| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Reproductive Endocrinology |
Division of Reproductive Medicine, Department of Obstetrics and Gynecology (I.S., P.M.T.H., B.C.J.M.F.), and Department of Endocrinology and Reproduction (F.F.G.R.), Dijkzigt Academic Hospital and Erasmus University Medical School, 3015 GD Rotterdam, The Netherlands
Address all correspondence and requests for reprints to: Dr. B. J. C. M. Fauser, Division of Reproductive Medicine, Department of Obstetrics and Gynecology, Dijkzigt Academic Hospital, Dr Molewaterplein 40, 3015 GD Rotterdam, The Netherlands.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In the presence of 10% vol/vol serum from regularly cycling women (n = 8), FSH-stimulated cAMP production was inhibited to 42 ± 2% (mean ± SEM of 2 experiments, each performed in duplicate) of cAMP production in the absence of serum, whereas a similar cAMP level (up to 38 ± 4% of the serum-free level) was observed at higher concentrations of serum (30–90% vol/vol). The inhibition of FSH-stimulated cAMP production in the presence of serum samples from normogonadotropic anovulatory patients, without (n = 13) or with (n = 16) PCOS, was similar to controls. Follicular fluid samples (n = 57) obtained during the follicular phase in 25 regularly cycling women and follicular fluid samples (n = 25) from 5 PCOS patients were tested in a slightly modified assay system. In the presence of 10 or 30% (vol/vol) follicular fluid, FSH-stimulated cAMP levels were decreased to 68 ± 2% and 55 ± 2% (mean ± SEM of a single experiment in triplicate) of the cAMP levels in the absence of follicular fluid, respectively. There was no correlation between the degree of cAMP inhibition and follicle size, steroid content (androstenedione or estradiol concentrations), or menstrual cycle phase. Furthermore, no differences in inhibition were found, comparing PCOS follicles with size- and steroid content-matched follicles obtained during the normal follicular phase.
It is concluded that inhibition of FSH receptor activation by proteins present in serum or follicular fluid is constant (60 and 40%, respectively) and independent from the developmental stage of the follicle, either during the normal follicular phase or in patients with normogonadotropic anovulation. Inhibition of FSH receptor activation may be of limited significance for normal and arrested follicle development.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
There are many indications that local regulation of FSH action may play a role in normal and disturbed follicular development (9, 10, 11, 12). Growth factors, such as insulin-like growth factors (IGF) (13) or activin (14), act as potentiators of FSH action in vitro. Ongoing growth of the dominant follicle, despite a decrease in FSH serum levels in the late follicular phase (LFP) (15), may be attributed to the enhancement of FSH action by these growth factors, acting in a para- or autocrine fashion. On the other hand, growth factors may also exert inhibitory actions, as has been described for epidermal growth factor (16, 17). Although stimulatory or inhibitory effects of growth factors are mediated by their specific receptors and pathways, inhibition of FSH action may also be caused by specific FSH receptor inhibitors (12). Several studies report the presence of specific FSH receptor-binding inhibitors of unknown origin in human serum (18, 19) and follicular fluid (20, 21, 22, 23). Part of these inhibitors may be of immunological nature (24) and could lead to premature ovarian failure. It may be postulated that partial inhibition of FSH receptor activation causes arrested follicular growth and absent dominant follicle selection in PCOS patients.
The aim of this study was to assess the level of inhibitors of FSH receptor activation in serum and follicular fluid of normogonadotropic anovulatory patients, with or without PCOS, as compared with normal controls. For this purpose, a Chinese hamster ovary (CHO)-cell line, transfected with the human FSH receptor (25), has been used. Inhibition of FSH-stimulated cAMP production has been studied at physiological concentrations of FSH and at a range of concentrations of serum or follicular fluid.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Recombinant human FSH (recFSH: Org 32489, bioactivity 8413 IU/mg, as assessed by in vivo bioassay relative to reference preparation IS 70/45; immunoactivity 12,000 IU/mg) was a generous gift from NV Organon (Oss, The Netherlands). Isobutyl-methyl-xanthine (IBMX) was purchased from Sigma Chemie (Bornhem, Belgium).
