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Factors Influencing Metabolic Activity of Bull Spermatozoa. IV. pH, Osmotic Pressure, and the Cations, Sodium, Potassium, and Calcium
F A C T O R S I N F L U E N C I N G M E T A B O L I C A C T I V I T Y OF B U L L S P E R M A T O Z O A . IV. p H , O S M O T I C P R E S S U R E , A N D T H E C A T I O N S , SODIUM, P O T A S S I U M , A N D C A L C I U M R. G. CRAGI~E1 AND G. W. SALISBURY Department of Dairy Science, University of Illinois, Urbana SUMMARY The combined effects of increasing pH from 4 to 8, of increasing potassium concentration, at the expense of sodium, and of varying osmotic pressure on the aerobic metabolic responses of washed bovine spermatozoa provided with fructose have been determined in one experiment. Low pI4 and high potassium levels were found to be inhibitory of all measures of metabolism (oxygen uptake, fructose utilization, and lactic acid accumulation). Osmotic pressure had no significant effect. In a second e~:periment, variations in the calcium level of the diluent replaced the variations in the osmotic pressure. The inhibitory effect of potassium was eliminated by the presence of calcium. Consequently, ptI was the only variable affecting metabolic response. The uptake of fructose in the first few minutes after resuspension of the washed cells in a fructose-containing diluent is more rapid if the diluent has a pH of 5 than if it has a pH of 7.5, though on continued exposure the utilization ceases at the low pH but continues unabated at the higher one.
O p t i m u m preservation of the fertility function of spermatozoa a f t e r ejaculation is dependent upon reduction of their metabolic activity to m i n i m u m levels during storage and complete reversal of the inhibitory processes at insemination. T e m p e r a t u r e control has been the inhibitory method of choice, with the diluent designed to provide protection against cold, and at body t e m p e r a t u r e to provide o p t i m u m conditions for motility and metabolic activity (4). These techniques have provided the basic advances responsible for the development of commercial artificial insemination of cattle since the discovery by Phillips (12) of the value of hens '-egg yolk for these purposes in 1939. However, observations made in a n u m b e r of laboratories in the course of studies on the epididymis and in measurements of metabolic activity of spermatozoa have indicated that other means of metabolic control were possible. These include the finding of a metabolic regulator in epididymal spermatozoa (9), of a metabolic inhibitor ill ejaculated semen closely associated with the spermatozoa (2), and of the effects on metabolism of p H (10, 16), osmotic pressure (17), of the concentration of the cations, calcium and potassium (10, 22), the concentration of anions (3, 17), and of the partial pressure of carbon dioxide (18, 19). Ill addition, new evidence on the environment afforded spermatozoa in the epididymis, where they are n o r m a l l y quiescent, shows that the fluids are higher in potassium, lower in sodium and in calcium concentration than seminal plasma (7, 15). Received for publication April 13, 1959. Present address: UT-AEC Agricultural Research Laboratory, P. O. Box 142, Oak Ridge, Tennessee. 1302
FACTORS I N F L U E N C I N G )/.[ETABOLIC A C T I V I T Y OF SPERMATOZOA. IV.
1305
The independent effects of some of these factors on aerobic metabolism of spermatozoa have been reported. However, no data are available on the effect of a combination of some of them on the several measures of metabolic activity. The experiments reported here were conducted to determine the interacting effects, if they existed, o'f pH, osmotic pressure, calcium concentration, and potassium concentration on the aerobic metabolism of washed bovine sperm cells. A preliminary report of these experiments appeared in 1957 (6). h,[ETHODS AND M A T E R I A L S
Semen used in these experiments was collected from mature dairy bulls by an artificial vagina. The spermatozoa were twice washed with 0.154 M NaC1 (saline), using the following procedure: The raw semen was diluted to approximately twice the o'riginal ejaculate volume with saline. The spermatozoa were then centrifuged out of suspension and the supernatant removed. The cells were suspended again in saline to a volume equal to the original ejaculate, recentrifuged, the supernatant removed, and the cells resuspended in saline solution to the desired concentration of cells. Two-tenths milliliter of this cell suspension was added to 0.8 nil. of test solution containing 200 rag/100 ml. of fructose as substrate in the W a r b u r g reaction flasks. Oxygen uptake, fructose utilization, and lactic acid accumulation were measured during a 2-hr. incubation period at 37 ° C. Initial and final p H and motility of the suspended cells were recorded. Fructose was determined by the method of Roe (14), as modified by Mann (11). Lactic acid was determined by the method of Barker and Summerson (1). Three experiments were conducted in this study. The first two were three-factor composite type experiments (5) involving eight replications each. In Experiment One, the variables of pH, osmotic pressure, and potassium concentration were studied, whereas in the second experiment, the effects of pH, calcium concentration, and potassium concentration were studied. In these composite experiments, initial p H values of 4, 5, 6, 7, and 8 were obtained by combinations of citric acid and sodium bicarbonate. Molarities of the final diluents (before adding spermatozoa) expressed as NaC1 were 0.084, 0.119, 0.154, 0.189, and 0.224 M. The theoretical o'smotic pressure equivalents are approximately 195 to 417 milliosmols, assuming