Immunologic Studies on Human Follicular Fluid*

.

Vol. 22, No.6, June 1971

FERTD..ITY AND STERD..ITY

Copyright

©

1971 by The Williams & Wilkins Co.

Printed in U.S.A.

IMMUNOLOGIC STUDIES ON HUMAN FOLLICULAR FLUID* SOL MANARANG-PANGAN, M.D., M.S.,t

AND

ALAN C. MENGE, PH.D.

Center for Research in Reproductive Biology, Department of Obstetrics and Gynecology, University of Michigan Medical Center, Ann Arbor, Michigan

Recently, Herve, Sergent, and Roheyl reported the immunologic detection of numerous follicular fluid components in the cervical mucus of women collected on the day of ovulation. Using antisera prepared against human normal serum and follicular fluid, they showed that at times other than ovulation, the cervical mucus reacted with none to two antigens in agar-gel diffusion and immunoelectrophoresis. Three to four precipitation lines which remained after absorption of the antiserum with various body tissues were thought to be due to antigens specific to follicular fluid. The presence of antigens peculiar to follicular fluid and absent in blood plasma offers interesting possible applications. Other investigators, 2, 3 however, have not been able to demonstrate specific antigens. Studies on the physical, chemical, and immunologic properties of bovine, porcine, and mare follicular fluids have been reported. 2-6 Compared to serum, follicular fluid had less proteins, with some minor quantitative differences in the individual components. However, the major macromolecular components were electrophoretically and immunologically similar. One precipitation line common to follicular fluid and plasma, but not found in serum, was shown to be fibrinogen. The {31ipoproteins, which have about the largest molecular weights of the serum proteins, were not found in follicular fluid by Caravaglios and * This work was supported by the Agency for International Development, PIOIP 492-90165, and the Ford Foundation, Grant 37482. t Present address: Department of Obstetrics and Gynecology, University of the Philippines, Philippine General Hospital Medical Center, Taft Avenue, Manila, The Philippines.

Cilotti,4 upon staining the electrophoretic strips for lipids. Shivers, Metz, and LutwakMann 3 likewise noted two antigens in serum not found in porcine follicular fluid, one of which could be {3 lipoprotein. Using human material, this study compares follicular fluid to serum and plasma, with regard to total protein content and electrophoretically, and immunologically detectable components. One objective was to determine if there are specific antigens in follicular fluid not found in blood plasma. MATERIALS AND METHODS

Follicular fluid, free of blood was aspirated from ovaries of 10 women at laparotomy for various benign uterine conditions. The ovaries were all grossly normal except one with a large follicular cyst. This cyst fluid was of a deeper yellow compared to the light straw color ofthe other specimens. It was treated separately from the rest, which were pooled. An aliquot of this pooled sample was concentrated to half its original volume by vacuum dialysis in a collodion bag. The human sera and plasma samples, obtained by venipuncture, were pooled. Lyophilized human fibrinogen was dissolved in saline and used at a concentration of 4 mg. of protein/ml. The antisera to pooled human follicular fluid were prepared in four rabbits. After taking preimmunization blood samples, 1 ml. of an emulsion of equal volumes of pooled follicular fluid and Freund's complete adjuvant was injected in numerous intramuscular and subcutaneous sites, once weekly for 3 weeks. Two weeks later, a booster injection' was given in the same manner. Antiserum (AFF) obtained a week

367

368

Vol. 22

MANARANG-PANGAN AND MENGE

Two aliquots were taken from each antifollicular-fluid serum, one absorbed with pooled human serum, the other absorbed with pooled human plasma (AFF ab/S and AFF ab/P, respectively). Absorption was done by incorporating the antigens in polyacrylamide gel as described by Bemfeld and Wan. 8 The follicular fluid, serum, and plasma were electrophoresed on cellulose polyacetate (Sepraphore III, Gelman Instrument Co., Ann Arbor, Mich.) strips with Tris-Barbitol (Gelman HR) buffer, 0.05 M, pH 8.6. The strips were run for 2 hr. at 130 volts, 1 ma./strip, stained with Ponceau S, cleared and read in a Photovolt Densicord recording electrophoresis densitometer. Passive hemagglutination tests 9 using tanned sheep red blood cells coated with follicular-fluid antigens, agar-gel immunodiffusion,10 and immunoelectrophoresis l l were performed with the four antifollicular TABLE l. Hemagglutirwtion Tests of Antisera to

