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Reproductive Endocrinology |
Prince Henry’s Institute of Medical Research (D.M.R., N.C., J.K.F., H.G.B.), Monash Medical Centre, Clayton Victoria 3168, Australia and Oxford Brookes University (N.G.), Oxford, OX3 OBP United Kingdom
Address all correspondence and requests for reprints to: David Robertson, Ph.D., Prince Henry’s Institute of Medical Research, Monash Medical Centre, Clayton Victoria 3168, Australia.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit-directed immunoassays. It was shown,
however, that the ovine in vitro bioassay detected
inhibin B poorly. These findings are extended in the present study by
the determination of the molecular weight profile of in
vitro bioactivity using rat pituitary cells, which detects both
inhibin A and B, a specific inhibin B enzyme-linked immunosorbent assay
(ELISA), an RIA detecting the N region of the -subunit, an
-subunit ELISA (Pro-C) directed to the inhibin forms containing
the Pro sequence, and an C subunit immunofluorometric assay that
detects all inhibin forms. The profile in hFF of inhibin in
vitro bioactivity, using rat pituitary cells in culture,
significantly (P < 0.001) correlated with
in vitro bioactivity using ovine pituitary cells (r
= 0.85), inhibin A immunoactivity (r = 0.70), inhibin B
immunoactivity (r = 0.89), and the combination of inhibin A+B
immunoactivities (r = 0.93), with peaks of activity identified at
66K, 55K, 36K and 33K, consistent with presumed known mol wt forms of
inhibin. Inhibin B profiles in fractionated serum from women stimulated
with gonadotropins and male plasma consisted of two forms (66K and
36K), whereas inhibin A in female serum included, in addition, the 55K
form. These findings indicated that higher molecular weight forms of
inhibin B are present in biological samples, and their distribution
differs from that of inhibin A, suggesting a differential processing of
the precursor forms in the circulation. Pro-C immunoactivity was
identified in serum samples with prominent peaks at 36K and 29K (known
Pro-C subunit forms) and not with any high mol wt dimeric forms of
inhibin. If this observation applies to a wider range of serum samples,
the Pro-C ELISA may provide an appropriate and specific assay for
the measurement of free -subunit. To compare immunoactivity levels
between assays, the inhibins A, B, and Pro-C standards were
calibrated in terms of their C subunit content, as determined by an
C subunit immunoassay, with the inhibin B standard containing 60%
of the C subunit content compared with either the inhibin A or
Pro-C standard. After adjustments of the various standards for this
difference in C subunit content, a comparison was undertaken of the
combined levels of inhibins A, B, and Pro-C immunoactivity across
the hFF and serum chromatograms and compared with levels determined by
the -subunit-directed immunoassays. A high correlation (r =
0.59–0.96) was observed, indicating that the -subunit
immunoactivity in serum consists largely of a composite of presumed
known molecular weight forms of inhibins A, B, and Pro-C. It is
concluded that: 1) inhibin in vitro bioactivity in hFF
is largely attributed to the presence of 33–36K and 50–66K forms of
inhibins A and B; and 2) inhibin -subunit immunoactivity in hFF and
serum is a composite of presumed known forms of inhibin A, inhibin B,
and the -subunit. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and either ßA (inhibin A) or ßB (inhibin B). A large
number of molecular weight forms of inhibin have been identified
(5, 6, 7, 8, 9, 10), which are attributed to processing of its precursor subunits
at recognized basic amino acid cleavage sites after dimerization or to
differential glycosylation with the presence of one or two carbohydrate
chains located in the carboxy region of the -subunit. In addition,
various processed forms of the biologically inactive -subunit have
now been identified (11, 12, 13, 14). The biological activity of many of the
forms of inhibin seems to reside in the mature 30K form. However, an
NH2-terminal region of the -subunit (termed N
subunit) seems to exhibit a different activity to inhibin (15).
Furthermore, it has been shown (8) that a dimer of the full-length
unprocessed - and ß-subunits, where the processing sites have been
mutated to prevent cleavage, was bioinactive in suppressing FSH
in vitro.
