Prince Henrys Institute of Medical Research, PO Box 5152, Clayton, Victoria, 3168, Australia
1 To whom correspondence should be addressed. e-mail: david.robertson{at}med.monash.edu.au
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Abstract |
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Key words: inhibin -subunit/inhibin B/pro-
C/spermatogenic failure/total inhibin ELISA
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Introduction |
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Previous studies (Robertson et al., 1996; 1997) have shown that inhibin B is present in male serum as both high mol wt precursor forms (believed to be
C/pro
-
B) and mature (
C/
B) forms (Figure 1). Previous mutagenesis studies (Mason et al., 1996
) have shown that the precursor forms of inhibin A are much less bio-active as assessed by in-vitro bioassay compared with the mature forms. These findings suggest that the high molecular weight (mol wt) inhibin forms require processing either in the circulation or at the pituitary in order to be become bio-active. Furthermore, current immunoassays do not differentiate between processed and non processed forms (Robertson et al., 1997
).
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This study has two objectives, the first to investigate the mol wt distribution of precursor and mature forms of inhibin B and -subunit in fertile and infertile men using a previously described serum fractionation procedure (Robertson et al., 1996
; 1997; Thirunavukarasu et al., 2002
) with a view to establishing whether precursor and mature forms differed in disease states. Secondly, these studies provide the opportunity to investigate the specificity of the inhibin B and
-subunit ELISAs by comparing the patterns of pro-
C and inhibin B in fractionated male plasma samples with that of total inhibin as measured by an ELISA that detected all inhibin
-subunit and dimeric forms.
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Materials and methods |
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Fractionation procedure
Plasma/serum was fractionated by previously described methods (Robertson et al., 1996; 1997) with modifications (Thirunavukarasu et al., 2002
) which enables the detection of precursor and mature forms of inhibin
-subunit and inhibin dimers. This procedure consists of an initial immunoaffinity step to isolate inhibin forms from large volumes of plasma prior to mol wt fractionation by preparative SDSPAGE. The various inhibin forms are eluted from the gel by an electro-elution procedure and assayed by inhibin ELISAs.
The fractionation procedure was applied to 11 fertile control men and 17 men in the various infertility groups. In a typical fractionation run, serum or plasma (1720 ml) was rapidly thawed in the presence of two sample volumes of 0.2 mol/l phosphate buffer pH 7.4 containing 0.02 mol/l EDTA, 0.002 mol/l phenylmethylsulphonyl fluoride (PMSF), and 1.67% Triton X-100. The mixture (5060 ml) was added to an immunoaffinity column consisting of an IgG fraction of sheep antiserum raised against a human inhibin C-subunit fusion protein and incubated at 4°C overnight on a rotating wheel. The gel suspension was then washed with 0.1 mol/l phosphate buffer pH 7.4, 0.01 mol/l EDTA and eluted with 6 mol/l guanidine hydrochloride (GnHCl). The GnHCl was then removed by fractionation on pre-washed disposable reverse phase cartridges. The inhibin fractions were eluted with 60% acetonitrile/0.1% trifluoroacetic acid, SDS (final concentration, 0.5%), and the sample lyophilised for 3 days. Aliquots for assessment of recoveries were lyophilised separately in BSA. The lyophilised sample was dissolved in 1200 µl, 1 mol/l TRIS pH 8.0, 0.002 mol/l PMSF, 0.1% Tween-40, bromophenol blue (assisted by sonication), and applied to a 10% polyacrylamide gel (20 x 20 cm gels) in Tris/Trycine/SDS buffers. Pre-stained mol wt standards (10250 k, Cat 1610372 Biorad, Hercules, CA, USA) were loaded alone in an outside lane. These standards migrated at a lower Rf such that the apparent mol weights determined are 0.88-fold those obtained with the standards used in earlier studies (Thirunavukarasu et al., 2002
). The gel was run overnight at 35 mA until the 13.5 k mol wt standard reached the bottom of the gel. At completion of the run, the polarity was reversed for 1 min. The electro-elution process was undertaken at 250 mA and continued until the voltage increased to 35 v. Thirty fractions (34 ml) were collected into weighed tubes containing 500 µl 0.75% NaN3, 0.8 mol/l NaCl, 5% BSA. The recovered fractions were assayed by specific ELISAs for inhibin B, pro-
C and total inhibin.
