Biochem. J. (1972) 127, 705-713Printed in Great Britain

Oestradiol Inhibits Collagen Breakdown in the Involuting Rat Uterus

By JANET N. RYAN* and J. FREDERICK WOESSNER, Jr.Departments ofBiochemistry and Medicine and Program in Cellular and Molecular Biology,

University ofMiami School of Medicine, Miami, Fla. 33152, U.S.A.

(Received 6 December 1971)

1. The earlier observation (Woessner, 1969) ofoestradiol inhibition ofcollagen breakdownis confirmed and extended. Administration of 100,ug of oestradiol-17,B/day to parturientrats strongly inhibits the loss of collagen from the involuting uterus. Three experimentsshow that this effect is due to an inhibition of collagen degradation rather than to astimulation of collagen synthesis. 2. Uterine collagen was labelled with hydroxy[14C]-proline by the administration of [14C]proline near the end of pregnancy. By 3 days postpartum, control uteri lost 83% of their collagen and 90% of their hydroxy['4C]proline.Uteri from oestradiol-treated rats lost only 50% of both total and labelled hydroxy-proline, with no decrease in the specific radioactivity of the hydroxyproline. 3. Incorpora-tion of [14C]proline into uterine collagen hydroxyproline in vivo was not affected byoestradiol treatment. 4. Urinary excretion ofhydroxyproline was increased in post-partumcontrol rats and decreased in oestradiol-treated rats. 5. An enzyme capable of cleaving4-phenylazobenzyloxycarbonyl-L-prolyl-L-leucylglycyl-L-prolyl-D-arginine (a substratefor clostridial collagenase) increased in activity in the post-partum uterus and wasunaffected by oestradiol treatment. 6. Uterine homogenates digested uterine collagenextensively at pH 3.2. This digestion was unaffected by the oestradiol treatment. 7.Lysosomal fractions prepared by density-gradient centrifugation of uterine homogenatescontained coincident peaks of cathepsin D activity and peptide-bound hydroxyproline.The cathepsin D and hydroxyproline contents of this peak were unaffected by oestradioltreatment.

Involution of the rat uterus during the post-partum period is an extensive remodelling processaccompanied by the rapid removal of collagen; asmuch as 85% of the total collagen present in theuterus at the end of pregnancy is removed within 4days (Harkness & Moralee, 1956; Woessner, 1962).This loss of collagen is dramatically decreased bythe administration of oestradiol-17# at the time ofparturition (Woessner, 1969). The fact that the uterifrom oestrogen-treated rats contain much morecollagen than do uteri from control rats by 3 dayspost partum might be due either to an inhibition ofcollagen breakdown or to the stimulation of new

collagen synthesis. The oestrogenic hormones gener-ally display anabolic rather than anticatabolicactivities. However, the amount of free hydroxy-proline in the involuting uterus was markedly de-creased by oestradiol treatment, and this was takento be good evidence for an inhibition of collagendegradation (Woessner, 1969). Other evidence for aninhibitory effect ofoestrogens on collagen breakdownin rat skin and bones has been presented by Skosey &

* Present address: Department of ExperimentalSurgery, University of Arizona, Tucson, Ariz. 85721,U.S.A.

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Kappas (1967) and Orimo et al. (1971). However, weconsidered that the finding of an anticatabolic effectof oestradiol on collagen metabolism might beplaced on a firmer basis by the use of isotopiclabelling. This seemed especially important in viewof reports (Henneman, 1968, 1971) suggesting thatoestradiol stimulates both synthesis and degradationof collagen. In the present paper we adduce evidencefrom the study of the breakdown of labelled collagen,the synthesis of labelled collagen and the appearanceof collagen catabolites in urine, which points toan anticatabolic action of oestradiol on collagenresorption.The physiological mechanisms by which collagen

is degraded during uterine involution have not beenfully elucidated. Nor has it been possible, thus far,to show at what point in these mechanisms oestradiolexerts its effect. It was shown earlier that lysosomalhydrolase activities increased during involution(Woessner, 1965) and that lysosomal cathepsinscould attack uterine collagen at acid pH values(Woessner, 1962). More recently, a number ofworkers(Brandes & Anton, 1969a,b; Parakkal, 1969;Schwarz & Guldner, 1967; Dessouky, 1971) haveprovided electron-microscopic evidence that uterine

