Download a time course study

Molecular Human Reproduction Vol.7 No.2 pp. 129–135, 2001
Effect of different FSH isoforms on cyclic-AMP production
by mouse cumulus–oocyte–complexes: a time course study
C.Yding Andersen1,3, L.Leonardsen1, A.Ulloa-Aguirre2, J.Barrios-De-Tomasi2,
K.S.Kristensen1 and A.G.Byskov1
1Laboratory
of Reproductive Biology, University Hospital of Copenhagen, Copenhagen, Denmark, and 2Research Unit in
Reproductive Medicine, Hospital de Ginecobstetricia ‘Luis Castelazo Ayala’, Instituto Mexicano del Seguro Social, Mexico DF,
Mexico
3To
whom correspondence should be addressed at: Laboratory of Reproductive Biology, Section 5712, Juliane Marie Center for
Children, Women and Reproduction, University Hospital of Copenhagen, Blegdamsvej 9, DK-2100 Copenhagen, Denmark. E-mail:
[email protected]
The ability of different isoforms of follicle stimulating hormone (FSH) to induce accumulation of cAMP in cultured
mouse cumulus–oocyte–complexes (COC) was evaluated in a time course study. Using isoform fractions representing
less acidic (pI 6.43–5.69), mid-acidic (pI 5.62–4.96) and acidic (pI 4.69–3.75) isoforms, the accumulation of cAMP
was monitored after an exposure time of 0, 5, 10, 15, 30, 60, 120 and 180 min. In addition, cAMP production was
monitored for 0, 5, 10, 15 and 30 min following a 5 min exposure to FSH isoform fractions. Based on FSH
measurements using radioimmunoassays, the less and mid-acidic isoforms caused almost twice as much cAMP to
be accumulated than the acidic isoform fraction, thereby confirming an enhanced biological activity of FSH isoforms
with a isoelectric point (pI) of >5.0. For all isoform fractions, maximal accumulation of cAMP was achieved after
30 min of exposure, after which the production declined to background levels. After a 5 min exposure to isoform
fractions, levels of cAMP were significantly higher in the less acidic isoform fractions, but after isoform removal,
the decline in cAMP production to background levels followed a similar time course. The results demonstrate that
FSH isoforms with a pI of >5.0 induced significant biological responses within a period of 30 min and that prolonged
exposure caused attenuated signal transduction. The present results, set in the context of the pulsatile characteristics
of FSH release from the pituitary and the reported half-life of less acidic isoforms of ~35 min, make it conceivable
that isoforms with a pI ⬎5.0 actually possess important physiological functions during the periovulatory period.
Key words: cAMP/cumulus–oocyte–complexes/FSH/isoforms
Introduction
The pituitary hormone, FSH, is a heterodimer composed of an
α- and a β-subunit involved in a number of gonadal functions.
Each of the two subunits exhibit two glycosylation sites
allowing N-linked oligosaccharides to be attached to the FSH
peptide back-bone at specific sites on the molecule. The
oligosaccharide structures present on FSH are highly variable
showing dibranched structures and even tri- or tetrabranched
structures (for review, see Ulloa-Aguirre et al., 1995, 1999).
Each branch of the oligosaccharides often terminates in a
negatively charged sialic acid residue and to a lesser extent in
a negatively charged sulphate residue (Dahl and Stone, 1992).
Separation of human FSH derived from the pituitary, serum
and urine by charge-based procedures has shown that this
gonadotrophin is composed of several isoforms that migrate
within a broad spectrum of isoelectric pHs (i.e. isoelectric
point pIs) between 4 and 8 (Ulloa-Aguirre et al., 1988, 1992;
© European Society of Human Reproduction and Embryology
Wide, 1985; Stanton et al., 1992). Thus, heterogeneity of the
charged oligosaccharide structures constitutes the main basis
for the existence of many different FSH isoforms.
