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The effect of carbon dioxide level and temperature on the in vitro metabolism of testis tissue
Comp. Biochem. Physiol., 1967, VoL 23, pp. 475 to 482. Pergamon Press. Printed in Great Britain
T H E E F F E C T OF CARBON D I O X I D E LEVEL T AND E M P E R A T U R E ON T H E I N V I T R O M E T A B O L I S M OF T E S T I S T I S S U E J. L. FLEEGER,* N. L. VANDEMARK and A. D. JOHNSON Animal Reproduction Teaching and Research Center, t The Ohio State University (Received 18 M a y 1967)
Abstract--1. Normal testis tissue from sixty mature rabbits was studied to determine the effect of 0, 2, 5 and 10% COs levels, temperature (33 and 37°C) and length of incubation time, at hourly intervals from 1 to 4, on glucose and Os uptake and lactate and COs production. 2. There was generally a significant effect of the time of incubation due to a high activity during the first hour and no evident effect thereafter. This was possibly due to either tissue damage or substrate depletion during preparation. 3. Increased temperature caused increased metabolic activity as shown by other workers in vivo. 4. At 0 and 10% CO2 there was marked decrease in metabolic activity, indicating a need for some COs by the testis tissue and an inhibitory effect by COs at higher levels. At these two levels there was a shift in metabolism from respiration to glycolysis. 5. The maximum metabolic activity occurred at 2 and 5% COs which is near the levels previously reported in vivo. INTRODUCTION SPERMATOGENICarrest has long been known to occur with elevated testis temperature (Glover & Young, 1963). Recent studies have shown that with the increasing temperature there is an increase in gas tension. Increased oxygen (O~) tension has been shown with local warming (Cross & Silver, 1962; Free & VanDemark, 1966) or with short-term cryptorchidism (Applebaum & VanDemark, 1964, unpublished). With the increased O3 tension there is also increased carbon dioxide (COs) tension as shown by Free & VanDemark (1966). Carbon dioxide has been shown to be essential to metabolism in sperm (Lodge et al., 1963) and in embryonic development (Spratt, 1940). On the other hand, high levels of CO2 have been shown to inhibit metabolism and respiration of sperm (Salisbury et al., 1960) and other tissues (Danes & Kieler, 1958; Loomis, 1959a; Kieler & Bicz, 1962). This principle has been used to inhibit sperm and yet preserve motility and fertility (VanDemark & Sharma, 1957).
* Present address: Department of Animal Science, Texas A & M University, College Station, Texas. t Department of Dairy Science, 735 Stadium Drive, Columbus, Ohio 43-210. x6
475
476
J.L. Ftamom~,N. L. VANDSMASKANt)A. D. JOHNSON
Stagnation of blood flow and the build up of COs in the testis have been suggested as results produced in artificial cryptorchidism (Moore, 1924). However, the association of these two phenomena and the effects of varying CO s levels on testis tissue metabolism have not been reported. The study reported here was undertaken to determine if COs is necessary for normal respiration and metabolism in rabbit testis tissue and, if so, to determine optimum in vitro levels of CO s. MATERIALS AND METHODS Untreated testes from sixty rabbits of mixed breeding were the source of tissue for the study. After the animals were anesthetized with sodium pentabarbital, the testes were removed through a scrotal incision, trimmed of extraneous tissue, placed in ice-cold saline and sliced with a Stadie-Riggs hand microtome. The slices (40-70 mg) were blotted, weighed and placed in a modified Dixon-Keilin flask. Samples for dry weight determinations were handled in the same way and dried in an oven at 100-105°C for 12 hr. A modified Locke's solution with 30 mM Tris buffer and sodium bicarbonate adjusted so that a pH of 7"2 was obtained was used in all flasks. The sodium chloride content of the buffer was varied to maintain a constant ionic strength depending on the amount of sodium bicarbonate used (Umbreit et al., 1959). Each flask also contained 0.004 M glucose and 500 i.u. of penicillin G. The gas phase was established in the flask by alternating vacuum with an aspirator and pressure from the gas cylinder. After this exchange had been made ten times, the flask was evacuated using 15 lb of vacuum; then the gas from the tank was allowed to flow through the flask while it was being attached to the Warburg unit. Three of the gas mixtures used contained, 2, 5 and 10% carbon dioxide, with a constant 20% oxygen, and the remainder as nitrogen. The fourth gas was air as it prevailed in the flask without using any gassing procedure. Under the latter condition the potasium hydroxide was turned in at zero time to render the flask COs free. This is referred to here as the 0% COs treatment. Tissues from the testes of a single rabbit were pooled and duplicate flasks for buffer control samples, 4-hr incubation at 0% CO~ and for each hourly interval from 1--4 for one other gas mixture were set up. Thus, the tissues from each rabbit were incubated at either 33 or 37°C (Harrison & Weiner, 1949) in 0% as well as one other gas mixture. At a single temperature and gas level tissues from ten rabbits were represented except at 0% COs where there were thirty at each temperature. Oxygen uptake and COs production were determined by the procedure outlined by Umbreit et al. (1959) and lactic acid production by the method of Barker & Summerson (1941). Glucose levels were determined enzymatically with a glucose oxidase reagent (Worthington Biochem Corp., 1963). Statistical analysis consisted of normal analysis of variance procedures for the entire experiment, then within temperature where temperature by COs level interactions was significant. Duncan's new multiple range test was used to determine differences between CO2 levels (Steel & Torrie, 1960).