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J. N. RYAN AND J. F. WOESSNER, Jr.

collagen is extensively phagocytized by macrophagesand is brought into digestive vacuoles containinglysosomal hydrolases.On the other hand, collagenase activity has been

detected in cultures of involuting uterus (Jeffrey &Gross, 1970). This collagenase is capable ofdegradingcollagen fibres at neutral pH values; it would presum-ably act outside the cell rather than inside digestivevacuoles. Although attempts to demonstrate colla-genase activity directly in uterine homogenates havemet with little success (Schaub, 1963; Morrione &Seifter, 1962; Woessner & Brewer, 1963; Jeffrey &Gross, 1970), we have recently shown that the en-zyme is present and is presumably bound firmly tothe collagen fibres (Ryan & Woessner, 1971a). Asecond enzyme, which cleaves a pentapeptide usedfor the assay of bacterial collagenase, has been foundin the involuting rat uterus by Strauch (1970). Thisenzyme is sometimes designated as collagenase,although there is no evidence that it digests collagen.

In the present study we have looked for effects ofoestradiol on the lysosomal system of the rat uterusand on the enzyme that cleaves the pentapeptide(PZ-peptidase).

Materials and Methods

Treatment ofanimalsFemale white rats at 17 days of gestation were

purchased from Sprague-Dawley, Inc., Madison,Wis., U.S.A. They were kept in individual cages andfed on Purina Rat Chow and given water ad lib. Thetime of delivery was recorded and the young wereleft in the cage to permit the mother to nurse. Ratswhich failed to nurse or to deliver live young, orwhich were later found to have retained placentaeor foetuses, were discarded. Animals receivingoestradiol-17,B [oestra-1,3,5(10)-triene-3,17fl-diol;Mann Research. Laboratories, New York, N.Y.,U.S.A.] were injected intraperitoneally with 0.lOmlofa suspension ofthe hormone (100,ug) in 0.9% NaClshortly after delivery was complete and on each suc-cessive day until death. Controls received injectionsof 0.9% NaCl only. Rats were first stunned with ablow, then killed by cervical dislocation. Uteri weredissected free of fatty tissue and placental sites,washed in cold 0.9% NaCl, blotted and weighed.

Studies ofcollagen metabolismTo measure the resorption of prelabelled collagen,

L-[U-'4C]proline (New England Nuclear Corp.,Boston, Mass., U.S.A.; 209mCi/mmol) was dissolvedin 0.9% NaCl and adjusted to pH7 with a finalradioactivity of 50,tCi/ml. Rats were given a seriesof three intraperitoneal injections over a 24h periodon the estimated days 18 and 19 of gestation for atotal dose of approx. 3,uCi/100g body wt. Several

injections over a period of time were used in anattempt to minimize individual variations in theincorporation of label. For the studies of collagensynthesis, rats were given a single intraperitonealdose of 3 or 12,uCi of [14C]proline 3 h before removalof the uteri. These studies were done on day 19 ofgestation and the second day after parturition.Incorporation of label was found to be proportionalto the administered dose, so the results are presentedas d.p.m./4Ci of [14C]proline injected.

In both types of metabolic study the uterine tissuewas minced, homogenized in 5vol. of cold water,then dialysed exhaustively against water in the cold.The homogenates were then hydrolysed in 6M-HCIand analysed for total hydroxyproline (Woessner,1961) and for hydroxy[4C]proline (Juva & Prockop,1966). Samples were counted for radioactivity in aPackard Tri-Carb model 574 liquid-scintillationspectrometer (machine efficiency 75-78 %) and thencorrected for background and to 100% efficiency bythe use of external standards. The scintillation fluidcontained 80g of naphthalene, 7g of 2,5-diphenyl-oxazole and 300mg of 1,4-bis-(4-methyl-5-phenyl-oxazol-2-yl)benzene in 1 litre of toluene-dioxan-ethanol (4:4:2.4, by vol.). The incorporation oflabel into non-collagenous protein was calculatedfrom the total radioactivity (d.p.m.) of the hydro-lysate by subtracting the contribution from colla-gen, assuming a proline/hydroxyproline ratio of1 :1.11 for rat uterine collagen (Kao et al., 1968).Rat urine was collected continuously in metabolic

