Clin. exp. Immunol. (1990) 82, 359-362
Complement, complement activation and anaphylatoxins in human ovarian follicular fluid R. PERRICONE, C. DE CAROLIS, C. MORETTI*, E. SANTUARIt, G. DE SANCTIS & L. FONTANA Cattedra di Immunologia Clinica, Dipartimento di Chirurgia, II University of Rome 'Tor Vergata,' Rome, * V Clinica Medica, I University of Rome 'La Sapienza,' Policlinico Umberto I, Rome, and tSIFT, Rome, Italy (Acceptedfor publication 30 April 1990)
SUMMARY Functionally active complement was sought and detected in human follicular fluids obtained during the pre-ovulatory period. All the functional complement activities tested, including total haemolytic complement, classical pathway activity and alternative pathway activity were present in nine fluids from four different donors with values within the normal serum range. The immunochemical analysis demonstrated the presence of complement factors from C1 to C9, of B and of Cl INH, H, I. Complement anaphylatoxins were found employing RIA techniques in amounts significantly higher than in human plasma, thus demonstrating that follicular fluid complement, at least during the preovulatory period, is partially activated. A possible role for urokinase-like substances in such an activation was indicated by further in vitro experiments. The presence of active complement in follicular fluid can be relevant for the function of the enzymatic multi-factorial mechanism of ovulation. Keywords human ovarian follicular fluid ovulation complement complement activation anaphylatoxins INTRODUCTION Ovulation is the result of complex inter-related mechanisms initiated by the surge of luteinizing hormone and characterized by resumption of meiosis and germinal vesicle breakdown, initiation of luteinization of the granulosa cells, rupture of the follicle wall and release of a fertilizable ovum (Lipner, 1988). Hormonal control of ovulation and biochemical and morphological changes of the preovulatory follicle, including collagenolysis and vascular changes, have been extensively studied; however, many details concerning some of the mechanisms leading to follicular rupture are not clear. Many investigators directed their attention to follicular fluid (FF) as an enzyme reservoir controlling the permeability of the capillaries of the antrum and proteolitically degrading collagen thus leading to the rupture of the follicle wall. The plasminogenactivator-plasminogen hypothesis is the most recent and most likely explanation of the mechanism initiating the cascade that leads to follicular rupture (Beers, 1975; Strickland & Beers, 1976). The pre-ovulatory follicle stimulated by gonadotropins secretes an urokinase-type plasminogen activator; this can convert plasminogen to plasmin, a latent collagenase is activated by plasmin, attacks collagen and the resulting telopeptide-
free collagen can be attacked and degraded by non-specific proteases (Lipner, 1988). The decrease of the tensile strength of the follicle wall allows the rupture of the follicle by the action of the existing intrafollicular pressure (Blandau & Rumery, 1963; Bronson et al., 1979). Although this hypothesis of enzymatic multi-factorial mechanism of ovulation is corroborated by the study of some single steps of the cascade, it is conceivable that other steps will be discovered, thus leading to a more complete model for the pathophysiological events of ovulation. Several multi-factorial systems (e.g. kinins and fibrinolysis) that are present in human plasma have been demonstrated and characterized in human FF. It is well known that in human plasma kinins and fibrinolysis interact with the complement system (Ross, 1986). Complement, once activated through the classical or the alternative pathway, releases several biologically active molecules, anaphylatoxins, and is important in inflammation and in tissue damage (Ross, 1986). We report here that functionally active complement is present in FF and may mediate events leading to follicular rupture. MATERIALS AND METHODS Follicularfluids Collection of human FF was performed by laparoscopy, under direct observation, by means of the 'follicle aspiration set,'
Correspondence: Dr Roberto Perricone, Cattedra di Immunologia Clinica, Dipartimento di Chirurgia, II University of Rome 'Tor Vergata,' c/o Ospedale S. Eugenio, P. le Umanesimo 10, 1-00 144, Rome, Italy.