The different isoforms all bind to and activate the FSHreceptor (FSH-R), which is exclusively located on the granulosa
cells. The FSH-R is a cell surface membrane-bound G-proteincoupled receptor, that uses cAMP as a second messenger for
signal transduction (Padmanabhan et al., 1991). The isoforms
differ in their biological activity, the more acidic isoforms
showing lower receptor binding activity and lower in-vitro
biological activity compared with the less acidic analogues
(de Leeuw et al., 1996; Yding Andersen et al., 1999; Zambrano
et al., 1999). It is still unclear whether the different biological
responses relate to an altered association/dissociation constant
and/or whether isoform specific activation of different biological responses occurs upon activation of the FSH-R (Timossi
et al., 2000).
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C.Yding Andersen et al.
The isoforms also differ in their metabolic clearance rate;
the acidic isoforms show a longer circulatory half-life compared
with that of less acidic isoforms. It appears that the concentration of circulating oestradiol is a major regulator of the isoform
mixture released by the pituitary. Different endocrinological
conditions with high concentrations of oestradiol have been
found to be highly correlated with the release of less acidic
isoforms, whereas conditions characterized by low concentrations of oestradiol induce the release of acidic isoforms
(Padmanabhan et al., 1988; Wide and Bakos, 1993; Zambrano
et al., 1995; for review, see Fauser, 1996).
Furthermore, it has recently been shown that oestradiol
down-regulates pituitary expression of α 2,3-sialyltransferase
mRNA, which encodes one of the enzymes responsible for
the incorporation of sialic acid residues into the FSH molecule,
thereby demonstrating one mechanism by which oestradiol
may induce the release of a greater amount of less acidic
isoforms (Damian-Matsumura et al., 1999). In accordance with
the above observations, the composition of FSH isoforms in
circulation varies throughout the follicular phase, being more
acidic in the early follicular phase, becoming less acidic as
oestradiol rises and ovulation approaches (Wide and Bakos,
1993; Anobile et al., 1998). The biological effect of the less
acidic FSH isoforms released in the periovulatory period is,
therefore, enhanced by a presumably enhanced biological
activity, but counteracted by a relatively quick clearance.
Studies in rats suggest that a bolus of human acidic FSH
isoforms have at least a 20-fold higher in-vivo activity than
that of less acidic isoforms (de Leeuw et al., 1996; Mulders
et al., 1997). However, in the light of the pulsatile release of
gonadotrophins from the pituitary (Padmanabhan et al., 1997),
it is difficult to assess the relative in-vivo potency of FSH
isoforms, and only few studies have been able to address
whether less acidic isoforms have a half-life of sufficient
length to allow for a significant biological response to occur. We
have recently shown that cumulus–oocyte–complexes (COC)
obtained from large preovulatory follicles of mice undergoing
ovarian stimulation may represent a physiological intact model
to study the effect of FSH isoforms (Yding Andersen et al.,
1999). Cumulus cells express FSH-R, and FSH in physiological
concentrations induces oocytes to resume meiosis through
activation of FSH-R located on the cumulus cells (Byskov
et al., 1997). In addition, FSH isoforms with a pI of ⬎5.0
show a significantly enhanced ability to induce resumption of
meiosis in cumulus-enclosed oocytes compared with more
acidic/sialyated isoforms (Yding Andersen et al., 1999).
The aim of the present study was to evaluate the time
required for different FSH isoforms to elicit a maximal response
in COC. COC were stimulated in vitro with individual FSH
isoform fractions of a concentration similar to that observed
during the mid-cycle surge of gonadotrophins, and the time
required to elicit a maximal accumulation of cAMP was
monitored and compared with that of the reported half-life of
the isoform fractions. The effect of prolonged FSH exposure
on the accumulation of cAMP was also determined. In addition,
experiments were performed to evaluate how fast the accumulation of cAMP declined after the COC were transferred from
a medium with a particular isoform to a medium without FSH.
