Estimate of oxygen consumption and intracellular zinc concentration of human spermatozoa in relation to motility

Ralf R. Henkel¹, Kerstin Defosse¹, Hans-Wilhelm Koyro², Norbert Weissmann³, Wolf-Bernhard Schill¹

¹Center for Dermatology and Andrology, ²Institute for Plant Ecology, ³Center for Internal Medicine, Justus Liebig University, D-35385 Giessen, Germany

Asian J Androl 2003 Mar; 5: 3-8             

Keywords: oxygen; energy consumption; human spermatozoa; sperm motility; zinc

Abstract

Aim: To investigate the human sperm oxygen/energy consumption and zinc content in relation to motility. Methods: In washed spermatozoa from 67 ejaculates, the oxygen consumption was determined. Following calculation of the total oxygen consumed by the Ideal Gas Law, the energy consumption of spermatozoa was calculated. In addition, the zinc content of the sperm was determined using an atomic absorption spectrometer. The resulting data were correlated to the vitality and motility. Results: The oxygen consumption averaged 0.24 µmol/10⁶ sperm ^. 24h, 0.28 µmol/10⁶ live sperm ^. 24h and 0.85 µmol/10⁶ live & motile sperm ^. 24h. Further calculations revealed that sperm motility was the most energy consuming process (164.31 mJ/10⁶ motile spermatozoa ^. 24h), while the oxygen consumption of the total spermatozoa was 46.06 mJ/10⁶ spermatozoa ^. 24h. The correlation of the oxygen/energy consumption and zinc content with motility showed significant negative correlations (r= -0.759; P<0.0001 and r=-0.441; P<0.0001, respectively). However, when correlating sperm energy consumption with the zinc content, a significant positive relation (r=0.323; P=0.01) was observed. Conclusion: Poorly motile sperm are actually wasting the available energy. Moreover, our data clearly support the "Geometric Clutch Model" of the axoneme function and demonstrate the importance of the outer dense fibers for the generation of sperm motility, especially progressive motility.

1 Introduction

Zinc is a ubiquitous trace element, which plays an important role in a number of different physiological processes. Among others, it is a co-factor for a number of enzymes such as alcohol dehydrogenase or superoxide dismutase. The high amount of zinc, which is 100 times higher in the ejaculate than in blood serum [1], has also been of interest in connection with fertility. It has been shown that the majority of zinc present in the seminal plasma comes from the prostate [2]. The views on the relevance of zinc to male fertility, especially to sperm motility, are inconsistent. On one hand, it has been suggested that zinc obviously plays a role in normal development and function of the testes [3], spermatogenesis [4] and sperm motility [5]. On the other hand, no relationship was observed between zinc present in human seminal plasma and motility [6]. There is also no common agreement on the connection between extracellular zinc present in the seminal plasma and intracellular zinc [2, 7-9]. According to Björndahl et al. [8] spermatozoa take up zinc from the seminal plasma during ejaculation to reduce the vulnerability of the sperm head.

Within the spermatozoon, zinc is localized in the flagella to an extent of more than 93 % [6] and is bound in so-called zinc-mercaptide complexes to the sulfhydryl groups of the outer dense fiber proteins [10]. The outer dense fibers (ODF) are structural elements in mammalian sperm flagella that develop during spermiogenesis [11] and extend along 60 % of the principal piece [12]. Considering that ODF take up to 30 % of the total sperm proteins [13] and that spermatozoa with disturbed development or topology of the ODF are only poorly motile or even immotile, it is obvious that these flagellar substructures must be of paramount importance for sperm motility and male fertility, otherwise ODF would have been eliminated during evolution [14]. Therefore, it is interesting that ODF functions, especially their contribution to the generation of progressive motility of mammalian spermatozoa, have been discussed controversially. To date, no clear straightforward concept of ODF functions is generally accepted.

