1 Departments of Animal Science and 2 Obstetrics & Gynecology, University of MissouriColumbia, Columbia, MO 65211-5300, 3 Department of Environmental Health, Occupational Health Program, Harvard School of Public Health, Boston, MA 02115 and 4 Vincent Memorial Obstetrics & Gynecology Service, Andrology Laboratory and In Vitro Fertilization Unit, Massachusetts General Hospital, Boston, MA 02114, USA
5 To whom correspondence should be addressed at: University of MissouriColumbia, S141 ASRC, 920 East Campus Drive, Columbia, MO 65211-5300, USA. e-mail: SutovskyP{at}missouri.edu
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Abstract |
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Key words: male infertility/sperm/SUTI assay/toxicology/ubiquitin
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Introduction |
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Being the universal proteolytic chaperone, the high expression of ubiquitin accompanies many pathological conditions such as apoptosis, Alzheimers disease and inflammatory disease (reviewed by Glickman and Ciechanover, 2002; Sutovsky, 2003
). Ubiquitin, an 8.5 kDa highly conserved protein, forms covalently linked polyubiquitin chains on substrate proteins and targets such ubiquitinated substrates for endocytosis and/or proteolytic degradation by the multi-subunit protease, the 26-S proteasome (reviewed by Hershko and Ciechanover, 1998
; Glickman and Ciechanover, 2002
).
While numerous ubiquitinated proteins could be carried over from the testis (reviewed by Escalier, 2003), the bulk of surface ubiquitination of defective sperm occurs during epididymal passage in both animals (Sutovsky et al., 2001a
; Sutovsky, 2003
) and humans (Dalzell et al., 2003
). The extracellular ubiquitination pathway in the epididymal lumen is now being deciphered. Several independent reports have shown that ubiquitin is secreted by the principal cells of the epididymal epithelium into the epididymal lumen, where it can mingle with the passing-through sperm (Santamaria et al., 1993
; Fraile et al., 1996
; Hermo and Jacks, 2002
; Sutovsky, 2003
; Sutovsky et al., 2001a
, 2003). Ubiquitin is also a major protein of human seminal plasma (Lippert et al., 1993
). Ubiquitin-conjugating enzyme E2 (Sutovsky et al., 2000
), ubiquitin C-terminal hydrolase PGP9.5 (Sutovsky, 2003
) and the valosin-containing protein VCP93 required for the presentation of ubiquitinated proteins to proteasome (Geussova et al., 2002
) are expressed in mammalian sperm. Furthermore, mRNAs encoding for ubiquitin and various proteasomal subunits are highly abundant in the epididymis (Jervis and Robaire, 2001, 2002). The proteasomal subunits are particularly enriched in the cytoplasm of the endocytotic, clear cells of the epididymal epithelium (P.Sutovsky et al., unpublished data), a finding consistent with the proposed role of ubiquitin system in the removal of defective sperm and debris during epididymal passage (Sutovsky et al., 2001a
, 2003
). The clearance of defective sperm and debris in the epididymis is not complete, although the number of defective cells decreases appreciably during epididymal passage. Intraluminal liquefaction, phagocytosis and fragmentation of epididymal sperm have been described (Flickinger, 1982
; Barrat and Cohen, 1987
; Barth and Oko, 1989
). Phagocytosis by resident leukocytes or by specialized epithelial cells, as well as luminal liquefaction and epithelial endocytosis, could contribute to such sperm removal (reviewed by Sutovsky, 2003
).
Based upon the above observations, we developed an objective immunoassay (SUTI or sperm-ubiquitin tag immunoassay; Sutovsky et al., 2001b), designed to reveal defective sperm regardless of whether or not their defects are detectable by light microscopic evaluation. Increased levels of sperm ubiquitination were found in men with heritable male infertility syndromes (Rawe et al., 2002
), but also in several idiopathic infertility cases (Sutovsky et al., 2001b
). In farm animals (Sutovsky et al., 2002
), ubiquitin immunoreactivity measured by flow cytometric SUTI assay correlated closely with sperm count and % abnormal morphology, but similar correlations have not been examined in humans. Most sperm with DNA fragmentation, a sign of apoptosis or necrotic sperm degeneration (Sun et al., 1997
; Sakkas et al., 2002
), are recognized by anti-ubiquitin antibodies in bulls (Sutovsky et al., 2002
) and humans (P.Sutovsky, unpublished data). The present study aims to further validate sperm ubiquitin as a biomarker of human male infertility in a group of 43 fertile donors and infertility patients with various aetiologies. This study is part of a series of trials that will focus on small, yet statistically informative groups of patients from several collaborating infertility centres.
