COLLAGENOLYTIC ENZYME ACTIVITY IN HUMAN OVARY: AN OVULATORY ENZYME SYSTEM

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1 FERTILITY AND STERILITY Copyright c 1981 The American Fertility Society Vol. 36, No. 6, December 1981 Printed in U.SA. COLLAGENOLYTIC ENZYME ACTIVITY IN HUMAN OVARY: AN OVULATORY ENZYME SYSTEM MAN ABU FUKUMOTO, M.D. *t YASUO YAJIMA, M.D.* HITOSHI OKAMURA, M.D.* OSAMU MIDORIKA WA, M.D. t Department of Pathology and Department of Gynecology and Obstetrics, Faculty of Medicine, Kyoto University, Kyoto, Japan Collagenolytic enzyme activities presumed to play an important role in ovulation were investigated in the human follicular apex, base, and granulosa cell layer throughout the ovarian cycle. Those analyzed were human ovarian collagenase, 2,4-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg-OH peptidase, N-carbobenzoxy Gly-Pro-Gly-Gly-Pro-Ala peptidase, and a-n-benzoyl-dl-arginine ~-naphthylamide hydrolase. Collagenase activity was also measured in human granulosa cell cultures. The activities of all four enzymes showed a marked preovulatory decrease in the apex. The activities in the apex were slightly higher than those in the base throughout the ovarian cycle. However, the activities in the granulosa cell layer and collagenase activity in the granulosa cell cultures increased toward preovulation and decreased after ovulation. These findings suggest that collagenase is synthesized in the granulosa cells maximally at preovulation and is consumed in the follicular apex at the same time, resulting in collagen degradation and disruption of the follicular wall in collaboration with other collagenolytic enzymes investigated here. Fertil Steril 36:746, 1981 The disruption of connective tissue in the wall of the Graafian follicle is necessary for mammalian ovulation. Since Schochet 1 proposed that specific enzyme activity is responsible for the disruption of the swine follicle wall at ovulation, many studies on the relation between proteolytic enzyme activity and ovulation have been performed. Before rupture, the connective tissue of the rabbit follicle undergoes deterioration, and this change is associated with dissociation of the follicular collagen. 2 Collagenolytic enzyme activity in the rabbit ovary is observed with N-carbobenzoxy-Gly-Pro-Gly-Gly-Pro-Ala (GPGGPA) as the Received June 11, 1981; revised and accepted July 17, *Reprint requests: Manabu Fukumoto, M.D., Department of Pathology, Faculty of Medicine, Kyoto University, Sakyo-Ku, Kyoto 606, Japan. tdepartment of Pathology. *Department of Gynecology and Obstetrics. substrate, and this activity changes in relation to ovulation. 3 Moreover, collagen-analogous synthetic substrate, 2,4-dinitrophenyl (DNP) Pro-Leu-Gly-Ile-Ala-Gly-n-Arg peptide digestible activity is observed in the rat Graafian follicle. 4 Since collagen is the major component of connective tissue and the helical portion of native collagen at physiologic ph is initially degraded by "true" collagenase (EC ), the measurement of collagenase activity is essential for understanding the initial step of follicular rupture. Although experimental injection of bacterial collagenase into the rabbit follicles in the estrous stage induces rupture, 5 "true" collagenase activity has never been measured in the ovary. In this study, the enzyme activities of collagenase and a-n-benzoyl-dl-arginine 13-naphthylamide (BANA) hydrolase (BANA-hydrolase), which can degrade native collagen at acid ph, and of two other collagenolytic enzymes, DNP Pro-Gln-Gly-Ile-Ala-Gly-Gln-n-Arg-OH (DNP- 746

Vol. 36, No. 6 COLLAGENOLYTIC OVULATORY ENZYME SYSTEM 747 peptide) digestible activity (DNP-peptidase) and GPGGPA peptidase (G-Gase), were investigated in the human ovary throughout the ovarian cycle.

