Original Paper Urol Int 1992;48:391-394

Department of Urology, Technische Universität München, Klinikum rechts der Isar, Munich, FRG

Adenosine Triphosphate and Adenosine Diphosphate in Human Semen: Correlation with Sperm Count and Motility

Key Words

Abstract

Adenosine triphosphate Adenosine diphosphate Motility Sperm count Bioluminescence

Adenosine triphosphate (ATP) and adenosine diphosphate (ADP) have been correlated with traditional semen parameters such as forward motility and sperm count. ATP and ADP were determined by a bioluminescence assay using the luciferin-luciferase reaction. Short boiling of the ejaculate was per­ formed to inactivate phosphatases (ATPases) in the seminal plasma and the sperm tails. Fresh and deep-frozen semen samples from 45 men with oligozoospermia (n = 22) and normal sperm count (n = 23) were evaluated. Freez­ ing of the sperm for 12 weeks did not effect the ATP or ADP content in the spermatozoa as compared to fresh semen. ATP and ADP concentration was in the range of picomoles/microliter and showed a significant correlation with the number of normal sperm per milliliter and a less marked correlation with the sperm motility. ATP and ADP concentration in the spermatozoa can be measured relatively easy and is reproducible. Unlike the monotonous evalua­ tion of sperm motility by a technician, this biochemical method provides an objective parameter for semen quality. These qualities suggest that this method could be a way to determine the fertilizing potential of semen and to relate this to actual pregnancy rates.

Sperm viability and motility may be related to the adenosine triphosphate (ATP) and adenosine diphos­ phate (ADP) content in the sperm. Hydrolysis of ATP to ADP leads to generation of energy in the sperm, which can be one factor for sperm viability and motility. Sperm motility is considered the most eminent factor in the fer­ tility potential, while sperm count alone seems to be less important [1-3]. Conventional methods to determine the fertility po­ tential of spermatozoa are subjective. With traditional

Received: July 17,1991 Accepted after revision: December 4. 1991

methods, measuring sperm count, morphology and motil­ ity only, statistically low correlations to the fertilizing potential are encountered. Conventional parameters can be measured reproducibly by a skilled technician as a rou­ tine procedure, however, these methods are monotonous due to their repetitive nature. Several newer methods have been reported to assess sperm motility objectively and to provide accurate and reproducible patterns of sperm characteristics. Multiex­ posure photography [4], light scattering techniques [5],

Rainer Hofmann. MD Dcpartmcnl of Urology, Technische Universität München Klinikum rechts der Isar Ismaningcrstrasse 22, D-W-8000 München 90 (FRG)

© 1992 S. Karger AG, Basel 0042-1138/92/0484-0391 $2.75/0

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Rainer Hofmann Antonie Lehmer Edeltraud Gürster Rudolf Hartung

Material and Methods Semen specimens from 45 men were obtained by masturbation after 5 days of sexual abstinence. After liquefaction all semen sam­ ples were examined by the same trained technician. Motility was evaluated after 30, 60, 120 and 240 min and classi­ fied as good, median, poor or absent forward movement according to the WHO guidelines. Sperm count was assessed under the light microscope (400 X magnification). Sperm morphology, fructose con­ tent, pH, volume of the ejaculate and bacteriology were obtained as additional parameters. ATP and ADP content in the spermatozoa was determined with a bioluminescence assay. Light emission was monitored on an LKB Wallac luminometer 1251 coupled to a potentiometric recorder. Aliquots of the semen specimens were taken after 30 and 120 min for ATP and ADP monitoring. ATP was extracted from the sperm samples by boiling them in Tris buffer for 1 min. After heating, the sample was divided into two aliquots: one was deep-frozen for 12 weeks at -20 °C, while the other was directly used for determination of ATP/ADP content. 800 pi Tris buffer, 200 pi AMP and 10 pi phosphoenolpyruvate (PEP; Boehringer Mannheim 127970) were mixed in a polystyrol cuvette and background light emission evaluated. Thereafter, a 20-pl semen specimen was added and the light emission registered for 10 min (pH 7-7.5, 25 °C). ADP was measured by regeneration of ATP with pyruvate kinase (PK) (Boehringer Mannheim). 10 pi PK was added to the test sample and after 5 min prereaction time, the light emission was monitored for 20 min. ATP and ADP determinations were performed in two aliquots of each semen. A coefficient of variation was found to be 2.5%. From one semen sample, ten aliquots were independently determined for their ADP and ATP concentration. The coefficient of variation was found to be 4.3%. Analysis of an ATP standard (LKB Wallac 1250-122) was done by generating a standard solution of 10~5 M ATP, which could be diluted to other standard concentrations with Tris buffer of pH 7.75. Internal standardization was completed by adding 20 pi ATP stan­ dard (1:20 diluted) to the whole test specimen and measuring the light emission for 20 min. ATP and ADP content were calculated from the formula: ATP(ADP) - lum‘ semen specimen x , 0- 11 M ATP lum. ATP standard