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Materials and methods
Animals
Immature female mice (B6D2-F1 strain C57B1/2J) were kept under
controlled light and temperature conditions with free access to food
and water. Ovarian stimulation was performed when the mice weighed
11–16 g and consisted of an i.p. injection of Gonadoplex (Leo,
Copenhagen, Denmark) containing 7.5 IU per mouse (pregnant mare’s
serum gonadotrophin 5 IU and human serum gonadotrophin 2.5 IU)
or by 20 IU human menopausal gonadotrophin (Menogon, Ferring,
Denmark). The animals were killed by cervical dislocation 44–48 h
later. The experiments were performed according to the rules of the
Danish Authorities for Animal Care, Ministry of Justice.
Media
The medium used for the culture of oocytes consisted of α-minimum
essential medium (αMEM), with Earle’s balanced salt solution
(EBSS), 4 mmol/l hypoxanthine (HX), 3 mg/ml bovine serum albumin
(BSA), 0.23 mmol/l pyruvate, 2 mmol/l glutamine, 100 IU/ml
penicillin and 100 µg/ml streptomycin as described earlier (Byskov
et al., 1997). This medium served as control and wash medium
(i.e. HX medium). Test media consisted of control media supplemented
with FSH isoform fractions. HX served as a an inhibitor to phosphodiesterases, thereby preventing a spontaneous meiotic resumption by
avoiding a drop in levels of cAMP within the oocyte.
Isolation of oocytes
The ovaries were recovered and placed in HX medium where an
initial cleaning and removal of connective tissue was carried out.
Oocytes were isolated from the ovaries by puncturing individual
follicles using 25 gauge needles. Isolation of oocytes was carried out
in HX medium to prevent spontaneous resumption of meiosis. The
oocytes were separated into two pools. One pool contained naked
oocytes, oocytes only partly covered with cumulus cells or COC
containing a thin or irregular ring of cumulus cells. The other pool
contained COC of equal size with a prominent uniform layer of
cumulus cells around the oocyte. Only the latter pool was used for
measurement of cAMP accumulation upon stimulation with FSH
isoforms. The COC were washed twice in control medium before
being subjected to media containing FSH isoforms. COC were
cultured in 4-well dishes (Nunclon, Roskilde, Denmark), with each
well containing 0.4 ml HX medium or medium supplemented with
FSH isoform fractions in a 100% humidified atmosphere of 5% CO2
with 95% air at 37°C.
Time-dependent production of cAMP in COC
At the beginning of each experiment (i.e. time ⫽ 0 min), a sufficient
number of COC (usually ~150) was placed in the medium containing
10 IU/l FSH (based on radioimmunoassay measurements; see below).
After incubation COC were removed from the medium at the following
time points: 5, 10, 15, 30, 60, 120 and 180 min. At each time point,
~20 COC were removed from the FSH containing medium and
washed in HX medium before being distributed directly to the tubes
in which the cAMP measurements took place. Each tube contained
100 µl cAMP assay buffer (see below). The specific number of COC
transferred to each tube was recorded. Each tube received 5–10 COC
in order to ensure measurable amounts of cAMP. Thus, at each time
point and for each specific experiment, a total of 2–4 individual
measurements of cAMP was performed. Immediately after each
experiment all tubes were stored at –20°C until measurement of
cAMP. The measurements of cAMP therefore included the total
amount of cAMP present in the COC.
FSH isoforms and cAMP production by COC
Figure 1. Outline of how the experiments on mouse cumulus
enclosed oocytes with or without a continued presence of FSH
isoform were performed. For each time point and for control and
FSH-isoform medium, cumulus enclosed oocytes were removed for
measurement of cAMP.
cAMP production by COC with or without the continuous presence
of FSH isoforms
Each experiment was initiated (i.e. time ⫽ 0 min) by the transfer of
~140 COC to a medium containing FSH isoforms. As illustrated in
Figure 1, all COC were exposed to a particular FSH isoform during
the first 5 min of incubation. After 5 min of incubation, 20 COC
were removed for measurement of cAMP and ~60 COC were removed
to a HX medium without FSH and the culture was continued after
one wash in HX medium, whereas the remaining COC continued to
be exposed to FSH. After a total incubation time of 10, 15 or 30 min
respectively, 20 COC were removed for measurement of cAMP from
the pool of COC being exposed to a continuous presence of FSH
and from the pool of COC transferred to a HX medium without FSH.
