Ovarian Proteolytic Enzymes and Ovulation

Transcription

1 BIOLOGY OF REPRODUCTION 10, (1974) Ovarian Proteolytic Enzymes and Ovulation LAWRENCE L. ESPEY Trinity University, San Antonio, Texas Mammalian ovulation is a dynamic phenomenon which requires disruption of the vascular system and displacement of the connective tissue in the wall of the Graafian follicle as the ovum is released. In most mammals, the whole follicle protrudes more markedly from the ovarian surface at the time of ovulation, and in many instances a thin translucent stigma, the macula pellucida, forms at the apex of the follicle as the final sign of impending rupture. This unusual morphological change has left a striking impression on those who have actually observed it. Kelly (1931) was so fascinated while observing a rabbit follicle near rupture he stated as tension within the follicle increases, the transparent portion around the pole begins to bulge.... It now stands out like the nipple on a breast. The moment of rupture sometimes appears as an explosive event, leading observers to compare it to a volcano erupting (Hill et al., 1935) or a blister that bursts (Blandau, 1966). There are several reports which provide detailed accounts of the macroscopic and microscopic changes that occur during this process (Walton and Hammond, 1928; Blandau, 1966). Until the past decade little progress has been made in the elucidation of the actual mechanism of follicular rupture. Lack of knowledge On this important event in the reproductive process is probably due to the fact that the experimental approach of most studies conducted prior to 1960 was limited to visual and histological observations of the follicle. But even the reports based on more comprehensive studies yielded indecisive opinions that the results of these experiments were either negative or so inconsistent that [the investigators] were unable to produce any conclusive evidence (Guttmacher and Guttmacher, 1921), and the problem still remains in this condition of uncertainty (Kraus, 1947). As Hisaw (1961) counseled a dozen years ago, the solution of the problem of ovulation may be found by investigating the basic physiological processes that go on in the follicle. The information presented in this symposium, which Dr. Nalbandov has so timely organized, reveals that steps have been taken in the right direction. NONENZYMATIC THEORIES Many hypotheses to explain ovulation were formulated prior to 1960, even though sounil experimental data were lacking. Hisaw (1947) has thoroughly reviewed the theories up to the middle of the twentieth century. Blandau and Rumery (1963) have extended the review through the next 15 yr, and Rondell (1970a) has covered the important literature during the past decade. There are numerous other reports which include extensive reference material on the mechanism of ovulation (Asdell, 1962; Blandau, 1966, 1967, 1968; Espey, 1964; Espey and Betteridge, 1970; Espey and Lipner, 1965). I shall not endeavor to itemize all the theories which have been published in this material; however, several points are noteworthy. Intrafollicular pressure. Up to 1963, the most popular speculations incorporated the idea that rupture resulted from an increase in intrafollicular pressure. This deduction 216 Copyright #{174} 1974 by The Society for the Study of Reproduction. All rights of reproduction in any form reserved.

2 PROTEOLYTIC ENZYMES AND OVULATION 217 was based primarily on the dynamic changes on the surface of the follicle at the time of ovulation. It is surprising this pressure theory was not challenged more seriously before the past decade. As early as 1919, Corner (1919) pointed out that the theca externa of the follicle is composed chiefly of collagenous fibrils and their associated fibroblasts. Because of the high tensile strength of this collagenous layer of the follicle wall, Guttmacher and Guttmacher (1921) could not induce rupture in sow follicles by maintaining a constant head of pressure of well over 300 mmllg in the ovarian arteries for hours at a time. They noted that the follicular vessels could be seen to wash out clearly but rupture did not occur in a single instance, even though one braced himself against a wall and pushed the piston of the injection syringe with all of the physical strength available. Yet, serious question of the pressure theories did not come until the 1960s when three separate studies (Blandau and Rumery, 1963; Espey and Lipner, 1963; Rondell, 1964) demonstrated that follicular pressure does not increase prior to ovulation. (This is not to irnpl\ that the low intrafollicular pressure which exists is not an essential hydrostatic force in the dissociation of a weakened follicle wall.) Smooth muscle. In 1849, Kolliker (1849) first mentioned smooth muscle as a structural constituent of the ovary. Several years later the hypothesis was formulated (Rouget, 1858; Grohe, 1863) that, in contracting, the muscle fibers of the ovary compressed the blood vessels, and the congestion from impaired venous return led to the rupture of the mature follicles. This idea has been supported for the past 100 yr by numerous histological reports (see Espey, 1964) of smooth muscle cells in the thecal tissue. However, observations with a polarizing microscope (Claes son, 1947) do not reveal smooth musculature in the follicle wall in cow, swine, rabbit, or guinea pig. Nor have ultrastructural studies of the rabbit (Espey, 1967a) or frog (Anderson and Yatvin, 1970) revealed smooth muscle cells in the ovarian follicle. Further challenge to the smooth muscle theory came from evidence that a wide variety of smooth muscle stimulants failed to induce contractile activity in sow follicles (Espey, 1964). In addition, recording of intrafollicular pressures during ovulation do not indicate that smooth muscle contractions are important in this process (Espey and Lipner, 1963). In spite of all the evidence to the contrary, during the past several years there has been a resurging interest in the possible role of smooth muscle cells in the mechanism of ovulation. New reports support the presence of this type of cell and the theory that they may play a role in the process of ovulation (O Shea, 1970) by causing follicular dehiscence and atusia (Fumagalli et al., 1971) through a contractile force which seems to be effective in dissociating the connective tissue (Okamura et at., 1972) leading to an opening of the stigma and the extrusion of the follicular contents (Palti and Freund, 1972). However, it is my opinion that none of the recent ultrastructural studies clearly demonstrate smooth muscle cells in the thecal layer of the follicle wall. In one case (Okamura et at., 1972), the electron micrograph which reportedly demonstrates a smooth muscle cell appears actually to be taken from a region of the ovarian stroma, rather than the theca folliculi. In the other reports, for the most part the investigators have failed to recognize that it is not uncommon to observe cytoplasmic filaments in fibroblasts (see Haust and More, 1967). I would not deny that the follicle wall may occasionally contain what appear to be myofibroblasts, but these cells are rare, and probably represent an anomaly which has no significant functional role in the mechanism of ovulation. Mechanical (mathematical) models. The mature ovarian follicle is a rigid sphere surrounded by a dense layer of collagenous

3 218 ESPEY connective tissue. An idealized mathematical and physical model of this sphere has been used by Rodbard (1968) to analyze the process of ovulation. He concluded (1) that by using the physical characteristics of the model the precise conditions for ovulation could be stated mathematically, (2) that as follicles reach a critical size the stigma formation may be explained by mechanical factors, and, finally, (3) that by providing a mechanical final common pathway, this approach has been able to reconcile previous conflicting data and theories concerning the events of ovulaton. However, his model is not only oversimplified, but based on many gross assumptions regarding the physiological processes within the ovarian follicle. Any mechanical model which is to be compared with the follicle during ovulation must incorporate the known changes in distensibility and breaking strength of the follicle wall (Rondell, 1970a). Lardner (personal communication from Dr. Thomas Lardner, Dept. Mech. Eng., M.I.T.) supports this position of Rondell regarding mathematical models. I believe that, because of the many assumptions which are necessary in the conversion of a living system into a mathematical model, it is doubtful this analytical approach will provide any useful information on the mechanism of rupture. DEGRADATION OF THE FOLLICLE Macroscopic changes. It is not difficult to recognize from macroscopic observations that there is a gross change in the integrity of the connective tissue of a follicle near rupture (unpublished). After handling hundreds of thousands of sow ovaries, it becomes obvious that near ovulation follicles are distinctly more flaccid: Exertion of only slight manual pressure causes such follicles to burst. As another example, if the wall of a rabbit follicle is penetrated with a micropipette (for determination of intrafollicular pressure, or for injection of a solution into the follicle antrum), negligible force is required to penetrate the surface of the follicle if it is close to rupture. Thirdly, when attempts are made to dissect whole follicles from the surface of the rabbit ovary, the follicles in precoital rabbits are difficult to extirpate because of connective tissue adhesions which interlace the follicular theca with the ovarian stroma. However, near rupture this c:innective tissue appears decomposed and the follicles peel out with minimal surgery. Collectively, these qualitative observations suggest there is active decomposition of the connective tissue in the ovary near rupture. Microscopic changes. Microscopic techniques have elucidated some of the transitions in the fine structure of the follicle wall as it approaches rupture. During ovulation in the rabbit, the thecal tissue appears to undergo significant deterioration (Espey, 1967a). This involves dissolution of the extracellular ground substance and dissociation of the follicular collagen. Not only is there a separation of the collagen fibers, but the cells also appear to be sparse in comparison to those in mature folicles distant from ovulation. In the minutes preceding rupture, the fibrous outer layers of the follicle wall thin to less than one-fifth their original width. This preovulatory thinning of the follicle wall has also been observed in the rat (Blandau, 1967). These observations suggested that lysosomal hydrolases might be active in the ovulatory process. However, examination of the ultrastructure of mature rabbit follicles in this laboratory has revealed negligible lysosomal bodies (Stutts, 1968). With the exception of the germinal epithelium, lysosomes were sparse in the cells of the follicle vall, nor did those present undergo any conspicuous changes during ovulation. Susequent studies (Espey, 1971a) of the ultrastructure of the Graafian follicle revealed intriguing multivesicular bodies which protrude from the follicular fibroblasts in increasing quantities as ovulation