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Materials and methods |
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A semen sample was produced on-site by masturbation into a sterile plastic specimen cup. The sample was allowed to liquefy at 37°C for 20 min prior to analysis. Subjects were instructed to abstain from ejaculation for 48 h prior to producing the semen sample.
We analysed samples for sperm concentration and motion parameters by computer-aided semen analysis (CASA; Hamilton-Thorne Version 10HTM-IVOS). Setting parameters and the definition of measured sperm motion parameters for the CASA were established by Hamilton-Thorne Co. (frames acquired: 30; frame rate: 60 Hz; straightness (STR) threshold: 80.0%; medium VAP threshold: 25.0 µm/s; duration of the tracking time: 0.5 s). To measure both sperm concentration and motility, aliquots of semen samples (5 µl) were placed into a pre-warmed (37°C) Makler counting chamber (Sefi Medical Instruments, Israel). A minimum of 200 sperm from at least four different fields were analysed from each specimen. We defined % motile sperm as WHO grade a sperm (rapidly progressive with a velocity 25 µm/s at 37°C) plus b grade sperm (slow/sluggish progressive with a velocity
5 µm/s but <25 µm/s).
Sperm morphology was determined by utilizing the WHO (1987) criteria and expressed as % abnormal sperm. Samples were coded so that the donor remained anonymous to the authors of this study.
Semen samples from 15 fertile donors were purchased from Fairfax Cryobank (USA). Fertile donors were non-smoking men with no known history of occupational exposure to toxic substances. All samples, from the fertile and infertile clinic patients, were cryopreserved using conventional techniques and kept in liquid nitrogen until examination.
Flow cytometric SUTI assay
Semen samples from 28 men from couples seeking infertility treatment (nos. 130; further patients) and 15 fertile donors (further donors) recruited and screened by a major sperm bank, were evaluated by the flow cytometric sperm-ubiquitin tag immunoassay (SUTI; Figure 1AC; Sutovsky et al., 2001b). Samples from two remaining patients (nos. 5, 24) were eliminated from the trial due to extremely low sperm count, which did not allow us to perform flow cytometric processing (no visible sperm pellet after centrifugation). All patients were of similar age (2943 years), except for patients no. 18 (48 years) and no. 25 (47 years). The sperm samples from 15 fertile donors (nos. 3145; aged 2042 years) were used as standards.
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Samples were screened on the day of processing using FACS Calibur flow cytometer (Becton Dickinson, USA), set at 488 nm wavelength. Ten thousand cells were measured in each sample and the overall ubiquitin median values (the value of ubiquitin-induced fluorescence at which half the cells are dimmer and half the cells are brighter) were recorded. To compare fertile men with patients, histograms of ubiquitin-induced fluorescence from three donors, one representing the low and two representing the high ubiquitin median values within the fertile group, were superimposed. The resulting combination curve (Figure 1C) was used as a standard for visual comparison with the patients samples (Figure 2). In addition to overall Ubi-median values, the histograms were divided into low fluorescent and highly fluorescent, positive cells (Figure 1 B) by quartile markers M2, M4, M6 and M8. For each of those markers/subpopulations, values in a complementary, remaining subpopulation of low fluorescent cells (markers M1, M3, M5 and M7) were also measured. Therefore, the M1 population represented the cells on the left of the threshold, complementary to M2-cell population to the right of the same threshold. M3 was then complementary to M4, M5 to M6 and M7 to M8. Blank, negative control samples from fertile donors and patients were processed with secondary antibodies only and showed low levels of fluorescent signal (Figure 1A, B). Blank samples were measured in the same runs under identical conditions. Scatter diagrams of visible light, indicative of cell size distribution (Figures 1 and 2) were also evaluated.