Cell cultures
A CHO cell line, stably transfected with recFSH receptor complementary DNA, was generously provided by NV Organon). CHO cells were cultured as described previously (25). In brief, cells (104 cells/cm2) were plated on 48-well culture dishes in DMEM/Ham-F12 (1:1) (GIBCO Europe BV, Breda, The Netherlands) with the addition of 100 U/mL penicillin and 100 µg/mL streptomycin and 10% vol/vol FCS (Sebak GmbH, Adenbach, Germany). After 2 days, the CHO cells reached confluence and were used for the FSH receptor activation assay.
Serum samples
Serum samples were collected from 29 anovulatory patients, attending the Dijkzigt Hospital outpatient clinic for infertility evaluation. Inclusion criteria were: infertility, oligo- or amenorrhea, and serum FSH levels within normal limits (1–10 IU/L). The involvement of human subjects in these investigations was approved by the Ethics Review Committee of the Dijkzigt Academic Hospital and Erasmus University Medical School, and informed consent was obtained from all subjects participating. Patient samples were divided into 2 subgroups representing normogonadotropic anovulation, either without (n = 13) or with PCOS (n = 16). Patients were diagnosed as having PCOS on the basis of rigid criteria, including elevated serum LH levels (>8 IU/L) and elevated serum androgens [androstenedione (AD) more than 15.0 nmol/L and/or testosterone (T) more than 3.0 nmol/L and/or a free androgen index (FAI: [T x 100]/steroid hormone-binding globulin ratio) more than 5] (1) and polycystic ovaries, as assessed by transvaginal sonography (26). Patients diagnosed as anovulatory (without PCOS) had normal LH and androgen concentrations and normal ovaries on ultrasound. In addition, FSH levels in all serum samples were measured by immunoradiometric assay (IRMA-FSH; Medgenix, Fleurus, Belgium). Serum samples selected for this study had IRMA-FSH levels between 3 and 6 IU/L, to fulfill the FSH receptor activation assay requirements (see below).
Normal ovulatory women in the early follicular phase (EFP) of the
menstrual cycle (8–10 days before the LH surge) served as controls
(n = 8). These volunteers were recruited by advertisement and were
paid for their participation (15). Mean cycle length was 28 ± 2
SD days. Again, control samples exhibiting IRMA-FSH levels
between 3 and 6 IU/L were chosen. See Table 1
for
population and endocrine characteristics of all subjects studied.
|
Follicle fluid was obtained from 22 women undergoing laparotomy
for reversal of tubal sterilization or adhesiolysis and from 3 women
undergoing laparoscopic tubal ligation. Informed consent was obtained
from all subjects participating. All subjects were regularly cycling
women with a mean cycle length of 27 ± 2 SD days and
of normal weight (mean body mass index: 24 ± 2 SD
kg/m2). Mean age was 33 ± 4 SD yr, and
all patients had a history of proven fertility. The day of the
menstrual cycle, at the time of the follicle puncture, was assessed
from the day of onset of the last menstrual period. Follicle fluid was
obtained from individual follicles (between 1 and 8 per patient; Fig. 1) and assayed for E2 and AD as described
previously (27). Follicular fluid samples were classified into 5
different categories. The classification was on the basis of 3
criteria: 1) the menstrual cycle phase in which patients underwent
surgery (EFP: cycle day 1–8 or LFP: cycle day 9–15); 2) follicle size
(nondominant: <10 mm or dominant: 
10 mm) (15, 27); and 3)
AD/E2 ratio (healthy: <4, or atretic: 4) (27, 28).
|
for the
classification and endocrine characteristics of the follicular fluid
samples used.
|
Validation of this assay has been described previously (25). In brief, cultured CHO cells were incubated in 48-well culture dishes. The culture medium was replaced by FCS-free DMEM/Ham-F12 with 0.1 mmol/L IBMX and various concentrations of human serum or follicular fluid (3–90% vol/vol), and cells were stimulated with recFSH. After 4 h of incubation, medium was removed and stored at -20 C until analysis for cAMP.