A large male plasma pool (in 20 ml aliquots) was prepared for use as a quality control sample and fractionated similarly to the other plasma samples in the fractionation studies. An aliquot was included in the fractionation series after every 56 samples for a total of five runs in the study. The mol wt profiles for the various inhibin forms (data not shown) were highly comparable with the data obtained for the control group (n = 11). The reproducibility of the procedure was assessed from the coefficient of variation (CV) of peak mol wt values for one of the pro-C peaks (28.0 ± 1.81, n = 5). An acceptable CV value of 6.5% was obtained.
Recoveries of inhibin B, pro-C and total inhibin immunoactivities for the 35 serum/plasma fractionation runs were 5358% (pro-
C and total inhibin) for the immunoaffinity step and 3948% (inhibin B, pro-
C and total inhibin) for the prep-PAGE/electro-elution step, with overall recoveries of 1823% (pro-
C and total inhibin). These recoveries are comparable with those obtained earlier (Thirunavukarasu et al., 2002
). Recoveries of inhibin B were exaggerated (125%) for the immunoaffinity step and were excluded from the above calculations. The increased recovery is attributed to an overestimation in inhibin B values when measuring inhibin in serum and fractionated serum samples where the serum concentration is reduced.
Data analysis
The molecular weights of the eluted Prep-PAGE fractions were calculated using the above stated protein standard markers, which bracketed the 2090 k mol wt range of interest. Regression analysis (fraction number versus log mol wt) of three markers gave correlation coefficients >0.995. The resulting line of best fit was used to determine the mol wt of the eluted fractions (n = 35). As a measure of precision, the mol wt determinations of the above protein standard markers when measured against the line of best fit were within 5% of their stated value.
The profiles of immuno-activity were assessed for clear evidence of peaks. The mol wt values of peak tubes of immuno-activity from multiple runs were combined (mean ± SD) and from their distribution, activity regions were defined. The immuno-activity levels/fraction were also presented as a percentage of recovered activity.
Assays
Inhibin B, total inhibin and Pro-C ELISAs were used to detect the various inhibin forms.
Inhibin B ELISA
The method of Groome et al. (1996a) was employed with modifications. Plates [
B-subunit capture monoclonal antibody (Mab, C5)], inhibin B standard and an alkaline phosphatase-labelled second antibody (R1) directed against the
C region (aa 132) of the
-subunit were provided by Oxford Bio-innovation Ltd (Upper Heyford, UK). The assay procedure was modified to measure inhibin B in electro-elution buffer. Sample (100 µl) or standard (100 µl) in electro-elution buffer and buffer (100 µl, Groome assay buffer (Groome et al., 1996a
) with the addition of hydrogen peroxide (10 µl, 30%) were incubated overnight at room temperature. The steps that followed in the assay protocol remained unchanged. The sensitivity of the assay was 510 ng/l. The between and within assay variations were 7.9 and 8.6% respectively.
Pro-C ELISA
The method of Groome et al. (1996b) was employed using a kit provided by Oxford Bio-innovation Ltd. The Mab INPRO directed against the entire Pro- region was used as capture antibody (Figure 1), and an alkaline phosphatase conjugated monoclonal antibody (R1) used as label. The assay procedure used was similar to that presented for the inhibin B ELISA (above) without the addition of hydrogen peroxide, and a 2 h rather than a 3 h second antibody incubation. The sensitivity of the assay was 2 ng/l. The between and within assay variations were 12.2 and 7.5% respectively.
Total inhibin ELISA
Total inhibin was determined by ELISA using PO14 + PO23 Mabs as capture antibodies and an alkaline phosphatase linked -subunit antibody (R1) as a labelled second antibody as previously described (Robertson et al., 2001
). Human recombinant inhibin A (91/624) preparation provided by the National Institute of Biological Standards and Control (Potters Bar, UK) was used as standard. The assay was modified slightly by introducing a shorter labelled antibody incubation step (2 h at room temperature). The sensitivity of the assay was 6 ng/l. The within and between assay variations were 6.9 and 14.3% respectively. Other assay characteristics are similar to those previously published (Robertson et al., 2001
).
Statistics
Subject hormone data were log transformed before analysis. Difference between groups were determined by ANOVA followed by Fishers LSD multiple group test. All data are presented either as geometric mean ± 2SD or mean ± SD.