cages, and 24h samples were chromatographed onDowex 50W (X2) resin by the method of Koevoet &Baars (1969), hydrolysed in HCI and assayed forhydroxyproline (Woessner, 1961).

Studies with lysosomeUterine tissue was homogenized in lOvol. of

0.25M-sucrose containing 1 mM-EDTA and O.O1M-sodium phosphate, pH7.4. Samples were layeredon 35-65% (w/v) sucrose density gradients andcentrifuged in the SW25.1 rotor of the Beckmanmodel L ultracentrifuge at 60000g (ray. 9.1cm) for16h to reach equilibrium. Fractions (1 ml) werecollected from the bottom of the tube, and sampleswere assayed for cathepsin D (Woessner & Brewer,1963). The fractions were dialysed to remove sucrose,hydrolysed in HCI and assayed for hydroxyproline(Woessner, 1961, method II).

Digestion of collagen by uterine cathepsins

Tissue was homogenized in lOvol. of 0.9 % NaCl,the pH was adjusted to 3.2 and 2.5vol. of 0.5M-sodium acetate buffer, pH3.2, was added. Sampleswere incubated overnight at 37°C and 0°C withshaking. They were then centrifuged at 30000g (ray.

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OESTRADIOL INHIBITS COLLAGEN BREAKDOWN

5.9cm) for 30min. The supernatants and pellets werecarefully separated, hydrolysed and assayed forhydroxyproline. The digestion of the endogenousuterine collagen by the uterine cathepsins was de-termined from the amount of hydroxyproline thathad been transferred from the pellet to the superna-tant after incubation at 37°C.

Assay ofPZ-peptidaseTissue was homogenized in 2mM-calcium acetate

and centrifuged at low speed (600g). The super-natant was tested for its ability to cleave 4-phenylazo-benzyloxycarbonyl-L-prolyl-L-leucylglycyl-L-prolyl-D-arginine (PZ-peptide; Mann Research Labora-tories) by using the assay developed by Wunsch &Heidrich (1963). High-voltage paper electrophoresisand amino acid chromatography were used toestablish that uterine extracts cleaved the Leu-Glybond.

ResultsMetabolism of collagen during involutionWe studied the effect of oestradiol on collagen

metabolism during uterine involution by examiningthree parameters: collagen resorption, collagen syn-thesis and the excretion of hydroxyproline in theurine. To measure the extent of collagen resorption,uterine collagen was labelled in vivo with [14C]prolineduring the final 3-4 days of gestation, and theanimals were randomly assigned to three groups.The amount of total hydroxyproline and of hydroxy-

['4C]proline present in the uterus at parturition wasdetermined for group 1 (Table 1). The loss of totaland labelled hydroxyproline by the third day postpartum was measured in control (group 2) andoestrogen-treated rats (group 3). In agreementwith previous results (Woessner, 1965, 1969),approx. 80% of the total collagen present at thetime of parturition is lost within 3 days duringnormal involution. When rats are injected with 100,ugof oestrogen daily, this loss is only 50%. Similarresults are observed in the loss of labelled collagen,i.e. that material present at the end of pregnancy(approx. 90% of the hydroxy[14C]proline) is re-moved in the control group whereas only 48 % is lostduring treatment with oestrogen. Thus the oestrogen-mediated inhibition of resorption of prelabelledcollagen exactly matches the inhibition of resorptionof total collagen. The specific radioactivity of thecollagen from the uteri of rats treated with oestrogenfor 3 days is unchanged from the value at parturition.In contrast to the resorption of collagen, the re-sorption of prelabelled non-collagenous protein is notaffected by treatment with oestrogen.The rate of synthesis of uterine collagen was

compared in the two groups by measuring the in-corporation of [14C]proline in vivo (Table 2). Ratswere injected with label at noon of the second daypostpartum and were killed 3h later. The incorpora-tion of label into uterine collagen (hydroxyproline)during the late pre-partum period (day 19) was alsodetermined to compare collagen synthesis during thepost-partum period with that occurring at a time of