CARBON DIOXID]~ AND TBMPERATUR]I ls~r-u~JL~ O N TESTIS METABOLISM
477
RESULTS Respiration
Oxygen uptake (#l/mg of dry tissue/hr) by the tissues is shown in Fig. 1. There was no significant effect of the CO a level in the flask on O 2 uptake. There was a slight trend for a reduced O2 uptake at the 10% level; however, this was not significant. Temperature had a highly significant effect (P< 0.005) with OI uptake being increased by over 50 per cent at the higher level. The effect of incubation time significantly (P<0"025) affected O2 uptake with the greatest uptake being shown at hour 1 and generally decreasing for subsequent 1 hr intervals. Thus the greatest tissue activity was during the first hour; however, there was no generally pronounced decrease after the first 2 hr of incubation. This difference was most noticeable at 37°C. 4 []
lhr
mZhr ~
CO2 ternp
04hr
o
~
5 * 33°
IO
o
10
2 37°
FIo. 1. Oxygen utilization by normal rabbit testicular tissue as affected by COj, temperature level and length of incubation in vitro. Carbon dioxide production (/~l/mg of dry tissue/hr; Fig. 2) was unaffected by level of COz whereas temperature had the expected highly significant (P< 0"005) increasing effect on respiration. The length of incubation likewise affected CO~ production in a highly significant (P< 0.005) manner. Again this was due to the high activity during the first hour but no predominant decrease occurred after hour 2. Further evaluation showed that this effect was significant (P< 0"005) only at the 37°C temperature. The respiratory quotients (R.Q.) for each CO2 level at each temperature, ignoring time of incubation effect, are shown in Table 1. These values indicated a slight upward trend with increasing CO2 level but differences were not significant. Glucose utilization
Glucose utilization (pl/mg of dry tissue/hr) was significantly (P < 0.01) affected by COs level (Fig. 3). At 33°C glucose utilization was significantly (P<0.01)
478
J. L. Fi~zoes, N. L. V~DE~o.m~ ANDA. D. JOHNSON
higher at the 5% level than at the other CO2 levels. However, at 10% CO2, glucose uptake was significantly higher than at 0%. At 37°C with both the 2 and 5% CO2 levels there was a significant ( P < 0"01) increase of glucose utilization over the 0 and 10% levels.
[ ] Jhr
D4h,
g C02
2
8
letup
Io
a
~o
Jo
37°
FIG. 2. Effect of COt, temperature level and incubation time on COs production by normal rabbit testicular tissue in vitro. []
Ihr
25
ml2hr
~2o
~3hr D4hr
.~ 15
''01 ¥
H
/
CO2 temp
o
Z
5 33°
io
0
5
uo
37°
F z o . 3. G l u c o s e utili=-atlon i n n o r m a l r a b b i t t e s t i c u l a r tissue in v i t r o as a l t e r e d b y
changesin COt, temperature and incubation time. Temperature increase caused increased ( P < 0.05) utilization of glucose. The length of incubation again had significant ( P < 0.05) effects due to increased utilization during the first hour at the higher temperature except at the 10% CO2 level.
479
CARBON DIOXIDE AND TEMPERATURE ]~FECTS O N TESTIS METABOLISM
In this case there was very little utilization during the first hour; however, recovery was evident after this initial period. Changes in the lactic acid accumulated (pg/mg dry tissue/hr) are shown in Fig. 4. There were no overall effects of CO= level or incubation time on lactate accumulation. Temperature caused an increased ( P < 0.01) lactate accumulation. Trends existed which suggested a temperature by COo level interaction but this was not significant. T A B L E 1 - - R E S P I R A T O R Y AND METABOLIC RATIOS AS AFFECTED BY TREATMENTS
CO= level (%)
Temperature (°C)
Ratio CO= --= O=
R.Q.