4 PROTEOLYTIC ENZYMES AND OVULATION 219 approaches. These unusual structures are present in the tunica albuginea and theca externa of all stages of ovulatory follicles; however, there is a ninefold increase in their concentration prior to rupture. (Similar structures are occasionally present in the cells of the stratum granulosum, but there is no apparent change in their numbers during ovulation.) The frequency with which digested ground substance can be observed around the multivesicular structures indicates they might contain a chemical which can decompose follicular connective tissue. Current studies in this laboratory reveal that the contents in these structures are not comparable to lysosomes because they do not elicit a positive Gomori reaction for acid phosphatase. The evidence also shows that the multivesicular structures are frequently located in the leading edge of cytoplasmic processes which extend from the fibroblasts of follicular tissue (Espey, 1971b). This is especially apparent just after ovulation, when the thecal fibroblasts are proliferating into the lutein granulosa, suggesting they could be important in facilitating the amoeboid movement of fibroblasts through dense collagenous tissue in the follicle wall. Further support for a specific role for these structures in the decomposition of dense connective tissue comes from the recent observation that the collagen of the relaxed symphysis pubis of the guinea pig is also digested by their contents (Chihal and Espey, 1973). Vascular changes. The conspicuous changes in the vascular system of the follicles near rupture have been overlooked as evidence of active decomposition of the follicular tissue during ovulation. During the hours preceding rupture, there is an increase in the vascularization of the follicular dome in pigs (Birger, 1952; Betteridge and Raeside, 1962; Hunter, 1967), in rabbits (Burr and Davies, 1951; Espey, 1964; Blandau, 1967), in monkeys (jewett and Dukelow, 1971), and presumably in other mammals. In addition, as the time of nipture nears, it is common to observe petechiae (Birger, 1952; Espcy, 196Th; Blandau, 1967) and extravasation of blood into the follicular wall or antrum (Heape, 1905; Hill et al., 1933; Corner, 1919; Espey and Lipner, 1963). This traumatic change in follicular arterioles must include disruption of collagenous tissue because collagen fibers are found in all vessels, spread over the whole wall (see Bader, 1963), and the vessels of the Graafian follicle are no exception. Robb-Srnith (1952) found ovarian collagen to be most abundant in the blood vessels, tunica albuginea, theca folliculi, and loose meditilary stroma. Thus, the weakened condition of the vascular compartment during ovulation is probably a consequence of the same biochemical processes which lead to the decomposition of the rest of the connective tissue in the follicle wall. This deduction is supported by the report (Robb-Smith, 1952) that the collagen capsules of the ovarian blood vessels, tunica albuginea, and follicles (but not a fine interlacing network of fibrils in the medullary stroma) are all dissolved by collagenase. Tensile strength changes. Studies on the tensile strength of the follicle wall have revealed that the collagenous connective tissue in the follicle wall does indeed deteriorate as rupture nears. In 1964, I demonstrated that strips of prerupture follicles from sows are more easily stretched than sections of mature follicles more distant from ovulation (Espev, 1964). In that same year, Rondell (1964) used a completely different technique to repo;t an increase in distensibility of rabbit folhcles near ovulation. However, instead of measuring the distensibility of the follicle wall, his procedure may actually have detected changes in the permeability of the blood-liquor barrier in the follicle. Such a vascular change does occur (Zachariae, 1958), and his experimental technique appears to l)e designed to measure this change. In any case,

5 220 ESPEY it is now clear that the tensile strength of the collagenous tissue in the follicle wall does decrease near the time of rupture (Espey, 1967b). ENZYME THEORY Early studies. More than one-half century ago Schochet (1916) first suggested that proteolvtic enzymes weaken the follicle wall by digesting the theca folliculi in the region of the stigma. In testing this idea, Rugh (1935) found that the external application of solutions of pepsin and hydrochloric acid initiated follicular rupture in frogs, but neither pepsin alone nor trypsin produced this effect. Eleven years later, Moricard and Gothie (1946) revived Schochet s original hypothesis with tenuous evidence that gonadotropins cause the secretion of a diastase (in French usage this word can mean any kind of enzyme) containing proteolytic activity that digests the various follicular coatings and results in the opening of the ovarian follicle. In a major effort to clarify the issue, Kraus (1947) utilized the experimental approach of Rugh and confirmed that the follicle wall in the frog ovary is disrupted by immersion in a solution of pepsin-hci (but not in trypsin-sodium sulfate). However, her attempts to identify a proteolytic enzyme in frog follicles were without results, nor did the application of proteolytic enzymes to the surface of follicles of hens and rabbits produce any effect. She concluded that nothing in her results indicated that ovulation could be attributed to ordinary proteolytic enzyme activity. Intrafollicular injections of enzymcs. More recently, we have found that the injection of small quantities of concentrated enzyme preparations directly into the antrum of the rabbit Graafian follicle can cause morphological changes similar to normal swelling, stigma formation, and rupture of the Graaflan follicle (Espey and Lipner, 1965). Clostridiopeptidase-A (bacterial collagenase), nagarse and pronase (also microbial enzymes) were the most effective in inducing rupture. In the initial study, trypsin was only moderately effective, but more recently we have found that concentrated preparations of trypsin from bovine pancreas (Sigma, stock no. T-8253) are highly effective in causing rupture of rabbit follicles (unpublished). No response was elicited from chymotrypsin, crude peptidase, amino-peptidase, ficin, papain, lysozyme, hyaluronidase (hyase), and elastase (Espey and Lipner, 1965). Injections of the nonenzymic agents 5-hydroxytryptamine, polyvinylpyrrolidone, plasminogen, ascorbic acid, histamine, and heparin were without effect (unpublished). Effect of enzymes on tensile strength. In addition, a variety of chemicals have been tested for their effect on follicular connective tissue by incubating them with strips of sow follicles and then measuring the tensile strength of the follicular tissue (Espey, 1970). Under these conditions, preparations of collagenase, elastase, general protease, trypsin, alpha-chymotrypsin, and to a lesser extent beta-chymotrypsin, were effective in reducing the tensile strength of the follicle wall. Amino-peptidase, hyaluronidase, and alpha-amylase had no effect. In a group of nonenzymic agents that were tested, L-ascorbic acid was highly effective in decomposing the follicular tissue and reduced the tensile strength of the follicle wall to essentially zero after 10 h of incubation. However, the action of this vitamin required a very high concentration of hydrogen ions (ph 3.2). L-diketogulonic acid and L-cysteine both appeared to be slightly effective in reducing the tensile strength of the sow follicle, but L- 1,4-gulonolactone, D-gulonic acid, glutathione, serotonin, and histamine were without effect. These data provide two conclusions that are important in future evaluations of the physiology of ovulation: (1) it is clear that a variety of proteolytic enzymes can weaken the tensile strength of the follicle wall, and (2) at least one nonenzymic agent, ascorbic acid, has the capacity to decompose follicular connective

