Cell Tiss. Res. 162,531--539 (1975) 9 by Springer-Verlag 1975
Oxytocinase-Immunohistochemical Demonstration in the Immature and Term Human Placenta * C. W. Small and W. B. W a t k i n s Postgraduate School of Obstetrics and Gynaecology, University of Auckland, Auckland, New Zealand Received May 30, 1975 Summary. Oxytocinase (cystine aminopeptidase) was purified from human retroplacental serum by a combination of fractional precipitation, hydroxylapatite chromatography and gel exlusion chromatography on Sephadex G-200. The purified enzyme possessed a specific activity of 980 mIU/mg using L-cystine-di-p-nitroanilide as substrate. This represented a 3200 fold concentration from the starting material in an overall yield of 12%. Antibodies against oxytocinase were raised in rabbits and the y-globulin fraction labelled with fluorescein isothiocyanate prior to its use in the immunofluorescenee histechemical localization of the enzyme in human placental tissue. Oxytocinase was confined to the syncytiotrophoblastic cells of normal term, and immature placentas as well as in placentas from patients suffering from severe toxaemia. Specific immunofluorescence was also present in the outer margins of the chorion and to a lesser extent in the amnion. Key words: Human placenta - - Syncytiotrophoblast - - Oxytocinase - - Anti-oxytocinase serum - - Immunofluorescence histochemistry.
Introduction The presence, in the peripheral serum of pregnant women, of an enzyme capable of destroying the biological activity of the neurohypophysial hormone, oxytoein, was first realized b y Fekete (1930, 1932). Later in 1956, H a w k e r demonstrated t h a t placental extracts inactivated oxytoein, the p H o p t i m u m of which was higher (pH 7-8) t h a n t h a t resulting from inactivation due to general tissue enzymes (pit o p t i m u m of 6.0) (Ryd6n, 1966). Although it is now generally accepted t h a t the placenta is the origin of h u m a n pregnancy serum oxytoeinase there has, in the past, been much debate as to the source of this enzyme (for a review see Tuppy, 1968). P r e g n a n c y serum oxytocinase has been shown to be an aminopeptidase with specificity towards the cleavage of amide bouds of eystine and eysteine derivatives e.g. L-eystine-di-#-naphthylamide (Tuppy and Nesvadba, 1957), L-cystine-di-pnitroanilide (Tovey, 1969; Small and Watkins, 1971a) and S-benzyl-L-cysteinep-nitroanilide (Small and Watkins, 1974). I n the earliest histochemieal demonstration of oxytocinase in the h u m a n placenta Semm and Waidl (1962) used L-cystine-di-#-naphthylamide as a substrate. This m e t h o d was recently modified b y Seelig and Roemheld (1969) and J a m e s (1966) who also employed the/~-naphthylamide derivative of L-leucine as Send o//print requests to: Dr. W. B. Watkins, Postgraduate School of Obstetrics and Gynaeeology, University of Auckland, Auckland, :New Zealand.
* This work was financed by a grant from The Medical Research Council of New Zealand.
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C.W. Small and W. B. Watkins
a substrate. These latter workers concluded from their findings that both the leucine- a n d c y s t i n e a m i n o p e p t i d a s e a c t i v i t y r e s i d e d in t h e t r o p h o b l a s t a n d X - c e l l s of t h e h u m a n p l a c e n t a . I n t h i s p r e s e n t w o r k we h a v e s t u d i e d t h e l o c a l i z a t i o n of o x y t o c i n a s e in t h e p l a c e n t a u s i n g a n i m m u n o c y t o c h e m i c a l t e c h n i q u e in a s s o c i a t i o n w i t h a n antis e r u m r a i s e d a g a i n s t a p u r i f i e d p r e p a r a t i o n of p l a c e n t a l o x y t o c i n a s e . A s h o r t a c c o u n t of t h i s w o r k has p r e v i o u s l y b e e n p u b l i s h e d ( S m a l l a n d W a t k i n s , 1971 a).
M a t e r i a l s and M e t h o d s
Purification o/Oxytocinase The method of purification of oxytocinase is similar to that described by Kulling and Yman (1970). Retroplacental serum was collected at normal deliveries and stored at --20~ for periods up to 3 months before processing. Serum obtained from the blood covering the trophoblastic layer of the placenta was also collected and used. To the stirred, cooled (4~ retroplacental serum (800 ml) was added a mixture of 95% (w/v) ethanol (800 ml), water (2400 ml) and 8 ml of sodium acetate (0.8 M, p H 4.0). The pH of the resultant mixture was adjusted to 6.0 with either 1.0 N acetic acid or 1.0 N sodium hydroxide depending upon whether the final mixture was basic or acidic respectively. After removing the precipitate by centrifugation (1400 g for 15 min) a solution of 400 ml of zinc acetate (0.25 M, p H 6.0) in 19% (v/v) ethanol at --5~ was added to the supernatant. The mixture was maintained at --5~ for 4 hr centrifuged at --5~ (1400 g for 20 min) and the precipitate discarded. Hydroxylapatite (100 gm) (Bio Rad, l~ichmond, U.S.A.) was equilibrated with phosphate buffer (0.001 M, pH 7.4) and after air-drying, was added to the supernatant (aminopeptidase fraction). The suspension was stirred at 4~ for 3 hr and supernatant obtained after centrifugation at 1400 g for 20 rain was discarded. The hydroxylapatite was extracted with phosphate buffer (0.02 M, pH 7.4) (4 • 100 ml) and the combined washings concentrated to 30-40 ml in an ultrafiltration cell (Amicon model 420, Amicon Corporation, Lexington, U.S.A.) fitted with an XM-100 membrane. Purification of the aminopeptidase concentrate was initially achieved by chromatography on a column (2.8• cm) of hydroxylapatite equilibrated in phosphate buffer (0.001 M, pH = 7.4). Development of the column was carried out with a linear gradient of phosphate buffer (pH : 7.4) from 0.001 M --> 0.1 1V[over a total volume of 1L at a flow rate of 18 ml/hr. The material in the peak containing oxytocinase activity was collected and concentrated to 3 ml by vacuum dialysis against polyethyleneglycol 20000 for 16 hr at 4~ Further purification of the enzyme fraction was carried out at 4~ by gel filtration on a column (1.7 • 40 cm) of Sephadex G-200 at a flow rate of 5.2 ml/hr. The gel filtration was repeated until a single co-incident protein and enzyme peak was obtained.