For the measurement of the inhibitory effect in the serum samples, a fixed concentration of 6 mIU/mL FSH was used. To obtain this concentration of FSH in each dilution of the serum samples, endogenous FSH serum levels (range: 3–6 IU/L) were corrected by the addition of recFSH.
A pool of hypogonadotropic serum, obtained from high-dosed (50 µg ethinyl-E2 daily) combined oral contraceptive pill users, served as a control in each assay. The FSH level of this serum pool was less than 0.5 IU/L, as assessed by IRMA.
Each serum sample was tested in duplicate in two independent FSH receptor activation assays. Results are expressed as the percentage of the cAMP production, relative to the cAMP response in serum free conditions, which was set at 100% at the given stimulatory dose of recFSH (6 mIU/mL).
Because of small volumes available, the CHO assay was adapted, to allow analysis of follicular fluid samples. For this purpose, CHO cells were cultured in 96-multiwell dishes, at a cell density of 104 cells/cm2, and incubated in a vol of 50 µL/well. The sensitivity for recFSH and the degree of stimulation of the cAMP production in this assay, applying small volumes, was similar to the assay performed in 48-wells (data not shown).
The small volumes of individual follicular fluid samples allowed for testing at only two concentrations (10 and 30% vol/vol) in triplicate. The inhibitory effect of follicular fluid was assessed by stimulating CHO cells with 3 mIU/mL recFSH, instead of 6 mIU/mL. This FSH concentration was taken, to approximate the endogenous FSH levels in the follicular fluid samples (4, 8, 29, 30). Because volumes of the tested follicular fluid samples were small (in some cases only 30 µL), it was not possible to measure the FSH content.
In all experiments, DNA content of the wells was measured by a fluorometric method, as described previously (31). The intraassay coefficient of variation for DNA content/well was less than 12%. In all FSH receptor activation assays, the cAMP levels were normalized on the basis of the DNA content of the wells.
RIAs
cAMP in the media was assayed as described previously (32). In brief, after acetylation, the samples were incubated overnight with cAMP antibody (purchased from Prof. Dr. J. Stoof, Free University, Amsterdam, The Netherlands). The assay was validated for the use of culture media and corrected for addition of serum in the samples. All samples were assayed in duplicate. Sensitivity of the assay was 0.125 pmol/mL. Inter- and intraassay coefficients of variation were 20% and 8%, respectively.
E2 and AD in follicular fluid were measured in a 1:100 dilution of the samples, as described previously (27). E2 concentrations were estimated using an RIA kit (Diagnostic Products Corporation, Los Angeles, CA); AD concentrations were estimated using antisera and diethylether-hexane extraction, as described previously (33).
Data analysis
Experimental data are presented as the mean ± SEM if they are normally distributed and as median and range if distributed otherwise. The inhibition of FSH-stimulated cAMP production is expressed as the percentage of the cAMP response in absence of serum or follicular fluid, which is set as 100%. Results were evaluated using one-way ANOVA, comparing responses as percentages of the serum free response. Correlation of follicle characteristics with experimental results were analyzed using Spearman’s rank order test. P values given are two-sided, with 0.05 taken as the limit for statistical significance.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The dose-dependent stimulation of adenylate cyclase in CHO cells
is shown in Fig. 2. Basal levels of cAMP were 0.9–2.1
pmol/µg DNA, and maximally stimulated levels were 350–420x higher.