complete dissociation of ions. (A solution which has an osmotic pressure of 290 milliosmols or a 0.154 M NaC1 solution is isotonic and has appro"ximately the same freezing point depression as seminal plasma.) T h e actual osmotic pressures were 150 to 392 milliosmols. Potassium was included in the experiments in levels of 40, 120, 200, 280, and 360 rag. % final dilution. Calcium was included in the seco~ld experiment at levels of 2, 12, 22, 32, and 42 mg. % final dilution. Potassium or calcium additions were made at the expense of sodium. MgCI,, • 6 HfO was included in all of the diluents at the rate of 0.5 ml. of a 1.54 21i solution in 50 ml. of diluent. The third aerobic experiment was a 32 factorial involving five replications, with the objective of determining the relatimlship of p H to metabolic activity at various intervals. The p H levels studied were 5, 6.25, and 7.5. The intervals at which samples were taken for fructose and lactic acid measurements were
130(~
R . G . CRAGLE AND G. W. SALISBURY
0, 1, and 2 hr. A 10-miu. period for equilibration of the spermatozoa and diluent preceded 0 hr. I n this experiment, therefore, fructose utilization and lactic acid accumulation were measured to include the 10-min. equilibration perio'd, whereas Oe uptake measurements began at 0 hr. F i f t y per cent of the diluent used in this experiment was made up with a combination of Na2ttP04 • 12 H 2 0 and N a H e P 0 4 • H20, depending on the p i t desired ; whereas, the other half contained 0.154 M NaC1 with 0.5 ml. of 0.11 M MgCle • 6 H 2 0 per 50 ml. and fructose solutions (200 rag. % final dilution). I n the composite experiments, the metabolic responses are expressed as the conventional Z values (13) whereas, in E x p e r i m e n t 3, they are expressed as total mieroliters or micrograms/10 s spermatozoa. RESULTS
The results of the first experiment involving five p H levels, five levels of osmotic pressure, and five potassium concentrations are given in F i g u r e 1. B y 0.224~ i-
I03~t'1
-11.2 -64 4 39
- 12,4 -256
0
,8454
-
1.7
1 0.109
tlJ
%_ -~_~,zo \
i
-8.8 :t -zz, 14B
-33 -2~
I
\ ~" °~t.~o
3
32
-4.4
--t50
i.3
~
i~ O.6o.154
.9
z,
~_\~6o -1~:~2"~ • Ot UPTAKE (Z 0 2) FRUCTOSE UTILIZATION ( Z r ) LACTATE FORMATION (Z L) MOTILITY (% AND RATE)
[0
0.8
IE
\x -50
-92 61 17
3.9
I.I
0,084
-88 49
4~
5~
~
g,
",
0.6
i
-7.8 ÷~ 29
0.119
"\
624.5
7
_ _
8
X I = pH OF DILUENT (INITIAL)
~I(L 1. Effects of p H , molarity of diluent, and p o t a s s i u m concentration on spermatozoan activity ( a v e r a g e of eight r e s p o n s e s ) .
study o'f this figure, one notes that the lower values of fructose utilization and of lactic acid production tend to be on the lower left side of the figure, at a low p H , a low osmotic pressure, and a high concentration of potassimn. Statistical analysis of the combined data show that not all of the differences shown in the figure are significant ones. F o r example, considering the oxygen uptake only, the effect of p H was pronounced. The Z-o was small at p H 4, increasing to a m a x i n m m in 2
the center of the figure and decreasing again at the highest p H .
This trend is
FACTORS
INFLUENCING
METABOLIC
ACTIVITY
OF SPERMATOZOA.
1307
IV.
best fitted by a quadratic expression. The potassium level also influenced the oxygen uptake. The effect was least at lo~v concentrations of potassium and greatest with the higher concentrations, but v a r y i n g in degree with the p H level. On the other hand, the molarity of the diluent did not exhibit a significant effect on oxygen uptake. The metabolic behavior of the washed spermatozoa as influenced by the three controlled variables can be expressed by regression equations, where X I is the effect o'f p H ; Xe, t h a t of v a r y i n g molarity of the solutions ; and X3, t h a t of potassium concentration. Only those regression coefficients (in either E x p e r i m e n t 1 or 2) to which a significant sum of squares could be attributed are given in the following equations : Z% = --10.6 + 1.1 X3 + 1.5 XI"
ZF ZL
= --119.8 -- 60.2 X1 + 19.9 X3 = 88.5 + 43.1 X1 --18.4 X1X3
The five levels for p H (X1) and the five levels for potassium (Xs) have been coded as --2, --1, 0, 1, and 2, respectively. F o r example, at a potassium content of 200 mg. % (X3 = 0) and a p H of 5 (X1 = - 1 ) , one would expect a Z% of - 9 . 1 . (Instead of the usual plus values o~
Zo2, minus
values are used here to indicate a
utilization of O2.) B y use of these equations, estimates of response can be made for a n y set of conditions of the variables and ranges studied. The equations indicate that for fructose utilization, the effects of p H and of potassium concentration are linear and independent. However, for lactic acid accumulation, in addition to a p H effect, a significant interaction between p H and potassium concentration was found.
12
250
J
200
- -
I0
ZF • 8
150 ZL
o, / 50
4
5
6
7
pH
~rG. 2. The effect of pH on metabolic activity.
8
Zo z
1308
R.G.
CRAGLE A N D
G. W. SALISBURY
12
250 _
200
ZF
i
~
o
~
8 ZOz
ZL
100 ZL 50
4 i
o
I
40
120 POTASSIUM
]J'IG,
[
200 CONCENTRATION -
3. The effect of potassium concentration
0 360
280 MG. /
IOOML.
on m e t a b o l i c ~lctivity.
The p r i m a r y effects of pH, of osmotic pressure, and of potassium concentration on the three metabolic responses in this experiment are illustrated in Figures 2, 3, and 4. The combined results of the second experiment involving p H (X1), calcium concentration (X2), and potassium eo~lcentration (X3) are given in F i g u r e 5.
12
25C
% I0
200
ZF
B Zoz
150
ZL
ZF
Ioo 4
5O
0
i 1"73
251 OSMOTIC
290
348
PRESSURE -- M I L L I O S M O L S
:FIG. 4. The effect of osmotic pressure on metabolic activity.
=0 406
1309
FACTORS INFLUENCING xSIETABOLIC ACTIV1TY OF SPERMATOZOA. IV.