392

Results

Comparison of ATP and ADP contents from fresh and deep-frozen semen specimens showed no statistically sig­ nificant difference. After 12 weeks of storage the ATP concentration as a percentage of the fresh concentration was 102.6% after storage. The ADP content was on the average 97.0%. Correlation of sperm count and ATP and ADP content was high (table 1, fig. 1,2). For the correla­ tion of sperm motility with the ATP and ADP concentra­ tion the patients were subgrouped into 8 groups according to their sperm count. Motility was evaluated as percent­ age of sperm with good forward motion after 30 and 120 min (table 2, fig. 3, 4). ATP and ADP concentration showed less correlation with the motility of spermatozoa than with the sperm count. However, decrease of ATP concentration between 30 and 120 min and ADP increase showed high correlation with the decrease of sperm motil­ ity within this time interval.

Discussion

Metabolism of ATP is one aspect of energy conversion and may provide the energy for sperm viability and pro­ pulsion. ATP is synthesized in the mitochondria of the spermatozoa, located in the middle part close to the tail. ATP diffuses to the tip of the tail, where surplus ATP is concentrated. ATP is hydrolyzed to ADP by an ATPase, thus providing energy for an ‘actin-myosin similar com­ plex’. As sperm motility and viability is directly dependent on the energy provided by the ATP hydrolysis, ATP and ADP monitoring can be used for an objective measure­ ment of sperm motility [14, 15]. As the ATP content in semen is in the order of micromoles/liter, biolumines­ cence using the luciferin-luciferase reaction is the method of choice. ADP cannot be measured directly but by resynthetization of ADP to ATP via ATP monitoring [16-18]. As ATP in the sperm is located intercellularly and luciferase cannot penetrate the cell membrane, the nucleotide has to be extracted from the cell. Both the seminal plasma and the spermatozoa contain ATP-degrading enzymes, these ATPase have to be inactivated instantly together with the lysis of the cell membrane in order to permit accurate assessment of ATP content. Several methods have been applied for ATP extraction using perchloric acid, trichloroacetic acid or boiling in Tris buffer for a short period of time [ 19],

Hofmann/Lehmer/GUrster/Hartung

ATP and ADP in Human Semen

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turbidometry [6], sperm motion analyzer, laser light scat­ tering techniques and photographic tracking [7-12] seem to provide more objective data and are not as time con­ suming as manual analyzing. Comparisons between data from different laboratories however lack standardization and show methodological differences [ 13]. Thus it seemed to be reasonable to correlate traditional sperm parameters as sperm count and motility with easily measurable objective data as the ATP and ADP content in human sperm.

Fig. 1. Correlation of sperm count and ATP content in the sper­ matozoa after 30 min (r = 0.87). Fig. 2. Correlation of sperm count and ADP content in the sper­ matozoa after 30 min (r = 0.82).

Table 2

ATP t, min r

30 0.87

ADP 120 0.72

30 0.825

120 0.82

Patients Sperm count, 106/ml r ATP r ADP

We used the method of boiling in Tris buffer. Tris con­ tains EDTA, which stabilizes the nucleotides and im­ proves the extradtion of ATP from the cells. Moreover, potassium and magnesium in Tris buffer react as cofac­ tors for the catalyzation of ADP to ATP by PK. An inter­ nal ATP standard has been used in each semen sample in order to rule out effects of endogenous or exogenous inhi­ bition, which would influence the assay. Human sperm are relatively resistant to cooling. ATP and ADP content was not significantly changed by freezing in comparison

7 -5 0.90 0.94

6 5-10 0.80 0.92

3 10-20 0.90 0.81

3 20-30 0.60 0.72

3 30-40 0.75 0.59

5 40-50 0.92 0.98

9 50-80 0.72 0.94

9 80 0.91 0.82

to fresh samples, so that in a hospital laboratory frozen samples could be used equally well. ATP and ADP concentration in the spermatozoa cor­ related well with sperm count, whereas the correlation with forward motility was less pronounced. The number of spermatozoa in the ejaculate correlates well with the pregnancy rate; however, sperm motility seems to be a better marker for male fertility, especially when encoun­ tering a low sperm count.

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Table 1

Fig. 3. Correlation of sperm motility (5—106 sperm/ml) and the ATP content. Fig. 4. Correlation of sperm motility (5—106 sperm/ml) and the ADP content.

Our data show a high correlation of ADP/ATP content with the number, but no significant correlation with sperm motility, indicating that ATP metabolism is only one of several factors contributing to energy production in spermatozoa. As there is no single factor alone respon­ sible for determining the fertilizing potential of sperm, ATP/ADP content can only provide one aspect of a multi­ factorial event. In combination with traditional semen parameters however, this marker could add additional objective information for evaluating sperm quality. In untreated samples, ATP content decreases and ADP content increases in spermatozoa over a period of time at room temperature. ADP increase and ATP decrease cor­ related well with the decreased motility measured in the same time interval (30-120 min).

