andrologia 9 (1) : 95-105 (1977)
Received January 1, 1976
Department for Andrology (Head: Prof. Dr. C. SCHIRREN) University Clinic Hamburg-Eppendorf Fachbereich Klinische Forschung u. Fachbereich Physikochemie und Informatik Schering AG, Berlin
Studies of the Correlation of Morphological and Biochemical Parameters in Human Ejaculate in Various Andrological Diagnoses 2nd Report Biochemical Parameters C. SCHIRREN, G . LAUDAHN, E. HARTMANN and I . HEINZE
1.
Introduction
In continuation of our studies we report in this paper on the results of biochemical analyses in human ejaculate. The parameters measured were the concentrations of several substrates and the activities of various enzymes of intermediary metabolism, some of which are associated with fructose metabolism. Of importance for the biochemical analysis is the fact that ejaculates cannot be processed like other body fluids or tissues: Since the sperm is ejaculated in a coagulated form, liquefaction must first of all be allowed to occur (this usually takes about 10 minutes). Unfortunately, it is almost certain that various biochemical reactions take place during this phase of liquefaction, which bring about, for example, an alteration in the initial concentrations of so-called energy-rich phosphates. Even if the ejaculate were to be collected in liquid nitrogen to avoid these processes, an accurate assessment could still not be performed because the degree of motility and the spermatozoal density cannot be determined under these conditions. At the present time, however, these values are still indispensable to the andrologist, even if he wishes to depart from purely morphological parameters. All studies were therefore performed 10 minutes after ejaculation, since it was possible at this time to divide the liquefied ejaculate into aliquot portions for the various studies. It must be expressly emphasized that the modus operandi described under “Methods” can only permit a statement to be made about the actual metabolic situation of the spermatic fluid 10 minutes after ejaculation. 2.
Methods
2. I Study material The studies were performed on the ejaculates of 525 andrological patients. The patients involved were the same as those in whom the physical and morphological parameters discussed in the 1st report were studied. The size of the individual groups, arranged according to clinical diagnoses, is shown in Table 1. Key wrds: ATP - ADP - AMP - pyruvate - lactate - a-ketoglutarate - Fructose phosphoglucoseisomerase - phosphoglyceratekinase - ATPase - acid phosphatase
-
myokinase -
C. SCHIRREN,G. LAUDAHN,E. HARTMANN and I. HEINZE
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Table 1: Size of groups
Diagnoses
n
Normozoospermia Asthenozoospermia Oligozoospermia Azoospermia
196 92 181 56
Total
5 25
2.2 Study methods The following parameters were determined and evaluated for this 2nd report:
Substrates Enzymes
ATP*, ADP, AMP,pyruvate, lactate, a-ketoglutarate, fructose. Myokinases, phosphoglucoseisomerases,phosphoglyceratekinases, ATPases, acid phosphatases.
The first report should be referred to for the assessment of the spermiogram.
2.3 Biochemical methods Substrates After allowing 10 minutes for liquefaction, the spermatic fluid for ATP, ADP, AMP, pyruvate, lactate and a-ketoglutarate is mixed with twice the amount of ice-cold 0.6 N perchloric acid and immediately centrifuged for 15 minutes in a cooling centrifuge (t 4OC) at 3 100 g. The clear supernatant is then poured into a test tube. Deproteinization for fructose: 1.0 ml ice-cold 0.33 N perchloric acid t 0.1 ml ejaculate, centrifuge. The measurements were made on a Zeiss photometer PMQ I1 at 340 nm in glass cuvettes with a layer thickness of 1 cm. The results were calculated according to the following formula: AExVxMW C=
= pg/ml (in the sample).
Exdxv = Extinction coefficient for NADH or NADPH at 340 nm (cm2/pMol) E = Extinction alteration = Test volume (ml) V v = Sample volume in the test d = Layer thickness (cm) MW = Weight of a micromol (pg) of the substrate
E A
Finally, take into account for the calculation: deproteinization, buffering and supernatant dilution.
* List of abbreviations see page 114. andrologia 9, Heft 1 (1977)
Studies of the Correlation of Morphological and Biochemical Parameters
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ATP (1) G-3-P t ATP PGK G-1.3-P t ADP (2) G-l.3-P2 t NADH t H' GAP H GAP t NAD' t Pi
6
2.0 m10.5 M TRA pH 7.6 (0.5 M TRA; 4 mM Mg SO4; 6 mM glycerate-3-phosphate), 0.20 ml NADH (2.5 mM); 0.20 ml deproteinized supernatant, mix, measure E l , 0.02 ml GAPDH/PGK/TIM (7 mg GAPDH/ml; 1 mg PGK/ml; 2 mg GDH/TIM/ml); mix, wait for reaction to cease (about 15 minutes), measure E2. If the reaction does not cease after 15 minutes, perform 3-5 further readings at intervals of 2 minutes and extrapolate E2 to the time of addition of the enzyme suspension. E l - E2 = A E. ADP/AMP (1) AMPtATP MK 2ADP (2) 2 A D P t 2 P N P PK 2 A T P t 2 (3) 2 Pyruvate + 2 NADH t 2 H+ LDIzmvate 2 Lactate t 2 NAD+
Adjust deproteinized supernatant with 0.5 M TRA (0.5 M TRA; 2.0 M K2CO3) to a pH value of 7.3-7.5 (pH meter check). The perchlorate which precipitates as a result is removed by brief centrifugation. 1.75 mlO.1 M TRA pH 7.5; 0.25 ml deproteinized supernatant; 0.20 ml PEP (10 mM PEP; 1.3 M KCI; 0.4 M MgSO4); 0.20 ml NADH (2.5 mM); 0.02 ml LDH (1 mg/ml); mix, measure Elafter 5 minutes; 0.02 ml PK (1 mg/ml); mix, wait for the reaction to cease, or perform 3-5 further readings at intervals of 2 min. and extrapolate E2 to the time of addition of PK; 0.02 ml MK (2 mg/ml); mix, wait for the reaction to cease, measure E3, or perform further readings as above. Blank value: sustitute 0.1 M TRA, pH 7.5, for the buffered supernatant in the test preparation. ADP = (El main - E2 main) - (El blank - E2 blank) AMP = (E2 main - E3 main) - (E2 blank - E3 blank) The fact that 1 Mol AMP corresponds to 2 Mol NAD must be taken into account when calculating AMP. Py ruva te Pyruvate t NADH + H+ LDH Lactate t NADt Adjust deproteinized supernatant with 0.7 M tribasic potassium phosphate to a pH of 6-7, then centrifuge. 1.9 ml aqua bidest.; 0.1 ml buffered supernatant; 0.20 ml 2.5 mM NADH; mix, measure E l ; 0.02 ml LDH (2 mg/ml); mix, measure E2 after 5 minutes or perform 3-5 further readings at intervals of 1 min. and extrapolate E2 to the time of addition of LDH. Lactate Lactate t NADt
LDH Pyruvate + NADH t H+
andrologia 9, Heft 1 (1977)
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C. SCHIRREN, G . LAUDAHN,E. HARTMANN and I. HEINZE
Main value: 2.00 ml glycin buffer (0.5 M glycin buffer pH 9.0; 0.4 M hydracin); 0.10 ml deproteinized su ernatant; 0.20 ml NAD (27 mM); 0.2 ml LDH (2 mg/ml), mix, allow to stand at 25gC in a water bath for exactly 1 hour. Pour into cuvettes. Measure extinction of the sample and of the blank value against air. Ep - EL = A E. Blank value: Substitute 0.10 ml perchloric acid in the test preparation for 0.10 ml deproteinized supernatant. a-KGS a-Ketoglutarate + NADH t E+ t NH;
GlDH Glutamate t NADt + H20
1S O ml TRA (0.1 M pH 7.5); 0.50 ml buffered supernatant (as with ADP/AMP); 0.05 ml NADH (7 mg/ml); mix, measure E l ; 0.05 ml GlDH (2 mg/ml); mix,measure E2 after 10 min. or perform 2-3 other readings at intervals of 1 min. and extrapolate E2 to the time of addition of GlDH.