42 -134
'~ -7.3 -40 30 22 L4
'; \40
-14.3
t -10.6 I~723 _191 ~170 3! ;L4
I \
"111
32
-II -210 153 20° 1.5
134 :~3 1.7
;:'o,\ o
I
__ I
-io3 \
:_-,,..,
22
.J .( z ;T
t~ ~; Z z 0
~r
-I10 108
\
* 0 z UPTAKE { Z 0 z) FRUCTOSE UTILIZATION (ZF) LACTATE FORMATION (Z L) MOTILITY (%
AND RATE)
4
-6.7 -32 ~J 17 o.g
5 XI =
~
-166 II I
-9.6
-I00 122 2g 1.6
6 pH
OF D I L U E N T
8
~
2g 2,0
Zl
?
m
l.Z
8
(INITIAL)
~IG. 5. JEffccts Of p H , caleimn concentration, a n d p o t a s s i u m c o n c e n t r a t i o n on s p e r m a tozoan a c t i v i t y ( a v e r a g e of eight r e s p o n s e s ) .
In this experiment, alt interesting fact appears. Calcimn not only has no marked effect on the metabolic response of the cells, but the previously observed marked inhibitory effects of increasing the concentration of potassium are virtually eliminated by its presenee. Thus, in the regression equations derived from the second experiment, only the effects of p H (X1) are included, for no other effect contributed a significant portion of the stun of squares.
Zo 2 = --9.6 - 1 . 8 X1 Z~, = --132.7 --64.4 X1 ZL = 101.4 + 46.0 X1 The effect of calcimn concentration as found in this experiment is illustrated in F i g u r e 6. The curves are drawn from the equations calculated for linear and quadratic effects, none of which was significant, so that the effects could equally as well be represented by straight lines across the chart represe]~ting the means for each metabolic measure. The third experiment was designed to cast some f u r t h e r light oll the rate of substrate uptake, as influenced by pt{ during the period of time in which temperature and gas pressure equilibration in the manometers and flask contents took place. The results are given in Table 1. The F values and the significant levels of the responses are given in Table 2. The highly significant interactions of the linear effects of time and the linear effects of p H for bo"ch fructose utilization and lactic acid accumulation are the result of the fact that spermatozoa at p H 5 in a phosphate buffer take up a sizable
R.
1310
G. C R A G L E
AND
G. W .
SALISBURY
12
250
I0
ZF 8 Z%
150 ZL. I00 ZL
4
50
0
12
2
CALCIUM
22
32
CONCENTRATION
-
MG. /
42
0
100 ML.
FIG. 6. The effects of calcium concentration on metabolic activity.
TABLE i Total oxygen uptake, f r u c t o s e utilization, and lactic acid accumulation per 10 8 spermatozoa as influenced by time and p H in p h o s p h a t e buffer ( E x p e r i m e n t 3, m e a n of five replications) Time
pit
D u r i n g equilibration period of 10 min.
5.00 6.25 7.50 5.00 6.25 7.50 5.00 6.25 7.50
E n d of I hr. a f t e r equilibration E n d of 2 hr. a f t e r equilibration
O~
Fructose
Lactate
(M.) ...... ......
tLg. 77 4e7 53 71 67 92 ~9 79 163
t~g. 2 5 6 3 27 24 10 34 40
8.6 9.4 10.3 13.4 12.7 17.1
TABLE 2 Analysis of variance of d a t a f r o m E x p e r i m e n t 3 ( F values)
Time P Time qb pH 1 plt q Time 1 × p H 1
P < .05. ~ P < .01.
linear. bq = quadratic. a 1=
02 u p t a k e
Fructose utilization
Lactic Acid accumulation
179.5 ~* 5.66* NS NS NS
8.86** NS NS NS 10.28 *~
59.38** NS 35.81 ~* 8.03 ~* 11.20 ~*
FACTORS I N F L U E N C I N G M E T A B O L I C ACTI-VITY OF SPERMATOZOA. IV.
1311
quantity of fructose in the initial 10 min. (Time 0) of exposure to substrate. Thereafter, the substrate is metabolized at a slow rate, whereas at a higher pH, they exhibited much higher metabolic responses after a slower initial uptake of substrate. These results show clearly that spermato'zoa removal from the seminal plasma soon after collection, and which have undergone a period of substrate deprivation during washing and the p r e p a r a t o r y stages of the experiment, take up hexose substrate at an initial rate substantially greater than the utilization rate. These results also show that at the lowest pH, after the initial uptake, which occurred within 10 rain. and possibly before the intracellular p H was reduced below that ~f normal semen, no uptake occurs. These facts have great significance in the interpretation of other experiments and in the design of f u t u r e ones. DISCUSSION
A number of difficulties face investigators in this area of research. An important one deals with the relation of metabolism to visual estimates of spermatozoan motility. Our interpretation has been that if spermatozoan metabolic activity was inhibited and final motility was inhibited to a similar degree, no logical conclusion could be reached. Either inhibition a n d / o r death of the cells may have taken place. Only in cases where final motility is the same as the control or only slightly depressed, and the metabolic measures are substantially decreased, can we conclude that metabolic inhibition has actually taken place. A second, equally fundamental difficulty has been the large coefficients of variation in substrate utilization associated with experiments of this nature preventing precise measurement of treatment effects. In the first and second of these experiments, the eo'effieients of variation for fructose utilization were 40% or higher bef()re ejaculate effects were removed. F r o m the third experiment, it would appear that the preconditioning of spermatozoa as affected by the time from ejaculation to removal of the cells from the seminal plasma, the initial p H of the semen and, possibly, many more such factors, are concerned in such variability. A third is a question primarily of semantics. In the first two experiment:~, the predominating cations in the diluents were sodium and potassium. When potassium was increased the sodium was decreased, and vice versa. This leaves the question of which ion should be considered as having the p r i m a r y effect and the naming of that effect. We have chosen to eo~nsider the p r i m a r y effect as due to potassium, because of its higher concentration in the cells (7) and the fact that the response of the cells to extracellular potassium was the one sought, namely, reversible inhibition of metabolic activity. The reversal o'f motility occurred upon removal of the ceils from the W a r b u r g flasks, dilution with 0.9% NaCI, and placing them under a cover slip on a microscope slide. Low concentrations of potassium have been shown to be necessary for optimum metabolism of spermatozoa (22); whereas, high concentrations have been reported as detrimental (23). It now appears that in the absence of calcium (Experiment 1), reversible inhibition of metabolic activity at 37 ° C. takes place