It has become apparent that inhibin and its -subunit may exist in
biological samples as a range of molecular weight forms. In a previous
companion paper (7), inhibin from human follicular fluid (hFF) and
serum/plasma from men and women was fractionated according to molecular
size and the profiles of inhibin in vitro bioactivity, and
immunoactive inhibin A and -subunit containing inhibin forms
identified. Inhibin was found to be present in these fluids in all the
recognized forms with some evidence of additional cleavage products.
Several issues arose out of this study that required further
clarification. Firstly, this study showed that the in vitro
bioassay employed using ovine pituitary cells, though highly sensitive
to inhibin A, was relatively insensitive to inhibin B, and thus, the
in vitro bioassay pattern was probably reflecting inhibin A
only. Secondly, the pattern of inhibin B was unknown. These aspects are
examined in the present study, with the addition of an in
vitro bioassay (using rat pituitary cells), which detects both
inhibins A and B, an RIA that detects the N region of the
-subunit and the recent availability of a specific inhibin B
enzyme-linked immunosorbent assay (ELISA). An -subunit ELISA
(Pro-C) directed to the inhibin C subunit containing the Pro-
sequence and an -subunit immunofluorometric assay (IFMA) that
detects all inhibin forms containing the -subunit were also
examined.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Molecular weight designations employed in this study are similar
to those used previously (7). For a further elaboration of molecular
weight forms of human inhibin, see Mason et al. (8). Known
forms of the inhibin -subunit are Pro-N-C (50–60K), N-C
(45K), and Pro-C (26–30K). Known forms of dimeric inhibin A and B
are (Pro)-N-C/proß-ß (90–105K), C/proß-ß (75K),
(Pro)-N-C/ß (55–66K), and C/ß (30–34K). Two glycosylated
forms of the C subunit have been identified that result in
additional molecular weight forms differing by 3K. These glycosylated
forms are more evident in the 20–40K region of the chromatograms. In
this and the previous study (7), mol wt for some inhibin forms were
different from that reported (8), e.g. inhibin /ßA
(30–34K) is shown in this study with mol wt of 33–36K and
C/proß-ß (75K) migrates at 66K. These differences are attributed
to methodological differences, including choice of protein standards
used in the assessment of molecular weights in the SDS-PAGE.
Preparations
The first international standard for inhibin A, (human
recombinant, 91/624, 30–34K) was obtained from the NIBSC, Potters Bar,
UK. To compare data with the previous study, in vitro
bioactivity and immunoactivity were expressed in terms of its nominal
vial content (5 µg). To relate data to the first international
inhibin standard, an adjustment factor of 30,000 IU/µg is used (16).
Recombinant human inhibin B (30K) was a gift from Dr. J. Mather and
used in the previous study (7). The Groome inhibin B standard was
purified from hFF on an immunoaffinity column consisting of immobilized
C subunit directed monoclonal antibody (R1), eluted in guanidinium
hydrochloride, diluted in water containing 1% BSA and lyophilized
(17). Pro-C preparation used as standard was a purified preparation
consisting of two mol wt forms (24K and 27K) (18). Some aspects of the
purity of these preparations are included in Table 1
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The samples investigated originated from an earlier study (7).
hFF, serum from women undergoing gonadotropin treatment as part of an
in vitro fertilization program (IVF serum), plasma from
women after menopause, and male plasma were fractionated by a combined
procedure consisting of an inhibin C subunit immunoaffinity
purification step, reversed phase high performance liquid
chromatography (HPLC) and preparative PAGE (Prep-PAGE). The gel slices
were electroeluted and the inhibin removed from the SDS elution buffer
by methanol precipitation. The same sample aliquots used in the
previous study (7) were used in the present study, to allow direct
comparisons.
In vitro bioassay using rat pituitary cells in culture
The method of Scott et al. (19) was employed, except FSH release, rather than FSH content, was used as assay endpoint. Inhibin A (91/624) was used as standard.