The sensitivity, defined as the dose of recFSH eliciting a response of
more than twice the standard deviation of the basal cAMP production,
was 0.4 mIU/mL. The half-maximal stimulation of cAMP production
(ED50 value) was obtained at a dose of 24.9 mIU/mL FSH. The
intraassay coefficient of variation, at different stimulatory
concentrations of FSH, was less than 16%. To study the effects of
serum at different doses of recFSH, CHO cells were incubated in the
presence of different concentrations of human hypogonadotropic serum
(10, 30, and 90% vol/vol) and stimulated with increasing doses of
recFSH (3–100 mIU/mL). Basal cAMP production was not affected by the
addition of hypogonadotropic serum, whereas in the presence of 90%
vol/vol hypogonadotropic serum, stimulated cAMP production decreased to
49–68% of responses under serum-free conditions (Fig. 2).
|
). At a high FSH concentration (30 mIU/mL), a
competition between FSH and serum components is apparent over the whole
serum concentration range, whereas at low FSH concentrations (3 or 6
mIU/mL), this competition occurs only at serum concentrations less than
3% vol/vol. At serum concentrations more than 10% vol/vol, a
saturation of the inhibitory effect is observed. Despite the slight
inhibition by serum factors, CHO cells remain sensitive to stimulation
by FSH. Even in the presence of 90% vol/vol of hypogonadotropic serum,
addition of 3 mIU/mL recFSH results in a 5- to 8-fold stimulation of
the cAMP production (data not shown).
|
Serum samples obtained from normally cycling individuals or
anovulatory patients, with or without PCOS, inhibited cAMP production
up to 39 ± 4%, 35 ± 2%, and 34 ± 3% of the
response under serum free conditions, respectively, when tested at 90%
vol/vol. Within each group, a significant further increase in
inhibition was absent (P > 0.38) when serum
concentrations were increased from 10 to 90% vol/vol. Furthermore, no
statistically significant differences were found between patient groups
and normal controls (P > 0.08), with regard to the
inhibition of FSH-stimulated cAMP production at each concentration of
serum (Fig. 4).
|
Adapted bioassay conditions were used to measure the effects of
small amounts of follicular fluid (see Materials and Methods
section). The intraassay coefficient of variation of the adapted assay
was greater (<21%), compared with the 48-well assay (<16%).
FSH-stimulated cAMP production was clearly inhibited in the presence of
10 or 30% vol/vol of follicular fluid, although in several samples, a
concentration-dependent decrease in cAMP was not observed. The mean
relative cAMP production was lower at a concentration of 30% vol/vol,
as compared with 10% vol/vol (55 ± 2% and 68 ± 2%,
respectively; P < 0.001). No differences in inhibition
of the cAMP response were found when comparing various classes of
follicular fluid samples from normally developing follicles (Fig. 5, upper panel). The inhibitory effect of
follicular fluid did not correlate with follicle size, with the
AD/E2 ratio of the follicular fluid, nor with the menstrual
cycle phase in which the samples were obtained (data not shown).
Similar results were obtained with follicular fluid from PCOS patients,
as compared with follicular fluid from regularly cycling women (Fig. 5, lower panel).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Primary cultures of rat granulosa (35) or Sertoli cells (36) have been used as in vitro bioassays for FSH. These bioassays are hampered by a strong inhibition of the FSH response caused by unspecified factors in serum. An inhibition of more than 80% of the induced E2 response can be observed in the presence of only 4% serum (25, 35). In contrast, currently used CHO cells show only a limited inhibition of FSH-induced cAMP production by serum. Even in the presence of 90% vol/vol human serum, the cAMP response is inhibited up to 60% only. Although this inhibition might be caused by metabolic changes in the cells, this seems less likely because prolonged incubation of the cells in serum affected neither cell viability nor basal cAMP production (25). In addition, the cells can be stimulated to produce large amounts of cAMP despite preincubation in 90% serum (25). It is of interest that in the presence of serum concentrations more than 10% vol/vol, the percentage inhibition of FSH-dependent receptor activation (at a fixed FSH concentration) is independent of the concentration of serum. This fixed magnitude of inhibition over a wide range of serum concentrations (10–90% vol/vol) suggests a saturating effect of the inhibitory components in serum. On the other hand, the CHO cells still respond to elevation of the FSH levels, irrespective of serum concentrations. This suggests that a competition exists between inhibiting factors and FSH for the FSH receptor. Although the mechanism of inhibition is unclear, this may be attributed to a nonspecific protein matrix effect, because bovine and equine sera show similar inhibition curves (25). Comparing sera from regularly cycling women with normogonadotropic anovulatory patients (with or without PCOS) showed no differences in inhibitory activity. This argues against the presence of increased levels of inhibitors of FSH receptor activation in serum from these patients.