Oxytocinase Assay Oxytocinase activity was measured by a previously described method (Small and Watkins, 1971b) using L-cystine-di-p-nitroanilide as substrate. One international unit of oxytocinase activity (IIU) is defined as that quantity of enzyme that will release one nanomole of p-nitroanilide/min at 37~
Antibodies to Human Oxytocinase An antiserum to the purified preparation of oxytocinase was raised in New Zealand albino rabbits by the subcutaneous injection of an emulsion containing Freund's complete adjuvant (Difco Laboratories, Detroit, U.S.A.) (1 ml) and the enzyme (1 mg) dissolved in phosphate buffer (0.01 M, pH : 7.4) (1 ml). The immunization was repeated at fortnightly intervals with Freund's incomplete adjuvant. After six weeks, the animals were bled three to four times on alternative days, and the serum stored at --20~ Antibodies were also raised against an impure preparation of oxytocinase i.e. against the fraction from the hydroxylapatite column.
Immunohistochemistry of Placengal Oxytocinase
533
Preparation o/ Labelled Anti.Oxytocinase Serum Antiserum (12 ml) raised against oxytocinase was labelled with fluoreseein isothioeyanate. Rabbit v-globulin was prepared by the addition of ammonium sulphate to 40% saturation. The precipitated v-globulins obtained by centrifugation (10000 g for 15 min) were redissolved in phosphate buffered saline (pH 7.1) (6 ml) and dialysed against phosphate buffered saline (2 • 5L) for 24 hr. Disodium hydrogen phosphate (0.2 M, 1 ml) was added to the v-globulin (8 mg in 4 ml) followed by fluorescein isothiocyanate solution (1 mg in 2 ml of disodium hydrogen phosphate 0.1 M). The pH was rapidly adjusted to 9.5 with trisodium orthophosphate (0.1 ~) and diluted to 8 ml with saline (0.9% w/v). After standing at 25~ for 30 min and subsequent cooling to 4~C, the reaction mixture was centrifuged (1400 g for 10 min). Unreacted fluorescent material was removed from the supcrnatant by gel filtration through a column (2 • 30 cm) of Sephadex G-25 at a flow rate of 18 ml/hr with the collection of 4 ml fractions. The labelled globulins were purified by ion-exchange chromatography on a column (1 • 30 cm) of ])EAE cellulose (Whatman, DE 32) equilibrated with phosphate buffer (0.01 M, pH 7.4) in order to remove highly charged molecules which would otherwise give rise to non-specific staining. Development of the column was achieved with a linear potassium chloride gradient from 0 --~ 0.5 M in phosphate buffer (0.01 M, pH 7.4) over a total volume of 500 ml. A flow rate of 9.6 ml/hr was used and 5 ml fractions collected. The peak that was eluted first was collected and concentrated against polyethylene glycol 20000 by vacuum dialysis. The molar ratio of fluorescein to protein was obtained from the absorption at 495 nm (A) and the protein concentration (P mg/ml) by substituting in the formula: Fluorescein to protein ratio = 2.8 • A/P. Where the factor 2.8 is derived from the molecular weight of fluorescein isothiocyanate and v-globulin and the molar extinction coefficient (73 • 103) of fluorescein isothiocyanate (Nairu, 1969).
Preparation o/ Tissue Sections and Fluorescent Staining Placental membranes, normal placentas and the placentas from women suffering from toxaemia were obtained at vaginal deliveries. The immature placentas (12-16 weeks) were obtained by suction curettage at termination of pregnancy. Small pieces of placental tissue were snap frozen by floating in an aluminium container on a mixture of dry ice-ethanol at --76~ Sections were cut on a cryostat (International Equipment Co., mass, U.S.A.) at 6-8 [z. membranes were rolled up prior to snap-freezing and sections were cut at a right angle to the axis of the roll. The sections attached to microscope slides were fixed by immersion in 95% (v/v) ethanol for 1 min and then air dried. In the "direct" staining procedure for the demonstration of oxytocinase, the tissue sections were covered with a drop of the fluorescein-labelled anti-oxytocinasc serum and incubated at room temperature for 15 rain under humid conditions. After washing the sections free of excess antiserum with phosphate-buffered saline (pH 7.4), they were mounted in glycerolsaline and viewed using a Leitz Wetzlar fluorescent microscope fitted with a K430 secondary filter. Control experiments were carried out using preimmune serum in place of anti-oxytociBase serum.
Results The results o b t a i n e d d u r i n g the various stages in the purification of oxytocinase are s u m m a r i z e d in Table 1. C h r o m a t o g r a p h y of the aminopeptidase fraction on a c o l u m n of h y d r o x y l a p a t i t e (Fig. 1) results i n the elution of a single m a j o r oxytocinase peak, over a phosphate c o n c e n t r a t i o n of 0-+0.005 raM. A second area of e n z y m e a c t i v i t y was also observed at a phosphate c o n c e n t r a t i o n of 0.015-+ 0.023 raM. The enzyme peak eluted first from the c o l u m n of h y d r o x y l a p a t i t e was rec h r o m a t o g r a p h e d on a c o l u m n of Sephadex G-200 (Fig. 2a) which resulted in a p a r t i a l separation of the oxytocinase fraction from other protein material. This procedure was repeated to give a coincident oxytocinase a n d protein peaks
534
C.W. Small and W. B. Watkins [po4] 3-
~160
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o.9.0.4 0.2
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Fig. 1. Ion-exchange chromatography of a retroplacental serum aminopeptidase fraction on a column (2.8• cm) of hydroxylapatite at a flow rate of 18 ml/hr. Fractions 5 ml were collected, (e-e) protein concentration, ( 9169 buffer concentration (mM), (o . . . . o) oxytocinase activity
Table 1. Summary of the purification of oxytocinase from human retroplacental serum Fraction
Volume (ml)
Activity (mIu/ml)
Activity Protein a recovered (mg/ml) (%)
Specific Puriactivity fica(mIu/mg) tion
Retroplacental serum Aminopeptidase fraction ex Hydroxylapatite 1st G-100 column 2nd G-100 column
800 36 2.7 4 3
215 450 2250 775 660
100 95 36 20 12
0.31 19.2 510 820 980
70 23.5 4.4 0.95 0.67
1.0 62 1650 2 650 3200
a Protein content was determined by the method of Lowry et al. (1951) using Versatol-A (General Diagnostics Division, Warner-Chilcott Laboratories, N.J. U.S.A.) as standard protein.