0
2
5
10
[33
--
0.77 ± 0.08
0.79 ± 0.52
0.85 ± 0-42
t
--
0.75 ±0.07
0.83 ±0.12
0.94±0.91
37
Gluco~
f33
1.45±0.32t 1.46±0.25t
2.27_+0.45 1.92±0.28"
Lactate
"~37
1"14 ± 0"27t ~ 2.43 ±0.45
2.31±0-33
0.93±0"16t~
* P<0"05, significandy different than the 5 per cent CO= level. t P<0"01, significantly different than the 5 per cent CO= level. P<0"01, significantly different than the 2 per cent COs level. I0.0
z
[]lh, [ ] 2 hr
9.0
N3h, e.~
I""l 4hr
g o
~ 6,Q ._u
3
5.0
CO2
temp
~ 0
Z
5
~o
I0
2
3? °
10
FIG. 4. Changes in lactic acid accumulation b y rabbit testicular tissue as affected b y COrn, temperature and incubation time in vitro.
The ratios between glucose uptake and lactic acid production are presented in Table 1. At 33°C the ratio at 5% was significantly greater than at 0 and 2% ( P < 0.01) or 10% ( P < 0"05). At 37°C both the 2 and 5% COl levels resulted in significantly ( P < 0-01) higher ratios than at either the 0 or 10% levels.
480
J.L. FLOOred,N. L. V~Dm~RK ANDA. D. JomcsoN
DISCUSSION As shown in this study CO2 level appears to have major effects on testis tissue metabolism reflected in both glycolytic and respiratory changes. The necessity of some COs in the environment for normal metabolism of different types of cells has been pointed out. Spratt (1940) showed a degeneration or inhibition of differentiation and morphogenesis in the developing chick embryo with CO2 deficiency. His results showed that the greater the deficiency the greater was the degeneration. Lodge et al. (1963) showed that extensive dilution inhibited metabolism and motility of sperm cells, but this dilution effect was overcome by incubating under 5% CO2, 95% Ns as compared with 100% N2. Loomis (1959b) found that this same grouping or CO2 accumulation effect is evident in many marine species. For a normal development rate to take place, the developing embryos must be in groups. Danes & Kieler (1958) and Kieler & Bicz (1962) studied the effects of CO s on different tissue and suggested COs levels above 0.5% should be maintained for maximum activity. High levels as well as low levels of CO2 appear to affect metabolism. When rats were subjected to an environment of 6% COs, 20% 02, 74% N s (Haring, 1960) deformations in the offspring occurred suggesting a critical COs effect in mammalian development. Again sperm metabolism, respiration and motility have been shown to be increasingly inhibited by rising levels of COs (Salisbury et al., 1960). Others (Danes & Kieler, 1958; Loomis, 1959a; Kielcr& Bicz, 1962), working with manY kinds of cells and tissues, support this general concept of inhibition by higher levels of COs. They suggest that COs is a physiological regulator of energy metabolism and cell multiplication. Since increasing levels of COs inhibit metabolism and respiration, error could result in interpretation of in vitro results where one system or tissue evolved a large level of COs and the other did not. This would result in inhibition of the more active system andresult in perhaps no ultimate difference between treatments or an actual metabolic decrease in the more active system. A temperature increase from 33-37°C increased the entire scope of activity regardless of any other factor involved. In the perfused rabbit testis decreased glucose uptake was found with increased temperature (Ewing & VanDemark, 1963). Decreased blood flow was also found, however, which would have decreased the glucose available. Waites & SetcheU (1964) showed that temperature increase in the testis in vivo caused increases in Os uptake and glucose utilization, but not lactate production. These workers further suggested that this increased metabolism without an increased supply of Os may be the cause of testicular degeneration. However, Free & VanDemark (1966) have shown an increased Os tension with in vivo temperature increase and COs tension increases as well. The effect of incubation time indicates that during the first hour the tissue is the most active in both glycolysis and respiration. After this initial period there was a general drop in activity with no time of incubation effect thereafter. Most of the glucose taken up is not accounted for in this first hour by the lactate and COs produced or by the Os uptake suggesting that it is probably held passively. During
CARBON DIOXIDE AND TEMP]~I~ATURE EFFECTS O N TESTIS METABOLISM
481