Table 1. Effect of oestradiol on collagen resorption during uterine involution

Figures in parentheses are the number of rats in each group. Values are presented as means+s.D. Significantdifferences from control: *P<0.001 and **P<0.05.

Wt. of rat (g)Wt. of uterus (mg)Content of hydroxyproline

(,ug/uterus)Radioactivity of hydroxyproline

(d.p.m./uterus)tSp. radioactivity of hydroxyproline

(d.p.m./,ug of hydroxyproline)Radioactivity of non-collagen

protein (d.p.m./uterus)Percentage of value at parturition

Hydroxyproline (jug/uterus)Hydroxyproline (d.p.m./uterus)Non-collagen protein

(d.p.m./uterus)

Parturition:day 0 (14)280 ± 30

2740 ± 4608660 ± 1160

32500 ± 1970

3.90 ± 2.50

182900 ±87300

Control:day 3 (16)300 ± 40620 ± 701500 ± 460

3300 ± 2300

2.30 ± 1.40

24800 ±15500

171014

Oestradiol-treated:day 3 (14)

275 ± 351000 ± 145*4300 ± 670*

16900 ± 9700*

3.85 ± 1.95**

33500 ± 11300

505218

t Label was introduced on days 18 and 19 of pregnancy.

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J. N. RYAN AND J. F. WOESSNER, Jr

Table 2. Effect of oestradiol on collagen synthesis during uterine involution

[14C]Proline was injected 3h before the animals were killed. Values are presented as means+ S.D.; the numberof animals is given in parentheses. Significant differences from control: *P<0.001 and **P<0.05.

Wt. of rat (g)Wt. of uterus (mg)Content of hydroxyproline

(,ug/uterus)Radioactivity of hydroxyproline

(d.p.m./g of uterus)Sp. radioactivity of hydroxyproline

(d.p.m/p,g of hydroxyproline)Radioactivity of non-collagen

protein (d.p.m./g of uterus)

Control:day 2 (5)307 ± 14885 ± 150

3450 ± 1050

500 + 210

0.143 ± 0.091

4613 ± 1528

Oestradiol-treated:day 2 (7)265 ± 10*1065 ± 90**4610 ± 670**

640 ± 170

0.147 ± 0.032

5903 ± 1109

Pre-partum(7)

3120 ± 380*8970 ± 2140*

1600 ± 1110**

0.560 ± 0.379*

10984 ± 3273*

rapid growth and synthesis. The synthesis of bothcollagen and non-collagen protein appeared to beslow at 2 days post partum compared with the pre-partum synthesis rate. Therewas no significant changein the incorporation of labelled proline into uterinecollagen after oestrogen administration.The excretion of hydroxyproline in the urine was

determined during involution (Table 3). By the secondday post partum, the amount of hydroxyprolineexcreted within a 24h period is significantly elevatedabove the average value observed during the final3 days of pregnancy. This increase in hydroxyprolinein the urine occurs at the time that collagen is beingmost rapidly lost from the uterus (Woessner, 1965).Oestrogen treatment results in a very significantdecrease in hydroxyproline in the urine relative tothat observed during normal involution as well asduring the pre-partum period.