6 PROTEOLYTIC ENZYMES AND OVULATION 221 tissue (although it is doubtful this acid is present in sufficient concentration in the follicle to induce ovulation). Analyses of Ovarian Proteolytic Enzymes Diverse reports. Very few investigators have attempted to identify specific proteolytic enzymes in ovarian tissue. Acid protease, with optimal activity in the range of ph 3 to ph 4, is reportedly present in the follicular fluid of mature sow follicles (Jung and Held, 1959), in the follicular fluid of humans near ovulation (Jung, 1969), in the ovary of rats (Reichart, 1962), and in follicular homogenates of sows (Lee and Malvin, 1970). Blandau has also detected acid protease (proteinase) in the stigmal area (personal comniunication). Gonadotropins possibly cause a decrease in this activity (Reichart, 1962). Ovarian tissue also contains an acid protease which can cause autolytic decomposition of follicular tissue, with maximum activity at ph 5, as evidenced by appearance of soluble ninhydrin-positive particles in incubation media (Lee and Malvin, 1970) and reduction of tensile strength of follicles incubated in Tris-maleate buffer (Espey, 1970). Possibly this follicular activity is similar to collagenolytic activity (maximal at ph 5.5) found in various rat tissues (Etherington, 1973; Houck et at., 1967, 1970; Shaub, 1964). There are only two reports (Espey and Rondell, 1967; Reichart, 1962) of ovarian proteolytic enzymes with optimal activity around ph 8. Different substrates were used to study these enzymes, and the possibility that they are not of the same species is evidenced by the fact that one enzyme increases in the tissue in response to gonadotropin stimulation (Reichart, 1962), whereas the other decreases (Espey and Rondell, 1968). Several other reports briefly mention hydrolytic enzymes in ovarian tissue. These include hyaluronidase (Zachariae and Jensen, 1958), acid phosphatase and esterase (Banon et al., 1964), general protease (Lipner, 1965), indopeptidase, leucine aminopeptidase, and dipeptidase (Unbehaun et al., 1963), and possibly DNA-ase and RNA-ase (Guraya, 1971). Assays of ovulatory tissue. In view of the limited data on ovarian proteolytic enzymes, and especially the lack of information on enzyme activity at intervals near ovulation, we have assaycd sow follicles and fluid for a variety of enzymes during the past seven years (unpublished). Greatest attention has been on the types of enzymes which arc known to decompose follicular tissue in vitro (Espey, 1970). The follicles were collected in quantity from a local packing house and routinely staged at 20, 5, and 1 h from ovulation (follicles estimated to be 1 h from ovulation were selected only from ovaries which contained at least one ruptured follicle). Follicular fluid was collected by syringe from follicles at each of the three stages. The fluid was centrifuged at 4O3Og for 10 mm and the supernatant fluid was stored at 1#{176}Cuntil the follicular walls were also ready to be assayed. The walls from each stage of follicles were minced, homogenized in distilled water (or buffer, depending on the assay) at 10% w/v, centrifuged at 4000g for 10 mm, and then assayed. The results (Table 1) indicate that the follicle contains trypsin (in the wall, but not the fluid), cathepsin (especially at ph 3.5), and a coflagenolytic enzyme which digests the synthetic substrate carbobenzoxy - glycyl - prolyl - glycyl-glycyl-prolylalanine (CBZ-GPGGPA). Only the enzyme( s) which digested the synthetic hexapeptide changed during ovulation. As rupture neared, there was a slight decrease in this activity in the follicle wall, but a concomitant increase in the follicular fluid. These changes in measurable activity may reflect physiological labilization and subsequent dissipation from the follicle of the enzyme determined by this procedure. (It is important to point out that the enzyme activity detected with this synthetic cal-

7 222 ESPEY TABLE 1 hydrolytic ENZYMEs IN OVARIAN FoL1.ICLIS DURING OvULA1 ION Relative ac tivity in Relative activity in Enzyme pit n wall before 20 h 5 h rupture 1 h fluid before rupture 20 h?i h 1 h Assay (Ref.) General proteolytic k Rick, 1963a Trypsin Rick, 1963b Elasta.se Sachar eta!., 1953 Cathepsin Anson, 1939 Cathepsin ) Anson, 1939 ilyase Tolksdorf et a!., 1949 Collagenase Espey and Rondell, 1968 * Since all assays involved a colorolnetric analysis the values are given as optical density measurements for convenience of comparison. lagenase substrate may not represent a true collagenase. Detailed information on the reasons the substrate specificity may be questionable is given elsewhere (Espey and Rondell, 1967; Rondell, 1970a; Harper and Gross, 1970).) Assay of plasma (luring ovulation. The evidence that an ovarian collagenolytic enzyme might be released from follicular tissue leads to an effort to determine if an increase in such enzyme activity could be detected in the plasma of rabbits during ovulation (unpublished). As a preliminary test of the technique, 200 units of bacterial collagenase (Sigma, stock no. C-0130) were injected into the ear vein of four rabbits, and subsequently the CBZ-GPGGPA substrate was used to assay samples of plasma taken at 10, 30, 60, and 120 mm after injection. There was a measurable increase in activity at 10 and 30 mm, but the enzyme disappeared from the blood within an hour. Blood samples from the ovarian vein of ten rabbits at intervals of 10, 5, 2, 1, and 0 h from ovulation did not show a detectable change in plasma collagenolytic activity as ovulation neared. However, it is possible the failure to observe an increase in activity could be due to (1) too small an amount of enzyme, (2) too much dilution of the enzyme by the vascular compartment, or (3) inactivation of the enzyme in the blood. In an effort to increase the sensitivity of the assay, 50 ml of blood were taken via cardiac puncture from ten precoital rabbits and ten rabbits that were 9-10 h postcoitus. The plasma proteins were fractionated and concentrated by ammonium sulfate precipitation, and then assayed for collagenolytic activity. By increasing the sensitivity of the assay with this modification, a 47% increase in enzyme concentration was measured in rabbits undergoing ovulation in comparison with unmated control animals. In nine precoital animals, the activity was 0.36 ± 0.053, (SEM) as compared to 0.53 ±.082 in ten animals within an hour of rupture. These results support the idea that a proteolytic enzyme may be released in response to gonadotropin stimulation of the ovary. (The method of Gries et at. (1970) might be helpful in any future efforts to confirm or extend this study.) Efforts to Isolate an Ovarian Collagenolytic Enzyme Lutein activity. Among the chemcals examined, a collagenolytic enzyme has appeared to be the most likely causative factor in the decomposition of the follicle during ovulation. Consequently, an extensive effort was made during the past 5 yr to extract such an enzyme from ovarian tissue (unpublished). The substrate CBZ- GPGGPA was used to monitor enzyme

8 PROTEOLYTIC ENZYMES AND OVULATION 223 activity during the development of an extraction procedure. Sow corpora lutea were used in the initial efforts to work out an extraction procedure because this tissue is available in much greater quantity than mature follicles. After considerable testing, the following procedure was adopted: (1) homogenize 50 g of lutein tissue in 500 ml DW for 5 X 2 mm at a temperature below 10#{176}C, (2) dialyze the homogenate for 15 h, against r CaCl to precipitate many inactive proteins, (3) centrifuge for 1 h at 30,000g and decant, (4) add (NH4) 2SO4 to the supernatant fluid to make a 50% saturated solution, (5) stir for 30 mm and then centrifuge for 20 mm at 30,000g, (6) add (NH4)2S04 to the supernatant fluid to make a 60% saturated solution, (7) stir for 30 mm and then centrifuge for 20 mm at 30,000g. (8) resuspend the pellet in 10 ml of Tris buffer (ph 8) containing 0.01 i i CaCl2, (9) dialyze the suspension for 40 h against M CaCI2, (10) centrifuge for 1 h at 30,000g. and lyophilize the supernatant fluid to concentrate the preparation before storage. Lyophiized material from ten extractions (i.e., from 500g of corpora lutea) was consolidated and resuspended in Tris buffer (ph 8) to determine if this preparation could alter the tensile strength of mature sow follicles. Strips of tissue from the walls of 14 follicles were incubated for 20 h in the extract, and then stretched 10% of their original length by a technique described elsewhere (Espey, 1967b). Under this stress the treated tissue developed a tension of only 5.9 g in comparison with an average tension of 15.4 g in 13 controls that were stretched in the same manner. The results meant the luteal extract caused a 62% reduction in the tensile strength of the follicle wall. This information was encouraging support for the idea that follicular rupture is induced by an ovarian proteolytic enzyme. Autolytic activity. A major concern during the early work, which utilized the technique of stretching follicular tissue to determine the effect of extracts and other test materials, was the observation that, after 20 h of incubation, even the control tissue frequently lost tensile strength, especially when incubated in buffered physiological salt solutions. To examine the possibility that autolytic decomposition might be occurring in the tissue, an evaluation was made on the relationship between the duration of incubation and the tensile strength of the follicle strips (unpublished). This check showed that as early as 14 h after incubation in Tris-Ringer solution (ph 7.4) the control tissue sometimes decomposes, and, in essentially all tests, decomposition appeared within 24 h after incubation. Furthermore, strips of the follicle wall which were incubated with follicular homogenates (or distilled water extracts) usually lost tensile strength only an hour or two before control tissue in buffered Ringer Solution. This information reveals that autolysis occurs in follicular tissue, in vitro, usually within 20 h after incubation, and that homogenates or simple extracts from follicular tissue slightly facilitate this autolytic decomposition. These findings may explain why Rondell (1970a, 1970b) detected a reduction in tensile strength of tissue incubated at neutral ph for 20 h in aqueous extracts of follicular tissue. Because of these observations, in this laboratory, later studies which have tested the effect of extracts and other preparations on the tensile strength of sow follicles have been based on a 10-h incubation time. Follicular activity. In more recent experiments (Espey and Stacy, 1970), mature sow follicles were run through the (NH4) 2SO4 fractionation method for lutein tissue as outlined above. The lyophilized extract was resuspended in Tris buffer (ph 7.6) and fractionated further on a column of Sephadex G-200. The fractions from the Sephadex column which most actively digested the substrate CBZ-GPGGPA were lyophilized and stored at -10#{176}C. After 500 g of follicular tissue were extracted, the