the first hour the R.Q. was higher even though respiration had increased, which could mean that biosynthetic processes requiring COs were not active. This may all be caused by tissue damage or depletion of vital substrates during the preparation processes. In bovine sperm subjected to high levels of COs the initial inhibition of motility is gradually overcome (VanDemark et aL, 1965). Whether a similar initial inhibition and later recovery occurred with the testis tissues in this study can only be determined by further investigation. The data suggest that the length of the in vitro studies may be critical. It appears that during the initial period of incubation, though the activity was the greatest, true glycolytie and respiratory activity of the tissue may not be evident. It appears that this initial period should serve as part of the preparation and equilibration. On the other hand, though not evident in this study, long-term incubations may result in an accumulation of COs which would inhibit glycolysis and respiration as seen in this study with increased COs levels. There are two conclusions from this study concerning the effect of COs on testis tissue. First, some COs appears necessary for maximum glycolysis and respiration of testis tissue at the level of Os used in the study. Secondly, increased levels of COs inhibit glycolysis and respiration. In both cases there is qualitative change in metabolism. This suggests COs levels around 2 and 5 per cent for maximum activity i n / n v/tro studies. This range of values results in COs tensions comparable to those estimated for normal ram testes in vivo (37 mm Hg) (Free & VanDemark, 1966). Acknow/edgements--The authors wish to thank Mrs. C. L. Jenkins for her technical assistance. This work was supported by USPHS Grant GM-12247. REFERENCES BARK~ S. B. & SUMMm~ONW. H. (1941) The colorimetric determination of lactic acid in biological materials, y. biol. Chem. 138, 535-554. Caoss B. A. & SXLVSRI. A. (1962) Neurovascular control of oxygen tension in the testis and epididymis. ~. Reprod. Fert. 3, 377-395. DANESB. S. & Kmtam J. (1958) The influence of COs tension on cellular respiration studied by the cartesian diver technique. C. r. Lab. Tray. Lab. Carlsburg 31, 61-75. EWINO L. L. & VArcDEMARKN. L. (1963) Factors affecting testicular metabolism and perfused testes in the rabbit, y. Repro& Fert. 6, 17-24. F a ~ M. J. & VANDEMAaKN. L. (1966) The effect of serotal warming on spermatic venous gas tensions. Fedn. tim. Socs. exp. Biol. 25, 313. GLov~a T. D. & YOUNO B. H. (1963) Temperature and the production of spermatoza. Fert. Steril. 14, 441-450. HAmNGO. M. (1960) Cardiac malformations in rats induced by exposure of the mother to carbon dioxide during pregnancy. Circulat. Res. 8, 1218-1227. HARRISONR. G. & Wmrnta J. S. (1949) Vascular patterns of the mammalian testis and their functional significance. ~. exp. Biol. 26, 304-316. KmLESJ. & BxczW. (1962) The function of COj in cell metabolism and growth. Henry Ford Hosp. International Symposium: Biological Interactions in Normal and Neoplastic Growth. Little, Brown and Co.
482
J. L. FLI~GER, N. L. VANDmVL~KAND A. D. JOHNSON
LODGE J. R., G ~ v E s C. N. & SALISBURYG. W. (1963) Carbon dioxide in prevention of the deleterious dilution effect on metabolism and motility of mammalian spermatazoa. Proc. Soc. exp. Biol. Med. 113, 824-827. LOOMIS W. F. (1959a) pCOs inhibition of normal and malignant growth, je. natn. Cancer Inst. 22, 207-217. LOOMIS W. F. (1959b) Feedback control of growth and differentiation by carbon dioxide tension and related metabolic variables. Cell, Organism and Milieu, pp. 253-294. Ronald Press Co., New York. MOORS C. R. (1924) Properties of the gonads as controllers of somatic and psychical characteristics--VIII. Heat application and testicular degeneration. The function of the s c r o t u m . . 4 m . j~. Anat. 34, 337. SALmeURY G. W., VANDmv~RKN. L., LODGEJ. R. & CRAGLe R. G. (1960) Inhibition of spermatozoan metabolism by pCOl, pH, K ion and antibacterial compounds. Am. ~. Physiol. 198, 659--664. SPSAz'r M. T., JR. (1949) Carbon dioxide requirements of the early chick embryo. Anat.
Re¢. 105, 583-584. S'rm~ R. G. D. & Tomtxe J. H. (1960) Principles and Procedures of Statistics. McGraw-Hill, New York. UMBREIT W. W., BURRISR. H. & STAUFFERJ. F. (1959) Manometric Techniques. Burgess Publishing Co., Minneapolis, Minn. V A N D m ~ z N. L., KOYAMAK. & LODOSJ. R. (1965) Factors affecting immobilization of bovine spermatozoa with COl and their subsequent reactivation, j~. Dairy Sci. 48, 586591. VANDEMARKN. L. & SHARMAU. D. (1957) Preliminary fertility results from the preservation of bovine semen at room temperatures. ~. Dairy Sci. 40, 438--439. WAITESG. M. H. & SETCHELLB. P. (1964) Effect of local heating on blood flow and metabolism in the testis of the conscious ram. j~. Relwod. Fert. 8, 339-349. Worthington Biochemical Corp. Bulletin (1963) Glucostat. Freehold, New Jersey.