Uterine lysosomesThe total activity of several uterine lysosomal

hydrolases such as cathepsin D, acid phosphataseand fl-glucuronidase was essentially unchangedby treatment with oestradiol during involution(Woessner, 1965). Nevertheless, the possibility stillremained that oestrogen might inhibit collagen de-gradation by altering lysosomal stability or by inter-fering with phagocytosis or some step in intralysoso-mal digestion. We prepared lysosome-rich fractionsfrom uteri of oestrogen-treated and untreated rats tocompare the stability of lysosomes prepared fromboth tissues. The recovery of intact lysosomes fromboth sources was approx. 20%; no increase in thesusceptibility of lysosomes to mechanical disruptionwas apparent after oestrogen treatment. Further,oestrogen did not increase lysosomal fragility as

studied by incubation at 37°C at pH 7.4 in iso-osmoticsucrose. Approx. 17% of the total cathepsin Dpresent in isolated lysosomes prepared from uteriof control rats was released after incubation for40min; this proportion was not altered by oestradioltreatment.

Lysosome-rich fractions were then centrifugedto equilibrium in 35-65% linear sucrose densitygradients. The individual fractions were assayed forcathepsin D and a sharp peak of activity was con-sistently observed at a density of approx. 1.18g/ml.A typical separation is illustrated in Fig. 1. Afterdialysis and hydrolysis of pooled fractions, hydroxy-proline was consistently found to be associated withthis peak. Treatment of the lysosome-rich fractionwith Triton X-100 before gradient centrifugationprevented most of the cathepsin D and the non-diffusible hydroxyproline-containing material fromentering the gradient (Fig. 1). Lysosomes were pre-pared from uteri removed from oestrogen-treatedrats on the second or third day ofinvolution and com-pared with those from untreated animals; no differ-ences were found in the equilibrium density of thepeak or in the ratio of hydroxyproline to cathepsinD in this peak.

It has also been shown that uterine collagen couldbe digested by endogenous cathepsins at acid pHvalues (Woessner, 1962). These studies were repeatedwith tissue from control and oestradiol-treated rats(Fig. 2). In controls, 90% of the collagen is digestedin overnight incubation; this percentage decreases asinvolution progresses. In tissue from the oestradiol-treated rats, the digestion of collagen remains at90% throughout the 5-day period. Thus the digestivecapacity of the uterus at acid pH values is not im-paired by oestrogen treatment, nor is the collagenrendered less susceptible to digestion.

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OESTRADIOL INHIBITS COLLAGEN BREAKDOWN

Table 3. Hydroxyproline excretion in the urine

Results are presented as means±S.D. Column (A) lists values of P for results significantly different from thepre-partum value and column (B) lists values of P for results significantly different from the control valuesafter treatment with oestradiol (Student's t test).

p

GroupPre-partumControlDay 1Day 2Day 3

Oestradiol-treatedDay 1Day 2Day 3

No. of animals22*

14139

10108

Hydroxyproline excreted(ug/24h)740±210

880±2001130± 3501090± 460

470± 200510±240510± 260

(A) (B)

0.01

0.0050.010.05

0.0010.0010.01

* Seven animals were studied daily during the last 34 days of gestation. The daily excretion remained constant duringthis period.

0.25 0.-

0.20 - la 0.

.21

* 0.5 _

_ 0.10 -

0.05 0.

0.100~~~~~0

0.05 0.

O L

2

0

5 10 Is 20 25

Fraction no.

65 A60 0

4540 835 0

Fig. 1. Localization of hydroxyproline and cathepsinD activity after centrifugation of homogenates ofuterus for 3h at 60000g through a 35-65% sucrose

density gradient

For experimental details see the text. Fractionvolume is 1 ml. (a) Pattern of homogenate of 3-daypost-partum uterus; (b) pattern of the same homo-genate treated with Triton X-100 before centrifuga-tion..... Concn. of sucrose; , hydroxyprolinecontent; *, cathepsin D activity.

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PZ-peptidase activity

The activity of a peptidase capable of cleaving thepentapeptide substrate for bacterial collagenase wasdetermined in homogenates of the involuting uterus(Fig. 3). The total activity per uterus was highestimmediately after parturition and then rapidly de-creased. Oestrogen treatment did not affect eitherthe total peptidase activity or the decrease in activitythat occurred during involution. However, since theuteri from the treated animals remained larger thanthose from the controls, the concentration of PZ-peptidase activity was considerably lower than in theuteri of untreated rats by the second daypostpartum.

