THE UTILIZATION OF GLUCOSE AND FRUCTOSE BY MOUSE TESTIS IN PHOSPHATE-CONTAINING MEDIA. [Manuscript receiverl.!uly

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1 THE UTLZATON OF GLUCOSE AND FRUCTOSE BY MOUSE TESTS N PHOSPHATE-CONTANNG MEDA By A. W. BLACKSHAW* [Manuscript receiverl.!uly Summary The respiratory activity of mouse testis is stimulated by added glucose or fructose. Under both aerobic and anaerobic conditions. glucose is rapidly metabolized with the production of lactic acid. Under the same conditions fructose is utilized to a much less extent and only small amounts of lactic acid are formed. norganic phosphate markedly stimulates aerobic and anaerobic glycolysis but inbibits respiration.. NTRODUCTON Over the last fifteen years extensive studies have revealed many details of the metabolism of spermatozoa. However, the metabolic function of the testis, from which these cells originate, has received relatively little attention. n comparative studies of various mammalian tissues, Dickens and Greville (1932, 1933a, 1933b) showed that glucose was rapidly metabolized by rat testis tissue. Although both glucose and fructose maintained respiratory activity, fructose was only partially converted to lactic acid. The results of Elliott, Greig, and Benoy (1937) showed a high rate of anaerobic glycolysis, and Schuler (1941) found that respiration was dependent on glucose and other substrates. More recently Tepperman, Tepperman, and Dick (1949) indicated that the effect of glucose on the respiration of rat testis was strongly influenced by the age of the animal. The respiration and glycolysis of spermatozoa and some other tissues are markedly affected by high levels of inorganic phosphate. n general, the respiration of spermatozoa is inhibited and the accumulation of lactic acid increased (Bishop and Salisbury 1955; Salisbury and Nakabayashi 1957; Blackshaw 1960). The present experiments were designed to investigate the metabolic activity of testis tissue from adult mice and to determine itr rerponse to glucose and fructmle in the presence of inorganic phosphate.. MATERALS AND ME'l'HODS Adult male mice were kept under summer conditions at the time of the experiments. They were killed by a blow on the head and the testes removed. The epididymis and attached fat were dissected away and the testes weighed to the nearest milligram. Each testis was placed in 1 0 ml M N ac until all dissections were completed. mmediately before placing the tissue in the appropriate diluent the testis was bisected and the tubules teased out. * Department of Physiology, University of Queensland, Brisbane.

2 Average 208 A. W. BLACKSHAW The testes were suspended in 2 0 ml of the medium contained in Thunberg tubes for the anaerobic experiments, and standard Warburg flasks for the aerobic TABLE 1 ANA,mOBlC GLYCOLYSS O}' MOUSE TES~'S N PHOSPHATE CONC['ANNG MEDA, WerH GLUCOSE AND FRUCTOSE AS SUBSTRATES Results given are means of 4 experiments Sodium chloride Phosphate - + ' _ Substrate No sugar Glucose Fructose Glucose and fructose Average phosphate effect Lactic Acid Produced (flg/100 mg/hr) Sugar Effect R _ R3 2 experiments. To obtain anaerobic conditions, the Thunberg tubes were evacuated for 10 min with a water-pump and then flushed with N 2. n the aerobic experiments TABLE 2 HEnnC'!'ON OF 2,3,5-TRPHENYLTETRAZOLUM CHLORDE BY MOUS~~ TESTS N PHOSPHATE CONTANNG MEDA, WTH GLUCOSE AND FRUCTOSE AS SUBSTRATES Results given are means of 6 replications _n.._, 1 i Sodium chloride -t + PhOsPhat-'"- -=- ~J= : =1 "_ Substrate Formazan Produced Average Sugar (flg/100 mg/hr) Effect No Rugar ) Glucose ) F,",'"~ 33",! <e,l 43'7 'H Glucose and fructose _ Average phosphate effect the gas phase was air, and CO 2 was absorbed by 20% KOH in the centre well. The incubation temperature was 37 C in both series of tests with a 3-hr incubation period for the anaerobic, and a 2-hr period for the aerobic experiments.