1312
R. G. C R A G L E AND G. W. S A L I S B U R Y
at potassium concentrations of front 200 to 280 rag. %. The inhibition is particularly evident at pH 5, but the K-level × pH interaction was statistically significant only for the lactic acid accumulation. These potassium levels are higher than are reported for seminal plasma (a mean of 161 mg. %) (8), but not so high as are the amounts found in the epididymis, where the cells are normally quiescent (15). However, in the presence of calcium, the high potassium levels do not Cause any measurable effect on sperm cell metabolism (Experiment 2). It does have a deleterious effect on motility. The interference of calcium with the inhibitory effects of potassium may have its counterpart in nature. The epididymal secretions are high in potassium and quite low in calcium ; whereas, on ejaculatou, the high potassium is diluted with sodium and the calcium level is increased by addition of other products of the accessory glands, thus paving the way for the burst of activity associated with ejaculated semen. The inhibition of metabolic activity by the potassium ion strongly suggests that this is at least one of the agents, if not the only one, responsible for depressing the oxygen uptake in whole semen reported by Bishop and Salisbury (2). These workers presented evidence that the inhibitor was associated with the spermatozoa. Subsequent work has shown that potassium is more concentrated within the spermatozoa than it is in the seminal plasma (8). Finally, the sodium/potassium ratio deserves some consideration in view of the emphasis placed on it by other workers with bull spermatozoa (15, 21) and in the inhibition of trout sperm (20). It appears from the results reported here that the sodium/potassium ratio is a useful expression only under specified conditions of pH, low levels of calcium, and where sodium and potassium make up nearly all of the cations present. REFERENCES
(1) BAIg.I.:ER,S. B., AND SUMMERSON~, W. H. The Colerimetric Determination of Lactic Acid in Biological Material. J. Biol. Chem., 138: 535. 1941. (2) BISHOP, M. ~'. H., AND SALISBURY, G. W. Effect of Sperm Concentration oll the Oxygen Uptake of Bull Semen. Am. J. Physiol., 180: 107. 1955. (3) BISHOP, M. W. H., AND SALmSURY, G. W. Effect of Dilution with Saline and Phosphate Solutions on Oxygen Uptake of Bull Semen. Am. J. Physiol., 181 : 114. 1955. (4) BL~CKSEAW, A. W., SALISBURY, G. W., AND VANDEMARK, N. L. Factors Influencing Metabolic Activity of BulI Spermatozoa. I. 37 °, 21 °, and 5 ° C. J. Dairy Sci., 40: 1093. 1957. (5) Box, G. E. P., A~D WILSOn, K. B. On the Experimental Attaimnent of Optimum Conditions. J. Roy Stat. Sou., B. 13: 1. 1951. (6) C~AGbE, R. G., AND SALrSSU~Y, G. W. Effects of pH, Osmotic Pressure, and Bulk Cations on the Metabolic Activity of Bull Sperm. J. Dairy Sci., 40: 621. 1957. (7) CI~AGLE~R. G., S.txLISBURY, G. W., AbeD MUNTZ, T. J-[. Distribution of Bulk and Trace Minerals in Bull Reproductive Tract Fluids and Semen. J. Dairy Sci., 41 : 1273. 1958. (8) CRAGL~, R. G., SALISBUI~Y, G. W., AND VANDEMARK, N. L. Sodium, Potassium, Calcium, and Chloride Distribution in Bovine Semen. J. Dairy Sci., 41: 1267. 1958. (9) LAI~DY, H. A., GHOSH, D., AND PLAUT, G. W. E. A Metabolic Regulator in Mammalian Spermatozoa. Science, 109: 365. 1949. (10) LARDY, I-I. A., AND PHILLIPS, P. H. Effect of p i t and Certain Electrolytes on the Metabolism of Ejaculated Spermatozoa. A ~ . J. Physiol., 138: 741. 1943.
FAOTOI%S INFLUENCING ]VIETABOLI0 ACTIVITY OF SPEI~IV[ATOZOA. IV.
1313
(11) MicrON, T. Fructose Content and Fructolysis in Semen. Practical Application in the Evaluation of Semen Quality. J. Agr. Sci., 38: 323. 1948. (12) PHmLres, P. H. The Preservation of Bull Semen. J. Biol. Chen~., 130: 415. 1939. (13) E~.VENZ, E. Ober den Spaltungsstoffweehsel der Saiigetier Spermatozoen in Zusammenhang mit der Bewegliehkiet. Biochem. Z., 257: 234. 1933. (14) Ro~, J. H. A Colorimetrie Method for the Determination of Fructose in Blood and Urine. J. Biol. Chem., 107: 15. 1934. (15) SALISBURY, G. W., AND C~AaLE, R. G. Freezing P o i n t Depressions and Mineral Levels of Fluids of the Ruminant Male Reproductive Tract. IIIrd Intern. Congr. Animal l~eproduction, Sec. I. 25. 1956. (16) SALISBURY, G. W., A~D K I ~ E Y , W. C., Ja. Factors Influencing Metabolic Activity of Bull Spermatozoa. I I i . pH. J. Dairy Sci., 40: 1343. 1957. (17) SALISBVRY, G. W., A~D NXKABAYASHI; N. T. Effect of Phosphate and Chloride Ions on Aerobic Metabolism of Bovine Spermatozoa. J. l~xptl. Biol., 34: 52. 1957. (18) SALISBURY, G. W., AND V~ND~AR~¢, N. L. Carbon Dioxide as a Reversible Inhibitor of Spermatozoal~ Metabolism. Nature, (London), 180: 989. 1957. (19) S~LmBURY, G. W., AND VA.NDEiVIAI~K, N. L. Sulfa Compounds in Reversible Inhibition of Sperm Metabolism by Carbon Dioxide. Science, 125: 1118. 1957. (20) SCKLF2~K, W., JR., AND KAH~I¢~¢, H. Die Chemisehe Zusammensetzung des Spermaliquors und ihre Physiologische Bedeutung, Untersuchung a n Forellensperma. Bioehem. Z., 295: 283. 1938. (21) S o r m ~ s ~ , EI), A~D A~I)ERS0N, S. The Influence of Sodium and Potassium Ions upon the Motility of Sperm Cells. IIIrd Intern. Co~tgr. Animal l~eproduction, Sect. I : 45. 1956. (22) WHITe, I. G. The Effect of Potassium on the Washing and Dilution of Mammalian Spermatozoa. Australian J. Exptl. Biol. Med. Sci., 31: 193. 1953. (23) WHITe, I. G. Studies on the Alkali Metal Requirements of Ram and Bull Spermatozoa. Australian J. Biol. Sci., 6: 716. 1953.