At the beginning of each experiment, prior to FSH exposure, 30–50
COC (10 COC in each tube) were taken to obtain a baseline level of
cAMP per COC. Collection of COC in tubes for cAMP measurement
was done as described for the above experiment.
Measurement of cAMP production by COC
Concentrations of cAMP in the culture medium were monitored by
radioimmunoassay. COC were transferred to 100 µl 0.05 mol/l sodium
acetate buffer pH 5.8 (i.e. assay buffer) and stored at –20°C. Tubes
containing COC were frozen and thawed three times in total to break
the cells and allow release of cAMP prior to the cAMP assay.
Standards spanning 2–128 fmol per tube were prepared, and sample
acetylation was performed by the addition of 5 µl of a 1:2 (v/v)
mixture of acetic anhydride and triethylamine. Adenosine 3⬘,5⬘-cyclic
phosphoric acid, 2⬘-O-succinyl [125I]-iodotyrosine methyl ester (NEX130; NEN Life Science Products Inc, Boston, MA, USA) was used
as tracer and a specific rabbit cAMP antibody preparation diluted
1:10 000 was used as antiserum (McNatty et al., 1990). After overnight
incubation at 4°C, bound and free [125I]-cAMP was separated by
adding 100 µl Sac-Cel (Wellcome Reagents Ltd, Beckenham, UK).
The supernatant was discarded and the pellet was counted. Measurements from a single experiment were performed within one assay
and the intra-assay variation was 8%.
Isolation of FSH isoforms
FSH isoforms were isolated from human pituitary extracts as previously described (Yding Andersen et al., 1999). Briefly, total glycopro-
tein extracts from anterior pituitary glands were obtained using a
previously described method (Jones et al., 1970). A chromatofocusing
column (dimensions 50⫻1 cm) was used to isolate the FSH isoform
fractions (Ulloa-Aguirre et al., 1992; Timossi et al., 1998b). Each
isoform fraction was then transferred to a dialysis membrane (molecular weight cut-off 12 kDa) and dialysed against deionized water and
10 mmol/l ammonium carbonate (pH 7.5). After freeze-drying, the
isoforms were redissolved in 100 mmol/l ammonium bicarbonate (pH
7.4) and applied to an affinity column with monoclonal anti-LH
antibodies in order to remove any LH that had co-eluted with the
FSH isoform fractions (Timossi et al., 1998b). This procedure removed
⬎90% of the immunoreactive LH present in the original concentrate
(Timossi et al., 1998b, 2000). Before using the FSH isoform fractions
for experiments, they were thoroughly dialysed against the HX
medium, divided into aliquots and kept at –20°C until use. In the
present study, three FSH isoform fractions were used: (i) less acidic,
having a pI of 6.43–5.69; (ii) mid-acidic, having a pI of 5.62–4.96;
and (iii) acidic having a pI of 4.69–3.75. Results of the purification
procedure for this particular batch of FSH isoforms have been
published (Yding Andersen et al., 1999).
Measurement of FSH concentration
Radioimmunoassay
The reagents used for the measurement of human FSH were provided
by the National Institute of Diabetes and Digestive and Kidney
Diseases (NIDDK). Iodination of the purified human FSH (human
FSH-I-4) was performed as previously described using the Iodogen
method (Yding Andersen et al., 1999). The FSH isoform fractions
were diluted in a buffer consisting of 0.05 mol/l sodium phosphate
buffer (pH 7.4) and 5 mg/ml BSA. As a standard, the reference
preparation LER-907 (1 mg LER-907 contains 53 IU FSH, 2nd
International Reference Preparation) was used. This standard, and
crude pituitary extracts, show a similar degree of charge heterogeneity
(Chappel et al., 1986). The rabbit polyclonal anti-human FSH
antiserum (human FSH-6) was used in a final dilution of 1:150 000.