Fructose D-Fructose + ATP HK F6-P + ADP F-6-P PGI G-6-P G-6-P t NADP M-PDH Gluconate-6-Phosphate + NADPH + Ht 2.58 ml TRA pH 7.5 (0.3 M TRA; 4 mM MgS04); 0.10 ml deproteinized supernatant;0.10mlNADP(12mM);0.10mlATP(16mM);O.O2mlPGI(2mglml);mix, measure E l ; 0.02 ml HK/G6-PDH (1 mg/ml; 1 mg/ml); mix, wait for reaction to cease (about 15 min.) or perform 3-5 further readings at intervals of 2 min. and extrapolate E2 to the time of addition of HK/Gh-PDH. E2-E1 =AE.
Enzymes After allowing 10 min. for liquefaction, the ejaculate samples were centrifuged for 15 min. at 3 100 g and a temperature of 4OC. The seminal plasma (SP) obtained in this way was tempered before being used in the tests. Any necessary dilution was performed with aqua bidest. The measurements for MK, PGI and PGK were performed on a Zeiss photometer PMQ I1 at 366 nm and a constant temperature of 25OC. Following an initial run of 0.050-0.100 the time for a further extinction alteration of 0.100 was stopped. Linearity existed under the study conditions. Study conditions for acid phosphatases: wave l e n p 405 nm, temperature 37OC; for ATPases: wave length 420 nm, temperature 25 C. Unless otherwise stated, the enzyme activity is expressed in international units (units, milli-units): 1 U is that enzyme activity which converts 1 pMol substrate per min.
MK Auxilliary and indicator reaction
AMPtATP MK 2ADP 2 ADP t 2 PEP PK 2 ATP + 2 Pyruv. 2 P ~ N v+. 2 NADH + 2 H+ LDH 2 Lact. + 2 NAD andrologia 9, Heft 1 (1977)
Studies of the Correlation of Morphological and Biochemical Parameters
Test preparation: 2.17 m10.05 M TRA pH 7.5; 0.20 ml ATP (10 mg/ml TRA pH 7.5); 0.15 ml MgS04/KC1(0.5 M Mg SO4 in 2 M KC1); 0.10 ml PEP (5 mg tricyclohexylammonium salt/ml); 0.10 ml NADH (10 mg/ml); 0.05 ml SP; 0.01 ml PK (10 mg/ml); 0.02 ml LDH (5 mg/ml). Start with 0.20 ml AMP (10 mg/ml TRA pH 7.5; Na salt). When calculating the activity it must be borne in mind that 2 pMol NADH react per pMol substrate (AMP). PG I F-6-P PGI G-6-P Indicator reaction G-6-P t NADP G-6-pDH Gluconate-6-P t NADPH t Ht 2.53 m10.05 M TRA pH 7.5; 0.10 ml F-6-P solution approx. 0.025 M; 0.20 ml MgSO4 0.1 M; 0.10 ml NADP (10 mg/ml); 0.02 ml G-6-PDH (1 mg/ml). Start with 0.05 ml SP 1:2. Production of the F-6-P solution: Dissolve 130 mg F-6-P barium salt in 3 ml aqua bidest. while adding 2 mlO.1 N HC1, add 0.5 ml9.2% K2SO4 solution, mix well, centrifuge. Neutralize supernatant with 0.1 N NaOH and make up to 10 ml with aqua bidest. This solution will keep for about 14 days in the refrigerator. PGK Glycerate-3-P + ATP PGK Glycerate-l.3-P2 + ADP Indicator reaction Glycerate-1.3-P2 t NADH t Ht GAPDH + NADt t Pi 2.15 m10.05 M TRA pH 7.5; 0.15 ml EDTA (5 mg/ml); 0.10 ml NADH (10 mg/ml); 0.20 ml ATP (10 mg/ml TRA pH 7.5); 0.10 ml3-PGS (50 mg tri-cyclohexylammonium salt/ml); 0.05 ml MgSO4 0.1 M; 0.05 ml GAPDH (10 mg/ml). Start with 0.2 ml SP 1:2. ATP-ases ATP t H20
ATP-ase ADP t Pi + H+
Main value: 2.90 ml buffer substrate solution and 0.1 ml SP are incubated at 25OC for 1 hour. Interruption of the reaction by the addition of 1.0 m l 2 W trichloro-acetic acid, centrifuge. Determination of the anorganic phosphate in the centrifuge supernatant. Blank value: pipette 1.0 ml 20% trichloro-acetic acid, 0.1 ml SP and 2.9 ml buffer substrate solution together (sequence!), centrifuge, phosphate determination. Phosphate determination with the Boehringer Biochemica Testcombination: 0.75 ml aqua bidest.; 0.25 ml centrifuge supernatant; 1.O ml ammoniummolybdate (40 mM ammoniummolybdate; 2.5 N H2SO4); 1.0 ml ammoniumvanadate (21 mM ammoniumvanadate; 0.28 N HNO3); mix, take extinction reading after 10 min. Measurement is made against the blank value: 1.O ml aqua bidest.; 1.O ml ammoniummoblybdate; 1.Oml ammoniumvanadate. Measured radiation: 420 nm;depth of layer: 1 cm. The amount of phosphate released as a result of ATP-ases activity represents the difference between the phosphate content of the blank and main values. The amount of phosphate - read off in pMol from a calibrated curve - divided by 60 results in pMol/min. Substrate buffer solution: 41 m10.05 M TRA pH 7.4 t 10 ml ATP/KCl solution (0.03 M Na2H2-ATP in 0.45 M KCI, with 1 N KOH to pH 7.4) t 4 m10.075 M MgS04. andrologia 9, Heft 1 (1977)
99
C. SCHIRREN, G. L A U D A H NE., HARTMANN and I. HEINZE
100
Acid phosphatase p-Nitrophenylphosphate + H20
Acid PhosPhatas p-Nitrophenol - Pi
Dilute SP 1:10 000 with ph siological NaC1. Measured radiation 405 nm; thickness of layer 1 cm; temperature 37 C. Measurement is made against the blank value.
B
Pipette into test tubes
P1
Buffer/substrate solution (50 mM citrate buffer, pH 4.8; 5.5 mM sodium-p-nitro-phenylphosphate) 0.2 M sodium tartrate SP 1: 10000 mix, 30 min. water bath 37OC
p2
1.0 ml -
NaOH, 0.02 N
1.0 ml 0.1 ml
0.2 ml
0.2 ml
10.0 ml
10.0 ml
Blank value: 1.0 ml buffer/substrate solution; 10.0 ml NaOH, 0.02 N; 0.2 ml SP 1: 10000. Ext. P i = Total acid phosphatases Ext. P2 = Prostatic phosphatases Calculation:
E x V x dilution txexv
[ U/mlI.