O p t i m u m preservation of the fertility function of spermatozoa a f t e r ejaculation is dependent upon reduction of their metabolic activity to m i n i m u m levels during storage and complete reversal of the inhibitory processes at insemination. T e m p e r a t u r e control has been the inhibitory method of choice, with the diluent designed to provide protection against cold, and at body t e m p e r a t u r e to provide o p t i m u m conditions for motility and metabolic activity (4). These techniques have provided the basic advances responsible for the development of commercial artificial insemination of cattle since the discovery by Phillips (12) of the value of hens '-egg yolk for these purposes in 1939. However, observations made in a n u m b e r of laboratories in the course of studies on the epididymis and in measurements of metabolic activity of spermatozoa have indicated that other means of metabolic control were possible. These include the finding of a metabolic regulator in epididymal spermatozoa (9), of a metabolic inhibitor ill ejaculated semen closely associated with the spermatozoa (2), and of the effects on metabolism of p H (10, 16), osmotic pressure (17), of the concentration of the cations, calcium and potassium (10, 22), the concentration of anions (3, 17), and of the partial pressure of carbon dioxide (18, 19). Ill addition, new evidence on the environment afforded spermatozoa in the epididymis, where they are n o r m a l l y quiescent, shows that the fluids are higher in potassium, lower in sodium and in calcium concentration than seminal plasma (7, 15). Received for publication April 13, 1959. Present address: UT-AEC Agricultural Research Laboratory, P. O. Box 142, Oak Ridge, Tennessee. 1302
FACTORS I N F L U E N C I N G )/.[ETABOLIC A C T I V I T Y OF SPERMATOZOA. IV.
1305
The independent effects of some of these factors on aerobic metabolism of spermatozoa have been reported. However, no data are available on the effect of a combination of some of them on the several measures of metabolic activity. The experiments reported here were conducted to determine the interacting effects, if they existed, o'f pH, osmotic pressure, calcium concentration, and potassium concentration on the aerobic metabolism of washed bovine sperm cells. A preliminary report of these experiments appeared in 1957 (6). h,[ETHODS AND M A T E R I A L S
Semen used in these experiments was collected from mature dairy bulls by an artificial vagina. The spermatozoa were twice washed with 0.154 M NaC1 (saline), using the following procedure: The raw semen was diluted to approximately twice the o'riginal ejaculate volume with saline. The spermatozoa were then centrifuged out of suspension and the supernatant removed. The cells were suspended again in saline to a volume equal to the original ejaculate, recentrifuged, the supernatant removed, and the cells resuspended in saline solution to the desired concentration of cells. Two-tenths milliliter of this cell suspension was added to 0.8 nil. of test solution containing 200 rag/100 ml. of fructose as substrate in the W a r b u r g reaction flasks. Oxygen uptake, fructose utilization, and lactic acid accumulation were measured during a 2-hr. incubation period at 37 ° C. Initial and final p H and motility of the suspended cells were recorded. Fructose was determined by the method of Roe (14), as modified by Mann (11). Lactic acid was determined by the method of Barker and Summerson (1). Three experiments were conducted in this study. The first two were three-factor composite type experiments (5) involving eight replications each. In Experiment One, the variables of pH, osmotic pressure, and potassium concentration were studied, whereas in the second experiment, the effects of pH, calcium concentration, and potassium concentration were studied. In these composite experiments, initial p H values of 4, 5, 6, 7, and 8 were obtained by combinations of citric acid and sodium bicarbonate. Molarities of the final diluents (before adding spermatozoa) expressed as NaC1 were 0.084, 0.119, 0.154, 0.189, and 0.224 M. The theoretical o'smotic pressure equivalents are approximately 195 to 417 milliosmols, assuming complete dissociation of ions. (A solution which has an osmotic pressure of 290 milliosmols or a 0.154 M NaC1 solution is isotonic and has appro"ximately the same freezing point depression as seminal plasma.) T h e actual osmotic pressures were 150 to 392 milliosmols. Potassium was included in the experiments in levels of 40, 120, 200, 280, and 360 rag. % final dilution. Calcium was included in the seco~ld experiment at levels of 2, 12, 22, 32, and 42 mg. % final dilution. Potassium or calcium additions were made at the expense of sodium. MgCI,, • 6 HfO was included in all of the diluents at the rate of 0.5 ml. of a 1.54 21i solution in 50 ml. of diluent. The third aerobic experiment was a 32 factorial involving five replications, with the objective of determining the relatimlship of p H to metabolic activity at various intervals. The p H levels studied were 5, 6.25, and 7.5. The intervals at which samples were taken for fructose and lactic acid measurements were
130(~
R . G . CRAGLE AND G. W. SALISBURY
0, 1, and 2 hr. A 10-miu. period for equilibration of the spermatozoa and diluent preceded 0 hr. I n this experiment, therefore, fructose utilization and lactic acid accumulation were measured to include the 10-min. equilibration perio'd, whereas Oe uptake measurements began at 0 hr. F i f t y per cent of the diluent used in this experiment was made up with a combination of Na2ttP04 • 12 H 2 0 and N a H e P 0 4 • H20, depending on the p i t desired ; whereas, the other half contained 0.154 M NaC1 with 0.5 ml. of 0.11 M MgCle • 6 H 2 0 per 50 ml. and fructose solutions (200 rag. % final dilution). I n the composite experiments, the metabolic responses are expressed as the conventional Z values (13) whereas, in E x p e r i m e n t 3, they are expressed as total mieroliters or micrograms/10 s spermatozoa. RESULTS
The results of the first experiment involving five p H levels, five levels of osmotic pressure, and five potassium concentrations are given in F i g u r e 1. B y 0.224~ i-
I03~t'1
-11.2 -64 4 39
- 12,4 -256
0
,8454
-
1.7
1 0.109
tlJ
%_ -~_~,zo \
i
-8.8 :t -zz, 14B
-33 -2~
I
\ ~" °~t.~o
3
32
-4.4
--t50
i.3
~
i~ O.6o.154
.9
z,
~_\~6o -1~:~2"~ • Ot UPTAKE (Z 0 2) FRUCTOSE UTILIZATION ( Z r ) LACTATE FORMATION (Z L) MOTILITY (% AND RATE)
[0
0.8
IE
\x -50
-92 61 17
3.9
I.I
0,084
-88 49
4~
5~
~
g,
",
0.6
i
-7.8 ÷~ 29
0.119
"\
624.5
7
_ _
8
X I = pH OF DILUENT (INITIAL)
~I(L 1. Effects of p H , molarity of diluent, and p o t a s s i u m concentration on spermatozoan activity ( a v e r a g e of eight r e s p o n s e s ) .
