Amino Acid and Protein Concentrations of Human Follicular Fluid*

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2S, No.1, January 1977 Print€d in U.s.A.

AMINO ACID AND PROTEIN CONCENTRATIONS OF HUMAN FOLLICULAR FLUID*

ANSELMO VELAzQUEZ, M.Sc. ALEJANDRO REYES, M.D. JAIME CHARGOY, M.D.t ADOLFO ROSADO, M.D., PH.D.

SecciOn de Bioquimica de la ReproducciOn, Departamento de InvestigaciOn en Medicina Experimental, Centro Medico Nacional Instituto Mexicano del Seguro Social, Apartado Postal 12-184, Mexico 12, D. F.

The amino acid and protein composition ofhuman follicular fluid, obtained during surgery from women with polycystic ovaries, and ofa simultaneously obtained sample of blood plasma were studied. In general, amino acid concentrations were higher in follicular fluid than in blood plasma: only the concentration ofCys was significantly lower in follicular fluid than in plasma, while Asp, Thr, Glu, Glu-NH2 , 'my, Ala, and Met showed concentrations that were not signifu:antly different in either biologic fluid. The concentration of basic amino acids, taken as group, was almost twice as high in follicular fluid as in plasma. The total protein concentration in follicular fluid was not significantly different from that in blood plasma. However, the follicular fluid albumin concentration was higher and globulin concentration lower than the respective concentrations in plasma. Polyacrylamide gel disc electrophoresis offollicular fluid showed some consistent differences, particularly in the a-globulin region, with the pattern observed in blood plasma. These findings are discussed in relation to the possible role of follicular fluid in capacitation and egg segmentation.

Follicular fluid is a transcellular fluid composed partly of plasma exudate and partly of the secretory activity of the granulosa and theca intema layers. The source of the fluid, its properties and composition, and its possible participation in various physiologic processes have been recently reviewed. 1 However, information on the composition of human follicular fluid is scanty and on occasions contradictory,2. 3 and no data have been published on the amino acid composition of this important component of the reproductive process. The participation of follicular fluid in some important steps of the reproductive process is well documented. Follicular fluid has been shown to be an active sperm-capacitating agent in the hamster,4.5 rat, mouse,6.7 and probably in man,S. 9 and to produce the so-called acrosomal reaction in the cow. 10 Sperm become vigorously motile when incubated with follicular fluid,4.9 which is capa-

ble of inducing a striking increase in oxygen uptake and in selective utilization of some substrates in human sperm cells.ll Incubation with follicular fluid is also known to produce important changes in the membrane characteristics of mammalian spermatozoa. 12 . 13 MATERIALS AND METHODS

Follicular fluid was obtained during surgery by puncture of follicles 0.5 cm in diameter or less, in six cases of follicles in polycystic ovaries. In each case the punctured follicles were labeled for later histologic observation in order to show the presence of a normal, mature, follicular structure. l l The fluid obtained from each woman was pooled and used for amino acid and protein analyses. For determination of serum composition, a sample of venous blood was obtained simultaneously and processed as previously described. 14 For free amino acid determination, 300 ILl of follicular fluid were diluted with 200 ILl of distilled water, and 15 mg of sulfosalicylic acid (powder)

Accepted July 20, 1976. *Supported in part by a grant from The Ford Foundation. tPresent address: Hospital de Especialidades del Instituto Mexicano del Beguro Social, Puebla, Pue., Mexico.