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Results |
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Flow cytometric evaluation of sperm ubiquitin
With one exception (donor no. 44; high ubiquitin medians, shifted histogram), sperm samples from fertile donors invariably showed normal, Gaussian distribution of fluorescent cells (Figure 1AC), low ubiquitin medians (Table I) and normal clinical semen parameters (Table I). Visible light-scatter diagrams (Figures 1 and 2) in donors invariably showed a tight focus of presumably normal-sized and -shaped sperm in the centre of the diagram. Within the patient group, the five patients with known male factor (abnormally low sperm count and motility by WHO criteria) had the highest average of ubiquitin median values for M2, M4, M6 and M8. Six patients with a self-reported history of occupational exposure had the highest overall median values of ubiquitin-induced fluorescence (Table I), followed by male factor patients, active smokers and ex-smokers. Male factor patients had highest ubiquitin medians M2, M4, M6 and M8. High ubiquitin levels were also recorded when the group of men with possible occupational exposures was combined with current smokers. Four of the six patients with self-reported occupational exposures had high ubiquitin medians and high % sperm abnormalities, though none of them was aged >40 years. In such samples, the histograms of ubiquitin-induced fluorescence were shifted to the right, reflecting an increase in the number of highly fluorescent, highly ubiquitinated sperm. In many of those patients, high proportion of cellular debris, documented by a large number of dots in the lower left corner of light-scatter diagrams (Figure 2; patients nos. 7 and 25), and lower sperm count were found. Importantly, in some patients with high sperm ubiquitin, the implied poor sperm quality was not revealed by subjective morphological analysis (e.g. only 28% of abnormal sperm, comparable with most fertile donors, were reported in patient no. 25; Figure 2B). Some of the patients (e.g. patient no. 7; Figure 2A) had low overall ubiquitin medians, but their histograms were unusually flat and the scatter diagrams indicated the prevalence of small cells and cellular debris over the normal-sized sperm. This corresponded with low sperm count and motility, and high % abnormal sperm. Some other patients showed atypical distribution of fluorescent cells with a plateau and a distinct peak in their histogram (e.g. Figure 2A; patient no. 19). Finally, some patients (e.g. Figure 2A; patient no. 11) had nearly normal clinical semen parameters and low ubiquitin medians, suggesting that they were fertile men from infertile couples with female factor infertility. We were unable to confirm this assumption since we did not collect data on the female partners of our patients, or on the outcome of infertility treatment.
Relationship of the sperm ubiquitin values and clinical semen parameters
In general, the ubiquitin median values showed strongest correlations with clinical data when they were taken from cell subpopulations determined by the quartile markers M2, M4, M6 and M8 rather than from the overall sample median measured in all screened cells without arbitrary subdivision. In the pooled analysis of all 43 subjects, ubiquitin median values increased with the declining sperm count (Figure 3A) and motility (Figure 3B), and increased proportionally with an increase in the percentage of abnormal sperm, evaluated by WHO criteria (Figure 3C), showing moderate-to-stronger correlations with sperm count (r = 0.63), % abnormalities (r = +0.55) and motility (r = 0.39). There was no correlation between donor age and either ubiquitin medians or % abnormalities. When fertile donors (men nos. 2943) were evaluated separately from patients, ubiquitin medians correlated well with sperm count (r = 0.41) and motility (r = 52). In the patient subgroup (men nos. 128), the highest correlations were found between ubiquitin medians and sperm count (r = 0.52). Similarly, ubiquitin medians correlated moderately with % abnormalities (r = +0.37) and to a lesser extent with motility (r = 0.26) and age (r = 0.28). Clinical sperm morphology (% abnormal sperm by WHO criteria) correlated moderately with sperm count (r = 0.47) and motility (r = 0.33) in all subjects. The correlation between sperm count and motility was also moderate (r = 0.40). Overall, the correlation coefficients for clinical semen parameters with ubiquitin were higher than those among individual clinical semen parameters. Smaller correlations between individual clinical parameters were found in subgroups of fertile men and infertility patients.