study o'f this figure, one notes that the lower values of fructose utilization and of lactic acid production tend to be on the lower left side of the figure, at a low p H , a low osmotic pressure, and a high concentration of potassimn. Statistical analysis of the combined data show that not all of the differences shown in the figure are significant ones. F o r example, considering the oxygen uptake only, the effect of p H was pronounced. The Z-o was small at p H 4, increasing to a m a x i n m m in 2
the center of the figure and decreasing again at the highest p H .
This trend is
FACTORS
INFLUENCING
METABOLIC
ACTIVITY
OF SPERMATOZOA.
1307
IV.
best fitted by a quadratic expression. The potassium level also influenced the oxygen uptake. The effect was least at lo~v concentrations of potassium and greatest with the higher concentrations, but v a r y i n g in degree with the p H level. On the other hand, the molarity of the diluent did not exhibit a significant effect on oxygen uptake. The metabolic behavior of the washed spermatozoa as influenced by the three controlled variables can be expressed by regression equations, where X I is the effect o'f p H ; Xe, t h a t of v a r y i n g molarity of the solutions ; and X3, t h a t of potassium concentration. Only those regression coefficients (in either E x p e r i m e n t 1 or 2) to which a significant sum of squares could be attributed are given in the following equations : Z% = --10.6 + 1.1 X3 + 1.5 XI"
ZF ZL
= --119.8 -- 60.2 X1 + 19.9 X3 = 88.5 + 43.1 X1 --18.4 X1X3
The five levels for p H (X1) and the five levels for potassium (Xs) have been coded as --2, --1, 0, 1, and 2, respectively. F o r example, at a potassium content of 200 mg. % (X3 = 0) and a p H of 5 (X1 = - 1 ) , one would expect a Z% of - 9 . 1 . (Instead of the usual plus values o~
Zo2, minus
values are used here to indicate a
utilization of O2.) B y use of these equations, estimates of response can be made for a n y set of conditions of the variables and ranges studied. The equations indicate that for fructose utilization, the effects of p H and of potassium concentration are linear and independent. However, for lactic acid accumulation, in addition to a p H effect, a significant interaction between p H and potassium concentration was found.
12
250
J
200
- -
I0
ZF • 8
150 ZL
o, / 50
4
5
6
7
pH
~rG. 2. The effect of pH on metabolic activity.
8
Zo z
1308
R.G.
CRAGLE A N D
G. W. SALISBURY
12
250 _
200
ZF
i
~
o
~
8 ZOz
ZL
100 ZL 50
4 i
o
I
40
120 POTASSIUM
]J'IG,
[
200 CONCENTRATION -
3. The effect of potassium concentration
0 360
280 MG. /
IOOML.
on m e t a b o l i c ~lctivity.
The p r i m a r y effects of pH, of osmotic pressure, and of potassium concentration on the three metabolic responses in this experiment are illustrated in Figures 2, 3, and 4. The combined results of the second experiment involving p H (X1), calcium concentration (X2), and potassium eo~lcentration (X3) are given in F i g u r e 5.
12
25C
% I0
200
ZF
B Zoz
150
ZL
ZF
Ioo 4
5O
0
i 1"73
251 OSMOTIC
290
348
PRESSURE -- M I L L I O S M O L S
:FIG. 4. The effect of osmotic pressure on metabolic activity.
=0 406
1309
FACTORS INFLUENCING xSIETABOLIC ACTIV1TY OF SPERMATOZOA. IV.
42 -134
'~ -7.3 -40 30 22 L4
'; \40
-14.3
t -10.6 I~723 _191 ~170 3! ;L4
I \
"111
32
-II -210 153 20° 1.5
134 :~3 1.7
;:'o,\ o
I
__ I
-io3 \
:_-,,..,
22
.J .( z ;T
t~ ~; Z z 0
~r
-I10 108
\
* 0 z UPTAKE { Z 0 z) FRUCTOSE UTILIZATION (ZF) LACTATE FORMATION (Z L) MOTILITY (%
AND RATE)
4
-6.7 -32 ~J 17 o.g
5 XI =
~
-166 II I
-9.6
-I00 122 2g 1.6
6 pH
OF D I L U E N T
8
~
2g 2,0
Zl
?
m
l.Z
8
(INITIAL)
~IG. 5. JEffccts Of p H , caleimn concentration, a n d p o t a s s i u m c o n c e n t r a t i o n on s p e r m a tozoan a c t i v i t y ( a v e r a g e of eight r e s p o n s e s ) .