Human Follicular Fluid FIG. l. Electrophoretic patterns of human (a) serum, (b) plasma, (c) normal follicular fluid, and (d) follicular cyst fluid. Arrow indicates probable location of fibrinogen.

after the booster injection was used in the immunologic tests. The sera were studied individually. All the above materials were centrifuged at 15,000 r.p.m. for 10 min. at 0° C., dialyzed against 0.85% saline for 2 days in the cold (except the follicular-fluid aliquot used as antigen in the hemagglutination tests), and stored at - 20° C. until used. Rabbit antihuman fibrinogen (Hyland Laboratories, Los Angeles, Calif.) was made specific by absorption with pooled human serum. The total protein contents of the follicular fluids, serum, plasma, and fibrinogen solution were determined by the biuret procedure described in Kabat and Mayer, 7 using a bovine serum albumin standard.

Serum

No. of tubes

Preimmunization serum: No.1 No.2 No.3 No.4

positive*

o

o o o

Immune serum: No.1 No.2 No.3 No.4

17 17 16 16

Immune serum absorbed/serum: No.1 No.2 No.3 No.4

15 14 14 15

Immune serum absorbed/plasma: No.1 No.2 No.3 No.4

11 7 10 8

* Serum dilution in the first tube is 1: 50.

r

June 1971

precipitation lines were allowed to form for 3 days, washed in 0.85% saline for 3 days, and then stained with Amido black. RESULTS

FFe

A

•

1

369

HUMAN FOLLICULAR FLUID

B FIG. 2. A, immunodiffusion in agar gel. Center well; antifoilicular fluid (AFF). Peripheral wells; follicular fluid twice concentrated (FFc) , plasma (P), pooled follicular fluid (FF) , and fibrinogen (F). B, immunodiffusion in agar gel. Center well; antifollicular fluid absorbed with serum (AFFab/S). Peripheral wells; serum (S), follicular fluid twice concentrated (FFc), antifibrinogen absorbed with serum (AFab/S), pooled follicular fluid (FF), and fibrinogen (F).

fluid sera, before and after absorption with serum and plasma. The antigens used were pooled follicular fluid, pooled follicular fluid twice concentrated, follicular cyst fluid, serum, plasma, and fibrinogen. The

Protein Content. The biuret method for total protein determination gave values of 35.8, 57.7, and 75.7 mg. of protein/ml. for the pooled follicular fluids, follicular cyst fluid, and twice concentrated fluid, respectively. The pooled sera and plasma samples had 67.5 and 75.7 mg./ml, respectively. Electrophoresis. The electrophoretic patterns on cellulose polyacetate strips (Fig. 1) show the relative quantities and mobilities of major components in serum, plasma, normal follicular fluid, and follicular cyst fluid. There is a striking similarity in the patterns produced by the normal follicle fluid and the cyst fluid. The follicular fluid components paralleled those in plasma, although plasma had quantitatively much more protein. Immunologic Tests. The hemagglutination titers of the four rabbit antisera to follicular fluid are shown in Table 1. All the preimmunization sera were negative. With a 1: 50 initial dilution, the postimmunization sera were positive up to 16-17 tubes. Absorption with pooled human serum reduced the titers by 1-2 tube dilutions. Absorption with pooled human plasma brought titers down by 5-9 tube dilutions. Completeion of absorption was checked by agar-gel diffusion and immunoelectrophoresis. The titers did not become negative although no more lines remained against follicular fluid or plasma, after plasma absorption. Double diffusion in agar showed varying numbers of precipitation lines with the different antisera. The serum with the greatest activity formed at least eight, nine, and nine lines against serum, plasma and follicular fluid, respectively (Fig. 2). It formed only one line with fibrinogen. All the lines against follicular fluid apparently fused

370

MANARANG-PANGAN AND MENGE

Vol. 22

+ AF~

FIG. 3. Immunoelectrophoresis. Antigens in circular wells, plasma (P), pooled follicular fluid (FF), and serum (S). Antisera in long troughs; antifollicular fluid (AFF), antifollicular fluid absorbed with serum (AFFab/S), and antifollicular fluid absorbed with plasma (AFFab/P).