3 ---~-~--- TESTS METABOLSM 209 The diluents used throughout were (1) 0 15M NaC; (2) an isotonic phosphate buffer (ph 7'0) containing 72ml 0'13M Na2HP04.l2H20, and 28ml 0'17M NaH2P04.2H20; and (3) equal volumes of the saline and phosphate solutions. As required, 200 mg/loo ml of either glucose or fructose or 100 mg/loo ml of both were added to the media to give a pseudofactorial plan with four sugar combinations and three levels of phosphate. n the anaerobic experiments 0 25 mg/ml of 2,3,5- triphenyltetrazolium chloride was added to each tube. At the conclusion of each experiment the lactic acid content of each tube or flask was determined by the method of Barker and Summerson (1941). n the aerobic trials the O2 uptake was meafmred by the direct Warburg method (Umbreit, Burris, and Stauffer 1957). '['ABLle 3 F:~~'~JC''R OF PHOSPHA''B LBV~JL AND THE PRESENCE OF GLUCOSE AND FRUC'l'OSB ON 'J'HJ;; OXYGgN UPTAKE OF MOUSE TESTS Results given are means of 6 experiments Sodium chloride! + + Phosphate Substrate Oxygen Consumed Average Sugar (1'1/100 mg/hr). Effect No sugar 79, ,6 Glucose Fructose ,7 Glucose and fructose Average phosphate effect 93' The reducing activity of the testis, as indicated by the reduction of the tetrazolium salt to the red insoluble formazan, was estimated by extracting each testis with 10 ml acetone; the optical density of the extract was measured at 485 m,u (Blackshaw 1960). n the tables the results for the accumulation of lactic acid and formazan and the uptake of O2 are expressed as,ug and,u1/l00 mg/hr. The initial wet weight of the testis was used in these determinations. Analyses of variance were performed on the results and a summary of these is given in Table 5,. RESULTS The two experimental periods were separated by 9 months and the absolute values for the anaerobic and aerobic series are not directly comparable. The results for the anaerobic experiments are given in Tables 1 and 2. The analysis of variance of the lactic acid data in Table 5 showed that phosphate significantly increases its accumulation in a linear fashion and, as anticipated, in the absence of added substrate lactic acid production is severely depressed.

4 -~~~ 210 A. W. BLACKSHAW Although some lactic acid appears when fructose alone is present, the amount is only a fraction of that found when glucose is the substrate. A significant interaction between diluents and substrate suggested that the response to phosphate is greater with glucose than with fructose. The reduction of 2,3,5-triphenyltetrazolium chloride to formazan is not influenced by the presence of glucose or fructose, apparently being dependent on endogenous substrates (Tables 2 and 5). However, its reduction is stimulated by phosphate although the complete replacement of sodium chloride hy phosphate does not further increase reduction. TAB.LE 4 E~'FECTR OF GLUCOSE AND FRUCTOSE ON THE AEROBC GLYCOLYAR OF MOUSE TERTA N PHOSPHATE-CONTANNG MEDA Results given are means of 6 experiments Sodium chloride + + Phosphate - + Substrate Lactic Acid Produced Average Sugar (p.gj100 mgjhr) Effect No sugar Glucose Fructose Glucose and fructose Average phosphate effect The values for O2 uptake and lactic acid accumulation in the aerobic experiments are given in Tables 3 and 4. Phosphate significantly depresses O2 uptake in a linear fashion but stimulates lactic acid production as before, although the response falls off at higher levels (Table 5). n the absence of sugar there is a low respiration rate (Table 5) but both glucose and fructose stimulate respiration to an equal extent. As with anaerobic conditions' glycolysis is depressed in the absence of substrate. Glucose leads to the rapid accumulation of lactic acid while fructose is only partially utilized _.... V. DSCUSSON The metabolism of the testis has some features in common with brain tissue. Dickens and Greville (1933a, 1933b) showed that like brain the O2 uptake of the testis is dependent on the presence of sugar or other similar substrate. This was later confirmed by Elliott, Greig, and Benoy (1937) but it is apparent that the age of the animal influences this effect as Tepperman, Tepperman, and Dick (1949) have demonstrated that glucose depresses the O2 uptake of juvenile rat testis. Testis