In this experiment, alt interesting fact appears. Calcimn not only has no marked effect on the metabolic response of the cells, but the previously observed marked inhibitory effects of increasing the concentration of potassium are virtually eliminated by its presenee. Thus, in the regression equations derived from the second experiment, only the effects of p H (X1) are included, for no other effect contributed a significant portion of the stun of squares.
Zo 2 = --9.6 - 1 . 8 X1 Z~, = --132.7 --64.4 X1 ZL = 101.4 + 46.0 X1 The effect of calcimn concentration as found in this experiment is illustrated in F i g u r e 6. The curves are drawn from the equations calculated for linear and quadratic effects, none of which was significant, so that the effects could equally as well be represented by straight lines across the chart represe]~ting the means for each metabolic measure. The third experiment was designed to cast some f u r t h e r light oll the rate of substrate uptake, as influenced by pt{ during the period of time in which temperature and gas pressure equilibration in the manometers and flask contents took place. The results are given in Table 1. The F values and the significant levels of the responses are given in Table 2. The highly significant interactions of the linear effects of time and the linear effects of p H for bo"ch fructose utilization and lactic acid accumulation are the result of the fact that spermatozoa at p H 5 in a phosphate buffer take up a sizable
R.
1310
G. C R A G L E
AND
G. W .
SALISBURY
12
250
I0
ZF 8 Z%
150 ZL. I00 ZL
4
50
0
12
2
CALCIUM
22
32
CONCENTRATION
-
MG. /
42
0
100 ML.
FIG. 6. The effects of calcium concentration on metabolic activity.
TABLE i Total oxygen uptake, f r u c t o s e utilization, and lactic acid accumulation per 10 8 spermatozoa as influenced by time and p H in p h o s p h a t e buffer ( E x p e r i m e n t 3, m e a n of five replications) Time
pit
D u r i n g equilibration period of 10 min.
5.00 6.25 7.50 5.00 6.25 7.50 5.00 6.25 7.50
E n d of I hr. a f t e r equilibration E n d of 2 hr. a f t e r equilibration
O~
Fructose
Lactate
(M.) ...... ......
tLg. 77 4e7 53 71 67 92 ~9 79 163
t~g. 2 5 6 3 27 24 10 34 40
8.6 9.4 10.3 13.4 12.7 17.1
TABLE 2 Analysis of variance of d a t a f r o m E x p e r i m e n t 3 ( F values)
Time P Time qb pH 1 plt q Time 1 × p H 1
P < .05. ~ P < .01.
linear. bq = quadratic. a 1=
02 u p t a k e
Fructose utilization
Lactic Acid accumulation
179.5 ~* 5.66* NS NS NS
8.86** NS NS NS 10.28 *~
59.38** NS 35.81 ~* 8.03 ~* 11.20 ~*
FACTORS I N F L U E N C I N G M E T A B O L I C ACTI-VITY OF SPERMATOZOA. IV.
1311
quantity of fructose in the initial 10 min. (Time 0) of exposure to substrate. Thereafter, the substrate is metabolized at a slow rate, whereas at a higher pH, they exhibited much higher metabolic responses after a slower initial uptake of substrate. These results show clearly that spermato'zoa removal from the seminal plasma soon after collection, and which have undergone a period of substrate deprivation during washing and the p r e p a r a t o r y stages of the experiment, take up hexose substrate at an initial rate substantially greater than the utilization rate. These results also show that at the lowest pH, after the initial uptake, which occurred within 10 rain. and possibly before the intracellular p H was reduced below that ~f normal semen, no uptake occurs. These facts have great significance in the interpretation of other experiments and in the design of f u t u r e ones. DISCUSSION
A number of difficulties face investigators in this area of research. An important one deals with the relation of metabolism to visual estimates of spermatozoan motility. Our interpretation has been that if spermatozoan metabolic activity was inhibited and final motility was inhibited to a similar degree, no logical conclusion could be reached. Either inhibition a n d / o r death of the cells may have taken place. Only in cases where final motility is the same as the control or only slightly depressed, and the metabolic measures are substantially decreased, can we conclude that metabolic inhibition has actually taken place. A second, equally fundamental difficulty has been the large coefficients of variation in substrate utilization associated with experiments of this nature preventing precise measurement of treatment effects. In the first and second of these experiments, the eo'effieients of variation for fructose utilization were 40% or higher bef()re ejaculate effects were removed. F r o m the third experiment, it would appear that the preconditioning of spermatozoa as affected by the time from ejaculation to removal of the cells from the seminal plasma, the initial p H of the semen and, possibly, many more such factors, are concerned in such variability. A third is a question primarily of semantics. In the first two experiment:~, the predominating cations in the diluents were sodium and potassium. When potassium was increased the sodium was decreased, and vice versa. This leaves the question of which ion should be considered as having the p r i m a r y effect and the naming of that effect. We have chosen to eo~nsider the p r i m a r y effect as due to potassium, because of its higher concentration in the cells (7) and the fact that the response of the cells to extracellular potassium was the one sought, namely, reversible inhibition of metabolic activity. The reversal o'f motility occurred upon removal of the ceils from the W a r b u r g flasks, dilution with 0.9% NaCI, and placing them under a cover slip on a microscope slide. Low concentrations of potassium have been shown to be necessary for optimum metabolism of spermatozoa (22); whereas, high concentrations have been reported as detrimental (23). It now appears that in the absence of calcium (Experiment 1), reversible inhibition of metabolic activity at 37 ° C. takes place