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HUMAN FOLLICULAR FLUID AMINO ACIDS

were then added. The precipitated proteins were centrifuged at 10,000 x g for 10 minutes. Aliquots (300 JLI) of the clear supernatant were used for amino acid analysis in a Technicon TS-M amino acid analyzer after having been passed through Technicon cartridges to which 0.3 ml of Chromobeads A resin had been added previously.15 For serum deproteinization, 30 mg of solid sulfosalicylic acid (powder) were mixed with 1 ml of plasma,16 and the precipitated proteins were then packed by centrifugation at 10,000 x g for 10 minutes. Aliquots (250 JLl) of the clear supernatant were processed for amino acid analysis as described above. To avoid artifacts produced by amino acid interchange/ 6. 17 all samples were examined within 24 hours of collection. The amino acid studies were performed in a Technicon TS-M amino acid analyzer, following the standard procedure 15 and using norleucine (on some occasions a-amino-yguanidobutyric acid) as internal standard. The amino acid peaks were quantitated by the use of a model CRS-110 A Infotronics electronic integrator adapted to the TS-M amino acid analyzer. Protein concentrations were measured by an improved biuret method 18 using human serum albumin as standard. Polyacrylamide gel disc electrophoresis (7.5% running gel) and celluloseacetate paper electrophoresis were performed according to standard procedures. 19. 20 Purified proteins were run simultaneously to establish the position of the main bands.

RESULTS

All amIno aCid chromatograms showed a stable base line. Separation of amino acid peaks was always satisfactory, including an excellent separation of the amino acids in the first group (threonine, serine, glutamine, glutamic acid, and asparagine). It is important to mention that a clear hydroxyproline peak was identifiable in all samples of serum and follicular fluid, but it was particularly noticeable in follicular fluid. Cystine, a difficult amino acid to analyze, was always clearly identified, probably because of the short period of time the samples were stored. Among the basic amino acids, ornithine and lysine were frequently partially overlapped. The amino acid concentrations (in micrograms per milliliter) of follicular fluid and blood plasma are shown in Table 1. The order given is that of the amino acid appearance in the chromatographic procedure. In general, amino acid con-

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TABLE 1. Amino Acid Concentration of Human Venous Blood and Follicular Fluid Amino acid

Venous biood'i

Mean

Cysteic acid Asp Thr Ser GIu GIu-NH2 Asp-NH2

Pro GIy Ala Cys Val Met I-Leu Leu Tyr

Phe Lys His Arg

Om

Follicular fluid"

SD

Mean

SD

Follicular fluid/venous blood

1.4 3.4 13.0

0.4 0.6 2.5 2.7 1.9 4.3

1.26 0.83 1.55" 1.19 0.82

3.6 4.2 3.1 7.52b 2.2 18.2b 3.8 3.7 1.0 7.4 1.4 8.3 1.2 12.3 b 1.8 9.9 b 1.9 21.7 b 3.2 34.4 b 4.9 20.1b 3.0 2.5 b 0.6

1.45" 1.21 1.0 0.32" 2.29" 0.90 1.65" 1.47" 1.95" 1.47" 1.64" 2.84" 2.91" 0.48"

I'I1lml

2.7 15.6 9.8 11.3 38.7 7.2 14.0 16.9 23.0 23.6 9.7 5.2 5.7 7.7 6.3 6.7 13.2 12.1 6.9 5.7

0.5 2.0 1.4 2.1 4.6 1.6 2.8 2.0 1.9 3.5 2.1 1.1 1.3 2.8 0.9 1.3 2.3 2.0 1.4 1.2

15.2b

13.5 31.8 20.3 b 20.4 23.0

UMeans and standard deviations of six paired determinations. bp < 0.05 when compared by the paired Student's t-test with the data for venous blood. "Indicates a ratio different from 1 (P < 0.05) by the X2 test for proportions.

centrations were higher in follicular fluid than in blood plasma. Only the concentration of cysteine (calculated as twice the cystine concentration) was significantly lower in follicular fluid than in plasma, while Asp, Thr, GIu, GIu-NH2 , Ala, and Met showed concentrations in follicular fluid that were not significantly different from those observed in venous blood serum. The total amino acid concentration was also significantly higher in follicular fluid (Table 2). When the amino acids were grouped according to ionization characteristics into acid-neutral and basic, it was possible to notice that, although the concentration of the acid-neutral amino acids was similar for venous TABLE 2. Acid-NeutralandBasicAminoAcidConcentrations in Human Venous Blood and Follicular Fluid Amino acid

Venous blood"

