Characterization of plasma inhibin forms in fertile and infertile men

D.M. Robertson1, T. Stephenson and R.I. McLachlan

Prince Henry’s 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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: The reciprocal relationship between plasma FSH and inhibin B generally reflects the state of spermatogenesis but data in some settings indicate further complexity in their relationship. Inhibin circulates as a range of higher molecular weight (mol wt) forms of varying bioactivity such that the serum profile of inhibin forms may differ between normal men and those with varying types of spermatogenic failure. The aim of this study was to establish if the inhibin B mol wt distribution was altered in men with infertility. METHODS: The mol wt profiles of inhibin B and free {alpha}-subunit were determined in plasma of fertile (n = 11) and infertile (n = 17) men using a combined immunoaffinity chromatography, preparative SDS–PAGE and electro-elution procedure and fractions assayed using ELISAs for inhibin B, total inhibin (all forms containing the {alpha}-subunit) and free {alpha}-subunit. RESULTS: Inhibin B was identified as precursor (60–65 k) and mature (26–30 k) forms in plasma in similar proportions (29%) in fertile men and oligospermic men (25%), but was undetectable in azoospermic men. The free {alpha}-subunit detected by the pro-{alpha}C ELISA was identified as both the precursor and processed (pro-{alpha}C) forms with similar proportions in fertile (8%) and all infertile (4–14%) men. The pro-{alpha}C ELISA did not detect the precursor forms of inhibin B in plasma while the inhibin B ELISA detected all total inhibin forms following removal of pro-containing forms by immunoabsorption. CONCLUSIONS: (i) the proportions of precursor inhibin B and {alpha}-subunit forms in the circulation are unchanged in men with spermatogenic disorders indicating there is no alteration of the Sertoli cell inhibin secretory pattern; (ii) these fractionation studies indicate that pro-{alpha}C and inhibin B ELISAs specifically detect the free {alpha}-subunit and inhibin B forms present in male plasma.

Key words: inhibin {alpha}-subunit/inhibin B/pro-{alpha}C/spermatogenic failure/total inhibin ELISA


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
It is recognized that there is a reciprocal relationship between plasma FSH and inhibin B, providing a biological basis for the gonadal regulation of FSH in the male (Nieschlag et al., 1999Go; Anderson and Sharpe, 2000Go; Hayles et al., 2001Go). However, this relationship appears complex as a number of gonadal factors, including testosterone, and germ cell elements are believed to contribute to the feedback process. In some settings, e.g. following chemotherapy (Wallace et al., 1997Go) or irradiation (Petersen et al., 1999Go), where spermatogenesis is severely compromised, a clear inverse relationship is evident between plasma FSH and inhibin B with a marked suppression of circulating inhibin B. However, in situations in which FSH/LH levels were suppressed to very low levels by prolonged androgen or androgen plus progestin treatment for contraceptive purposes, inhibin B was only partially reduced despite induction of azoospermia (Anderson et al., 1997Go; Martin et al., 2000Go; Behre et al., 2001Go).

Previous studies (Robertson et al., 1996Go; 1997) have shown that inhibin B is present in male serum as both high mol wt precursor forms (believed to be {alpha}C/pro{beta}-{beta}B) and mature ({alpha}C/{beta}B) forms (Figure 1). Previous mutagenesis studies (Mason et al., 1996Go) 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., 1997Go).



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Figure 1. Schematic diagram of the precursor forms of the human inhibin {alpha}-subunit and inhibin B. The binding sites for the various monoclonal antibodies are also included. The stars refer to potential glycosylation sites.

 
Thus, if the precursor forms of inhibin B are bio-inactive or of reduced bio-activity then a closer correlation between plasma FSH and the plasma levels of mature inhibin B forms would be expected than with total inhibin B which includes both mature and precursor forms. The extent of processing of inhibin B may differ between fertile and infertile men. It is interesting to note that in the published scatterplots comparing serum FSH and inhibin B in fertile and infertile men (Pierik et al., 1998Go; Bohring and Krause, 1999Go; von Eckardstein et al., 1999Go) the selection of a serum FSH concentration in the normal fertile range is associated with a 5–6 fold range in serum inhibin B levels. One explanation for this wide distribution in inhibin B may relate to differences in its bioactivity and thus proportion of mature forms between individuals or clinical states.

