Molecular Human Reproduction, Vol. 6, No. 3, 215-218,
March 2000
© 2000 European Society of Human Reproduction and Embryology
Testis and spermatozoa |
Prostatic origin of a zinc binding high molecular weight protein complex in human seminal plasma
1 Dipartimento di Biologia Cellulare, Università degli Studi della Calabria, 87030 Arcavacata di Rende, Cosenza, and 2 Centro Trattamento della Sterilità, Diagnostica Medica, 86100 Avellino, Italy
Abstract
The profile of the zinc ligand high molecular weight proteins was investigated in the seminal plasma of 55 normozoospermic subjects by size exclusion high performance liquid chromatography (HPLC). The proteins were recovered from Sephadex G-75 gel filtration of seminal plasma in three zinc-containing fractions which were then submitted to HPLC analysis. The results were, that in all the samples, the protein profiles showed two peaks with apparent molecular weight of ~660 and ~250 kDa. Dialysis experiments revealed that both ~660 and ~250 kDa proteins were able to uptake zinc against gradient indicating their zinc binding capacity. The HPLC analysis of the whole seminal plasma evidenced only the ~660 kDa protein complex as a single well quantifying peak, furthermore a positive correlation between its peak area and the seminal zinc values (P < 0.001) was observed. This suggested a prostatic origin of the ~660 kDa protein complex which was then confirmed by the seminal plasma HPLC analysis of a subject with agenesis of the Wolffian ducts. Finally the study demonstrated the presence of two zinc binding proteins, ~660 and ~250 kDa respectively, in human seminal plasma and the prostatic origin of the ~660 kDa.
high molecular weight proteins/human semen/prostatic secretion/protein profile/zinc ligands
Introduction
In human seminal plasma zinc is bound to high (HMW), intermediate (IMW) and low (LMW) molecular weight ligands originated by vesicular and prostatic secretions (Arver 1980
, 1982; Arver and Eliasson, 1982). The seminal ligand content and their interactions strongly influence the biological availability of zinc and this, in turn, influences metabolic, physiological and functional sperm properties (Huacuja et al., 1973; Bettger et al., 1981; Björndahl and Kvist, 1982; Foresta et al., 1990).
The HMW ligands are high molecular weight proteins with very high affinity for the cation. In fact, they bind the free and loosely bound zinc during the ejaculation, reducing the zinc fraction available for sperm cells (Björndahl and Kvist, 1990; Björndahl et al., 1991). Furthermore, the HMW proteins, when abundant, could deplete zinc from spermatozoa (Kvist et al., 1990). Therefore, the zinc amount bound to these high sized ligands (HMW–Zn %) has been determined in the evaluation of sperm functional properties (Kjellberg et al., 1992; Carpino et al., 1998).
The aim of this paper was to study the composition of the zinc binding high molecular weight proteins by size exclusion HPLC of seminal plasma in a group of normozoospermic donors. At that same time, zinc and fructose concentrations were measured in the samples as prostatic and vesicular secretion markers.
Materials and methods
Subjects
The ejaculates of 55 healthy donors (aged 20–35 years) and of a patient with agenesis of the Wolffian ducts were analysed. The diagnosis of agenesis was confirmed by surgical exploration.
Semen collection and analysis
Semen was collected from patients after 4 days of sexual abstinence and conventional semen analysis was assessed according to the World Health Organization (WHO, 1992) criteria. The liquefied semen was centrifuged at 1000 g for 15 min and seminal plasma was used for biochemical determinations.
Total protein assay
The Biuret or the Lowry's methods (Lowry et al., 1951; Yatzidis, 1977) were used for protein concentration measurement.
Sephadex G-75 gel chromatography
Sephadex (Pharmacia) G-75 gel filtration of seminal plasma was performed according to Arver (Arver, 1980) as previously reported (Carpino et al., 1998). All the chromatography fractions were investigated for protein (A280 nm) and for Zn concentration.