(Fig. 2 b). A f t e r t h e second c h r o m a t o g r a p h y on S e p h a d e x G-200 t h e o x y t o c i n a s e o b t a i n e d possessed a specific a c t i v i t y of 980 m I u / m g which corresponded to a 3200 fold purification as c o m p a r e d to t h e s t a r t i n g r e t r o p l a c e n t a l s e r u m w i t h a n overall r e c o v e r y of a c t i v i t y of 12 %. T h e homogeneous n a t u r e of t h e p r o d u c t was confirmed b y i m m u n o e l e c t r o phoresis a g a i n s t a n i m p u r e p r e p a r a t i o n of a n t i - o x y t o c i n a s e . As p r e v i o u s l y dem o n s t r a t e d ( W a t k i n s a n d Small, 1972) a single p r e c i p i t i n line was observed with t h e purified p r o t e i n whereas m u l t i p l e b a n d s were seen with a s a m p l e of p r e g n a n c y serum. T h e fluorescein-labelled a n t i - o x y t o c i n a s e serum possessed a fluorescein t o p r o t e i n r a t i o of 1:3 a n d a n a n t i b o d y t i t r e of 1 in 4. T h e l a t t e r was d e t e r m i n e d b y m i c r o i m m u n o d i f f u s i o n a g a i n s t a s t a n d a r d a n t i g e n a t a c o n c e n t r a t i o n of 1 m g / m l .
Immunohistochemistry of Placental Oxytocinase 1.6
535
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~
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~
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20 40 60 80 100 Elutionvolume(ml) i
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~
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o 0.4
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,
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.~
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.
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i
Fig. 2. (a) Gel filtration of the active fraction from Fig. 1 on a column (1.7 • 40 cm) of Sephadex G-200 at a flow rate of 5.2 ml/hr. Fractions 2 ml (o-o) protein concentration, (9 . . . . 0) oxytocinase activity. (b) l~echromategraphy of the oxytecinase peak from (a) on a column of G-200 using the same conditions as in Fig. 2a. Phosphate buffer (0.001 m, pH 7.4) was used as solvent
Immunofluorescence histology of oxytocinase in the normal (Fig. 3a), toxaemic (Fig. 3b) and immature (16 weeks gestation) (Fig. 4a) placentas demonstrates the presence of specific fluorescence confined to the syncytial tissue of the trophoblast. There was no apparent differences in the staining intensity observed in immature, term or toxaemic placentas. In the sections of placental membranes (Fig. 4b) the immunofluorescence was found to be mainly associated with the chorion and to a lesser extent with the amnion.
Discussion
Oxytocinase was first purified from retroplacental serum by Tuppy and ~Vintersberger (1960) who used ammonium sulphate fractionation, rivanol precipitation and absorption of the impurities onto bentonite. A later procedure used by Barth et al. (1967) involving ethanol fractionation, gel filtration and DEAE-cellulose ion exchange chromatography resulted in a 3 000 fold purification of the enzyme with an overall yield of 9 %. This yield was subsequently increased to 25-38% by modification of the ethanol fractionation (Barth et al., 1971). The method used for the purification of oxytociuase in this present paper, however, is based upon that procedure described by Kulling and Yman (1970). Although our product possessed a lower purification factor than that obtained by Yman and his colleagues (Sjoholm and Yman, 1966) its immunological homogenity indicated its suitability for the present studies. The observations that specific immunofluorescence is confined to the syncytium of the placenta is in accord with the available histochemical evidence of Semm and Waidl (1962) and James (1966). However, since the substrate used by these workers, L-eystine-di-fl-naphthylamide, would be expected to be at-
536
C.W. Small and W. B. Watkins
Fig. 3a and b. Immunofluorescence histological localization of oxytocinase in the syncytium of (a) normal and (b) toxaemic placenta ( • 155)
tacked by aminopeptidases other than oxytocinase, a positive reaction product cannot necessarily be attributed to eystine aminopeptidase (oxytocinase) alone. Similarly, the histochemical inhibition studies carried out by Seelig and Roemheld (1969) does not completely preclude the contribution of aminopeptidases towards a positive histochemical reaction. Our immunocytological findings provide conclusive evidence for the placental origin of serum oxytocinase. That oxytocinase is localized in the synetium supports the earlier conclusions of Page (1946) who showed that rise in oxytocinase activity during pregnancy followed the increase in syncytial mass rather than of cytotrophoblastic bulk which diminishes after mid-pregnancy. Mathur and Walker (1970), using a novel morphometrical technique, showed that there was a positive correlation between oxytocinase activity and the proportion of trophoblast in the placenta and thereby concluded that oxytocinase is located in the syncytio-
Immunohistochemistry of Placental Oxytocinase
537
Fig. 4a and b. Specific localization of oxytocinase in (a) the cells of the outer layer of an immature placenta trophoblast and (b) in the chorion (C) and amnion (A). ( X 155)
trophoblast. I t is interesting to note that other placental components t h a t are released in increasing amounts into the maternal peripheral system as pregnancy progresses are also localized in the syncytium viz, human placental lactogen (Beck et al., 1969) and heat stable alkaline phosphatase (Hempel and Geyer, 1969; Watkins and Anderson, 1971; Watkins and Small unpublished observations). I n studying the effect of p H on the hydrolysis of L-cystine-di-fl-naphthylamide by extracts of human amnion and chorion, Ryd6n (1966) showed that the highest cystine aminopeptidase activity occurred at a p H of approximately 6.0, which is indicative of the action of non-specific tissue aminopeptidases alone. Our findings, however, have shown t h a t the chorionic tissue is associated with an enzyme that possesses immunological determinants cross-reactive against an antiserum produced against placental oxytocinase. The presence of oxytocinase
538
C.W. Small and W. B. Watkins
in the a m n i o n m a y be expected to result from either local synthesis, t r a n s p o r t from the chorion or a b s o r p t i o n from the amniotic fluid. I t is d o u b t f u l if the latter m e c h a n i s m operates in view of the findings t h a t h u m a n amniotic fluid appears to c o n t a i n only traces of cystine aminopeptidase a c t i v i t y (Miiller-Hartburg et al., 1959 ; Itashimoto, 1961 ; Riad, 1962).
