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From the Clinical Research Centers |
University of Washington (N.A.K., D.E.B., W.J.B., M.R.S.), Seattle, Washington 98105; Northwestern University (T.K.W.), Chicago, Illinois 60208; University of Michigan (V.P.), Ann Arbor, Michigan 48109; and Stanford University (L.C.G.), Stanford, California 94305
Address all correspondence and requests for reprints to: Nancy A. Klein, M.D., 4225 Roosevelt Way NE, Suite 305, Seattle, Washington 98105. E-mail: nklein{at}u.washington.edu
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Inhibin and activin are glycoprotein dimers produced by granulosa cells that are defined by their ability to suppress (inhibin) (6, 7) and stimulate (activin) (6, 8) FSH synthesis and secretion. Follistatin is a single chain polypeptide that is also an inhibitor of FSH (6), but primarily functions as a potent binding protein of both activin and inhibin (9). The net effect of these hormones is dependent on the relative amounts present, the amount of bioavailable ligand, and the available receptors. After the development of specific, two-site ELISAs for dimeric inhibin, it was demonstrated that serum inhibin B was decreased in the early follicular phase of older, ovulatory women with FSH elevation (10, 11, 12, 13, 14). Reame and colleagues reported that as well as decreased inhibin B, older ovulatory women had increased circulating levels of total activin A (12). In addition to their endocrine roles as FSH modulators, inhibin and activin appear to have multiple autocrine/paracrine roles in follicle development, including promotion of follicle growth by inhibin (15) and promotion of follicle atresia (15, 16), premature ovulation, and oocyte degeneration (16) by activin.
Other putative intraovarian regulators of follicle growth include the GH/insulin-like growth factor (IGF) family of proteins. FF IGF-II levels positively correlate with follicle size and cycle day and inversely correlate with the androgen/estrogen ratio (17). IGFBPs (which primarily inhibit IGF actions by sequestering the peptides) have been identified in human FF in levels that vary with the functional status of the follicles (18). We previously demonstrated that normal older reproductive age women have reduced concentrations of IGF-I (but not IGF-II) in both serum and the FF of the dominant follicle (19). However, binding proteins were not measured in this earlier study, and the significance of this finding is unknown.
Vascular endothelial growth factor (VEGF) is an angiogenic cytokine produced by granulosa cells, and its expression appears to be enhanced in response to hypoxic stimuli (20). Oocytes from follicles with decreased dissolved oxygen FF content demonstrate abnormalities in the organization of chromosomes within the meiotic spindle similar to those observed in older, ovulatory women (21). FF VEGF concentrations may therefore vary in response to age-related differences in ovarian perfusion and intrafollicular oxygen content.
The current study was carried out to further evaluate the roles of dimeric inhibin, activin, follistatin, IGF-I, IGF-II [as well as IGF-binding protein-2 (IGFBP-2) and IGFBP-3], and VEGF in older reproductive age women by determining their relative FF concentrations. We hypothesized that in the older women, the preovulatory dominant follicle contains altered concentrations of one or more of these regulatory hormones. Considering that activin can cause premature ovulation and oocyte degeneration in rodents, we postulated that older ovulatory women would have increased levels of activin in their dominant follicles.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Normal female volunteers, aged 40–45 yr (subjects; n = 20) and 20–25 yr (controls; n = 19), were recruited for the study. All women were required to have regular ovulatory menstrual cycles [luteal phase progesterone (P), >10 nmol/L], be healthy with normal body mass index, be taking no medications or exogenous hormones, have no past or current reproductive endocrine problems, participate in no more than 5 h/week of aerobic exercise, and have normal serum PRL (<20 µg/L) and testosterone (T; <3.5 nmol/L) levels. FF steroid and IGF concentrations have been reported previously for 5 of these subjects (2), and FF inhibin concentrations have been reported for 10 of these subjects (10), who were included in our previous studies. The study protocol was approved by the University of Washington human subjects committee, and written consent was obtained from all volunteers. All participants were provided monetary compensation for their time and inconvenience.
Study procedures
Dominant follicle development was monitored in a natural menstrual cycle with serial estradiol (E2) levels and transvaginal ultrasound. When the dominant follicle measured at least 16 mm and/or serum E2 was at least 550 pmol/L, 10,000 IU hCG were administered im. Thirty-two hours after hCG, transvaginal aspiration of the dominant follicle was performed under ultrasound guidance. FF samples were centrifuged and frozen for subsequent assay. For this study, aliquots of the FF were assayed for dimeric inhibin A and B, total activin A, total follistatin, IGF-I, IGF-II, IGFBP-2, IGFBP-3, VEGF, E2, androstenedione (A), T, and P.
