The impact of epididymal and accessory sex gland function on sperm motility

S. Elzanaty1,4, J. Richthoff2, J. Malm3 and A. Giwercman1

1 Scanian Andrology Centre, Fertility Centre, 2 Department of Urology and 3 Department of Clinical Chemistry, Malmö University Hospital, SE 205 02 Malmö, Sweden


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Little is known about the regulation of sperm motility, which is an important predictor of male fertility. However, both testicular and post-testicular factors may be involved, although the impact of the latter has been relatively poorly investigated. METHODS: In semen samples from 301 young men from the general Swedish population (mean ± SD age 18.2 ± 0.4 years), we assessed sperm motility by use of a manual method as well as computer-assisted sperm analysis (CASA) and correlated these values to seminal levels of neutral {alpha}-glucosidase (NAG), prostate-specific antigen (PSA), zinc and fructose. RESULTS: There were significant positive correlations between seminal levels of NAG, and PSA and CASA percentage motile sperm (r = 0.158, P = 0.009; r = 0.155, P = 0.01 respectively), and significant negative correlations with CASA percentage immotile sperm (r = –0.206, P = 0.001; r = –0.157, P = 0.009 respectively). In a multiple regression analysis it was found that, apart from sperm concentration, the level of PSA was the most significant and independent parameter in predicting percentage motile sperm (ß = 0.220, P = 0.037). CONCLUSION: Our study demonstrated the regulatory effect of post-testicular glands on the motility of sperm. This is to our knowledge the first study showing a direct correlation between the seminal PSA levels and sperm motility in a group of men representing the general population. In future investigations and searches for specific treatment modalities in male infertility, more attention should be paid to the epididymis and accessory sex gland function.

Key words: accessory sex glands/epididymis/prostate-specific antigen/seminal plasma/sperm motility


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Sperm motility has been shown, in several studies, to be a good predictor of human male fertility in vivo and in vitro (Auger et al., 1994Go). Therefore, an insight into factors which may have an impact on the motility characteristics of sperm may give us a better understanding of the mechanisms behind male infertility and lead to the development of new, specific treatment modalities. However, our knowledge about the impact of testicular and post-testicular mechanisms on sperm motility is limited. It is well known that defects of spermatogenesis are not only associated with oligozoospermia but also with asthenozoospermia (Nistal and Paniagua, 1984Go; Okuyama et al., 1984Go; Viviani et al., 1991Go). On the other hand, it has also been shown that the epididymis and accessory sex glands also play a role for the functional status of the male gametes (Gonzales et al., 1992Go).

The epididymis has been shown to play an important role for sperm maturation including gaining the ability to be motile (Longo, 1987Go). Several biochemical markers of epididymal function can be detected in seminal fluid (Frenkle et al., 1974Go; Guerin et al., 1990Go), neutral {alpha}-glucosidase (NAG) being considered as one of the most useful from the clinical point of view (Roland et al., 1979Go; Kret and Milad, 1995Go). Although a number of studies have shown positive correlations between the seminal levels of NAG and sperm motility (Viljoen et al., 1990Go; Fourie et al., 1991Go), these results have been contradicted by other reports (Guerin et al., 1990Go; Krause and Bohring, 1999Go).

Even less knowledge exists regarding the impact of accessory sex glands on sperm motility characteristics. The gel-forming proteins—semenogelins—are mainly synthesized in the seminal vesicles (Lilja et al., 1989Go) and are believed to have an inhibitory effect on the ability of sperm to move (Robert and Gagnon, 1996Go). On the other hand, another vesical product, fructose, has been reported to be a source of energy for the motility of the gametes (Mann, 1964Go). Patel et al. demonstrated a positive correlation between seminal levels of fructose and percentage motile sperm (Patel et al., 1988Go), but other studies (Lewis-Jones, et al., 1996Go; Andrade-Rocha, 1999Go; Zöpfgen et al., 2000Go) did not find such a correlation.

