Initiation of human parturition

Initiation of human parturition XI. Lysosomal from laboring BARRY

enzyme release in vitro from and nonlaboring women

amnions

obtained

E. SCHWARZ

PAUL

CT. MACDONALD

JOHN

M.

JOHNSTON

nn llos, ?‘PXU.\ In the present investigation, we sought to ascertain whether an alteration in the physiology of amnionic lysosomes could be demonstrated in association with normal human labor at term. We reasoned that perturbattion of the lysosomes of amnion would be associated with a greater rate of release of known lysosomal marker enzyme activity from this tissue. In order to investigate this possibility, we emwyed discs of amnion tissue which were incubated in medium containing sucrose (0.25M). We observed that the rate of release of lysosomal marker enzyme activity, N-acetyl glucosaminidase, into the incubation medium from arnnion tissue obtained from laboring women was graater than the rate of release of the same enzyme activity from amnion tissue obtained from women who were not in labor. During incubation, there was no detectable release of lactate dehydrogenase, a cytosofii marker enzyme, which attests to the viability of the amnion tissue during the experiment. These findings are consistent with the view that there is an alteration in the physiology of lysosomes of the amnion associated with human labor at term, a finding which is supportive of a role for lysosomes of the human fetal membranes in the initiation of human parturition. (AM. J. OBSTET. GYNECOL. 137:21, 1980.)

SEVER A L investigators have hypothesized that lysosomes and lysosomal enzymes have a crucial role in the initiation of human labor.‘-3 This role is presumed to be subserved by the activity of the enzyme phospholipase Az, the enzyme that catalyzes the hydrolysis of glycerophospholipids, thereby effecting the release of the fatty acid present in the sn-2 position, which is enriched in polyunsaturated fatty acids, e.g., arachidonic acid. The liberated arachidonic acid can be uti-

From thr Cecil H. and Ida Green Center for Reproductive Biology Sciences, and the Departments of Ob,stetrio-Gynecology and Biochemist?, The University of Texas Southwestern Medicnl School. Supported in part by United Grant No. 5-P50-HDl1149.

States Public

Presented at the Twenty-sixth Annual Society,for Gynecologir Investigation, California, March 21-24, 1979. ReceiuedJor Accepted

publication September

April

Service

Meeting of the San Diego,

29, 1979.

1, 1979.

Repint requests: Barry E. Schwarz, M.D., Obstetrics and Gynecology, Ths University Southwestern Medical School, 5323 Harq Boulevard, Dallas, Trxns 75235. 0002-9378/80/090021+04$00.40/0~

Health

Department of Texas Hines

1980TheC.V.Mosby

Co.

of

lized in the biosynthesis of prostaglandins or prostaglandin-related substances, which, in turn, may initiate the myometrial contractions that lead to labor. An alteration in the physiology of lysosomes, as reflected by a leakage of lysosomal enzyme(s) into amnionic fluid4 or cytoplasm of decidual cells,” has been demonstrated in studies of pregnant women undergoing elective induced termination of pregnancy in the second trimester. The purpose of the present study was to ascertain whether similar changes in the physiology of lysosomes of amnion could be identified in association with spontaneous human labor at term. Employing human amnion tissue discs, we measured the rate of release of’ a lysosomal marker enzyme, N-acetyl glucosaminidase, to an incubation medium. We chose to evaluate the activity of N-acetyl glucosaminidase because the available assays for this enzyme are exquisitely sensitive, whereas those tin phospholipase A2 activity are not. We found a significant difference between the rate of rclease of this lysosomal marker enzyme from amnion tissue obtained from laboring women at term and that from amnion tissue obtained from women who were at term but not in labor. 21

22

Schwarz, MacDonald, and Johnston

Fig. 1. Photograph of human amnion dissected from chorion laeve and the circular knife used to cut discs of amnion tissue.