This antiserum is characterized by having ⬍0.1% cross-reactivity
with highly purified human LH and undetectable reactivity with the
free α-subunit. Bound and free [125I]-FSH were separated by adding
100 µl Sac-Cel (Wellcome Reagents Ltd). The supernatant was
discarded and the pellet was counted. The inter- and intra-assay
coefficients of variation of a sample containing 25 IU/l were 7 and
5% respectively.
Chinese hamster ovary (CHO) cell assay
The CHO cell assay for FSH bioactivity (Albanese et al., 1994) was
developed by constructing a CHO cell line, which stably expresses
the recombinant human FSH receptor and which, upon stimulation
with FSH, releases cAMP. Briefly, a sufficient number of cells were
propagated in α-MEM supplemented with 10% (v/v) fetal calf serum
(FCS), penicillin/streptomycin and Geneticin 418 (all Gibco Life
Technologies, Paisley, Scotland, UK) to allow for a transfer of
250 000 cells to each well in the actual assay. After overnight
incubation, the cells were washed twice with phosphate-buffered
saline (PBS) containing 10 mg/ml BSA and incubated with 250 µl
α-MEM supplemented with 0.25 mmol/l IBMX and 1 mg/ml BSA.
Standards (LER-907, NIDDK, Bethesda, USA) and unknowns were
diluted in PBS containing 10 mg/ml BSA, added in 20 µl aliquots
and tested in triplicate for each dilution. After an incubation period
of exactly 4 h at 37°C, the medium from each well was pipetted into
tubes and stored at –20°C until assayed for cAMP. Measurement of
cAMP was performed as described above.
Statistical analysis
Results are presented as mean ⫾ SEM. Statistical evaluations were
performed using Student’s t-test after log-transformation where appropriate.
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C.Yding Andersen et al.
Figure 2. Time-dependent cAMP production by cumulus–oocyte–
complexes (COC) stimulated with different FSH isoforms. Based
on FSH measurements using radioimmunoassay. Cumulus enclosed
oocytes were removed for measurement of cAMP at the following
time points: 5, 10, 15, 30, 60, 120 and 180 min. Open squares ⫽
less acidic isoforms (pI 6.43–5.69); closed triangles ⫽ mid-acidic
isoforms (pI 5.62–4.96) and open diamonds ⫽ acidic isoforms
(pI 4.69–3.75). Values represent six experiments and are shown as
mean ⫾ SEM.
Results
Each of the three different FSH-isoform fractions was tested
for their ability to induce cAMP accumulation in the COC
using a total of 2550 COC, including controls. The timedependent accumulation of cAMP per COC per 10 IU/l of
FSH (as determined by radioimmunoassay) was tested in at
least three separate experiments (Figure 2). Overall, the three
isoform fractions elicited accumulation of cAMP in a similar
fashion. Maximal cAMP levels were detected after 30 min of
FSH exposure; thereafter, they decreased progressively during
the ensuing 90 min, reaching the lowest concentrations at
~120 min of exposure and remaining unchanged up to 180 min
of incubation. Accumulation of cAMP provoked by the less
acidic and mid-acidic isoform fractions was more rapid and
more pronounced than that elicited by the acidic FSH isoform
fraction. Already at 5 min of incubation, cAMP accumulation
induced by less- and mid-acidic isoforms was significantly
higher and the concentrations remained almost twice as high
as those observed for COC exposed to the acidic isoform
during the remaining incubation periods. The areas under the
cAMP curve yielded by the less- and mid-acidic isoforms
were respectively 82 and 74% higher than that shown by the
acidic isoform.
The time-dependent accumulation of cAMP for each isoform
fraction whose FSH concentrations were determined by the
CHO cell assay is shown in Figure 3. After an incubation time
of 30 min, a maximal production of cAMP was observed;
nevertheless, the activities of the three isoform fractions were
similar. It must be emphasized that the CHO cell assay also
measures the FSH concentration by cAMP accumulation as
an end-point. Thus, FSH induces production of cAMP with
equal efficacy in COC (mouse FSH-R) and in CHO cells
(human FSH-R).