June 1971

HUMAN FOLLICULAR FLUID

with those in plasma and serum with the exception of the fibrinogen line which was absent in serum. After absorption with serum, one line common to both follicular fluid, plasma, and fibrinogen remained. This formed a line of identity with the single line formed between antihuman fibrinogen and follicular fluid and plasma. Absorption with plasma removed all precipitation lines. Immunoelectrophoresis essentially confirmed these results, but gave a better resolution of the number of lines formed with the unabsorbed antisera (Fig. 3). At least 11 lines appeared against serum; and at least 12 against both follicular fluid and plasma, in the antiserum with the most lines. This particular serum however, did not show antibodies to albumin, whereas the other three antisera produced heavy precipitin lines against albumin. DISCUSSION

The protein content of human follicular fluid was about half that of plasma. The immunologic and electrophoretic procedures performed did not demonstrate any antigen in the follicular fluid not found in plasma. The single component in both plasma and follicular fluid, and absent in serum, was shown to be fibrinogen. These findings are in agreement with the results of Shivers et at.,3 on porcine, and of Desjardin, Kirton, and Hafs 2 on bovine follicular fluid. The concept that follicular fluid is a transudate from blood, rather than a secretion of the granulosa cells, is gaining more experimental support. Clark 12 described the structural basis for this in his detailed study of the blood vessels of the human ovary from infancy to menopause. He describes a rich "follicular wreath" of vessels surrounding each growing follicle like a net, between the theca interna and the granulosa. This capillary wreath reaches maximum vascularization at ovulation. The ves-

371

sels do not invade the granulosa until after ovulation, when luteinization starts. Similar patterns have been described in the ovaries of other animals. 13·16 Von Kaulla, Aikawa, and Pettigrew 17 showed that radioactive tracers and labeled drugs administered intravenously to women prior to hysterectomy penetrated into the follicular fluid and may even be concentrated therein. Peckham and Kiekhofer 18 demonstrated the movement of tritiated water from the blood into the follicular fluid of women at laparotomy. Equilibration occurred rapidly, between 15 and 25 min. after intravenous administration of the tritiated water. On the basis of such speed of equilibration, they suggested that the diffusion rate was probably limited by the vascularity of the structure rather than by any "membrane effect" between blood and follicular fluid. Fluorescence-labeled proteins, 19. 20 likewise easily traverse through the granulosa of a growing follicle. In heifers immunized against semen, specific immune globulins were present in the follicular fluid. 21 Yatvin and Leathem 22 clearly demonstrated transudation of protein into experimentally induced follicle cysts in the rat. They made use of the abnormal changes in the serum proteins in rats made hypothyroid, fed labeled amino acids, and then injected with gonadotropins. The appearance of and electrophoretic distribution of radioactivity in the serum proteins paralleled that in the follicular fluid proteins. Likewise, CR51_ labeled proteins injected intracardially, appeared within 4 hr. in the cyst fluid fractions corresponding to those labeled in the serum. With the results of this study, it is not possible to discount the theoretical possibility of granulosa cell-specific proteins getting into the follicular fluid. However, if this does happen, it must be to a minor degree and not the primary source of follicular fluid proteins. The hemagglutina-

372

MANARANG-PANGAN AND MENGE

tion tests done on unabsorbed and absorbed antisera are of limited value in this respect. As these tests are more sensitive than immunologic precipitation, the positive titers remaining after absorption with plasma may have resulted from incomplete absorption. Another possibility, however, is the presence of nonprecipitating antibodies to certain follicular fluid antigens not found in plasma.

chemistry. Charles C Thomas, Springfield, TIl., 1961, p. 559. 8. BERNFELD, P., AND WAN, J. Antigens and enzymes made insoluble by trapping them into lattices of synthetic polymers. Science 145:678, 1963. 9. CAMPBELL, D. H., GARVEY, J. S., CREMER, N. E., AND SUSSDORF, D. H. Methods in Immunology. W. A. Benjamin, Inc., New York, 1963, p. 161. 10. OUCHTERLONY, O. Diffusion-in-gel methods for immunological analysis. Progr Allerg 5:1, 1958. 11. HIRSCHFELD, J. "Immunoelectrophoresis: procedure and application to the study of group specific variations in sera." In O. Ouchterlony's Handbook

of Immunodifussion and Immunoelectrophoresis.