Assays
FSH. The FSH assay employed was a solid phase two-site fluoroimmunometric (Delfia) assay in which two monoclonal antibodies, supplied by Wallac, Inc. (Gaithersburg, MD), are directed against two separate antigenic determinants on the human gonadotropin molecule. The assay cross-reacts less than 1% with LH. The intra- and interassay coefficients of variation were 2.3% and 4.6%, respectively.
E2. E2 was measured by RIA performed using reagents supplied by ICN Biomedicals, Inc. (Costa Mesa, CA). The antibody cross-reacts 9% with estrone, 1.5% with estriol, and less than 1% with all other steroids. The inter- and intraassay coefficients of variation were 16% and 7%, respectively.
Inhibin A and B. FF levels of inhibin A and inhibin B were
measured by enzyme-linked immunosorbent assay (ELISA) as described
previously (22, 23). Briefly, the assays are two-site
ELISAs based on the use of multiwell plates coated with specific
monoclonal antibodies to the ßA- and
ßB-subunits as capture antibodies. The Fab
fraction of a mouse monoclonal antibody to the N-terminal portion of
the 20-kDa 
-subunit (R1) conjugated to alkaline phosphatase is used
for detection in both assays. Recombinant inhibin A (National Institute
of Biological Standards and Control, Hertfordshire, UK)
and inhibin B (Genentech, Inc., South San Francisco, CA)
were used as standards. Intra- and interassay coefficients of variation
were 8% and 11%, and the assay detection limit was 7.8 pg/mL for both
assays.
Total activin A. Total activin A was measured using a
two-site enzyme immunoassay procedure that incorporates an analyte
denaturation and oxidation step (24). Recombinant human
activin A (rh-activin A) is used as an assay standard, diluted from
frozen aliquots of a 250 ng/mL stock. Monoclonal antibodies against a
synthetic peptide corresponding to residues 82–114 of the mature
ßA-subunit of human inhibin are covalently
coupled to 96-well hydrazide plates (24).
Cross-reactivities of less than 0.5% are observed with inhibin A,
inhibin B, and activin B, and cross-reactivities of less than 0.1% are
observed with bovine pro-C and follistatin. The assay detection
limit is 10 pg/well, and coefficients of variation within and between
plates are 4.9% and 9.1%, respectively.
Follistatin. Total follistatin concentrations were measured using a two-site ELISA (25). The assay uses a pair of mouse monoclonal antibodies generated against follistatin 288. The assay is specific for follistatin and cross-reacts minimally (<0.3%) with inhibin A, inhibin B, activin A, activin B, and activin AB. The cross-reactivity of follistatin-315 in the assay is 9.9%.
FF steroids. Follicular fluid E2, P, T, and A were extracted and separated by Celite chromatography and then measured by RIA as previously described (26).
Western analysis. Qualitative analysis of FF protein
complexes was performed as follows. rh-follistatin, rh-activin A (both
provided by Al Parlow, National Hormone and Pituitary Program,
Torrance, CA), and rh-inhibin A standards (positive controls)
and FF samples were subjected to SDS-PAGE in a 4–20% or 12%
Tris-glycine gel. Total protein amounts were equivalent per gel lane
loaded. Samples were reduced in 10% SDS sample buffer with 2.5%
ß-mercaptoethanol, then heated at 90 C for 2 min. The gels were
transferred to polyvinylidene difluoride membranes for immunoblot
analysis. The monoclonal antibodies used to detect follistatin via
immunoblot (Western) analysis were 7FS30 and 6FS6 (provided by Pat
Sluss, Massachusetts General Hospital, Boston, MA), either alone or in
combination, and immunoreactivity was determined using ECL detection
(Amersham Pharmacia Biotech, Arlington Heights, IL). The
follistatin antibodies detect both the 288- and 315-amino acid forms.
Inhibin subunits were detected with the polyclonal chicken
antiinhibin antibody, and the ß-subunits were detected with a
monoclonal antibody, 6H5 (provided by Genentech, Inc.).