T H E E F F E C T OF CARBON D I O X I D E LEVEL T AND E M P E R A T U R E ON T H E I N V I T R O M E T A B O L I S M OF T E S T I S T I S S U E J. L. FLEEGER,* N. L. VANDEMARK and A. D. JOHNSON Animal Reproduction Teaching and Research Center, t The Ohio State University (Received 18 M a y 1967)
Abstract--1. Normal testis tissue from sixty mature rabbits was studied to determine the effect of 0, 2, 5 and 10% COs levels, temperature (33 and 37°C) and length of incubation time, at hourly intervals from 1 to 4, on glucose and Os uptake and lactate and COs production. 2. There was generally a significant effect of the time of incubation due to a high activity during the first hour and no evident effect thereafter. This was possibly due to either tissue damage or substrate depletion during preparation. 3. Increased temperature caused increased metabolic activity as shown by other workers in vivo. 4. At 0 and 10% CO2 there was marked decrease in metabolic activity, indicating a need for some COs by the testis tissue and an inhibitory effect by COs at higher levels. At these two levels there was a shift in metabolism from respiration to glycolysis. 5. The maximum metabolic activity occurred at 2 and 5% COs which is near the levels previously reported in vivo. INTRODUCTION SPERMATOGENICarrest has long been known to occur with elevated testis temperature (Glover & Young, 1963). Recent studies have shown that with the increasing temperature there is an increase in gas tension. Increased oxygen (O~) tension has been shown with local warming (Cross & Silver, 1962; Free & VanDemark, 1966) or with short-term cryptorchidism (Applebaum & VanDemark, 1964, unpublished). With the increased O3 tension there is also increased carbon dioxide (COs) tension as shown by Free & VanDemark (1966). Carbon dioxide has been shown to be essential to metabolism in sperm (Lodge et al., 1963) and in embryonic development (Spratt, 1940). On the other hand, high levels of CO2 have been shown to inhibit metabolism and respiration of sperm (Salisbury et al., 1960) and other tissues (Danes & Kieler, 1958; Loomis, 1959a; Kieler & Bicz, 1962). This principle has been used to inhibit sperm and yet preserve motility and fertility (VanDemark & Sharma, 1957).
* Present address: Department of Animal Science, Texas A & M University, College Station, Texas. t Department of Dairy Science, 735 Stadium Drive, Columbus, Ohio 43-210. x6
475
476
J.L. Ftamom~,N. L. VANDSMASKANt)A. D. JOHNSON
Stagnation of blood flow and the build up of COs in the testis have been suggested as results produced in artificial cryptorchidism (Moore, 1924). However, the association of these two phenomena and the effects of varying CO s levels on testis tissue metabolism have not been reported. The study reported here was undertaken to determine if COs is necessary for normal respiration and metabolism in rabbit testis tissue and, if so, to determine optimum in vitro levels of CO s. MATERIALS AND METHODS Untreated testes from sixty rabbits of mixed breeding were the source of tissue for the study. After the animals were anesthetized with sodium pentabarbital, the testes were removed through a scrotal incision, trimmed of extraneous tissue, placed in ice-cold saline and sliced with a Stadie-Riggs hand microtome. The slices (40-70 mg) were blotted, weighed and placed in a modified Dixon-Keilin flask. Samples for dry weight determinations were handled in the same way and dried in an oven at 100-105°C for 12 hr. A modified Locke's solution with 30 mM Tris buffer and sodium bicarbonate adjusted so that a pH of 7"2 was obtained was used in all flasks. The sodium chloride content of the buffer was varied to maintain a constant ionic strength depending on the amount of sodium bicarbonate used (Umbreit et al., 1959). Each flask also contained 0.004 M glucose and 500 i.u. of penicillin G. The gas phase was established in the flask by alternating vacuum with an aspirator and pressure from the gas cylinder. After this exchange had been made ten times, the flask was evacuated using 15 lb of vacuum; then the gas from the tank was allowed to flow through the flask while it was being attached to the Warburg unit. Three of the gas mixtures used contained, 2, 5 and 10% carbon dioxide, with a constant 20% oxygen, and the remainder as nitrogen. The fourth gas was air as it prevailed in the flask without using any gassing procedure. Under the latter condition the potasium hydroxide was turned in at zero time to render the flask COs free. This is referred to here as the 0% COs treatment. Tissues from the testes of a single rabbit were pooled and duplicate flasks for buffer control samples, 4-hr incubation at 0% CO~ and for each hourly interval from 1--4 for one other gas mixture were set up. Thus, the tissues from each rabbit were incubated at either 33 or 37°C (Harrison & Weiner, 1949) in 0% as well as one other gas mixture. At a single temperature and gas level tissues from ten rabbits were represented except at 0% COs where there were thirty at each temperature. Oxygen uptake and COs production were determined by the procedure outlined by Umbreit et al. (1959) and lactic acid production by the method of Barker & Summerson (1941). Glucose levels were determined enzymatically with a glucose oxidase reagent (Worthington Biochem Corp., 1963). Statistical analysis consisted of normal analysis of variance procedures for the entire experiment, then within temperature where temperature by COs level interactions was significant. Duncan's new multiple range test was used to determine differences between CO2 levels (Steel & Torrie, 1960).