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(Sweden Lab), or by ultrasonically guided (by means of a vaginal transducer) puncture technique. FF were obtained from four women undergoing either in vitro fertilization (IVF) or gamete intrafallopian tube transfers (GIFT). They participated in the study for unexplained infertility (n = 2) or for infertility caused by endometriosis (n = 2). Patients' follicular function was normal as demonstrated by clinical, hormonal and ultrasonical studies. In view of IVF or GIFT procedures, ovarian stimulation for multiple follicular growth was induced in three donors with either human menopausal gonadotropins (Pergonal) or clomiphene citrate (Clomid) alone or in combination. In one donor ovulation was not stimulated by the use of any drug. For all complement studies, except for C3a, C4a and C5a assays, FF was used immediately after collection (without the addition of any substance); immediately after collection, aliquots of FF received EDTA (EDTA-FF) and were employed for the measurement of C3a, C4a and C5a concentrations. Sera Venous blood was collected and immediately centrifuged at 2000 rev/min. Plasma fractions were separated and allowed to clot at room temperature for 60 min. After coagulation, the clot was removed by means of a Pasteur pipette and the serum samples were used immediately. C3a, C4a and C5a were measured employing EDTA-plasma samples. Complement studies Buffers. Veronal-buffered saline, pH 75, 0-015 M, containing 0-1% gelatin, 0-00015 M Ca" and 0-0005 M Mg++ (GVB++) and veronal-buffered saline containing 0-01 M EDTA and 0.1% gelatin (EDTA-GVB) were prepared by standard methods (Rapp & Borsos, 1970). Veronal-buffered saline, pH 7.5, containing 0-02 M Mg+ + and 0-008 M EGTA (EGTA-VB) was prepared according to the method of Platts-Mills & Ishizaka (1974). Haemolytic assays. Total haemolytic complement (THC) was assayed by the method of Mayer (1961). Classical pathway activity (CPA) was determined by the technique of Takada et al. (1978). Briefly, 1 ml of optimally sensitized sheep erythrocytes (1 x 108) was incubated with 0 5 ml of serial dilutions of the serum sample for 15 min at 30°C, and subsequently with 1 ml of guinea pig serum diluted 1/25 with EDTA-GVB for a further 60 min at 37°C. After this, 2 5 ml of cold EDTA-GVB were added, the mixtures were centrifuged at 2000 rev/min for 10 min and the supernatant optical densities were evaluated at 414 nm. Alternative pathway activity (APA) was determined using rabbit erythrocytes in EGTA-GVB by the method Platts-Mills & Ishizaka (1974). A previously reported modification of this method measured the APA C3 convertase (2sAPA) (Perricone et al., 1987; Carini et al., 1989). Haemolytic C4 activity (hC4) was measured using agarose plates containing sensitized erythrocytes and ammonia-treated guinea pig serum (C4 depleted) (Brown & Hobart, 1977). Haemolytic complement factor B activity (hB) was measured by the haemolytic radial diffusion technique of Martin et al. (1976). Immunochemical analysis. Levels of Cl INH, C3, C4 and B were measured by the single radial immunodiffusion technique (RID) with monospecific antisera (Mancini, Carbonara & Heremans, 1965). The antisera directed against C I NH, C3, C4 were purchased from the Behring Institute. The antiserum directed against B was purchased from Miles. The presence of
split products ofC3 and B was investigated with immunoelectrophoresis (IEP) and counter-immunoelectrophoresis (CIE). IEP was performed according to the method of Scheidegger (1955) in 1% agarose containing 1 0 mm EDTA, for 120-150 min at 5 mA constant current/slide. CIE was carried out according to Arroyave & Tan (1976, 1977) in 0-6% agarose containing 40 mM EDTA. The antisera employed in IEP were anti-C3 and anti-B (Behring Institute); the antiserum employed in CIE was anti-C3. These antisera were found to contain antibodies directed against the native molecules as well as the respective split products. Anaphylatoxins concentrations. Levels of C3a, C4a and C5a were measured in EDTA-plasma samples and in EDTA-FF samples by means of RIA commercially available assays (Amersham). In vitro study Urokinase (5000 U in 0-1 ml volume) was added to 0 4 ml volume of human FF and incubated in a water bath at 37°C for 60 min. Controls were performed by replacing FF with the respective donor sera or by replacing urokinase preparations with solutions identical in salt concentrations but urokinasefree. These experiments were performed in triplicate. After incubation, THC, CPA and APA were assayed, as well as the presence of cleavage fragments of C3.
Statistical analysis A computer-assisted statistical analysis program was used for the statistical evaluation of the data. Statistical evaluation of the data included paired and unpaired Student's t-tests where appropriate (P < 0 05).