Activin bound to follistatin is not recognized by the ß-subunit
antibody, and the complex, once formed, is irreversible
(27). All primary antibodies were used at a dilution of
1:2000. The primary antibodies used in the experiment were
biotinylated, and incubation of the blot with horseradish
peroxidase-strepavidin by itself did not result in the detection of any
protein bands (negative control).
IGFs and IGFBPs. IGF-I concentrations were measured using a two-site immunoradiometric assay (IRMA; Active, Diagnostic Systems Laboratories, Inc., Webster, TX). The intra- and interassay coefficients of variation were 7% and 7.4%, respectively. IGF-II concentrations were measured using a two-site IRMA (Active, Diagnostic Systems Laboratories, Inc.) that includes an extraction step to separate the IGF-II from its binding protein. The intra- and interassay coefficients of variation were 6.5% and 6.3%, respectively. IGFBP-2 concentrations were measured using a RIA (Diagnostic Systems Laboratories, Inc.). The intra- and interassay coefficients of variation were 8.5% and 7.6%, respectively. IGFBP-3 concentrations were measured using a two-site IRMA (Active, Diagnostic Systems Laboratories, Inc.). The intra- and interassay coefficients of variation were 3.9% and 1.9%, respectively.
VEGF. FF concentrations of VEGF were quantified using a quantitative sandwich enzyme immunoassay that uses a horseradish peroxidase-linked VEGF polyclonal antibody for detection (Quantikine, R and D Systems, Inc. Minneapolis, MN) with recombinant human VEGF as a standard (21). Optical density readings were obtained at 450 nm in a microtiter plate reader (TiterTek Multiskan MC, ICN Biomedicals, Inc.). The sensitivity of the assay ranges from 5–9 pg/mL, and the intra- and interassay coefficients of variation are less than 10%.
Statistics
The mean and SEM for all hormone concentrations were calculated for each age group. Mean values were then compared between groups by Mann-Whitney U test. For each analysis, P < 0.05 was considered to represent a significant difference.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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). Results for FF hormone concentrations
(mean ± SEM) are shown in Figs. 1-3. Not all subjects had sufficient
aliquots of FF to perform all assays; therefore, the numbers of
subjects represented by each mean value are indicated in the
figures.
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). As previously reported (2), there was a trend
toward higher E2 and lower T concentrations in
the older subjects with a higher E2/T ratio;
however, this did not reach statistical significance (1.0
vs. 0.7; P = 0.09). There were also no
differences in FF levels of inhibin A or inhibin B, whereas older
subjects had significantly higher FF concentrations of total activin A
and total follistatin (Fig. 2).
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). The
abundance of the inhibin-follistatin complex did not change between FF
samples. Upon reduction, free follistatin of the expected size of 34
kDa was detected, and inhibin was released from a complex and detected
at the expected mature size of 32 kDa. The detected inhibin did not
reduce to - and ß-subunits under these conditions. No free activin
A was detected in any of the samples; therefore, activin A levels were
either below the sensitivity of the method or present only in a bound
form [once formed, the follistatin-activin complex is irreversible and
not recognized by the ß-subunit antibody (27)].
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. The intrafollicular concentration of
IGF-I was lower in the older subjects, and there was a trend toward
higher concentrations of IGFBP-2 (which would further decrease the
available IGF-I), but the difference did not reach statistical
significance (P = 0.18). Concentrations of IGF-II and
IGFBP-3 were similar in the two age groups.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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-subunit messenger ribonucleic acid is
expressed in small antral follicles in the macaque; therefore, activin
may be preferentially secreted by these smaller follicles, whereas
inhibin is secreted by the more mature antral (Graffian) follicles
(28). Granulosa cells possess activin receptors
(15), and in vitro and in vivo
studies in rodent models have demonstrated both stimulatory and
inhibitory effects of activin on follicle development and function
(8, 15, 16, 29). It is likely that activin has variable
effects on the follicle that are dependent on the stage of follicle
development and/or other local factors (e.g. relative levels
of other intraovarian hormones and growth factors). In the present
study we have begun to characterize the potential contributions of
activin, follistatin, and the IGF system to the process of reproductive
aging by examining their FF concentrations in the preovulatory
follicle. Our findings indicate that both inhibin and activin are present in the FF as a complex with follistatin, and that inhibin is the predominant FF form of the inhibin/activin dimers. There was no evidence of free (bioactive) activin in the FF samples from either our older or younger subjects. Using gel permeation chromatography, previous investigators have reported that the detectable activin A in human FF (eluted with apparent molecular mass >70 kDa) is present in a bound form, with little if any free activin A (25 kDa) present (28). The fact that both total activin A and total follistatin were elevated in the FF of older subjects suggests that elevated follistatin production compensates for the increased FF levels of activin A, thus potentially conferring a protective effect against activin. The fact that the preovulatory dominant follicle does not appear to contain significant levels of free (bioactive) activin does not eliminate the possibility that activin plays an important paracrine role in the early stages of follicle selection and development. In human FF, follistatin is found in levels far exceeding those in serum, and there appears to be a relatively high steady state concentration of follistatin that does not differ among normal dominant, atretic, or small antral follicles (30). This argues against changes in follistatin production as a major factor in the regulation of follicle selection and atresia.