Follicular fluid" I'I1lml

Acid-neutral Basic

191 ± 24 45 ± 10

196 ± 28 98 ± 18 b

Total

236 ± 34

294 ± 39 b

4.24

1.98"

Acid-neutrallbasic

Means ± standard deviations of six paired determinations. bp < 0.05 when compared by the paired Student's t-test with the data for venous blood. "P < 0.01 when compared by the X2 test for proportions with the data for venous blood. U

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TABLE 3. Protein Concentration in Human Blood Serum and Follicular Fluid Protein

Human serum"

Follicular fluid"

Serumlfollicular fluid

mglml

36.5 12.8 8.9 ~-Globulins 6.8 a.-Globulins 4.2 a.-Globulins

Albumin ")I-Globulins

Total

± ± ± ± ±

3.2 1.4 1.2 1.1 0.6

68.4 ± 4.1

48.2 10.3 5.9 5.5 2.7

± ± ± ± ±

2.6b

1.8 0.4 b

1.0 0.6 b

72.8 ± 4.2

0.75 1.24 1.51 1.24 1.56 0.93

u Means

± standard deviations of six paired determinations. bp < 0.05 when compared by the paired Student's t-test with the data for serum.

blood serum and follicular fluid, the concentration of basic amino acids was almost twice as high in follicular fluid. This difference was reflected in a decrease in the ratio of acid-neutral to basic amino acids from 4.24 in blood serum to 1.98 in follicular fluid.

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The total protein concentration in follicular fluid was not significantly different from that in the serum of the same woman (Table 3). However, the cellulose-acetate electrophoresis distribution (Fig. 1) showed some important differences. The concentration of albumin was 35% higher in follicular fluid than in plasma, while a- and {3globulins were less concentrated in follicular fluid than in plasma; the difference was more noticeable in the case of the a-globulins, which were about one-half as concentrated in the liquor folliculi as in plasma. On the other hand, the y-globulin fraction showed the same concentration in follicular fluid as in plasma (Table 3). When the disc electrophoresis patterns were studied (Fig. 1) it was possible to see that, in general, blood plasma and follicular fluid had very similar pr.otein patterns. However, some differ-

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VELAzQUEZ ET AL.

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FIG. 1. Representative diagram of the electrophoretic behavior of human follicular fluid and blood plasma. The continuous line represents the densitometric analysis of the cellulose acetate electrophoretic pattern of human follicular fluid. The line patterns represent a diagram, adjusted to the densitometric trace, of the acrylamide gel electrophoresis of the follicular fluid and the blood plasma obtained simultaneously from the same woman. Equal amounts of protein were used for both fluids, and electrophoresis was carried out in 7.5% acrylamide according to the method of Davis. 20

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HUMAN FOLLICULAR FLUID AMINO ACIDS

ences were consistently seen that were particularly noticeable in the ')I-globulin region. DISCUSSION

Our results showing that the protein concentration of human follicular fluid is similar to that found in blood plasma are in agreement with those recently reported by Shalgi et al. 2 Some earlier studies,3, 11 however, showed that the protein concentration in human follicular fluid was approximately 50% of that in serum. Our results and those of Shalgi et al,2 seem to be more in accord with the kinetic studies of several groups21,22 which have shown that serum proteins are rapidly transported into the follicular fluid. The results presented in Table 3 on the relative concentration of the different plasma protein species are also in accord with those of Shalgi et al.,2 confirming the hypothesis that follicular fluid seems to be formed by selective filtration of the blood plasma. The observed differences in amino acid concentration imply a differential, selective function in the formation of follicular fluid. However, the fluids analyzed in this study were obtained from polycystic ovaries-albeit from follicles the size of normal large follicles rather than from cysts per se-and it is possible that these fluids may differ from the content of large-size follicles in normal ovaries. Incubation of human spermatozoa in the presence of some amino acids, specifically Cys and His, induces significant increases in oxygen uptake and in progressive motility.23 This procedure also produces a shift to the preferential utilization by these cells of endogenous phospholipids instead of exogenous substrates. 24 These metabolic changes have been supposed to participate in the mechanism of sperm capacitation 1!' 25 and can be correlated with the induced release of zinc from the sperm cells by the chelating properties of these amino acids. More recently, Keller and Polakoskp6 have shown that another amino acid, L-arginine, also has the property of enhancing sperm motility in vitro. Human follicular fluid has been reported to induce changes 11 very similar to those mentioned above owing to the presence in the sperm milieu of some specific amino acids. Since His and Arg are two of the amino acids present in follicular fluid in high concentrations (Table 1), it is possible to propose that at least some of the observed effects offollicular fluid on sperm

