Ovarian Proteolytic Enzymes and Ovulation

BIOLOGY OF REPRODUCTION 10, 216-235 (1974)

Copyright #{174}1974 by The Society for the Study of Reproduction.All rights of reproduction in any form reserved.

Ovarian Proteolytic Enzymes and Ovulation

LAWRENCE L. ESPEY

Trinity University, San Antonio, Texas 78284

Mammalian ovulation is a dynamic phe-

nomenon 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 in-

stances a thin translucent stigma, the

macula pellucida, forms at the apex of the

follicle as the final sign of impending rup-

ture. 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 fol-

licle 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 nip-

ple on a breast.” The moment of rupture

sometimes appears as an explosive event,

leading observers to compare it to a “vol-

cano 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 obser-

vations 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 evi-

dence” (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

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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 com-

posed 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 rup-

ture in sow follicles by maintaining a con-

stant 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 in-

stance, 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 struc-

tural constituent of the ovary. Several years

later the hypothesis was formulated

(Rouget, 1858; Grohe, 1863) that, in con-

tracting, 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 fol-

licle 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 chal-

lenge 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 intrafol-

licular 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 con-

trary, during the past several years there

has been a resurging interest in the possible

role of smooth muscle cells in the mecha-

nism 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” (Fum-

agalli et al., 1971) through “a contractile

force which seems to be effective in

dissociating the connective tissue” (Okam-

ura et at., 1972) leading to “an opening of

the stigma and the extrusion of the folli-

cular contents’ (Palti and Freund, 1972).

However, it is my opinion that none of the

recent ultrastructural studies clearly dem-

onstrate smooth muscle cells in the thecal

layer of the follicle wall. In one case

(Okamura et at., 1972), the electron micro-

graph 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 cyto-

plasmic 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 func-

tional role in the mechanism of ovulation.

Mechanical (mathematical) models. The

mature ovarian follicle is a rigid sphere

surrounded by a dense layer of collagenous

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218 ESPEY

connective tissue. An idealized mathemati-

cal 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 ovu-

lation 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 the-

ories concerning the events of ovulat�on.

However, his model is not only oversimpli-

fied, 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 com-

munication 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 mathe-

matical model, it is doubtful this analytical

approach will provide any useful informa-

tion 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 folli-

cles 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), negli-

gible force is required to penetrate the sur-

face of the follicle if it is close to rupture.

Thirdly, when attempts are made to dissect

whole follicles from the surface of the rab-

bit ovary, the follicles in precoital rabbits

are difficult to extirpate because of connec-

tive tissue adhesions which interlace the

follicular theca with the ovarian stroma.

However, near rupture this c:innective tis-

sue appears decomposed and the follicles

“peel out” with minimal surgery. Collec-

tively, these qualitative observations sug-

gest there is active decomposition of the

connective tissue in the ovary near rupture.

Microscopic changes. Microscopic tech-

niques have elucidated some of the transi-

tions in the fine structure of the follicle

wall as it approaches rupture. During ovu-

lation in the rabbit, the thecal tissue ap-

pears 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 pre-

ceding 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 lyso-

somal hydrolases might be active in the

ovulatory process. However, examination

of the ultrastructure of mature rabbit fol-

licles in this laboratory has revealed negli-

gible 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 theultrastructure of the Graafian follicle re-

vealed intriguing multivesicular bodies

which protrude from the follicular fibro-

blasts in increasing quantities as ovulation

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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. (Simi-

lar 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 struc-

tures indicates they might contain a chemi-

cal which can decompose follicular connec-

tive tissue. Current studies in this labora-

tory reveal that the contents in these struc-

tures are not comparable to lysosomes be-

cause they do not elicit a positive Gomori

reaction for acid phosphatase.