E = Extinction coefficient of 4-nitrophenol in alkaline solution at 405 nm = 18.5 cm2/Mol. V = Testvolume(ml) v = Sample volume in the test t = Time in minutes 2.4 Statistical analysis See first report. 3.
Results
3.1 Relationship of the parameters to age As with the morphological parameters, the first check made in the biochemical analyses was for the existence of any relationship of the parameters to age. It was found that there was a distinct relationship between age and the fructose content of the seminal fluid in as much as lower fructose values were measured with increasing age (p < 0.05) (Fig. l)*. This applies equally for pyruvate (Fig. 2), whilst the converse was observed for lactate (Fig. 3). No age-related behaviour was observed for the other substrates. Lower activities were established in advanced age for the enzymes PGI and PGK, whilst higher activities were observed for the enzymes ATPase and acid prostatic phosphatase. ~~
* All Figures see page 102/103. andrologia 9 , Heft 1 (1977)
Studies of the Correlation of Morphological and Biochemical Parameters
101
3.2 Relationship of the parameters to the diagnosis groups and to one another The mean values’of the concentrations of AMP, ADP, ATP and of fructose, pyruvate and lactate are presented in Figs. 4-7. It was observed that no difference exists for ATP between the individual diagnoses. In contrast to this, lower values can be demonstrated for ADP in the diagnosis oligozoospermia (Fig. 4) (p < 0.05),whilst there is again no difference for AMP.If the values determined for these three substrates in the group normozoospermia and the pooled groups asthenozoospermia, oligozoospermia and azoospermia are now compared, then it can be seen for ATP and ADP that distinctly higher values are present in normozoospermia; in contrast, the converse can be demonstrated for AMP (Fig. 5). A break-down of the values for fructose, pyruvate and lactate according to the various diagnoses results in a relationship only in the case of fructose, for which lower values can be demonstrated (p < 0.05) in the case of normozoospermia and asthenozoospermia - which can be regarded as identical in respect of the sperm count (cf. Fig. 15, first report) (87.5 million sperms/ml) - than in oligozoospermia and azoospermia (Fig. 6). No difference between the diagnosis groups can be demonstrated for lactate; in the case of pyruvate the mean value is distinctly lower in the diagnosis asthenozoospermia than in all other diagnoses (Fig. 7) (p < 0.05). A study of the extent to which the enzyme activities in spermatic plasma are associated with the respective pH value measured shows that pH values above 7.2 correlate with distinctly lower activities for acid prostatic phosphatase, PGI and MK (Fig. 8). These studies were conducted on 120 ejaculates in which the pH value was determined by the situation in the unprepared seminal fluid; the three enzyme activities were studied under these conditions. In this connection we have also conducted in vitro studies which are presented in Figs. 9 and 10. These figures show the activity optima of MK, PGK, PGI and ATPase in human spermatic plasma at various pH values. The object of these studies was to determine the respective optimum of the enzyme activity; we know that pH values above 8.0 are a particularly rare finding in adrological examinations. It is clear from both figures that the pH range of 7.4-7.7 must be regarded as the optimum for PGK and ATPase. Of particular interest in this connection is the relationship between the pH value of the ejaculate and the fructose and ATP concentrations (Fig. 11). According to this, under conditions of unprepared spermatic fluid pH values of 7.1-7.7 are accompanied by decreasing ATP concentrations and increasing fructose concentrations. A comparison of the concentrations of ADP and AMP shows that, at higher ADP values, higher AMP concentrations are also measured. If, furthermore, the lactate and ADP concentrations are placed in relationship to the fructose concentration in the ejaculate, lower values for ADP and lactate can be observed at higher fructose concentrations (p < 0.05). Correlations of this nature - between individual substrates, between substrates and enzymes, and between individual enzymes - are shown in Figs. 16-21. It can be seen here (Fig. 12) that, at higher pyruvate concentrations in the ejaculate, lower concentrations for AMP and ADP but higher lactate concentrations can be demonstrated. If pyruvate, ADP and ATP are placed in relationship to the KGS concentration (Fig. 13), a respectively higher share for pyruvate, ADP and ATP concentrations can be observed at higher values for KGS (p < 0.05). If, on the other hand, the MK activity in spermatic plasma is placed in relationship to the concentration of ADP, pyruvate and fructose (Fig. 14), a lower fructose and pyruvate concentration must be expected at higher MK activity, whilst the concentration for ADP shows higher values. Further, higher ATPase activities correlate with lower measured values for lactate, pyruvate and KGS and with higher ADP values (Fig. 15). andrologia 9, Heft 1 (1977)
andrologia 9, Heft 1 (1977)
Studies of the Correlation of Morphological and Biochemical Parameters
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The activity of PGI has been placed in relationship to the concentration of ATP, ADP, pyruvate and fructose. Lower fructose and pyruvate concentrations can be demonstrated at higher PGI activity, whilst higher concentration values are observed for ATP and ADP. Finally, in Fig. 16, the enzymes examined are placed in relationship to one another, whereby the measured values of PGI are compared with those of ATPase, KM and PGK. In this case, at higher PGI activity values, higher activities are also obtained for other enzymes. Discussion Although the division of the patients into certain diagnosis groups chosen by us is somewhat arbitrary, there is still no better alternative than to make the morphology of the spermatozoa the basis of diagnostic differentiation. It must also be possible to place the biochemical findings in relation to the morphological parameters. The object is to improve the clinical possibilities of differentiation and also to improve the accuracy of conclusions about the ability to procreate made on the basis of the correlation of morphological and biochemical findings. When interpreting our data it must be borne in mind that all the studies were carried out after the ejaculate had been allowed to stand for 10 minutes at room temperature, the reasons for which were reported in the introduction. It is our intention in the future to build up on these results and, using a limited amount of patient material, to investigate the question of what the biochemical status looks like if the spermatic fluid is ejaculated directly into liquid nitrogen, for example. For the initial studies we shall use ejaculates from "normal cases" known to us, for whom it must be assumed that they will exhibit their usual morphologically normal findings at the time of use of the nitrogen method as well. The evaluation will, of course, be made with this assumption in mind. Our present studies confirm earlier fmdings (Schirren - 1961) that lower fructose values are to be observed in the ejaculate at advanced ages. This applies also to pyruvate, whilst the opposite behaviour can be observed in the case of lactate. The increased presence of lactate as a final product of fructolysis suggests that the spermatozoa of older men have a lower capacity for the final oxidative degeneration of carbohydrates than the spermatozoa of younger men. This provides material for complementary studies in which the question of a hormone dependence of fructolysis should also be investigated. Since the mean values for the various substrates differ from one another when they are placed in relationship to the individual andrological diagnoses, we have formed two