99

cells are due to a direct stimulating effect of these two amino acids. Human sperm cells possess a sperm specific isozyme of pyruvate kinaseY This enzyme-one of the key enzymes in the regulation of the glycolytic pathway-is inhibited by low concentrations of calcium and zinc 28 and is stimulated by low concentrations of serine and phenylalanine. 27 The high concentrations of these two amino acids in human follicular fluid (Table 1) could partially explain the sperm metabolic stimulation produced by this fluid. This stimulating action also may be due, in part, to the histidine-induced release of zinc from the sperm cells,23 with the concomitant release of pyruvate kinase from a partially zincinhibited state. 28 Cys is one of the amino acids present in lower concentrations in follicular fluid (Table 1). However, in analyzing the results of Huacuja et al.,23 it may be seen that Cys is a stronger stimulant of the motility and the oxygen uptake of human spermatozoa than is His or Arg. This fact argues against the hypothesis that the amino acid composition of follicular fluid contributes to the formation of an appropriate environment for the occurrence of the sperm capacitation process. However, since small amounts of -SH compounds interfere with the process offertilization (probably by affecting the structure of the zona pellucid a or the processes of attachment and binding29 ), the low concentration of Cys in follicular fluid might prove to be an advantage if one considers that the sperm-stimulating properties can be carried out by His and Arg and perhaps by Ser and Phe. Follicular fluid is clearly involved in some physical processes involved in the migration of the mature oocyte to the oviduct. 30 Its role in such physiologic functions as nourishment ofthe oocyte and of the granulosa cells, in the hormone reservoir, and as agent in some reproductive steps that occur in the oviduct has not been clearly established. A role for follicular fluid in the capacitation of spermatozoa has been proposed repeatedly.4, 9,11 Edwards,1 after examining the results of some in vitro capacitation and fertilization studies, concluded that follicular fluid seems to be an unlikely candidate for inducing capacitation. However, it is clear that results obtained in vitro cannot be correlated directly with the in vivo occurrence of a physiologic process. In fact, there is some evidence 11 showing that in vivo and in vitro capacitation can be considered different processes. The data presented herein on the amino

VELAzQUEZ ET AL.