CARBON DIOXID]~ AND TBMPERATUR]I ls~r-u~JL~ O N TESTIS METABOLISM
477
RESULTS Respiration
Oxygen uptake (#l/mg of dry tissue/hr) by the tissues is shown in Fig. 1. There was no significant effect of the CO a level in the flask on O 2 uptake. There was a slight trend for a reduced O2 uptake at the 10% level; however, this was not significant. Temperature had a highly significant effect (P< 0.005) with OI uptake being increased by over 50 per cent at the higher level. The effect of incubation time significantly (P<0"025) affected O2 uptake with the greatest uptake being shown at hour 1 and generally decreasing for subsequent 1 hr intervals. Thus the greatest tissue activity was during the first hour; however, there was no generally pronounced decrease after the first 2 hr of incubation. This difference was most noticeable at 37°C. 4 []
lhr
mZhr ~
CO2 ternp
04hr
o
~
5 * 33°
IO
o
10
2 37°
FIo. 1. Oxygen utilization by normal rabbit testicular tissue as affected by COj, temperature level and length of incubation in vitro. Carbon dioxide production (/~l/mg of dry tissue/hr; Fig. 2) was unaffected by level of COz whereas temperature had the expected highly significant (P< 0"005) increasing effect on respiration. The length of incubation likewise affected CO~ production in a highly significant (P< 0.005) manner. Again this was due to the high activity during the first hour but no predominant decrease occurred after hour 2. Further evaluation showed that this effect was significant (P< 0"005) only at the 37°C temperature. The respiratory quotients (R.Q.) for each CO2 level at each temperature, ignoring time of incubation effect, are shown in Table 1. These values indicated a slight upward trend with increasing CO2 level but differences were not significant. Glucose utilization
Glucose utilization (pl/mg of dry tissue/hr) was significantly (P < 0.01) affected by COs level (Fig. 3). At 33°C glucose utilization was significantly (P<0.01)
478
J. L. Fi~zoes, N. L. V~DE~o.m~ ANDA. D. JOHNSON
higher at the 5% level than at the other CO2 levels. However, at 10% CO2, glucose uptake was significantly higher than at 0%. At 37°C with both the 2 and 5% CO2 levels there was a significant ( P < 0"01) increase of glucose utilization over the 0 and 10% levels.
[ ] Jhr
D4h,
g C02
2
8
letup
Io
a
~o
Jo
37°
FIG. 2. Effect of COt, temperature level and incubation time on COs production by normal rabbit testicular tissue in vitro. []
Ihr
25
ml2hr
~2o
~3hr D4hr
.~ 15
''01 ¥
H
/
CO2 temp
o
Z
5 33°
io
0
5
uo
37°
F z o . 3. G l u c o s e utili=-atlon i n n o r m a l r a b b i t t e s t i c u l a r tissue in v i t r o as a l t e r e d b y
changesin COt, temperature and incubation time. Temperature increase caused increased ( P < 0.05) utilization of glucose. The length of incubation again had significant ( P < 0.05) effects due to increased utilization during the first hour at the higher temperature except at the 10% CO2 level.
479
CARBON DIOXIDE AND TEMPERATURE ]~FECTS O N TESTIS METABOLISM
In this case there was very little utilization during the first hour; however, recovery was evident after this initial period. Changes in the lactic acid accumulated (pg/mg dry tissue/hr) are shown in Fig. 4. There were no overall effects of CO= level or incubation time on lactate accumulation. Temperature caused an increased ( P < 0.01) lactate accumulation. Trends existed which suggested a temperature by COo level interaction but this was not significant. T A B L E 1 - - R E S P I R A T O R Y AND METABOLIC RATIOS AS AFFECTED BY TREATMENTS
CO= level (%)
Temperature (°C)
Ratio CO= --= O=
R.Q.
