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Modifications of Human Spermatozoa Glycolysis by Cyclic Adenosine Monophosphate (c AMP), Estrogens, and Follicular Fluid*†
)
Vol.
FERTILITY AND STERILITY
2;~,
No. 12. December 1972
Printed in U.S.A.
Copyright © 1972 by the Williams & \Vilkins Co.
MODIFICATIONS OF HUMAN SPERMATOZOA GLYCOLYSIS BY CYCLIC ADENOSINE MONOPHOSPHATE (cAMP), ESTROGENS, AND FOLLICULAR FLUID*t JUAN JOSE HICKS, M.D.,:j: NIEVES PEDRON, M.D.,
AND
ADOLFO ROSADO, M.D., PH.D.
Departamento de Investigacion Cientifica, Centro Medico Nacional, Instituto Mexicano del Seguro Social. Apdo. Postal 73-032, Mexico 73, D. F.
Energy metabolism in human spermatozoa is predominantly a glycolytic process, although the presence of significant oxidative metabolism has been clearly established recently. 1 We have been able to demonstrate that, as in other species, the metabolism of the human sperm cell is modified during incubation with follicular fluid. 2 Also we have shown that cyclic adenosine monophosphate (cAMP) plays an important role in the regulation of the oxidative metabolism of the human spermatozoa. 2 Since no clear-cut experimental evidence has been presented to date, it is not possible to speak of capacitation of primate spermatozoa as a prerequisite for sperm penetration or to speak definitively of the mentioned changes induced by follicular fluid 2 as capacitation of human spermatozoa. However, the evidence put forth by Shettles,3 by Edwards, Bavister, and Steptoe,4 and by Seitz et al. 5 suggests a need for human sperm capacitation. Furthermore, as Brackett 6 states, the results of Shettles 3 and of Edwards et al. 4 indicate that "the mechanism of human sperm capacitation may be similar to that of the hamster." Received March 9, 1972; revised June 28, 1972. Presented in part at the 28th Annual Meeting of The American Fertility Society, February 28March 1, 1972, New York N.Y. t Supported in part by a grant from the Ford Foundation. :j: This paper will be included in a thesis to be submitted to the Graduate Council of E.N.C.B. Instituto Politecnico Nacional Mex., in partial fulfillment of the requirements for the Sc.D. degree. .*
In this paper we examine by means of the technic of radiorespirometry the effect of some of the components of follicular fluid and of cyclic AMP on the selective utilization of labeled substrates. MATERIALS AND METHODS
Human spermatozoa were obtained from fresh ejaculates by centrifugation of the liquified semen for 10 min. at 3000 g. The pellets were resuspended to the original volume with potassium phosphate buffer pH 7.4, 0.085 M and washed once. The spermatozoa were finally resuspended in the same phosphate buffer in such a way as to obtain a final concentration of 2 x 10 8 spermatozoa ml. -1. Sperm numbers were read from a standard curve established by relating optical density at 550 nm. to sperm counts made with a hemocytometer. A Gilson differential respirometer adjusted to 37 ± 0.10 C. was used for radiorespirometry. Warburg flasks, 5 ml., were used with the following additions: 0.1 ml. of 2 M KOH to the center well, 0.1 ml. of 0.5 M perchloric acid to the side arm, and 0.5 ml., equivalent to 10 8 , of the resuspended spermatozoa to the principal chamber. Measurements were carried out in air at a shaking rate of 70 oscillations/ min. Each one of the following substrates obtained from New England Nuclear Co. was used in different experiments: glucose-U-L-C 14, gluclose-l-C 14, glucose 3-4Cl., glucose-6-C I4 , pyruvate-l-C 14 , pyruvate 2_C I4 •
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December 1972
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HUMAN SPERMATOZOA GLYCOLYSIS
These were adjusted to a specific activity of 33.3 nC./~mole and added to the flasks at a final concentration of 3 ~moles/ml.
Incubations were carried out in the presence of the following hormones or cofactors, all obtained from Sigma Chemical Co.: estrone, 17-,B-estradiol, estriol, progesterone, cyclic AMP. On some occasions follicular fluid was also used. This fluid was obtained by puncture of follicles 0.5 cm. or less in diameter, in cases of polycystic ovary in whose walls histologic observation showed almost normal follicular structure. Finally the volume was adjusted to 1.0 ml. in all analytical procedures by the addition of the same buffer used to resuspend the spermatozoa. After a lO-min. period allowed for temperature equilibration, the flasks were closed and oxygen uptake was measured manometrically at 15-min. intervals in the Gilson differential respirometer. After the chosen time elapsed, the reaction was stopped by tipping in the perchloric acid solution. After a period of 30 min., allowed for complete trapping of the C 14 0 2, the flasks were opened. Substrate oxidation was calculated by counting the center well filter paper with the KOH trapped C 14 0 2, using a liquid scintillation spectrometer and correcting counts per minute to disintegrations per minute using the ratio method. The flasks' contents were homogenized in an all glass Potter-Elvejhem homogenizer and the homogenate spun-down at 3000 r.p.m. in a clinical centrifuge. Proteins and deoxyribonucleic acid (DNA)7 were measured in the precipitate, the former according to the method of Lowry,8 and lactate 9 and residual glucose 10 were measured in the supernatant by usual analytic procedures. In addition, the biochemical composition of the utilized follicular fluids was studied. Total estrogen was measured in
an aliquot of the unfractionated liquid by the fluorometric procedure of Ittrich. 11 For the determination of macromolecules, an aliquot of the follicular fluid was precipitated by the addition of ice cold perchloric acid to obtain a 0.2 M final concentration. The mixture was centrifuged in the cold at 3000 g for 15 min. and the supernatant carefully siphoned out and used to measure glucose,lo lactate,9 ketohexoses,12 and ions, the last by means of a Unicam SP-90 Atomic Absorption Spectrometer. 13 The pellet was washed twice more with 0.2 M perchloric acid, and finally resuspended in 1 M perchloric acid and heated at 70° C. for 20 min. DNA 8 and ribonucleic acid (RNA) 14 were measured in the supernatant of the perchloric acid hydrolyzate and proteins by Lowry's 7 procedure in the precipitate. Significance of differences between means (p value) was obtained by the use of the Student's nonpaired "t" test. RESULTS
Table 1 shows the composition of follicular fluid. Follicular fluid seems to be acellular since no DNA is present, and proteins are extremely low. However, an appreciable amount of RNA. exists. A high estrogen concentration (2.0 ~g./ml.), that represents a difference of about four or five orders of magnitude in relation to blood serum, is noticeable. The concentration of glucose was comparable to the amount in blood serum and that of lactate about half that of plasma. Ionic concentration is also near that found in plasma, although the values tend to be in the highest limits or even higher as in the case of calcium. The over-all production of C 14 0 2 from glucose is indicated in Table 2. Follicular fluid produced a 6-fold increase in C 14 0 2 production from C-6-labeled glucose
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HICKS ET AL.