IGF-I has been shown to serve as an intraovarian regulator of follicle function in rodents, and it exerts direct effects on human and rodent granulosa cell function (31, 32). In vitro, IGF-I, in conjunction with gonadotropins, appears to have a role as a promoter of follicle growth (33) and steroid secretion (34, 35) and as an antiatretic hormone (36). In vivo, adjuvant therapy with GH increased sensitivity to exogenous gonadotropins in women undergoing controlled ovarian hyperstimulation associated with elevated FF levels of IGF-I (37). In the current study older subjects had lower FF concentrations of IGF-I and a trend toward higher concentrations of IGFBP-2. Together, these changes would result in a reduced local hormone effect. If there is a decrease in the bioavailable IGFs, this may have some relevance to alterations in oocyte maturation, as it has been shown that IGF-I can accelerate oocyte maturation in vitro (38). The possibility that activin A action could be mediated in part through the IGF system is supported by the finding that activin A promoted IGFBP production by cultured human granulosa cells (39).
The finding of elevated VEGF in the FF of older subjects is interesting, given that VEGF production may be enhanced in response to hypoxic stimuli (20). Van Blerkom and co-workers demonstrated that oocytes from follicles with reduced FF oxygen content after controlled ovarian hyperstimulation have an increased rate of abnormalities of the meiotic spindle in the metaphase II oocyte (21). Whether there is a relationship between ovarian perfusion, intrafollicular oxygen content, VEGF secretion, and the meiotic spindle abnormalities observed in older women deserves further study.
To assure uniform exposure among subjects to a luteinizing stimulus (as well as to make follicle aspiration and oocyte retrieval logistically possible), it was necessary to administer exogenous hCG before follicle aspiration. In a prior study using separate groups of normal women selected in the same manner, we noted that the groups demonstrated no differences in follicle size or peak E2 levels at the time of the spontaneous LH surge (1). Although the dose, criteria for administration, and duration of exposure were uniform among subjects, the possibility that differential responses to exogenous hCG could either obscure or exaggerate physiological differences between groups cannot be entirely excluded.
In conclusion, the dominant follicle in older reproductive age women with FSH elevation appears to be compensated in terms of hormone secretion despite accelerated follicular development. This supports the concept that early follicular phase inhibin B deficiency reflects decreased numbers of antral follicles rather than reduced granulosa cell secretory capacity. One could speculate that similar to the premature ovulation and follicle atresia induced experimentally by exogenous activin in the rat (16), an enhanced paracrine effect of activin A may promote the accelerated follicle development, follicle depletion, early ovulation, and oocyte abnormalities observed in older women. Further studies are needed to clarify the roles of activin and inhibin dimers and the IGF hormones in aging of the female reproductive system, particularly in the early stages of follicle selection and development.
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Acknowledgments |
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Footnotes |
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Received January 4, 2000.
Revised April 21, 2000.
Revised August 28, 2000.
Accepted September 2, 2000.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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and activin A promote insulin-like growth factor-binding protein-2 and
-4 accumulation by human luteinizing granulosa cells, and
interferon- promotes their apoptosis. J Clin Endocrinol Metab. 83:179–186.This article has been cited by other articles:
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S. L. Franklin, R. J. Ferry Jr., and P. Cohen Rapid Insulin-Like Growth Factor (IGF)-Independent Effects of IGF Binding Protein-3 on Endothelial Cell Survival J. Clin. Endocrinol. Metab., February 1, 2003; 88(2): 900 - 907. [Abstract] [Full Text] [PDF]
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