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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2005-1192
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 11 6275-6282
Copyright © 2005 by The Endocrine Society

Human Sperm Sex Hormone-Binding Globulin Isoform: Characterization and Measurement by Time-Resolved Fluorescence Immunoassay

David M. Selva, Lluis Bassas, Francina Munell, Ana Mata, Francis Tekpetey, John G. Lewis and Geoffrey L. Hammond

Departments of Obstetrics and Gynaecology, University of British Columbia, and Child and Family Research Institute (D.M.S., G.L.H.), Vancouver, Canada V5Z 4H4; Laboratory of Andrology and Embryology (L.B., A.M.), Fundacio Puigvert, Barcelona 08025, Spain; Grup de Recerca Endocrinologia Molecular (F.M.), Hospital Vall d’Hebron, Barcelona 08035, Spain; Department of Obstetrics and Gynecology (F.T.), University of Western Ontario, Ontario, Canada N6H 1C9; and Canterbury Health Laboratories (J.G.L.), Christchurch, New Zealand

Address all correspondence and requests for reprints to: Geoffrey L. Hammond, Ph.D., Child and Family Research Institute, 950 West 28th Avenue, Vancouver, British Columbia, Canada V5Z 4H4. E-mail: ghammond{at}cw.bc.ca.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context: SHBG gene expression in human testis results in an SHBG isoform that accumulates in the sperm head.

Objective: The objective of this study was to further characterize the SHBG isoform in human sperm and to assess its biological relevance.

Design, Setting, and Patients: A time-resolved immunofluorometric assay was established to measure SHBG isoform concentrations in sperm samples from patients and sperm donors attending in vitro fertilization clinics.

Results and Conclusions: Molecular characterization of SHBG transcripts in human testis and sperm and biochemical analyses of the sperm SHBG isoform indicate that its smaller size compared with plasma SHBG is due to a lack of amino-terminal residues. The SHBG isoform is lost from sperm by one freeze and thaw cycle and during capacitation, which suggests it is located in or between the outer acrosomal and sperm plasma membranes. Sperm SHBG levels were proportional to the number of sperm analyzed and within assay variability in samples taken on different occasions from seven of nine individuals. Intra- and interassay variability (coefficient of variation) was 5.8 and 8.5%, respectively. Sperm SHBG levels ranged from 6–49 pM/106 sperm in 13 donor samples and did not correlate with serum SHBG levels. Sperm SHBG levels were lowest in fertile men and highest in patients with untreated varicocele, but these differences were not significant. Patients studied for couple infertility and those with surgically treated varicocele showed intermediate values. Sperm SHBG isoform levels correlate significantly with age and sperm motility and may influence sperm function in relation to male fertility.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
EXPRESSION OF THE SHBG gene in the testis has been studied extensively in the rat (1). In this species, it encodes the SHBG homolog known as the testicular androgen-binding protein, which is produced by Sertoli cells and secreted into the seminiferous tubules where it is thought to regulate testosterone availability in the male reproductive tract (1).

Although SHBG transcripts are present in the human testis (2), our studies in transgenic mice (3, 4, 5) and testicular cell lines (5) have shown that the human SHBG gene is not expressed in Sertoli cells. Human SHBG transcripts in the testes of our transgenic mice are confined to germ cells and developing sperm. They contain a noncoding alternative exon 1 sequence that lacks a typical translation initiation codon (4), but they encode an SHBG isoform that is 4–5 kDa smaller than SHBG in blood. More importantly, this SHBG isoform accumulates in the acrosome of developing sperm and binds both androgens and estradiol with high affinity (4).