TABLE 1. Composition of Human Follicular Fluid* I. Cations Sodium Potassium Calcium Magnesium II. Substrates Glucose Ketoses Lactate III. Macromolecules DNA RNA Proteins IV. Hormones Estrogens
165.30 5.31 6.72 2.63
mEq./L. mEq./L. mEq./L. mEq./L.
0.66 mg./ml. O. 10 mg./ml. 0.48 Jlmoles/ml. 0.0 281 Jlg./ml. 2.8 mg./ml. 2.0 Jlg./ml.
,* All values represent the mean of three different samples. TABLE 2. Production of C- 14 02 from Glucose Selectively Labeled* No. of experiments
Additions
(8) None (7) Follicular fluid (10 Jl1 ·10' Sz) (9) 17 - ~-estradiol (10 ng .. 10' Sz) (9) Estrone (10 ng. ·10' Sz) (4) Progesterone (10 ng .. 10' Sz)
l_C 1 4
3-4-Cu
6_C14
1241 1832
2121 6921t
660 3586t
2712t
4179:j:
915
2411t
381O:j:
1111:j:
1198
913
* Average of the results obtained in the indicated number of experiments. t p < 0.05 when compared with the control. :j: p < 0.01 when compared with the control.
and a 3-fold increase from glucose-3, 4-C 14 with no increase in the C 14 0 2 from C-l. On the contrary, 17 -,a-estradiol stimulates C-1 conversion to C I4 0 2 with no increase in C-6 conversion. Estrone produced an increase in all parameters. Since radiorespirometry permits a determination of which of the alternative metabolic pathways, normally opened to a particular substrate, is preferentially followed within the cellular metabolism, the data presented in Tables 3, 4, and 5 are important. Basal utilization of glucose by untreated spermatozoa shows that all
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ratios 3,4: 1; 3,4:6; and 1:6 of carbon atoms are higher than one, and are accompanied by an oxygen uptake of 2.45 Jll./hr./10 8 spermatozoa and a lactate production of 1.52 Jlmoles/hr./flask (Tables 3 and 5). A comparison of the effects on the glycolytic sperm metabolism of liquid from ovarian follicles, estrogens, progesterone, and 3'-5' cyclic AMP is presented in Tables 3, 4, and 5. In Table 3 a significant increase of C-1 over C-6 utilization when estrone and 17 -,a-estradiol are present is evident from the increase in the C-1: C-6 ratio. In contrast, an important decrease of the same ratio is produced when the incubation media contain follicular fluid, progesterone, or cyclic AMP. The ratio of glucose-C-3,4 utilization to that of C-6 is also increased by estradiol and estrone, while no important modification is produced in the C-1:C-3,4 ratio by these steroids. Follicular fluid, 17- ,a-estradiol, and cyclic AMP produced a significant increase in oxygen uptake. Sperm cells seem to be active in producing pyruvate decarboxylation probably converting it into acetylCoA; however, further metabolism of acetyl-CoA seems to be carried out imperfectly as is reflected in the C 1: C 2 ratio of 3.22, (Table 4). Follicular fluid, as well as 17 -,a-estradiol, is able to reduce this ratio significantly indicating an increase of pyruvate usage by means of the Krebs cycle. Although cyclic AMP also tends to decrease the pyruvate C-1: C2 ratio, the difference is not statistically significant. Table 5 shows the lactate produced during sperm cell metabolism using pyruvate or glucose as substrates. When pyruvate is used, lactate liberation to the medium is increased by follicular fluid, estradiol, and estrone, 17-,a-estradiol being the most active. On the other hand when glucose is used as substrate, the only
I
December 1972
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HUMAN SPERMATOZOA GLYCOLYSIS
TABLE 3. Glucose Utilization by Human Spermatozoa Glucose (3 jlmoles/10 8 sperm cells
No. of ex.periments
Oxygen uptake ZO,
Production of C 1-(0 2
Treatment
1 :3-4
1:6
X
8 Control 7 Follicular fluid (10 ILl.) 9 17 -~- Estradiol * 9 Estrone* 4 Estriol* 4 3'5' Cyclic-AMP* 4 Progesterone*
X
S.E.
p<
1.86 0.52
0.06 0.03
0.001
2.96 2.18 1.47 1.02 1.30
0.43 0.005 0.61 0.27 0.010 0.63 0.48 N.S. 0.68 0.07 0.001 0.09 0.001
3-4:6
S.E.
p<
0.571 0.05 0.33 0.07
0.01
0.04 N.S.t 0.11 N.S. 0.12 N.S.
X
S.E.
p<
X
S.E.
p<
3.22 0.35 1.93 0.40 0.01
2.45 3.81
0.093 0.14 0.00 1
4.24 0.86 0.025 4.58 0.92 0.025 3.61 0.82 N.S.
3.37 2.21 2.59 3.11
0.13 0.00 1 0,10 N .S. 0.09 N .S. 0.08 0.0 5
* Hormones, 10 ng., were added. t N.S., not significant * Nucleotide, 500 ng., was added in presence of 0.008 M theophylline. TABLE 4. Pyruvate Utilization by Human Spermatozoa pyrruvate (8 .umoles/108 sperm cells)
No. of experiments
8 6 7 12 7 10
l-C140 z : 2-ClfOZ
Treatment
Control Follicular fluid (10 ILl.) 17-~-Estradiol*
Estrone* Estriol* 3' -5' -Cyclic-AMP 500 ng./ 10' sperm
Oxygen uptake ZO,
X
S.E.
p
X
S.E.
p
3.22 1.96 0.90 3.03 3.63 2.70
0.42 0.48 0.18 0.30 0.51 0.34
0.005 0.001 N.S.t N.S. N.S.
2.81 3.03 3.32 2.35 2.72 3.12
0.08 0.11 0.19 0.21 0.11 0.09
N.S. N.S. N.S. N.S. N.S.
* Hormones, 10 ng., were added. t N.S., not significant. TABLE 5. Lactate Produced (Micromoles/Hour/Flask) Substrates (3 J.Lmoles/10 8 sperm cells
Treatment
Pyruvate
Glucose
No.
Control Follicular fluid (10 ILl.) 17-~-Estradiol * Estrone* Estriol*
8 6 7 12 7
No.
X
S.E.
p<
1.08 1.23 1. 70 1.22 0.91
0.02 0.08 0.05 0.04 0.02
0.050 0.01 0.01 N.S.
8 7 9 9 4
X
S.E.
p<
1.52 1.41 2.41 1.66 1.56
0.02 0.06 0.04 0.05 0.09
N.S.t 0.01 N.S. N.S.