Size exclusion HPLC
The chromatography HMW protein fractions and the whole seminal plasma (a 50-fold sample dilution) were analysed by size exclusion HPLC as follows: a small portion of the sample was injected (20 µl loop) onto a Progel-TSK G3000 SWXL HPLC column (Supelco-Inc., Supelco Park, Bellefonte, USA) (7.8x300 mm i.d.) equipped with a Progel-TSK SW guard column (7.5x75 mm i.d.). Elution was performed under isocratic condition in 0.05 mol/l Tris buffer containing 0.15 mol/l NaCl, pH 7.4, at a flow rate of 0.5 ml/min. Proteins were monitored by the UV absorbance at 280 nm and the peak areas were recorded. Thyroglobulin (660 kDa), catalase (240 kDa), albumin (68 kDa) and ribonuclease (13.7 kDa) were eluted as molecular weight standards. All the samples were assayed in duplicate.
Dialysis experiments
The first (A), the second (B) and the third (C) HMW–Zn protein fraction were submitted to dialysis experiments. Ten pooled samples of each fraction were lyophilized, resuspended in distilled water (1:10 of the pooled volume) and then dialysed in a cellulose bag (Sigma, molecular weight cut of ~10 kDa) against a buffer solution containing 150 mmol/l NaCl, 50 mmol/l Tris, pH 7.4 at 4°C for 48 h for equilibration. Pooled fractions were then dialysed against a zinc supplemented buffer 0.01 mmol/l Zn, 150 mmol/l NaCl, 50 mmol/l Tris, pH = 7.4. The zinc concentration in the bag was measured at the end of each dialysis.
Zinc and fructose assay
Zinc analysis in seminal plasma was performed by atomic absorption spectrophotometry (Pleban and Mei, 1983) while seminal fructose was measured from a deproteinized sample according to the WHO manual (WHO, 1992)
Statistical analysis
The Pearson correlation test was used to analyse the data. Significance was designed for P < 0.01.
Results
The 55 donors showed the following ejaculate parameters: pH = 7.6 ± 0.3; volume = 4 ± 1.7 ml; sperm count = 78 ± 16x106/ml; progressive sperm motility = 50 ± 5%; normal sperm form = 48 ± 7%. Their seminal zinc, fructose and total protein levels were in the normal range (zinc = 220 ± 75 µg/ml; fructose = 3.2 ± 1.1 mg/ml; total protein = 27 ± 8 mg/ml). The agenesic patient showed no coagulation, pH = 5.9; vol = 0.5 ml, azoospermia, zinc = 900 µg/ml, fructose = 0, total protein = 12 mg/ml.
Sephadex G-75 gel filtration
The HMW–Zn proteins were recovered in three fractions of the void volume (A, B, C) in all the donors. The zinc detected in the first, in the second and in the third fraction was respectively 24 ± 7, 47 ± 8 and 29 ± 11% of the total HMW zinc while the protein concentrations were 7.7 ± 1.6, 12.5 ± 1.9 and 8.6 ± 1.2 mg/ml respectively. In the agenesic patient the seminal zinc was associated only to low-sized ligands.
Size exclusion HPLC
HPLC analysis of the single HMW protein fractions in donors gave the following results: the first fraction (A) contained only a protein peak with apparent molecular weight ~660 kDa, while both the second (B) and the third (C) fractions showed two protein peaks at ~660 kDa and at ~250 kDa (Figure 1).
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HPLC analysis of the whole seminal plasma in donors showed a first single protein peak ~660 kDa followed by a peak cluster from ~250 to ~10 kDa (Figure 2
). A positive correlation (r = 0.76, P < 0.001) between the seminal zinc concentration and the ~660 kDa peak area (percentage of total area) was observed (Figure 3).
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Seminal plasma HPLC analysis of the agenesic patients showed a protein profile characterized by a first peak at ~660 kDa followed by two peaks at medium molecular weight (Figure 2
).