References Barth, T., Pli~ka, V., Rychlik, I., ~orm, F. : Enzymatic inactivation of oxytocin V. Purification and some properties of enzymes from human retroplacental serum. Coll. Czech. chem. Comm. 32, 2327-2336 (1967) Barth, T., Rychllk, I., Mannsfeldt, H. G. : Human pregnancy oxytocinase isolation and substrate characteristics. Coll. Czech. chem. Comm. 36, 2540-2546 (1971) Beck, J. S., Gordon, R. L., Donald, D., Melvin, J. M. 0.: Characterisation of antisera to a growth-hormone-like placental antigen (human placental lactogen): Immunofluorescence studies with these sera on normal and pathological syncytiotrophoblast. J. Path. 97, 545555 (1969) Fekete, K. : Beitr~ge zur Physiologie der Gravidlt~t. Endokrinologie 7, 364-369 (1930) Fekete, K.: Gibt es w~hrend der Schwangerschaft ein aktives Hypophysen-Hinterlappenhormon im Blute? Endokrinologie 1O, 16-23 (1932) Hashimoto, T. : Studies on 1-cystine-aminopeptidase. Part II. Clinical observations. J. Jap. obstet, gynaec. Soc. 8, 96-104 (1961) Hawker, R. W. : Inactivation of antidiuretic hormone and oxytocin during pregnancy. Quart. J. exp. Physiol. 41, 301-308 (1956) Hempel, Von E., Geyer, G.: Submikroskopische Verteilung der alkalischen Phosphatase in der mensehlichen Placenta. Acta histochem. (Jena) 34, 138-147 (1969) James, N. T.: Histochemical demonstration of oxytocinase in the human placenta. Nature (Lend.) 210, 1276-1277 (1966) Kulling, B., Yman, L.: Purification and some properties of leucine aminopeptidases from retroplacental serum. Acta pharma, sueeica 7, 65-74 (1970) Lowry, 0. H., Rosebrough, N. J., Farr, A. L., Randall, R. J. : Protein measurement with the Folin Phenol reagent. J. biol. Chem. 193, 265-275 (1951) Mathur, V. S., Walker, J. M. : The origin of human placental oxytocinase. J. Physiol. (Lend.) 208, 291-298 (1970) Mfiller-Hartburg, W. H., Nesvabda, H., Tuppy, H. : Die Anwendung einer chemischen Methode zur Bestimmung des Oxytocinasespiegels im Schwangerenserum. Arch. Gyn~k. 191, 442-456 (1959) Nairn, R. C. : Fluorescent protein tracing, 3rd edn. London: Livingstone 1969 Page, E. W. : The value of plasma pitocinase determinations in obstetrics. Amer. J. Obstet. Gynec. 52, 1014-1022 (1946) Riad, A.M.: Studies on pregnancy serum eystine aminopeptidase activity "oxytocinase". J. Obstet. Gynaec. Brit. Comm. 69, 409-416 (1962) Ryd@n, G.: Cystine aminopeptidase and oxytocinase activity in pregnancy. Acta. obstet. gynaec, scand. Vol. 45, Suppl. No. 3 (1966) Scelig, H.-P., Roemheld, 1~.: Untersuchungen zur histochemischen Lokalisation der Leucinund Cystinaminopeptidase (Oxytocinase) in der Placenta. Histoehemie 18, 30-39 (1969) Semm, K., Waidl, U. E.: Histochemische Untersuehungen fiber die Serumoxytocinase-Bildung im menschlichen Trophoblasten. Z. Geburtsh. Gyn~k. 158, 165-171 (1962) SjSholm, I., Yman, L. : Preparation of highly purified oxytocinase (cystine aminopeptidase) from retroplacental serum. Acta pharma, suecica 3, 377-388 (1966) Small, C. W., Watkins, W. B. : Immunochemieal localisation of some placental enzymes. N. Z. Med. J. 74, 338 (1971a) Small, C. W., Watkins, W. B. : An improved method for the determination of human pregnancy serum oxytoeinase activity. Enzymologia 41, 121-128 (1971b) Small, C. W., Watkins, W. B. : S-benzyl-L-cysteine-p-nitroanilide.A new substrate for the determination of oxytocinase with improved specificity. Biochem. Med. 9, 103-112 (1974)
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Tovey, J. E.: Serum oxytocinase. Clin. Biochem. 2, 289-310 (1969) Tuppy, H. : The influence of enzymes on neurohypophysial hormones and similar peptidcs. In: Handbook of experimental pharmacology, ed. Berde, B., p. 67-129. Berlin-HeidelbergNew York: Springer 1968 Tuppy, H., Nesvadba, U . H . : ~ber die Aminopeptidaseaktivit~t des Schwangerenserums und ihre Beziehung zu dessen Verm5gen, Oxytocin zu inaktivieren. Mh. Chem. 88, 977-988 (1957) Tuppy, H., Wint~rsberger, E.: Reinigung und Eigenschaften der Serumoxy~cinase. Mh. Chem. 91, 1001-1010 (1960) Watkins, W. B., Anderson, S. R. A. : The origin of heat-stable placental alkaline phosphatase. J. Obstet. Gynaec. Brit. Comm. 78, 691-693 (1971) Watkins, W. B., Small, C.W.: Immunologic inactivation of human pregnancy serum oxytocinase activity. Amer. J. Obstet. Gynec. 118, 973-978 (1972)
Oxytocinase-Immunohistochemical Demonstration in the Immature and Term Human Placenta * C. W. Small and W. B. W a t k i n s Postgraduate School of Obstetrics and Gynaecology, University of Auckland, Auckland, New Zealand Received May 30, 1975 Summary. Oxytocinase (cystine aminopeptidase) was purified from human retroplacental serum by a combination of fractional precipitation, hydroxylapatite chromatography and gel exlusion chromatography on Sephadex G-200. The purified enzyme possessed a specific activity of 980 mIU/mg using L-cystine-di-p-nitroanilide as substrate. This represented a 3200 fold concentration from the starting material in an overall yield of 12%. Antibodies against oxytocinase were raised in rabbits and the y-globulin fraction labelled with fluorescein isothiocyanate prior to its use in the immunofluorescenee histechemical localization of the enzyme in human placental tissue. Oxytocinase was confined to the syncytiotrophoblastic cells of normal term, and immature placentas as well as in placentas from patients suffering from severe toxaemia. Specific immunofluorescence was also present in the outer margins of the chorion and to a lesser extent in the amnion. Key words: Human placenta - - Syncytiotrophoblast - - Oxytocinase - - Anti-oxytocinase serum - - Immunofluorescence histochemistry.
