| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Articles |
a Department of Immunology, Hospital Universitario ``12 de Octubre", Universidad Complutense, Madrid, 28041 Madrid, Spain
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
C on the one hand, and seminal plasma levels of inhibin B, activin, and -inhibin on the other, were also measured. Inhibin B and related peptides were quantitated using a novel two-site assay with monoclonal antibodies to the and ß subunits of inhibin. Our results showed that HLA class I and class II molecules were expressed on the spermatozoa's surface, following a cyclic pattern, and that there was a simultaneous and coordinated expression of both types of molecules (r = 0.801, P < 0.0001). Furthermore, when the expression of these molecules was plotted against the different hormone levels, serum inhibin B showed a clear inverse correlation with HLA class I (r = -0.612, P < 0.0001) and class II (r = -0.534, P < 0.0001). This finding reveals unexpected functions of inhibin B, which may be relevant in the fertilization process and on male fertility control.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The presence of HLA molecules on the surface of the seminal cells and spermatozoa has been a matter of controversy, and several techniques have been used to address this issue [3, 4]. The expression of these molecules on sperm cells may play a role in the fertilization process [5]. In infertile women, a significantly higher incidence of circulating anti-HLA antibodies was found [6], and the existence of mechanisms inhibiting the fertilization of MHC-compatible gametes have been postulated in tunicates [7] and mammals [8]. Indeed, HLA heterozygosity is favored in certain human populations [9], and an excess of HLA antigen sharing has been found in couples with repetitive abortions [10].
Recent work from our group clearly showed the expression of HLA class I and class II molecules on purified sperm cells [11]. The expression seemed diploid, followed a cyclic pattern, and apparently showed an inverse correlation with inhibin concentration [11].
Inhibin is a glycoprotein hormone that inhibits the production or secretion of the pituitary gonadotropins, preferentially FSH [12]. It was first purified from ovarian sources in the female [13], whereas the major sources in the male are Sertoli and Leydig cells [14]. Two major forms of inhibin have been isolated: inhibin A and inhibin B share a common subunit () and have dissimilar subunits (ßA and ßB, respectively), which form disulfide-linked dimeric proteins with the capacity to suppress FSH secretion [13]. Inhibin A is thus secreted as ßA and inhibin B as ßB. Besides the well-established regulatory effect on FSH levels, it is known that inhibin B also influences a wide array of effects, either reproductive (inhibition of spermatogonial mitosis, as assessed by a decrease in tritiated thymidine incorporation) or nonreproductive (inhibition of erythroid differentiation) [12, 14].
Several assays have been developed to measure circulating inhibin levels, such as RIA [15] or ELISA [16], but they were either not sensitive enough or unable to discriminate between inhibins A and B levels. It was not until very recently that Groome and colleagues developed a two-site assay utilizing monoclonal antibodies to both the and ß subunits, to specifically measure inhibin A and inhibin B in a sensitive and reliable manner [17].
Since in our previous work [11] inhibin B levels were measured using an assay that recognizes distinct epitopes on the chain, we wished to unequivocally confirm with this new methodology our preliminary findings, by measuring inhibin B and other related peptides, namely activin (dimers of inhibin B subunits, with an opposite effect to that of inhibin) and pro-C (dimer of the C-terminal part of the -subunit, C, covalently linked to the N-terminal part of the same subunit), in serum and seminal plasma. The results obtained are presented in this work.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Four healthy volunteers were studied. From each individual, semen and blood samples were obtained weekly over a period of 3 mo (15 wk). Sexual intercourse 2 to 3 times weekly was recorded for each donor.
Acquisition and Retention of Purified Sperm Cells
Semen samples were collected from the donors and allowed to liquefy at room temperature for 30 min. Samples were centrifuged at 600 x g for 15 min to obtain a supernatant of pure seminal plasma. Then sperm cells were washed twice with PBS at 600 x g for 10 min at 4°C, the supernatants were discarded, and the pellets were gently resuspended. Totally pure motile spermatozoa (SPZ) and nonspermatozoal seminal cells (SCnonSPZ) were obtained as described previously [11]. SPZ were identified on the basis of sperm motility, swim-up, and supernatant collection; thus only motile cells were collected. Each sperm sample was counted and carefully evaluated by microscopy to ensure the purity of the preparations. Three Neubauer chambers were mounted per sample, and 400 cells were counted per chamber. Furthermore, the final preparation was labeled with a CD45 monoclonal antibody, which stains only lymphocytes, to confirm that no lymphoid cells contaminated the sample; the flow cytometer (Facscalibur; Becton Dickinson, San Jose, CA) counted at least 4000 cells that had the specific spermatozoal size and shape, as visualized in the forward-scatter and side-scatter screen. In no instance were contaminating somatic cells observed. SCnonSPZ were mostly germinal diploid cells (80%), and the rest were leukocyte cells [18].