Results of the cAMP accumulation in COC during and after
exposure to FSH isoforms are shown in Figure 4. The amount
of cAMP per COC at each time point is expressed relative to
the time zero value (i.e. 100%). A continuous presence of
FSH caused increasing amounts of cAMP to be accumulated
132
Figure 3. Time-dependent cAMP production by cumulus–oocyte–
complexes (COC) stimulated with different FSH isoforms. Based
on FSH measurements using the Chinese hamster ovary (CHO) cell
assay. Cumulus enclosed oocytes were removed for measurement of
cAMP at the following time points: 5, 10, 15, 30, 60, 120 and
180 min. Open squares ⫽ less acidic isoforms (pI 6.43–5.69); open
triangles ⫽ mid-acidic isoforms (pI 5.62–4.96); and closed
diamonds ⫽ acidic isoforms (pI 4.69–3.75). Values represent six
experiments and are shown as mean ⫾ SEM.
Figure 4. cAMP accumulation in cumulus–oocyte–complexes
(COC) with or without the continuous stimulus of FSH isoforms.
All COC were exposed to FSH isoform for 5 min after which some
COC were transferred to control medium, whereas the remaining
continued with FSH exposure. Open symbols ⫽ data pooled from
the less acidic isoform fraction (pI 6.43–5.69; one experiment) and
the mid-acidic isoform fraction (pI 5.62–4.96; two experiments)
(open squares: continued presence of FSH and open circles:
removed from FSH isoform containing media after 5 min). Closed
symbols ⫽ data for the acidic isoform fraction (pI 4.69–3.75; three
experiments) (closed triangles: continued presence of FSH and
closed diamonds: removed from FSH isoform containing media
after 5 min). Values are mean ⫾ SEM.
over the 30 min time period; cAMP accumulation was higher
in the presence of less and mid-acidic isoforms than during
exposure to the more acidic analogue. When COC were
removed from the FSH-containing medium after a 5 min
exposure time, cAMP accumulation stopped and rapidly
dropped thereafter to control levels. The pattern of decline of
cAMP concentrations after COC removal from either the
less acidic or more acidic FSH isoforms-containing medium
occurred in a similar manner. Results for the acidic isoform
represent data from three individual experiments. Results from
the less and mid-acidic isoform fractions were similar and
demonstrated exactly the same pattern, and the data in Figure
4 were derived from one set of experiments with the less
FSH isoforms and cAMP production by COC
acidic isoform fraction and two sets of experiments with the
mid-acidic isoform fraction.
Discussion
This study demonstrates that FSH isoforms induced maximal
accumulation of cAMP in COC within 30 min. The maximal
production was unrelated to whether less acidic, mid-acidic or
acidic isoforms were used. However, looking at the total
production of cAMP over a 3 h period, less acidic and midacidic isoforms were almost twice as effective in inducing
accumulation of cAMP than the acidic isoform fraction, and
just a 5 min exposure to less acidic and mid-acidic isoforms
was sufficient to elicit a marked production of cAMP, thereby
confirming and extending previous studies showing an
enhanced in-vitro bioactivity of less acidic isoforms (for
reviews, see Ulloa-Aguirre et al., 1995, 1998; Timossi et al.,
1998a; Vitt et al.,1998; Yding Andersen et al., 1999, Zambrano
et al., 1999). For each isoform fraction, a continued presence
of FSH of ⬎30 min attenuated the accumulation of cAMP
and caused levels to decline to little above control values. The
plasma half-life of pituitary FSH isoforms with a pI of 5.1–
5.9 (which resembles the less- and mid-acidic isoform fractions
used in this study) has been reported to be ~35 min, whereas
the corresponding half-life of acidic isoforms was twice as
high, being ~70 min (for review, see Ulloa-Aguirre et al.,
1995). Therefore, the plasma half-life of FSH isoforms with a
pI of ⬎5.0 corresponds quite accurately to the period of time
required for FSH to induce a maximal accumulation of cAMP
in COC. This makes it conceivable that the less acidic isoforms
exert important biological functions in vivo before being
cleared from circulation, and support other studies suggesting
that the increased biological activity exhibited by the less
acidic isoforms compensates for their relatively short plasma
half-lives (Timossi et al., 1998a).