Because factors inhibiting FSH receptor activation may be present exclusively in the follicular compartment, follicular fluid obtained from individual follicles throughout the follicular phase of the menstrual cycle and from PCOS patients also were tested. Follicular fluid samples were classified according to the stage of the menstrual cycle in which they were obtained, as well as by size and steroid content (expressed as the AD/E2 ratio). Different classes of normally developing follicles did not reveal differences with regard to inhibition of FSH receptor activation. Neither did follicular fluid from PCOS patients, compared with matched control follicles, show any difference in inhibition of FSH receptor activation. Results obtained with follicular fluid displayed a larger degree of variation, compared with results obtained from the addition of serum samples. Part of this variation may be caused by the higher intraassay coefficient of variation of the assay applying small volumes. Another explanation may be found in differences in the endogenous FSH levels of the individual follicular fluid samples. In the present study, FSH content in follicular fluid could not be assessed, because of the small volumes available. It was therefore not possible to normalize for potential differences in FSH levels. In an attempt to limit the variation in FSH levels in the follicular fluid assays, a small amount of FSH (3 mIU/mL) was added to each follicular fluid dilution. The choice for this FSH concentration was on the basis of data from the literature (4, 8, 29) that report levels of intrafollicular immunoreactive FSH ranging between 0.3 and 6 IU/L. This uncertainty in the final FSH concentration could explain the reversal of inhibition observed in some samples at increased concentration. However, despite these uncertainties, the observed responses did not correlate with any follicle characteristic, such as size or steroid content, or with menstrual cycle phase or PCOS diagnosis. Although inhibitory effects of subfractions in serum or follicular fluid have not been tested separately, the total net effect of potentially different combinations is similar in both controls and anovulatory patients. Therefore, it seems unlikely that FSH receptor inhibition plays a significant role in the (patho)physiology of normal or disturbed follicle development.
Specific FSH receptor-binding inhibitors have been described in partly purified fractions of human serum and follicular fluid (19, 23, 37). However, not all FSH-binding inhibitory activity results in inhibition of FSH, because part of these binding inhibitors have FSH agonist activity (22). Results of the present study show that the biological activity of FSH in the context of serum proteins is approximately 50% of the activity in the absence of serum. The mechanism underlying this reduction in bioactivity is not known, but it may be postulated that FSH is partly bound to plasma proteins with low affinity, thereby reducing the amount available for receptor activation. On the other hand, serum proteins also may bind reversibly to the receptor, thereby reducing the number of receptors available for interaction with FSH. One other aspect to be considered is that, in the present study, only adenylate cyclase activity has been taken as the endpoint of FSH receptor activation. It is known that FSH receptor activation results in formation of intracellular messengers other than cAMP, like calcium ions (38, 39). Although it is not yet clear whether this Ca2+ pathway functions independently from cAMP (39, 40), it could be postulated that specific FSH receptor inhibitors have a preference for one pathway, leaving the other less inhibited, which could thus result in expression of different cellular functions. Possible inhibition of different pathways is illustrated by findings that immunoglobulins in serum from women with premature ovarian failure block FSH-induced DNA synthesis in granulosa cells in vitro (24), although they do not affect FSH-induced steroidogenesis (41).