* Estrogens, 10 ng., were added. t N.S., not significant.
agent capable of increasing lactate production is 17-,B-estradioL DISCUSSION
There is no direct evidence that metabolic changes in sperm may be of func-
tional significance in capacitation. However, the initial report of Hamner and Williams 15 of the production of a 4-fold increase in the oxygen uptake of rabbit spermatozoa incubated in the rabbit uterus coincident with an increase in its fertil-
890
HICKS ET AL.
lzmg capacity has been confirmed by others. 16 Sperm are also known to become motile at ejaculation, and it has been reported that sperm become "vigorously motile" at about the time of capacitation. 17 , 18 It is then interesting that follicular fluid is able to induce a significant increase in the oxygen uptake of human spermatozoa (Tables 3 and 4), and that this same fluid produces a marked increase in sperm motility. 2 Although 17{j-estradiol and 3'-5'-cAMP also increased the oxygen uptake, their effects are less important than those induced by follicular fluid (Tables 3 and 4). In principle the preference of any given cell for using one or more of the metabolic paths open to glucose will be easily established by the use of isotopic tracers. 19 Nevertheless, the complexity of the metabolism of carbohydrates makes this determination less simple than might be supposed. Considering the normal values of the selective glucose utilization by human spermatozoa (Table 3), it can be concluded that the predominance of conversion of carbons 3 and 4 to CO 2 is a clear indication that glucose is preferentially utilized by way of the glycolytic pathway. Nevertheless, the presence of an important alternative metabolic path is demonstrated by the fact that the yields of CO 2 from carbons 1 and 6 of glucose are not the same. The excess in the yield of C-l shows the importance of the pentoses shunt as an alternative route of glucose metabolism in the human spermatozoa (Table 3). It is also necessary to point out that the differential utilization of C-l- or C-2-labeled pyruvate indicates that even though the glucose is used preferentially through the glycolytic pathway, the pyruvate formed can follow two different routes. The predominance of C-l to be converted to CO 2 over C-2 in the spermatozoa shows that the pyruvate is decarboxylated, but very poorly utilized in the Krebs cycle.
Vol. 23
The direct utilization of pyruvate in the Krebs cycle requires a ratio equal to 1. The ratio found higher than 3 indicates that 3 times more pyruvate is decarboxylated than utilized by Krebs cycle (Table 4). Melrose and Terner 20 and Terner 21 postulated the existence of a mechanism of pyruvate decarboxylation independent of respiration based on the dis mutation of 2 molecules of pyruvate to form 1 molecule each of CO 2, lactate, and acetate. If such a reaction exists one would expect the amount of C 14 0 2 evolved from pyruvate-1-C14 to be much greater than that evolved from pyruvate-2-C 14. Hoskins and Patterson,22 in monkey spermatozoa found some results that will be in accord with the presence, in this animal species, of this dismutation of pyruvate, since twice as much C 14 0 2 was formed from pyruvate-1-C 14 than from pyruvate-3-C 14 and also because the addition of antimycin-A resulted in 30 and 98% inhibition, respectively, of C 14 0 2 formation from 1-C 14 and 3-C 14 pyruvate. Our results indicate that human spermatozoa produce C 14 0 2 more readily from pyruvate-1-CI4 than from pyruvate-2-C14 (ratio C-1: C-2 = 3.22), which seems to be in accord with the presence in human spermatozoa of the dis mutation of pyruvate proposed by Melrose and Terner 20 and Terner.21 However, it must be remembered that part of the acetyl-CoA produced by the oxidative decarboxylation of pyruvate may be used for fatty acid synthesis 23 and only a part enters the Krebs cycle. Of the acetyl carbons entering the cycle, only a fraction will finally be liberated as CO 2, since a part will be incorporated into compounds such as aspartate and glutamate. 24 In liver slices, the yield of CO 2 from C-3 of lactate has been shown to be only from 8.5-33% of that from carbon 1 and in mammary gland it drops to 3.4%.19 So it seems that no conclusive evidence about the presence of pyruvate dis mutation can be obtained from the data presented herein.
December 1972
891
HUMAN SPERMATOZOA GLYCOLYSIS
The follicular fluid from different mammalian species has been used during the last years as a capacitating agent in vitro. Yanagimachi 18. 25 has consistently obtained results in the hamster similar to the ones obtained when the process of capacitation is realized in vivo in the genital tract of the female; 26. 27 while Iwamatsu and Chang 28 have shown that the acrosome reaction of mouse epididymal sperm can be induced and the capacitation of mouse sperm can be achieved in vitro in the presence of bovine follicular fluid. Edwards'4 work in humans seems to indicate that the presence of follicular fluid is also necessary to produce capacitation, since preincubation of spermatozoa in follicular fluid led to the attachment of more spermatozoa to the zona pellucida, and to a higher incidence of penetrated and pronucleate eggs. In our results, follicular fluid seems to improve the utilization of glucose via the Embden-Meyerhof pathway (ratio 1: 3; 4 = 0.33 ± 0.12) and also via the Krebs cycle (ratio 1: 6 = 0.52 ± 0.03). This same increase in energy-producing metabolic activity is observed when spermatozoa are incubated with cAMP (ratio 1: 6 = 1.02 ± 0.07). On the other hand, analysis of the results obtained in relation to the metabolism of differentially labeled pyruvate shows that 17-t1-estradiol, as well as follicular fluid are able to increase utilization of C-2. This implies a better usage of pyruvate through the citric acid cycle. However, since the ratio C1: C2 almost reaches 1, indicating that all the pyruvate that is decarboxylated is used in the Krebs cycle, the estrogen activity is greater than that of the follicular fluid (Table 4). All these results point toward the induction of a more efficient machinery for the utilization of available substrates by the spermatozoa in order to produce the energy required by this cell to support its motility, a fact that is doubtless related
to the increase in motility reportedly induced by some of the mentioned agents. 2 Mounib and Chang 29 observed in freshly ejaculated rabbit spermatozoa a preferential oxidation of glucose-1-C14 over glucose-6-C 14 obtaining a C-1: C-6 oxidation ratio that varied from 1.33-2.54, values that are very similar to our 1.86. This preferential oxidation of glucose 1-C 14 was further strengthened by incubation of the spermatozoa in the uterus of the estrous rabbit during 6 hr., reaching values as high as 16.23 for the C-1: C-6 ratio. This remarkable increase may perhaps be related to the small but very significant increase produced by estrogens (Table 3), since these steroids must be in high concentration in the estrous uterus, but must be contrasted with the mentioned action of follicular fluid and cAMP. Capacitation has been postulated to occur in two stages,2 the first apparently taking place at the endometrium and the second completing the process in the fallopian tube, probably through the contact of sperm cells with the follicular fluid expelled during ovulation. Our results seem to support this hypothesis, suggesting that possibly the two different effectors are cyclic AMP and follicular fluid, the latter having effect partly through the direct action of estrogens. In conclusion, follicular fluid seems to be able to produce marked changes or modifications in human spermatozoa metabolism. These changes cannot be attributed only to the presence of certain substrates or of estrogens by themselves, because a comparison of the effects of the substrates or estrogens with those produced by follicular fluid shows that, although there are some similarities, there are important differences in some of the studied parameters. SUMMARY
Glycolytic metabolism under basal conditions and its modifications in the presence of follicular fluid, estrogens, and
892
HICKS ET AL.