Initial studies assumed that the outer dense fibers possess ATPase activity [15], thus being actively involved in the generation of sperm motility. This was supported by Brito et al.[16], who found phosphatase activity and phosphoproteins present in the ODF. On the other hand, Henkel et al. [17] did not detect phosphorus in these flagellar substructures. Due to this fact and because ODF proteins are stabilized by numerous disulphide bridges [18], recent studies assume that ODF rather have passive-elastic functions [6, 19]. An important aspect for this assumption is that ODF apparently protect the flagella from shear forces [20]. Another reasonable cha-racteristic for the important function of ODF is given by the "Geometric-Clutch Model" of axoneme function [21]. According to this model, the kinetic energy produced by the axoneme is transferred by the ODF as stiff structural elements to the flagellar basis. By means of the much bigger effective diameter of flagella containing ODF, a much higher torque can be generated than in ordinary cilia, thus resulting in an amplification of tubular sliding and energy utilization. Accordingly, progressive sperm motility will be generated from the interaction between the axoneme and the ODF that are stiffened by their extraordinary high amount of disulphide bridges [14].

It is thought that ODF incorporate a high amount of zinc during spermatogenesis in order to protect ODF proteins from premature oxidation [7, 22]. As a result, the fibrillar structure and accurate topology of ODF can be formed properly. For final stabilization by the formation of disulphide bridges [23], however, zinc must be eliminated again [6, 14, 24]. In addition, the flagellar zinc content is negatively correlated with human sperm motility [6], implicating that the quality of energy conversion might be responsible for generation of motility, especially of progressive motility [25].

From this, it appears that the flagellar zinc content might be closely correlated with male fertility, sperm motility and ODF function. ODF are probably some kind of a pivot for sperm movement and we aimed at further investigating their importance in energy conversion for generation of motility.

2 Materials and methods

For this study, ejaculates from 41 healthy donors and 26 patients, aged 19~50 years, visiting the Andrological Outpatient Clinic, the Center for Dermatology and Andrology of this University were used. Ejaculates were collected in polystyrene beakers after a period of sexual abstinence of 3 to 5 days. After liquefaction, the sperm count and vitality were determined. An aliquot of 1.0~1.5 mL was diluted 1:5 with human tubal fluid medium according to Quinn et al. [26], supplemented with 1 % human serum albumin (HTF-HAS, Centeon Pharma, Marburg, Germany) and centrifuged for 10 minutes at 400 g. Subsequently, the pellet was resuspended in fresh HTF-HSA to a sperm concentration of 2010⁶/mL. This sample was used for motility measurement using the Strömberg-Mika Cell Motion Analyser (Mika Medical Equipment, Bad Feilnbach, Germany) and for determination of the oxygen consumption. Finally, another aliquot of 500 µL semen was taken for the determination of zinc. These processed samples were frozen at -20 until further use.

2.2 Measurement of oxygen consumption

Following adjustment of sperm concentration to 2010⁶/mL with HTF-HSA, 4 aliquots of 1.3 mL were filled into 1.8 mL sample vials (3212 mm; MAGV, Rabenau-Londorf, Germany) and overlaid with mineral oil (Sigma, St. Louis, USA). The vials were carefully closed with screw caps with a silicone-PTFE seal (MAGV, Rabenau-Londorf, Germany). Any air bubbles inside the vials must be avoided. Subsequently, two of these samples were analyzed with a blood gas analyzer "Acid Base Laboratory" (ABL 330; Radiometer A/S, Munich, Germany). These samples were referred to as controls (t_0h). The other two samples were incubated for 24 hours at 37 before this analysis (t_24h). All measurements were performed at 37 .

By using the blood gas analyzer, the partial pressure of oxygen (p_O2) was measured. After careful resuspen-sion of the spermatozoa in the sample vial, an aliquot of 400 mL was taken for each measurement with a syringe where the canula was inserted through the silicone-PTFE seal. The remaining sample was used to determine the cell viability and motility. According to equation (I), the difference of the partial pressure of oxygen (Dp_O2) before and after incubation of the spermatozoa was calculated.