In vitro bioassay using ovine pituitary cells in culture
The method of Tsonis et al. (20) was employed. Data presented in this paper using this method was presented previously (7) and is included for comparison purposes. Inhibin A (91/624) was used as standard.
Inhibin A (ßA) ELISA
The method of Groome and O’Brien (21) was employed. Data presented in this paper using this method was presented previously (7) and is included for comparison purposes. Inhibin A (91/624) was used as standard.
Inhibin B (ßB) ELISA
The method of Groome et al. (17) was employed without
modifications. The monoclonal antibody (C5) to a carboxy terminal
peptide of the inhibin ßB subunit was employed as capture antibody
with the Fab-alkaline phosphatase conjugate of monoclonal antibody (R1)
to the -subunit peptide (1–32) of human inhibin C subunit as
label. The alkaline phosphatase activity was amplified using the Gibco
kit (Life Technologies, Gaithersburg, MD). Samples were initially
boiled in 2% SDS and treated with hydrogen peroxide. The Groome
inhibin B preparation was used as standard. The sensitivity of the
assay was 15 pg/mL. The specificity of the assay is presented in Table 1
.
Pro-C subunit ELISA
The ELISA of Groome et al. (18) was employed without
modifications. The monoclonal antibody (INPRO13) directed against the
entire Pro region of human -subunit was used as capture antibody
with the Fab-alkaline phosphatase conjugate of monoclonal antibody (R1)
to the C subunit peptide of human inhibin as label. The alkaline
phosphatase activity was amplified using the Gibco kit. The Groome
Pro-C preparation was used as standard. The sensitivity of the assay
was 6 pg/mL. The specificity of the assay is presented in Table 1.
N subunit RIA
The RIA consisted of an antiserum (no. 52) raised in sheep to a
fusion protein fragment of the bovine N-subunit precursor region
(amino acids 1–26) with iodinated peptide (amino acids 1–26 of bovine
N) as tracer and unlabeled peptide as standard (15). The sensitivity
of the assay was 16 pg/tube. Recombinant human inhibin A showed no
cross-reaction with the addition of 2500 pg/well (<0.6%) in the
assay. Insufficient sample was available to apply this assay across the
serum Prep-PAGE chromatograms.
C subunit IFMA
A two-site IFMA was developed with the aim of detecting all C
subunit-containing inhibin forms. The capture antibody used was a
caprylic acid IgG cut of a sheep polyclonal antibody (no. 128) raised
initially against human inhibin C subunit fusion protein (22) and
subsequently boosted with recombinant inhibin A. The antibody used as
label was a sheep polyclonal antibody (no. 41) raised against human
inhibin C subunit fusion protein (22), immunopurified against bovine
inhibin C subunit fusion protein, and biotinylated (4 h at room
temperature) using the biotin-isothiocyanate procedure (Wallac, Turku,
Finland) in 50 mmol/L Na2CO3 buffer, pH 9.8, at
a 60- to 90-fold molar excess of biotin reagent. The sheep antisera and
fusion protein were gifts from Biotech Australia P/L, Sydney,
Australia. Ninety-six well microtitre plates (Maxisorb, Nunc, Roskilde,
Denmark) were coated with 2 µg/well capture antibody in glycine
buffer, pH 4.4, and blocked for 1 day at room temperature with 50
mmol/L Tris/HCl, 1% BSA, pH 7.5. Assay buffer used was 50 mmol/L
Tris/HCl, 0.154 mol/L NaCl, 0.1% NaN3, 0.5% BSA, pH 7.4
(TSA.BSA) containing 0.1% ovine IgG. Sample and standard (200 µL)
were incubated in the antibody-coated microtitre plate for 2 h at
room temperature with shaking. The plate was then washed and
biotinylated antibody (200 ng/well) was added and incubated a further
2 h at room temperature. After washing, Eu-streptavidin
(50ng/well, Wallac) was added, incubated 30 min, washed 6 times, and
enhancement solution (23) was added. The plates were read on a Wallac
1234 Fluorometer. The dose response curves were log-log transformed
and, where appropriate, potencies determined using parallel-line
bioassay statistics. The sensitivity of the assay was 30 pg/mL (or 0.9
IU/mL) in terms of the inhibin A (91/624) standard (16) with a working
range of 30–50,000 pg/mL. Inhibin A (91/624) was used as standard.