cyclic AMP were studied in human sperm cells. Respirometric data indicate that this cell metabolizes glucose by means of the glycolytic pathway in a· preferential manner. An important alternative is decarboxylation through the hexose-monophosphate pentose pathway (Cl: C6 ratio = 1.86). Pyruvate is efficiently decarboxylated, but it is poorly used in the tricarboxylic acid cycle by the spermatozoa (Cl: C2 ratio = 3.22). Follicular fluid and estrogens increase the utilization of pyruvate, while cyclic AMP does not. Follicular fluid increases glucose catabolism by means of the glycolytic pathway (C 1: C6 ratio = 0.52), while estrogenic substances produce an opposite change (Cl: C6 ratio = 2.96. Cyclic AMP acts in a way similar to follicular fluid, in that it increases oxygen uptake. In contrast it decreases the basal C1 : C6 ratio (C 1 : 6 = 1.0). As a conclusion, we postulate that endometrial cyclic AMP may be able to initiate the capacitation process, which is ended in the fallopian tube by estrogens and follicular fluid. REFERENCES 1. PETERSON, R. N., AND FREUND, M. An evaluation of the respiratory capacity of human spermatozoa. J Reprod Fertil17:357, 1968. 2. HICKS, J. J., PEDR6N, N., MARTINEZ-MANAUTOU, J., AND ROSADO, A., Metabolic changes in human spermatozoa related to capacitation. Fertil SteriI23:172,1972. 3. SHETTLES, L. B. A morula stage of human ova developed in vitro. Fertil SteriI6:287, 1955. 4. EDWARDS, R. G., BAVISTER, B. D., AND STEPTOE, P. C. Early stages of fertilization in vitro of human oocytes matured in vitro. Nature (London) 221:632, 1969. 5. SEITZ, H. M., JR., ROCHA, G., BRACKETT, B. G., AND MASTROIANNI, L. Cleavage of human ova in vitro. Fertil Steril 22:255, 1971. 6. BRACKETT, B. G. "Recent Progress in Investigations of Fertilization in Vitro." In The Biology of the Blastocyst, Blandau, R. J., Ed. Univ. Chicago Press, Chicago, 1971, p. 329.
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7. GILES, K. W., AND MYERS, H. An improved diphenylamine method for the estimation of deoxyribonucleic acid. Nature (London) 206: 93,1965. 8. LAYNE, E. "Spectrophotometric and Turbidimetric Methods for Measuring Proteins." In Methods of Enzymology (Vol. 111), Colowick, S. P., and Kaplan, N. 0., Eds. Academic Press, New York, 1957, p. 448. 9. HOHORST, H. J. "L(+)-Lactate. Determination with Lactic Dehydrogenase and DPN." In Methods of Enzymatic Analysis, Bergmeyer, H. V., and Bernt, E., Eds. Academic Press, New York, 1963, p. 266. 10. DAHLQVIST, A. "Intestinal Disaccharidases." In Methods of Enzymology (Vol VIII), Colowick, S. P. and Kaplan, N. 0., Eds. Academic Press, New York, 1966, p. 589. 11. ITTRICH, G. Extraction of the red Kober matter by organic solvents in the determination of estrogens in the urine. Acta Endocr 35:34, 1960. 12. ASHWELL, G."Colorimetric Analysis of Sugars." In Methods of Enzymology (Vol ill), Colowick, S. P., and Kaplan, N. 0., Eds. Academic Press, New York, 1957, p. 75. 13. ROSADO, A., HICKS, J. J., AZNAR, R., AND MARTiNEZ-MANAUTOU, J. Effect of the I.U.D. upon the biochemical composition of human endometrium. Amer J Obstet Gynec 114:88, 1972. 14. SCHNEIDER, W. C. "Determination of Nucleic Acids in Tissues by Pentose Analysis." In Methods of Enzymology (Vol rrn, Colowick, S. P., and Kaplan, N. 0., Eds. Academic Press, New York, 1957, p. 680. 15. HAMNER, C. E., AND WILLIAMS, W. L. Effect of the female reproductive tract on sperm metabolism in the rabbit and fowl. J Reprod FertiI5:143, 1963. 16. IRITANI, A., GOMES, W. R., AND VAN DEMARK, N. L. The effect of whole, dialysed and heated female genital tract fluids on respiration of rabbit and rat spermatozoa. Bioi Reprod 1:77, 1969. 17. YANAGIMACHI, R. The movement of golden ham· ster spermatozoa before and after capacitation. J Reprod Fertil23:193, 1970. 18. YANAGIMACHI, R. In vitro capacitation of hamster spermatozoa by follicular fluid. J Reprod FertiI18:275, 1969. 19. KATZ, J., AND WOOD, H. G. The use of glucoseC" for the evaluation of the pathways of glucose metabolism. J Bioi Chem 235:2165, 1960. 20. MELROSE, D. R., AND TERNER, C. The metabolism of pyruvate in bull spermatozoa. Biochem J 53:296, 1953. 21. TERNER, C. The effects of 2.4-dinitrophenol and p-nitrophenol on the aerobic and anaerobic me-
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22.
23.
24.
25.
HUMAN SPERMATOZOA GLYCOLYSIS
tabolism of bull spermatozoa. Biochim Biophys Acta 36:479, 1959. HOSKINS, D. D., AND PATTERSON, D. L. Metabolism of Rhesus monkey spermatozoa. J Reprod Fertil16:183, 1968. MINASSIAN, E. S., AND TERNER, C. Biosynthesis of lipids by human and fish spermatozoa. Amer J Physiol 210:615, 1966. PUMPIANSKI, R., AND SHARON, A. Transaminase activity of human semen: corre~ation with sperm concentration. Int J FertiI1O:253, 1965. YANAGIMACHI, R. In vitro acrosome reaction and capacitation of Golden Hamster spermatozoa by bovine follicular fluid and its fractions. J Exp Zool 170:269, 1969.
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26. BEDFORD, J. M. Sperm capacitation and fertilization in mammals. Bioi Reprod 2 (Suppl 2):128, 1970. 27. HARPER, M. J., AND CHANG, M. C. "Some Aspects of the Biology of Mammalian Eggs and Spermatozoa." In Advances in Reproductive Physiology (Vol V), Bishop, M. W. H., Ed. Academic Press, New York, 1971, p.167. 28. IWAMATSU, T., AND CHANG, M. C. In vitro fertilization of mouse eggs in the presence of bovine follicular fluid. Nature (London) 224:919, 1969. 29. MOUNIB, M. S., AND CHANG, M. C. Effect of in utero incubation on the metabolism of rabbit spermatozoa. Nature (London) 201:943, 1964.