Introduction The presence, in the peripheral serum of pregnant women, of an enzyme capable of destroying the biological activity of the neurohypophysial hormone, oxytoein, was first realized b y Fekete (1930, 1932). Later in 1956, H a w k e r demonstrated t h a t placental extracts inactivated oxytoein, the p H o p t i m u m of which was higher (pH 7-8) t h a n t h a t resulting from inactivation due to general tissue enzymes (pit o p t i m u m of 6.0) (Ryd6n, 1966). Although it is now generally accepted t h a t the placenta is the origin of h u m a n pregnancy serum oxytoeinase there has, in the past, been much debate as to the source of this enzyme (for a review see Tuppy, 1968). P r e g n a n c y serum oxytocinase has been shown to be an aminopeptidase with specificity towards the cleavage of amide bouds of eystine and eysteine derivatives e.g. L-eystine-di-#-naphthylamide (Tuppy and Nesvadba, 1957), L-cystine-di-pnitroanilide (Tovey, 1969; Small and Watkins, 1971a) and S-benzyl-L-cysteinep-nitroanilide (Small and Watkins, 1974). I n the earliest histochemieal demonstration of oxytocinase in the h u m a n placenta Semm and Waidl (1962) used L-cystine-di-#-naphthylamide as a substrate. This m e t h o d was recently modified b y Seelig and Roemheld (1969) and J a m e s (1966) who also employed the/~-naphthylamide derivative of L-leucine as Send o//print requests to: Dr. W. B. Watkins, Postgraduate School of Obstetrics and Gynaeeology, University of Auckland, Auckland, :New Zealand.
* This work was financed by a grant from The Medical Research Council of New Zealand.
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C.W. Small and W. B. Watkins
a substrate. These latter workers concluded from their findings that both the leucine- a n d c y s t i n e a m i n o p e p t i d a s e a c t i v i t y r e s i d e d in t h e t r o p h o b l a s t a n d X - c e l l s of t h e h u m a n p l a c e n t a . I n t h i s p r e s e n t w o r k we h a v e s t u d i e d t h e l o c a l i z a t i o n of o x y t o c i n a s e in t h e p l a c e n t a u s i n g a n i m m u n o c y t o c h e m i c a l t e c h n i q u e in a s s o c i a t i o n w i t h a n antis e r u m r a i s e d a g a i n s t a p u r i f i e d p r e p a r a t i o n of p l a c e n t a l o x y t o c i n a s e . A s h o r t a c c o u n t of t h i s w o r k has p r e v i o u s l y b e e n p u b l i s h e d ( S m a l l a n d W a t k i n s , 1971 a).
M a t e r i a l s and M e t h o d s
Purification o/Oxytocinase The method of purification of oxytocinase is similar to that described by Kulling and Yman (1970). Retroplacental serum was collected at normal deliveries and stored at --20~ for periods up to 3 months before processing. Serum obtained from the blood covering the trophoblastic layer of the placenta was also collected and used. To the stirred, cooled (4~ retroplacental serum (800 ml) was added a mixture of 95% (w/v) ethanol (800 ml), water (2400 ml) and 8 ml of sodium acetate (0.8 M, p H 4.0). The pH of the resultant mixture was adjusted to 6.0 with either 1.0 N acetic acid or 1.0 N sodium hydroxide depending upon whether the final mixture was basic or acidic respectively. After removing the precipitate by centrifugation (1400 g for 15 min) a solution of 400 ml of zinc acetate (0.25 M, p H 6.0) in 19% (v/v) ethanol at --5~ was added to the supernatant. The mixture was maintained at --5~ for 4 hr centrifuged at --5~ (1400 g for 20 min) and the precipitate discarded. Hydroxylapatite (100 gm) (Bio Rad, l~ichmond, U.S.A.) was equilibrated with phosphate buffer (0.001 M, pH 7.4) and after air-drying, was added to the supernatant (aminopeptidase fraction). The suspension was stirred at 4~ for 3 hr and supernatant obtained after centrifugation at 1400 g for 20 rain was discarded. The hydroxylapatite was extracted with phosphate buffer (0.02 M, pH 7.4) (4 • 100 ml) and the combined washings concentrated to 30-40 ml in an ultrafiltration cell (Amicon model 420, Amicon Corporation, Lexington, U.S.A.) fitted with an XM-100 membrane. Purification of the aminopeptidase concentrate was initially achieved by chromatography on a column (2.8• cm) of hydroxylapatite equilibrated in phosphate buffer (0.001 M, pH = 7.4). Development of the column was carried out with a linear gradient of phosphate buffer (pH : 7.4) from 0.001 M --> 0.1 1V[over a total volume of 1L at a flow rate of 18 ml/hr. The material in the peak containing oxytocinase activity was collected and concentrated to 3 ml by vacuum dialysis against polyethyleneglycol 20000 for 16 hr at 4~ Further purification of the enzyme fraction was carried out at 4~ by gel filtration on a column (1.7 • 40 cm) of Sephadex G-200 at a flow rate of 5.2 ml/hr. The gel filtration was repeated until a single co-incident protein and enzyme peak was obtained.