Dialysis experiments
After dialysis against Tris buffer the zinc concentration measured in the cellulose bags containing the pooled HMW–Zn protein fractions was respectively 1.3 µg/ml for A, 2.4 µg/ml for B and 1.6 µg/ml for C. A zinc level of 2.5 µg/ml for A, 7 µg/ml for B and 3.2 µg/ml for C was then detected in the bag after dialysis against the zinc supplemented Tris buffer, with an increase of the 90% for A, of 190% for B and of 100% for C (Figure 4).
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Discussion
In the present investigation the zinc binding high molecular weight (HMW–Zn) proteins were obtained by seminal plasma Sephadex G-75 gel filtration experiments (Arver and Eliasson, 1982). HPLC analysis of the HMW–Zn protein chromatography fractions showed two peaks with an apparent molecular weight of ~660 and ~250 kDa in all the normozoospermic samples.
The zinc binding capacity of the two HMW protein complexes was then demonstrated by dialysis experiments. In fact, a zinc uptake against gradient was demonstrated when the single ~660 kDa protein complex or both the ~660 and ~250 kDa proteins were analysed. Furthermore, a Zn uptake increasing proportionally to the protein areas was observed.
Therefore, our results showed two zinc binding protein complexes in human seminal plasma while the HMW–Zn ligands were previously reported only as 250 kDa proteins (Arver and Eliasson, 1982). This was probably due to the high resolution chromatography method used.
By HPLC analysis of the whole seminal plasma only the ~660 kDa peak showed a resolution to be quantified and a positive correlation between its area and the seminal zinc level was observed in donors. This suggested a prostatic origin of the protein complex which was then confirmed by the seminal plasma HPLC analysis of a patient with agenesis of the Wolffian ducts, having a predominantly prostatic seminal fluid. In fact, the ~660 kDa peak was one of the few peak areas observed in the protein profile of this patient, and only two other peaks at medium molecular weight probably corresponding to prostatic acid phosphatase, albumin (second peak) and prostate specific antigen (third peak) were detected. Furthermore, according to the absence of vesicular secretion, the low sized components were lacking, as well as the ~250 kDa proteins, confirming their previous reported vesicular origin (Arver and Eliasson, 1982; Kwist et al., 1990).
According to Kwist, the seminal zinc of the agenesic patient was not detected in the HMW protein fractions, but it was bound only to low sized ligands (Kwist et al., 1990). Therefore in this patient the ~660 kDa protein complex showed a hindered binding capacity, probably for changes in the macromolecular conformation due to the low pH value of this semen (Arver, 1980).
The ~660 kDa protein complex is a prostatic component which could influence sperm motility for the zinc ligand properties; therefore its relation with the prostatosomes, prostate-derived organelles with sperm motility stimulant effect (Fabiani et al., 1994; Carlsson et al., 1997) would have to be investigated. Finally, this study, after many years, puts the attention on the composition of the zinc binding HMW proteins, which has been little studied despite their influence on functional sperm properties. This study showed two seminal high molecular weight zinc ligands: (i) a ~250 kDa protein complex, already previously reported, and (ii) a prostatic ~660 kDa protein complex which was, until now, completely unknown. Further studies are needed to evaluate their biochemical characteristics (homogeneity and specificity of the protein areas) and their behaviour in semina with pathological spermiograms.
Acknowledgments
To the memory of Eleonora Vizza, a young biologist, recently deceased, whose talent was particularly present in this paper which was also the object of her degree, goes our special thanks. We thank Doct. Sergio Mazzulla for technical assistance. Financial support was obtained from `Ministero dell'Università e della Ricerca Scientifica e Tecnologica' (MURST 60%).
Notes
3 To whom correspondence should be addressed 
References
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Submitted on August 31, 1999; accepted on December 3, 1999.
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