9 224 ESPEY lyophilized material was resuspended in 10 ml of Tris buffer (ph 7.6) and tested for its ability to decompose follicular tissue. Ten strips of the follicle wall, which were incubated for 10 h at 37#{176}Cwith the resuspended extract, had a tensile strength of 17.6 g when stretched by 10% of their original length. This value was not appreciably different from the average tension of 19.1 g developed by control tissue. In contrast, a solution of clostridiopeptidase-a caused a complete loss in the tensile strength of other follicle strips incubated during the same 10-h interval. In conjunction with these tensile-strength experiments, the incubation solutions of both the resuspended follicular extract and the clostridiopeptidase-a were assayed for enzymes that digest CBZ-GPGGPA. The resuspended extract contained 17.1 relative units of enzyme activity in comparison to only 4.8 units of activity in the solution of bacterial collagenase. In other words, the follicular extract possessed more than three times as much collagenolvtic activity as the clostridiopeptidase-a solution, and yet it did not reduce the tensile strength of the follicular strips. This important observation was confirmed by complete duplication of the entire experiment, starting with fresh tissue in the extraction process. The results suggest that the follicular factor which digests the CBZ-GPGGPA peptide may not be comparable to a true collagenase. This conclusion is in agreement with Rondell s (1970a) analysis of experimental data obtained by a different procedure. His extracts from follicles also digested the synthetic substrate CBZ- GPGGPA, but not reconstituted collagen. Current Research Considerable difficulty has been encountered in efforts to understand the enzymatic processes involved in the remodeling of collagenous tissues. As Strauch and Venceli (1967) noted several years ago, in spite of evidence that higher animals are able to degrade native collagen, the search in extracts and homogenates of mammalian tissue for collagenases has been rewarded with but little success However, significant advances have been made in the past few years. A major breakthrough occurred in 1968 (Eisen et at., 1968), when a collagenolytic enzyme-one which could not be detected in tissue extracts-was isolated from the culture medium of tissue cultures of normal human skin. Since that important discovery, similar collagenase activity has been detected in the media of other cultured tissues (Eisen et at., 1970a). These findings could explain why so much difficulty has been encountered in efforts to extract an ovarian collagenolytic enzyme. The reason the enzyme is present in tissue culture, but not in homogenates of whole tissue, is not clear. Tissue homogenates and extracts might contain (1) enzyme at too low of a concentration, (2) enzyme in the form of an inactive zymogen, (3) enzyme bound to its endogenous collagen substrate, or (4) enzyme inhibitors. Another interesting point, regarding the enzyme(s) which can be obtained only by the tissue culture technique, is that activity can be detected in the culture media only after h of incubation (Bauer et at., 1970). This requirement of a long incubation time may explain why Rondell (1970a) was unable to detect collagenolytic activity in follicular material which was also cultured, but only for 2-36 h. In view of this new information on animal collagenases, this laboratory has begun a reexamination of follicular tissue for collagenolytic enzyme(s). The three major approaches include: (1) the original tissue culture method developed by Gross and Lapiere (1962) which utilizes a reconstituted collagen gel as the substrate, (2) the incubation of follicular tissue in culture flasks containing a fluid medium, which is assayed 48 h later for enzymes that digest the synthetic substrate CBZ-GPGGPA, and

10 PROTEOLYTIC ENZYMES AND OvULATION 225 (3) an assay based on the detection of soluble l4caabeled glycine, which is released from reconstituted collagen fibrils in culture medium (Jeffrey et al., 1971a). These experiments were still in progress at the time this review was written; nevertheless, preliminary data are summarized below because of their bearing on the working hypothesis at the end of this report. Reconstituted collagen gel as substrate. The substrate for this method was prepared by extracting collagen from rabbit skin. The soluble collagen was processed in a manner which led to the formation of an opaque gel of reconstituted collagen at the bottom of a culture dish. After the surface of the collagen gel was rinsed with a standard culture medium (e.g., Dulbecco s, Trowell s, or Kreb s-ringer at ph 7.4), it was ready for tissue incubation. A positive reaction for collagenase was recorded if a transparent halo developed in the opaque gel indicating the tissue secreted an enzyme which digested the collagen. A total of 221 tissue samples have been incubated on reconstituted collagen gels. Both follicular tissue and ovarian stroma caused lysis of the gels. Digestion occurred more often on gels rinsed with Kreb s-ringer solution rather than Dulbecco s Modified Eagle s Medium, or Trowell s Medium. When lysis occurred, it was always complete within the first 15 h of incubation. No tissue induced transparency in gels that were thicker than 1.5 mm, even if the incubation time was extended to 72 h. Follicles that had not been stimulated by gonadotropin (i.e., follicles from precoital rabbits) were about as effective in digesting the reconstituted collagen (5 out of 8 gels showed lysis) as tissue that was taken within 1 h of rupture (26 out of 33 gels showed lysis). Neither LH, cyclic AMP, progesterone, nor estrogen appeared to facilitate lysis when added to the incubation media; however, different concentrations of these compounds are still being tested. The observations that (1) there is insufficient enzyme to digest thick gels of reconstituted collagen, (2) lysis does not continue beyond the first hr of incuba-. tion, (3) LH did not facilitate lytic action, and (4) precoital tissue is as effective as prerupture tissue in causing lysis has led to the tentative conclusion that mature follicles contain only a small amount of collagenolytic enzyme (or its zymogen); and, gonadotropin may not induce synthesis of this enzyme, but only initiate the sequence of events that facilitate the release of stored enzyme. Blandau (personal communication) has recently found that if the stigmal area is dissected from the surface of a preovulatory follicle, it causes depolymerization of a gelatin membrane in culture, and a future report on his investigation should help enlighten the enzyme process. Synthetic peptide as substrate. Ova!ian follicles from rabbits have been incubated in disposable culture flasks containing 2.0 ml of Kreb s-ringer solution. The flasks were exposed to a mixture of 95% 02-5% CO2 and incubated at 37#{176}Cin a shaker bath for 48 h. Samples of the fluid media were withdrawn and tested for enzymes which decompose the synthetic peptide CBZ- GPGGPA. Kreb s-ringer solution was used primarily because the more complete culture media (e.g., Dulbecco s and Trowell s) contain amino acids which react with ninhydrin during the colorimetric analysis of the substrate solution. Preliminary results are available on the assays of 155 culture flasks. After 48 h of incubation, the culture medium contained only a very slight amount of enzyme activity capable of digesting CBZ-GPGGPA. There is even less activity in follicles which are removed from the ovary during the hour preceding rupture. The addition of cyclic AMP to the culture medium caused a threefold increase in activity in both precoital and prerupture follicles. LH, progesterone, and estrogen did not increase the activity. No conclusion is warranted at this time.