-,
AOP lMml
Fig. 14
I
Rym
"ale rnd
I t&
2951 280
=:Y :
,220
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15! 250
235
450
180
350
1
4w
I
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andrologia 9, Heft 1 (1977)
PI3 U/%
104
C. SCHIRREN, G . LAUDAHN, E. HARTMANN and I. HEINZE
groups, one of which is composed of cases of normozoospermia and the other of the diagnoses asthenozoospermia, oligozoospermia and azoospermia. This results in the finding that distinctly higher values for ATP and ADP are present in the cases of normozoospermia. Since ATP and ADP are not present in free form in seminal plasma, but are bound to the spermatozoa (Peterson and Freund - 1971), the higher content of energy-rich phosphates in the spermatozoa in normozoospermia could also explain, for example, the superior motility characteristics in this group. A look at the substrates fructose, pyruvate and lactate in relationship to the diagnosis groups revealed a similar situation to that already established in the discussion of the relationship to age. The known correlation between fructose and spermatozoal density (Schirren - 1961) was apparent again here too. So far we have not reported on any new possibility of interpretation which improves on our old statement about a possible connection with the oestrogen content of the spermatozoa and a corresponding feed-back to the anterior lobe of the pituitary. Of interest are the data on the enzyme activity at the respective pH value in the ejaculate, whereby our in vitro findings obtained with different experimentally adjusted pH valuesmust be compared with the values determined in vivo. It can be seen from this comparison that the optima for certain enzymes determined in vitro do not exist in vivo - at least not immediately after ejaculation. On the other hand, we know that the pH value of the ejaculate undergoes a shift to the alkaline side after it has stood for a while. Moreover, for the significance of this factor in vivo the biochemical or physicochemical milieu of the vagina and other components of the female genital tract (e.g. cervical mucus, endometrial and tubal secretion), i.e. influences which have not previously been examined in this manner, must also be taken into account. Summary In this report the authors present the results about biochemical analyses in human ejaculate. The following parameters were determined ATP, ADP, pyruvate, lactate, a-ketoglutarate, fructose, myokinase, phosphoglucoseisomerase, phosphoglyceratekinase, ATPase, acid phosphatase. It was found a relationship between age and fructose content, this was equally for pyruvate, whilst the converse was observed for lactate. N o age dependend relationship was observed for the other substrates. For ATP and ADP distinctly higher values can be demonstrated in normozoospermia. A special study of the extent to which the encyme activities in sperm plasma are associated with the respective pH value measured shows that pH values above 7.2 correlate with distinctly lower activities for acid phosphatase, PGI and MK. Un&nuchungen zur Korrelation rnorphologixher und biochemischer MeEgroRen irn menschlichen Ejakulat bei verschiedenen andrologischen Diagnosen
I I. Mitteilung: Biochemische MeBgroBen
Zusammenfassung In Fortsetzung friiherer Untersuchungen teilen wir in dieser Arbeit das Ergebnis biochemischer Analysen im menschlichen Ejakulat mit. Gemessen wurden die Konzentrationen einiger Substrate sowie die Aktivitaten verschiedener Enzyme des intermediaren Stoffwechsels, die teilweise Beziehungen zum Fruktosestoffwechsel besitzen. Fur die biochemische Analytik ist von Bedeutung, dal3 sich Ejakulate nicht wie andere Korperfliissigkeiten oder -gewebe verarbeiten lassen: so mu5 zunachst die Verflussiandrologia 9, Heft 1 (1977)
Studies of the Correlation of Morphological and Biochemical Parameters
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gung des coaguliert entleerten Sperma abgewartet werden (in der Regel etwa 10 Minuten). In dieser Verflussigungsphase laufen mit gro5ter Wahrscheinlichkeit bereits verschiedene biochemische Umsetzungen ab, die 2.B. eine h d e r u n g in den Anfangskonzentrationen sogenannter energiereicher Phosphate bewirken. Bei der Interpretation unserer Daten ist zu beriicksichtigen, da5 alle Untersuchungen erst nach zehnminiitigem Stehenlassen des Ejakulates bei Raumtemperatur vorgenommen wurden. Unsere jetzigen Untersuchungen bestatigen friihere Befunde, d d in hoheren Lebensaltern niedrigere Fruktosewerte im Ejakulat zu messen sind. Identisch verhalt sich Pymvat, wahrend bei Lactat das entgegengesetzte Verhalten zu beobachten ist. Die Anhaufung von Lactat als Endprodu I der Fruktolyse legt den Gedanken nahe, d d Spermatozoen alterer Manner eine geringere Kapazitat zum oxydativen Endabbau der Kohlenhydrate haben als die Spermatozoen Jiingerer. Hier ergeben sich Ansatzpunkte fur Erganzungsuntersuchungen, in denen auch die Frage einer Hormonabhangigkeit der Fruktolyse zu priifen ist. Wenn man die Mittelwerte fur die verschiedenen Substrate in Beziehung setzt zu den einzelnen andrologischen Diagnosen, ergibt sich, daD bei der Normozoospermie eindeutig hohere, Werte fur ATP und ADP vorliegen. Da ATP und ADP nicht frei im Seminalplasma vorkommen, sondern an die Spermatozoen gebunden sind, konnte der hohere Gehalt an energiereichen Phosphaten in den Spermatozoen bei Normozoospermie 2.B. auch die uberlegenen Motilitatscharakteristika in dieser Gruppe erklaren. Von Interesse sind die Daten zur Enzymaktivitat bei dem jeweiligen pH-Wert im Ejakulat. Man mu5 hienu unsere in vitro-Befunde mit experimentell unterschiedlich eingestelltem pH den in vivo ermittelten Werten gegeniiberstellen. Es ergibt sich dabei, d d die in vitro ermittelten Optima fur bestimmte Enzyme in vivo zumindestens unmittelbar nach der Ejakulation nicht gegeben sind. Wir wissen andererseits, d d sich der pH-Wert des Ejakulates nach langerem Stehenlassen zur alkalischen Seite verschiebt. Dariiberhinaus mu5 man fur die Bedeutung dieses Faktors in vivo sicherlich auch das biochemische bzw. physiko-chemische Milieu der Scheide und der weiteren Teile des weiblichen Genitaltraktes (2.B. Cervixschleim, Endometriums- und Tubarsekret) beriicksichtigen, d.h. Einfliisse, die in dieser Weise bisher noch nicht untersucht wurden.
References Peterson, R.N. and M. Freund: Glycolysis of human spermatozoa: Levels of glycolytic intermediates. Biol. Reprod. 5, 221-227 (1971). Schirren, C.: Fertilititsstorungen des Mannes. Diagnostik, Biochemie des Spermaplasmas, Hormontherapie. Stuttgart: F. Enke 1961. Schirren, C.: Practical andrology. Berlin: Briider Hartmann 1971. Schirren, C., G. Laudahn, E. Hartmann, I. Heinze und E. Richter: Untersuchungen zur Korrelation morphologischer und biochemischer MeDgroBen im menschlichen Ejakulat bei verschiedenen andrologischen Diagnosen. I. Mitt.: Beziehungen zwischen Ejakulatvolumen, Zahl, Motilitat und Morphologie der Sperrnatozoen unter Beriicksichtigungdes Lebensalters. andrologia 7, 117-125 (1975). Address: Prof. Dr. C. SCHIRREN, Dept. for Andrology, University Hospital, Martinistr. 52, D-2000 Hamburg 20, Germany.