Reductive alkylation
Two to three samples of each preparation (Table 1) were diluted
in an equal volume of 0.2 mol/L Tris/HCl, 6 mol/L guanidinium
hydrochloride, 2 µmol/L EDTA, pH 7.5. Dithiothreitol (2:1 wt/wt
protein) was added and incubated at 37 C for 3 h. Iodoacetic acid
(2:1 wt/wt dithiothreitol) in 1 mol/L Tris/HCl, pH 7.9, was added and
incubated for 15 min in the dark. The samples were gel filtered in
TSA.BSA buffer before C-subunit determination by IFMA.
Previous studies (24) had shown that the oxidized form of inhibin A,
which is present to a variable extent in inhibin preparations,
exhibited a higher affinity for an inhibin monoclonal antibody in an
ß-ELISA, resulting in spurious immunoassay results. To ensure that
differences in measured C content between preparations were not
caused by this effect, the reduced and alkylated samples (100 µL)
also were oxidized with H2O2 (50 µL, 5%) and
incubated at room temperature for 30 min. TSA.BSA buffer (200 µL) was
added and samples assayed in the C subunit IFMA.
Statistical analyses
Prep-PAGE fractions were assayed at one dose level in all immunoassays. Prep-PAGE samples assayed in the inhibin in vitro bioassay and samples used in the specificity studies were assessed at multiple doses and assayed, where appropriate, by parallel-line statistics.
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Results |
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The specificity of the various immunoassays (Table 1) was
investigated. In the inhibin B ELISA, no significant cross-reaction
(<0.1%) was observed with inhibin A or Pro-C, although the two
inhibin B preparations showed different levels of activity. The inhibin
A ELISA also showed high specificity, as shown previously (7); however,
the two inhibin A preparations also showed different levels of
activity. The Groome inhibin B preparation contained significant
amounts of inhibin A and some Pro-C.
The differences in activity between the two inhibin A and the two
inhibin B preparations suggested that the designated mass estimates of
some of these preparations were not correct. If this is the case, then
the use of some of these preparations as standards in the immunoassays
will lead to an incorrect assessment of the levels of the various
inhibins in the samples. Because inhibins A, B, and Pro-C all
contain the C subunit, an C subunit IFMA was used that
quantitates the levels of the inhibin C subunit. Inhibins A, B, and
Pro-C were reduced and alkylated to release the -subunit from the
dimer, and the -subunit content was determined in the C IFMA
using inhibin A (91/624) as reference preparation. The immunoactivity
of the various preparations was increased 2- to 4-fold after reductive
alkylation, although similar relative values, compared with the
untreated samples (Table 1), were obtained. When standardized against
inhibin A (91/624) standard, large differences in -subunit content
were noted between preparations, ranging from 16–2914% of stated
values. The addition of the oxidative step did not change the
immunoactivities of the various preparations (Table 1).
Fractionation of hFF (Fig. 1)
A comparison of the inhibin in vitro bioactivity, as
determined using ovine pituitary cells (specific for inhibin A) and rat
pituitary cells (specific for inhibins A and B) across the hFF
Prep-PAGE chromatogram, showed highly comparable profiles (r =
0.85, P < 0.001) between activities. Similar peaks of
bioactivity were identified at 66K, 55K, 36K, and 33K, with 5-times
higher levels of bioactivity, using the rat in vitro
bioassay (Fig. 1).
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). The Pro-C immunoactive profile consisted
of peaks at 
55K, 33–36K, and 29K with no evidence of higher mol wt
forms. An immunoactive N subunit peak consistent with either
(Pro)-N-C or (Pro)-N-C/ß was identified as a relatively
broad peak at 55K. C subunit immunoactivity, as determined by C
IFMA, showed a similar pattern of immunoactivity to that seen with the
other -subunit immunoassays (7) with major peaks at 66K, 55K,
33–36K, and 29K (Fig. 1).