2 Vol. 36, No. 6 COLLAGENOLYTIC OVULATORY ENZYME SYSTEM 747 peptide) digestible activity (DNP-peptidase) and GPGGPA peptidase (G-Gase), were investigated in the human ovary throughout the ovarian cycle. Collagenase activity in human granulosa (G) cell cultures throughout the ovarian cycle was also determined to clarify further the significance of the enzyme activity in human ovulation. MATERIALS AND METHODS Ovarian tissues were obtained from women at their reproductive age, with their written consent, during hysterectomy for myoma uteri, carcinoma in situ, etc. Their menstrual cycles were confirmed to be regular. The staging of the ovaries was determined according to cycle date, diameter of the follicles, macroscopic findings, and histochemical findings 6 (Table 1). Enzyme Activities in the Ovarian Tissues A follicle, corpus hemorrhagicum, or corpus luteum was enucleated from the ovary under a stereoscopic dissecting microscope. The follicle and corpus hemorrhagicum were further separated into G cell layer, apical wall of the follicle (apex), and basal wall of the follicle (base). Macroscopically yellowish corpus luteum was taken en bloc. The obtained tissues were further divided in two, one part for collagenase and DNP-peptidase activity assay and the other for G-Gase and BAN A hydrolase activity assay. Collagenase and DNP-Peptidase Activity Assay The tissues were thoroughly washed with 50 mm Tris-HCl buffer (ph 7.6) containing 0.15 M NaCl and 5 mm CaCl 2 (assay buffer) and homogenized in the assay buffer with a glass homogenizer and a Teflon homogenizer to a concentration of 50 mg tissue/mi. The homogenates were incubated at 42 C for 30 minutes for heat shock 7 and cooled, then centrifuged at 15,000 x g for 30 minutes. The supernatants were passed through Millex-HA filter (pore size 45!J.m, Millipore Corp., Bedford, Mass.). The filtrates were lyophilized and stored at - 20 C. When enzyme assay was performed, preparations were dissolved in the assay buffer to fivefold concentration of the initial homogenates and used as crude enzyme solution for collagenase and DNP-peptidase. [ 14 C]glycine-labeled collagen for collagenase activity assay e 4 C-collagen) was prepared by the method of Nagai and Hori. 7 Substrate, 2000 cpm, was preincubated at 37 C for 1.5 hours for fibril TABLE 1. Staging of the Follicles Stage Middle follicle (MF) Late follicle (LF) Preovulation (Pre-ov) Ovulation (Ov) Early corpus luteum (EL) Corpus luteum spurium (CL) Criteria Diameter of follicle < 1 em Cycle date is considered Diameter of follicle > 1 em 3J3-HSOO activity in Gb cell layer (-) Apex: not transparent Diameter offollicle > 1.5 em 3J3-HSD activity in G cell layer (+) Apex: thin, transparent Bleeding in follicle or G cell layer(-) _ Diameter of follicle > 1.5 em Apex: thin, transparent Bleeding in follicle or G cell layer ( +) Apex: disrupted Shape of follicle is preserved Yellowish corpus luteum 3J3-HSD activity ( +) Cycle date is considered a3j3-hydroxysteroid hydrogenase activity was histochemically determined on frozen sections by the method of Goldberg et al. 6 bgranulosa_ formation, then incubated with 0.2 ml of the enzyme solution for 4 hours. After centrifugation at 1500 x g for 20 minutes, 0.2 ml of the supernatant was mixed with 0.2 ml of 1 M NaOH, and radioactivity was counted in a Tri-Carb liquid scintillation spectrometer (Packard Instrument Co., Inc., Downers Grove, Ill.) using 10 ml of Bray's solution. DNP-peptidase activity was assayed by a modified method of Morales et al. 4 The synthetic DNPpeptide (Peptide Institute, Protein Research Foundation, Osaka, Japan) was dissolved in the assay buffer to a concentration of 4.5 x 10-4 M and stirred for 72 hours in a cold room. The solution was centrifuged at 15,000 x g for 30 minutes, and the supernatant was used as the substrate for DNP-peptidase. The substrate solution (0.1 ml) was incubated with 0.1 ml of the enzyme solution at 37 C for 2 hours, the reaction was stopped by the addition of 0.2 ml of 1 M HCl, and cleaved peptide was extracted with 1 ml of ethyl acetate. The absorbance at 365 nm of the organic phase was measured. G-Gase and BANA-Hydrolase Activity Assay The tissues were washed with physiologic saline and homogenized in 0.25 M sucro~e containing 0.1% Triton-X 100 to a concentration of30 mg tissue/mi. The homogenates were centrifuged at 6000 x g for 10 minutes, and the supernatants were used as crude enzyme solutions. G-Gase ac-