The human testis also contains alternatively spliced SHBG transcripts (2), but their relative abundance is not known. As in mice that express human SHBG transgenes, human sperm contains an SHBG isoform that is about 5 kDa smaller than SHBG in plasma (4). Our goal has therefore been to further characterize this SHBG isoform in human sperm and to develop a robust, ultrasensitive immunoassay (6) for its measurement in sperm from fertile men or men whose fertility is in question.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects and samples

Serum and/or semen samples from 22 healthy semen donors (21–34 yr old) were obtained for assay development. Samples were also obtained from 70 patients undergoing semen analysis, as well as semen donors (n = 9) with more than a 20% pregnancy rate per insemination cycle. Indications for semen analysis included request for vasectomy (n = 4), couple infertility (n = 52), and assessment of varicocele presurgery (n = 7) and postsurgery (n = 7). This pilot study was approved by the Institutional Review Board of Fundacio Puigvert, and all the participants gave written consent to the procedures of the study.

Semen specimens produced by masturbation after 3–7 d of abstinence were processed for analysis within 2 h. Spermiograms were performed in accordance with World Health Organization (7) and European Society of Human Reproduction and Embryology (8) guidelines, and included volume, pH, motility, sperm concentration, vitality, and morphology assessment. Kinematics of motile sperm was assessed by computer-assisted sperm analysis HTM 2030, software version 7.0 (Hamilton-Thorn Research, Beverly, MA). Because we required a minimum number of sperm for SHBG analysis, specimens with less than 106 sperm/ml or less than 1 ml were excluded.

Semen samples were diluted 1:1 (vol/vol) with modified human tubular fluid (HTF) medium (Irvine Scientific, Santa Ana, CA), and sperm was separated by centrifugation (350 x g for 10 min) and washed with the HTF. After recentrifugation, sperm was resuspended in the HTF to a count of 100 x 106/ml. Aliquots (100 µl) were frozen at –20 C until analysis. In a subset of samples, 100 x 106 washed sperm were diluted 1:9 (vol/vol) with HTF supplemented with 2% BSA and incubated under capacitating conditions (37 C, 5% CO2) for 18–20 h, before analysis.

Immunohistochemisty

A formaldehyde-fixed human testis was prepared for immunohistochemistry, and deparaffinized sections were boiled in citrate buffer (pH 9.9) for 10 min. Primary rabbit antihuman SHBG antiserum (Dako, Carpinteria, CA) was diluted 1:500 and applied at 4 C for 16 h. Primary antibodies were removed by washing in PBS, and sections were incubated for 1 h with rhodamine-conjugated goat antirabbit antibodies (Pierce Chemical Company, Rockford, IL). After washing in PBS, sections were mounted with Immuno-Fluore mounting medium (ICN Pharmaceuticals Inc.. Costa Mesa, CA). The positions of sperm were recorded by transmission microscopy.

RNA analysis

Total RNA was extracted from human testis and sperm, as well as from livers and testicular germ cells of mice that express an 11-kb human SHBG transgene (3), using TRIzol reagent (Invitrogen Corp., Burlington, Ontario, Canada). Poly(A)+RNA was isolated, separated by agarose electrophoresis in the presence of formaldehyde, and transferred to Zeta-Probe nylon membrane (Bio-Rad Laboratories Inc., Mississauga, Canada), as described (4). Membranes were hybridized with various 32P-labeled human SHBG coding sequences, i.e. a 3' EcoRI fragment of a human SHBG cDNA (9), the SHBG exon 1 sequence encoding the SHBG leader sequence for secretion (2), the SHBG alternative exon 1 sequence (2), or the SHBG exon 7 sequence (2).

Human SHBG transcripts in RNA extracts of testicular cells (Sertoli and germ cells) from the transgenic mice (4) and from human testis and sperm were characterized in RT-PCR experiments, as described previously (4). For this purpose, we used an oligonucleotide corresponding to a 5' sequence (5'-GCGGTTCAAAGGCTCCC) within the SHBG alternative exon 1 and a reverse primer oligonucleotide complementary to a sequence (5'-TGGCTTCTGTTCAGGGCC) within exon 8 of the human SHBG gene (2). The PCR was performed for 40 cycles at 94 C for 15 sec, 59 C for 30 sec, and 72 C for 45 sec. To control for the integrity and relative amounts of mRNA in the samples, mouse and human cyclophilin A (CypA) cDNAs were coamplified using specific primers (5'-CAGATGGGGTAGGGACG and 5'-ATGGTCAACCCCACCACCGTG for mouse CypA and 5'-ATGGTCAACCCCACCGTG and 5'-TGCAATCCAGCTAGGCATG for human CypA).

