fold decline over the following
8-h period. In contrast, total ir- levels in the F1 granulosa were
maintained over the same 8-h period. As the preovulatory surge of LH
occurs about 6 h before ovulation in the hen (41), we hypothesized
that the LH surge promotes an abrupt cessation of inhibin A synthesis
without a corresponding reduction in total ir- synthesis by
selectively blocking the expression of ßA-subunit. Direct support for
this hypothesis was recently provided by Chen and Johnson (42), who
reported that LH negatively regulates the expression of ßA-subunit
mRNA, but not that of -subunit in the F1 granulosa of the hen.
The present study revealed that activin A predominates in the thecal
layer of the preovulatory follicles of the hen, consistent with the
recently reported detection of ßA mRNA expression in this compartment
(10). However, despite the exposure of follicles to preovulatory surges
of LH at approximately 24-h intervals, concentrations showed little
variation (both within and between different follicle positions) in
either the thecal or granulosa layer. In the granulosa, activin A was
present at much lower levels than inhibin A, and such a low level of
synthesis may be less affected than inhibin A by reduction in
ßA-subunit expression. Such observations provide further evidence for
differential regulation of inhibin/activin dimer assembly from - and
ßA-subunits.
In mammals, inhibins and activins have been shown to modulate LH-induced androgen production by thecal cells (43, 44). Activin has also be shown to up-regulate FSH receptor expression (45), enhance FSH-induced aromatase activity/estrogen production (46, 47), and promote functional differentiation of granulosa cells (48). Receptors for activin have been identified on mammalian granulosa cells, thecal cells, and oocytes (49). It remains to be established whether inhibin and activin have comparable effects in the ovary of the hen, although Rombauts et al. (27) recently reported that inhibin and activin have antagonistic effects on androgen production by primary cultures of ovarian and testicular cells derived from chicken embryos. It should be noted that the steroidogenic activity of chicken ovarian follicles differs markedly from that of their mammalian counterparts, and that progesterone, rather than estradiol, is the principal follicular steroid involved in the generation of the preovulatory LH surge (1, 2). The granulosa layer of large yolk-filled follicles contains much more progesterone and much less androgen and estrogen than the thecal layer (50). The capacity of granulosa cells to produce progesterone increases as the follicle progresses through the hierarchy, and the F1 granulosa layer is considered to be the main source of the circulating progesterone that triggers the LH surge. The dramatic increase in inhibin A production by F1 granulosa cells may be an important factor in the acquisition of a fully mature and ovulable status. The capacity of thecal cells to convert granulosa-derived progesterone into androgen is lost when the follicle assumes the F1 position (2, 49), and it is conceivable that this could reflect an inhibitory paracrine action of granulosa-derived inhibin A. Clearly, further investigations are required to evaluate these possibilities.
It has been shown that follicular FSH receptor-binding activity (51) and mRNA levels (52) decrease as the follicle progresses through the hierarchy to the F1 stage. Although there is no indication from the present findings that activin A has a regulatory role in large (F4–F1) preovulatory follicles, the possibility should be explored that activin A may play a role in the maintenance of healthy follicles at an earlier stage of development, as chicken FSH receptor mRNA is lower in postselection atretic follicles in comparison with normal 3- to 5-mm prehierachical follicles (52).
In conclusion, the present study has shown that differential changes in
inhibin A, total ir-, and activin A production occur during
preovulatory follicular development in the hen, with the F2–F1
transition being associated with a selective 20-fold increase in
granulosa content of inhibin A. These observations support the view
that these proteins subserve functional roles in the chicken ovary, but
further in vivo and in vitro experiments are now
needed to identify these putative roles.
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Acknowledgments |
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Footnotes |
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Received July 31, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit
of inhibin from chicken ovarian granulosa cells. Biol Reprod 49:453–458[Abstract]
- and ßA-subunits in the domestic
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) and serial dilutions of pooled
chicken granulosa (
and •) and thecal (
and 
) extracts.
Values are the means of duplicate determinations.
) and granulosa (