Fig. 2. Total enzymatic activities of N-acetyl glucosaminidase and lactate dehydrogenase in discs of human amnion tissue. The activities of N-acetyl glucosaminidase (mean and standard errors) are presented in the left half of the diagram, and the activities of lactate dehydrogenase (mean and standard errors) are presented in the right half of the diagram. Total enzymatic activities in discs of amnion tissues obtained from women in labor are represented by solid bars, and total enzymatic activities in discs of amnion tissues obtained from women not in labor are represented by stippIed bars. Numbers in parentheses above each bar indicate the number of amnion tissue discs in each group.

Material and methods Amnions were obtained at the time of cesarean section from nine women in active labor and from 15 women at term prior to the onset of labor. Each of these cesarean sections was performed between the thirty-eighth and forty-first week of gestation. Cesarean section was performed electively prior to the onset of labor in women who had been delivered previously by cesarean section. Cesarean section was performed in laboring women for fetal distress or for cephalopelvic disproportion. The amnion tissue was dissected from the chorion laeve. Discs of amnion tissue of uniform

size, 1.9 cm in diameter, were cut from wideI\, sp;~ced areas of each amnion, blotted, and weighed. This JXOcedure is illustrated in Fig. 1. These tissue fragments were incubated in an oxygenated medium containingsucrose (0.25M, pH 7.0) with gentle shaking undcl, an atmosphere of 95%’ oxygen and 5’% CO, at 37” C fi)r 60 minutes. Aliquots of the incubation medium were removed at 15-minute intervals and assayed t’or Iysosomal marker enzyme activity, N-acet>l glucosaminidase (EC 3.2. I .30), and cytosolic marker enzyme acrivity, lactate dehydrogenase (EC 1.1.1.2i). N-Acetyl glucosaminidase activity was assayed by the method of Levvy and Conchie’ with the use of p-nitrophenyl-Nacetyl-P-D-glucosaminide as the substrate. Lactate dchydrogenase activity was assayed with the use ot pyruvic acid and reduced /3-nicotinamide adenine dinucleotide as substrates.; After incubation, each amnionic disc was homogenized vigorously (Polytron P- 10 homogenizer, five 30-second bursts at 4” C) in a solution of sucrose (0.25M), and the homogenates were assayed for the same two enzymatic activities. Total enzymatic activity of each amnionic disc was calculated as the sum of the activity released to the incubation medium plus the activity measured in the whole homogenate after incubation. Total enzymatic activities were assumed to be distributed normally among discs of amnion tissue, and comparison of means of enzyme activity was performed by means of Student’s t test. The measured enzymatic activity released to the incubation medium was not assumed to be normally distributed, and comparison of means of enzyme activity was performed with the use of the nonparametric Mann-Whitney test of significance.R Analysis was made of a total of 36 discs of amnion tissue prepared from nine amnions obtained from women in labor and 57 discs of amnion tissue prepared from 15 amnions obtained from women nor in labor.

Results The total N-acetyl glucosaminidase activities and lactate dehydrogenase activities of the discs of amnion tissue of the two groups are illustrated in Fig. 2. The total N-acetyl glucosaminidase activity of the discs of amnion tissue obtained from laboring women was 160 f 19.4 (mean and SE) nmoles of product X min-’ x g-’ tissue, a value that was similar to the total N-acetyl glucosaminidase activity of the discs of amnion tissue obtained from women not in labor, 142 + 11.6 nanomoles. Similarly, the total lactate dehydrogenase activity of the discs of amnion tissue obtained from laboring women, 2,625 ? 327.4 (mean and SE) nanomoles X min-’ x g-’ tissue, was similar to the total lactate dehydrogenase activitv of the discs of amnion tis-