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and 3)
Inhibin B immunoactivity was determined with peaks at 60–66K and
36K only, with little or no immunoactivity at 55K (as seen with inhibin
A). Pro-C immunoactivity was detectable at 36K and 29K with low, but
detectable, levels elsewhere.
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C immunoactivity and -subunit immunoactivity, using the C
IFMA, were detectable as a broad range between 30K and 60K. In
addition, a peak at 20K, consistent with the presence of the C
subunit, was identified with the C IFMA.
Fractionation of male plasma (Fig. 4)
Male plasma showed two peaks of inhibin B immunoactivity at 66K
and 36K, similar to that seen for IVF serum. It was shown previously
(7) that no inhibin A activity was detected across this chromatogram.
Pro-C immunoactivity showed a similar pattern to that seen with hFF
and IVF serum, with peaks at 55K, 36K, and 29K. In addition, minor
immunoactive peaks determined by the C IFMA were observed at 100K
and 20K.
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C values across the
chromatograms according to the C subunit content of the respective
standards in the various assays
The relative levels of the inhibins A, B, and -subunit profiles
detected in the chromatograms are dependent, in part, on the designated
unitage of the standards employed in the various assays. To compare
values obtained between assays, the inhibins A, B, and Pro-C
standards were re-calibrated in terms of their C subunit content
using the C subunit IFMA (Table 1). The Pro-C standard showed
levels of C subunit similar to the inhibin A (91/624) standard. In
the case of the Groome inhibin B standard, because of its large content
of -subunit-like immunoactivity, its inhibin B levels were initially
measured in the inhibin B ELISA in terms of the highly purified
Genentech inhibin B preparation, which in turn, was adjusted for its
C subunit content after assay in the C subunit IFMA. These
adjustments in the calibration of the various standards resulted in a
change in the relative levels of the inhibin A, B, and Pro-C across
the various chromatograms. The largest change was with inhibin B, which
was 60% of its original value compared with inhibin A (100%) and
Pro-C (109%). A comparison between the hFF profile of inhibin
in vitro bioactivity using rat pituitary cells and the
profile of the sum of inhibin A and inhibin B immunoactivities showed a
very good correlation (r = 0.93) with a slope of the regression
line close to unity (Table 2, Fig. 1). A comparison between the
combined levels of inhibin A, B, and Pro-C immunoactivities and
-subunit immunoactivity determined with the various -subunit
immunoassays of both hFF and plasma/serum profiles also showed high
correlations, although the slopes of the regression lines varied
markedly between assays (Table 3,
Figs. 1–4).
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C in hFF and
serum/plasma (Table 4)
The levels of inhibin A, B, and Pro-C in hFF and serum, when
the standards are adjusted for their C subunit content and compared
with their C IFMA values, are presented in Table 4. The Pro-C
levels in hFF and IVF serum (Table 4) represent immunoactivity
identified in the 29–36K region only, excluding the 45–55K region
because the 45–55K region may be either -subunit monomer
(Pro-N-C) or ß-subunit dimer (Pro-N-C/ß).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Calibration of standards
Results from the specificity studies (Table 1) indicated that the
levels of C subunit in the various inhibin preparations were at a
variance with their stated mass values. For example, Genentech inhibin
B gave an C subunit content value of 16% compared with the inhibin
A (91/624) standard, indicating that the stated inhibin content of this
preparation was overestimated. This 16% value compares favorably with
the cross-reaction values previously observed (7) with the inhibin
in vitro bioassay (17.2%) and two immunoassays (Medgenix
- ELISA, 16.7% and inhibin RIA, 12.3%). These low values are in
contrast to that obtained with the Genentech Ck/Ck ELISA, where a value
of 82% was obtained (7). It should be noted that the Genentech Ck/Ck
ELISA measurements were undertaken at Genentech, whereas all other
stated assays were undertaken in Melbourne. These findings suggest that
for reasons unknown, the inhibin B preparation tested in Melbourne was
of reduced activity compared with the preparation tested by Genentech.