The evidence also shows that the multi-

vesicular structures are frequently located

in the leading edge of cytoplasmic pro-

cesses 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 fol-

licles near rupture have been overlooked

as evidence of active decomposition of the

follicular tissue during ovulation. During

the hours preceding rupture, there is an in-

crease in the vascularization of the follicu-

lar dome in pigs (Birger, 1952; Betteridge

and Raeside, 1962; Hunter, 1967), in rab-

bits (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 nip-

ture nears, it is common to observe pete-

chiae (Birger, 1952; Espcy, 196Th; Blan-

dau, 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 excep-

tion. Robb-Srnith (1952) found ovarian col-

lagen to be most abundant in the blood

vessels, tunica albuginea, theca folliculi,

and loose meditilary stroma.

Thus, the weakened condition of the vas-

cular compartment during ovulation is

probably a consequence of the same bio-

chemical processes which lead to the de-

composition 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 net-

work 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 de-

teriorate as rupture nears. In 1964, I dem-

onstrated 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 ovula-

tion. However, instead of measuring the

distensibility of the follicle wall, his proce-

dure may actually have detected changes

in the permeability of the blood-liquor bar-

rier in the follicle. Such a vascular change

does occur (Zachariae, 1958), and his ex-

perimental technique appears to l)e de-

signed to measure this change. In any case,

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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 cen-

tury ago Schochet (1916) first suggested

that proteolvtic enzymes weaken the folli-

cle 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 hy-

drochloric 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 se-

cretion 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 at-

tempts 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 ovula-

tion could be attributed to ordinary pro-

teolytic enzyme activity.

Intrafollicular injections of enzymcs.

More recently, we have found that the in-

jection of small quantities of concentrated

enzyme preparations directly into the an-

trum 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 (bac-

terial collagenase), nagarse and pronase

(also microbial enzymes) were the most

effective in inducing rupture. In the initial

study, trypsin was only moderately effec-

tive, 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 pep-

tidase, amino-peptidase, ficin, papain, lyso-

zyme, 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, gen-

eral protease, trypsin, alpha-chymotrypsin,

and to a lesser extent beta-chymotrypsin,

were effective in reducing the tensile

strength of the follicle wall. Amino-pep-

tidase, 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 follicu-

lar 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 con-

centration of hydrogen ions (pH 3.2).

L-diketogulonic acid and L-cysteine both

appeared to be slightly effective in reduc-

ing 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 con-

clusions that are important in future eval-

uations of the physiology of ovulation: (1)

it is clear that a variety of proteolytic en-

zymes can weaken the tensile strength of

the follicle wall, and (2) at least one non-

enzymic agent, ascorbic acid, has the

capacity to decompose follicular connective

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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 proteo-

lytic 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 comniunica-

tion). Gonadotropins possibly cause a de-

crease in this activity (Reichart, 1962).

Ovarian tissue also contains an acid

protease which can cause autolytic decom-

position of follicular tissue, with maximum

activity at pH 5, as evidenced by appear-

ance 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 ac-

tivity 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 possi-

bility that they are not of the same species

is evidenced by the fact that one enzyme

increases in the tissue in response to go-

nadotropin stimulation (Reichart, 1962),

whereas the other decreases (Espey and

Rondell, 1968).

Several other reports briefly mention hy-

drolytic enzymes in ovarian tissue. These

include hyaluronidase (Zachariae and Jen-

sen, 1958), acid phosphatase and esterase

(Banon et al., 1964), general protease

(Lipner, 1965), indopeptidase, leucine

aminopeptidase, and dipeptidase (Un-

behaun 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 en-

zymes, and especially the lack of informa-

tion 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). Great-

est attention has been on the types of en-

zymes which arc known to decompose fol-

licular 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 ovula-

tion 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, ho-

mogenized in distilled water (or buffer,

depending on the assay) at 10% w/v, centri-

fuged 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 carbo-

benzoxy - glycyl - prolyl - glycyl-glycyl-prolyl-

alanine (CBZ-GPGGPA). Only the en-

zyme( 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 con-

comitant increase in the follicular fluid.