5 TESTS METABOLSM 211 tissue also resembles brain in having a high rate of anaerobic glycolysis but differs from it and other normal tissues as the rate of aerobic glycolysis is also high (Elliott, Greig, and Benoy 1937). Although conversion of fructose to lactic acid is very much less than that of. glucose in the testis, the former does maintain respiration in the absence of other substrates. Other tissues do not utilize fructose to as great an extent as the testis under anaerobic conditions, with the exception of the kidney (Dickens and Greville TABLE 5 SUMMARY ANALYSES OF VARANCE FOR THE DATA OF TABLES 1, 2, 3, AND 4 The appropriate error mean squares are given at the foot of the variance ratio columns Source of Variation..- _. Anaerobic Tests Lactic Acid Tetrazolium Variance Variance D.F. D.F. Ratio Ratio Aerobic Tests Lactic Acid Oxygen Variance Ratios Replications 3 5'7* ** 3'1* 18'5** Phosphate levels (2) - (2) Linear 1 19'6** ** 40'2** 9'8** Quadratic '9** 12'7** 0 1 Sugars (3) - (3) Sugar v. no sugar ** '8** 22 1 ** Glucose v. fructose ** ** 0 9 Glucose and fructose v. glucose or fructose '8* nteractions All replicate interactions Phosphate X sugar 6 3'7* 6 4 2** 2'7* 0 6 Residual (error) 17t * P<0 05. ** P<O Ol. t One missing plot. 1932). Even spermatozoa, for which the usual natural substrate is fructose, preferentially metabolize glucose when the two sugars are present together (Vantienhoven et al. 1952). t is well known that inorganic phosphorus plays an important role in the Embden-Meyerhof glycolytic scheme and it has been shown that phosphate stimulates the accumulation of lactic acid by spermatozoa (Salisbury and Nakabayashi 1957; Blackshaw 1960). Similar results have been obtained with mouse testis under aerobic and anaerobic conditions, and with either glucose or fructose as the substrate. On the other hand phosphate greatly inhibits the respiration of spermatozoa (Bishop and Salisbury 1955) and to a lesser extent inhibits that of testis tissue. t has been suggested (Mann and White 1957) that the oxidation of lactic acid is inhibited by phosphate but it has little effect on the activity of sperm lactic dehydrogenase (Blackshaw, unpublished data) and the inhibition probably occurs later in the

6 212 A. W. BLAOKSHAW breakdown. t may be noted that phosphate also inhibits glucose 6-phosphate dehydrogenase, the enzyme catalysing the first reaction of the hexose monophosphate shunt (Teorell 1935; Kravitz and Guarino 1958). The importance of this pathway in testis metabolism is unknown but in ejaculated spermatozoa a proportion of the carbohydrate utilized may pass through this cycle (Flipse 1956) and in testicular spermatozoa this pathway is considerably more important (Wu et at. 1959). V. REFERENOES BARKER, 8. B., and SUMMERSON, W. H. (1941).-J. Bioi. Chem. 138: 535. BSHOP, M. W. H., and SALSBURY, G. W. (1955).-Amer. J. Phys,:ol. 181: BLACKSHAW, A. W. (1960).-Aust. J. Bioi. Sci. 13: 37l. DCK~JNS, F., and GREvrr,L~J, G. D. (1932).-Biochem. J. 26: 11)46. DCKENS, F., and GREVLLE, G. D. (1933a).-Biochem. J. 27: 832. DCKENS, F., and GREVLLE, G. D. (1933b).-Biochem. J. 27: ELLOTT, K. A. C., GREG, M. E., and BENOY, M. P. (1937).-Biochem. J. 31: FLPSE, R. J. (1956).-J. Dairy Sci. 39: KRAVTZ, E. A., and GUARNO, A. J. (1958).-Science 128: MANN, T., and WHTE,!. G. (1957).-Biochem. J. 65: 634. SALSBURY, G. W., and NAKABAYA;'lH, N. T. (1957).-J. Exp. Biol. 34: 52. SCHULER, W. (1941).-Helv. Chim. Acta 27: TEORELL, H. (1935).-Biochem. Z. 275: 416. TEPPERMAN, J., TEPPERMAN, H. M., and DCK, H. J. (1949).-Endocrinology 45: 49l. UMBRET, W. W., BURRS, R. H., and STAUFFER, J. F. (1957).-"Manometric Techniques." (Burgess Publ. Co.: Minneapolis.) VANTENHOVEN, A., SALSBURY, G. W., VANDEMARK, N. L., and HANSEN, R. G. (1952).-J. Dairy Sci. 35: 637. Wu, S. H., McKENZE, F. F., FANG, S. C., and BUTTS, J. S. (1959).-,1. Dairy Sci. 42: 110.

Effects of the Pre-incubation in a Na + -free Medium on the O 2 Uptake and Glucose Utilization by the Intestine *

REVISTA ESPAÑOLA DE FISIOLOGIA R. esp. Fisiol., 25, n. 4, págs. 225-232, 1969 Department of Physiology and Biochemistry Faculty of Sciences University of Navarra Pamplona (Spain) Effects of the Pre-incubation