Based on the C subunit content of inhibin A and inhibin B, the
cross-reaction of inhibin B in the rat in vitro bioassay and
the various immunoassays is increased (now ranging between 77 and 104%
of that of inhibin A). Thus, inhibin B cross-reacts equally well with
inhibin A in the rat in vitro bioassay and various
-subunit immunoassays (Monash RIA and Medgenix - ELISA).
Nonetheless, the cross-reaction of inhibin B in the ovine in
vitro bioassay, with correction for C subunit content, is still
low (5.6%), indicating that ovine pituitary cells, in contrast to rat
pituitary cells, are relatively insensitive to inhibin B. These
findings highlight the need for high quality standards, particularly in
situations where comparisons between assays are intended. Thus, there
is a need for an international reference preparation for inhibin B, and
possibly Pro-C, as in the case for inhibin A (16).
hFF
The combined profile of inhibin A and B immunoactivity in hFF
correlated closely (r = 0.93) with inhibin in vitro
bioactivity, as determined with the rat bioassay, suggesting that the
sum of the two immunoassays provides a good measure of inhibin
bioactivity in this sample. However, based on the slope of the
regression line (Table 2), the combined inhibin A+B levels were 1.58
times higher. When the standards were corrected for their C subunit
content, the slope of the regression line was 1.1. These data strongly
suggest that the inhibin in vitro bioactivity in hFF is
largely attributed to the presence of various known molecular weight
forms of inhibin A and B.
The four peaks of inhibin immunoactivity and bioactivity (66K, 55K,
36K, and 33K) in the hFF chromatogram can now be broadly identified.
The 66K peak most likely consists of C/proß-ß, as assessed from
its mol wt, its bioactivity, and the absence of N subunit. No
discernible levels of either Pro-N-C/proß-ß or
Pro-N-C/ß were evident. The 55K region consists of
Pro-N-C and N-C/ß, based on the coelutions of dimer
activity, N subunit, and 55K Pro-C immunoactivity, although it is
not possible to establish the relative proportions. Free N would not
be detected in this chromatogram, because it would not bind to the C
subunit immunoaffinity column used in the fractionation procedure. The
36K form is attributed to ßA or ßB. In the previous study
(7), the 33K peak was attributed to either monoglycosylated ßA or
B or a cleavage product of 36K inhibin, based on the absence of 33K in
serum and the observation that an inhibin A immunoassay failed to
detect it. This aspect is discussed further below. The failure to see
significant amounts of 95–105K forms of inhibin is attributed to the
particular hFF batch examined in this study. Higher proportions of
these inhibins have been identified in chromatograms in an earlier
study (6), indicating that these forms are detectable in hFF in this
combined fractionation/assay system.
The Pro-C ELISA, though previously shown (20) to detect higher
molecular weight forms of inhibin containing Pro-C [e.g.
Pro-N-C/proß-ß (90–105K) or Pro-N-C/ß (55–65K)],
detected primarily 36K and 29K forms of Pro-C in hFF. The Pro-C
immunoactivity identified at 55K may be either Pro-N-C or
Pro-N-C/ß.
IVF serum, postmenopausal and male plasma
The mol wt profiles of inhibin B immunoactivity in IVF and male
plasma/serum share similarities with peaks at 66K and 36K, whereas
the inhibin A profile in IVF serum showed an additional peak at 55K.
Based on the similar inhibin A and B profiles in hFF and the different
patterns of inhibins A and B in plasma, it is concluded that inhibins A
and B are differentially processed in the circulation. Inhibin A, with
the exception of the 33K form absent in serum, has a similar profile to
that found in hFF and reflects processing of both - and ß-subunit
precursor forms. Inhibin B in serum, however, shows an absence of the
55K form, which is believed to be N-C/ß, whereas the 66K form
(C/proß-ß) is retained. A similar absence of the 55K form of
inhibin B was evident in male serum.