These changes in measurable activity may

reflect physiological labilization and subse-

quent 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-

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222 ESPEY

TABLE 1

hYDROLYTIC ENZYMEs IN OVARIAN FoL1.ICLI�S DURING OvULA1’ION

Enzyme pIT n

Relative ac

wall before

20 h 5 h

tivity in

rupture

1 h

Relative activity in

fluid before ruptureAssay

(Ref.)20 h ?i h 1 h

General proteolytic 7.3 4 .01k .01 .01 .01 .01 .01 Rick, 1963a

Trypsin 7.8 3 . 16 . 18 . 17 .00 .00 .00 Rick, 1963b

Elasta.se 8.8 4 .04 .04 .03 .03 .033 .04 Sachar eta!., 1953

Cathepsin 4.7 4 .10 .09 .08 .02 .02 .02 Anson, 1939

Cathepsin 3.3 3 .36 .34 .36 .31) .31 .39 Anson, 1939

ilyase 3.3 2 .00 .00 .01 .00 .04 .01 Tolksdorf et a!., 1949

Collagenase 8.0 7 .23 .26 .18 .18 .32 .46 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 en-

zyme might be released from follicular tis-

sue 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 rab-

bits, 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 in-

crease 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 de-

tectable change in plasma collagenolytic

activity as ovulation neared. However, it

is possible the failure to observe an in-

crease 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 frac-

tionated and concentrated by ammonium

sulfate precipitation, and then assayed for

collagenolytic activity. By increasing the

sensitivity of the assay with this modifica-

tion, a 47% increase in enzyme concentra-

tion was measured in rabbits undergoing

ovulation in comparison with unmated con-

trol animals. In nine precoital animals, the

activity was 0.36 ± 0.053, (SEM) as com-

pared 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 ex-

amined, a collagenolytic enzyme has ap-

peared to be the most likely causative fac-

tor in the decomposition of the follicle dur-

ing 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”

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activity during the development of an ex-

traction 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

0.001 �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 cen-

trifuge 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 0.001 M CaCI2, (10) centrifuge

for 1 h at 30,000g. and lyophilize the super-

natant fluid to concentrate the preparation

before storage.

Lyophiized material from ten extrac-

tions (i.e., from 500g of corpora lutea)

was consolidated and resuspended in Tris

buffer (pH 8) to determine if this prepara-

tion could alter the tensile strength of ma-

ture 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 de-

scribed 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 en-

couraging support for the idea that follicu-

lar rupture is induced by an ovarian pro-

teolytic enzyme.

Autolytic activity. A major concern dur-

ing the early work, which utilized the tech-

nique of stretching follicular tissue to de-

termine 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 oc-

curring in the tissue, an evaluation was

made on the relationship between the dura-

tion 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 decom-

poses, and, in essentially all tests, decom-

position appeared within 24 h after incuba-

tion. 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. Be-

cause of these observations, in this labora-

tory, 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 ex-

periments (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 di-

gested the substrate CBZ-GPGGPA were

lyophilized and stored at -10#{176}C. After

500 g of follicular tissue were extracted, the

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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 resus-

pended extract, had a tensile strength of

17.6 g when stretched by 10% of their origi-

nal 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 activ-

ity” as the clostridiopeptidase-A solution,

and yet it did not reduce the tensile

strength of the follicular strips. This impor-

tant observation was confirmed by com-

plete duplication of the entire experiment,

starting with fresh tissue in the extraction

process. The results suggest that the follicu-

lar factor which digests the CBZ-GPGGPA

peptide may not be comparable to a “true”

collagenase. This conclusion is in agree-

ment 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 encoun-

tered in efforts to understand the enzymatic

processes involved in the remodeling of col-

lagenous 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, sig-

nificant advances have been made in the

past few years. A major breakthrough oc-

curred in 1968 (Eisen et at., 1968), when

a collagenolytic enzyme-one which could

not be detected in tissue extracts-was iso-

lated 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 encoun-

tered in efforts to extract an ovarian col-

lagenolytic enzyme. The reason the enzyme

is present in tissue culture, but not in ho-

mogenates of whole tissue, is not clear. Tis-

sue homogenates and extracts might con-

tain (1) enzyme at too low of a concentra-

tion, (2) enzyme in the form of an inactive

zymogen, (3) enzyme bound to its en-

dogenous collagen substrate, or (4) en-

zyme 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 24-48 h of incubation (Bauer et at.,

1970). This requirement of a long incuba-

tion time may explain why Rondell

(1970a) was unable to detect col-

lagenolytic activity in follicular material

which was also cultured, but only for 2-36

h.

