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Original Studies |
Reproductive Sciences Program and Department of Pediatrics and Pathology and Nursing (V.P., D.S.M., N.B., N.E.R., A.R.M.), University of Michigan, Ann Arbor, Michigan 48109-0404; and the Reproductive Endocrine Unit (P.M.S., Q-F.W., R.H.K., A.L.S., W.F.C.), Massachusetts General Hospital, Boston, Massachusetts 02114
Address all correspondence and requests for reprints to: Vasantha Padmanabhan, Ph.D., Reproductive Sciences Program, University of Michigan, 300 N. Ingalls Building, Room 1110, Ann Arbor, Michigan 48109-0404. E-mail: vasantha{at}umich.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-2-macroglobulin do not
cross-react or interfere in this assay. Preincubation of FS with
activin results in dose-dependent loss of immunoreactivity, confirming
specificity of the assay for free FS. Human follicular fluid, pituitary
extract, and serum with added FS dilute parallel with the recombinant
human FS-288 standard. Recovery of recombinant human FS-288 from serum
is quantitative. Using this assay, we found circulating concentrations
of free FS to be at or below the detection limit of the assay
throughout the menstrual cycle. Comparison of circulating total and
free FS levels in postmenopausal or cycling women and normal men
suggested that at least 90% is activin-bound. In contrast, measurable
quantities of free FS were found in follicular fluid and pituitary
extracts. The results of this study, showing that most circulating FS
is normally activin-bound, argue against an endocrine role for FS and
suggest that a major role of circulating FS is to bind and neutralize
the bioactivity of circulating activin. The roles of FS as a local
autocrine or paracrine regulator of activin in target tissues, where FS
exists in free form, or as an endocrine regulator in human
pathophysiology, warrants further investigation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Like inhibin and activin, FS seems to be synthesized not only in gonadal but also in other tissues, including hypothalamus, pituitary, kidney, adrenal, and placenta (11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22). In addition to its postulated endocrine effects (23), FS seems to act in an autocrine/paracrine manner at both the gonadal and pituitary levels (11, 24, 25, 26, 27, 28, 29). All of the bioactivity of FS described to date seems to be caused by its high-affinity binding and subsequent inactivation of activin (15, 28, 30, 31, 32). FS also binds inhibin through the common ß-subunit (33), although it has a higher apparent affinity for activin than inhibin (Kd in the range of 10-9 to 10-10 mol/L) (34). FS can completely block the effects of activin both at the ovarian and pituitary levels (15, 25, 26, 27, 28, 30, 31). Because no independent effect for FS has been identified, its biological actions seem to be mediated solely through neutralization of activin activity. The relative amounts of activin-free and activin-bound FS are important considerations in determining the bioavailability of activin and, therefore, evaluating the potential endocrine role of circulating FS.
Using a recently developed polyclonal RIA that measures total FS (activin-bound + free), we (35), like others (36, 37, 38, 39), found relatively high (10–20 ng/mL) circulating total FS levels in both men and women. Furthermore, the levels of total FS were found not to change during different phases of the menstrual cycle (35, 37). Total FS levels also have been reported to increase during pregnancy (38, 40), aging (38), after in vitro fertilization (IVF) treatment (39), and after GnRH antagonist treatment of GnRH-deficient men (41). To determine whether the circulating FS existed in the free or activin-bound state, we have used two monoclonal antibodies with nonoverlapping epitopes to develop and validate an immunoradiometric assay that detects free FS (42). In this report, we used a second-generation solid-phase immunochemiluminescent assay (SPICA) to show that if any human FS circulates free of activin, it must be in low concentrations (<1 ng/mL) that represent less than 10% of the total FS measured in peripheral serum from men and women. These results question any endocrine role for FS in men and normally cycling women.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recombinant human FS (rc-hFS-288) was prepared by Dr. Nicholas
Ling under contract NO1-HD-0–2902 of the National Institute of Child
Health and Human Development and generously provided through the
National Hormone Pituitary Program of the NIH. Recombinant human
inhibin-A (rc-hINH A) and human activin-A (rc-hACT A) were provided by
Genentech (San Francisco, CA). Two monoclonal antibodies, with
nonoverlapping epitopes raised to rc-hFS-288, were used in this study.
The generation and specificity of these monoclonal anti-FS antibodies
were described previously (42). Horse serum-based diluent used in FS
calibrators, superparamagnetic particles, the dimethyl acridinium ester
used for labeling antibodies, and the magnetic rack for particle
separation (Corning Magic Rack, Cat. no. 472231) were obtained from
Chiron Diagnostics Inc. (Walpole, MA). Human 2-macroglobulin
(2M), transforming growth factor-ß (TGF-ß), and GnRH were
obtained from Sigma (St. Louis, MO). Human LH (hLH) and hFSH were
provided through the National Hormone Distribution Program of the NIH.