Discussion

Inhibition of collagen degradation by oestradiol-17PThe original observation (Woessner, 1969) was

that at 3 days post partum uteri from rats that hadreceived oestrogen from the time of parturition con-tained significantly more collagen than did uteri fromcontrol animals. This observation is subject to twopossible interpretations. The first is that oestrogeninhibits collagen degradation during this period; thesecond is that degradation is unaffected whereas thesynthesis of new collagen is increased to replace partof that lost by degradation. On the basis of thedecrease in free hydroxyproline in uteri from oestro-gen-treated animals, it appeared that the main effectof oestrogen was to block the breakdown of collagen.The results presented in the present paper are con-sistent with this hypothesis.

23

6 (a)

5-

4 ~~~~03 -.. ...

2-

I * * 0e00a0 00.

00 '9

5 10 1 5 20 25

1~~~~~~~~~~~~~~~~~~~~~F (b)

Th,~0

sloomoose

709

J. N. RYAN AND J. F. WOESSNER, Jr.

90 F

80

70 p-4

la

60

50F

Pre-partumO 2 3Days postpartum

4 5

Fig. 2. Digestion of collagen at acid pH values inhomogenates of uteri

For experimental details see the Materials andMethods section. o, Control rats; *, oestradiol-treated rats. The pre-partum value was obtainedapproximately 1 day before parturition.

20

$-5K

0

4)

ZscaS

vPre-partumO 2 3

Days post partum4 5

Fig. 3. PZ-peptidase activity at pH7.1 in uteri

For experimental details see the text. o, Control rats;*, oestradiol-treated rats; each point representsthree animals.

Oestrogen treatment partially blocks the loss oflabel from uterine collagen during involution (Table1). By 3 days post partum uteri from oestrogen-treated rats contain 50% of the labelled hydroxy-proline present at the time of parturition; in con-trast to this, untreated animals retain only 10%.Further, the loss of hydroxyproline labelled beforeparturition exactly parallels the net loss of totalhydroxyproline. These results agree well with thepostulate that the degradation ofcollagen is decreasedby oestrogen treatment. They do not agree with thehypothesis that oestrogen stimulates new collagensynthesis since there is no decrease in the specificradioactivity of the collagen and since only 50%of the label is lost as compared with 90% lost fromthe controls.The incorporation of label during the post-partum

period into previously unlabelled collagen is notstimulated by oestrogen treatment (Table 2). It mightbe argued that the oestrogen-treated animals had alarger metabolic pool of free proline, which dilutedthe added label and masked an increase in collagensynthesis. Although this possibility cannot be ex-cluded, it does not seem likely. A considerable partof the uterine proline pool is presumed to arise from

the extensive breakdown of uterine protein. Table 1

indicated that non-collagen protein was breakingdown at control rates, or slightly slower in theoestrogen-treated animals, whereas collagen wasbreaking down much more slowly. Thus these twosources would, if anything, provide decreased prolinepools in oestrogen-treated animals. The finding of alack of stimulation of collagen synthesis by oestradiolis consistent with the failure to observe any dilutionof the specific radioactivity of collagen duringinvolution in the oestrogen-treated rat (Table 1).The amount ofhydroxyproline excreted in the urine

was used as an indicator of collagen metabolismthroughout the body. An increase in the excretion ofhydroxyproline could be due to an increase in eithercollagen degradation or collagen synthesis, since someof the newly synthesized forms of collagen are parti-cularly susceptible to degradation and turn overrapidly (Nimni et al., 1967). Conversely, decreasedamounts of hydroxyproline in the urine would in-dicate decreased collagen metabolism (Aer, 1968;Kohler & Pechet, 1966). The decrease in the excretionof hydroxyproline shown in Table 2 supports thecontention that oestrogen decreases the degradationofcollagen. It is not known how much ofthe hydroxy-