Oxytocinase Assay Oxytocinase activity was measured by a previously described method (Small and Watkins, 1971b) using L-cystine-di-p-nitroanilide as substrate. One international unit of oxytocinase activity (IIU) is defined as that quantity of enzyme that will release one nanomole of p-nitroanilide/min at 37~
Antibodies to Human Oxytocinase An antiserum to the purified preparation of oxytocinase was raised in New Zealand albino rabbits by the subcutaneous injection of an emulsion containing Freund's complete adjuvant (Difco Laboratories, Detroit, U.S.A.) (1 ml) and the enzyme (1 mg) dissolved in phosphate buffer (0.01 M, pH : 7.4) (1 ml). The immunization was repeated at fortnightly intervals with Freund's incomplete adjuvant. After six weeks, the animals were bled three to four times on alternative days, and the serum stored at --20~ Antibodies were also raised against an impure preparation of oxytocinase i.e. against the fraction from the hydroxylapatite column.
Immunohistochemistry of Placengal Oxytocinase
533
Preparation o/ Labelled Anti.Oxytocinase Serum Antiserum (12 ml) raised against oxytocinase was labelled with fluoreseein isothioeyanate. Rabbit v-globulin was prepared by the addition of ammonium sulphate to 40% saturation. The precipitated v-globulins obtained by centrifugation (10000 g for 15 min) were redissolved in phosphate buffered saline (pH 7.1) (6 ml) and dialysed against phosphate buffered saline (2 • 5L) for 24 hr. Disodium hydrogen phosphate (0.2 M, 1 ml) was added to the v-globulin (8 mg in 4 ml) followed by fluorescein isothiocyanate solution (1 mg in 2 ml of disodium hydrogen phosphate 0.1 M). The pH was rapidly adjusted to 9.5 with trisodium orthophosphate (0.1 ~) and diluted to 8 ml with saline (0.9% w/v). After standing at 25~ for 30 min and subsequent cooling to 4~C, the reaction mixture was centrifuged (1400 g for 10 min). Unreacted fluorescent material was removed from the supcrnatant by gel filtration through a column (2 • 30 cm) of Sephadex G-25 at a flow rate of 18 ml/hr with the collection of 4 ml fractions. The labelled globulins were purified by ion-exchange chromatography on a column (1 • 30 cm) of ])EAE cellulose (Whatman, DE 32) equilibrated with phosphate buffer (0.01 M, pH 7.4) in order to remove highly charged molecules which would otherwise give rise to non-specific staining. Development of the column was achieved with a linear potassium chloride gradient from 0 --~ 0.5 M in phosphate buffer (0.01 M, pH 7.4) over a total volume of 500 ml. A flow rate of 9.6 ml/hr was used and 5 ml fractions collected. The peak that was eluted first was collected and concentrated against polyethylene glycol 20000 by vacuum dialysis. The molar ratio of fluorescein to protein was obtained from the absorption at 495 nm (A) and the protein concentration (P mg/ml) by substituting in the formula: Fluorescein to protein ratio = 2.8 • A/P. Where the factor 2.8 is derived from the molecular weight of fluorescein isothiocyanate and v-globulin and the molar extinction coefficient (73 • 103) of fluorescein isothiocyanate (Nairu, 1969).
Preparation o/ Tissue Sections and Fluorescent Staining Placental membranes, normal placentas and the placentas from women suffering from toxaemia were obtained at vaginal deliveries. The immature placentas (12-16 weeks) were obtained by suction curettage at termination of pregnancy. Small pieces of placental tissue were snap frozen by floating in an aluminium container on a mixture of dry ice-ethanol at --76~ Sections were cut on a cryostat (International Equipment Co., mass, U.S.A.) at 6-8 [z. membranes were rolled up prior to snap-freezing and sections were cut at a right angle to the axis of the roll. The sections attached to microscope slides were fixed by immersion in 95% (v/v) ethanol for 1 min and then air dried. In the "direct" staining procedure for the demonstration of oxytocinase, the tissue sections were covered with a drop of the fluorescein-labelled anti-oxytocinasc serum and incubated at room temperature for 15 rain under humid conditions. After washing the sections free of excess antiserum with phosphate-buffered saline (pH 7.4), they were mounted in glycerolsaline and viewed using a Leitz Wetzlar fluorescent microscope fitted with a K430 secondary filter. Control experiments were carried out using preimmune serum in place of anti-oxytociBase serum.
Results The results o b t a i n e d d u r i n g the various stages in the purification of oxytocinase are s u m m a r i z e d in Table 1. C h r o m a t o g r a p h y of the aminopeptidase fraction on a c o l u m n of h y d r o x y l a p a t i t e (Fig. 1) results i n the elution of a single m a j o r oxytocinase peak, over a phosphate c o n c e n t r a t i o n of 0-+0.005 raM. A second area of e n z y m e a c t i v i t y was also observed at a phosphate c o n c e n t r a t i o n of 0.015-+ 0.023 raM. The enzyme peak eluted first from the c o l u m n of h y d r o x y l a p a t i t e was rec h r o m a t o g r a p h e d on a c o l u m n of Sephadex G-200 (Fig. 2a) which resulted in a p a r t i a l separation of the oxytocinase fraction from other protein material. This procedure was repeated to give a coincident oxytocinase a n d protein peaks
534
C.W. Small and W. B. Watkins [po4] 3-
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Fig. 1. Ion-exchange chromatography of a retroplacental serum aminopeptidase fraction on a column (2.8• cm) of hydroxylapatite at a flow rate of 18 ml/hr. Fractions 5 ml were collected, (e-e) protein concentration, ( 9169 buffer concentration (mM), (o . . . . o) oxytocinase activity
Table 1. Summary of the purification of oxytocinase from human retroplacental serum Fraction
Volume (ml)
Activity (mIu/ml)
Activity Protein a recovered (mg/ml) (%)
Specific Puriactivity fica(mIu/mg) tion
Retroplacental serum Aminopeptidase fraction ex Hydroxylapatite 1st G-100 column 2nd G-100 column
800 36 2.7 4 3
215 450 2250 775 660
100 95 36 20 12
0.31 19.2 510 820 980
70 23.5 4.4 0.95 0.67
1.0 62 1650 2 650 3200
a Protein content was determined by the method of Lowry et al. (1951) using Versatol-A (General Diagnostics Division, Warner-Chilcott Laboratories, N.J. U.S.A.) as standard protein.