BSA and all other reagents used in preparation of buffers were obtained
from Aldrich (Milwaukee, WI). Assay buffer was prepared using reverse
osmosis- and Milli-Q-filtered, double-distilled water
(ddH2O) and was comprised of 0.01 mol/L sodium phosphate,
0.2 mol/L NaCl, 0.01 mol/L EDTA, 0.5% Tween-20, and 0.1% BSA at pH
7.0.
Immunoassays
Total FS assay. Concentrations of total FS (activin-free plus activin-bound) in human circulation, pituitary extracts, and follicular fluid were measured using a previously validated double-antibody, competitive RIA (35). Circulating levels of total FS in men and women have been reported earlier (35). Concentrations of total FS in a subset of the samples from the original study that were also measured in the free FS assay are reported here for comparative purposes. Activin and inhibin neither cross-react in the assay nor interfere with FS measurements, suggesting that the assay detects activin-bound, as well as free, FS. The limit of detection of the total FS assay is 0.4 ng/tube, with intra- and interassay coefficients of variation each averaging less than 12% (35).
Free FS (activin-free FS) assay. The assay format involves an anti-FS monoclonal detection antibody conjugated to the dimethyl acridinium ester (anti-FS-7FS30 DMAE) and an anti-FS monoclonal capture antibody conjugated to superparamagnetic particles (anti-FS-6FS7 PMP), to effect separation. Briefly, a sample is incubated with anti-FS-7FS30 DMAE, followed by a second incubation with anti-FS-6FS7 PMP, and then, after washing, treated with acid and then peroxide anion to initiate light emission (43). Standard curves are generated, based on increasing relative light units (RLU) determined in a Magic Lite Analyzer-II (MLA-II, Chiron Diagnostics Inc.).
Preparation of anti-FS-7FS30 DMAE
An Ig-rich fraction of anti-FS, clone 7FS30 (Massachusetts General Hospital), was purified by affinity chromatography using protein G (Immuno Plus; Pierce Chemical Co., Chicago, IL) and conjugated to N-hydroxysuccinamide-activated DMAE (44, 45, 46) using the method of Weeks et al. (47). The resulting labeled antibody was diluted to 50 mL in assay buffer, resulting in an IgG concentration of 7.2 µg/mL. The working concentration (a further 1:700 dilution) for the assay was calculated to provide 350,000 RLU/100 µL.
Preparation of anti-FS-6FS7 PMP
An Ig-rich fraction of anti-FS, clone 6FS7 (Massachusetts General Hospital), was purified by affinity chromatography on protein G and conjugated to superparamagnetic particles using the method of Groman et al. (48). The particles were washed and suspended in 25 mL assay buffer. The working concentration was a 1:10 dilution of this concentrate, which provided 8.56 µg antibody coupled to 54.0 µg PMP per tube.
Assay description
FS standard was prepared from rc-hFS-288 in horse serum diluent (Chiron Diagnostics Multi Diluent). The horse serum was used to minimize assay matrix effects by standardizing the protein concentration in each assay tube. Other similar products (Diagnostic Products Corporation, for example) are readily available and may be substituted in the assay. Using 12 x 75-mm glass test tubes, 100 µL of standard or test sample (human serum, follicular fluid, or culture incubates) was added and diluted to 200 µL with assay buffer. This was followed by the addition of anti-FS-7FS30 DMAE (100 µL). The tubes were shaken by hand and incubated overnight (18–20 h) at room temperature. Anti-FS 6FS7-PMP (100 µL) was added, and the tubes were again shaken by hand and incubated overnight at room temperature. Twenty-four hours later, tubes were shaken by hand to resuspend the particles. Separation of the bound antibody complex was performed magnetically, supernatant was decanted, and the particles were washed with a further two 1.0-mL aliquots of assay buffer, followed by 1.0 mL ddH2O. The tubes were briefly vortexed, 100 µL of ddH2O was added, and the tubes were read in an MLA-II by injecting 300 µL of 0.1 mol/L HNO2 and 0.5% H2O2, followed by 300 µL of a solution containing 0.5% Arquad, a surfactant (Azko Chemical Co., Chicago, IL), and 0.25 mol/L NaOH, to initiate light emission. The MLA-II was programmed to read for a 2-sec integral, with data output as RLU’s.
hLH Assay. Circulating concentrations of hLH were measured using the Ciba Corning ACS:180, two-site, chemiluminometric (sandwich) immunoassay, a commercially available clinical assay (Chiron Diagnostics Inc.) that uses two antibodies with specificity for intact hLH, a polyclonal sheep anti-LH antibody labeled with acridinium ester, and a monoclonal mouse anti-LH antibody covalently coupled to paramagnetic particles. Values are expressed in terms of the WHO 2nd IRP 80/552 reference material. The detection limit and intra- and interassay coefficients of variation of the assay were 0.6 mIU/mL, 3.2%, and 8.5%, respectively.