Prostate-specific antigen (PSA) has been reported to be involved in degradation of semenogelins (Lilja et al., 1989Go; Robert and Gagnon, 1996Go) and might therefore be expected to have a positive impact on sperm motility. Two studies (Ahlgren et al., 1995Go; Charlens et al., 1999Go) found that seminal PSA was low in patients with low sperm motility. Zinc, which to a high degree also originates from the prostate (Arver and Eliasson, 1982Go), also plays an important role in sperm function (Lewis-Jones et al., 1996Go), mainly via its effect on chromatin stability (Kvist et al., 1987Go). The results of reports on the association between seminal zinc levels and sperm motility have been conflicting, indicating a positive correlation (Fuse et al., 1999Go; Sin-Eng et al., 2000Go), a negative correlation (Carreras and Mendoza, 1990Go) or no association at all (Behne et al., 1988Go; Lin et al., 2000Go).

Some of the reasons behind this disagreement may be related to the selection of the study population and methodology for motility assessment. The majority of the studies are based on cohorts of infertile men in whom a mixture of different pathophysiologies may affect the motility of the sperm. Furthermore the traditional—laboratory technician-based—motility assessment is usually characterized by a significant inter- and intra-observer variation. The modern methods of computer-assisted sperm analysis (CASA) are giving a more subtle and more reproducible description of sperm motility (Larsen et al., 2000Go).

We have, therefore, in a group of Swedish military conscripts, correlated the seminal levels of different markers of epididymal, prostatic and seminal vesicle function with sperm motility assessed by use of the manual method as well as CASA. The aim of this study was to evaluate the impact of the function of these three organs on sperm motility by focusing on a more homogeneous male population, thereby avoiding some of the methodological problems associated with the assessment of sperm motility.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Subjects
Approximately 95% of all 18 year old Swedish men undergo a medical health examination prior to military service. Only men with serious chronic diseases are excluded from this conscript examination. Thus, the conscripts are representative of the general population of young Swedish males. A group of 2255 consecutive men living <60 km from the city of Malmö underwent the compulsory medical board examination between May and December 2000, and were asked to take part in this study, aiming to evaluate the reproductive function of young Swedish males from the general population. Of these, 305 (13.5%) agreed to participate in the study, which took place at Malmö University Hospital.

The volunteers were asked about their reproductive history including cryptorchidism and other diseases of the male reproductive organs. These data are reported in another publication (Richthoff et al., 2002Go). Following an andrological examination, the subjects were asked to deliver a semen sample in a separate room in the laboratory area. The volunteers were paid CISOdia=55. The study was approved by the local ethics committee.

The mean ± SD age of the conscripts was 18.2 ± 0.4 years and their mean body mass index was 23 ± 3.1 kg/m2. Two men did not deliver semen samples and an additional two men were azoospermic. Consequently, data from 301 men were used for the study.

Semen analysis
The semen samples were obtained by masturbation after 12–500 h of sexual abstinence (mean 83.4 h). After 30 min of liquefaction, 450 µl of the ejaculate was removed and mixed with 50 µl of bezamidine (0.1 mol/l) in order to stop the biochemical processes involved in liquefaction. The mixture was centrifuged for 20 min at 4500 g, and the seminal plasma was decanted and stored at –20°C until analysed for the levels of NAG, and the concentrations of PSA, zinc and fructose. The seminal volume in 10 of the 301 men was too low to perform biochemical analysis of seminal fluid. Therefore, biochemical markers were only assessed in 291 semen samples. After liquefaction, within 1 h of ejaculation, the ejaculates were analysed for the following characteristics: semen volume, sperm concentration, and sperm motility (defined as WHO motility grades A, B, C and D). All semen tests were performed according to published recommendations (World Health Organization, 1999Go). Thereafter computer-assisted analysis of sperm motility using the ‘CRISMAS’ system (Image House, Copenhagen, Denmark) was performed.