(Fig. 2 b). A f t e r t h e second c h r o m a t o g r a p h y on S e p h a d e x G-200 t h e o x y t o c i n a s e o b t a i n e d possessed a specific a c t i v i t y of 980 m I u / m g which corresponded to a 3200 fold purification as c o m p a r e d to t h e s t a r t i n g r e t r o p l a c e n t a l s e r u m w i t h a n overall r e c o v e r y of a c t i v i t y of 12 %. T h e homogeneous n a t u r e of t h e p r o d u c t was confirmed b y i m m u n o e l e c t r o phoresis a g a i n s t a n i m p u r e p r e p a r a t i o n of a n t i - o x y t o c i n a s e . As p r e v i o u s l y dem o n s t r a t e d ( W a t k i n s a n d Small, 1972) a single p r e c i p i t i n line was observed with t h e purified p r o t e i n whereas m u l t i p l e b a n d s were seen with a s a m p l e of p r e g n a n c y serum. T h e fluorescein-labelled a n t i - o x y t o c i n a s e serum possessed a fluorescein t o p r o t e i n r a t i o of 1:3 a n d a n a n t i b o d y t i t r e of 1 in 4. T h e l a t t e r was d e t e r m i n e d b y m i c r o i m m u n o d i f f u s i o n a g a i n s t a s t a n d a r d a n t i g e n a t a c o n c e n t r a t i o n of 1 m g / m l .
Immunohistochemistry of Placental Oxytocinase 1.6
535
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~
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i
~
.~
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~
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o 0.4
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,
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.
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i
Fig. 2. (a) Gel filtration of the active fraction from Fig. 1 on a column (1.7 • 40 cm) of Sephadex G-200 at a flow rate of 5.2 ml/hr. Fractions 2 ml (o-o) protein concentration, (9 . . . . 0) oxytocinase activity. (b) l~echromategraphy of the oxytecinase peak from (a) on a column of G-200 using the same conditions as in Fig. 2a. Phosphate buffer (0.001 m, pH 7.4) was used as solvent
Immunofluorescence histology of oxytocinase in the normal (Fig. 3a), toxaemic (Fig. 3b) and immature (16 weeks gestation) (Fig. 4a) placentas demonstrates the presence of specific fluorescence confined to the syncytial tissue of the trophoblast. There was no apparent differences in the staining intensity observed in immature, term or toxaemic placentas. In the sections of placental membranes (Fig. 4b) the immunofluorescence was found to be mainly associated with the chorion and to a lesser extent with the amnion.
Discussion
Oxytocinase was first purified from retroplacental serum by Tuppy and ~Vintersberger (1960) who used ammonium sulphate fractionation, rivanol precipitation and absorption of the impurities onto bentonite. A later procedure used by Barth et al. (1967) involving ethanol fractionation, gel filtration and DEAE-cellulose ion exchange chromatography resulted in a 3 000 fold purification of the enzyme with an overall yield of 9 %. This yield was subsequently increased to 25-38% by modification of the ethanol fractionation (Barth et al., 1971). The method used for the purification of oxytociuase in this present paper, however, is based upon that procedure described by Kulling and Yman (1970). Although our product possessed a lower purification factor than that obtained by Yman and his colleagues (Sjoholm and Yman, 1966) its immunological homogenity indicated its suitability for the present studies. The observations that specific immunofluorescence is confined to the syncytium of the placenta is in accord with the available histochemical evidence of Semm and Waidl (1962) and James (1966). However, since the substrate used by these workers, L-eystine-di-fl-naphthylamide, would be expected to be at-
536
C.W. Small and W. B. Watkins
Fig. 3a and b. Immunofluorescence histological localization of oxytocinase in the syncytium of (a) normal and (b) toxaemic placenta ( • 155)
tacked by aminopeptidases other than oxytocinase, a positive reaction product cannot necessarily be attributed to eystine aminopeptidase (oxytocinase) alone. Similarly, the histochemical inhibition studies carried out by Seelig and Roemheld (1969) does not completely preclude the contribution of aminopeptidases towards a positive histochemical reaction. Our immunocytological findings provide conclusive evidence for the placental origin of serum oxytocinase. That oxytocinase is localized in the synetium supports the earlier conclusions of Page (1946) who showed that rise in oxytocinase activity during pregnancy followed the increase in syncytial mass rather than of cytotrophoblastic bulk which diminishes after mid-pregnancy. Mathur and Walker (1970), using a novel morphometrical technique, showed that there was a positive correlation between oxytocinase activity and the proportion of trophoblast in the placenta and thereby concluded that oxytocinase is located in the syncytio-
Immunohistochemistry of Placental Oxytocinase
537
Fig. 4a and b. Specific localization of oxytocinase in (a) the cells of the outer layer of an immature placenta trophoblast and (b) in the chorion (C) and amnion (A). ( X 155)
trophoblast. I t is interesting to note that other placental components t h a t are released in increasing amounts into the maternal peripheral system as pregnancy progresses are also localized in the syncytium viz, human placental lactogen (Beck et al., 1969) and heat stable alkaline phosphatase (Hempel and Geyer, 1969; Watkins and Anderson, 1971; Watkins and Small unpublished observations). I n studying the effect of p H on the hydrolysis of L-cystine-di-fl-naphthylamide by extracts of human amnion and chorion, Ryd6n (1966) showed that the highest cystine aminopeptidase activity occurred at a p H of approximately 6.0, which is indicative of the action of non-specific tissue aminopeptidases alone. Our findings, however, have shown t h a t the chorionic tissue is associated with an enzyme that possesses immunological determinants cross-reactive against an antiserum produced against placental oxytocinase. The presence of oxytocinase
538
C.W. Small and W. B. Watkins
in the a m n i o n m a y be expected to result from either local synthesis, t r a n s p o r t from the chorion or a b s o r p t i o n from the amniotic fluid. I t is d o u b t f u l if the latter m e c h a n i s m operates in view of the findings t h a t h u m a n amniotic fluid appears to c o n t a i n only traces of cystine aminopeptidase a c t i v i t y (Miiller-Hartburg et al., 1959 ; Itashimoto, 1961 ; Riad, 1962).