Equation (I) p_O2/t_0h - p_O2/t_24h = Dp_O2

From this equation, the oxygen consumption per 24 hours was calculated for 10⁶ sperm/mL. Afterwards, the energy consumption of the spermatozoa was estimated by a calculation according to the chemical equation (II) for aerobic metabolism in combination with Dalton's Law and the Ideal Gas Law (equation III). After transformation of equation (III), the total amount of oxygen consumed by the spermatozoa was calculated by equation (IV).

Equation (II) C₆H₁₂O₆ + 6O₂ + 38ADP + 38P ----> 6CO₂ + 6H₂O + 38ATP

Equation (III) p ^. V = n ^. R ^. T

p = total gas pressure (pa)

V = volume (l)

n = amount of gas compounds (Mol)

R = molar gas constant (= 8.31 J . mol^-1.K^-1)

T = temperature (K)

Equation (IV) n=p^.V/(R^.T)

From equation (IV) and (II) and the energy content of ATP (30.56 KJ/Mol ATP) a calculation of the energy consumption (mJ/10⁶ sperm 24 h) of spermatozoa was possible. Finally, the power (µW/10⁶ spermatozoa) was calculated. Energy consumption and power was then calculated for 10⁶ sperm/ml, 10⁶ viable sperm/mL, 10⁶ motile sperm/mL and 10⁶ progressively motile sperm/mL.

2.3 Zinc determination

Since zinc is a ubiquitous element, special care has to be taken when handling the samples to avoid contami-nation. Therefore, for all procedures only chemicals of the highest purity (supra pure or pro analytical) were used. Semen samples of 0.5 mL were taken immediately after liquefaction and were centrifuged at 350 g for 15 minutes. The supernatant was discarded and the pellet was washed with 0.5 mL isotonic KCl (supra pure; Merck, Darmstadt, Germany) in water (analytical grade) (Merck) twice. After sperm count, the samples were frozen at -20 until zinc determination, for which samples were thawed, dissolved in 1 % nitric acid (supra pure; Merck) and incubated for 30 minutes at room temperature. For preparation of a calibration curve, a zinc standard solution (0, 1, 3, 5 µg/L; Merck) was diluted in 1 % nitric acid. Samples were measured with an atomic absorption spectrometer (M 2100, Perkin-Elmer, Überlingen, Germany) using a graphite furnace (Perkin-Elmer GRK/PP 2100) under argon at a wavelength of 213.9 nm. For each measurement, 5 µL of the diluted sample was used.

During the observation period of 24 hours, the oxygen consumption per 10⁶ sperm averaged 0.2382 (range: 0.0711~0.4663) µmol/10⁶ spermatozoa 24 h and 0.2811 (range: 0.0889~0.8968) µmol/10⁶ live sperm 24 h and 0.8488 (range: 0.1270~8.2587) µmol/10⁶ live & motile sperm 24 h. From these data, the energy consumption/power was calculated and related to 10⁶ spermatozoa as well. The summarized data of energy consumption/power are depicted in Table 1. The range was 13.77~90.27 mJ/10⁶ spermatozoa 24 h. Comparing energy consumption/power related to the total number of sperm with the total number of live spermatozoa, only a minor increase (about threefold) was observed for the total number of live spermatozoa. However, a dramatic increase was detected when the energy consumption/power was related to motility. Correlation of global and progressive motility with energy consumption/power revealed a highly significant relationship between motility and power related to live & motile sperm. However, no or only weak correlations were observed for the total amount of spermatozoa or for live spermatozoa (Table 2, Figure 1). The calculated curves asymptotically approached a base line value of about 0.5 µW/10⁶ motile spermatozoa. Since the power is only calculated from the data of oxygen consumption per time unit, data are only given in power (µW/10⁶ spermatozoa).

Table 1. Oxygen and energy consumption and power of human spermatozoa related to number of spermatozoa (n=67). For motile spermatozoa a dramatic increase (about threefold) is observed compared with the total number of sperm or vital sperm.

Table 2. Mean power of human spermatozoa correlated with different motility parameters (n=67).