Western blot analysis

Frozen pellets of washed human donor sperm samples were subjected to a single freeze/thaw cycle to release SHBG into 50 µl of 0.25 M Tris-HCl (pH 8.0). Half of the extract was then treated with N-glycosidase F (Roche Diagnostics, Laval, Quebec, Canada) at 37 C overnight in parallel with a diluted (1:50) human serum sample, and the reaction mixtures and the untreated samples were subjected to Western blotting (4). Blots were blocked in PBS containing 0.01% Tween 20 and 5% skim milk and then incubated overnight at 4 C with 11F11 mouse monoclonal antibody against human SHBG. They were then washed, and antibody-antigen complexes were identified using horseradish peroxidase-labeled donkey antimouse IgG and chemiluminescent substrates (Pierce Biotechnology Inc.) by exposure to x-ray film.

Time-resolved fluorometric immunoassay

Proteins released from sperm samples into 100 µl HTF were diluted 1:2.5 in filtered sample buffer (1%BSA, 50 mM Tris-HCl, pH 7.5, and 150 mM NaCl) for a time-resolved fluorometric immunoassay (6). In brief, duplicate 100-µl aliquots of each sample or human SHBG standards were added to the wells of a MaxiSorp microtiter plate (Fisher Scientific Ltd., Nepean, Ontario, Canada), precoated with rabbit polyclonal anti-serum against human SHBG, and incubated for 1 h at room temperature. Reaction mixtures were then aspirated, and the wells were washed three times before the addition of 100 µl Europium-labeled monoclonal antihuman SHBG antibody (kindly provided by Orion Diagnostica, Oulunsalo, Finland) in a DELFIA assay buffer (PerkinElmer Life Sciences, Turku, Finland). After 1 h at room temperature, the Europium-labeled antibody was removed and the wells were washed, as above. DELFIA enhancement solution (100 µl) was then added 10 min before time-resolved fluorescence measurements in a Victor plate reader, as recommended by PerkinElmer Life Sciences.

Serum SHBG measurements

Serum SHBG was measured using an established saturation steroid-binding capacity assay (10).

Statistical analyses

The effects of clinical and semen categories on sperm SHBG isoform concentrations were assessed by the Kruskal-Wallis test. Correlations between quantitative parameters were computed by the Spearman’s {rho} (rs). Multiple regression analysis was performed considering the SHBG concentration in sperm as the dependent variable. Values of P < 0.05 were considered significant. All the calculations were done with the statistical package SPSS version 12.0 (SPSS Inc. Chicago, IL).


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Identification and localization of SHBG in human sperm

Immunohistochemical analysis of a human testis section shows SHBG localized to sperm heads that were identified by transmission microscopy within a seminiferous tubule (Fig. 1Go). In this and other sections, no immunoreactivity was detected in structures corresponding to Sertoli cells.



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  		FIG. 1. Identification of immunoreactive SHBG in human sperm heads. Human testis sections incubated with primary antiserum against human SHBG showed that fluorescence-labeled secondary antibodies reacted only with sperm heads (identified by transmission microscopy) within a seminiferous tubule. No immunolabeling was detectable in sections incubated only with rhodamine-labeled secondary antibodies.