Biochemical analysis
Neutral {alpha}-glucosidase
Seminal plasma NAG was measured using a commercially available kit (Episcreen®; Fertipro, Gent, Belgium) according to the instructions given by the manufacturer. The test is based on the measurement of the intensity of a colour change evoked by the reaction between {alpha}-glucosidase and 0.125 of reagent 1 (0.09 % Na-azide) which was added to 0.125 ml of thawed seminal plasma. The mixture was mixed well by pipetting, one diagnostic tablet (p-nitophenyl-{alpha}-D-glucopyranoside) was then added, and the mixture was remixed and vortexed for 60 s and incubated for 4 h at 37°C. After incubation, 3 ml of reagent 2 (0.02 mol/l NaOH) was added to the solution, which was centrifuged for 6 min at 3000 g. The absorbance value, obtained by reading the supernatant against reagent 2 as a blank, was measured by use of a spectrophotometer at 405 nm. This value was plotted on a standard curve and the corresponding total {alpha}-glucosidase activity was read on the abscissa. The NAG level was estimated by the use of the corresponding table provided by the manufacturer.

Prostate-specific antigen
The concentration of PSA in seminal plasma was determined with the ProstatusTM kit (Wallac Oy, Finland). This is a DelfiaTMmethod, i.e. an immunofluorometric method, using three monoclonal antibodies against PSA. The coefficient of variation was 12% for control samples with a mean PSA concentration of 660 mg/l.

Zinc
The concentration of zinc in seminal plasma was determined with a colorimetric method (Makino et al., 1982Go). The proteins in the sample were precipitated with trichloroacetic acid, the supernatant mixed with a water-soluble pyridylazo dye and the absorbance measured at 560 nm. The coefficient of variation was 7% for control samples with a mean zinc concentration of 2.0 mmol/l.

Fructose
The concentration of fructose in seminal plasma was determined with a spectrophotometric method, essentially as previously described (Wetterauer and Heite, 1976Go), run on a Beckman Synchron LX20 instrument. Proteins in the sample were precipitated with perchloric acid and the absorbance of the supernatant measured. After addition of phosphoglucose isomerase, resulting in conversion of fructose to glucose, the absorbance was measured again. The absorbance difference corresponds to the concentration of fructose in the sample. The coefficient of variation was 5.0% for control samples with a mean fructose concentration of 12.7 mmol/l.

CASA sperm motility analysis
CASA sperm motility analysis was performed by the use of the CRISMAS system as previously described (Larsen et al., 2000Go). The system comprises a video camera connected to standard C-mount equipped microscope. A framegrabber installed in a standard PC operating under Windows NT converts the video signal obtained at 25 MHz to raw digital data. Images are available for random access in digital form for analysis and playback. For analysis, a 5 µl aliquot of the ejaculate was pipetted into a Makler counting chamber 10 µm deep, which was placed on a heated microscope stage (37°C). Video recordings were made from four different fields of the chamber using a x20 magnification objective. CASA analysis was based upon capturing sequences of 64 frames per field and counting a minimum of 100 sperm.

For each analysed sequence the following parameters were obtained: curvilinear velocity (VCL), straight-line velocity (VSL), average path velocity (VAP), amplitude of lateral head displacement (ALH), and linearity (LIN). These parameters were defined according to published guidelines (World Health Organization, 1999Go). The total population of sperm was divided into three categories: motile (VCL >25 µm/s), ‘locally’ motile (VCL = 5–25 µm/s), and immotile (VCL <5 µm/s). Sperm motility characteristics are only given for motile sperm since the percentage of these cells was previously found to be an independent predictor of male fertility (Larsen et al., 2000Go).

For technical reasons, samples from only 285 of the 291 ejaculates on which the biochemical analysis was performed were available for the CASA analysis of the main motility characteristics (percentage motile, locally motile, and immotile sperm), and from 270 subjects for the motility characteristics, VCL, VSL, VAP, ALH and LIN. However, there was no selection bias since the difference between the 21 ejaculates which were not fully analysed and the 270 which underwent the analysis, was not statistically significant for standard sperm characteristics, including motility and concentration.