Figure 1. Correlation of global (A) and progressive motility (B) with the power of motile spermatozoa. In both cases, a significant negative relationship can be observed. The calculated curves approach asymptotically a base line value of about 0.5 µW/10⁶ motile spermatozoa. n=67.

The mean concentration of zinc was 71.1 ng/10⁶ spermatozoa (median: 40.41 and range: 1.07~334.75 ng/10⁶ spermatozoa). Significant negative correlations were found for the zinc concentration with global motility (r= -0.441, P<0.0001, Figure 2A) and progressive motility (r=-0.302, P=0.017, Figure 2B). Correlation of sperm zinc concentration with energy consumption/power was only significant with the power related to live & motile sperm (Table 3, Figure 3).

Figure 2. Spermatozoal zinc concentration correlated with global (A) and progressive motility (B). Significant negative correlations can be seen in both cases. n=67.
Figure 3. Mean spermatozoal zinc concentration correlated with power of motile spermatozoa. A significant positive correlation can be seen. n=67.

Table 3. Mean power of human spermatozoa correlated with spermatozoal zinc concentration (n=67). Only the correlation with the number of motile spermatozoa is significant. However, a tendency towards this significance throughout the parameter is visible.

Spermatozoa are the only cells that fulfill their purpose outside the body in another individual. Moreover, they are the smallest cells in the body with only a very thin cytoplasmic border, which does not contain high amounts of energy resources. Therefore, the energy must be taken up from the environment. In addition, in mature and highly differentiated spermatozoa, where almost the entire available energy will be invested for sperm movement [27, 28], special adaptation must have been developed during evolution in order to use the external energy most efficiently. Only an economic use of energy can guarantee an effective movement of spermatozoa, which is the most important sperm function.

A common characteristic of higher vertebrates is the similar use of ATP reserves. Generation of flagellar movement takes place by a relatively low ATP concentration and comparatively high amounts of ADP and AMP [27,29, 30]. In boar spermatozoa, Kamp et al. [27] showed that the phosphagenic systems, by which energy-rich phosphates are transported from the mitochondria to distal dynein-ATPases, do not exist as phospho-creatinine or creatinine kinase. Spermatozoa from bull, stallion and rat exhibit extraordinarily low creatine kinase activity [27, 31]; phosphagenic systems are not present. By contrast, human spermatozoa show a phosphagenic shuttle to overcome the transport of energy-rich metabolites by diffusion only [32]. On the other hand, in different mammalian species the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase is bound to the fibrous sheath [33]. This would explain the ATP supply in distal parts of the flagellum.

Our data demonstrate that sperm movement is the most oxygen/energy consuming process in spermatozoa (0.85 µmol/10⁶ motile spermatozoa 24 hours, which equals a power of 1.9 µW/10⁶ motile spermatozoa). Relating oxygen/energy consumption to sperm vitality, an oxygen consumption of only 0.28 µmol/10⁶ spermatozoa 24 hours (=54.4 mJ/10⁶ spermatozoa 24 hours) was calculated. A basic metabolism of human spermatozoa was estimated at about 0.24 µmol/10⁶ spermatozoa 24 hours (=0.5 µW/10⁶ spermatozoa). Moreover, it can be seen that ejaculates with a higher percentage of motile spermatozoa consume a relatively lower amount of oxygen/energy than those with poorly motile spermatozoa. It appears that poorly motile spermatozoa are actually wasting the available energy. This phenomenon could be explained by the fact that glycolytic ATP production is required for vigorous motility of human spermatozoa [34] and will therefore not consume oxygen. On the other hand, highly motile spermatozoa could utilize the available energy much more efficiently. Such a mechanism was already proposed by Storey & Kayne [35], Kamp et al. [27] and Minelli et al. [28] in different animal models. However, a correlation analysis between sperm oxygen/energy consumption and different motility parameters has not yet been performed.