Monoclonal Antibodies and Cytofluorometric Analysis
Stained cells (see below) were analyzed with the Fascalibur flow cytometer gated to exclude nonviable cells. Fluorescence intensities above the upper limit of the negative control distribution were considered positive. The following monoclonal antibodies were used: 22.63.4 anti-HLA-ABC fluorescein isothiocyanate (FITC)-conjugated (Ylem, Rome, Italy); L243 anti-HLA-DR phycoerythrin (PE)-conjugated (Becton Dickinson, Mountain View, CA); YTH 66.9HL "CAMPATH-12" anti-CD52 FITC-conjugated (Serotec, Oxford, UK); and BRIC 110 anti-CD55 PE-conjugated. These two last antibodies are used as positive controls [19, 20]. The negative control used was M1F2aPT mouse anti-IgG1 FITC-conjugated (Caltag, San Francisco, CA). All conjugates were preabsorbed with a pool of normal human cells. To avoid nonspecific staining, rabbit serum was used as a blocking agent. Thirty thousand viable cells were counted in each experiment.
Labeling procedures were carried out as previously described [11]. Briefly, sperm cells were washed twice at 600 x g in Hanks' buffered salt solution (Gibco-Biocult, Scotland, UK). Sperm cells were first treated by incubating the pellet for 30 min at 4°C with 100 µl rabbit serum inactivated and absorbed with human cells before use. Three x 105 cells were then pipetted into one well of a microtiter plate and washed twice with PBS at 4°C, and then incubated with 20 µl of monoclonal antibody for 30 min at 4°C. After three more washes with PBS, 5 µl of normal rabbit serum was added to each well. The cells were finally washed with PBS, resuspended in 200 µl of PBS containing 1% formaldehyde, and subjected to cytofluorometric analysis.
Blood Samples
On the day the semen samples were obtained, a serum blood sample was also drawn by venopuncture. This sample was used to measure several hormone levels.
LH, FSH, and Testosterone Serum Levels
Serum concentrations of LH, FSH, and testosterone were measured by an RIA as previously described [21]. Results are expressed in international units per milliliter.
Inhibin B, Activin, and pro-C Levels in Serum and Seminal Plasma
Inhibin B, activin, and pro-C levels were measured in serum and seminal plasma samples, using a novel two-site ELISA [17] (Serotec, Oxford, UK). This test is based on the use of plates coated with specific capture antibodies to the inhibin ßA and ßB subunits and the pro-region of the subunit, respectively. Briefly, the protocol involves the incubation of samples (all used undiluted, except when inhibin B is measured in seminal plasma, in which case a 1/10 dilution is carried out) and the standards in the wells of the appropriate plate, in which a monoclonal antibody specific for the molecule of interest (either inhibin A or B, activin, or pro-C) has been placed. During the incubation step, the antigen binds to the captured or immobilized antibody. After a washing step, a second or detection antibody is added. This is the Fab fragment of a monoclonal antibody, specific for the subunit of inhibin, coupled to alkaline phosphatase. Any unreacted material is then removed by washing before the detection of alkaline phosphatase using a sensitive substrate reaction. The reaction is finally read with a plate reader at 490 nm, and the concentrations in the samples are deduced from the regression line obtained with the standards. Results are expressed in pg/ml for inhibin B or pro-C, and in ng/ml for activin.
Statistics
Mean value ± standard error of the mean (SEM), standard deviation, range, and median values of the expression of HLA molecules, hormones, inhibin B, and related peptides concentrations, were calculated using the InStat statistics software (GraphPad, San Diego, CA). Comparisons between serum and seminal plasma concentrations of inhibin B and related peptides were carried out using a two-tailed Mann-Whitney two-sample test. A linear regression test was used for correlating HLA class I or class II expression on spermatozoa with hormone concentrations in serum or seminal plasma. In all comparisons, the corresponding level of significance (P value) was calculated.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
In keeping with our previous results [11], HLA expression was found on the surface of purified spermatozoa. It ranged, depending on the individual studied, between 1% and 28% of spermatozoa for HLA class I (with a mean ± SEM expression value of 11.2 ± 0.6%), and between 1% and 21% of spermatozoa for HLA II (mean ± SEM, 6.7 ± 0.5%). The level of expression consistently differed from one individual to another (inter-individual variation), and thus in some donors the percentage of expression found was always higher than in other donors, in all the samples obtained throughout the experiment (see Table 1). Moreover, changes in the level of expression in the weekly semen samples obtained from each donor (intra-individual variation) were also detected (Table 1). This suggests the existence of a cycle in the expression of HLA class I and class II molecules on sperm cells. The variations in the expression level of both types of molecules showed a parallel pattern during the 15 wk, and their level of expression rose or declined simultaneously (Fig. 1, r = 0.801, P < 0.0001, linear regression test).