References Barth, T., Pli~ka, V., Rychlik, I., ~orm, F. : Enzymatic inactivation of oxytocin V. Purification and some properties of enzymes from human retroplacental serum. Coll. Czech. chem. Comm. 32, 2327-2336 (1967) Barth, T., Rychllk, I., Mannsfeldt, H. G. : Human pregnancy oxytocinase isolation and substrate characteristics. Coll. Czech. chem. Comm. 36, 2540-2546 (1971) Beck, J. S., Gordon, R. L., Donald, D., Melvin, J. M. 0.: Characterisation of antisera to a growth-hormone-like placental antigen (human placental lactogen): Immunofluorescence studies with these sera on normal and pathological syncytiotrophoblast. J. Path. 97, 545555 (1969) Fekete, K. : Beitr~ge zur Physiologie der Gravidlt~t. Endokrinologie 7, 364-369 (1930) Fekete, K.: Gibt es w~hrend der Schwangerschaft ein aktives Hypophysen-Hinterlappenhormon im Blute? Endokrinologie 1O, 16-23 (1932) Hashimoto, T. : Studies on 1-cystine-aminopeptidase. Part II. Clinical observations. J. Jap. obstet, gynaec. Soc. 8, 96-104 (1961) Hawker, R. W. : Inactivation of antidiuretic hormone and oxytocin during pregnancy. Quart. J. exp. Physiol. 41, 301-308 (1956) Hempel, Von E., Geyer, G.: Submikroskopische Verteilung der alkalischen Phosphatase in der mensehlichen Placenta. Acta histochem. (Jena) 34, 138-147 (1969) James, N. T.: Histochemical demonstration of oxytocinase in the human placenta. Nature (Lend.) 210, 1276-1277 (1966) Kulling, B., Yman, L.: Purification and some properties of leucine aminopeptidases from retroplacental serum. Acta pharma, sueeica 7, 65-74 (1970) Lowry, 0. H., Rosebrough, N. J., Farr, A. L., Randall, R. J. : Protein measurement with the Folin Phenol reagent. J. biol. Chem. 193, 265-275 (1951) Mathur, V. S., Walker, J. M. : The origin of human placental oxytocinase. J. Physiol. (Lend.) 208, 291-298 (1970) Mfiller-Hartburg, W. H., Nesvabda, H., Tuppy, H. : Die Anwendung einer chemischen Methode zur Bestimmung des Oxytocinasespiegels im Schwangerenserum. Arch. Gyn~k. 191, 442-456 (1959) Nairn, R. C. : Fluorescent protein tracing, 3rd edn. London: Livingstone 1969 Page, E. W. : The value of plasma pitocinase determinations in obstetrics. Amer. J. Obstet. Gynec. 52, 1014-1022 (1946) Riad, A.M.: Studies on pregnancy serum eystine aminopeptidase activity "oxytocinase". J. Obstet. Gynaec. Brit. Comm. 69, 409-416 (1962) Ryd@n, G.: Cystine aminopeptidase and oxytocinase activity in pregnancy. Acta. obstet. gynaec, scand. Vol. 45, Suppl. No. 3 (1966) Scelig, H.-P., Roemheld, 1~.: Untersuchungen zur histochemischen Lokalisation der Leucinund Cystinaminopeptidase (Oxytocinase) in der Placenta. Histoehemie 18, 30-39 (1969) Semm, K., Waidl, U. E.: Histochemische Untersuehungen fiber die Serumoxytocinase-Bildung im menschlichen Trophoblasten. Z. Geburtsh. Gyn~k. 158, 165-171 (1962) SjSholm, I., Yman, L. : Preparation of highly purified oxytocinase (cystine aminopeptidase) from retroplacental serum. Acta pharma, suecica 3, 377-388 (1966) Small, C. W., Watkins, W. B. : Immunochemieal localisation of some placental enzymes. N. Z. Med. J. 74, 338 (1971a) Small, C. W., Watkins, W. B. : An improved method for the determination of human pregnancy serum oxytoeinase activity. Enzymologia 41, 121-128 (1971b) Small, C. W., Watkins, W. B. : S-benzyl-L-cysteine-p-nitroanilide.A new substrate for the determination of oxytocinase with improved specificity. Biochem. Med. 9, 103-112 (1974)
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Tovey, J. E.: Serum oxytocinase. Clin. Biochem. 2, 289-310 (1969) Tuppy, H. : The influence of enzymes on neurohypophysial hormones and similar peptidcs. In: Handbook of experimental pharmacology, ed. Berde, B., p. 67-129. Berlin-HeidelbergNew York: Springer 1968 Tuppy, H., Nesvadba, U . H . : ~ber die Aminopeptidaseaktivit~t des Schwangerenserums und ihre Beziehung zu dessen Verm5gen, Oxytocin zu inaktivieren. Mh. Chem. 88, 977-988 (1957) Tuppy, H., Wint~rsberger, E.: Reinigung und Eigenschaften der Serumoxy~cinase. Mh. Chem. 91, 1001-1010 (1960) Watkins, W. B., Anderson, S. R. A. : The origin of heat-stable placental alkaline phosphatase. J. Obstet. Gynaec. Brit. Comm. 78, 691-693 (1971) Watkins, W. B., Small, C.W.: Immunologic inactivation of human pregnancy serum oxytocinase activity. Amer. J. Obstet. Gynec. 118, 973-978 (1972)