3 748 FUKUMOTO ET AL. December ~ 60 ~'l. o M F LF Pno Ov EL CL (j) <Z> "' <Z> <Z> <Z> <Z> G-GASE!:~ ' 0 M F LF P,. Ov EL CL.. <ll a> Q) (j) <Z> Cll "1 SANA-HYDROLASE ~50 ~ ~ 40 ~~ 30 & 20 o MF LF Pno Ov EL CL MF LF Pno 0v EL CL e "i>oa> <~> <~>. "i><~>~~> Ill FIG. 1. Collagenolytic enzyme activities throughout the ovarian cycle. Enzyme activity in the apex (o), the base (e), and in the granulosa cell layer (x). Bars, mean - standard deviation. Circled numbers, number of materials. tivity was measured by the method of Espey and Rondell. 8 The enzyme solution was incubated with 0.5% GPGGPA (Sigma Chemical Co., St. Louis, Mo.) buffered with Tris (ph 8.0). G-Gase activity was expressed as absorbance at 570 nm of ninhydrin reacting peptide released from GPGGPA. BANA-hydrolase activity was assayed by the method of Barrett. 9 The enzyme hydrolyzes BANA at ph 6.0, and released 13-naphthylamine was coupled with fast garnet GBC (Sigma Chemical Co., St. Louis, Mo.). The activity was expressed as absorbance at 520 nm of resulting azo dye extracted by n-butanol. Collagenase Activity in Cultures G cells or corpus luteum cells were harvested from human ovaries by the method of Channing and Ledwitz-Rigby. 10 Cells (5 x 10 5 ) were seeded into plastic dishes (60 x 10 mm, Falcon Products, Oxnard, Calif.) containing 2 ml of Eagle's minimal essential medium and were incubated at 37 C in an atmosphere of5% C0 2 in air. The medium was changed every 24 hours, and the conditioned medium was centrifuged at 300 x g for 10 minutes to remove cell debris. The supernatant was dialyzed against 30!J.M Tris (ph 7.2) and lyophilized. The preparation was dissolved in 0.3 ml of the assay buffer, and collagenase activity was assayed by the above method with the use of 14 C collagen. RESULTS The stoichiometric study of collagenase and DNP-peptidase activity was confirmed by the use of a preovulatory follicle (cycle day 13) as the enzyme source. Collagenase was totally inactivated by boiling for 10 minutes and was inhibited by 25 mm ethylenediaminetetraacetic acid (EDTA) or 12 mm cysteine, and linearity remained within 3 mg of protein for 4 hours of incubation. DNPpeptidase was also inactivated by boiling and was inhibited by 50 mm EDTA or 12 mm cysteine, and linearity remained up to 0.5 mg of protein for 2 hours of incubation. As shown in Figure 1, the activity of the four enzymes in the apex tended to be somewhat higher than those in the base, and we found that the decrease at preovulation and the increase to the restoration levels after ovulation were significant in the apex (collagenase; P < 0.05, n = 4, DNPpeptidase; P < 0.10, n = 7, BANA-hydrolase; P < 0.02, n = 8, G-Gase; P < 0.05, n = 16). The decrease at preovulation in the G cell layer was not clear. BANA-hydrolase and G-Gase activity reached a maximum during the luteal phase. Collagenase activity in the G cell cultures appeared at the late follicular stage, reached a maximum at preovulation, then decreased and disappeared during the corpus luteum stage (Table 2). DISCUSSION Ovulation is the result of complex events that occur sequentially: follicular maturation, selection of the preovulatory follicle(s), and disruption of the follicular wall. These events are coordinately controlled by gonadotropins, which induce ovulation. Because some of the local ovarian factors are essential to achieve disruption of the follicle, a proteolytic ovulatory enzyme that acts on the collagen framework of the follicle has been postulat- TABLE 2. Collagenase Activity in Human Ovarian Granulosa and Corpus Luteum Cell Cultures Stage No. Collagenase activity 1" 2 3 cpmlmg proteinlhr Middle follicle 1 - b Late follicle Preovulation Ovulation Corpus luteum 4 0 Days in culture. bnot detectable.