|
|
LH, FSH, and Testosterone Serum Levels
Serum levels of LH, FSH, and testosterone yielded a mean value of 4.7 ± 0.2 IU/ml, 4.2 ± 0.2 IU/ml, and 722.5 ± 30.3 IU/ml, respectively, as in our previous data (Table 2).
|
Inhibin B, Activin, and pro-C Levels in Serum and Seminal Plasma
The levels of inhibin B, activin, and pro-C in serum and seminal plasma are shown in Tables 2 and 3. These samples were obtained on the same day the cytofluorometric analysis of spermatozoa was carried out. The mean value of inhibin B in serum was 336.4 ± 11.3 pg/ml, whereas in seminal plasma a value of 750.6 ± 77.6 pg/ml was found. Thus, the mean value differed when both body fluids were compared (P < 0.001, Mann-Whitney test). There were relevant inter-individual variations, especially in the seminal plasma, for which donor 2 presented very high concentration values. Moreover, different values were obtained in a given individual throughout the 15 wk during which the samples were obtained (i.e., intra-individual variations). These values, when plotted, showed a cyclic behavior (see below). Activin levels in serum (0.05 ± 0.004 ng/ml) were lower than in seminal plasma (0.6 ± 0.04 ng/ml, P < 0.001). As for inhibin B, inter-individual differences were found (see Tables 2 and 3), but no extreme intra-individual differences appeared. Pro-C levels in serum showed a mean value of 400 ± 8.8 pg/ml, significantly higher than in seminal plasma (8.4 ± 0.5 pg/ml, P < 0.01). As for activin, inter- but not intra-individual differences were found.
Inverse Relation Between HLA Expression and Inhibin Levels
The levels of expression of HLA class I and class II molecules on sperm cells were plotted against the serum and seminal plasma levels of all hormones tested. The analysis carried out with all four donors revealed that serum inhibin B levels showed an inverse correlation with the expression of HLA class I (r = -0.612, P < 0.0001) or class II (r = -0.534, P < 0.0001) molecules on the spermatozoa surface. Thus, when HLA levels peaked, the inhibin B levels decreased, whereas low HLA expression was accompanied by higher inhibin B concentration. Concordantly, HLA expression and inhibin B levels showed a cyclic pattern. Figure 1 shows the result obtained with all four donors when HLA class I and class II expression on spermatozoa was plotted against serum B inhibin levels. None of the remaining hormones tested showed such a clear relationship, and only FSH yielded a linear regression value of r = 0.368 with HLA class I and r = 0.374 with class II (P < 0.01 in both instances).
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
In the study reported here, the expression of HLA molecules was measured on purified spermatozoa from four donors throughout a 15-wk period. Figure 1 shows the results obtained with the four donors, with cyclic expression detected. Although the level of expression clearly differed from one individual to another, it was always higher for HLA class I than for class II molecules. These data are in keeping with our previous work [11]. The pattern of expression of both types of HLA molecules when all four donors were considered showed a tight relationship (r = 0.801, P < 0.0001), and a simultaneous increase or decrease in their expression was clearly observed along the 15-wk period. This is concordant with the finding of a common regulation in the transcription of HLA class I and class II genes [22]. The fact that this expression follows a cyclic pattern may explain the apparently puzzling results obtained by different researchers on this topic: depending on the time at which the samples were obtained, a positive or negative result could be obtained.
LH, FSH, Testosterone, Inhibin B, Activin, and pro-C Serum Levels (Tables 2 and 3)
The inhibin B, activin, and pro-C values obtained differed from one individual to another, ranging from a mean concentration of 274 ± 16.1 pg/ml in individual 1 to 432 ± 15.7 pg/ml in individual 3 for inhibin B, from 0.04 ± 0.005 ng/ml in individual 1 and 0.06 ± 0.01 ng/ml in individual 2 for activin, and from 351.3 ± 16.6 pg/ml in individual 1 to 453 ± 12.5 pg/ml in individual 4 for pro-C. Further, inhibin B showed marked variations in a given individual, depending on the time at which the samples were taken, suggesting the existence of a cyclic pattern in the secretion of inhibin (see below). Considering the inhibin hypothesis (i.e., inhibin secretion by the testis would serve as a selective regulator of FSH, [12]), serum inhibin B levels in all four tested individuals were plotted against their corresponding serum FSH levels. Surprisingly, the linear regression value obtained was somewhat lower than expected, although it was, nevertheless, significant (r = -0.446, P < 0.01). Previous work by Illingworth and colleagues [23] failed to correlate inhibin B levels with FSH levels in a group of adult healthy men, although correlation was found in a group of young semen donors [23].