This study has two objectives, the first to investigate the mol wt distribution of precursor and mature forms of inhibin B and {alpha}-subunit in fertile and infertile men using a previously described serum fractionation procedure (Robertson et al., 1996Go; 1997; Thirunavukarasu et al., 2002Go) 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 {alpha}-subunit ELISAs by comparing the patterns of pro-{alpha}C and inhibin B in fractionated male plasma samples with that of total inhibin as measured by an ELISA that detected all inhibin {alpha}-subunit and dimeric forms.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Subjects
Healthy men aged 25–45 years who had fathered children were used as controls. Infertile men attending an infertility clinic were invited to donate blood for this study. Details of the various infertility groups are presented in Table I. The study was approved by the Human Research and Ethics Committee of Monash Medical Centre. All men gave informed consent. Clinical data included serum testosterone, FSH, LH and histology assessed on open biopsy material and classified according to de Kretser and Holstein (1976)Go.


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Table I. Plasma hormone levels in fertile and infertile men. Data are presented as geometric means ± 2SD
 
Male plasma/serum collection
Blood was collected on ice in EDTA-coated tubes (Greiner, Labortechnik, Kremsmunster, Austria), centrifuged at 4°C within 2 h of collection and the plasma stored at –80°C. Plasma (20 ml) was collected for all men. In addition, serum and plasma were collected from six normal men. Blood (60 ml) was collected in serum-gel tubes (Greiner), placed immediately on ice, allowed to clot at 4°C and centrifuged within 2 h. Plasma was collected as described above. Serum and plasma were stored at –80°C. Equal aliquots of plasma and serum from each subject were then thawed and combined to form the plasma 4°C and serum 4°C pools. An additional serum pool (serum 37°C) was prepared consisting of serum incubated at 37°C for 24 h in the presence of 0.1% sodium azide.

Fractionation procedure
Plasma/serum was fractionated by previously described methods (Robertson et al., 1996Go; 1997) with modifications (Thirunavukarasu et al., 2002Go) which enables the detection of precursor and mature forms of inhibin {alpha}-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 SDS–PAGE. 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 (17–20 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 (50–60 ml) was added to an immunoaffinity column consisting of an IgG fraction of sheep antiserum raised against a human inhibin {alpha}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 (10–250 k, Cat 161–0372 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., 2002Go). 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 (3–4 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-{alpha}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 5–6 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-{alpha}C peaks (28.0 ± 1.81, n = 5). An acceptable CV value of 6.5% was obtained.

Recoveries of inhibin B, pro-{alpha}C and total inhibin immunoactivities for the 35 serum/plasma fractionation runs were 53–58% (pro-{alpha}C and total inhibin) for the immunoaffinity step and 39–48% (inhibin B, pro-{alpha}C and total inhibin) for the prep-PAGE/electro-elution step, with overall recoveries of 18–23% (pro-{alpha}C and total inhibin). These recoveries are comparable with those obtained earlier (Thirunavukarasu et al., 2002Go). 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 20–90 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-{alpha}C ELISAs were used to detect the various inhibin forms.

Inhibin B ELISA
The method of Groome et al. (1996aGo) was employed with modifications. Plates [{beta}B-subunit capture monoclonal antibody (Mab, C5)], inhibin B standard and an alkaline phosphatase-labelled second antibody (R1) directed against the {alpha}C region (aa 1–32) of the {alpha}-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., 1996aGo) 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 5–10 ng/l. The between and within assay variations were 7.9 and 8.6% respectively.