11 226 ESPEY Reconstituted collagen containing Clabeled glycine as substrate. Results are not yet available from this assay procedure. However, I want to briefly point out that this approach is important for three reasons: (1) it will serve as a reliable test for a true collagenase in ovulatory tissue; (2) it should allow final clarification of the question of whether the proteolytic activity from follicular tissue which digests CBZ-GPGGPA is different from true collagenase; and (3) it will allow the tissue to be cultured in a medium more complete than Kreb s-ringer and, therefore, should improve the viability of the tissue. PROPERTIES OF ANIMAL COLLAGENASES It is useful to examine additional information about animal collagen and collagenase before attempting to develop a hypothesis to summarize the current status of knowledge on the mechanism of ovulation. Inhibition Serum antiproteases. The discovery that collagenase from human skin is inhibited by human serum led to the conclusion that collagenase activity is controlled, at least in part, by factors present in the blood (Eisen et at., 1970b). This hypothesis was supported by evidence that the alphaimmunoglobulin fraction of serum, specifically alphai-antitrypsin and alpha2- macroglobulin, inhibit animal collagenases (Eisen et al., 1970b; Hawley and Faulk, 1970). This means that, in normal tissues undergoing connective tissue remodeling, collagenase is probably present in sufficient quantity to digest endogenous substrate lying close to the cells which produce the enzyme, but enzyme that diffuses to remote sites is prevented from acting by serum inhibitors. The specific method of action of these inhibitors has not been clarified. Native alpha2-macroglobulin might cause inactivation by irreversibly binding animal collagenase) (Abe and Nagai, 1973). However, it has been reported that active collagenase can be chromatographically separated from the serum antiproteases (Eisen et at., 1971). This new information on natural collagenase inhibitors in animal tissues is an important consideration in the design of future studies on the mechanism of ovulation. The existence of these antiproteases might explain the difficulty encountered in searches for collagenolytic enzymes in homogenates and extracts of follicular tissue. These inhibitors could also impair the detection of collagenolytic activity during the first h of tissue culture experiments (Eisen et at., 1970a). (As a footnote, it would be interesting to know if alphaimmunoglobulins are elaborated by leukocytes, because shortly after ovulation there is an increase of granulocytes resembling basophils in the follicle wall (unpublished; Zachariae et at., 1958). Progesterone. When postpartum tissue from rats is cultured at physiological ph the tissue produces a specific collagenase for up to 10 days in culture (Jeffrey et al., 1971a). This activity can be detected only in the medium of cultures which contain uterine tissue removed from the animal within the first 72 h after parturition, i.e., the period during which there is active degradation and reabsorption of uterine collagen. It is of particular interest that uterine collagenase activity is completely abolished when progesterone is added to the culture medium in a concentration of 5 X 10 M (Jeffrey et at., 1971b), because this implies that progesterone might be a specific regulator of collagenase activity in at least one reproductive tissue. More than a decade ago Hisaw (1961) stated that steroid action may be the answer [to the mechanism of ovulation]. In recent years, evidence has been presented to support the idea that progesterone might facilitate the decomposition of the follicle wall (Lipner and Greep, 1971; Rondell, 1970b, 1974). This hy-

12 PROTEOLYTIC ENZYMES AND OVULATION 227 pothesis is not consistent with the report above that progesterone inhibits uterine collagenase. It may be relevant that brief tests in this laboratory (unpublished) have shown that: (1) progesterone in ethanol, corn oil, or water does not induce ovulation if injected into mature rabbit follicles in vivo, (2) progesterone possibly inhibits the lysis of collagen gels by follicular tissue, and (3) progesterone does not facilitate collagenolysis of CBZ-GPGGPA incubated with rabbit follicles in fluid culture media. Other inhibitors. Although cysteine activates some proteases (White et at., 1968), it inhibits clostritiopeptidase-a (Harper, 1966; Seifter, 1970), corneal collagen ase (Hook et at., 1972), and skin collagenase (Eisen et at., 1968), but not uterine collagenase (Jeffrey and Gross, 1970); the latter observation suggests collagenase in reproductive tissue might be different from that in other tissues. The effect of antiinflammatory agents on collagenolytic activity is varied, presumably depending on the histological origin and ph optima. Rat skin collagenase is inhibited by chioroquine (Cowey and Whitehouse, 1966), salicylate, and soybean trypsin inhibitor (Houck et at., 1967). However, soybean trypsin inhibitor is not effective against human skin collagenase (Eisen et at., 1968). Collagenolytic enzymes in rat liver lysosomes are inhibited by the antiinflammatory drugs phenylbutazone and ibufenac (Anderson, 1969). Gingival collagenase is inhibited by cortisone and histamine (Taylor, 1971). However, Houck et at. (1970) reported that cortisol and indomethacn induce the release of active collagenase from mouse and human fibroblasts. It would be interesting to know if these two agents could activate collagenolysis in the fibroblasts in the follicle. Although contradictory, it may be relevant that Tsafriri et al. (1972) believe indomethacin exerts an antiovulatory action directly on the follicle and prevents follicular rupture. The effect of trypsin and trypsin inhibitors on follicular decomposition deserves more attention in the future because it is known that (1) small quantities of trypsin injected directly into rabbit follicles can induce rupture (Espey and Lipner, 1965), and (2) trypsin reduces the tensile strength of strips of sow follicles, in vitro (Espey, 1970). It could be relevant that a natural trypsin inhibitor (Kunitz inhibitor) is present in mammalian ovaries (Chauvet and Acher, 1972). Activation It is well known that most proteolytic enzymes are synthesized as precursors called zymogens (e.g., pepsinogen and trypsinogen) and then stored in granules to protect the tissues from self-destruction by their own enzymes. it is now recognized that activation of these precursors can be accomplished by (1) action of another enzyme (e.g., interokinase activation of trypsinogen), (2) auto-activation (e.g., after a small amount of pepsinogen is converted to pepsin in the presence of HCI, the active enzyme causes further conversion of the proenzyme into pepsin), and (3) endogenous enzymic activity within the individual zymogen molecule which allows these precursors to activate themselves in some instances. Regarding the latter example, it is now evident that this intrinsic self-activating property is possessed by a wide variety of proteolytic enzymes (Kassell and Kay, 1973). Zymogen precursors of collagenase. An inactive zymogen proenzyme of tadpole collagenase has been recently isolated (Harper et at., 1971). Activation of the zymogen can be accomplished by incubation with collagenase-free tailfin culture media, but not with trypsin or chymotrypsin. Additional studies (Harper and Gross, 1972) have led to the conclusion that lack of expression of collagenase activity in cultured tissue during the first h may not be due to the presence of inhibitors such as the aiphaimmunoglobulins, but, instead, the result of a time lag before the

13 228 ESPEY appearance of activation factors which convert the zymogen into active enzyme. This conclusion is based on evidence that a procollagenase is secreted into the culture media at maximal levels beginning with the first day after tissue incubation, but an activator is not detectable until later. The activator present in culture media has not yet been identified, but it is heat labile and nondialyzable. There are several types of collagenase enzymes, and it is not vet clear whether the zymogen in amphibian tissue has the same activation requirements as that of mammalian procollagenases (Bauer et al., 1972). There is one report of a collagenase proenzyme in crude human leukocyte extracts which is activated by some component of rheumatoid synovial fluids taken from knee joints (Kruze and Wojtecka_ 1972). Ascorbic acid. It has been known for some time that gonadotropin initiates the release of ovarian ascorbic acid (Foreman, 1963; Mukerji et at., 1965; Parlow, 1958), and that this ovarian response can be detected near the time of ovulation (Pillay, 1940; Astrada and Caligaris, 1966; Paeschke, 1967). This information raises the question of whether ascorbic acid has a significant role in the mechanism of ovulation, or whether its depletion in response to gonadotropin is primarily related to steroidogenesis (Leatham, 1961). The latter possibility is supported by evidence of changes in ascorbic acid metabolism in the adrenal gland in response to corticotropin stimulation of steroidogenesis (Kitabchi, 1967). Nevertheless, the idea that this vitamin might have a more direct role in the mechanism of ovulation remains intriguing. Such a functional role is supported by evidence that ascorbic acid causes a complete loss in the tensile strength of sow follicles, in vitro (Espey, 1970). However, this response requires a relatively high acid concentration (ph 3.2) in the incubation medium. Therefore, it is doubtful ascorbic acid can elicit this effect under physiological conditions, because the ph in the follicular wall (and the follicular fluid) remains slightly above neutral during the ovulatory process (Espey, 1970; Lee, 1970). It could be relevant that ascorbic acid depolymerizes tropocollagen, even at neutral ph (Miyata et al., 1970), leaving open the question of whether this vitamin might expedite the digestion of thecal tissue after other agents initially degrade the collagen matrix. Furthermore, we have found that small quantities of ascorbic acid cause digestion of reconstituted collagen gels (unpublished), a fact which also points out that ovarian follicles could induce the lysis of gels by the release of ascorbic acid. In addition, ascorbic acid can induce the depolymerization of hyaluronic acid (Niedermeier et al., 1967), a major component of the glycoaminoglycans in collagenous tissue. This same report presents evidence that diketogulonic acid, the metabolite of ascorbic acid, is the derivative responsible for depolymerization of hyaluronic acid. This action might explain wily diketogulonic acid is also effective in reducing the tensile strength of the follicle wall, in vitro (Espey, 1970). Theoretically, ascorbic acid could have a completely different role in ovulation. Several years ago at the physiology meetings (Espey and McDavid, 1969) we discussed the possibility of this vitamin serving as an enzyme activator. This consideration was based on the knowledge that some animal proteases are active only in the presence of strong reducing agents such as ascorbic acid (White et at., 1968; Loh and Wilson, 1971; Kassell and Kay, 1973). The hypothesis has been supported by reports that (1) ascorbic acid appears to activate ovarian proteases, in vitro (Lee, 1970), and (2) by activating a zymogen, ascorbic acid causes a five-fold increase in a collagen hydroxylase enzyme elaborated from cultured mouse fibroblasts (Stassen et at., 1973).