1312
R. G. C R A G L E AND G. W. S A L I S B U R Y
at potassium concentrations of front 200 to 280 rag. %. The inhibition is particularly evident at pH 5, but the K-level × pH interaction was statistically significant only for the lactic acid accumulation. These potassium levels are higher than are reported for seminal plasma (a mean of 161 mg. %) (8), but not so high as are the amounts found in the epididymis, where the cells are normally quiescent (15). However, in the presence of calcium, the high potassium levels do not Cause any measurable effect on sperm cell metabolism (Experiment 2). It does have a deleterious effect on motility. The interference of calcium with the inhibitory effects of potassium may have its counterpart in nature. The epididymal secretions are high in potassium and quite low in calcium ; whereas, on ejaculatou, the high potassium is diluted with sodium and the calcium level is increased by addition of other products of the accessory glands, thus paving the way for the burst of activity associated with ejaculated semen. The inhibition of metabolic activity by the potassium ion strongly suggests that this is at least one of the agents, if not the only one, responsible for depressing the oxygen uptake in whole semen reported by Bishop and Salisbury (2). These workers presented evidence that the inhibitor was associated with the spermatozoa. Subsequent work has shown that potassium is more concentrated within the spermatozoa than it is in the seminal plasma (8). Finally, the sodium/potassium ratio deserves some consideration in view of the emphasis placed on it by other workers with bull spermatozoa (15, 21) and in the inhibition of trout sperm (20). It appears from the results reported here that the sodium/potassium ratio is a useful expression only under specified conditions of pH, low levels of calcium, and where sodium and potassium make up nearly all of the cations present. REFERENCES
(1) BAIg.I.:ER,S. B., AND SUMMERSON~, W. H. The Colerimetric Determination of Lactic Acid in Biological Material. J. Biol. Chem., 138: 535. 1941. (2) BISHOP, M. ~'. H., AND SALISBURY, G. W. Effect of Sperm Concentration oll the Oxygen Uptake of Bull Semen. Am. J. Physiol., 180: 107. 1955. (3) BISHOP, M. W. H., AND SALmSURY, G. W. Effect of Dilution with Saline and Phosphate Solutions on Oxygen Uptake of Bull Semen. Am. J. Physiol., 181 : 114. 1955. (4) BL~CKSEAW, A. W., SALISBURY, G. W., AND VANDEMARK, N. L. Factors Influencing Metabolic Activity of BulI Spermatozoa. I. 37 °, 21 °, and 5 ° C. J. Dairy Sci., 40: 1093. 1957. (5) Box, G. E. P., A~D WILSOn, K. B. On the Experimental Attaimnent of Optimum Conditions. J. Roy Stat. Sou., B. 13: 1. 1951. (6) C~AGbE, R. G., AND SALrSSU~Y, G. W. Effects of pH, Osmotic Pressure, and Bulk Cations on the Metabolic Activity of Bull Sperm. J. Dairy Sci., 40: 621. 1957. (7) CI~AGLE~R. G., S.txLISBURY, G. W., AbeD MUNTZ, T. J-[. Distribution of Bulk and Trace Minerals in Bull Reproductive Tract Fluids and Semen. J. Dairy Sci., 41 : 1273. 1958. (8) CRAGL~, R. G., SALISBUI~Y, G. W., AND VANDEMARK, N. L. Sodium, Potassium, Calcium, and Chloride Distribution in Bovine Semen. J. Dairy Sci., 41: 1267. 1958. (9) LAI~DY, H. A., GHOSH, D., AND PLAUT, G. W. E. A Metabolic Regulator in Mammalian Spermatozoa. Science, 109: 365. 1949. (10) LARDY, I-I. A., AND PHILLIPS, P. H. Effect of p i t and Certain Electrolytes on the Metabolism of Ejaculated Spermatozoa. A ~ . J. Physiol., 138: 741. 1943.
FAOTOI%S INFLUENCING ]VIETABOLI0 ACTIVITY OF SPEI~IV[ATOZOA. IV.
1313
(11) MicrON, T. Fructose Content and Fructolysis in Semen. Practical Application in the Evaluation of Semen Quality. J. Agr. Sci., 38: 323. 1948. (12) PHmLres, P. H. The Preservation of Bull Semen. J. Biol. Chen~., 130: 415. 1939. (13) E~.VENZ, E. Ober den Spaltungsstoffweehsel der Saiigetier Spermatozoen in Zusammenhang mit der Bewegliehkiet. Biochem. Z., 257: 234. 1933. (14) Ro~, J. H. A Colorimetrie Method for the Determination of Fructose in Blood and Urine. J. Biol. Chem., 107: 15. 1934. (15) SALISBURY, G. W., AND C~AaLE, R. G. Freezing P o i n t Depressions and Mineral Levels of Fluids of the Ruminant Male Reproductive Tract. IIIrd Intern. Congr. Animal l~eproduction, Sec. I. 25. 1956. (16) SALISBURY, G. W., A~D K I ~ E Y , W. C., Ja. Factors Influencing Metabolic Activity of Bull Spermatozoa. I I i . pH. J. Dairy Sci., 40: 1343. 1957. (17) SALISBVRY, G. W., A~D NXKABAYASHI; N. T. Effect of Phosphate and Chloride Ions on Aerobic Metabolism of Bovine Spermatozoa. J. l~xptl. Biol., 34: 52. 1957. (18) SALISBURY, G. W., AND V~ND~AR~¢, N. L. Carbon Dioxide as a Reversible Inhibitor of Spermatozoal~ Metabolism. Nature, (London), 180: 989. 1957. (19) S~LmBURY, G. W., AND VA.NDEiVIAI~K, N. L. Sulfa Compounds in Reversible Inhibition of Sperm Metabolism by Carbon Dioxide. Science, 125: 1118. 1957. (20) SCKLF2~K, W., JR., AND KAH~I¢~¢, H. Die Chemisehe Zusammensetzung des Spermaliquors und ihre Physiologische Bedeutung, Untersuchung a n Forellensperma. Bioehem. Z., 295: 283. 1938. (21) S o r m ~ s ~ , EI), A~D A~I)ERS0N, S. The Influence of Sodium and Potassium Ions upon the Motility of Sperm Cells. IIIrd Intern. Co~tgr. Animal l~eproduction, Sect. I : 45. 1956. (22) WHITe, I. G. The Effect of Potassium on the Washing and Dilution of Mammalian Spermatozoa. Australian J. Exptl. Biol. Med. Sci., 31: 193. 1953. (23) WHITe, I. G. Studies on the Alkali Metal Requirements of Ram and Bull Spermatozoa. Australian J. Biol. Sci., 6: 716. 1953.