Pro-{alpha}C ELISA
The method of Groome et al. (1996bGo) 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 {alpha}-subunit antibody (R1) as a labelled second antibody as previously described (Robertson et al., 2001Go). 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., 2001Go).

Statistics
Subject hormone data were log transformed before analysis. Difference between groups were determined by ANOVA followed by Fisher’s LSD multiple group test. All data are presented either as geometric mean ± 2SD or mean ± SD.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Plasma hormone levels in fertile and infertile men
Plasma levels of FSH, LH, testosterone, inhibin B, pro-{alpha}C and total inhibin were determined in 11 fertile men and in infertile men with Sertoli cell-only (SCO, n = 6), severe hypospermatogenesis (HypoSG, n = 8), Klinefelter’s syndrome (n = 7), following chemotherapy (n = 5) and germ cell arrest (GCA, n = 2), (Table I). Plasma FSH levels were significantly (P < 0.05) elevated in all infertility groups while plasma LH was significantly (P < 0.05) increased in the chemotherapy, Klinefelter’s syndrome and GCA groups.

Inhibin B levels were significantly (P < 0.05) lower in SCO, chemotherapy and Klinefelter’s syndrome groups compared with controls. No differences in pro-{alpha}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 Klinefelter’s syndrome group. Testosterone was lower in the Klinefelter’s 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), Klinefelter’s 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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Figure 2. Molecular weight profiles of total inhibin, inhibin B and pro-{alpha}C in plasma from control men and men with hypospermato genesis. The mol weights of the various regions, are Region 1: 10.1–15 k; 2: 15.1–20 k; 3: 20.1–25 k; 4: 25.1–30 k; 5: 30.1–35 k; 6: 35.1–40 k; 7: 40.1–45 k; 8: 45.1–50 k; 9: 50.1–55 k; 10: 55.1–60 k; 11: 60.1–65 k; and 12: 65.1–70 k etc. The vertical dashed line corresponds to the mol wt region where 30 kDa inhibin is located.

 


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Figure 3. Molecular weight profiles of total inhibin and pro-{alpha}C in plasma from men with Sertoli cell-only and chemotherapy. See legend to Figure 2 for code for mol wt regions.

 
Inhibin B
The mol wt profile of inhibin B in fertile men consisted of three main peaks of immuno-activity (25.7, 30.2 and 60.4 k) consistent with the known two glycosylated 30 k ({alpha}C/{beta}B) forms and the precursor form, {alpha}C/pro{beta}.{beta}B (Figure 2). Mol wt profile of inhibin B in HypoSG men showed some similarities (27, 30.6 and 62.4 k) with normal subjects, although an additional 38.8 k peak was identified of unknown structure. Inhibin B was not present in sufficiently high levels in serum samples from men with other forms of infertility to be detected in the fractionation procedure.

Pro-{alpha}C
Three mol wt forms (21.6, 27.5 and 45.6 k) of pro-{alpha}C were detected in fertile subjects, consistent with the two glycosylated pro-{alpha}C forms and the precursor form, pro-{alpha}N-{alpha}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 (35–50 k) mol wt forms of pro-{alpha}C present in plasma in the various fertility and infertility groups (Table II).


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Table II. Proportion (%) of high mol wt forms of inhibin B (50–70 k) and inhibin {alpha}-subunit (35.5–50 k) in plasma from control men and men with various infertilities
 
Inhibin B and pro-{alpha}C forms in plasma and serum
To establish if the profile of plasma inhibin was modified by collection and storage conditions, inhibin B and pro-{alpha}C forms were compared in plasma and serum collected from normal men under defined (temperature and time) conditions. Plasma and serum collected fresh at 4°C, and serum which was incubated at 37°C overnight, were processed through the fractionation procedure and the inhibin B and pro-{alpha}C profiles determined. The mol wts of inhibin B and pro-{alpha}C peaks were unchanged by the sample treatment (data not shown). Significantly (P < 0.05) more 30 k inhibin B was observed in plasma stored at 4°C (51%) or serum incubated at 37°C (46%) compared with serum stored at 4°C (35%) (Table III), suggesting that proteolysis of high mol wt inhibin B was facilitated in plasma and by temperature in serum implicating different proteolytic processes.