0
2
5
10
[33
--
0.77 ± 0.08
0.79 ± 0.52
0.85 ± 0-42
t
--
0.75 ±0.07
0.83 ±0.12
0.94±0.91
37
Gluco~
f33
1.45±0.32t 1.46±0.25t
2.27_+0.45 1.92±0.28"
Lactate
"~37
1"14 ± 0"27t ~ 2.43 ±0.45
2.31±0-33
0.93±0"16t~
* P<0"05, significandy different than the 5 per cent CO= level. t P<0"01, significantly different than the 5 per cent CO= level. P<0"01, significantly different than the 2 per cent COs level. I0.0
z
[]lh, [ ] 2 hr
9.0
N3h, e.~
I""l 4hr
g o
~ 6,Q ._u
3
5.0
CO2
temp
~ 0
Z
5
~o
I0
2
3? °
10
FIG. 4. Changes in lactic acid accumulation b y rabbit testicular tissue as affected b y COrn, temperature and incubation time in vitro.
The ratios between glucose uptake and lactic acid production are presented in Table 1. At 33°C the ratio at 5% was significantly greater than at 0 and 2% ( P < 0.01) or 10% ( P < 0"05). At 37°C both the 2 and 5% COl levels resulted in significantly ( P < 0-01) higher ratios than at either the 0 or 10% levels.
480
J.L. FLOOred,N. L. V~Dm~RK ANDA. D. JomcsoN
DISCUSSION As shown in this study CO2 level appears to have major effects on testis tissue metabolism reflected in both glycolytic and respiratory changes. The necessity of some COs in the environment for normal metabolism of different types of cells has been pointed out. Spratt (1940) showed a degeneration or inhibition of differentiation and morphogenesis in the developing chick embryo with CO2 deficiency. His results showed that the greater the deficiency the greater was the degeneration. Lodge et al. (1963) showed that extensive dilution inhibited metabolism and motility of sperm cells, but this dilution effect was overcome by incubating under 5% CO2, 95% Ns as compared with 100% N2. Loomis (1959b) found that this same grouping or CO2 accumulation effect is evident in many marine species. For a normal development rate to take place, the developing embryos must be in groups. Danes & Kieler (1958) and Kieler & Bicz (1962) studied the effects of CO s on different tissue and suggested COs levels above 0.5% should be maintained for maximum activity. High levels as well as low levels of CO2 appear to affect metabolism. When rats were subjected to an environment of 6% COs, 20% 02, 74% N s (Haring, 1960) deformations in the offspring occurred suggesting a critical COs effect in mammalian development. Again sperm metabolism, respiration and motility have been shown to be increasingly inhibited by rising levels of COs (Salisbury et al., 1960). Others (Danes & Kieler, 1958; Loomis, 1959a; Kielcr& Bicz, 1962), working with manY kinds of cells and tissues, support this general concept of inhibition by higher levels of COs. They suggest that COs is a physiological regulator of energy metabolism and cell multiplication. Since increasing levels of COs inhibit metabolism and respiration, error could result in interpretation of in vitro results where one system or tissue evolved a large level of COs and the other did not. This would result in inhibition of the more active system andresult in perhaps no ultimate difference between treatments or an actual metabolic decrease in the more active system. A temperature increase from 33-37°C increased the entire scope of activity regardless of any other factor involved. In the perfused rabbit testis decreased glucose uptake was found with increased temperature (Ewing & VanDemark, 1963). Decreased blood flow was also found, however, which would have decreased the glucose available. Waites & SetcheU (1964) showed that temperature increase in the testis in vivo caused increases in Os uptake and glucose utilization, but not lactate production. These workers further suggested that this increased metabolism without an increased supply of Os may be the cause of testicular degeneration. However, Free & VanDemark (1966) have shown an increased Os tension with in vivo temperature increase and COs tension increases as well. The effect of incubation time indicates that during the first hour the tissue is the most active in both glycolysis and respiration. After this initial period there was a general drop in activity with no time of incubation effect thereafter. Most of the glucose taken up is not accounted for in this first hour by the lactate and COs produced or by the Os uptake suggesting that it is probably held passively. During
CARBON DIOXIDE AND TEMP]~I~ATURE EFFECTS O N TESTIS METABOLISM
481