SUMMARY

Immunodiffusion and immunoelectrophoresis, using rabbit antisera, and acetatestrip electrophoresis of human follicular fluid showed components identical to those in plasma. Total protein content of follicular fluid was approximately one-half that of plasma. These results indicate that follicular fluid is mainly derived from blood by transudation. Acknowledgments. The authors wish to thank Dr. S. J. Behrman for his guidance and Mrs. Mara Lieberman and Dr_ Y. Amano, for their technical advice and review of the manuscript. REFERENCES 1. HERVE, R, SERGENT, P., AND RoHEY, M. Anti· genic properties of the ovary. Their demonstration in ovarian tissue, follicular fluid and cervical mucus. Presse Med 76:2421, 1968. 2. DESJARDIN, C., KIRTON, K. R, AND HAFS, H. D. Some chemical, immilllOchemical and electrophoretic properties of bovine follicular fluid. J

12.

13.

14.

15.

16. 17.

18.

19.

Reprod Fertilll:237, 1966. 3. SHIVERS, C. A., Metz, C. B., AND LUTWAK-MANN, C. Some properties of pig follicular fluid. J Reprod FertiI8:115, 1964. 4. CARAVAGLIOS, R, AND CILOTTI, R A study of the proteins in the follicular fluid of the cow. J Endocr 15:273, 1957. 5. SHORT, R V. Steroids present in the follicular fluid ofthe mare. J Endocr 20:147, 1960. 6. ZACHARlAE, F., AND JENSEN, C. E. Studies on the mechanism of ovulation: Histochemical and physicochemical investigations on genuine follicular fluids. Acta Endocr (Kobenhavn) 27:343, 1958. 7. KABAT, E., AND MAYER, M. Experimental Immuno-

Vol. 22

20.

Science Pub., Inc., Ann Arbor, Mich., 1968, p. 139. CLARK, J. G. The origin, development and degeneration of the blood vessels of the human ovary. Johns Hopkins Hosp Rep 9:593, 1900. Basset, D. L. Changes in the vascular pattern of the ovary of the albino rat during the estrous cycle. Amer J Anat 73:251, 1943. BOLING, J. L., BLANDAU, R J., SODERWALL, A. L., AND YOUNG, W. C. Growth of the Graafian follicle and the time of ovulation in the albino rat. Anat Rec 79:313, 1941. DEANESLY, R The development and vascularization of the corpus luteum in the mouse and rabbit. Proc Roy Soc London [Bioi.) 107:60, 1930. MARKEE, J. E., AND HINSEY, J. C. Observations on ovulation in the rabbit. Anat Rec 64:309, 1936. VON KAULLA, K. N., AIKAWA, J. K., AND PETTIGREW, J. D. Concentration in the human ovarian follicular fluid of radioactive tracers and drugs circulating in the blood. Nature, (London) 182: 1238, 1958. PECKHAM, B., AND KIEKHOFER, W. The movement of tritium-labelled water in the human ovarian follicle. AmerJ Obset Gynec 78:1012, 1959. MANCINI, R E., VILAR, 0., GOMEZ, C., DELLACHA, J. M., DAVIDSON, O. W., AND CASTRO, A. Histological study of distribution of fluorescent serum proteins in connective tissue. J Histochem Cytochem 9:356, 1961. MANCINI, R E., VILAR, 0., DELLACHA, J. M., DAVIDSON, O. W., AND ALVAREZ, B. Extravascular distribution of fluorescent albumin, globulin, and fibrinogen in connective tissue. J Histochem Cyto-

chem 10:194, 1962. 21. MENGE, A. C. Immune reactions and infertility.

J Reprod Fertil {Suppl. 10):171, 1970. 22. YATVIN, M. B., AND LEATHEM, J. H. Origin of ovarian cyst fluid: Studies on experimentally induced cysts in the rat. Endocrinology 75:733, 1964.