Vol.
FERTILITY AND STERILITY
2;~,
No. 12. December 1972
Printed in U.S.A.
Copyright © 1972 by the Williams & \Vilkins Co.
MODIFICATIONS OF HUMAN SPERMATOZOA GLYCOLYSIS BY CYCLIC ADENOSINE MONOPHOSPHATE (cAMP), ESTROGENS, AND FOLLICULAR FLUID*t JUAN JOSE HICKS, M.D.,:j: NIEVES PEDRON, M.D.,
AND
ADOLFO ROSADO, M.D., PH.D.
Departamento de Investigacion Cientifica, Centro Medico Nacional, Instituto Mexicano del Seguro Social. Apdo. Postal 73-032, Mexico 73, D. F.
Energy metabolism in human spermatozoa is predominantly a glycolytic process, although the presence of significant oxidative metabolism has been clearly established recently. 1 We have been able to demonstrate that, as in other species, the metabolism of the human sperm cell is modified during incubation with follicular fluid. 2 Also we have shown that cyclic adenosine monophosphate (cAMP) plays an important role in the regulation of the oxidative metabolism of the human spermatozoa. 2 Since no clear-cut experimental evidence has been presented to date, it is not possible to speak of capacitation of primate spermatozoa as a prerequisite for sperm penetration or to speak definitively of the mentioned changes induced by follicular fluid 2 as capacitation of human spermatozoa. However, the evidence put forth by Shettles,3 by Edwards, Bavister, and Steptoe,4 and by Seitz et al. 5 suggests a need for human sperm capacitation. Furthermore, as Brackett 6 states, the results of Shettles 3 and of Edwards et al. 4 indicate that "the mechanism of human sperm capacitation may be similar to that of the hamster." Received March 9, 1972; revised June 28, 1972. Presented in part at the 28th Annual Meeting of The American Fertility Society, February 28March 1, 1972, New York N.Y. t Supported in part by a grant from the Ford Foundation. :j: This paper will be included in a thesis to be submitted to the Graduate Council of E.N.C.B. Instituto Politecnico Nacional Mex., in partial fulfillment of the requirements for the Sc.D. degree. .*
In this paper we examine by means of the technic of radiorespirometry the effect of some of the components of follicular fluid and of cyclic AMP on the selective utilization of labeled substrates. MATERIALS AND METHODS
Human spermatozoa were obtained from fresh ejaculates by centrifugation of the liquified semen for 10 min. at 3000 g. The pellets were resuspended to the original volume with potassium phosphate buffer pH 7.4, 0.085 M and washed once. The spermatozoa were finally resuspended in the same phosphate buffer in such a way as to obtain a final concentration of 2 x 10 8 spermatozoa ml. -1. Sperm numbers were read from a standard curve established by relating optical density at 550 nm. to sperm counts made with a hemocytometer. A Gilson differential respirometer adjusted to 37 ± 0.10 C. was used for radiorespirometry. Warburg flasks, 5 ml., were used with the following additions: 0.1 ml. of 2 M KOH to the center well, 0.1 ml. of 0.5 M perchloric acid to the side arm, and 0.5 ml., equivalent to 10 8 , of the resuspended spermatozoa to the principal chamber. Measurements were carried out in air at a shaking rate of 70 oscillations/ min. Each one of the following substrates obtained from New England Nuclear Co. was used in different experiments: glucose-U-L-C 14, gluclose-l-C 14, glucose 3-4Cl., glucose-6-C I4 , pyruvate-l-C 14 , pyruvate 2_C I4 •
886
December 1972
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HUMAN SPERMATOZOA GLYCOLYSIS
These were adjusted to a specific activity of 33.3 nC./~mole and added to the flasks at a final concentration of 3 ~moles/ml.
Incubations were carried out in the presence of the following hormones or cofactors, all obtained from Sigma Chemical Co.: estrone, 17-,B-estradiol, estriol, progesterone, cyclic AMP. On some occasions follicular fluid was also used. This fluid was obtained by puncture of follicles 0.5 cm. or less in diameter, in cases of polycystic ovary in whose walls histologic observation showed almost normal follicular structure. Finally the volume was adjusted to 1.0 ml. in all analytical procedures by the addition of the same buffer used to resuspend the spermatozoa. After a lO-min. period allowed for temperature equilibration, the flasks were closed and oxygen uptake was measured manometrically at 15-min. intervals in the Gilson differential respirometer. After the chosen time elapsed, the reaction was stopped by tipping in the perchloric acid solution. After a period of 30 min., allowed for complete trapping of the C 14 0 2, the flasks were opened. Substrate oxidation was calculated by counting the center well filter paper with the KOH trapped C 14 0 2, using a liquid scintillation spectrometer and correcting counts per minute to disintegrations per minute using the ratio method. The flasks' contents were homogenized in an all glass Potter-Elvejhem homogenizer and the homogenate spun-down at 3000 r.p.m. in a clinical centrifuge. Proteins and deoxyribonucleic acid (DNA)7 were measured in the precipitate, the former according to the method of Lowry,8 and lactate 9 and residual glucose 10 were measured in the supernatant by usual analytic procedures. In addition, the biochemical composition of the utilized follicular fluids was studied. Total estrogen was measured in
an aliquot of the unfractionated liquid by the fluorometric procedure of Ittrich. 11 For the determination of macromolecules, an aliquot of the follicular fluid was precipitated by the addition of ice cold perchloric acid to obtain a 0.2 M final concentration. The mixture was centrifuged in the cold at 3000 g for 15 min. and the supernatant carefully siphoned out and used to measure glucose,lo lactate,9 ketohexoses,12 and ions, the last by means of a Unicam SP-90 Atomic Absorption Spectrometer. 13 The pellet was washed twice more with 0.2 M perchloric acid, and finally resuspended in 1 M perchloric acid and heated at 70° C. for 20 min. DNA 8 and ribonucleic acid (RNA) 14 were measured in the supernatant of the perchloric acid hydrolyzate and proteins by Lowry's 7 procedure in the precipitate. Significance of differences between means (p value) was obtained by the use of the Student's nonpaired "t" test. RESULTS
Table 1 shows the composition of follicular fluid. Follicular fluid seems to be acellular since no DNA is present, and proteins are extremely low. However, an appreciable amount of RNA. exists. A high estrogen concentration (2.0 ~g./ml.), that represents a difference of about four or five orders of magnitude in relation to blood serum, is noticeable. The concentration of glucose was comparable to the amount in blood serum and that of lactate about half that of plasma. Ionic concentration is also near that found in plasma, although the values tend to be in the highest limits or even higher as in the case of calcium. The over-all production of C 14 0 2 from glucose is indicated in Table 2. Follicular fluid produced a 6-fold increase in C 14 0 2 production from C-6-labeled glucose
888
HICKS ET AL.