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Table III. Effects of blood sample collection (plasma or serum) and storage temperature (4°C, 24 h at 37°C) on mol wt distribution of inhibin B and inhibin {alpha}-subunit immuno-activities. Data are presented as percentage of recovered activity from three replicate fractionations, mean ± SD
 
The proportion of the pro-{alpha}N-{alpha}C (~60 k) form was similar between fresh serum and plasma (7.3 and 8.3%) but this proportion decreased to 2.4% (P < 0.001) when the serum was incubated for 24h at 37°C (data not shown). This data indicates that pro-{alpha}N-{alpha}C subunit is sensitive to serum-induced proteolytic cleavage.

Identification of pro-{alpha}C forms in serum/plasma
Theoretically the Pro-{alpha}C ELISA will detect all mol wt forms of inhibin B containing the pro- region as well as the free {alpha}-subunit containing the pro- region (pro-{alpha}C or pro-{alpha}N-{alpha}C), (Figure 1). Thus the Pro-{alpha}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-{alpha}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-{alpha}C immuno-activity was detectable. The INPRO-stripped plasma was then immuno-absorbed with an {alpha}-subunit antiserum (as described in the fractionation procedure) to remove the remaining inhibin forms (including all dimeric and free {alpha}N-{alpha}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-{alpha}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 {alpha}C region of the {alpha}-subunit (Figure 4A and B). No inhibin B was detected across the chromatogram. These data indicate that in male serum, the Pro-{alpha}C ELISA is detecting the free {alpha}-subunit forms and not the dimeric inhibin forms.



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Figure 4. (A) and (B): Molecular weight profiles of total inhibin and pro-{alpha}C in male plasma immuno-absorbed with an antibody (INPRO) to the pro- region of the inhibin {alpha}-subunit. (C) and (D): Molecular weight profiles of total inhibin and inhibin B remaining in normal male plasma after it had been immuno-absorbed with an antibody (INPRO) to the pro- region of the inhibin {alpha}-subunit. Horizontal dashed line refers to the sensitivity of the respective assays.

 
Similarly, a good correspondence was seen between total inhibin ELISA and inhibin B in the fractionated inhibin sample. A small peak of ~40 k was observed with the total inhibin ELISA which may represent {alpha}N-{alpha}C (Figure 4C) which was not detected by either Pro-{alpha}C or inhibin B ELISAs, however its contribution to the total was small. Together these data are taken to show that the Pro-{alpha}C and inhibin B ELISAs are detecting the majority of inhibin forms present in male serum.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This study has shown that the proportion of precursor inhibin B forms in plasma is unchanged in men with spermatogenic disorders indicating there is no alteration on the Sertoli cell inhibin secretory pattern and that Pro-{alpha}C and inhibin B ELISAs provide an unambiguous assessment of the levels of the free {alpha}-subunit and inhibin B forms in the circulation of men. Along with the data regarding the effects of sample storage condition on assay outcomes, these data provide a basis for further studies on the physiology of inhibin in health and disease.

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 SDS–PAGE 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., 1996Go; 1997; Thirunavukarasu et al., 2002Go) 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., 1996Go; Illingworth et al., 1996Go; Wallace et al., 1997Go; Pierik et al., 1998Go; Bohring and Krause, 1999Go; Foppiani et al., 1999Go; Foresta et al.,1999Go; von Eckardstein et al., 1999Go; Kolb et al., 2000Go; Fujisawa et al., 2001Go).

Pro-{alpha}C
The proportion of high mol wt forms of pro-{alpha}C detected in fertile and infertile men was much lower (4–14%) 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-{alpha}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-{alpha}C forms in late pregnancy serum (Thirunavukarasu et al., 2002Go) which was attributed to serum/tissue protease activity. We have no evidence to support this contention in the current study.