The secretory pattern of pituitary FSH release has been
studied in detail in the sheep (Padmanahban et al., 1997). This
study showed that FSH, in parallel with LH, is released through
both a tonic (basal secretion) and episodic type of secretion
(in pulses). These investigators observed 1–2 FSH pulses/h,
found the half-time disappearance rate of FSH in circulation
to be 25 min, and suggested that FSH secreted in pulses may
be cleared faster from circulation than the hormone secreted
through the basal mode (Padmanahban et al., 1997). In the
present study, accumulation of cAMP began to decline after a
continuous exposure to FSH of ⬎30 min. The pulsatile
characteristics of FSH secretion suggest that cumulus cells in
this way actually receive a near maximal stimulation in vivo,
since FSH secreted in pulses, which due to their short halflife presumptively contain large amounts of less acidic/sialyated
isoforms, is present for ~30 min, one or two times per hour.
In a previous study, we showed that exposure of COC to
unfractionated FSH for just 30 min was sufficient to provoke
oocytes to resume meiosis evaluated after a total culture period
of 24 h (Byskov et al., 1997). Thus, the overall results
concurrently demonstrate that, not only is the accumulation of
cAMP maximal after 30 min of exposure, but also that signal
transduction down-stream of cAMP generation may operate
and induce specific biological events within this time frame.
Taken together, the present results suggest that less acidic
isoforms actually play important physiological functions and
that the pulsatile release of FSH may secure an optimal
stimulation of cumulus cells and/or granulosa cells in vivo.
Following removal of COC from a FSH-containing medium
after an exposure of 5 min, the production of cAMP rapidly
declined to around background levels. Although there was a
large difference between the increase of less acidic and acidic
isoforms, the cAMP production induced by both isoforms
returned to background levels within the same time period.
This suggests that a continuous presence of FSH stimulus is
necessary to achieve maximal signal transduction, and indicates
that the receptor dissociation of FSH in our system is similar
for the less acidic and acidic FSH isoforms, and that the
enhanced activity of less acidic isoforms may be exerted
through different mechanisms.
The effect of unfractionated FSH on FSH-R mRNA and
FSH binding has been studied in cultured rat Sertoli cells
(Themmen et al., 1991). This study found that FSH induced
an almost complete down-regulation of the FSH-R mRNA and
reduced binding of [125I]-FSH to Sertoli cell membranes after
4 h of FSH exposure. This effect was mimicked by dibuturyl
cAMP, suggesting that the effect of FSH on its receptor mRNA
was mediated by cAMP. In addition, maximal production of
cAMP by the Sertoli cells was attained only after 15–30 min
of FSH exposure. These results are in agreement with those
of the present study which demonstrate an almost identical
temporal relationship of cAMP production by cumulus cells
and also indicate that the responsiveness of the cumulus
cells may be down-regulated. Furthermore, the present study
confirms and extends previous observations that homologous
FSH-R desensitization is a relatively rapid process, which in
just 30 min becomes manifest when monitored as a decreased
cAMP output. Results of the present study were obtained using
a concentration of 10 IU/l FSH in the culture medium. A FSH
concentration of this magnitude is highly correlated with
that detected in circulation of women undergoing ovarian
stimulation and that to which the granulosa cells are exposed
(Yding Andersen et al., 1997). It is well known that granulosa
cells obtained in connection with oocyte retrieval following
ovarian stimulation are refractory to FSH stimulation immediately after recovery and that a culture period of at least 1 day
is required for the granulosa cells to regain their responsiveness
to FSH (Lambert et al., 2000). This impaired responsiveness
may, in addition to the FSH-R down-regulation occurring in
connection with the ovulatory process, also be related to a
prolonged exposure to high concentrations of FSH, such as
may occur during ovarian stimulation. These observations may,
therefore, be correlated with the results of the present study,
and raise the question of whether pulsatile administration of
FSH, possibly using less acidic isoforms, may induce a more
physiological response combined with a reduced consumption
of FSH.