RESULTS
The results obtained measuring functional complement by means of a variety of techniques, including THC, CPA, APA, 2sAPA, hC4, hB, are reported in Table 1. As can be seen, all the functional complement activities tested were present in all the FF studied. The values obtained were not different (except CPA, P
Complement, complement activation and anaphylatoxins in human ovarian follicular fluid R. PERRICONE, C. DE CAROLIS, C. MORETTI*, E. SANTUARIt, G. DE SANCTIS & L. FONTANA Cattedra di Immunologia Clinica, Dipartimento di Chirurgia, II University of Rome 'Tor Vergata,' Rome, * V Clinica Medica, I University of Rome 'La Sapienza,' Policlinico Umberto I, Rome, and tSIFT, Rome, Italy (Acceptedfor publication 30 April 1990)
SUMMARY Functionally active complement was sought and detected in human follicular fluids obtained during the pre-ovulatory period. All the functional complement activities tested, including total haemolytic complement, classical pathway activity and alternative pathway activity were present in nine fluids from four different donors with values within the normal serum range. The immunochemical analysis demonstrated the presence of complement factors from C1 to C9, of B and of Cl INH, H, I. Complement anaphylatoxins were found employing RIA techniques in amounts significantly higher than in human plasma, thus demonstrating that follicular fluid complement, at least during the preovulatory period, is partially activated. A possible role for urokinase-like substances in such an activation was indicated by further in vitro experiments. The presence of active complement in follicular fluid can be relevant for the function of the enzymatic multi-factorial mechanism of ovulation. Keywords human ovarian follicular fluid ovulation complement complement activation anaphylatoxins INTRODUCTION Ovulation is the result of complex inter-related mechanisms initiated by the surge of luteinizing hormone and characterized by resumption of meiosis and germinal vesicle breakdown, initiation of luteinization of the granulosa cells, rupture of the follicle wall and release of a fertilizable ovum (Lipner, 1988). Hormonal control of ovulation and biochemical and morphological changes of the preovulatory follicle, including collagenolysis and vascular changes, have been extensively studied; however, many details concerning some of the mechanisms leading to follicular rupture are not clear. Many investigators directed their attention to follicular fluid (FF) as an enzyme reservoir controlling the permeability of the capillaries of the antrum and proteolitically degrading collagen thus leading to the rupture of the follicle wall. The plasminogenactivator-plasminogen hypothesis is the most recent and most likely explanation of the mechanism initiating the cascade that leads to follicular rupture (Beers, 1975; Strickland & Beers, 1976). The pre-ovulatory follicle stimulated by gonadotropins secretes an urokinase-type plasminogen activator; this can convert plasminogen to plasmin, a latent collagenase is activated by plasmin, attacks collagen and the resulting telopeptide-
free collagen can be attacked and degraded by non-specific proteases (Lipner, 1988). The decrease of the tensile strength of the follicle wall allows the rupture of the follicle by the action of the existing intrafollicular pressure (Blandau & Rumery, 1963; Bronson et al., 1979). Although this hypothesis of enzymatic multi-factorial mechanism of ovulation is corroborated by the study of some single steps of the cascade, it is conceivable that other steps will be discovered, thus leading to a more complete model for the pathophysiological events of ovulation. Several multi-factorial systems (e.g. kinins and fibrinolysis) that are present in human plasma have been demonstrated and characterized in human FF. It is well known that in human plasma kinins and fibrinolysis interact with the complement system (Ross, 1986). Complement, once activated through the classical or the alternative pathway, releases several biologically active molecules, anaphylatoxins, and is important in inflammation and in tissue damage (Ross, 1986). We report here that functionally active complement is present in FF and may mediate events leading to follicular rupture. MATERIALS AND METHODS Follicularfluids Collection of human FF was performed by laparoscopy, under direct observation, by means of the 'follicle aspiration set,'
Correspondence: Dr Roberto Perricone, Cattedra di Immunologia Clinica, Dipartimento di Chirurgia, II University of Rome 'Tor Vergata,' c/o Ospedale S. Eugenio, P. le Umanesimo 10, 1-00 144, Rome, Italy.
359
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R. Perricone et al.