In view of this new information on ani-

mal collagenases, this laboratory has begun

a reexamination of follicular tissue for col-

lagenolytic enzyme(s). The three major

approaches include: (1) the original tissue

culture method developed by Gross and

Lapiere (1962) which utilizes a reconsti-

tuted 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

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PROTEOLYTIC ENZYMES AND OvULATION 225

(3) an assay based on the detection of

soluble l4CAabeled glycine, which is re-

leased from reconstituted collagen fibrils

in culture medium (Jeffrey et al., 1971a).

These experiments were still in progress

at the time this review was written; never-

theless, 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 stan-

dard 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-Rin-

ger solution rather than Dulbecco’s Mod-

ified Eagle’s Medium, or Trowell’s Me-

dium. When lysis occurred, it was always

complete within the first 15 h of in-

cubation. No tissue induced transparency

in gels that were thicker than 1.5 mm,

even if the incubation time was ex-

tended to 72 h. Follicles that had not been

stimulated by gonadotropin (i.e., follicles

from precoital rabbits) were about as effec-

tive 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 ap-

peared 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 in-

sufficient enzyme to digest thick gels of re-

constituted collagen, (2) lysis does not

continue beyond the first 10-15 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 fol-

licles contain only a small amount of col-

lagenolytic 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 communica-

tion) has recently found that if the stigmal

area is dissected from the surface of a pre-

ovulatory follicle, it causes depolymeriza-

tion 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” cul-

ture media (e.g., Dulbecco’s and Tro-

well’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 activ-

ity 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 pre-

coital and prerupture follicles. LH, proges-

terone, and estrogen did not increase the

activity. No conclusion is warranted at this

time.

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226 ESPEY

Reconstituted collagen containing �C-

labeled 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 ac-

tivity from follicular tissue which digests

CBZ-GPGGPA is different from “true” col-

lagenase; 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 infor-

mation about animal collagen and col-

lagenase 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 alpha-

immunoglobulin fraction of serum, spe-

cifically 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 inactiva-

tion by irreversibly binding animal col-

lagenase) (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 col-

lagenase inhibitors in animal tissues is an

important consideration in the design of

future studies on the mechanism of ovula-

tion. The existence of these antiproteases

might explain the difficulty encountered in

searches for collagenolytic enzymes in

homogenates and extracts of follicular tis-

sue. These inhibitors could also impair the

detection of collagenolytic activity during

the first 24-48 h of tissue culture experi-

ments (Eisen et at., 1970a). (As a footnote,

it would be interesting to know if alpha-

immunoglobulins are elaborated by leuko-

cytes, 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 con-

tain 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), becausethis 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 pre-

sented to support the idea that proges-

terone might facilitate the decomposition

of the follicle wall (Lipner and Greep,

1971; Rondell, 1970b, 1974). This hy-

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pothesis is not consistent with the report

above that progesterone inhibits uterine col-

lagenase. 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 ac-

tivates 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 col-

lagenase (Jeffrey and Gross, 1970); the lat-

ter observation suggests collagenase in re-

productive 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). How-

ever, soybean trypsin inhibitor is not effec-

tive against human skin collagenase (Eisen

et at., 1968). Collagenolytic enzymes in rat

liver lysosomes are inhibited by the anti-

inflammatory drugs phenylbutazone and

ibufenac (Anderson, 1969). Gingival col-

lagenase is inhibited by cortisone and his-

tamine (Taylor, 1971). However, Houck

et at. (1970) reported that cortisol and in-

domethac�n induce the release of active

collagenase from mouse and human fibro-

blasts. It would be interesting to know if

these two agents could activate col-

lagenolysis in the fibroblasts in the follicle.