Results from a number of studies on the biological potency
of FSH isoforms have been questioned because of the possibility that the immunological methods employed to determine
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C.Yding Andersen et al.
the FSH mass may be affected by the particular structures of
FSH isoforms (for reviews, see Chappel, 1995; Ulloa-Aguirre,
1995). However, the radioimmunoassay used in this study has
been shown to measure different FSH isoforms with almost
equal efficacy (Zambrano et al., 1996). In addition, a recent
study showed that the immunopotency of FSH is unrelated to
the tested FSH charge and isoform fraction, thus suggesting
that immunological methods actually measure the molarity of
the different FSH isoforms (Oliver et al., 1999). This also
implies that a number of previous studies were correct in
predicting that the less acidic isoforms possessed an enhanced
biological activity compared with the acidic analogues (Timossi
et al., 1998a; Vitt et al., 1998; Yding Andersen et al., 1999).
The CHO cell assay of the present study measures FSH
activity through the production of cAMP by cells expressing
the recombinant human FSH-R. Results based on the CHO
cell assay showed no differences in cAMP production between
the three isoform fractions. However, this was expected since
both monitoring systems employed cAMP production, as a
result of FSH-R stimulation, as the output parameter. In
essence, therefore, FSH is equally effective in inducing accumulation of cAMP in cells of COC and in CHO cells. In a
previous study we showed that the less and mid-acidic isoforms
used in this study induced resumption of meiosis more effectively than acidic isoforms, even when FSH measurements
were based on the present CHO cell assay (Yding Andersen
et al., 1999). The discrepancy between the ability to induce
resumption of meiosis and accumulation of cAMP by FSH
based on the CHO cell measurements, therefore, indicates that
FSH (in addition to cAMP) employs other signal transduction
pathways for reinitiation of meiosis in cumulus enclosed
oocytes. This also illustrates that there may be other, and
perhaps more appropriate, FSH bioassay response parameters
to be studied once a more detailed knowledge is gained with
respect to the interaction of human FSH isoforms with mouse
gonadotrophin receptors.
The present study uses COC from large preovulatory follicles
collected just prior to the mid-cycle surge of gonadotrophins.
The time of collection therefore corresponds to the period
during which the less acidic isoforms dominate the natural
menstrual cycle and emphasizes the present model as a suitable
way of monitoring the physiological regulation of events taking
place in the preovulatory follicle. However, compared with
the natural menstrual cycle, follicles and COC have experienced
ovarian stimulation with exogenous FSH before further exposure to FSH in the course of the bioassay.
In conclusion, the present study shows that unmanipulated
oocyte cumulus complexes from large preovulatory follicles
respond to different FSH isoforms with a rapid and immediate
production of cAMP, with the less- and mid-acidic isoforms
being around twice as effective as the acidic isoforms. Exposure
to FSH for ⬎30 min caused production of cAMP to decline,
possibly due to FSH receptor desensitisation. These data are
correlated with the pulsatile release of FSH in vivo, every 30–
60 min, making it conceivable that the predominant release of
less acidic isoforms occurring just around ovulation may play
a significant role in exerting near maximal stimulation of the
cumulus/granulosa cells of preovulatory follicles.
134
Acknowledgements
The technical assistance by Tiny Roed and Anette Winkel is gratefully
acknowledged. Dr A.F.Parlow and the National Hormone and Pituitary
Program, USA is thanked for providing the reagents for FSH RIA
measurements, and Dr A.R.La Barbera, University of Cincinnati,
Cincinnati, OH, USA for providing the cAMP antiserum. This study
was supported by the Danish Medical Research Council, (grant nos.
9400824, 9502022 and 9602272).
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Received on September 14; accepted on November 30, 2000
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