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Evaluation of the relationship between medical history, clinical semen analysis and SUTI results
Diagrams summarizing clinical and SUTI measurements in 10 selected patients and five donors are shown in Figure 4. Average ubiquitin median M8 of male factor patients, defined as having abnormally low sperm counts and/or motility, was 383.0, compared to 331.0 for current smokers, 327.0 for men with self-reported solvent exposures and 301.9 for fertile donors (Table II). The P-values were P = 0.01 for ubiquitin comparison between exposed and non-exposed patients, P = 0.05 between non-exposed patients and donors, and P = 0.05 between exposed patients and donors. According to clinical semen analysis that accompanied the samples (Table III), none of the patients would be considered infertile due to poor sperm morphology, expected to be >20% of normal sperm in a normal, fertile semen sample. It should also be considered that many sperm abnormalities revealed by SUTI analysis are not readily detectable by subjective morphology evaluation under the light microscope (Sutovsky et al., 2001b, 2002). Five patients had a history of testicular injury. Of those five patients, two also indicated a change in testicular size following the injury. Four patients had mumps before puberty. All other patients had normal clinical semen parameters by WHO criteria and no indication of medical history with potential to affect reproductive functions.
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Discussion |
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Reduced sperm count of high ubiquitin sperm samples could be a result of increased sperm degradation in the epididymis, as described previously (e.g. Flickinger, 1982; Barrat and Cohen, 1987
; Barth and Oko, 1989
). We have shown in animal models that the overall proportion of the surface-ubiquitinated, defective sperm decreases during epididymal passage, suggesting that some defective sperm undergo passive breakdown and/or active proteolysis during their descent from caput to cauda epididymis (Sutovsky et al., 2001a
). Alternatively, the reduction of the sperm count in the infertility patients could be a result of an overall reduction in sperm output by apoptosis of germ cells in the testis (Maeda et al., 2002
), or a combination of testicular and epididymal factors. In the present study, we found an increase in the proportion of small cells/cellular debris in the samples with high ubiquitin levels. Our previous studies indicate that such debris is mostly of spermatogenic origin (cytoplasmic droplets, residual bodies and fragments thereof; Sutovsky et al., 2001b
). It is thus possible that instead of reduced testicular output, there is an increase in sperm degradation rates in the epididymis, where the binding of ubiquitin to defective sperm occurs. Recent studies of the apoptotic process in the testis and epididymis suggest that the bulk of DNA fragmentation, suggestive of apoptosis, is seen in the epididymal sperm, rather than in the testis (Sakkas et al., 2002
). A feasible explanation is that the apoptosis could be induced during the final steps of spermiogenesis, while the fragmentation of DNA, a late/terminal apoptotic event, only occurs once the cells are relocated to the epididymis. Alternatively, necrosis, rather than caspase-mediated apoptosis, could be occurring in the epididymal sperm.
Motility is a highly variable semen characteristic that may reflect the sample quality/fertility to some extent. However, sperm motility declines significantly with time between sample donation and evaluation, and within the length of time for evaluation of motility (Elliason, 1981; Drobnis, 1992
; Jørgensen et al., 2001
). It is therefore not surprising that both clinical sperm morphology values and ubiquitin values show smaller correlations with motility than with sperm count or sperm morphology. Others noted that while there is significant overlap between clinical semen parameters of fertile and infertile men, poor sperm morphology is a stronger infertility predictor than poor motility (Guzick et al., 2001
).
Increased sperm abnormalities, assessed by subjective light microscopic analysis, should and do correlate positively with ubiquitin median values. However, the anti-ubiquitin antibodies bind to many sperm with intrinsic, hidden defects that would not be detected by light microscopic evaluation (Sutovsky et al., 2001b, 2002). Morphology in the present study showed slightly better correlation with the sperm motility than with the sperm count, which can be expected because of two reasons. First, secondary sperm defects such as sperm coiling occur in some samples concomitantly with the decline in sperm motility in the period between sample donation and clinical evaluation. Such defects are not a result of abnormal spermatogenesis and thus are not recognized by SUTI assays. Second, sperm motility is less reflective of fertility than sperm count or sperm morphology (Guzick et al., 2001
).