List of abbreviations see page 114 andrologia 9, Heft 1 (1977)
Received January 1, 1976
Department for Andrology (Head: Prof. Dr. C. SCHIRREN) University Clinic Hamburg-Eppendorf Fachbereich Klinische Forschung u. Fachbereich Physikochemie und Informatik Schering AG, Berlin
Studies of the Correlation of Morphological and Biochemical Parameters in Human Ejaculate in Various Andrological Diagnoses 2nd Report Biochemical Parameters C. SCHIRREN, G . LAUDAHN, E. HARTMANN and I . HEINZE
1.
Introduction
In continuation of our studies we report in this paper on the results of biochemical analyses in human ejaculate. The parameters measured were the concentrations of several substrates and the activities of various enzymes of intermediary metabolism, some of which are associated with fructose metabolism. Of importance for the biochemical analysis is the fact that ejaculates cannot be processed like other body fluids or tissues: Since the sperm is ejaculated in a coagulated form, liquefaction must first of all be allowed to occur (this usually takes about 10 minutes). Unfortunately, it is almost certain that various biochemical reactions take place during this phase of liquefaction, which bring about, for example, an alteration in the initial concentrations of so-called energy-rich phosphates. Even if the ejaculate were to be collected in liquid nitrogen to avoid these processes, an accurate assessment could still not be performed because the degree of motility and the spermatozoal density cannot be determined under these conditions. At the present time, however, these values are still indispensable to the andrologist, even if he wishes to depart from purely morphological parameters. All studies were therefore performed 10 minutes after ejaculation, since it was possible at this time to divide the liquefied ejaculate into aliquot portions for the various studies. It must be expressly emphasized that the modus operandi described under “Methods” can only permit a statement to be made about the actual metabolic situation of the spermatic fluid 10 minutes after ejaculation. 2.
Methods
2. I Study material The studies were performed on the ejaculates of 525 andrological patients. The patients involved were the same as those in whom the physical and morphological parameters discussed in the 1st report were studied. The size of the individual groups, arranged according to clinical diagnoses, is shown in Table 1. Key wrds: ATP - ADP - AMP - pyruvate - lactate - a-ketoglutarate - Fructose phosphoglucoseisomerase - phosphoglyceratekinase - ATPase - acid phosphatase
-
myokinase -
C. SCHIRREN,G. LAUDAHN,E. HARTMANN and I. HEINZE
96
Table 1: Size of groups
Diagnoses
n
Normozoospermia Asthenozoospermia Oligozoospermia Azoospermia
196 92 181 56
Total
5 25
2.2 Study methods The following parameters were determined and evaluated for this 2nd report:
Substrates Enzymes
ATP*, ADP, AMP,pyruvate, lactate, a-ketoglutarate, fructose. Myokinases, phosphoglucoseisomerases,phosphoglyceratekinases, ATPases, acid phosphatases.
The first report should be referred to for the assessment of the spermiogram.
2.3 Biochemical methods Substrates After allowing 10 minutes for liquefaction, the spermatic fluid for ATP, ADP, AMP, pyruvate, lactate and a-ketoglutarate is mixed with twice the amount of ice-cold 0.6 N perchloric acid and immediately centrifuged for 15 minutes in a cooling centrifuge (t 4OC) at 3 100 g. The clear supernatant is then poured into a test tube. Deproteinization for fructose: 1.0 ml ice-cold 0.33 N perchloric acid t 0.1 ml ejaculate, centrifuge. The measurements were made on a Zeiss photometer PMQ I1 at 340 nm in glass cuvettes with a layer thickness of 1 cm. The results were calculated according to the following formula: AExVxMW C=
= pg/ml (in the sample).
Exdxv = Extinction coefficient for NADH or NADPH at 340 nm (cm2/pMol) E = Extinction alteration = Test volume (ml) V v = Sample volume in the test d = Layer thickness (cm) MW = Weight of a micromol (pg) of the substrate
E A
Finally, take into account for the calculation: deproteinization, buffering and supernatant dilution.
* List of abbreviations see page 114. andrologia 9, Heft 1 (1977)
Studies of the Correlation of Morphological and Biochemical Parameters
97
ATP (1) G-3-P t ATP PGK G-1.3-P t ADP (2) G-l.3-P2 t NADH t H' GAP H GAP t NAD' t Pi
6
2.0 m10.5 M TRA pH 7.6 (0.5 M TRA; 4 mM Mg SO4; 6 mM glycerate-3-phosphate), 0.20 ml NADH (2.5 mM); 0.20 ml deproteinized supernatant, mix, measure E l , 0.02 ml GAPDH/PGK/TIM (7 mg GAPDH/ml; 1 mg PGK/ml; 2 mg GDH/TIM/ml); mix, wait for reaction to cease (about 15 minutes), measure E2. If the reaction does not cease after 15 minutes, perform 3-5 further readings at intervals of 2 minutes and extrapolate E2 to the time of addition of the enzyme suspension. E l - E2 = A E. ADP/AMP (1) AMPtATP MK 2ADP (2) 2 A D P t 2 P N P PK 2 A T P t 2 (3) 2 Pyruvate + 2 NADH t 2 H+ LDIzmvate 2 Lactate t 2 NAD+
Adjust deproteinized supernatant with 0.5 M TRA (0.5 M TRA; 2.0 M K2CO3) to a pH value of 7.3-7.5 (pH meter check). The perchlorate which precipitates as a result is removed by brief centrifugation. 1.75 mlO.1 M TRA pH 7.5; 0.25 ml deproteinized supernatant; 0.20 ml PEP (10 mM PEP; 1.3 M KCI; 0.4 M MgSO4); 0.20 ml NADH (2.5 mM); 0.02 ml LDH (1 mg/ml); mix, measure Elafter 5 minutes; 0.02 ml PK (1 mg/ml); mix, wait for the reaction to cease, or perform 3-5 further readings at intervals of 2 min. and extrapolate E2 to the time of addition of PK; 0.02 ml MK (2 mg/ml); mix, wait for the reaction to cease, measure E3, or perform further readings as above. Blank value: sustitute 0.1 M TRA, pH 7.5, for the buffered supernatant in the test preparation. ADP = (El main - E2 main) - (El blank - E2 blank) AMP = (E2 main - E3 main) - (E2 blank - E3 blank) The fact that 1 Mol AMP corresponds to 2 Mol NAD must be taken into account when calculating AMP. Py ruva te Pyruvate t NADH + H+ LDH Lactate t NADt Adjust deproteinized supernatant with 0.7 M tribasic potassium phosphate to a pH of 6-7, then centrifuge. 1.9 ml aqua bidest.; 0.1 ml buffered supernatant; 0.20 ml 2.5 mM NADH; mix, measure E l ; 0.02 ml LDH (2 mg/ml); mix, measure E2 after 5 minutes or perform 3-5 further readings at intervals of 1 min. and extrapolate E2 to the time of addition of LDH. Lactate Lactate t NADt
LDH Pyruvate + NADH t H+
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C. SCHIRREN, G . LAUDAHN,E. HARTMANN and I. HEINZE
Main value: 2.00 ml glycin buffer (0.5 M glycin buffer pH 9.0; 0.4 M hydracin); 0.10 ml deproteinized su ernatant; 0.20 ml NAD (27 mM); 0.2 ml LDH (2 mg/ml), mix, allow to stand at 25gC in a water bath for exactly 1 hour. Pour into cuvettes. Measure extinction of the sample and of the blank value against air. Ep - EL = A E. Blank value: Substitute 0.10 ml perchloric acid in the test preparation for 0.10 ml deproteinized supernatant. a-KGS a-Ketoglutarate + NADH t E+ t NH;
GlDH Glutamate t NADt + H20
1S O ml TRA (0.1 M pH 7.5); 0.50 ml buffered supernatant (as with ADP/AMP); 0.05 ml NADH (7 mg/ml); mix, measure E l ; 0.05 ml GlDH (2 mg/ml); mix,measure E2 after 10 min. or perform 2-3 other readings at intervals of 1 min. and extrapolate E2 to the time of addition of GlDH.