 
Characterization of SHBG transcripts and the SHBG isoform in human testis and sperm

To characterize the SHBG transcripts in the human testis, Northern blots of poly(A)+ RNA from human testis, liver, and isolated germ cells from mice expressing the 11-kb human SHBG transgene were performed using exon-specific human SHBG probes (Fig. 2AGo). The blots show that SHBG transcripts in the human testis contain the alternative exon 1 sequence, which is also present in the human SHBG transcripts extracted from germ cells of the transgenic mice. As expected, the SHBG transcript in the liver of these mice includes the exon 1 sequence that contains the translation initiation codon for the SHBG precursor polypeptide and the leader sequence for secretion, but this exon is not present in the SHBG transcripts from human testis or germ cells from the transgenic mice (Fig. 2AGo). Interestingly, the majority of SHBG transcripts in the human testis lack exon 7 sequences, whereas most of the transcripts in the testicular germ cells of the transgenic mice contain these sequences (Fig. 2AGo). When a human SHBG cDNA that recognizes exons 6–8 sequences was used as a probe, human SHBG transcripts were detected in liver and testicular germ cells from the transgenic mice, as well as in the human testis. However, the human testis SHBG transcripts appear smaller than those in germ cells of transgenic mice (Fig. 2AGo), and this could be due to the lack of exon 7 sequences in most of the SHBG transcripts extracted from human testis.



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  		FIG. 2. Characterization of human SHBG transcripts in the liver and testicular germ cells from 11-kb human SHBG transgenic mice and from human testis and sperm. A, Duplicate Northern blots (left and right panels) were probed with a 3'EcoRI fragment (i.e. spanning exon 6–8 sequences) of a human SHBG cDNA in the top panels, human SHBG alternative exon 1 (middle left panel) and exon 1 (middle right panel)-specific sequences (2 ), and the human SHBG exon 7 sequence (bottom panels). The relative abundance of various human SHBG transcripts in liver and germ cells from the transgenic mice and human testis is evident from the signals in the Northern blots in the top panels. Human SHBG transcripts containing the alternative exon 1 sequence were only detected in germ cells from the transgenic mice and the human testis (middle left panel), whereas human SHBG transcripts containing the exon 1 sequence were only detected in the transgenic mouse liver (middle right panel). When the blots were probed with the exon 7-specific sequence (bottom panels), human SHBG transcripts were most abundant in the transgenic mouse liver and testis. B, Human SHBG transcripts were detected in isolated testicular cells (Sertoli cells and germ cells) from 11-kb human SHBG transgenic mice, as well as from human testis and sperm by RT-PCR using specific oligonucleotides for the human SHBG alternative exon 1 and 8 sequences. These results confirm that most of the SHBG transcripts in human testis and sperm lack the exon 7 sequence, whereas the majority of the human SHBG transcripts in testicular cells from the 11-kb human SHBG transgenic mice contain the exon 7 sequence.

 
The SHBG transcripts were also analyzed in human testis and sperm RNA extracts, as well as RNA extracts of testicular cells from the 11-kb human SHBG transgenic mice by RT-PCR. This was done using specific oligonucleotide primers corresponding to human SHBG alternative exon 1 and human SHBG exon 8 sequences and mouse and human CypA-specific primers as controls for RNA abundance and integrity. In these experiments, human SHBG transcripts were detected in the testicular cells of 11-kb human SHBG transgenic mice as well as in the human testis and sperm, and the majority of human SHBG transcripts in the mouse testis contain exon 7 sequences, whereas most of those in the human testis and sperm do not (Fig. 2BGo).

When human serum and sperm samples are treated with N-glycosidase F to remove N-linked oligosaccharides before Western blot analysis, it is apparent that the human sperm SHBG isoform is N-glycosylated (Fig. 3Go). Although the reduction in the apparent size of the sperm SHBG isoform after N-glycosidase F treatment is smaller than that observed for serum SHBG, the deglycosylated sperm SHBG still appears to be 4–5 kDa smaller than deglycosylated serum SHBG (Fig. 3).



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  		FIG. 3. Western blots demonstrating the presence of N-linked oligosaccharides in the human sperm SHBG isoform. Western blots show differences in the electrophoretic mobility of serum SHBG and the sperm SHBG isoform before (–) and after (+) treatment with N-glycosidase F to remove N-linked oligosaccharides. The positions of protein size markers are shown on the left.