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acid composition of human follicular fluid can be taken as indirect evidence of the participation of this biologic fluid in the final steps in the capacitation of spermatozoa. REFERENCES 1. Edwards RG: Follicular fluid. J Reprod Fertil37: 189, 1974 2. Shalgi R, Kraicer PF, 80ferman N: Human follicular fluid (abstr). J Reprod Fertil 31:515, 1972 3. Manarang-Pangan S, Menge AC: Immunobiologic studies on human follicular fluid. Fertil Steril 22:367, 1971 4. Yanagimachi R: In vitro capacitation of hamster spermatozoa by follicular fluid. J Reprod Fertil18:275, 1969 5. 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 6. MukheIjee AB, Lippes J: Effect of human follicular and tubal fluids on human, mouse and rat spermatozoa in vitro. Can J Genet Cytol14:167, 1972 7. Iwamatsu T, Chang MC: In vitro fertilization of mouse eggs in the presence of bovine follicular fluid. Nature 224:919, 1969 8. Edwards RG, Bavister BD, Steptoe PC: Early stages of fertilization in vitro of human oocytes matured in vitro. Nature 221:632, 1969 9. Hicks JJ, Martinez-Manautou J, Pedron N, Rosado A: Metabolic changes in human spermatozoa related to capacitation. Fertil Steril 23:172, 1972 10. Hicks JJ, Rosado A: Steroid hormones and spermatozoa metabolism. Adv Biochem Pharmacol Steroids 5:263,1976 11. Hicks JJ, Pedron N, Rosado A: Modifications of human spermatozoa glycolysis by cyclic adenosine monophosphate (cAMP), estrogens, and follicular fluid. Fertil Steril 23:886, 1972 12. Rosado A, Velazquez A, Lara-Ricalde R: Cell polarography. II. Effect of neuraminidase and follicular fluid upon the surface characteristics of human spermatozoa. Fertil Steril 24:349, 1973 13. Johnson MH: The macromolecular organization of membranes and its bearing on events leading up to fertilization. J Reprod Fertil 44:167, 1975 14. Velazquez A, Rosado A, Bernal A, Noriega L, Arevalo N: Amino acid pools in the feto-maternal system. BioI Neonate 29:28, 1976 15. Operation Manual for the Technicon TS-M System. Tarrytown NY, Technicon Instrument Corporation, 1971

January 1977 16. Block WD, Markovs ME, Steele BF: Comparison between free amino acid level in plasma deproteinated with picric acid and with sulfosalicylic acid. Proc Exp BioI Med 122: 1089, 1966 17. Armstrong MD, Stave V: A study of plasma free amino acid levels. I. Study of factors affecting validity of amino acid analyses. Metabolism 22:549,1970 18. Itzhaki RF, Gill DM: A micro-biuret method for estimating proteins. Anal Biochem 9:401, 1964 19. Felgennauer K: Molecular size of human serum proteins determined by exclusion gel electrophoresis. Clin Chim Acta 32:53, 1971 20. Davis BJ: Disc electrophoresi&-II Method and application to human serum proteins. Ann NY Acad Sci 121:404 1964 ' 21. Mancini RE, Vilar 0, Heinrich JJ, Davidson OW, Alvarez B: Transference of circulating labeled serum proteins to the follicle ofthe rat ovary. J Histochem Cytochem 11:80 1963 ' 22. Yatvin MB, Leathem JH: Origin of ovarian cyst fluid: studies on experimentally induced cysts in the rat. Endocrinology 75:733, 1964 23. Huacuja L, 80sa A, Delgado NM, Rosado A: A kinetic study of the participation of zinc in human spermatozoa metabolism. Life Sci 13:1383, 1973 24. Delgado NM, Huacuja L, Pancardo RM, Rosado A: Modification of human sperm metabolism by the induced release of intracellular zinc. Life Sci 16:1483, 1975 25. Rosado A, Hicks JJ, Reyes A, Blanco I: Capacitation in vitro of rabbit spermatozoa with cyclic adenosine monophosphate and human follicular fluid. Fertil Steril 25: 821, 1974 26. Keller DW, Polakoski KL: L-Arginine stimulation of human sperm motility in vitro. BioI Reprod 13:154, 1975 27. Rosado A: Estudio del metabolismo del espermatozoide humano. MSc thesis, Facultad de Medicina, Universidad Nacional Autonoma de Mexico, Mexico DF, 1970 28. Rosado A, Hicks JJ, Martinez-Zedillo G Bondani A Martinez-Manautou J: Inhibition of human ~perm motilit; by calcium and zinc ions. Contraception 2:259, 1970 29. Reyes A, Mercado E, Rosado A: Inhibition of capacitation and of the fertilizing capacity of rabbit spermatozoa by blocking membrane sulfhydryl groups. In Recent Advances in Human Reproduction, Excerpta Medica Int Congr Ser 370, 1974, P 322 30. Blandau RJ: Gamete transport-comparative aspects. In The Mammalian Oviduct: Comparative Biology and Methodology, Edited by ESE Hafez, RJ Blandau. Chicago, University of Chicago Press, 1969, p 129