The Pro-C ELISA primarily detects monomeric -subunit forms in
serum with little evidence for the detection of higher molecular weight
dimeric inhibin forms (e.g. Pro-N-C/ß,
Pro-N-C/proß-ß). This assay previously has been shown to
detect Pro-C containing dimeric inhibin forms, thus indicating that
in serum, the levels of these high-molecular weight forms are low. If
this observation is confirmed in a wider range of serum samples, then
this assay seems to be unique in detecting the free Pro-(N)-C
subunit in serum, because other -subunit assays detect all C
subunit-containing proteins, including inhibin dimeric forms. Thus, the
Pro-C and inhibin A or B assays, when applied to serum under these
specified conditions, would be showing assay-independent effects.
However, the Pro-C ELISA would be unable to detect the free C
subunit (minus the Pro- region). There is a suggestion of the presence
of the free C subunit in the male and postmenopausal plasma
chromatograms, in particular, based on its mol wt (20K) and its
detection by the C IFMA but not by the Pro-C ELISA. This issue
requires further examination. Previous studies (17) have shown that
inhibin B, and not inhibin A, was detected in male serum, and this was
confirmed in these studies.
Pro-C and -subunit immunoactivity, but not inhibin A or B
immunoactivity, was detected across the Prep-PAGE profile of
postmenopausal plasma. Some of the forms (21K, 29K, 36K, and possibly
55K) are consistent with known, or presumed known, -subunit forms.
The identification of the 21K peak with the C IFMA suggests that
it is the free C subunit. The origin of this immunoactivity in
postmenopausal plasma, with the absence of inhibin A and B, has yet to
be established.
The availability of the C IFMA and other -subunit immunoassays
provides an opportunity to compare quantitatively in hFF and plasma the
profiles of C subunit immunoactivity with the combined or additive
levels of inhibin A, B, and Pro-C immunoactivity determined across
the same chromatograms. The inhibin B levels were adjusted for the C
content in the inhibin B standard. In hFF, with several of the
-subunit immunoassays (Table 3), a high correlation (hFF r =
0.90–0.96) was obtained with the combined inhibin A, B, and Pro-C
values, with slope values ranging between 0.1 and 3. In serum, the
correlations were lower (0.64–0.91), reflecting differences between
immunoactivity profiles (for example, in the 20K region, where C
subunit immunoactivity was detectable with the C IFMA but not with
the Pro-C ELISA). Not withstanding these differences, the close
correlation among levels determined with several of the immunoassays
(including the C IFMA) and the combined inhibin A, B, and Pro-C
values suggests that the profile of hFF and serum inhibin can be
described appropriately in terms of these inhibin A, B and -subunit
immunoactivities, although the precise structures of many of these
forms are unknown. These findings provide a useful platform from which
to explore situations where inhibin may be modified in health and
disease, for example, in the monitoring of various types of ovarian
cancer (25, 26).
In Table 4, the adjusted concentrations of inhibin A, B, and Pro-C
in the hFF and serum/plasma pools are presented. It is evident that in
these samples, the levels of Pro-C are elevated (1.3- to 20-fold),
compared with inhibin A and B and substantiate findings from several
studies (17, 18).
In summary, inhibin in vitro bioactivity is largely
attributed to the presence of different molecular weight forms of
inhibin A and inhibin B in hFF and serum. Inhibin A, B, and Pro-C
immunoactivity correlate closely with levels of inhibin determined by
-subunit-directed immunoassays, suggesting that these activities,
with some caveats, will provide a reasonable description of the inhibin
forms present in biological samples. These findings provide a basis for
understanding the patterns of circulating inhibins and related proteins
observed in various physiological conditions.
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Acknowledgments |
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-subunit fusion protein, and Monash IVF (Clayton, Victoria,
Australia) for provision of hFF and serum. Dr. Kim Pettersson
(University of Turku, Turku, Finland) is thanked for his advice in the
development of the C subunit IFMA. |
Footnotes |
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Received August 28, 1996.
Revised October 28, 1996.
Accepted November 13, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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subunit and its isolation from bovine follicular fluid. J
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