1972

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00 0

00

0 0

0

8

0

0

1- ---I~ f-

8

0

0~@

0i

@0

I

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OESTRADIOL INHIBITS COLLAGEN BREAKDOWN

proline in the urine derives from the uterus. If we usethe pre-partum value as a control, then of the 7000,ugof uterine hydroxyproline lost in the first 3 days, only900,ug (13%) would be accounted for by urinaryexcretion. This seems a reasonable value (cf.Kivirriko, 1970); however, if we accept this, then theoestrogen effect on hydroxyproline in the urine seemsextremely large in proportion to the inhibition ofcollagen breakdown in the uterus. Even if the entireurinary output of the control were derived from theuterus, the oestrogen effect would still be too large.It must be concluded therefore that the hydroxy-proline in the urine is derived from other sourcesin addition to the uterus, and that oestrogen inhibitsdegradation of collagen in these sources as well.Alternatively, oestrogen might modify the transportof hydroxyproline peptides through the kidney.There have been conflicting reports on the urinary

excretion ofhydroxyproline in the post-partum periodin women. Klein (1964) studied three patients andfound a maximum excretion of hydroxyproline at2-6 days post partum. This would agree with thepresent study and the known rate of involution of thehuman uterus (Woessner & Brewer, 1963). Klein &Yen (1970) found that only two of six patients showeda peak in excretion of hydroxyproline in the post-partum period whereas the other four reached a peakbefore parturition. Unfortunately, their study pres-ents almost no results for the period of greatestinterest, between 2 and 14 dayspostpartum. However,there is no question that the concentration of hydr-oxyproline in urine is well above normal valuesduring post-partum involution. Klein & Yen (1970)further argue that since the urinary amounts do notaccount for more than 10% of the uterine hydroxy-proline, and since the uterus is low in the prolidaseactivity required to cleave the hydroxyproline peptidesthat would otherwise appear in the urine, the uterinecollagen must therefore be removed by a non-degradative process that preserves the collagenmolecule more or less intact.

This argument is based on a faulty interpretationof the work of Smith (1948), who in fact showedconsiderable amounts of prolidase in the humanuterus. The argument also ignores the high prolidaseactivity and concentrations of free hydroxyprolinefound in the rat uterus (Woessner, 1962). We believethat most of the collagen is degraded to the levelof small peptides and free amino acids and that thefree hydroxyproline undergoes further metabolism(Wolf& Berger, 1958) so that only a small proportionof the breakdown products of collagen is found inthe urine.The observations that oestradiol decreases the loss

of labelled uterine collagen, does not alter the in-corporation of [14C]proline into uterine collagen anddecreases free hydroxyproline in the uterus and totalhydroxyproline in the urine, all lead to the inescapable

Vol. 127

conclusion that oestradiol inhibits collagen degrada-tion in the uterus and probably other tissues as well.Several lines of evidence support the contention thatoestrogen, at least in pharmacological doses, in-hibits collagen breakdown in a variety of tissues. Atleast four studies show that treatment of adultwomen with oestrogen leads to decreased urinaryexcretion of hydroxyproline (Young & Nordin,1967; Young et al., 1968; Katz & Kappas, 1968;Schwartz et al., 1969). Oestrogen has a similar effectin the treatment of osteoporotic women (Riggs et al.,1969), and the results of Orimo et al. (1971) showthat in rats there is a direct effect on the loss ofcollagen from bone. Ebling & Hale (1966) show thatthe hydroxyproline content of rat skin varies withthe hair cycle and that oestrogen decreases the fallin the collagen content. Other examples of oestrogeninhibition of collagen breakdown were cited byWoessner (1969).Henneman (1968, 1971) has suggested that

oestradiol stimulates collagen synthesis and break-down in various guinea-pig tissues. Guinea pigs weregiven [14C]proline over long periods of time to labelthe collagen; then oestrogen was administered for5 weeks. This caused the uterus to increase in weight,collagen content and [14C]hydroxyproline contentcompared with controls. The interpretation (Henne-man, 1971) is that oestrogen stimulated the de-gradation of labelled collagen from other tissuesources and stimulated the synthesis of new collagenfrom these degradation products. Such an explanationappears unlikely. The uterus collagen has one of theshortest half-lives known (Kao et al., 1961). It isdifficult to see how other tissues could contributeprecursors of higher specific radioactivity than thosealready in the uterus. On the other hand, if oestradioldecreases degradation, then it is easy to explain whythe treated uteri would contain more collagen at theend of 5 weeks and why they would then retain moreof their initial radioactivity than the controls at theend of this time. Similar arguments could be advancedfor the findings on guinea-pig bone (Henneman,1971). In any dynamic system of collagen metabolismit is essential to evaluate separately the relativecontributions of synthesis and degradation to thenet changes in collagen content.