 
Validation of the immunoassay of the SHBG isoform in human sperm

The efficiency of SHBG isoform extraction from human sperm was tested using one freeze and thaw cycle, one freeze and thaw cycle plus 15-min sonication, or two freeze and thaw cycles plus 15-min sonication. After these treatments, the samples were centrifuged and diluted for analysis, as indicated above. In addition, the pellets were resuspended in sample buffer, and after an additional freeze and thaw cycle plus 15-min sonication, the supernatants were collected for analysis. To demonstrate that SHBG is not released from sperm without these treatments, a washed sperm sample was centrifuged, and the supernatant was analyzed directly. The pellet of this sample was also suspended in sample buffer and subjected to one freeze and thaw cycle plus 15-min sonication, and the supernatant was taken for analysis. The results demonstrate that one freeze and thaw cycle releases essentially all the SHBG isoform from sperm into the assay buffer (Fig. 4Go).



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  		FIG. 4. Conditions for extraction of the SHBG isoform from human sperm. Different methods to release the sperm SHBG isoform were tested. These involved exposing a sperm sample to a single freeze and thaw cycle, one freeze/thaw cycle plus 15-min sonication, and two freeze/thaw cycles plus 15-min sonication. Sperm pellets from the untreated and treated samples were resuspended and subjected to an additional freeze/thaw cycle plus 15 min of sonication before analysis of SHBG in the supernatant. In the untreated sample, almost all of the SHBG was retained in the sperm pellet (open bar), whereas essentially all the SHBG was released into the supernatant after one freeze/thaw cycle, and additional freeze/thaw cycles and sonication did not increase the efficiency of the extraction of SHBG from sperm (solid bars).

 
Sperm SHBG measurements were also performed on samples before and after capacitation. The mean ± SEM concentration of the SHBG isoform in freshly prepared sperm was 22.1 ± 2.1 pmol/106 sperm and 0.6 ± 0.2 pmol/106 sperm after capacitation. These data indicate that capacitation results in 97% loss of SHBG from the sperm samples.

The lowest standard used to generate the standard curve was 0.19 pM, and the signal (counts per second) obtained was greater than +2 SD of a sample containing no human SHBG. To assess assay precision, the intraassay coefficient of variation, 5.8%, was determined by analyzing four to five samples in duplicate from two donors. The interassay variability (coefficient of variation of 8.5%) was also determined by analyzing duplicate aliquots from the same patient on five different occasions.

The accuracy of the assay was determined by measuring the SHBG isoform extracted from different amounts of sperm from the same donor, and the values were directly proportional to the number of sperm analyzed (Fig. 5Go). In addition, sperm SHBG isoform levels measured in samples collected on two different occasions from the same individual remained within assay variability in seven of nine subjects (Fig. 6Go). Serum SHBG levels were also determined in 13 donors whose sperm SHBG isoform levels ranged between 6 and 49 pM/106 sperm, but there was no correlation between these measurements (data not shown).



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  		FIG. 5. Correlation between sperm SHBG isoform levels and number of sperm. Linear regression analysis shows that the amount of SHBG present in human sperm measured in picomoles is proportional to the number of sperm analyzed.

 


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  		FIG. 6. Sperm SHBG isoform levels in semen samples collected from the same individuals on two different occasions. Sperm SHBG isoform levels in samples from seven of nine individuals were within assay variability.

 
Sperm SHBG isoform levels in men undergoing fertility evaluation

To assess the utility of the assay for SHBG isoform levels in sperm, we conducted a preliminary survey of semen samples representative of the routine workload of an andrology laboratory. This showed median (±SD) SHBG concentrations of 23.0 ± 17.0 pM/106 sperm, with a range of 3.0–112.3 pM/10 million sperm. Interestingly, sperm SHBG levels were significantly correlated to age, percentage of motile sperm, and the proportion of grade A motile sperm. Correlations with motility variables maintained their significance after adjusting for the effect of age (Table 1Go). Sperm SHBG isoform levels were significantly lower in semen samples with poor motility compared with specimens with good motility (Fig. 7Go). A multiple regression analysis identified total motility and age as independent predictors for SHBG concentration in sperm (multiple R2 = 0.144, P = 0.006). Other semen variables (sperm count, motile density, percentage of normal morphology, vitality, total number of deviations from reference values, and variables of sperm kinematics) were not significantly associated with SHBG levels.