Statistical methods
The statistical analysis was performed using the SPSS version 10.0 software (SPSS Inc., Chicago, IL, USA). Primarily, Pearson’s coefficients were calculated in order to assess the bivariate correlations between the levels of biochemical seminal markers and sperm parameters as well as the length of the abstinence period. Prior to this, logarithmic transformation of the abstinence period and sperm concentration was undertaken in order to obtain a normal distribution of the data. For the remaining parameters no transformation was necessary. In addition, corresponding analyses were performed by use of the non-parametric Spearman’s rank correlation test. As the two correlation tests gave identical results, and because when determining NAG the maximum activity that could be measured was 15 mU/ml, which means that samples with activity values >15 mU/ml were assigned a value of 16 mU/ml, hence possibly skewing the results, we decided to present only the results of the non-parametric bivariate analyses. After performing the bivariate analysis, the impact of seminal parameters found to have a significant correlation with sperm motility characteristics was tested in a multiple regression model with CASA percentage of motile sperm as the dependent variable. A correlation was considered statistically significant when P < 0.05.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The descriptive statistics for the abstinence period, and semen parameters are presented in Table IGo.


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Table I. Descriptive statistics for semen variables in a group of 301 young men from the general Swedish population
 
Relationship between the levels of biochemical markers
As shown in Table IIGo the levels of biochemical parameters in seminal fluid were closely correlated to each other. There were significantly positive correlations between the epididymal marker (NAG) and prostatic markers (PSA and zinc), and between PSA and zinc, and a significantly negative correlation between PSA and vesicle marker (fructose). Neither NAG nor zinc showed a correlation with fructose.


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Table II. Bivariate correlation coefficients (r) between levels of NAG, prostatic markers (PSA, zinc), and vesical marker (fructose) in 291 young men from the general Swedish population
 
Relationship between biochemical markers and sperm motility
The levels of NAG and the concentrations of PSA correlated positively with CASA percentage motile sperm (r = 0.158, P = 0.009; r = 0.155, P = 0.010 respectively) (Figure 1Go), and negatively with CASA percentage immotile sperm (r = –0.206, P = 0.001; r = –0.157, P = 0.009 respectively). Concentration of zinc showed a significantly negative correlation with CASA percentage immotile sperm (r = –0.138, P = 0.022). For other combinations of levels of biochemical markers and sperm motility characteristics, no statistically significant correlations were found (Table IIIGo).



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Figure 1. Correlation between concentrations of (A) neutral {alpha}-glucosidase (NAG, mU/ml), (B) prostate-specific antigen (PSA, mg/l), and computer-assisted sperm analysis (CASA) percentage motile sperm in 285 young men from the general Swedish population.

 

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Table III. Bivariate correlation coefficients (r) between levels of the NAG, PSA, zinc and fructose, and sperm motility characteristics in 291 young men from the general Swedish population
 
Relationship between sperm motility and other semen charactristics
Statistically significant positive correlations were found between sperm concentration and percentage of rapidly progressive sperm (grade A), percentage of total progressively motile sperm (A + B) and CASA percentage motile sperm, whereas the correlations between sperm concentration and percentage non-progressively motile sperm (C), immotile sperm (D), and CASA percentage immotile sperm were negative (Table IVGo). The length of abstinence period did not correlate with the percentage of CASA motile sperm or the percentages of categories A or A + B, as assessed manually (data not shown).


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Table IV. Bivariate correlation coefficients (r) between semen volume, sperm concentration and sperm motility characteristics in 291 young men from the general Swedish population
 
When the concentrations of sperm, the levels of NAG, and the concentrations of PSA and zinc were tested as independent variables in a linear multiple regression model, with CASA percentage motile sperm as the dependent variable, both the sperm concentration and PSA were found to be independent predictors of sperm motility (ß = 0.298, P < 0.001; (ß = 0.220, P = 0.037 respectively) (Table VGo).