Fructose D-Fructose + ATP HK F6-P + ADP F-6-P PGI G-6-P G-6-P t NADP M-PDH Gluconate-6-Phosphate + NADPH + Ht 2.58 ml TRA pH 7.5 (0.3 M TRA; 4 mM MgS04); 0.10 ml deproteinized supernatant;0.10mlNADP(12mM);0.10mlATP(16mM);O.O2mlPGI(2mglml);mix, measure E l ; 0.02 ml HK/G6-PDH (1 mg/ml; 1 mg/ml); mix, wait for reaction to cease (about 15 min.) or perform 3-5 further readings at intervals of 2 min. and extrapolate E2 to the time of addition of HK/Gh-PDH. E2-E1 =AE.
Enzymes After allowing 10 min. for liquefaction, the ejaculate samples were centrifuged for 15 min. at 3 100 g and a temperature of 4OC. The seminal plasma (SP) obtained in this way was tempered before being used in the tests. Any necessary dilution was performed with aqua bidest. The measurements for MK, PGI and PGK were performed on a Zeiss photometer PMQ I1 at 366 nm and a constant temperature of 25OC. Following an initial run of 0.050-0.100 the time for a further extinction alteration of 0.100 was stopped. Linearity existed under the study conditions. Study conditions for acid phosphatases: wave l e n p 405 nm, temperature 37OC; for ATPases: wave length 420 nm, temperature 25 C. Unless otherwise stated, the enzyme activity is expressed in international units (units, milli-units): 1 U is that enzyme activity which converts 1 pMol substrate per min.
MK Auxilliary and indicator reaction
AMPtATP MK 2ADP 2 ADP t 2 PEP PK 2 ATP + 2 Pyruv. 2 P ~ N v+. 2 NADH + 2 H+ LDH 2 Lact. + 2 NAD andrologia 9, Heft 1 (1977)
Studies of the Correlation of Morphological and Biochemical Parameters
Test preparation: 2.17 m10.05 M TRA pH 7.5; 0.20 ml ATP (10 mg/ml TRA pH 7.5); 0.15 ml MgS04/KC1(0.5 M Mg SO4 in 2 M KC1); 0.10 ml PEP (5 mg tricyclohexylammonium salt/ml); 0.10 ml NADH (10 mg/ml); 0.05 ml SP; 0.01 ml PK (10 mg/ml); 0.02 ml LDH (5 mg/ml). Start with 0.20 ml AMP (10 mg/ml TRA pH 7.5; Na salt). When calculating the activity it must be borne in mind that 2 pMol NADH react per pMol substrate (AMP). PG I F-6-P PGI G-6-P Indicator reaction G-6-P t NADP G-6-pDH Gluconate-6-P t NADPH t Ht 2.53 m10.05 M TRA pH 7.5; 0.10 ml F-6-P solution approx. 0.025 M; 0.20 ml MgSO4 0.1 M; 0.10 ml NADP (10 mg/ml); 0.02 ml G-6-PDH (1 mg/ml). Start with 0.05 ml SP 1:2. Production of the F-6-P solution: Dissolve 130 mg F-6-P barium salt in 3 ml aqua bidest. while adding 2 mlO.1 N HC1, add 0.5 ml9.2% K2SO4 solution, mix well, centrifuge. Neutralize supernatant with 0.1 N NaOH and make up to 10 ml with aqua bidest. This solution will keep for about 14 days in the refrigerator. PGK Glycerate-3-P + ATP PGK Glycerate-l.3-P2 + ADP Indicator reaction Glycerate-1.3-P2 t NADH t Ht GAPDH + NADt t Pi 2.15 m10.05 M TRA pH 7.5; 0.15 ml EDTA (5 mg/ml); 0.10 ml NADH (10 mg/ml); 0.20 ml ATP (10 mg/ml TRA pH 7.5); 0.10 ml3-PGS (50 mg tri-cyclohexylammonium salt/ml); 0.05 ml MgSO4 0.1 M; 0.05 ml GAPDH (10 mg/ml). Start with 0.2 ml SP 1:2. ATP-ases ATP t H20
ATP-ase ADP t Pi + H+
Main value: 2.90 ml buffer substrate solution and 0.1 ml SP are incubated at 25OC for 1 hour. Interruption of the reaction by the addition of 1.0 m l 2 W trichloro-acetic acid, centrifuge. Determination of the anorganic phosphate in the centrifuge supernatant. Blank value: pipette 1.0 ml 20% trichloro-acetic acid, 0.1 ml SP and 2.9 ml buffer substrate solution together (sequence!), centrifuge, phosphate determination. Phosphate determination with the Boehringer Biochemica Testcombination: 0.75 ml aqua bidest.; 0.25 ml centrifuge supernatant; 1.O ml ammoniummolybdate (40 mM ammoniummolybdate; 2.5 N H2SO4); 1.0 ml ammoniumvanadate (21 mM ammoniumvanadate; 0.28 N HNO3); mix, take extinction reading after 10 min. Measurement is made against the blank value: 1.O ml aqua bidest.; 1.O ml ammoniummoblybdate; 1.Oml ammoniumvanadate. Measured radiation: 420 nm;depth of layer: 1 cm. The amount of phosphate released as a result of ATP-ases activity represents the difference between the phosphate content of the blank and main values. The amount of phosphate - read off in pMol from a calibrated curve - divided by 60 results in pMol/min. Substrate buffer solution: 41 m10.05 M TRA pH 7.4 t 10 ml ATP/KCl solution (0.03 M Na2H2-ATP in 0.45 M KCI, with 1 N KOH to pH 7.4) t 4 m10.075 M MgS04. andrologia 9, Heft 1 (1977)
99
C. SCHIRREN, G. L A U D A H NE., HARTMANN and I. HEINZE
100
Acid phosphatase p-Nitrophenylphosphate + H20
Acid PhosPhatas p-Nitrophenol - Pi
Dilute SP 1:10 000 with ph siological NaC1. Measured radiation 405 nm; thickness of layer 1 cm; temperature 37 C. Measurement is made against the blank value.
B
Pipette into test tubes
P1
Buffer/substrate solution (50 mM citrate buffer, pH 4.8; 5.5 mM sodium-p-nitro-phenylphosphate) 0.2 M sodium tartrate SP 1: 10000 mix, 30 min. water bath 37OC
p2
1.0 ml -
NaOH, 0.02 N
1.0 ml 0.1 ml
0.2 ml
0.2 ml
10.0 ml
10.0 ml
Blank value: 1.0 ml buffer/substrate solution; 10.0 ml NaOH, 0.02 N; 0.2 ml SP 1: 10000. Ext. P i = Total acid phosphatases Ext. P2 = Prostatic phosphatases Calculation:
E x V x dilution txexv
[ U/mlI.