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Results |
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Inhibin B levels were significantly (P < 0.05) lower in SCO, chemotherapy and Klinefelters syndrome groups compared with controls. No differences in pro-C levels were observed between groups. Relative to the fertile group, total inhibin levels were significantly higher in the chemotherapy group and significantly lower in Klinefelters syndrome group. Testosterone was lower in the Klinefelters syndrome group. Plasma FSH (r = 0.67, P < 0.001, n = 39) and LH (r = 0.35, P < 0.05, n = 39) in combined fertile plus infertile groups were inversely correlated with plasma inhibin B. Plasma inhibin B and LH were poorly correlated (r < 0.20, P = not significant).
Fractionation of plasma from fertile men and men with various types of infertility
Plasma samples from fertile (n = 11, Figure 2) and infertile men (HypoSG (n = 4, Figure 2), SCO (n = 4, Figure 3), chemotherapy (n = 4, Figure 3), Klinefelters syndrome (n = 3 data not shown) and GCA (n = 2, data not shown) were individually fractionated by the combined immunoaffinity/preparative PAGE electro-elution procedure and the apparent mol wt profiles of immuno-activity determined using the various inhibin assays.
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Pro-C
Three mol wt forms (21.6, 27.5 and 45.6 k) of pro-C were detected in fertile subjects, consistent with the two glycosylated pro-
C forms and the precursor form, pro-
N-
C. Similar mol wt forms were identified in HypoSG men (22.5, 28.5 and 45.6 k) and men undergoing chemotherapy treatment (21.2, 26.4 and 44.7 k) compared with fertile men. However, significant differences (P < 0.001) were observed between normal men (21.6, 27.5 k) and men with SCO and GCA (24, 29 k). No significant differences were observed in the proportion of the high (3550 k) mol wt forms of pro-
C present in plasma in the various fertility and infertility groups (Table II).
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Identification of pro-C forms in serum/plasma
Theoretically the Pro-C ELISA will detect all mol wt forms of inhibin B containing the pro- region as well as the free
-subunit containing the pro- region (pro-
C or pro-
N-
C), (Figure 1). Thus the Pro-
C ELISA may detect both dimeric and monomeric inhibin forms.
To establish what types of inhibin are present and detected in male plasma by the Pro-C ELISA, the following immunoabsorption experiment was performed. Plasma was repeatedly stripped with the INPRO antiserum (thereby removing all pro- containing inhibin forms), until no more pro-
C immuno-activity was detectable. The INPRO-stripped plasma was then immuno-absorbed with an
-subunit antiserum (as described in the fractionation procedure) to remove the remaining inhibin forms (including all dimeric and free
N-
C forms). The pro-containing fractions and inhibin fractions were then separately fractionated by the Prep-PAGE/electro-elution procedure and the various inhibin mol wt forms present identified by the various ELISAs.
The profile of pro-C immuno-activity in the INPRO-absorbed sample was similar to that detected by the total inhibin ELISA which detects all inhibin forms containing the
C region of the
-subunit (Figure 4A and B). No inhibin B was detected across the chromatogram. These data indicate that in male serum, the Pro-
C ELISA is detecting the free
-subunit forms and not the dimeric inhibin forms.
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Discussion |
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In order to handle the relatively large volumes of plasma needed to perform this study, we utilised a serum fractionation procedure which included an immunoaffinity chromatography step as a preliminary purification/concentration procedure prior to the high resolution preparative SDSPAGE step. The inhibin forms were electro-eluted from the gel prior to quantitation by various ELISAs. This procedure has been extensively characterized (Robertson et al., 1996; 1997; Thirunavukarasu et al., 2002
) and while it involves procedural losses, recovery of inhibin forms is sufficient to permit their detection in the final fractions.
Inhibin B
The fractionation procedure was applied to plasma from a range of fertile men, and infertile men where inhibin B levels were largely decreased. In the latter setting, the low levels of inhibin B in the initial plasma samples precluded its detection after fractionation. The two groups where inhibin B was readily detectable (fertile and HypoSG) showed a similar proportion of high mol wt forms. In fertile men, 30% of plasma inhibin B was found as the 60 k precursor form, and a similar proportion was observed in HypoSG men, indicating that there is no major change in Sertoli cell inhibin biosynthesis or subsequent processing. These findings thus suggest that the feedback relationship between FSH and inhibin B is not modified in these infertile men compared with fertile men as the proportion of precursor forms of lower biological activity were similar in both cases. In relation to the HypoSG group and perhaps to the other infertile groups, it would appear that there is little added advantage in developing assays to specifically detect the mature inhibin B forms as a means to monitor the circulating forms of bioactive inhibin B.