The increased ratio of defective sperm in some patients could also be explained by the reduced capacity of the epididymis for their removal. Epigenetic factors and ageing can influence gene expression in the epididymal epithelium and the maturation of epididymal sperm (Robaire and Serre, 2000; Jervis and Robaire, 2002
) by reducing transcription of specific genes in the epididymal cells (Pera et al., 1996
). For instance, the transcription of genes related to ubiquitin-proteasome pathway, mainly those encoding for various proteasomal subunits, is markedly reduced in the rat epididymis during ageing (Jervis and Robaire, 2002
). Our new data demonstrate that the proteasomal subunits are most prominently expressed in the epididymal clear cells, responsible for the removal and degradation of proteins and cellular debris from the epididymal epithelium (P.Sutovsky et al., unpublished data). Thus the effect of occupational exposure and age on the epididymal sperm could be mediated by the down-regulation of specific mRNA for proteasomal subunits in the epididymal epithelium.
We have found some of the strongest correlations of ubiquitin levels and clinical sperm parameters in the respective subgroups of patients that were current smokers and patients with job titles with suggested occupational exposure to solvents. When evaluated separately, the highest average ubiquitin median values, highest % of ubiquitin-positive cells, the highest % of morphological abnormalities by WHO standards, lowest motility and low sperm counts were recorded in the group of patients with job titles with solvent exposure. Current smokers ranked second highest on ubiquitin median, had lowest sperm count, and high % ubiquitin-positive cells. Pooled evaluation of current smokers and potentially exposed workers yielded the overall highest correlation coefficients between ubiquitin median values and clinical sperm parameters. The link between male infertility and smoking or occupational exposure has been established by a number of studies (e.g. Rubes et al., 1998; Moline et al., 2000
). Using TUNEL assay, Sun et al. (1997
) reported higher incidence of DNA fragmentation in smokers. Surprisingly, this study and others (e.g. Wallock et al., 2001
) showed higher sperm motility in smokers. Adelusi et al. (1998
) even suggested that smoking may actually increase sperm motility in subfertile men. This could potentially lead to misdiagnosis if clinical motility data are taken into account as a determinant of fertility. Ubiquitin assay, in contrast, clearly recognized semen abnormalities in several patients who had high ubiquitin levels despite having good sperm motility.
In addition to epigenetic factors, the pre-existing conditions possibly affecting the male reproductive system could also affect sperm quality and sperm ubiquitin levels. Two high ubiquitin patients with normal or poor clinical semen parameters reported testicular injury (no. 7), pre-pubertal mumps (no. 2) and one of the five low count/high ubiquitin patients suffered from unilateral cryptorchidism (no. 28). Other patients reporting testicular injury (nos. 4, 11, 21) and mumps (nos. 10, 21) had low ubiquitin levels and good clinical semen parameters. We have observed increased ubiquitin levels in all five patients with previous indication of poor sperm quality (nos. 7, 23, 25, 27 and 28; low count in all five patients, combined with low motility in patient no. 28). Thus, SUTI assay clearly identified men with obvious male factor. Overall, neither the subjective morphology nor ubiquitin values showed strong correlations with age. However, most subjects of this study were in their prime reproductive age (average age 35 years for patients, 30 years for donors). Some of the fertile donors were in their low 20s, thus complicating the evaluation of the effect of age on ubiquitin in this group. Count, motility and % abnormal sperm did not correlate with age in any of the evaluated populations (patients, fertile donors, patients and donors together).
Our previous studies showed increased sperm ubiquitin levels in infertility patients with male factor and idiopathic infertility (Sutovsky et al., 2001b) and in teratospermic men suffering of heritable infertility syndrome (FSD; Rawe et al., 2002
). In the present study, the relative measures of sperm ubiquitin content correlated closely with sperm count and motility in a mixed population of infertility patients with varied aetiologies. While some of the correlations and averages in the present study are limited by unbalanced and small numbers of subjects in subgroups, they provide useful preliminary data for possible studies in reproductive toxicology and epidemiology. Even more importantly, measures of relative sperm ubiquitin levels appear to be more informative than the evaluation of sperm morphology by WHO criteria, as they correlate better with sperm count and motility.
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Acknowledgements |
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Submitted on February 10, 2003; resubmitted on October 16, 2003; accepted on November 21, 2003.