E = Extinction coefficient of 4-nitrophenol in alkaline solution at 405 nm = 18.5 cm2/Mol. V = Testvolume(ml) v = Sample volume in the test t = Time in minutes 2.4 Statistical analysis See first report. 3.
Results
3.1 Relationship of the parameters to age As with the morphological parameters, the first check made in the biochemical analyses was for the existence of any relationship of the parameters to age. It was found that there was a distinct relationship between age and the fructose content of the seminal fluid in as much as lower fructose values were measured with increasing age (p < 0.05) (Fig. l)*. This applies equally for pyruvate (Fig. 2), whilst the converse was observed for lactate (Fig. 3). No age-related behaviour was observed for the other substrates. Lower activities were established in advanced age for the enzymes PGI and PGK, whilst higher activities were observed for the enzymes ATPase and acid prostatic phosphatase. ~~
* All Figures see page 102/103. andrologia 9 , Heft 1 (1977)
Studies of the Correlation of Morphological and Biochemical Parameters
101
3.2 Relationship of the parameters to the diagnosis groups and to one another The mean values’of the concentrations of AMP, ADP, ATP and of fructose, pyruvate and lactate are presented in Figs. 4-7. It was observed that no difference exists for ATP between the individual diagnoses. In contrast to this, lower values can be demonstrated for ADP in the diagnosis oligozoospermia (Fig. 4) (p < 0.05),whilst there is again no difference for AMP.If the values determined for these three substrates in the group normozoospermia and the pooled groups asthenozoospermia, oligozoospermia and azoospermia are now compared, then it can be seen for ATP and ADP that distinctly higher values are present in normozoospermia; in contrast, the converse can be demonstrated for AMP (Fig. 5). A break-down of the values for fructose, pyruvate and lactate according to the various diagnoses results in a relationship only in the case of fructose, for which lower values can be demonstrated (p < 0.05) in the case of normozoospermia and asthenozoospermia - which can be regarded as identical in respect of the sperm count (cf. Fig. 15, first report) (87.5 million sperms/ml) - than in oligozoospermia and azoospermia (Fig. 6). No difference between the diagnosis groups can be demonstrated for lactate; in the case of pyruvate the mean value is distinctly lower in the diagnosis asthenozoospermia than in all other diagnoses (Fig. 7) (p < 0.05). A study of the extent to which the enzyme activities in spermatic plasma are associated with the respective pH value measured shows that pH values above 7.2 correlate with distinctly lower activities for acid prostatic phosphatase, PGI and MK (Fig. 8). These studies were conducted on 120 ejaculates in which the pH value was determined by the situation in the unprepared seminal fluid; the three enzyme activities were studied under these conditions. In this connection we have also conducted in vitro studies which are presented in Figs. 9 and 10. These figures show the activity optima of MK, PGK, PGI and ATPase in human spermatic plasma at various pH values. The object of these studies was to determine the respective optimum of the enzyme activity; we know that pH values above 8.0 are a particularly rare finding in adrological examinations. It is clear from both figures that the pH range of 7.4-7.7 must be regarded as the optimum for PGK and ATPase. Of particular interest in this connection is the relationship between the pH value of the ejaculate and the fructose and ATP concentrations (Fig. 11). According to this, under conditions of unprepared spermatic fluid pH values of 7.1-7.7 are accompanied by decreasing ATP concentrations and increasing fructose concentrations. A comparison of the concentrations of ADP and AMP shows that, at higher ADP values, higher AMP concentrations are also measured. If, furthermore, the lactate and ADP concentrations are placed in relationship to the fructose concentration in the ejaculate, lower values for ADP and lactate can be observed at higher fructose concentrations (p < 0.05). Correlations of this nature - between individual substrates, between substrates and enzymes, and between individual enzymes - are shown in Figs. 16-21. It can be seen here (Fig. 12) that, at higher pyruvate concentrations in the ejaculate, lower concentrations for AMP and ADP but higher lactate concentrations can be demonstrated. If pyruvate, ADP and ATP are placed in relationship to the KGS concentration (Fig. 13), a respectively higher share for pyruvate, ADP and ATP concentrations can be observed at higher values for KGS (p < 0.05). If, on the other hand, the MK activity in spermatic plasma is placed in relationship to the concentration of ADP, pyruvate and fructose (Fig. 14), a lower fructose and pyruvate concentration must be expected at higher MK activity, whilst the concentration for ADP shows higher values. Further, higher ATPase activities correlate with lower measured values for lactate, pyruvate and KGS and with higher ADP values (Fig. 15). andrologia 9, Heft 1 (1977)
andrologia 9, Heft 1 (1977)
Studies of the Correlation of Morphological and Biochemical Parameters
103
The activity of PGI has been placed in relationship to the concentration of ATP, ADP, pyruvate and fructose. Lower fructose and pyruvate concentrations can be demonstrated at higher PGI activity, whilst higher concentration values are observed for ATP and ADP. Finally, in Fig. 16, the enzymes examined are placed in relationship to one another, whereby the measured values of PGI are compared with those of ATPase, KM and PGK. In this case, at higher PGI activity values, higher activities are also obtained for other enzymes. Discussion Although the division of the patients into certain diagnosis groups chosen by us is somewhat arbitrary, there is still no better alternative than to make the morphology of the spermatozoa the basis of diagnostic differentiation. It must also be possible to place the biochemical findings in relation to the morphological parameters. The object is to improve the clinical possibilities of differentiation and also to improve the accuracy of conclusions about the ability to procreate made on the basis of the correlation of morphological and biochemical findings. When interpreting our data it must be borne in mind that all the studies were carried out after the ejaculate had been allowed to stand for 10 minutes at room temperature, the reasons for which were reported in the introduction. It is our intention in the future to build up on these results and, using a limited amount of patient material, to investigate the question of what the biochemical status looks like if the spermatic fluid is ejaculated directly into liquid nitrogen, for example. For the initial studies we shall use ejaculates from "normal cases" known to us, for whom it must be assumed that they will exhibit their usual morphologically normal findings at the time of use of the nitrogen method as well. The evaluation will, of course, be made with this assumption in mind. Our present studies confirm earlier fmdings (Schirren - 1961) that lower fructose values are to be observed in the ejaculate at advanced ages. This applies also to pyruvate, whilst the opposite behaviour can be observed in the case of lactate. The increased presence of lactate as a final product of fructolysis suggests that the spermatozoa of older men have a lower capacity for the final oxidative degeneration of carbohydrates than the spermatozoa of younger men. This provides material for complementary studies in which the question of a hormone dependence of fructolysis should also be investigated. Since the mean values for the various substrates differ from one another when they are placed in relationship to the individual andrological diagnoses, we have formed two