4 Vol. 36, No.6 COLLAGENOLYTIC OVULATORY ENZYME SYSTEM 749 ed. For instance, plasminogen activators and other collagenolytic enzymes have been investigated in detaily Rondell 12 could not demonstrate collagenase activity in hog follicles, presumably because he failed to lyophilize the enzyme solutions. Although DNP-peptide, which contains Gly-Ile bond, is regarded to be the specific substrate for collagenase, 13 we could not find any significant correlation between DNP-peptidase and collagenase activity (correlation coefficient r = 0.36, n = 34). Vaes et al. 14 have found a proteinase in cultures of mouse bone explants which degrades denatured collagen at neutral ph and is inhibited by EDTA or cysteine. DNP-peptide is composed of a single chain, which is much shorter than native collagen. Therefore, DNP-peptidase prqbably contains a peptidase(s) similar to that discovered by V aes et al. that serves as a collagenolytic enzyme after native collagen is initially attacked by collagenase. BANA-hydrolase is thought to be mixed activity of lysosomal collagenolytic enzymes, because BANA is sensitive to cathepsin H (EC ) as well as cathepsin B (EC ), 15 and both are known to be collagenolytic at acid ph. 16 BANA-hydrolase has maximal activity in the luteal phase; this coincides with the fact that lysosomal enzyme activity is closely related to degeneration and steroidogenesis in the corpus luteump The activity of BAN A-hydrolase also fluctuated throughout the ovarian cycle and was lowest at preovulation, like the other collagenolytic enzymes investigated. This also suggests that BANA-hydrolase has functional activities in ovulation. The ph for the G-Gase activity assay was 8.0, different from the general properties of lysosomal proteolytic enzymes. However, there are several reports in which certain lysosomal enzymes are considerably collagenolytic even at neutral ph,l 8 19 and GPGGPA can be digested by hyaluronidase (EC ) at ph Together with our findings that G-Gase activity was in parallel with BANA-hydrolase activity and was maximal during the luteal phase, it is speculated that G Gase is a lysosomal enzyme(s). As was shown by G cell cultures, the production of active collagenase was highest in the preovulatory phase, while collagenase activity in the follicle was lowest in the same phase, implying that collagenase is produced in the G cells maximally at preovulation and immediately transported extracellularly, where it is consumed at the site of collagen degradation. This assumption is in good agreement with the morphologic findings in the postpartum rat uterus, where collagen breakdown is drastically accelerated. 20 Ultrastructural studies revealed that fibroblasts containing microvesicles and lysosomelike granules appear, and collagen fiber seems to be digested by their contents during ovulation in the rabbit 21 and human follic;les The three other enzyme activities investigated were closely coupled with that of collagenase, because they were also considered to be consumed at preovulation. It is thus concluded that the collagenolytic enzyme activity investigated in this study is part of a multienzyme system of ovulatory enzymes and that this system works actively on the disruption of the follicular wall during ovulation. However, it must be emphasized that the rate-controlling step in collagen degradation by the ovulatory enzyme system is the initial cleavage of native collagen by ovarian collagenase and that the other three peptidases observed here play a role in a sequential degradation mechanism of the reaction products of collagenase. Acknowledgment. We thank Dr. Shigeru Okada (Department of Pathology, Faculty of Medicine, Kyoto University) for pertinent advice and encouragement. REFERENCES 1. Schochet SS: A suggestion as to the process