In principle, the Pro-{alpha}C ELISA detects all inhibin forms containing pro- and {alpha}C fragments. These forms include high mol wt inhibin B (pro-{alpha}N-{alpha}C/pro{beta}-{beta}B or pro-{alpha}N-{alpha}C/{beta}B) as well as pro-{alpha}N-{alpha}C and pro-{alpha}C, but would exclude {alpha}N-{alpha}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-{alpha}C ELISAs, the Pro-{alpha}C ELISA is detecting all free {alpha}-subunit forms. Thus the Pro-{alpha}C ELISA is detecting all known inhibin {alpha}-subunit forms in male plasma. Since the pro-{alpha}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-{alpha}C levels are not different between fertile and infertile groups (Anawalt et al., 1996Go; Illingworth et al., 1996Go; Kolb et al., 2000Go) although increases following chemotherapy have been previously observed (Wallace et al., 1997Go).

Processing of inhibin B and pro-{alpha}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 {alpha}C/pro{beta}-{beta}B and thus it is postulated that it is the full length {beta}B-subunit that is subject to this plasma-associated proteolytic cleavage.

In contrast pro-{alpha}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-{alpha}N-{alpha}C) decreased from 7 to 2% after 24 h incubation at 37°C. These data are consistent with earlier studies (McLachlan et al., 1986Go) showing that bovine 58 k inhibin A ({alpha}N-{alpha}C/{beta}A) was converted to the 32 k ({alpha}C/{beta}A) form when incubated in serum and shown to be cleaved by a protease with furin-like properties (Robertson et al., 1987Go).

In summary, in this study we found no difference in the proportion of inhibin B or pro-{alpha}C forms in plasma as precursor and mature forms between fertile men and men with sub-fertility. The data also suggest that {alpha}- and {beta}B-subunit chains are processed in blood by different mechanisms. The present study provides evidence that the inhibin B and Pro-{alpha}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.


    Acknowledgements
 
The authors acknowledge the excellent technical support of Enid Pruysers and the gift of immobilised INPRO antibody from Dr N.Groome. This study was funded by a Program Grant (983212) of the National Health and Medical Research Council of Australia.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Anawalt, B.D., Bebb, R.A., Matsumoto, A.M., Groome, N.P., Illingworth, P.J., McNeilly, A.S. and Bremner, W.J. (1996) Serum inhibin B levels reflect Sertoli cell function in normal men and men with testicular dysfunction. J. Clin. Endocrinol. Metab., 81, 3341–3345.[Abstract]

Anderson, R.A., Wallace, E.M., Groome, N.P., Bellis, A.J. and Wu, F.C.W. (1997) Physiological relationships between inhibin B, follicle stimulating hormone secretion and spermatogenesis in normal men and response to gonadotrophin suppression by exogenous testosterone. Hum. Reprod., 12, 746–751.[Abstract]

Anderson, R.A. and Sharpe, R.M. (2000) Regulation of inhibin production in the human male and its clinical applications. Int. J. Andrology, 23, 136–144.[CrossRef][ISI][Medline]

Behre, H.M., Kliesch, S., Lemcke, B., von Eckardstein, S. and Nieschlag, E. (2001) Suppression of spermatogenesis to azoospermia by combined administration of GnRH antagonist and 19-nortestosterone cannot be maintained by this non-aromatizable androgen alone. Hum. Reprod., 16, 2570–2577.[Abstract/Free Full Text]

Bohring, C. and Krause, W. (1999) Serum levels of inhibin B with different causes of spermatogenic failure. Andrologia, 31, 137–141.[CrossRef][ISI][Medline]

de Kretser, D.M. and Holstein, A.F. (1976) Testicular biopsy and abnormal germ cells. In Hafez, E.S.E. (ed) The human semen and fertility regulation in men. Mosby and Co. St Louis, Missouri, pp 332–343.

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Submitted on September 23, 2002; resubmitted on December 9, 2002; accepted on February 2, 2003.





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