Our conclusion from these in vitro studies that FSH receptor inhibition is a constant, and therefore may be of only limited significance for regulation of follicle development, is being supported by recent findings from clinical studies. Growth and selection of the dominant follicle is induced by increasing levels of FSH during the luteo-follicular transition in the menstrual cycle (42). Although normogonadotropic anovulatory patients display bioactive and immunoreactive FSH levels comparable with follicular phase levels during the normal menstrual cycle (1, 5), these patients lack the intercycle rise. Normal monofollicular development and ovulation can be induced in normogonadotropic anovulatory patients by the administration of exogenous FSH, applying a decremental-dose regimen (6, 43). Although the administration of gonadotropins in these patients suggests an increased FSH-threshold level that needs to be surpassed to ensure follicle development, it seems that normal follicle development can be achieved in normogonadotropic anovulatory patients without increasing FSH to supraphysiological concentrations (5). These findings dispute the presence of an increased FSH-threshold level in normogonadotropic anovulatory patients, which could be caused by locally acting factors blocking FSH action. It could well be that the absence of the transient increase in FSH levels in anovulatory patients is an important factor in the etiology of arrest of follicle development. It may be of interest that, in particular, bioactive FSH levels increase during the luteo-follicular transition, as has been demonstrated recently (34). Although the bioactive-to-immunoactive FSH ratio remains constant during the EFP in regular cycling women (5, 34) and is not different in anovulatory patients (5), the change in FSH levels and the bioactive-to-immunoreactive ratio in the late luteal phase may be of significance for follicle recruitment and selection. Furthermore, under normal conditions, ongoing growth of the dominant follicle occurs despite decreasing levels of FSH in the LFP (15). This suggests an enhancement of FSH action, allowing the dominant follicle to continue its development, whereas FSH levels drop below the threshold for the remaining, less mature follicles. This enhancement of FSH action may be the result of locally active growth factors, such as IGF-I and -II, and the IGF-binding proteins (13). These factors act through their own specific receptors and therefore exert their effect through intracellular pathways and not directly through the FSH-receptor. The potential significance of IGF-II, as an intraovarian factor involved in the development of the dominant follicle in the human, has been stressed recently (44).
In conclusion, analysis of inhibition of FSH receptor activation, by serum or follicular fluid samples from both regularly cycling women and PCOS patients, indicates that increased levels of FSH receptor inhibitors do not play a significant role in the regulation of normal and abnormal follicle development. This is in concert with findings that normogonadotropic anovulatory patients do not exhibit an increased FSH-threshold level per se. Both the dynamics in FSH levels in the EFP and locally active growth factors, acting through their own receptors, may be of greater significance for follicle growth and selection.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Received September 19, 1996.
Revised November 22, 1996.
Accepted January 30, 1997.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  S. Jonard and D. Dewailly The follicular excess in polycystic ovaries, due to intra-ovarian hyperandrogenism, may be the main culprit for the follicular arrest Hum. Reprod. Update, March 1, 2004; 10(2): 107 - 117. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. S. E. Laven, A. G. M. G. J. Mulders, J. A. Visser, A. P. Themmen, F. H. de Jong, and B. C. J. M. Fauser Anti-Mullerian Hormone Serum Concentrations in Normoovulatory and Anovulatory Women of Reproductive Age J. Clin. Endocrinol. Metab., January 1, 2004; 89(1): 318 - 323. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Schipper, W. C. J. Hop, and B. C. J. M. Fauser The Follicle-Stimulating Hormone (FSH) Threshold/Window Concept Examined by Different Interventions with Exogenous FSH during the Follicular Phase of the Normal Menstrual Cycle: Duration, Rather Than Magnitude, of FSH Increase Affects Follicle Development J. Clin. Endocrinol. Metab., April 1, 1998; 83(4): 1292 - 1298. [Abstract] [Full Text]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
, Healthy
follicles (AD/E2 ratio < 4); +, atretic follicles
(AD/E2 ratio 
, 30%; 
, 90% vol/vol).
) of
recFSH, incubated in the presence of increasing concentrations (3–90%
vol/vol) of hypogonadotropic human serum. Results (mean ±
SEM of three experiments in triplicate) are expressed as
the percentage of the response under serum-free conditions (100%).