14 PHOTEOLYTIC ENZYMES AND OVULATION 229 Cyclic AMP. It has been established that cyclic AMP mediates the action of gonadotropin in inducing luteinization of follicular tissue (Channing, 1974) by stimulating steroidogenesis (Marsh et at., 1973), particularly progesterone synthesis (Herniier et a!., 1972). Without going into repetitious detail of material covered in other papers in this symposium, it is appropriate to emphasize that cyclic AMP is known to stimulate collagenase and hyaluronidase activity in media of organ cultures of bullfrog tadpoles (Harper and Toole, 1973), because this observation raises the question of whether cyclic AMP might elicit a similar reaction in ovarian follicles. Other Inforniation There are a number of useful review articles on properties of collagenase enzymes. Mandl (1961) has reviewed the information prior to 1960 and again more recently (Mandl, 1972). Woessner has reviewed acid hydrolases in connective tissues (1965) and has presented a comprehensive report on the biological processes involved in collagen resorption in many normal and pathological conditions (1968). Valuable information can be obtained from a review by Eisen et al. (1970a) and a brief review by Bauer et at. (1972), the latter concentrating on the regulation of vertebrate collagenase activity. The reviews also include information on cytological localization and purification of various types of collagenase. CONCLUSIONS There is reasonabl e idence that the chemical changes which occur in the mahire ovarian follicle during ovulation involve a complex sequence of reactions. Deterioration of the thecal tissue in the follicle wall at the time of rupture may, or may not, depend upon proteolytic enzymes (and their precursors), steroids, ascorbate, cyclic AMP, and any number of other known factors or cofactors (possibly ineluding prostaglandins, see LeMaire et at., 1973). The sequence of biochemical events could prove to be highly complex. In fact, complexity should probably be anticipated because ovulation requires traumatic disruption of tissue; and, if the physiological integrity of the rest of the ovary is to be maintained, decomposition must be localized. The chemical sequence might be as complex, for example, as the blood-clotting mechanism, another biological process which requires a high degree of localization. On the positive side, however, because there are so many steps in the clotting mechanism, there are also numerous methods of controlling this phenomenon. The advantage is clear-the greater the complexity of a system, the greater the number of sites for potentially regulating the output of that system. And, I need not remind anyone at this symposium that the primary output of the ovulatory process is a fertile egg. One other point needs to be stressed before attempting to develop a general hypothesis: It has been overlooked that the follicle wall is fragile for only a short period of time during the ovulatory process. The weakened condition of the thecal tissue commences only a few hours prior to rupture, and tensile strength is regained during the early stages of luteinization (Espey, 196Th). Immediately following rupture, the fibroblasts in the thecal layer rapidly undergo functional reorientation (1) to initiate healing at the point of rupture and (2) to meet the support requirements of the proliferating lutcin cells. In other words, collagenolysis does not last long during the ovulatory process, and this factor in itself might be highly relevant to the difficulty encountered in attempts to identify a follicular collagenase. For instance, even when a tissue culture method is used as the assay for enzyme activity, if the incubation procedure is effective in mimicking the biochemical processes which normally occur in vivo, then the very same factors which suppress collagenolysis after ovulation should mask any enzyme in culture medium.

15 230 ESPEY A Working Hypothesis The following hypothesis on the mechanism of ovulation is formulated mainly from information covered in this review and is intended to serve as a working basis for future studies. Before gonadotro pin stimulation. Once an ovarian follicle has reached maturity it possesses all the metabolic components necessary for rupture. The theca interna and the membrana granulosa (to a lesser degree) are already producing steroids. Ascorbic acid is present in relatively high concentration in the theca interna (see Deane, 1952). The thecal fibroblasts are in a quiescent, stationary phase of activity. A zymogen with the potential for reducing the tensile strength of collagenous tissue is present in the fibroblasts (and possibly to a lesser degree in the cells of the membrana granulosa and theca interna). During ovulation. Appropriate gonadotropin stimulation is expressed by a substantial increase in cyclic AMP in the follicle and an acceleration of steroidogenic activity. In a preliminary step towards luteinization, there is a significant increase in mitotic activity in all the cells in the Graafian follicle, an increase initiated either by a rise in metabolic activity associated with cyclic AMP, or by the local changes in steroids. Concomitantly, the thecal fibroblasts are converted from the stationary state to the proliferative state. Within a relatively short period of time a significant quantity of zymogen is elaborated into the extracellular compartment, via multivesicular structures present in cytoplasmic processes of fibroblasts. In the extracellular spaces, the zymogen is activated either by itself, by ascorbic acid, or by some unknown factor. The activation begins slowly, but newly formed enzyme catalyzes a rapid acceleration of the conversion reaction. The connective tissue throughout the follicle wall (and possibly to a lesser extent in the ovarian stroma) is degraded, leading to a gross reduction in the tensile strength of the cohlagenous layers which encapsulate the follicle. By morphological design, the thin region at the apex of the follicle is the area most susceptible to distension under the stress of a small (but very important) intrafollicular pressure. Rupture is eminent as the degraded follicle wall begins to dissociate under this stress. By the time of rupture the enzymatic activity has also depolymerized the ground substance in the cumulus oopherus to the extent that the corona radiata is dislodged and ready for expulsion from the follicle. After ovulation. Shortly after ruptue, further collagenolysis is suppressed because (1) the zymogen is depleted and the active enzyme is dissipated from the follicle, (2) the extrayasation of blood at ovulation increases the serum antiproteases in the follicle wall, (3) the increase in basophil leukocytes in postovulatory follicles possibly deposit additional antiproteases, and (4) luteinization causes an excessvely high level of progesterone, which somehow inhibits further collagenolysis, possibly by direct action on the fibroblasts. Obviously, this hypothesis is based on both premise and presumption. Hopefully, it will age considerably during the next decade. Suggestions for Future Studies As an addendum, I would suggest that the following questions be given consideration in future studies. 1. What are the structural and chemical differences between an immature and mature Graafian follicle? Specifically, what changes occur in the cellular components during the final stages of follicle maturation to prime the follicle in a manner which allows it to respond to luteinizing hormone? 2. Which cells in the mature ovarian follicle are influenced by FSH and LH, and which ones are not? There is growing information on the responses of granulosa cells to gonadotropin stimulation, but what about the other cells? Do FSH and LII

16 PROTEOLYTIC ENZYMES AND OVULATION 231 directly act on the fibroblasts of the theca externa, the secretory fibrocvtes of the theca interna, or the ovum? If not, then what is the source of the stimulus which leads to the ovulatory changes in these cells? 3. What is the significance of ovarian ascorbic acid in ovulation? Does it have a direct effect on thecal collagen? Is it important in ovarian steroidogenesis? Is it a reducing agent for ovarian hydrolases? 4. What kinds of chemical agents are produced by proliferating fibroblasts to digest pathways through dense extracellular connective tissue? How important to ovulation are the multivesicular structures which protrude from thecal fibroblasts? What is their chemical composition? What is the cytoplasmic origin of this material? If they are responsible for follicular rupture, then how are they stimulated by the LII surge? 5. WThat is the basic chemical composition of the glycoaminoglycans and other components of the extracellular cement at the apex of the follicle wall? What types of enzymes will depolymerize this material? 6. What is the specific function of the follicular antrum and fluid in mammalian follicles? Are they for the purpose of protecting the ovum from hydrolytic enzymes produced in the thecal layers of the follicle at the time of ovulation? 7. What effect do various inhibitors of proteolytic enzymes have on ovulation when they are injected in small quantities directly into follicles (in vivo), or injected in larger quantities intraperitoneahly? 8. Can a collagenolytic enzyme, possessing the same properties as other animal collagenases, be isolated from follicular tissue? Of particular importance is the evidence that a purified collagenase from human skin can be used to produce an anti-human skin collagenase antibody (Bauer et al., 1970). If there is an ovarian collagenase, which has similar immunogenic properties, it would be extremely important to know whether the antibodies (presumably alphaglobulins) could inhibit ovulation. 9. Does the concentration of alpha1-antitrypsin or alpha2-macroglobulin in the serum of mammals change during the sexual cycle? What happens to the level of these anti-collagenases in the days following parturition, a time during which there is a significant increase in collagenolysis in the uterus. Can these serum immunoglobulins be raised to a titer that would inhibit follicular proteases during ovulation? Since there is evidence that tissue injury induces an increase in this type of circulatory collagenase inhibitor, is it possible for gross damage of tissues (incurred during accidents or surgery ) to cause a temporary increase in serum inhibitors capable of influencing ovulation and fertility? 10. Can knowledge on the sequential processes of ovulation and luteinization contribute useful information to the efforts to determine the mechanism of tumor growth? I present this possibility not simply because of the recent rearrangements in funding priorities, but because it is an idea which has fascinated me for some time. In several respects, the ovulatory changes in the Graafian follicle are comparable to tumorous growths. For examples: (1) ovulation is initiated by adenohypophyseal hormones, all of which induce grotvth of some tissue(s) in the mammalian system, (2) estrogens, which are highly concentrated in the follicle, are well known carcinogens, (3) postovulatory changes include the marked enlargement and proliferation of foihicular cells as they transform into a corpus lutcum (i.e., a physiological tumor ), and (4) ovulation involves decomposition of collagenous tissue, and, likewise, tumors stimulate fibroblasts to secrete enzymes which depolymerize the connective tissue surrounding the growth. The big difference, of course, is that the lutein tumor stops growing when it reaches a certain size; and, it under-