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Table V. Effect of sperm concentration, levels of NAG, PSA and zinc on CASA percentage motile sperm in 285 young men from the general Swedish population, obtained from multiple regression analysis
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In a study of semen samples from 301 young men from the general Swedish population, we found correlations between the seminal levels of markers of epididymal (NAG), prostate (PSA, zinc) function and sperm motility characteristics. Thus, in bivariate analysis we found statistically significant positive correlation between CASA percentage motile sperm and seminal levels of NAG, as well as the concentrations of PSA. Correspondingly, there was a negative correlation with CASA percentage immotile sperm. Furthermore zinc levels showed a significant negative correlation with CASA percentage immotile sperm. In the multivariate linear regression analysis, apart from sperm concentration PSA was the only statistically significant predictor of the CASA percentage of motile sperm.

The physiological role of PSA in seminal fluid is largely unknown. This enzyme was shown to degrade rapidly semenogelins I and II into multiple fragments of low molecular mass. It has been suggested that this degradation considerably reduces the inhibitory effect of semenogelins on motility of sperm (Lilja et al., 1989Go; Robert and Gagnon, 1996Go). Previous studies based on materials from men with spinal cord injury (Charlens et al., 1999Go) and infertile men (Ahlgren et al., 1995Go) indicated low PSA levels in patients with reduced percentage of motile sperm. The present study is the first to indicate a direct correlation between PSA and motility of sperm in a group of men recruited from the general population and not due to infertility problems.

Some of the enzymes important for the function of sperm are zinc metallo-enzymes and can thus become dysfunctional when zinc is deficient. One of these, sorbitol dehydrogenase (SoDH), utilizes sorbitol to provide sperm with fructose for energy, so that SoDH activity is correlated to motility (Dhanad et al., 1981Go). Similarly lactate dehydrogenase-X, another zinc metallo-enzyme, was also reported to have some relationship with motile sperm (Eliasson and Virgi, 1985Go). The results of our study add to our understanding of the diverging results regarding the impact of zinc on sperm motility, which can be found in the literature (Fuse et al., 1999Go; Henkel et al., 1999Go; Sin-Eng et al., 2000Go; Wong et al., 2001Go; Eggert-Kruse et al., 2002Go). Thus, in a bivariate analysis there was a significantly negative correlation with percentage of immotile sperm and a positive correlation with percentage motile sperm, close to the level of statistical significance. However, these findings might be partly due to the strong correlation between the seminal levels of zinc and PSA, also found by others (Ahlgren et al., 1995Go). In the multivariate analysis, when removing the effect of co-variation between the two markers, the effect of zinc on motility of sperm was negative, although not statistically significant. Robert et al. concluded that the concentration of free zinc in seminal plasma is likely to be an important factor in the processing of semenogelin after ejaculation (Robert et al., 1997Go). High concentrations of free zinc are likely to inhibit the PSA activity and thus degradation of the semenogelin, and lack of this metal may protect gel-forming proteins from proteolysis. These observations stress the need for a more holistic approach when evaluating the effect of the epididymis and accessory sex glands on sperm parameters.

NAG is secreted by the epididymal epithelium, probably to provide optimal levels of energy for sperm maturation. The importance of the epididymal compartments in the maintenance of an NAG pool is well demonstrated by the observation that its exclusion by vas ligation leads to a drastic decrease of {alpha}-glucosidase (Roland et al., 1979Go). The results of our study are in accordance with other studies demonstrating positive correlation between NAG and motility of sperm (Fourie et al., 1991Go; Roland et al., 1979Go; Viljoen et al., 1990Go). It has been reported that a significantly lower number of sperm are bound to the zona pellucida in a zona-binding assay in samples with a low {alpha}-glucosidase activity when compared with samples with a high activity (Ben Ali et al., 1994Go). Moreover, it has recently been suggested that {alpha}-glucosidase activity could be used to predict the outcome of IVF (Spiessen et al., 1998Go). However, the clinical value of NAG analysis is still unclear. Thus, Krause and Bohring previously concluded that the determination of {alpha}-glucosidase activity does not give additional information of the fertility status exceeding that of other clinical investigations or parameters of semen analysis (Krause and Bohring, 1999Go). Furthermore, we found a significant association between NAG levels and sperm motility in the bivariate but not in the multivariate analysis.