TABLE 1. Composition of Human Follicular Fluid* I. Cations Sodium Potassium Calcium Magnesium II. Substrates Glucose Ketoses Lactate III. Macromolecules DNA RNA Proteins IV. Hormones Estrogens
165.30 5.31 6.72 2.63
mEq./L. mEq./L. mEq./L. mEq./L.
0.66 mg./ml. O. 10 mg./ml. 0.48 Jlmoles/ml. 0.0 281 Jlg./ml. 2.8 mg./ml. 2.0 Jlg./ml.
,* All values represent the mean of three different samples. TABLE 2. Production of C- 14 02 from Glucose Selectively Labeled* No. of experiments
Additions
(8) None (7) Follicular fluid (10 Jl1 ·10' Sz) (9) 17 - ~-estradiol (10 ng .. 10' Sz) (9) Estrone (10 ng. ·10' Sz) (4) Progesterone (10 ng .. 10' Sz)
l_C 1 4
3-4-Cu
6_C14
1241 1832
2121 6921t
660 3586t
2712t
4179:j:
915
2411t
381O:j:
1111:j:
1198
913
* Average of the results obtained in the indicated number of experiments. t p < 0.05 when compared with the control. :j: p < 0.01 when compared with the control.
and a 3-fold increase from glucose-3, 4-C 14 with no increase in the C 14 0 2 from C-l. On the contrary, 17 -,a-estradiol stimulates C-1 conversion to C I4 0 2 with no increase in C-6 conversion. Estrone produced an increase in all parameters. Since radiorespirometry permits a determination of which of the alternative metabolic pathways, normally opened to a particular substrate, is preferentially followed within the cellular metabolism, the data presented in Tables 3, 4, and 5 are important. Basal utilization of glucose by untreated spermatozoa shows that all
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ratios 3,4: 1; 3,4:6; and 1:6 of carbon atoms are higher than one, and are accompanied by an oxygen uptake of 2.45 Jll./hr./10 8 spermatozoa and a lactate production of 1.52 Jlmoles/hr./flask (Tables 3 and 5). A comparison of the effects on the glycolytic sperm metabolism of liquid from ovarian follicles, estrogens, progesterone, and 3'-5' cyclic AMP is presented in Tables 3, 4, and 5. In Table 3 a significant increase of C-1 over C-6 utilization when estrone and 17 -,a-estradiol are present is evident from the increase in the C-1: C-6 ratio. In contrast, an important decrease of the same ratio is produced when the incubation media contain follicular fluid, progesterone, or cyclic AMP. The ratio of glucose-C-3,4 utilization to that of C-6 is also increased by estradiol and estrone, while no important modification is produced in the C-1:C-3,4 ratio by these steroids. Follicular fluid, 17- ,a-estradiol, and cyclic AMP produced a significant increase in oxygen uptake. Sperm cells seem to be active in producing pyruvate decarboxylation probably converting it into acetylCoA; however, further metabolism of acetyl-CoA seems to be carried out imperfectly as is reflected in the C 1: C 2 ratio of 3.22, (Table 4). Follicular fluid, as well as 17 -,a-estradiol, is able to reduce this ratio significantly indicating an increase of pyruvate usage by means of the Krebs cycle. Although cyclic AMP also tends to decrease the pyruvate C-1: C2 ratio, the difference is not statistically significant. Table 5 shows the lactate produced during sperm cell metabolism using pyruvate or glucose as substrates. When pyruvate is used, lactate liberation to the medium is increased by follicular fluid, estradiol, and estrone, 17-,a-estradiol being the most active. On the other hand when glucose is used as substrate, the only
I
December 1972
889
HUMAN SPERMATOZOA GLYCOLYSIS
TABLE 3. Glucose Utilization by Human Spermatozoa Glucose (3 jlmoles/10 8 sperm cells
No. of ex.periments
Oxygen uptake ZO,
Production of C 1-(0 2
Treatment
1 :3-4
1:6
X
8 Control 7 Follicular fluid (10 ILl.) 9 17 -~- Estradiol * 9 Estrone* 4 Estriol* 4 3'5' Cyclic-AMP* 4 Progesterone*
X
S.E.
p<
1.86 0.52
0.06 0.03
0.001
2.96 2.18 1.47 1.02 1.30
0.43 0.005 0.61 0.27 0.010 0.63 0.48 N.S. 0.68 0.07 0.001 0.09 0.001
3-4:6
S.E.
p<
0.571 0.05 0.33 0.07
0.01
0.04 N.S.t 0.11 N.S. 0.12 N.S.
X
S.E.
p<
X
S.E.
p<
3.22 0.35 1.93 0.40 0.01
2.45 3.81
0.093 0.14 0.00 1
4.24 0.86 0.025 4.58 0.92 0.025 3.61 0.82 N.S.
3.37 2.21 2.59 3.11
0.13 0.00 1 0,10 N .S. 0.09 N .S. 0.08 0.0 5
* Hormones, 10 ng., were added. t N.S., not significant * Nucleotide, 500 ng., was added in presence of 0.008 M theophylline. TABLE 4. Pyruvate Utilization by Human Spermatozoa pyrruvate (8 .umoles/108 sperm cells)
No. of experiments
8 6 7 12 7 10
l-C140 z : 2-ClfOZ
Treatment
Control Follicular fluid (10 ILl.) 17-~-Estradiol*
Estrone* Estriol* 3' -5' -Cyclic-AMP 500 ng./ 10' sperm
Oxygen uptake ZO,
X
S.E.
p
X
S.E.
p
3.22 1.96 0.90 3.03 3.63 2.70
0.42 0.48 0.18 0.30 0.51 0.34
0.005 0.001 N.S.t N.S. N.S.
2.81 3.03 3.32 2.35 2.72 3.12
0.08 0.11 0.19 0.21 0.11 0.09
N.S. N.S. N.S. N.S. N.S.
* Hormones, 10 ng., were added. t N.S., not significant. TABLE 5. Lactate Produced (Micromoles/Hour/Flask) Substrates (3 J.Lmoles/10 8 sperm cells
Treatment
Pyruvate
Glucose
No.
Control Follicular fluid (10 ILl.) 17-~-Estradiol * Estrone* Estriol*
8 6 7 12 7
No.
X
S.E.
p<
1.08 1.23 1. 70 1.22 0.91
0.02 0.08 0.05 0.04 0.02
0.050 0.01 0.01 N.S.
8 7 9 9 4
X
S.E.
p<
1.52 1.41 2.41 1.66 1.56
0.02 0.06 0.04 0.05 0.09
N.S.t 0.01 N.S. N.S.