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  		TABLE 1. Bivariate Spearman’s correlation coefficients (rs) between sperm SHBG isoform levels and selected variables in 82 men

 


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  		FIG. 7. Sperm SHBG isoform levels in samples grouped by the percentage of motile sperm. Box plot of SHBG concentrations. Horizontal lines correspond to 10th, 25th, median (thick line), 75th, and 90th percentiles, and extreme values are shown separately. Kruskal-Wallis test {chi}2 = 10.29; P = 0.006.

 
When the patient and donor samples were classified with respect to fertility status (Table 2Go), fertile men (i.e. prevasectomy patients and semen donors) seemed to have the lowest sperm SHBG levels, whereas the highest sperm SHBG levels were seen in untreated patients with varicocele. Interestingly, patients analyzed after surgical varicocele repair had lower SHBG than in untreated varicocele patients, but the differences between these groups were not significant (Table 2Go). Because semen quality was not considered a sufficiently sensitive criterion to define male infertility, patients with couple infertility were classified only on the basis of having a known female risk factor or a normal female evaluation of their partner. When this was done, infertile patients showed intermediate values of sperm SHBG isoform levels, but again there were no significant differences between groups (TableGo 2).


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  		TABLE 2. Sperm SHBG isoform levels (mean ± SD) and seminal variables in fertile men and men undergoing fertility evaluation

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Our previous studies have demonstrated that the human and rodent SHBG genes are expressed in different cell types within the testis (4). As in the rat, expression of the mouse SHBG gene appears to be confined to Sertoli cells (4), but expression of 11-kb human SHBG transgenes in the mouse testis (3) occurs only in germ cells (4), and the resulting transcripts contain an alternative exon 1 sequence (4). We have also shown that a human SHBG isoform accumulates in the acrosome of germ cells and developing sperm in these transgenic mice and that a similar SHBG isoform is present in human sperm (4). These studies have indicated that cell-specific differences in SHBG expression in the testis reflect the use of unique transcription units under the control of distinct promoter sequences in different species. In support of this, we have recently shown that USF-1 and USF-2 transcription factors bind to a cis-active element (footprint 4) within the human SHBG promoter and repress human SHBG gene expression in Sertoli cells (5). Thus, SHBG transcription in the human testis is likely confined to germ cells under the control of a promoter flanking the alternative exon 1 sequence, which is located 1.2 kb upstream of the promoter responsible for SHBG transcription in the liver (2).

In the present study, we confirm that SHBG transcripts in the human testis and sperm contain the alternative exon 1 sequence originally identified within cDNAs from a human testis library (2). Although most of these transcripts lack exon 7 sequences, the SHBG isoform in human sperm contains oligosaccharides that must be linked to Asn residues, the codons for which can only be present in mRNA containing exons 7 and 8 sequences. Thus, we conclude that the sperm SHBG isoform is encoded by a transcript containing an alternative exon 1 sequence as well as the exon 7 sequence.

As in our previous studies of the human SHBG isoform in the testis of transgenic mice (4), removal of N-linked oligosaccharides from the human sperm SHBG isoform results in a smaller reduction in its size when compared with serum SHBG treated in the same way, and this suggests a qualitative difference in N-glycosylation or the use of only one N-glycosylation site within the carboxy terminus of sperm SHBG. Moreover, the size difference (4–5 kDa) between the deglycosylated sperm SHBG isoform and serum SHBG suggests that the sperm isoform lacks amino-terminal residues, which include a site for O-linked glycosylation at Thr7 in the mature SHBG polypeptide (9). We therefore conclude that the human sperm SHBG isoform results from translation initiation at the in frame AUG codon in the exon 2 sequence of alternative SHBG transcripts that include exon 7 sequences.