Although contradictory, it may be relevant

that Tsafriri et al. (1972) believe indo-

methacin exerts an antiovulatory action di-

rectly on the follicle and prevents follicular

rupture.

The effect of trypsin and trypsin inhibi-

tors 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 en-

zyme (e.g., interokinase activation of tryp-

sinogen), (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) en-

dogenous enzymic activity within the indi-

vidual zymogen molecule which allows

these precursors to activate themselves in

some instances. Regarding the latter exam-

ple, it is now evident that this intrinsic

self-activating property is possessed by a

wide variety of proteolytic enzymes (Kas-

sell 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 incuba-

tion with collagenase-free tailfin culture

media, but not with trypsin or chymotryp-

sin. Additional studies (Harper and Gross,

1972) have led to the conclusion that lack

of expression of collagenase activity in cul-

tured tissue during the first 24-48 h may

not be due to the presence of inhibitors

such as the aiphaimmunoglobulins, but, in-

stead, the result of a time lag before the

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228 ESPEY

appearance of activation factors which con-

vert the zymogen into active enzyme. This

conclusion is based on evidence that a pro-

collagenase 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 ex-

tracts which is activated by some com-

ponent 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 (Pil-

lay, 1940; Astrada and Caligaris, 1966;

Paeschke, 1967). This information raises

the question of whether ascorbic acid has

a significant role in the mechanism of ovu-

lation, or whether its depletion in response

to gonadotropin is primarily related to

steroidogenesis (Leatham, 1961). The lat-

ter 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 mecha-

nism 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 re-

sponse requires a relatively high acid con-

centration (pH 3.2) in the incubation me-

dium. Therefore, it is doubtful ascorbic

acid can elicit this effect under physiologi-

cal conditions, because the pH in the fol-

licular wall (and the follicular fluid) re-

mains slightly above neutral during the

ovulatory process (Espey, 1970; Lee,

1970).

It could be relevant that ascorbic acid

depolymerizes tropocollagen, even at neu-

tral 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 diges-

tion of reconstituted collagen gels (unpub-

lished), a fact which also points out that

ovarian follicles could induce the lysis of

gels by the release of ascorbic acid. In ad-

dition, ascorbic acid can induce the depoly-

merization of hyaluronic acid (Nieder-

meier et al., 1967), a major component of

the glycoaminoglycans in collagenous tis-

sue. 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 diketo-

gulonic 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 meet-

ings (Espey and McDavid, 1969) we dis-

cussed the possibility of this vitamin serv-

ing as an enzyme activator. This considera-

tion 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 re-

ports that (1) ascorbic acid appears to ac-

tivate 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).

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PHOTEOLYTIC ENZYMES AND OVULATION 229

Cyclic AMP. It has been established that

cyclic AMP mediates the action of gonado-

tropin in inducing luteinization of follicular

tissue (Channing, 1974) by stimulating ste-

roidogenesis (Marsh et at., 1973), particu-

larly’ progesterone synthesis (Herniier et

a!., 1972). Without going into repetitious

detail of material covered in other papers

in this symposium, it is appropriate to em-

phasize that cyclic AMP is known to stimu-

late collagenase and hyaluronidase activity

in media of organ cultures of bullfrog tad-

poles (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 arti-

cles on properties of collagenase enzymes.

Mandl (1961) has reviewed the informa-

tion 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 concen-

trating on the regulation of vertebrate col-

lagenase 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 ma-

hire ovarian follicle during ovulation in-

volve a complex sequence of reactions. De-

terioration 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 in-

eluding prostaglandins, see LeMaire et at.,

1973). The sequence of biochemical

events could prove to be highly complex.

In fact, complexity should probably be an-

ticipated because ovulation requires trau-

matic 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 biologi-

cal process which requires a high degree

of localization. On the positive side, how-

ever, because there are so many steps in

the clotting mechanism, there are also

numerous methods of controlling this phe-

nomenon. 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 sym-

posium that the primary output of the ovu-

latory process is a fertile egg.