* Estrogens, 10 ng., were added. t N.S., not significant.
agent capable of increasing lactate production is 17-,B-estradioL DISCUSSION
There is no direct evidence that metabolic changes in sperm may be of func-
tional significance in capacitation. However, the initial report of Hamner and Williams 15 of the production of a 4-fold increase in the oxygen uptake of rabbit spermatozoa incubated in the rabbit uterus coincident with an increase in its fertil-
890
HICKS ET AL.
lzmg capacity has been confirmed by others. 16 Sperm are also known to become motile at ejaculation, and it has been reported that sperm become "vigorously motile" at about the time of capacitation. 17 , 18 It is then interesting that follicular fluid is able to induce a significant increase in the oxygen uptake of human spermatozoa (Tables 3 and 4), and that this same fluid produces a marked increase in sperm motility. 2 Although 17{j-estradiol and 3'-5'-cAMP also increased the oxygen uptake, their effects are less important than those induced by follicular fluid (Tables 3 and 4). In principle the preference of any given cell for using one or more of the metabolic paths open to glucose will be easily established by the use of isotopic tracers. 19 Nevertheless, the complexity of the metabolism of carbohydrates makes this determination less simple than might be supposed. Considering the normal values of the selective glucose utilization by human spermatozoa (Table 3), it can be concluded that the predominance of conversion of carbons 3 and 4 to CO 2 is a clear indication that glucose is preferentially utilized by way of the glycolytic pathway. Nevertheless, the presence of an important alternative metabolic path is demonstrated by the fact that the yields of CO 2 from carbons 1 and 6 of glucose are not the same. The excess in the yield of C-l shows the importance of the pentoses shunt as an alternative route of glucose metabolism in the human spermatozoa (Table 3). It is also necessary to point out that the differential utilization of C-l- or C-2-labeled pyruvate indicates that even though the glucose is used preferentially through the glycolytic pathway, the pyruvate formed can follow two different routes. The predominance of C-l to be converted to CO 2 over C-2 in the spermatozoa shows that the pyruvate is decarboxylated, but very poorly utilized in the Krebs cycle.
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The direct utilization of pyruvate in the Krebs cycle requires a ratio equal to 1. The ratio found higher than 3 indicates that 3 times more pyruvate is decarboxylated than utilized by Krebs cycle (Table 4). Melrose and Terner 20 and Terner 21 postulated the existence of a mechanism of pyruvate decarboxylation independent of respiration based on the dis mutation of 2 molecules of pyruvate to form 1 molecule each of CO 2, lactate, and acetate. If such a reaction exists one would expect the amount of C 14 0 2 evolved from pyruvate-1-C14 to be much greater than that evolved from pyruvate-2-C 14. Hoskins and Patterson,22 in monkey spermatozoa found some results that will be in accord with the presence, in this animal species, of this dismutation of pyruvate, since twice as much C 14 0 2 was formed from pyruvate-1-C 14 than from pyruvate-3-C 14 and also because the addition of antimycin-A resulted in 30 and 98% inhibition, respectively, of C 14 0 2 formation from 1-C 14 and 3-C 14 pyruvate. Our results indicate that human spermatozoa produce C 14 0 2 more readily from pyruvate-1-CI4 than from pyruvate-2-C14 (ratio C-1: C-2 = 3.22), which seems to be in accord with the presence in human spermatozoa of the dis mutation of pyruvate proposed by Melrose and Terner 20 and Terner.21 However, it must be remembered that part of the acetyl-CoA produced by the oxidative decarboxylation of pyruvate may be used for fatty acid synthesis 23 and only a part enters the Krebs cycle. Of the acetyl carbons entering the cycle, only a fraction will finally be liberated as CO 2, since a part will be incorporated into compounds such as aspartate and glutamate. 24 In liver slices, the yield of CO 2 from C-3 of lactate has been shown to be only from 8.5-33% of that from carbon 1 and in mammary gland it drops to 3.4%.19 So it seems that no conclusive evidence about the presence of pyruvate dis mutation can be obtained from the data presented herein.
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891
HUMAN SPERMATOZOA GLYCOLYSIS
The follicular fluid from different mammalian species has been used during the last years as a capacitating agent in vitro. Yanagimachi 18. 25 has consistently obtained results in the hamster similar to the ones obtained when the process of capacitation is realized in vivo in the genital tract of the female; 26. 27 while Iwamatsu and Chang 28 have shown that the acrosome reaction of mouse epididymal sperm can be induced and the capacitation of mouse sperm can be achieved in vitro in the presence of bovine follicular fluid. Edwards'4 work in humans seems to indicate that the presence of follicular fluid is also necessary to produce capacitation, since preincubation of spermatozoa in follicular fluid led to the attachment of more spermatozoa to the zona pellucida, and to a higher incidence of penetrated and pronucleate eggs. In our results, follicular fluid seems to improve the utilization of glucose via the Embden-Meyerhof pathway (ratio 1: 3; 4 = 0.33 ± 0.12) and also via the Krebs cycle (ratio 1: 6 = 0.52 ± 0.03). This same increase in energy-producing metabolic activity is observed when spermatozoa are incubated with cAMP (ratio 1: 6 = 1.02 ± 0.07). On the other hand, analysis of the results obtained in relation to the metabolism of differentially labeled pyruvate shows that 17-t1-estradiol, as well as follicular fluid are able to increase utilization of C-2. This implies a better usage of pyruvate through the citric acid cycle. However, since the ratio C1: C2 almost reaches 1, indicating that all the pyruvate that is decarboxylated is used in the Krebs cycle, the estrogen activity is greater than that of the follicular fluid (Table 4). All these results point toward the induction of a more efficient machinery for the utilization of available substrates by the spermatozoa in order to produce the energy required by this cell to support its motility, a fact that is doubtless related
to the increase in motility reportedly induced by some of the mentioned agents. 2 Mounib and Chang 29 observed in freshly ejaculated rabbit spermatozoa a preferential oxidation of glucose-1-C14 over glucose-6-C 14 obtaining a C-1: C-6 oxidation ratio that varied from 1.33-2.54, values that are very similar to our 1.86. This preferential oxidation of glucose 1-C 14 was further strengthened by incubation of the spermatozoa in the uterus of the estrous rabbit during 6 hr., reaching values as high as 16.23 for the C-1: C-6 ratio. This remarkable increase may perhaps be related to the small but very significant increase produced by estrogens (Table 3), since these steroids must be in high concentration in the estrous uterus, but must be contrasted with the mentioned action of follicular fluid and cAMP. Capacitation has been postulated to occur in two stages,2 the first apparently taking place at the endometrium and the second completing the process in the fallopian tube, probably through the contact of sperm cells with the follicular fluid expelled during ovulation. Our results seem to support this hypothesis, suggesting that possibly the two different effectors are cyclic AMP and follicular fluid, the latter having effect partly through the direct action of estrogens. In conclusion, follicular fluid seems to be able to produce marked changes or modifications in human spermatozoa metabolism. These changes cannot be attributed only to the presence of certain substrates or of estrogens by themselves, because a comparison of the effects of the substrates or estrogens with those produced by follicular fluid shows that, although there are some similarities, there are important differences in some of the studied parameters. SUMMARY
Glycolytic metabolism under basal conditions and its modifications in the presence of follicular fluid, estrogens, and
892
HICKS ET AL.