(Sweden Lab), or by ultrasonically guided (by means of a vaginal transducer) puncture technique. FF were obtained from four women undergoing either in vitro fertilization (IVF) or gamete intrafallopian tube transfers (GIFT). They participated in the study for unexplained infertility (n = 2) or for infertility caused by endometriosis (n = 2). Patients' follicular function was normal as demonstrated by clinical, hormonal and ultrasonical studies. In view of IVF or GIFT procedures, ovarian stimulation for multiple follicular growth was induced in three donors with either human menopausal gonadotropins (Pergonal) or clomiphene citrate (Clomid) alone or in combination. In one donor ovulation was not stimulated by the use of any drug. For all complement studies, except for C3a, C4a and C5a assays, FF was used immediately after collection (without the addition of any substance); immediately after collection, aliquots of FF received EDTA (EDTA-FF) and were employed for the measurement of C3a, C4a and C5a concentrations. Sera Venous blood was collected and immediately centrifuged at 2000 rev/min. Plasma fractions were separated and allowed to clot at room temperature for 60 min. After coagulation, the clot was removed by means of a Pasteur pipette and the serum samples were used immediately. C3a, C4a and C5a were measured employing EDTA-plasma samples. Complement studies Buffers. Veronal-buffered saline, pH 75, 0-015 M, containing 0-1% gelatin, 0-00015 M Ca" and 0-0005 M Mg++ (GVB++) and veronal-buffered saline containing 0-01 M EDTA and 0.1% gelatin (EDTA-GVB) were prepared by standard methods (Rapp & Borsos, 1970). Veronal-buffered saline, pH 7.5, containing 0-02 M Mg+ + and 0-008 M EGTA (EGTA-VB) was prepared according to the method of Platts-Mills & Ishizaka (1974). Haemolytic assays. Total haemolytic complement (THC) was assayed by the method of Mayer (1961). Classical pathway activity (CPA) was determined by the technique of Takada et al. (1978). Briefly, 1 ml of optimally sensitized sheep erythrocytes (1 x 108) was incubated with 0 5 ml of serial dilutions of the serum sample for 15 min at 30°C, and subsequently with 1 ml of guinea pig serum diluted 1/25 with EDTA-GVB for a further 60 min at 37°C. After this, 2 5 ml of cold EDTA-GVB were added, the mixtures were centrifuged at 2000 rev/min for 10 min and the supernatant optical densities were evaluated at 414 nm. Alternative pathway activity (APA) was determined using rabbit erythrocytes in EGTA-GVB by the method Platts-Mills & Ishizaka (1974). A previously reported modification of this method measured the APA C3 convertase (2sAPA) (Perricone et al., 1987; Carini et al., 1989). Haemolytic C4 activity (hC4) was measured using agarose plates containing sensitized erythrocytes and ammonia-treated guinea pig serum (C4 depleted) (Brown & Hobart, 1977). Haemolytic complement factor B activity (hB) was measured by the haemolytic radial diffusion technique of Martin et al. (1976). Immunochemical analysis. Levels of Cl INH, C3, C4 and B were measured by the single radial immunodiffusion technique (RID) with monospecific antisera (Mancini, Carbonara & Heremans, 1965). The antisera directed against C I NH, C3, C4 were purchased from the Behring Institute. The antiserum directed against B was purchased from Miles. The presence of
split products ofC3 and B was investigated with immunoelectrophoresis (IEP) and counter-immunoelectrophoresis (CIE). IEP was performed according to the method of Scheidegger (1955) in 1% agarose containing 1 0 mm EDTA, for 120-150 min at 5 mA constant current/slide. CIE was carried out according to Arroyave & Tan (1976, 1977) in 0-6% agarose containing 40 mM EDTA. The antisera employed in IEP were anti-C3 and anti-B (Behring Institute); the antiserum employed in CIE was anti-C3. These antisera were found to contain antibodies directed against the native molecules as well as the respective split products. Anaphylatoxins concentrations. Levels of C3a, C4a and C5a were measured in EDTA-plasma samples and in EDTA-FF samples by means of RIA commercially available assays (Amersham). In vitro study Urokinase (5000 U in 0-1 ml volume) was added to 0 4 ml volume of human FF and incubated in a water bath at 37°C for 60 min. Controls were performed by replacing FF with the respective donor sera or by replacing urokinase preparations with solutions identical in salt concentrations but urokinasefree. These experiments were performed in triplicate. After incubation, THC, CPA and APA were assayed, as well as the presence of cleavage fragments of C3.
Statistical analysis A computer-assisted statistical analysis program was used for the statistical evaluation of the data. Statistical evaluation of the data included paired and unpaired Student's t-tests where appropriate (P < 0 05).
RESULTS
The results obtained measuring functional complement by means of a variety of techniques, including THC, CPA, APA, 2sAPA, hC4, hB, are reported in Table 1. As can be seen, all the functional complement activities tested were present in all the FF studied. The values obtained were not different (except CPA, P