the first hour the R.Q. was higher even though respiration had increased, which could mean that biosynthetic processes requiring COs were not active. This may all be caused by tissue damage or depletion of vital substrates during the preparation processes. In bovine sperm subjected to high levels of COs the initial inhibition of motility is gradually overcome (VanDemark et aL, 1965). Whether a similar initial inhibition and later recovery occurred with the testis tissues in this study can only be determined by further investigation. The data suggest that the length of the in vitro studies may be critical. It appears that during the initial period of incubation, though the activity was the greatest, true glycolytie and respiratory activity of the tissue may not be evident. It appears that this initial period should serve as part of the preparation and equilibration. On the other hand, though not evident in this study, long-term incubations may result in an accumulation of COs which would inhibit glycolysis and respiration as seen in this study with increased COs levels. There are two conclusions from this study concerning the effect of COs on testis tissue. First, some COs appears necessary for maximum glycolysis and respiration of testis tissue at the level of Os used in the study. Secondly, increased levels of COs inhibit glycolysis and respiration. In both cases there is qualitative change in metabolism. This suggests COs levels around 2 and 5 per cent for maximum activity i n / n v/tro studies. This range of values results in COs tensions comparable to those estimated for normal ram testes in vivo (37 mm Hg) (Free & VanDemark, 1966). Acknow/edgements--The authors wish to thank Mrs. C. L. Jenkins for her technical assistance. This work was supported by USPHS Grant GM-12247. REFERENCES BARK~ S. B. & SUMMm~ONW. H. (1941) The colorimetric determination of lactic acid in biological materials, y. biol. Chem. 138, 535-554. Caoss B. A. & SXLVSRI. A. (1962) Neurovascular control of oxygen tension in the testis and epididymis. ~. Reprod. Fert. 3, 377-395. DANESB. S. & Kmtam J. (1958) The influence of COs tension on cellular respiration studied by the cartesian diver technique. C. r. Lab. Tray. Lab. Carlsburg 31, 61-75. EWINO L. L. & VArcDEMARKN. L. (1963) Factors affecting testicular metabolism and perfused testes in the rabbit, y. Repro& Fert. 6, 17-24. F a ~ M. J. & VANDEMAaKN. L. (1966) The effect of serotal warming on spermatic venous gas tensions. Fedn. tim. Socs. exp. Biol. 25, 313. GLov~a T. D. & YOUNO B. H. (1963) Temperature and the production of spermatoza. Fert. Steril. 14, 441-450. HAmNGO. M. (1960) Cardiac malformations in rats induced by exposure of the mother to carbon dioxide during pregnancy. Circulat. Res. 8, 1218-1227. HARRISONR. G. & Wmrnta J. S. (1949) Vascular patterns of the mammalian testis and their functional significance. ~. exp. Biol. 26, 304-316. KmLESJ. & BxczW. (1962) The function of COj in cell metabolism and growth. Henry Ford Hosp. International Symposium: Biological Interactions in Normal and Neoplastic Growth. Little, Brown and Co.
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J. L. FLI~GER, N. L. VANDmVL~KAND A. D. JOHNSON
LODGE J. R., G ~ v E s C. N. & SALISBURYG. W. (1963) Carbon dioxide in prevention of the deleterious dilution effect on metabolism and motility of mammalian spermatazoa. Proc. Soc. exp. Biol. Med. 113, 824-827. LOOMIS W. F. (1959a) pCOs inhibition of normal and malignant growth, je. natn. Cancer Inst. 22, 207-217. LOOMIS W. F. (1959b) Feedback control of growth and differentiation by carbon dioxide tension and related metabolic variables. Cell, Organism and Milieu, pp. 253-294. Ronald Press Co., New York. MOORS C. R. (1924) Properties of the gonads as controllers of somatic and psychical characteristics--VIII. Heat application and testicular degeneration. The function of the s c r o t u m . . 4 m . j~. Anat. 34, 337. SALmeURY G. W., VANDmv~RKN. L., LODGEJ. R. & CRAGLe R. G. (1960) Inhibition of spermatozoan metabolism by pCOl, pH, K ion and antibacterial compounds. Am. ~. Physiol. 198, 659--664. SPSAz'r M. T., JR. (1949) Carbon dioxide requirements of the early chick embryo. Anat.
Re¢. 105, 583-584. S'rm~ R. G. D. & Tomtxe J. H. (1960) Principles and Procedures of Statistics. McGraw-Hill, New York. UMBREIT W. W., BURRISR. H. & STAUFFERJ. F. (1959) Manometric Techniques. Burgess Publishing Co., Minneapolis, Minn. V A N D m ~ z N. L., KOYAMAK. & LODOSJ. R. (1965) Factors affecting immobilization of bovine spermatozoa with COl and their subsequent reactivation, j~. Dairy Sci. 48, 586591. VANDEMARKN. L. & SHARMAU. D. (1957) Preliminary fertility results from the preservation of bovine semen at room temperatures. ~. Dairy Sci. 40, 438--439. WAITESG. M. H. & SETCHELLB. P. (1964) Effect of local heating on blood flow and metabolism in the testis of the conscious ram. j~. Relwod. Fert. 8, 339-349. Worthington Biochemical Corp. Bulletin (1963) Glucostat. Freehold, New Jersey.