Role of lysosomes and ofPZ-peptidaseWe consistently found non-diffusible hydroxy-

proline associated with the peak of cathepsin Dactivity after isopycnic density-gradient centrifuga-tion (Fig. la). If the homogenates were treated withTriton X-100, both the hydroxyproline and cathepsinD remained in the supernatant and no longer movedon centrifugation to the original lysosome position(Fig. lb). These results are intriguing and suggestthat the hydroxyproline may be associated with

711

712 J. N. RYAN AND J. F. WOESSNER, Jr.

membranous structures such as the lysosomes. Thesebiochemical observations may correspond to theelectron-microscopic picture of the involuting uterus,which shows collagen fragments within lysosome-likestructures in macrophages (see the introduction).The function of lysosomes in degradation of colla-

gen appears to be unimpaired by treatment withoestrogen. No differences were detected in the ratio ofcathepsin D to hydroxyproline in the lysosomalpeak obtained from uteri of control or treated rats;this suggests that the ability of uterine macrophagesto phagocytize collagen is unaffected. Further,collagen from the uteri of oestrogen-treated rats isreadily digested by the tissue acid proteinases (Fig. 2).These results, together with the previous failure to findsignificant alterations in the activities ofthe lysosomalhydrolases (Woessner, 1969), would seem to eliminatethe lysosomal digestive system as a mediator of theoestrogen effects.The behaviour of the PZ-peptidase in normal

involution (Fig. 3) is similar to that reported byStrauch (1970). The uterine content shows an in-crease in the post-partum period and the activity perg of tissue reaches a peak at 2-3 days. Platt &Frackenpohl (1971) also showed an increase in thisenzyme in the post-partum rat uterus, but theyfailed to specify whether they measured total uterineenzyme activity or enzyme activity per g of tissue. Weconclude that this enzyme is not significantly affectedby oestrogen treatment. Its role in collagen break-down remains to be determined; Strauch (1970) callsit a collagenase. However, we have not been ableto digest collagen with preparations of this enzyme,and we have been able to separate it from a uterineenzyme that does digest collagen (J. N. Ryan andJ. F. Woessner, unpublished work). Harper & Gross(1970) have obtained similar results for the collagen-ase and peptidase activities from cultures of tadpoletail fin.

In conclusion, we have found that oestradiolinhibits collagen breakdown in the involuting uterus,and it does not appear to act on several enzymesystems postulated or believed to be implicated incollagen catabolism. The most likely remainingmechanism by which oestrogen could inhibit de-gradation of collagen would be through an effect onthe activity of uterine collagenase. We have foundthat uterine collagenase activity increases during thetime of most rapid collagen loss and that this activityis decreased by treatment with oestrogen (Ryan &Woessner, 1971b). However, the true explanation isprobably more complicated than this finding wouldsuggest, since uterine tissue in culture responds toadded oestradiol by the production ofmore collagen-ase (Jeffrey et al., 1971).

This work was supported by U.S. Public Health ServiceGrant HE 11035 from the National Heart and Lung

Institute. J. N. R. received fellowship support from U.S.Public Health Service Training Grants HE 05463(National Heart and Lung Institute) and GM00649(General Medical Sciences). J. F. W. is an Investigatorof the Howard Hughes Medical Institute. Part of thematerial in this paper was submitted in the dissertation forthe Ph.D. degree (J. N. R.) at the University of Miami,January, 1971. We thank Mrs. Carolyn Taplin for herexcellent technical assistance.

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