Sperm motility was always assessed by the same expe­ rienced technician. Nevertheless, the subjective determi­ nation of the microscopic sperm motility might add to the lower correlation with the ATP/ADP content. Semen samples with high sperm count were often highly viscous, which made dilution sometimes difficult. For the biolu­ minescence assay good mixing of the samples is also cru­ cial for reproducibility. The clinical significance of ATP and ADP concentration in spermatozoa has not yet been proved. Its importance as an objective, relatively inexpen­ sive, reproducible and investigator-independent method could lie in a better assessment of the fertilizing potential of semen. ATP and ADP concentration in combination with conventional semen parameters could lead to a bet­ ter assessment of sperm quality as compared to tradi­ tional semen parameters alone.

1 Dunphy BC, Neal LM. Cooke ID: The clinical value of conventional semen analysis. Fertil Steril 1989;51:324-329. 2 Aitken RJ. Best FSM, Richardson DW. Djahanbakch O. Templeton A, Lees MM: An anal­ ysis of semen quality and sperm function in Fertil Steril 1982;38:705-709, 3 Zaini A, Jennings MG, Baker HWG: Are con­ ventional sperm morphology and motility as­ sessments of predictive value in subfertile men? Int J Androl 1985;8:427-432. 4 Makler A: Use of elaborated multiple exposure photography method in routine sperm motility analysis and for research purposes. Fertil Steril 1980:33:160-165. 5 Jouanette P. Volocrine B, Deguent P: Light scattering determination of various character­ istic parameters of spermatozoa motility in a series of human sperm. Andrologia I977;9:3941. 6 Sokolowski JE. Blasco L. Storey BT: Turbidimetric analysis of human sperm motility. Fertil Steril 1977;28:1337-1342. 7 Overstreet JW, Price MJ. Blazak WF. Lewis EL, Katz DF: Simultaneous assessment of hu­ man sperm motility and morphology by vid­ eomicrography. J Urol 1981;126:357-360.

8 Vantman D. Banks STA. Koukoulis G, Denni­ son L, Sherins RJ: Assessment of sperm motion characteristics from fertile and infertile men using a fully automated computer-assisted se­ men analyzer. Fertil Steril 1989;51:156-161. 9 Katz DF. Overstreet JW: Sperm motility as­ sessment by videomicrography. Fertil Steril 1981;35:188-193. 10 Milligan MP, Harris SJ, Dennis KJ: The effect of temperature on the velocity of human sper­ matozoa as measured by time-lapse photogra­ phy. Fertil Steril 1978:30:592-596. 11 Holt WV, Moore HDM, HillierSG: Computerassisted measurement of sperm swimming speed in human semen: correlation of results with in vitro fertilization assays. Fertil Steril 1985;44:112-117. 12 Mahony MC, Alexander NJ, Swanson RJ: Evaluation of semen parameters by means of automated sperm motion analyzers. Fertil Steril 1988:49:876-880. 13 Knuth UA. Nieschlag E: Comparison of com­ puterized semen analysis with the conventional procedure in 322 patients. Fertil Steril 1988; 49:881-885.

14 Caldini AL, Orlando C, Fiorelli G, Cuomo S, Serio M: ATP and ADP content of human ejac­ ulated spermatozoa. II. Time-dependent changes after ejaculation. Int J Androl 1982:5: 579-584. 15 Orlando C. Caldini AL. Fiorelli G. Cuomo S. Serio M: ATP and ADP content of human spermatozoa. I. Relationship with semen phys­ ical parameters in normal donors and oligozoospermic patients. Int J Androl !982;5:497503. 16 Comhaire F, Vermeulen L, Ghedira K. Mas J. Irvine S, Callipolis G: Adenosine triphosphate in human semen: quantitative estimate of fer­ tilizing potential. Fertil Steril 1983:40:500504. 17 Ramquist G, Brody J, Gottfries A, Stegmayr B: An Mg: *-Ca2, stimulated adenosine triphos­ phatase in human prostate fluid. Andrologia 1978;10:261-264. 18 Gottlieb C. Svanborg K. Eneroth P. Bygdeman M: Effects of prostaglandins on human sperm function in vitro and seminal adenosine tri­ phosphate content. Fertil Steril 1988:49:322— 327. 19 Gottlieb C. Svanborg K, Eneroth P, Bygdeman M: Adenosine triphosphate in human semen: a study on conditions for a bioluminescence as­ say. Fertil Steril 1987;47:992-999.

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Hofmann/Lehmer/Giirster/Hartung

ATP and ADP in Human Semen

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References

Adenosine triphosphate and adenosine diphosphate in human semen: correlation with sperm count and motility.

Adenosine triphosphate (ATP) and adenosine diphosphate (ADP) have been correlated with traditional semen parameters such as forward motility and sperm...
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