One other point needs to be stressed be-

fore attempting to develop a general hy-

pothesis: It has been overlooked that the

follicle wall is fragile for only a short pe-

riod of time during the ovulatory process.

The weakened condition of the thecal tis-

sue 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 rup-

ture and (2) to meet the support require-

ments 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 pro-

cesses which normally occur in vivo, then

the very same factors which suppress col-

lagenolysis after ovulation should mask any

“enzyme” in culture medium.

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230 ESPEY

A “Working” Hypothesis

The following hypothesis on the mecha-

nism 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 de-

gree) are already producing steroids. As-

corbic acid is present in relatively high con-

centration 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 mem-

brana granulosa and theca interna).

During ovulation. Appropriate gonado-

tropin stimulation is expressed by a substan-

tial increase in cyclic AMP in the follicle

and an acceleration of steroidogenic activ-

ity. In a preliminary step towards luteiniza-

tion, there is a significant increase in mi-

totic 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 ste-

roids. 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 extracel-

lular 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 fac-

tor. The activation begins slowly, but

newly formed enzyme catalyzes a rapid ac-

celeration 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 un-

der the stress of a small (but very impor-

tant) 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 ruptu�e,

further collagenolysis is suppressed be-

cause (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 baso-

phil leukocytes in postovulatory follicles

possibly deposit additional antiproteases,

and (4) luteinization causes an excess�vely

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 considera-

tion in future studies.

1. What are the structural and chemical

differences between an immature and ma-

ture 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 fol-

licle are influenced by FSH and LH, and

which ones are not? There is growing infor-

mation on the responses of granulosa cells

to gonadotropin stimulation, but what

about the other cells? Do FSH and LII

Dow




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 im-

portant in ovarian steroidogenesis? Is it a

reducing agent for ovarian hydrolases?

4. What kinds of chemical agents are

produced by proliferating fibroblasts to di-

gest pathways through dense extracellular

connective tissue? How important to ovula-

tion 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 composi-

tion of the glycoaminoglycans and other

components of the extracellular cement at

the apex of the follicle wall? What types

of enzymes will depolymerize this ma-

terial?

6. What is the specific function of the

follicular antrum and fluid in mammalian

follicles? Are they for the purpose of pro-

tecting 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, pos-

sessing the same properties as other animal

collagenases, be isolated from follicular tis-

sue? Of particular importance is the evi-

dence 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 immuno-

genic properties, it would be extremely im-

portant to know whether the antibodies

(presumably alphaglobulins) could inhibit

ovulation.

9. Does the concentration of alpha1-anti-

trypsin or alpha2-macroglobulin in the

serum of mammals change during the sex-

ual cycle? What happens to the level of

these anti-collagenases in the days follow-

ing parturition, a time during which there

is a significant increase in collagenolysis

in the uterus. Can these serum immuno-

globulins be raised to a titer that would

inhibit follicular proteases during ovula-

tion? Since there is evidence that tissue

injury induces an increase in this type of

circulatory collagenase inhibitor, is it pos-

sible 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 sim-

ply 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 com-

parable to tumorous growths. For exam-

ples: (1) ovulation is initiated by adeno-

hypophyseal hormones, all of which induce

grotvth of some tissue(s) in the mam-

malian 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 tis-

sue, and, likewise, tumors stimulate fibro-

blasts to secrete enzymes which depoly-

merize 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-

Dow



232 ESPEY

BAUER, E. A., EISEN, A. Z., AND JEFFREY, J. J.(1972). Regulation of vertebrate collagenase

goes resorption, a phenomenon which

should be of special interest to cancer

researchers.

ACKNOWLEDGMENTS

The author acknowledges the dependable assis-

tance 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

02649 and HD-06371, and NIH Contract 69-2126.

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Ovarian Proteolytic Enzymes and Ovulation

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