Preparation of rc-hFS-315
The hFS-315 complementary DNA (cDNA) (kindly provided by Dr. Shimasaki, Whittier Institute, LaJolla, CA) was subcloned into PCDNA3 (InVitrogen, Carlsbad, CA) for expression in mammalian cells. COS cells (ATCC) were transfected using the diethylaminoethyl ether dextran method (Maniatus), and geneticin-resistant clones were screened for FS secretion using a solid-phase FS-binding assay and radiolabeled activin as trace, as previously described (34), as well as by FS SPICA. Two batches of conditioned medium from the highest secreting clone (58–2-22) were tested in this study. On SDS-PAGE, numerous bands between 32 and 41 kDa were observed that were recognized by anti-FS monoclonal antibodies (data not shown), suggesting that at least some of these FS315-derived proteins may be proteolytically processed after translation, yet recognizable by the assay.
Human samples
Collections of samples from all male and female subjects were reviewed by the Institutional Review Boards for human studies at the University of Michigan or the Massachusetts General Hospital. All serum samples were obtained from volunteers after obtaining informed consent. All blood and follicular aspirates from patients participating in an IVF program were collected at the time of routine blood sampling and aspirations for IVF, after obtaining informed consent from volunteers participating in the studies. Human serum samples were handled according to the guidelines of safe laboratory practices. Human pituitaries were obtained from the National Hormone and Pituitary Program. Pituitary extracts were prepared by dounce homogenization in PBS-BSA [10 mmol/L, pH 7.2, 3% BSA].
Statistical analysis
Standard curve parameters were calculated via a four-parameter, logistical function using AssayZap version 2.32 (BioSoft Inc., Cambridge, UK). Parallelism of assay curves was evaluated by ANOVA (F-test). Assay curves were judged to be parallel if the P-value for the test of the sum of squares associated with parallelism was greater than 0.05. FS concentrations in different physiological states were compared by ANOVA, followed by Scheffé’s test statistic. Changes between total and free FS during a given physiologic state were compared by paired t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Matrix effects. Comparison of dose-response relationships of
rc-hFS-288 (standard) in gel PBS, and heparin-stripped (removes FS)
human serum (35) showed that human serum interfered with and reduced
light output (Fig. 1
, top). To
eliminate this effect of serum, the standard curve was prepared in a
high-protein, horse serum diluent (Chiron Diagnostics Multi Diluent),
which closely mimics the protein concentration of human serum.
Comparison of rc-hFS-288 dose response curves in Multi Diluent and
heparin-stripped serum (35, 42) revealed no differences in RLU output
(P = 0.70) (Fig. 1, bottom).
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(left) shows FS dose-response curves using rc-hFS-288 as the
assay standard in the FS SPICA assay using Multi Diluent. A log,
dose-logit-response curve was linear from 1–1000 ng/mL (R2
= 0.993), with a minimum detection limit, defined as the concentration
that exceeded by 2 standard deviations the mean RLUs of tubes
containing no FS, of 1.0 ng/mL (0.1 ng/tube) (Fig. 2, left).
ED50 was 50 ng/mL. The intraassay coefficient of variation
at doses of 2.2 and 128.3 ng/mL averaged 8.1% (n = 20) and 8.0%
(n = 19), respectively. The interassay CV at the same dose levels
averaged 14.2 and 12.5% (n = 4 assays).
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2M (0.01–10 mg), rc-hACT A (1–1000 ng), rc-hINH A (1–100 ng),
hFSH (1–1000 ng), hLH (10–10,000 ng), TGF-ß (2.5–1500 ng), BSA
(1–1000 ng), or GnRH (1–1000 ng), indicating the high specificity of
the FS SPICA (Fig. 2
, left). To study the possibility that
other circulating substances might indirectly affect the measurement of
FS, a variety of peptides and proteins also were tested in the presence
of various concentrations of rc-hFS-288. Human FSH (1 µg), hLH (10
µg), GnRH (1 µg), and TGF-ß (1 and 250 ng) did not interfere in
the FS assay (data not shown). Proteins derived from cells expressing
the 315 splice variant of FS (rc-hFS-315, 2 batches of medium with
expressed protein, B1 and B2, respectively) showed parallel
displacement to the 288 form of the rc-hFS (Fig. 2, right;
P = 0.09 and 0.12 for B1 and B2, respectively),
suggesting that the assay measures FSs derived from the 315 amino acid
variant, as well.