-,
AOP lMml
Fig. 14
I
Rym
"ale rnd
I t&
2951 280
=:Y :
,220
-2M
15! 250
235
450
180
350
1
4w
I
1-
IBO
0 9 1'4 1'9 2'4 29 3 4 39
andrologia 9, Heft 1 (1977)
PI3 U/%
104
C. SCHIRREN, G . LAUDAHN, E. HARTMANN and I. HEINZE
groups, one of which is composed of cases of normozoospermia and the other of the diagnoses asthenozoospermia, oligozoospermia and azoospermia. This results in the finding that distinctly higher values for ATP and ADP are present in the cases of normozoospermia. Since ATP and ADP are not present in free form in seminal plasma, but are bound to the spermatozoa (Peterson and Freund - 1971), the higher content of energy-rich phosphates in the spermatozoa in normozoospermia could also explain, for example, the superior motility characteristics in this group. A look at the substrates fructose, pyruvate and lactate in relationship to the diagnosis groups revealed a similar situation to that already established in the discussion of the relationship to age. The known correlation between fructose and spermatozoal density (Schirren - 1961) was apparent again here too. So far we have not reported on any new possibility of interpretation which improves on our old statement about a possible connection with the oestrogen content of the spermatozoa and a corresponding feed-back to the anterior lobe of the pituitary. Of interest are the data on the enzyme activity at the respective pH value in the ejaculate, whereby our in vitro findings obtained with different experimentally adjusted pH valuesmust be compared with the values determined in vivo. It can be seen from this comparison that the optima for certain enzymes determined in vitro do not exist in vivo - at least not immediately after ejaculation. On the other hand, we know that the pH value of the ejaculate undergoes a shift to the alkaline side after it has stood for a while. Moreover, for the significance of this factor in vivo the biochemical or physicochemical milieu of the vagina and other components of the female genital tract (e.g. cervical mucus, endometrial and tubal secretion), i.e. influences which have not previously been examined in this manner, must also be taken into account. Summary In this report the authors present the results about biochemical analyses in human ejaculate. The following parameters were determined ATP, ADP, pyruvate, lactate, a-ketoglutarate, fructose, myokinase, phosphoglucoseisomerase, phosphoglyceratekinase, ATPase, acid phosphatase. It was found a relationship between age and fructose content, this was equally for pyruvate, whilst the converse was observed for lactate. N o age dependend relationship was observed for the other substrates. For ATP and ADP distinctly higher values can be demonstrated in normozoospermia. A special study of the extent to which the encyme activities in sperm plasma are associated with the respective pH value measured shows that pH values above 7.2 correlate with distinctly lower activities for acid phosphatase, PGI and MK. Un&nuchungen zur Korrelation rnorphologixher und biochemischer MeEgroRen irn menschlichen Ejakulat bei verschiedenen andrologischen Diagnosen
I I. Mitteilung: Biochemische MeBgroBen
Zusammenfassung In Fortsetzung friiherer Untersuchungen teilen wir in dieser Arbeit das Ergebnis biochemischer Analysen im menschlichen Ejakulat mit. Gemessen wurden die Konzentrationen einiger Substrate sowie die Aktivitaten verschiedener Enzyme des intermediaren Stoffwechsels, die teilweise Beziehungen zum Fruktosestoffwechsel besitzen. Fur die biochemische Analytik ist von Bedeutung, dal3 sich Ejakulate nicht wie andere Korperfliissigkeiten oder -gewebe verarbeiten lassen: so mu5 zunachst die Verflussiandrologia 9, Heft 1 (1977)
Studies of the Correlation of Morphological and Biochemical Parameters
105
gung des coaguliert entleerten Sperma abgewartet werden (in der Regel etwa 10 Minuten). In dieser Verflussigungsphase laufen mit gro5ter Wahrscheinlichkeit bereits verschiedene biochemische Umsetzungen ab, die 2.B. eine h d e r u n g in den Anfangskonzentrationen sogenannter energiereicher Phosphate bewirken. Bei der Interpretation unserer Daten ist zu beriicksichtigen, da5 alle Untersuchungen erst nach zehnminiitigem Stehenlassen des Ejakulates bei Raumtemperatur vorgenommen wurden. Unsere jetzigen Untersuchungen bestatigen friihere Befunde, d d in hoheren Lebensaltern niedrigere Fruktosewerte im Ejakulat zu messen sind. Identisch verhalt sich Pymvat, wahrend bei Lactat das entgegengesetzte Verhalten zu beobachten ist. Die Anhaufung von Lactat als Endprodu I der Fruktolyse legt den Gedanken nahe, d d Spermatozoen alterer Manner eine geringere Kapazitat zum oxydativen Endabbau der Kohlenhydrate haben als die Spermatozoen Jiingerer. Hier ergeben sich Ansatzpunkte fur Erganzungsuntersuchungen, in denen auch die Frage einer Hormonabhangigkeit der Fruktolyse zu priifen ist. Wenn man die Mittelwerte fur die verschiedenen Substrate in Beziehung setzt zu den einzelnen andrologischen Diagnosen, ergibt sich, daD bei der Normozoospermie eindeutig hohere, Werte fur ATP und ADP vorliegen. Da ATP und ADP nicht frei im Seminalplasma vorkommen, sondern an die Spermatozoen gebunden sind, konnte der hohere Gehalt an energiereichen Phosphaten in den Spermatozoen bei Normozoospermie 2.B. auch die uberlegenen Motilitatscharakteristika in dieser Gruppe erklaren. Von Interesse sind die Daten zur Enzymaktivitat bei dem jeweiligen pH-Wert im Ejakulat. Man mu5 hienu unsere in vitro-Befunde mit experimentell unterschiedlich eingestelltem pH den in vivo ermittelten Werten gegeniiberstellen. Es ergibt sich dabei, d d die in vitro ermittelten Optima fur bestimmte Enzyme in vivo zumindestens unmittelbar nach der Ejakulation nicht gegeben sind. Wir wissen andererseits, d d sich der pH-Wert des Ejakulates nach langerem Stehenlassen zur alkalischen Seite verschiebt. Dariiberhinaus mu5 man fur die Bedeutung dieses Faktors in vivo sicherlich auch das biochemische bzw. physiko-chemische Milieu der Scheide und der weiteren Teile des weiblichen Genitaltraktes (2.B. Cervixschleim, Endometriums- und Tubarsekret) beriicksichtigen, d.h. Einfliisse, die in dieser Weise bisher noch nicht untersucht wurden.
References Peterson, R.N. and M. Freund: Glycolysis of human spermatozoa: Levels of glycolytic intermediates. Biol. Reprod. 5, 221-227 (1971). Schirren, C.: Fertilititsstorungen des Mannes. Diagnostik, Biochemie des Spermaplasmas, Hormontherapie. Stuttgart: F. Enke 1961. Schirren, C.: Practical andrology. Berlin: Briider Hartmann 1971. Schirren, C., G. Laudahn, E. Hartmann, I. Heinze und E. Richter: Untersuchungen zur Korrelation morphologischer und biochemischer MeDgroBen im menschlichen Ejakulat bei verschiedenen andrologischen Diagnosen. I. Mitt.: Beziehungen zwischen Ejakulatvolumen, Zahl, Motilitat und Morphologie der Sperrnatozoen unter Beriicksichtigungdes Lebensalters. andrologia 7, 117-125 (1975). Address: Prof. Dr. C. SCHIRREN, Dept. for Andrology, University Hospital, Martinistr. 52, D-2000 Hamburg 20, Germany.
List of abbreviations see page 114 andrologia 9, Heft 1 (1977)