of ovulation and ovarian cyst formation. Anat Rec 10:447, Espey LL: Ultrastructure of the apex of the rabbit Graafian follicle during the ovulatory process. Endocrinology 81:267, Espey LL, Rondell P: Collagenolytic activity in the rabbit and sow Graafian follicle during ovulation. Am J Physiol 214:326, Morales Tl, Woessner JF, Howell DS, Marsh JM, LeMaire WJ: A microassay for the direct demonstration of collagenolytic activity in Graafian follicles of the rat. Biochim Biophys Acta 524:428, Espey LL, Lipner H: Enzyme-induced rupture of rabbit Graafian follicle. Am J Physiol 208:208, Goldberg B, Jones GES, Turner DA, Sarlos IJ, Horton EH: Steroid 3~-ol dehydrogenase activity in human endocrine tissues. Am J Obstet Gynecol 86:349, Nagai Y, Hori H: Vertebrate collagenase: direct extraction from animal skin and human synovial membrane. J Biochem 72:1147, Espey LL, Rondell P: Estimation of mammalian collagenolytic activity with a synthetic substrate. J Appl Physiol 23:757, Barrett AJ: A new assay for cathepsin B1 and other thiol proteinases. Anal Biochem 47:280, Channing CP, Ledwitz-Rigby F: Methods for assessing hormone-mediated differentiation of ovarian cells in culture and in short-term incubations. In Methods in Enzy-

5 750 FUKUMOTO ET AL. December 1981 mology, Vol 39, Edited by JG Hardmann, BW O'Malley. New York, Academic Press, 1975, p Bjersing L: Intraovarian mechanisms of ovulation. In Human Ovulation, Edited by ESE Hafez. Amsterdam, North Holland Publishing Co, 1979, p Rondell P: Biophysical aspects of ovulation. Biol Reprod (Suppl) 2:64, Masui Y, Takemoto T, Sakakibara S, Hori H, Nagai Y: Synthetic substrates for vertebrate collagenase. Biochem Med 17:215, Vaes G, Eeckhout Y, Lenaers-Claeys G, Fran~ois-Gillet C, Druetz J-E: The simultaneous release by bone explants in culture and the parallel activation of procollagenase and of a latent neutral proteinase that degrades cartilage proteoglycans and denatured collagen. Biochem J 172: 261, Singh H, Kalnitsky G: Separation of a new a-n-benzoylarginine-13-naphthylamide hydrolase from cathepsin B1: purification, characterization, and properties of both enzymes from rabbit lung. J Biol Chern 253:4319, Singh H, Kuo T, Kalnitsky G: Collagenolytic activity of lung BANA hydrolase and cathepsin Bl. In Protein Turnover and Lysosome Function, Edited by HL Segal, DJ Doyle. New York, Academic Press, 1978, p Strauss JF 111; Flickinger GL: A role for lysosomes in corpus luteum function. In Protein Turnover and Lysosome Function, Edited by HL Segal, DJ Doyle. New York, Academic Press, 1978, p Stevens FS, Torre-Blanco A, Hunter AA: A neutral protease in rheumatoid synovial fluid capable of attacking the telopeptide regions of polymeric collagen fibrils. Biochim Biophys Acta 405:188, Woessner JF Jr: Purification of cathepsin D from cartilage and uterus and its action on the protein-polysaccharide complex of cartilage. J Biol Chern 248:1634, Montfort I, Perez-Tamayo R: The distribution of collagenase in the rat uterus during postpartum involution: an immunohistochemical study. Connect Tissue Res 3:245, Espey LL: Decomposition of connective tissue in rabbit ovarian follicles by multivesicular structures of thecal fibroblasts. Endocrinology 88:437, Okamura H, Takenaka A, Yajima Y, Nishimura T: Ultrastructural study of the ovarian follicular apex in human tissue. Fertil Steril 30:729, Okamura H, Takenaka A, Yajima Y, Nishimura T: Ovulatory changes in the wall at the apex of the human Graafian follicle. J Reprod Fertil 58:153, 1980

The Distribution of Collagenous Connective Tissue in Rat Ovarian Follicles

BIOLOGY OF REPRODUCTION 14, 502-506 (1976). The Distribution of Collagenous Connective Tissue in Rat Ovarian Follicles LAWRENCE L ESPEY Reproductive Physiology Laboratory, Trinity University, San Antonio,