17 232 ESPEY goes resorption, a phenomenon which should be of special interest to cancer researchers. ACKNOWLEDGMENTS The author acknowledges the dependable assistance of Sue Stacy, Patrice Jordy, and Larry Coons in producing the original data cited, and the aid of Linda Katakalos in preparing this review. I am especially indebted to Dr. Andrew G. Cowles for his generous donation of the facilities essential to the conduct of this work. In addition, these studies have been supported by NIH Grants HD and HD-06371, and NIH Contract REFERENCES ABE, S., AND NACAI, Y. (1973). Evidence for th2 presenc of a complex of collagenase with e-macroglobulin in human rheumatoid synovial fluid: A possible regulatory mechanism of collagenase activity in vivo. J. Biochem. 73, ANDERSON, A. J. (1969). Effects of lysosomal collagenolytic enzymes, antiinfiammatory drugs and other substances on some properties of insoluble collagen. Biochem. J. 113, ANDERSON, J. W., ANt) YATVIN, M. B. (1970). Metabolic and ultrastructural changes in the frog ovarian follicle in response to pituitary stimulation. I. Cell Biol. 46, ANsox, M. L. (1939). The estimation of pepsin trypsin, papain, and cathepsin with hemoglobin I. Gen. Ph,jsiol. 22, ASDELL, S. A. (1962). Mechanism of ovulation. In The Ovary. (S. Zuckerman, ed), p. 436, Academic Press, London. ASTRADA, J. J., AND CAUCABIS, L. (1966). Ovarian ascorbic acid concentration and its modification during sexual and reproductive activity in the rat. Acta Physiol. Latinoam. 16, 1-5. BADER, II. (1963). The anatomy and physiology of the vascular wall. In Handbook of Physiology. Circulation. (W. F. Hamilton, ed), Vol. II, p. 865, Amer. Physiol Soc., Washington, D.C. BANON, P., BRANDES, D., AND FROST, J. K. (1964). Lysosomal enzymes in the rat ovary and endometrium during the estrous cycle. Acta Cytol. 8, BAyER, E. A., EIsEN, A. Z., AND JEFFREY, J. J. (1970). Immunologic relationship of a purified human skin collagenase to other human and animal collagenases. Biochim. Biophys. Acta 206, BAUER, E. A., EISEN, A. Z., AND JEFFREY, J. J. (1972). Regulation of vertebrate collagenase activity in vivo and in vitro. J. Invest. Dermat. 59, BETTEIUDCE,K. J., AND RAESXDE, J. I. (1962). Observation of th ovary by peritoneal cannulation in pigs. Rex. Vet. Sci. 3, BIRGER, J. F. (1952). Sex physiology of pigs. Onderstepoort I. Vet. Res. 25 (Suppl. 2), BLANDAIJ, R. J. (1966). The mechanisms of ovulation. In Ovulation: Stimulation Suppression Detection. (R. B. Greenblatt, ed), p. 3, J. B. Lippincott Co., Philadelphia. BLANDAU, R. J. (1967). Anatomy of ovulation. Clin. Obstet. Cynecol. 10, BI.ANDAU, R. J. (1968). Follicular growth, ovulation and egg transport. In The Ovary. (H. C. Mack, ed.), p. 3, C. C Thomas, Springfield. BLANDAU, R. J., AND RUMERY, R. E. (1963). Measurements of intrafollicular pressure in ovulatory and preovulatory follicles of the rat. Fert. Steril. 14, BURR, J. M., AND DAVIES, J. 1. (1951). The vascular system of the rabbit ovary and its relationship to ovulation. Anat. Rec. 111, CHAUVET, J., AND ACHER, R. (1972). Isolation of a trypsin inhibitor (Kunitz inhibitor) from bovine ovary by affinity chromatography through trypsin-sepharose. FEBS Lett. 23, Csrmi.&L, H. J., AND E5PEY, L. L. (1973). Utilizalion of the relaxed symphysis pubis of the guinea pig for clues to the mechanism of ovulation. Endocrinology 93, CLAESSON, L. (1947). Is there any smooth musculature in the wall of the Craafian follicle? Acta Anat. 3, CORNER, C. W. (1919). On the origin of the corpus luteum of the sow from both granulosa and theca interna. Amer. J. Anat. 26, COWEY, F. K., AND WHITEHOUSE. M. W. (1966). Biochemical properties of anti-inflammatory drugs. VII. Inhibition of proteolytic enzymes in connective tissue by chioroquine (resochin) and related antimalarial/antirheumatic drugs. Biochem. Pharmacol. 15, DEANE, H. W. (1952). Histochernical observations on the ovary and oviduct of the albino rat during the estrous cycle. Amer. J. Anat. 91, EISEN, A. Z., JEFFREY, J. J., AND Cnoss. J. (1968). Human skin collagenase. Isolation and mechanism of attack on the collagen molecule. Biochim. Biophys. Acta 151, EIsEN, A. Z., BAUER, E. A., AND JEFFREY, J. J. (1970a). Animal and human collagenases. (Special Review Article). I. Invest. Dermat. 55, EISEN, A. Z., BLOCH, K. J., AND SAKAI, T. (1970b). Inhibition of human skin collagenase by human serum. J. Lab. Clin. Med. 75, EISEN, A. Z., BAyER, E. A., AND JEFFREY, J. J.