Fructose has been reported to be a source of energy for the motility of the gametes (Mann, 1964Go). However, one might expect to find a negative correlation between the levels of fructose and motility of sperm, due to the sperm-immobilising effect of semenogelins (Robert and Gagnon, 1996Go; Robert and Gagnon, 1999Go), which are co-secreted by seminal vesicles. In accordance with the previous results (Lewis-Jones et al., 1996Go; Andrade-Rocha, 1999Go; Zöpfgen et al., 2000Go) our study did not demonstrate such association. Moreover, another group (Lay et al., 2001Go) found no significant difference between male fertility status and seminal plasma fructose level.

The bivariate correlations between the levels of biochemical markers and sperm motility characteristics were generally weak. However, sperm motility is dependent on a multitude of pre-testicular, testicular and post-testicular factors and it is hard to imagine a single factor as a strong predictor of the percentage of motile sperm. The motility of sperm has been reported to be one of the most important parameters in evaluating the fertilizing ability of ejaculated sperm both in vivo and in vitro (Gerris and Khan 1987Go; Auger et al., 1994Go; Amann 1995; Donnelly et al., 1998Go). Moreover, recent studies have indicated that sperm motility data obtained by CASA may also be predictive of fertility (Barratt et al., 1993Go; Yukihirano et al., 2001Go). In a study of Danish women planning their first pregnancy, performed using the same CASA (CRISMAS) system, the percentage of motile sperm, as assessed by CASA (but not manually) (Bonde et al., 1998Go; Larsen et al., 2000Go) was found to be an independent predictor of fecundability. Therefore, our data indicate that epididymal and prostatic function may play an important role for motility of sperm, and subsequently the fertility of an individual.

The design of the study did not allow us to draw conclusions that could be directly transferred to clinical practice. However, we obtained an insight into the biological mechanisms, which can help us in our understanding of the pathophysiology of male infertility, and be a starting point for designing more targeted studies of well-defined groups of patients.

The fact that we did not find any statistically significant correlation between the traditional sperm motility parameters and the levels of biochemical markers in seminal fluid may be due to a higher degree of intra- and inter-observer variation when sperm motility is assessed manually as compared with the CASA measurements (Krause and Viethen 1999Go).

Since the biochemical reactions taking place in the seminal fluid are not stopped before 30 min post ejaculation, our measurements may not exactly mirror the secretory activity of the epididymis and the accessory sex glands and thereby not give the full picture of the impact of these glands function on motility of sperm.

The participation rate in this study was only 13.5%, and one could ask whether this group of men was representative for the general population of young Swedish males. However, in a recent Danish study (Andersen et al., 2000Go) using the same approach with a similar overall number of participants, the proportion of men delivering a semen sample was considered representative of young males in the general population in terms of reproductive function. In that study, with a total participant rate of 80%, the conclusion was based on finding the same levels of reproductive hormones in those men delivering an ejaculate, as in subjects who were willing only to give a blood sample and not an ejaculate.

In conclusion, in a study of semen samples from 301 men from the general population, we found an indication of a positive impact of epididymal and prostate function on percentage of motile sperm. Since sperm motility is an important determinant of male fertility potential, more attention should be paid to the function of these organs in the future ‘work-up’ and in the search for therapeutic methods in male infertility.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We would like to thank Erna Stridsberg, Rita Hofvander and Dr Göran Svensson for their help in recruiting the participants in the study. Anna Bremer, Camilla Anderberg and Katarina Jepson are thanked for their technical assistance. The study was financially supported by Swedish government founding for clinical research Crafoordska Fund, Ove Tulefjords Fund and Foundation for Urological research.


    Notes
 
4 To whom correspondence should be addressed at: Scanian Andrology Centre, Fertility Centre, Malmö University Hospital, SE 205 02 Malmö, Sweden. E-mail: saad.elzanaty{at}kir.mas.lu.se Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on May 7, 2002; accepted on August 2, 2002.