According to the "Geometric-Clutch Model" [21], the ODF play an important role in this energy transmission system. These flagellar substructures, which take at least 30 % of the total protein amount in human spermatozoa [13], are rather stiff and passive-elastic elements [6, 20] and transfer the kinetic energy produced in the axoneme towards the junction of the flagella with the sperm head. Due to a bigger effective diameter of the flagellum at this point, a higher torque can be generated [21]. This process is thought to intensify and bundle the energy generated from numerous dynein bonds in a flagellar curvature. Thus, progressive sperm motility is generated by co-operative action of the axoneme and stiff structural elements, the ODF [6]. The data of Holcomb-Wygle et al. [36] support this hypothesis. However, despite of the correlations between flagellar zinc and motility on the one hand and zinc and oxygen consumption on the other hand, one should not forget that motility is a parameter that is not exclusively based on ODF-function, but depends on other factors like changes in the sperm membrane as well. This can be seen in the rather mediocre correlation coefficients.

Our data also clearly support this hypothesis. Sperm motility, especially progressive motility, can efficiently be generated because of the presence of these stiff structural elements in the flagellum. The stiffness of the ODF results from the extraordinarily high amount of the amino acid cysteine, which forms disulphide-bridges. In this connection, zinc present in the ODF is of enormous importance. During spermatogenesis at the time of spermatide elongation, zinc is actively incorporated in the ODF [7]. This trace element binds to the sulfhydryl groups of the cysteine by forming zinc-mercaptide complexes [10] and protects the ODF from premature oxidation [14]. Subsequently, during epididymal sperm maturation, more than 60 % zinc is removed from the ODF [22, 24] resulting in their final stiffening by oxidation of the sulfhydryl-groups to disulphide-bridges [23]. Thus, the element zinc has motility-modulating properties. Spermatozoa containing a too high amount of zinc will be only poorly motile. This element was not efficienthy removed from the ODF during epididymal maturation. Earlier results demonstrating this negative correlation between flagellar zinc content and motility [6] were clearly confirmed in the present study. Thus, the removal of zinc from the ODF is a mandatory step in epididymal maturation of spermatozoa.

In the flagellum, zinc is bound to an extent of at least 75 % to the ODF [22]. This enormous amount of the element present in the ODF and the fact that synthesis of ODF proteins makes up at least 30 % of the total protein synthesis in human spermatozoa [13] emphasize the exceptional role of the ODF for the development of sperm movement in species with internal fertilization such as mammals. Synthesis of such an amount of a single protein structure must definitely provide a marked selection advantage for those species with strongly developed ODF; otherwise, these structures would have been eliminated during the course of evolution.

Numerous functions have been attributed to the ODF since their first description. Initially it was supposed that these substructures possess ATPase activity [15]. In fact, phosphoproteins could be identified in ODF from bull spermatozoa [16]. However, since ODF show structural parallels to the cytoskeleton, today they are believed to have rather passive-elastic functions [19]. The enormous amount of cysteine and therefore the high number of disulphide-bridges are indicative of these stabilizing functions [7]. Apart from these stabilizing features, which eventually lead to an improved energy conversion, ODF seem to play an additional role in protecting the flagella from shear forces, that occur during ejaculation [20].

In conclusion, our data clearly support the Geome-tric-Clutch Model?and demonstrate the importance of the ODF for generation of sperm motility. Only a whip-like flagellar beat, which is caused by stiff ODF, will enable spermatozoa to a sufficient progressive movement, for which the elimination of the element zinc from the ODF is an essential step in epididymal sperm maturation. ODF appear to have two main functions: (i) improving energy conversion and thus improving the flagellar beat and (ii) providing an efficient system to protect the flagella from shear forces.

This study was supported by the Schering Research Foundation, Berlin, Germany. The authors wish to thank Ms. A. Hanschke for skillful technical assistance as well as Mrs. G. Scharfe and Mrs. S. Henkel for linguistic review.

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Correspondence to: PD Dr. Ralf Henkel, Center for Dermatology and Andrology, Gaffkystr. 14, D-35385 Giessen, Germany.
Tel: +49-641-99 43350,   Fax: +49-641-99 43368
E-mail: ralf.henkel@derma.med.uni-giessen.de
Received 2003-01-10      Accepted 2003-03-07

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