cyclic AMP were studied in human sperm cells. Respirometric data indicate that this cell metabolizes glucose by means of the glycolytic pathway in a· preferential manner. An important alternative is decarboxylation through the hexose-monophosphate pentose pathway (Cl: C6 ratio = 1.86). Pyruvate is efficiently decarboxylated, but it is poorly used in the tricarboxylic acid cycle by the spermatozoa (Cl: C2 ratio = 3.22). Follicular fluid and estrogens increase the utilization of pyruvate, while cyclic AMP does not. Follicular fluid increases glucose catabolism by means of the glycolytic pathway (C 1: C6 ratio = 0.52), while estrogenic substances produce an opposite change (Cl: C6 ratio = 2.96. Cyclic AMP acts in a way similar to follicular fluid, in that it increases oxygen uptake. In contrast it decreases the basal C1 : C6 ratio (C 1 : 6 = 1.0). As a conclusion, we postulate that endometrial cyclic AMP may be able to initiate the capacitation process, which is ended in the fallopian tube by estrogens and follicular fluid. REFERENCES 1. PETERSON, R. N., AND FREUND, M. An evaluation of the respiratory capacity of human spermatozoa. J Reprod Fertil17:357, 1968. 2. HICKS, J. J., PEDR6N, N., MARTINEZ-MANAUTOU, J., AND ROSADO, A., Metabolic changes in human spermatozoa related to capacitation. Fertil SteriI23:172,1972. 3. SHETTLES, L. B. A morula stage of human ova developed in vitro. Fertil SteriI6:287, 1955. 4. EDWARDS, R. G., BAVISTER, B. D., AND STEPTOE, P. C. Early stages of fertilization in vitro of human oocytes matured in vitro. Nature (London) 221:632, 1969. 5. SEITZ, H. M., JR., ROCHA, G., BRACKETT, B. G., AND MASTROIANNI, L. Cleavage of human ova in vitro. Fertil Steril 22:255, 1971. 6. BRACKETT, B. G. "Recent Progress in Investigations of Fertilization in Vitro." In The Biology of the Blastocyst, Blandau, R. J., Ed. Univ. Chicago Press, Chicago, 1971, p. 329.
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7. GILES, K. W., AND MYERS, H. An improved diphenylamine method for the estimation of deoxyribonucleic acid. Nature (London) 206: 93,1965. 8. LAYNE, E. "Spectrophotometric and Turbidimetric Methods for Measuring Proteins." In Methods of Enzymology (Vol. 111), Colowick, S. P., and Kaplan, N. 0., Eds. Academic Press, New York, 1957, p. 448. 9. HOHORST, H. J. "L(+)-Lactate. Determination with Lactic Dehydrogenase and DPN." In Methods of Enzymatic Analysis, Bergmeyer, H. V., and Bernt, E., Eds. Academic Press, New York, 1963, p. 266. 10. DAHLQVIST, A. "Intestinal Disaccharidases." In Methods of Enzymology (Vol VIII), Colowick, S. P. and Kaplan, N. 0., Eds. Academic Press, New York, 1966, p. 589. 11. ITTRICH, G. Extraction of the red Kober matter by organic solvents in the determination of estrogens in the urine. Acta Endocr 35:34, 1960. 12. ASHWELL, G."Colorimetric Analysis of Sugars." In Methods of Enzymology (Vol ill), Colowick, S. P., and Kaplan, N. 0., Eds. Academic Press, New York, 1957, p. 75. 13. ROSADO, A., HICKS, J. J., AZNAR, R., AND MARTiNEZ-MANAUTOU, J. Effect of the I.U.D. upon the biochemical composition of human endometrium. Amer J Obstet Gynec 114:88, 1972. 14. SCHNEIDER, W. C. "Determination of Nucleic Acids in Tissues by Pentose Analysis." In Methods of Enzymology (Vol rrn, Colowick, S. P., and Kaplan, N. 0., Eds. Academic Press, New York, 1957, p. 680. 15. HAMNER, C. E., AND WILLIAMS, W. L. Effect of the female reproductive tract on sperm metabolism in the rabbit and fowl. J Reprod FertiI5:143, 1963. 16. IRITANI, A., GOMES, W. R., AND VAN DEMARK, N. L. The effect of whole, dialysed and heated female genital tract fluids on respiration of rabbit and rat spermatozoa. Bioi Reprod 1:77, 1969. 17. YANAGIMACHI, R. The movement of golden ham· ster spermatozoa before and after capacitation. J Reprod Fertil23:193, 1970. 18. YANAGIMACHI, R. In vitro capacitation of hamster spermatozoa by follicular fluid. J Reprod FertiI18:275, 1969. 19. KATZ, J., AND WOOD, H. G. The use of glucoseC" for the evaluation of the pathways of glucose metabolism. J Bioi Chem 235:2165, 1960. 20. MELROSE, D. R., AND TERNER, C. The metabolism of pyruvate in bull spermatozoa. Biochem J 53:296, 1953. 21. TERNER, C. The effects of 2.4-dinitrophenol and p-nitrophenol on the aerobic and anaerobic me-
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22.
23.
24.
25.
HUMAN SPERMATOZOA GLYCOLYSIS
tabolism of bull spermatozoa. Biochim Biophys Acta 36:479, 1959. HOSKINS, D. D., AND PATTERSON, D. L. Metabolism of Rhesus monkey spermatozoa. J Reprod Fertil16:183, 1968. MINASSIAN, E. S., AND TERNER, C. Biosynthesis of lipids by human and fish spermatozoa. Amer J Physiol 210:615, 1966. PUMPIANSKI, R., AND SHARON, A. Transaminase activity of human semen: corre~ation with sperm concentration. Int J FertiI1O:253, 1965. YANAGIMACHI, R. In vitro acrosome reaction and capacitation of Golden Hamster spermatozoa by bovine follicular fluid and its fractions. J Exp Zool 170:269, 1969.
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26. BEDFORD, J. M. Sperm capacitation and fertilization in mammals. Bioi Reprod 2 (Suppl 2):128, 1970. 27. HARPER, M. J., AND CHANG, M. C. "Some Aspects of the Biology of Mammalian Eggs and Spermatozoa." In Advances in Reproductive Physiology (Vol V), Bishop, M. W. H., Ed. Academic Press, New York, 1971, p.167. 28. IWAMATSU, T., AND CHANG, M. C. In vitro fertilization of mouse eggs in the presence of bovine follicular fluid. Nature (London) 224:919, 1969. 29. MOUNIB, M. S., AND CHANG, M. C. Effect of in utero incubation on the metabolism of rabbit spermatozoa. Nature (London) 201:943, 1964.