Preincubation of rc-hFS-288 assay standards with 2M (0.01–100
µg/tube), which shares functional homology with FS (49) or heparin
sulfate (10–1000 units/tube), to which FS is known to bind (5), or 1
ng/tube TGF-ß did not interfere in this assay (data not shown).
Preincubation of the rc-hFS-288 for 3 h at room temperature with
rc-hACT A (1–100 ng), however, showed a dose-dependent interference in
the assay (Fig. 3, left), with
a calculated Ki for this interaction of 150 nmol/L ± 12.3 nmol/L
(n = 3). In contrast, preincubation of rc-hFS-288 for 3 h at
room temperature with rc-hINH A (1–100 ng) showed no interference in
this assay, nor did inhibin overcome the interference caused by activin
(Fig. 3, middle). The interference that the addition of
activin provided in detecting FS was linear (Fig. 3, right).
Interference studies carried out in human serum pool (with no
detectable free FS) also showed that recovery of added FS was
suppressed in a dose-related manner with increasing concentrations of
rc-hACT A (Fig. 4A). Furthermore,
addition of rc-hACT A (80 ng) eliminated completely the detection of
rc-hFS-288 from human serum samples of various physiologic states
(these samples had no detectable free FS before rc-hFS-288 addition)
(Fig. 4B).
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, top left; P
= 0.14, 0.16, and 0.16 for FF1, FF2, and pituitary extract). In
contrast, increasing volumes of human serum from men and women during
various physiologic states showed essentially no measurable free FS,
even with volumes as high as 100 µL (Fig. 5, top right).
However after adding rc-FS-288 to serum from various physiological
states, dilution of human serum generated response curves parallel to
the standard curve (Fig. 5, bottom; P = 0.31
female, 0.48 IVF, and 0.24 male).
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Levels of FS in various physiological states. The mean
circulating pattern of FS, free of activin, in daily samples of serum
collected at 1- to 2-day intervals from normal cycling women, is
presented in Fig. 6. Though all five
women studied exhibited the characteristic preovulatory LH surge,
circulating activin-free FS was near or below assay sensitivity
throughout the entire menstrual cycle. Circulating levels of total FS
in cycling women (during different stages of the menstrual cycle),
postmenopausal women, and men have been reported earlier (35). To
determine the relative proportion of circulating total FS and free FS
levels, a subset of these samples from the original study (35) also
were measured in the SPICA assay, which is specific for detecting
activin-free FS. Table 1 shows the
relative levels of total and free FS in human serum obtained from this
subset of cycling women, postmenopausal women, and men. Whereas
measurable levels of total FS were found in both women and men, free FS
was not measurable in cycling or postmenopausal women or in normal
men.
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and Table 1), the majority of the FS in the follicular
fluid aspirations during IVF was measurable, using the SPICA method
(Fig. 7). Measurable amounts of free FS
also were found in human pituitary extracts (n = 2) (data not
shown). Free FS concentrations in the two human pituitary extracts
measured were 32.4 and 36.4 ng/mL, respectively.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The newly developed immunoassay reported here: 1) was specific
for FS proteins derived from either of the 288 or 315 amino acid splice
variants (Fig. 2); and 2) did not measure FS bound to activin (Figs. 3 and 4). Thus, the assay is specific for detecting free FS that is not
bound to activin. The basis for the specificity of this two-site SPICA
assay is unknown. However, it seems that regions of the protein
encompassing epitopic sites to which one or both of the monoclonal
antibodies bind may be involved in the binding of activin to FS.
Alternatively, FS may, upon binding to activin, undergo conformational
changes that influence the ability of one or both antibodies to bind to
FS. The activin interference studies described in this manuscript were
carried out using rc-hACT A. Unavailability of activin B and activin AB
precluded us from undertaking similar studies with these activin
variants. On the contrary, although FS has been shown to bind inhibin
(33, 34), inhibin did not affect the ability of the immunoassay to
measure FS. This finding suggests that either the affinity of the
antibodies for FS is sufficiently greater than that of inhibin for FS
to allow measurement of FS in the presence of FS-inhibin complexes or
that the assay measures the FS-inhibin complex, as well as free FS
per se. The affinity of inhibin for FS has been shown to be
considerably weaker than that of activin for FS (34).