Inhibin B, pro-C, and Activin Seminal Plasma Levels (Table 2)
The same day on which blood serum samples were drawn, seminal plasma samples were also obtained, and inhibin B, pro-C, and activin levels measured (see Table 2). To the best of our knowledge, this is the first report on the levels of these hormones in this body fluid. Mean inhibin B values were extremely high in semen (750.6 ± 77.6 pg/ml), showing very disparate concentrations from one individual to another. Thus, individual 1 yielded a mean concentration of 279.3 ± 29.9 pg/ml, whereas a value of 1689.2 ± 73.9 pg/ml was obtained in individual 2. These differences were consistently maintained throughout the experiment, and individual 2 yielded higher results than individuals 1, 3, and 4 in the 15 samples analyzed (data not shown). It appears, then, that this hormone showed an uneven distribution between individuals in seminal plasma, and a homogeneous one in serum. These differences may well be explained by the methodology used to measure inhibin B levels. Two monoclonal antibodies are required in the ELISA test used: one directed against the chain and another against the ßA chain. This makes the test unable to differentiate fully processed forms of inhibin B from those not fully processed (i.e., 31-kDa and 58-kDa forms of inhibin). It may be possible that semen samples contain higher amounts of immature forms of inhibin B than do blood samples, which cannot be distinguished from mature ones with the methodology used herein. Should a test enabling differentiation of mature from immature forms be available, these discrepancies could probably be overridden.
HLA Expression and Inhibin B Concentration
When the levels of expression of HLA class I and class II molecules were correlated with the serum or seminal plasma levels of several hormones, serum inhibin B levels showed the most clear correlation with HLA class I (r = -0.612, P < 0.0001, all four donors considered) or class II (r = -0.534, P < 0.0001) expression on spermatozoa. The correlation is an inverse one: whenever the expression of HLA rose, inhibin B levels decreased, whereas, in contrast, when inhibin B peaked, the surface expression of HLA diminished (Fig. 1). It appears, then, that inhibin B may regulate the expression of HLA molecules on spermatozoa. Seminal plasma inhibin B did not yield such a correlation, probably because, as mentioned above, the methodology used is not able to distinguish fully mature, biologically active inhibin B from immature forms, which may be abundant in seminal plasma, according to the high values obtained. In keeping with the inhibin B hypothesis, FSH values showed a direct correlation with the expression of HLA molecules on spermatozoa, although the linear regression values obtained were clearly lower than those obtained with inhibin B (r = 0.368 for HLA class I and r = 0.374 for class II, P < 0.01).
The finding that inhibin B may regulate expression of HLA is intriguing, since it is not known how inhibin may exert such an effect. Inhibin B belongs to the transforming growth factor ß family, and it is documented that several members of this family down-regulate the expression of HLA class I or class II molecules by interacting with conserved proximal promoter elements [22, 24]. It seems likely, then, that inhibin B could also alter HLA expression, by influencing the transcription rate of the genes. In fact, it is known that this is the way inhibin B acts when regulating FSH levels [25].
Alternatively, inhibin B may exert this effect in an indirect manner. There is increasing evidence that the neuroendocrine and the immune systems are closely interrelated, influencing one another by means of soluble mediators: hormones, neurotransmitters, and cytokines [26]. Cells of the neuroendocrine and immune systems are known to produce and respond to these mediators. Thus, inhibin-induced alterations in the FSH levels could ultimately affect HLA expression on sperm cells. However, such an indirect mechanism would hardly explain the correlation values obtained between HLA expression and inhibin B levels in serum.
In conclusion, assuming that the expression of HLA molecules on germ cells could be a mechanism to avoid fertilization of ova by spermatozoa with similar or identical HLA in order to favor heterosis, our data reveal an unexpected function of inhibin B, which may be relevant to the fertilization process and to male fertility control.
|
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 The contributions of Martin-Villa and Longás are equal, and the order of authorship is arbitrary.
Accepted: July 26, 1999.
Received: February 23, 1999.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  L.A. Bergmeier and T. Lehner Innate and Adaptive Mucosal Immunity in Protection against HIV Infection Adv. Dent. Res., April 1, 2006; 19(1): 21 - 28. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. A. Bergmeier, K. Babaahmady, Y. Wang, and T. Lehner Mucosal alloimmunization elicits T-cell proliferation, CC chemokines, CCR5 antibodies and inhibition of simian immunodeficiency virus infectivity J. Gen. Virol., August 1, 2005; 86(8): 2231 - 2238. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