We have found that a single freeze and thaw cycle is sufficient to release essentially all the immunoreactive SHBG from human sperm samples. Because immunoreactive SHBG covers the entire head of immature testicular sperm, we suggest that it is located between the outer membrane of the acrosome and the sperm plasma membrane or is somehow loosely associated with these membranes. This is further supported by a loss of SHBG from sperm after incubation under conditions that induce capacitation and the acrosome reaction (11). Some glycoproteins of testicular origin are intercalated or anchored in the membrane, although their precise localization may change by the remodeling processes that take place during the passage through the epididymis (12). Why a human SHBG isoform accumulates in the sperm head remains unclear, but this seems to occur at an early stage of spermatogenesis and may therefore influence sperm maturation. At present, the only information regarding any possible function of sperm SHBG is that it binds steroids like plasma SHBG (4). Plasma membrane receptors for progesterone and estrogens have been described in spermatozoa, and they seem to mediate different nongenomic events that influence capacitation and the acrosome reaction, such as calcium influx into sperm and phosphorylation of several membrane proteins (13, 14, 15). It is therefore possible that sperm SHBG influences the physiological actions of estradiol on sperm through modulation of its availability to sperm estrogen membrane receptors.

To measure the relatively low concentrations of the SHBG isoform in human sperm, we adapted a time-resolved fluorometric immunoassay that allowed us to detect as little as 0.19 pM SHBG. This assay is at least 2 orders of magnitude more sensitive than commercially available time-resolved immunofluorometric assays. An additional feature of the assay is that values are expressed in terms of the number of sperm analyzed. For this purpose, a standard number of 10 million sperm was selected as an amount that contained sufficient concentrations of SHBG isoform that could be measured in a selection of sperm donors. It was also reasoned that this number of sperm could generally be obtained and accurately measured for analysis. Although it is clear that samples from patients with severe oligozoospermia often contain less than 10 million sperm, our assay evaluation studies indicate that the SHBG isoform could be reliably detected in as few as 2 million sperm. Therefore, as long as the amount of sperm exceeds this number, an assay can be performed because values can be adjusted to a standard concentration of 10 million sperm.

In addition to being ultrasensitive, the assay appears to be highly accurate and precise. Importantly, although SHBG isoform levels in human sperm samples vary considerably between individuals, they are remarkably consistent in samples taken from most individuals on different occasions. Moreover, blood SHBG and sperm SHBG isoform levels do not correlate, and we conclude that sperm SHBG isoform production is completely unrelated to the control of SHBG gene expression in the liver.

As a first step toward defining the function of the human sperm SHBG isoform, we measured its levels in relation to sperm viability and quality in fertile men and patients undergoing fertility assessment for various reasons. Although the levels were variable in most of the groups, and the intergroup differences were not significant, the lowest values were found in fertile patients, whereas men with untreated varicocele generally showed the highest concentrations. It was also noted that the sperm SHBG levels in patients treated surgically for varicocele showed lower sperm SHBG than most of the untreated patients. Interestingly, a significant negative correlation was found between sperm SHBG levels and age, despite little change in plasma SHBG levels in men within the age groups studied (16). More importantly, a significant association was observed between sperm SHBG levels and sperm motility, which is a key parameter reflecting the functional fitness of male gametes. Taken together, these data suggest that sperm SHBG levels may be influenced by physiological factors that modulate testicular function, and their relevance to sperm biology deserves further study.


   Acknowledgments
 
We thank Tracie Galbraith for assistance with the preparation of the manuscript.


   Footnotes
 
This work was supported by grants from the Canadian Institutes of Health Research (to G.L.H.) and the Serono-Fundación Salud 2000 (to L.B. and F.M.). G.L.H. holds a Canada Research Chair in Reproductive Health.