18 PROTEOLYTIC ENZYMES AND OVULATION 233 (1971). Human skin collagenase. The role of serum alpha-globulins in the control of activity in vivo and in vitro. Proc. Nat. Acad. Sci. 68, E5PEY, L. L. (1964). Mechanism of Mammalian Ovulation. Ph.D. Dissertation. Fla. State U., Tallahassee. ESPEY, L. L. (1967a). Ultrastructure of the apex of the ral)l)it Craafian follicle during the ovulatory process. Endocrinology 81, ESPEY, L. L. (19671)). Tenacity of porcine Graafian follicle as it approaches ovulation. Amer. I. Physiol. 212, ESPEY, L. L. (1970). Effect of various substances on tensile strength of sow ovarian follicles. Amer. I. Physiol. 219, ESPEY, L. L. (1971a). Decomposition of connective tissue in rabbit ovarian follicles by multivesicular structures of thecal fibroblasts. Endocrinology 88, ESPEY, L. L. (1971b). Multivesicular structures in proliferating fibroblasts of rabbit ovarian follicles during ovulation. I. Cell Biol. 48, ESPEY, L. L., AND BErrERIDGE, K. J. (1970). Bibliography on mechanism of follicular rupture in matnmals. Bibliogr. Reprod. 16, ESPEY, L. L., AND LIPNER, H. (1963). Measurements of irmtrafollicular pressures in the rabbit ovary. Amer. I. Physiol. 205, E5PEY, L. L., AND LIPNER, H. (1965). Enzymeinduced rupture of rabbit Craafian follicle. Amer. I. Physiol. 208, ESPEY, L. L., AND MCDAVID, W. D. (1969). The effect of various substances on the tensile strength of sow Graafian follicles. Physiologist 12, 217 (Abstr.). ESPY, L. L., AND RONDELL, P. (1967). Estimation of mammalian collagenolytic activity with a synthetic substrate. I. Appl. Physiol. 23, ESPEY, L. L., AND RONDELL, P. (1968). Collagenolytic activity in the rabbit and sow Graafian follicle during ovulation. Amer. I. Physiol. 214, ESPEY, L., AND STACY, S. (1970). Failure of an ovarian collagenolytic extract to decompose the connective tissue in the mature sow Graafian follicle. Fel. Proc. 29, 833 (Abstr.). ETIIERINGTON, D. J. (1973). Collagenolyticcathepsin and acid-proteinase activities in the rat uterus during post partum involution. Eur. J. Biochem. 32, FOREMAN, D. (1963). Effects of gonadotrophic hormones on the concentration of ascorbic acid of the rat ovary. Endocrinology 72, FUMACALLI, Z., M0TrA, P., AND CALVIERI, S. (1971). The presence of smooth muscular cells in the ovary of several mammals as seen under the electron microscop. Experimentia 27, CRIES, C., BURESCH, H., AND STRAUCH, L. (1970). Collagenolytic enzymes in human senmi. Experimentla 26, CROHE, F. (1863). Ueber den Bau und das Wachstum des menschlichen Elerstocks. Virchows Arch. 26, GRoss, J., AND LAPIEBE, C. M. (1962). Collagenolytic activity in amphibian tissues: A tissue culture assay. Proc. Nat. Acad. Sci. 48, GmJRAYA, S. S. (1971). Histochemical changes of nucleic acids in the follicular stroma of the rabl)its ovary before and during ovulation. I. Reprod. Fert. 24, CUrrMACHER, M. S., ANt) CUTrMACI-IEB, A. F. (1921). Morphological and physiological studies of the musculature of the mature Craafian follicle of the sow. John Hopkins Hosp. Bull. 32, HARPER, E. (1966). Mechanism of action of collagenase. Irreversible inhibition of cysteine, reversible inhibition by histidine or imizadole. Fed. Proc. 25, 790 (Abstr.). harper, E., AND CROSS, J. (1970). Sel)aration of collagenase and peptidase activities of tadpole tissues in culture. Biochim. Biophys. Acta 198, HARPER, E., BLOCH, K. J., AND GROSS, J. (1971). The zymogen of tadpole collagenase. Biochemis. try 10, HARPER, E., AND GROSS, J. (1972). Collagenase, procollagenase and activator relationships in tadpole tissue cultures. Biochem. Biophys. Res. Commun. 48, HARPER, E., AND TOOLE, B. P. (1973). Collagenase and hyaluronidase stimulation by dibutyryl adenosine cyclic 3 : 5 -monophosphate. I. Biol. Chem. 248, HAUST, M. D., AND MORE, B. H. (1967). Electronmicroscopy of connective tissues and elastogenesis. In The Connective Tissue. (B. M. Wagner, and D. E. Smith, eds.), p. 352, Williams and Wilkins Co., Baltimore. HAWLEY, P. R., AND FAULK, W. P. (1970). A circulatory collagenase inhibitor. Brit. J. Surg. 57, HEAPE, W. (1905). Ovulation and degenerating ova in the rabbit. Proc. Roy. Soc. Brit. 76, HERMR, C., SANTOS, A. A., WISNEWSKY, C., NErrER, A., AND JUSTISZ, M. (1972). Role de l AMPc et d une prot#{233}ine r#{233}gulatrice dans l action, in vitro, de la gonadotropine choriale humaine (HCG) stir le corps jaune humain. Compt. Rend. 275, HILL, R. T., ALLEN, E., AND KRAMER, T. C. (1935). Cinemicrographic studies of rabbit ovulation. Anat. Rec. 63, HISAW, F. L. (1947). Development of the Graafiart

19 234 ESPEY follicle and ovulation. Physiol. Rev. 27, HISAW, F. L. (1961). (quoted from the Discussion) In Control of Ovulation. (C. A. Villee, ed.), p. 50, Pergamon Press, New York. HOOK, C. W., BULL, F. C., IwANmJ, V., AND BRowN, S. I. (1972). Purification of corneal collagenases. Invest. Ophthal. 11, HoucE, J. C., PATEL, Y. M., AND GLADENER, J. (1967). The effects of anti-inflammatory drugs upon the chemistry and enzymology of rat skin. Biochem. Pharmacol. 16, HOUCK, J. C., SHAis ia, V. K., CARILLO, A. L. (1970). Control of cutaneous collagenolysis. Advan. Enz. Reg. 8, HUNTER, R. H. F. (1967). Porcine ovulation after injection of human chorionic gonadotrophin. Vet. Rec. July, JEFFREY, J. J., COFFEY, B. J., AND EISEN, A. Z. (1971a). Stulies on uterine collagenase in tissue culture. I. Relationship of enzyme production to collagen metabolism. Biochim. Biophys, Acta 252, JEFFREY, J. J., COFFEY, R. J., AND EISEN, A. Z. (197 lb). Studies on uterine collagenase in tissue culture. II. Effect of steroid hormones on enzyme production. Biochim. Biophys. Acta 252, JEFFREY, J. J., AND CROSS, J. (1970). Collagenase from rat uterus. Isolation and partial characterization. Biochemistry 9, JEWETF, D. A., AND DUKELOW, W. R. (1971). Follicular morphology in macaca fascicularis. Fed. Proc. 30, JUNG, G. (1969). Stoffwechselvorgange im Ovar im Hinblick auf Follikelreifung und Ovulation. Archly, fur Cynak. 207, JUNG, C., AND HELD, H. (1959). Uber Fermente in der Follikelfiussigkeit. Arc/sic, fur Gynak. 192, KASSELL, B., AND KAY, J. (1973). Zymogens of proteolytic enzymes. Science 180, KELLY, C. L. (1931). Direct observations of rupture of Graafian follicles in the mammal. J. Fla. Med. Ass. 17, KITABCHI, A. E. (1967). Ascorbic acid in steroidogenesis. Nature 215, KOLLIKER, A. VON (1849). Beitrage zur Kenntnis der glatten Muskein. Z. Wi.cs. Zool. 1, KRAUS, J. D. (1947). Observations on the mechanism of ovulation in the frog, hen and rabbit. Western J. Surg. 55, KEUZE, D., AND WoJmcKA, E. (1972). Activation of leucocyte collagenase proenzyme by rheumatoid synovial fluid. Biochim. Biophys. Acta 285, LEATHAM, J. H. (1961). Nutritional effects of endocrine secretion. In Sex and Internal Secretion. (3rd ed.) (W. C. Young, ed.), p. 686, Williams and Wilkins Co., Baltimore. LEE, C. Y. (1970). Proteolytic activity of sow ovarian follicle and its possible role in ovulation. Diss. Abstr. 31, 2942-B. LEE, C. Y., AND MALWN, B. (1970). Acid proteolytic activity of the sow ovarian follicle. Fed. Proc. 29, 643. (Abstr.). LEMAIRE, W. J., YANG, N. S. T., BEHRMAN, H. H., AND MARSH, J. M. (1973). Preovulatory changes in the concentration of prostaglandins in rabbit Graafian follicles. Prostaglandins 3, 367 (Abstr.). LIPNER, H. (1965). Induction of Craafian follicle proteolytic enzyme activity by human chorionic gonadotrophin (HCC) in rat Craafian follicles. Amer. Zool. 5, 167 (Abstr.). LIPNER, H., AND CREEP, R. 0. (1971). Inhibition of steroidogenesis at various sites in the biosynthetic pathway in relation to induced ovulation. Endocrinology 88, Los, H. S., AND WilsoN, C. W. M. (1971). Relationship of human ascorbic-acid metabolism to ovulation. Lancet 1, MANDL, I. (1961). Collagenases. Advan. Enzymol. 23, MANDL, I. (1972). Collagenase comes of age. In Collagenase. (I. Mandl, ed), p. 1, Gordin and Breach, Science Publishers, New York. MARSH, J. M., MILLS, T. M., AND LEMAIBE, W. J. (1973). Preovulatory changes in the synthesis of cyclic AMP by rabbit Graafian follicles. Biochim. Biophys. Acta 304, MIYATA, T., KAWAI, S., RUBIN, A. L., AND STENYEL, K. H. (1970). Tropocollagen: depolymerization by L-ascorbic acid. Biochim. Biophys. Acta 200, MorucAIw, R., AND COTHIE, S. (1946). Dissociation des cellules de la granulosa et probleme d un mechanisme diastasique dans Ia rupture du follicule ovarien de lapine. Compt. Rend. 140, MUKERJI, S., BELL, E. T., AND LORAINE, J. A. (1965). The effect of pregnant mare serum gonadotrophin and human chorionic gonadotrophin on rat ovarian ascorbic acid and cholesterol. J. Endocrinol. 31, NIEDERMEIER, W., LANKY, B. P., AND DoBsoN, C. (1967). The mechanism of action of ceruloplasmin in inhibiting ascorbic acil-induced depolymerization of hyaluronic acid. Biochim. Riophys. Acta 148, OKAMURA, H., VIRUTAMASEN, P., Wmcsrr, K. H., AND WALLACH, E. E. (1972). Ovarian smooth muscle in the human being, rabbit, and cat. Amer. I. Obstet. Gynecol. 112, O SIIRA, J. D. (1970). An ultrastructural study of smooth muscle-like cells in the theca externa of ovarian follicles in the rat. Anat. Rec. 167, PAESCHKE, K. D. (1967). Untersuchung des As-

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