Serum inhibin B levels in our subjects with spermatogenic failure were reduced, and their FSH levels elevated, consistent with their proposed inverse relationship as shown by others (Anawalt et al., 1996; Illingworth et al., 1996
; Wallace et al., 1997
; Pierik et al., 1998
; Bohring and Krause, 1999
; Foppiani et al., 1999
; Foresta et al.,1999
; von Eckardstein et al., 1999
; Kolb et al., 2000
; Fujisawa et al., 2001
).
Pro-C
The proportion of high mol wt forms of pro-C detected in fertile and infertile men was much lower (414%) than that observed for inhibin B. No significant differences were observed between fertile and infertile groups although the low number of samples in some groups prevents a definitive conclusion. However, significant differences in molecular weights (24, 29 k) of pro-
C were observed in SCO and GCA men compared with controls (21.7, 27.6 k). Earlier studies identified smaller forms of inhibin A and pro-
C forms in late pregnancy serum (Thirunavukarasu et al., 2002
) which was attributed to serum/tissue protease activity. We have no evidence to support this contention in the current study.
In principle, the Pro-C ELISA detects all inhibin forms containing pro- and
C fragments. These forms include high mol wt inhibin B (pro-
N-
C/pro
-
B or pro-
N-
C/
B) as well as pro-
N-
C and pro-
C, but would exclude
N-
C and other inhibin dimer forms which do not contain the pro- region. This study showed that immuno-absorption of plasma with the INPRO antibody does not lead to an absorption of inhibin B forms and furthermore, based on the similarity between the mol wt profiles produced by the total inhibin and Pro-
C ELISAs, the Pro-
C ELISA is detecting all free
-subunit forms. Thus the Pro-
C ELISA is detecting all known inhibin
-subunit forms in male plasma. Since the pro-
C forms were removed from plasma by immunoabsorption with the INPRO Mab, it enabled the direct comparison of mol wt profiles of inhibin B and total inhibin in the remaining sample. A close correspondence between the mol wt profile of these two immuno-activities indicates that the inhibin B ELISA is detecting both high mol wt and mature inhibin B forms.
The data supports previous studies that plasma pro-C levels are not different between fertile and infertile groups (Anawalt et al., 1996
; Illingworth et al., 1996
; Kolb et al., 2000
) although increases following chemotherapy have been previously observed (Wallace et al., 1997
).
Processing of inhibin B and pro-C
We found that the proportion of high mol wt inhibin B was significantly higher in serum (51%) than plasma (30%) suggesting that the proportion of high mol wt inhibin B in the circulation is at least 50%. The lower proportion observed in plasma, while surprising, suggests that the precursor forms are cleaved by proteases (e.g. those associated with the blood clotting process) present in plasma but not serum. In contrast, the processing of high mol wt (60 k) inhibin B in serum was limited with no differences observed between serum stored at 4°C compared with serum incubation at 37°C for 24 h. Previous studies (Robertson et al., 1997) had indicated that 60 k inhibin B is a partially processed form, consisting of C/pro
-
B and thus it is postulated that it is the full length
B-subunit that is subject to this plasma-associated proteolytic cleavage.
In contrast pro-C processing was evident when serum was incubated at 37°C while no differences were observed between plasma and serum when stored at 4°C. The proportion of the 45 k form (believed to be pro-
N-
C) decreased from 7 to 2% after 24 h incubation at 37°C. These data are consistent with earlier studies (McLachlan et al., 1986
) showing that bovine 58 k inhibin A (
N-
C/
A) was converted to the 32 k (
C/
A) form when incubated in serum and shown to be cleaved by a protease with furin-like properties (Robertson et al., 1987
).
In summary, in this study we found no difference in the proportion of inhibin B or pro-C forms in plasma as precursor and mature forms between fertile men and men with sub-fertility. The data also suggest that
- and
B-subunit chains are processed in blood by different mechanisms. The present study provides evidence that the inhibin B and Pro-
C ELISAs are providing an independent measure of their respective inhibin forms in plasma and can be applied in further studies on the physiology of inhibin in disorders of testicular function.
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Acknowledgements |
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References |
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Submitted on September 23, 2002; resubmitted on December 9, 2002; accepted on February 2, 2003.