First Published Online August 30, 2005

Abbreviations: CypA, Cyclophilin A; HTF, human tubular fluid.

Received May 27, 2005.

Accepted August 22, 2005.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

  1. Joseph DR 1994 Structure, function, and regulation of androgen-binding protein/sex hormone-binding globulin. Vit Horm 49:197–280[Medline]
  2. Hammond GL, Underhill DA, Rykse HM, Smith CL 1989 The human sex hormone-binding globulin gene contains exons for androgen-binding protein and two other testicular messenger RNAs. Mol Endocrinol 3:1869–1876[Abstract]
  3. Jänne M, Deol HK, Power SGA, Yee S-P, Hammond GL 1998 Human sex hormone-binding globulin gene expression in transgenic mice. Mol Endocrinol 12:123–136[Abstract/Free Full Text]
  4. Selva DM, Hogeveen KN, Seguchi K, Tekpetey F, Hammond GL 2002 A human sex hormone-binding globulin isoform accumulates in the acrosome during spermatogenesis. J Biol Chem 277:45291–45298[Abstract/Free Full Text]
  5. Selva DM, Hogeveen KN, Hammond GL 2005 Repression of the human sex hormone-binding globulin gene in Sertoli cells by USF transcription factors. J Biol Chem 280:4462–4468[Abstract/Free Full Text]
  6. Niemi S, Mäentausta O, Bolton NJ, Hammond GL 1988 Time-resolved immunofluorometric assay of human sex hormone binding globulin (SHBG). Clin Chem 34:63–66[Abstract/Free Full Text]
  7. World Health Organization 1999 Laboratory manual for the examination of human semen and sperm-cervical mucus interaction. 4th ed. Cambridge, UK: Cambridge University Press
  8. Kvist U, Bjorndahl L 2002 Manual on basic semen analysis. ESHRE Monographs ed. Oxford, UK: Oxford University Press
  9. Hammond GL, Underhill DA, Smith CL, Goping IS, Harley MJ, Musto NA, Cheng CY, Bardin CW 1987 The cDNA-deduced primary structure of human sex hormone-binding globulin and location of its steroid-binding domain. FEBS Lett 215:100–104[CrossRef][Medline]
  10. Hammond GL, Lähteenmäki PLA 1983 A versatile method for the determination of serum cortisol binding globulin and sex hormone binding globulin binding capacities. Clin Chim Acta 132:101–110[CrossRef][Medline]
  11. Byrd W, Tsu J, Wolf DP 1989 Kinetics of spontaneous and induced acrosomal loss in human sperm incubated under capacitating and noncapacitating conditions. Gamete Res 22:109–122[CrossRef][Medline]
  12. Schroter S, Osterhoff C, McArdle W, Ivell R 1999 The glycocalyx of the sperm surface. Hum Reprod Update 5:302–313[Abstract/Free Full Text]
  13. Bray C, Brown JCK, Publicover S, Barrat CLR 1999 Progesterone interaction with sperm plasma membrane, calcium influx and induction of the acrosome reaction. Reprod Med Rev 7:81–93
  14. Luconi M, Muratori M, Forti G, Baldi E 1999 Identification and characterization of a novel functional estrogen receptor on human sperm membrane that interferes with progesterone effects. J Clin Endocrinol Metab 84:1670–1678[Abstract/Free Full Text]
  15. Luconi M, Francavilla F, Porazzi I, Macerola B, Forti G, Baldi E 2004 Human spermatozoa as a model for studying membrane receptors mediating rapid nongenomic effects of progesterone and estrogens. Steroids 69:553–559[CrossRef][Medline]
  16. Khosla S, Melton LJ, Atkinson EJ, O’Fallon WM 2001 Relationship of serum sex steroid levels to longitudinal changes in bone density in young versus elderly men. J Clin Endocrinol Metab 86:3555–3561[Abstract/Free Full Text]




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