Although the precise isoform specificity of this assay is not fully
characterized, our measurements indicate that recombinant FSs derived
from either of the two recognized messenger RNA splice variants can be
measured (Fig. 2), and the antibodies bind to both glycosylated and
deglycosylated forms of these proteins (50). The SPICA assay allowed
the quantitative measurement of endogenous free FS in human follicular
fluid and of rc-hFS added to human serum (Fig. 5; recovery averaged 93
and 103% at 0.5 and 5 ng of added FS). In contrast, no endogenous free
FS was detectable in serum from normal men, normally cycling women, or
postmenopausal women. Measurable quantities of free FS were, however,
found in extracts of human pituitary glands, another recognized site of
FS synthesis and secretion in many species (11, 13, 15, 41, 51, 52).
Considering that the detection limit of the free FS SPICA assay is approximately 1 ng/mL and roughly 10 ng/mL of total FS is found in serum from men, postmenopausal women, or normally cycling women (35), it seems that less than 10% of the total FS circulates in an activin-free state. A higher percentage (24%) of free FS was estimated to be in circulation by Sakamoto et al. (53) using a competitive protein binding assay. The inconsistencies in estimates of free FS in the two reports may reflect, on the one hand, differences in the ability of the two assays to recognize various forms of FS, or alternatively, a function of the nature and purity of the standard used; rc-hFS-288 was used as the assay standard in the SPICA assay, and rc-hFS 315 was used as the assay standard by Sakamoto et al. (53). Another important methodological difference is the use of 125I-activin and detergents in the competitive assay (53). This may have led to displacement of some of the activins bound to the FS by the 125I-activin, resulting in an overestimation of the free FS levels in serum by the competitive assay. Irrespective of these differences, the majority of circulating FS seems to be in an activin-bound state in normal men and women. Because of the essentially irreversible nature of the FS-activin complex (34), the results of these studies also suggest that most of circulating FS is likely to be in a biologically inactive state.
In contrast to low concentrations of free FS in the circulation, in the ovary (e.g. follicular fluid) and pituitary, substantial amounts of free FS were found. Although more direct studies are required, this observation, though preliminary, suggests that free FS may be secreted from these sources but then complexes with activin in the circulation. These considerations are consistent with our previous suggestion, based on measurement of peripheral total FS (35), that the protein is an unlikely endocrine factor but seems to be an important element in limiting the bioavailability of circulating activin.
Support for the premise that the role of FS in circulation is to limit
activin availability also comes from studies quantifying activin A and
B in circulation. Using an immunoassay method specific for free activin
A and B, Woodruff and colleagues (54) report that very little
biologically active free activin A or B circulates in nonpregnant
women. Further support for this premise comes from chromatographic
studies of Muttukrishna et al. (55), which show that almost
all of the activin-A in pregnant women is in a bound state. In direct
contrast to these reports, Demura et al. (56), using a
competitive protein-binding assay, estimated much higher levels of
circulating free activin (
2 ng/mL). The differences between reports
in estimating free circulating activin (54, 56) may relate to
differences in the ability of these assays to measure various
circulating forms of activin (A, B, and AB). The difference between
free FS and total FS measurements in circulation suggest that
circulating activins should be high (8 ng/mL), far more than
estimated by the activin A assay alone. Circulating total activin A
concentrations average only 100–200 pg/mL during the human menstrual
cycle, with approximately 5-fold greater concentrations seen in
postmenopausal women (57).
Though it is premature, and may be erroneous, to make quantitative comparisons across published reports involving different patient populations, different assay formats and standards, these data bring to the forefront the complicated relationship existing between FSs and activins and perhaps other proteins in circulation. Thus, although it is becoming clear that FSs may not function as endocrine factors during reproductive cyclicity, the autocrine/paracrine relationship between activin and FS, in potentially regulating the bioavailability of activin, and a possible endocrine role for FS during human pathophysiology warrants further study.
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Acknowledgments |
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Footnotes |
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Received April 8, 1997.
Revised July 9, 1997.
Revised October 23, 1997.
Accepted November 12, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), 10 (
),
and 100 (
) ng of rc-hACT A (A). Middle, Effect of
preincubation (3 h) of FS with 0, 1, or 10 ng of rc-hINH A (I) or
rc-hINH A (0, 1, and 10 ng), and rc-hACT A (10 ng) on subsequent
detection of FS in the SPICA assay. Inhibin did not interfere with the
ability of the antibodies to recognize FS or with the ability of FS to
form FS-activin complexes. Right, Effect of increasing
doses of rc-hACT A on the detection of 10 ng FS in the activin-free FS
assay.