Volume Number

137 1

Table

I. N-Acetyl

Initiation of human parturition. XI

glurosaminidase

activity released

to incubation Period

sue obtained from women who were not in labor, 2,744 2 207.7 nanomoles. The rate of release of N-acetyl glucosaminidase activity to the incubation medium is presented in Table I. A significantly greater amount of N-acetyl glucosaminidase activity was released to the medium per unit time from discs of amnion tissue obtained from women who were in labor, compared to the rate of release of this enzyme activity from discs of amnion tissue obtained from women who were not in labor. Greater release of N-acetyl glucosaminidase activity from amnion tissue obtained from laboring women was found at each time of sampling of the medium during the 60 minutes of the experiment, compared to that released from amnion tissue obtained from women not in labor. The rate of release of N-acetyl glucosaminidase activity to the incubation medium was greatest during the first 15 minutes of incubation; thereafter, the rate of release became relatively constant and somewhat lower for the remaining 45 minutes of incubation in both groups of tissues. Lactate dehpdrogenase activity was readily measurable in the whole homogenates of the discs of amnion tissue after incubation; however, there was no detectable lactate dehydrogenase activity in the incubation medium of any of the discs of amnion tissue at any time during the incubation.

Comment The appearance of Iysosomal enzyme activity in extracellular spaces in the absence of concomitant cell death and autolysis has been taken by some to be a reflection of an alteration of intracellular lysosomal physiolog) ,g+” In the present investigation, we found an increased rate of release of the lysosomal marker enzyme, N-acetyl glucosaminidase, into an incubation medium from amnion tissues obtained from laboring women, compared to the rate of release of this lysosomal enzyme by amnion tissues obtained from women who were not in labor. We also found a similarity in the total N-ace@ glucosaminidase and lactate dehydrogenase enzyme activity content of the two groups of tissues, a finding that adds credence to the view that the greater release of lysosomal marker enzyme from amnion tissue obtained from laboring women was a

medium

of incubation

from discs of amnion

23

tissue

(minute)

reflection of a physiologic alteration ofthe lysosomes of the amnion. The release of lactate dehydrogenase to the incubation medium was evaluated in order- to monitor cell death and autolysis. It should be noted that, if there had been a fractional release of lactate tlehydrogenase comparable to the observed fractional release of the lysosomal marker, N-acetyl glucosaminidase, it would have been readily detectable by 1he assay method employed. Since no detectable lac.tatc dehydrogenase was released to the incubation medium, although the activity of this enzyme was readily detectable in the whole homogenates of the tissues atier incubation, we conclude that the observed altet&on in intracellular lysosomal physiologv was no1 a reflection of cell death. We proposed previously that metabolic processes in the fetal membranes (amnion and chorion laeve) ma) be important in the initiation of human parturition.‘” This proposition was based, in part, on the assumption that prostaglandin(s) occupy an importanr role in the initiation of the myometrial contractions of human labor. The results of a series of investigations are supportive of this hypothesis. Recall that free ,crachidonic acid is the obligate precursor in rhe biosynthesis of prostaglandin Ez and Fzo and related substances of the P-series, and that the rate of release of free arachidonic acid is believed to be the rate-limiting step in prostaglandin biosynthesisin most tissues.‘” In thiv regard, we have shown the following. (a) There is a striking increase in the concentration of free arachiclonic acid in the amnionic fluid of women in labor, compared to that in the amnionic fluid of women not in labor.‘2 (b) The increase in the concentration of free arachidonic acid in amnionic fluid of women in labor is disproportionately greater than that of other free fatty ac.ids.lZ (c) The introduction of free arachidonic acid into the amnionic sac of women in the second trimester of pregnancy and into the amnionic sac of wome11 in the third trimester with a dead fetus is followed promptly by labor and delivery.” (d) The arachidonic acid content of human amnion and chorion is great.‘* (e) The greatest amount of arachindonic acid in amnion and chorion is contained in phosphatidylethanolamine.LS (f) There is a striking reduction in the phosphatidyletha-

24

Schwarz, MacDonald, and Johnston

nolamine arachidonic acid content in amnion and chorion during labor.‘” (Curbelo and associates’6 have made similar observations.) (g) The phospholipase A2 activity of the human chorioamnion catalyzes preferentially the hydrolysis of phosphatidylethanolamines and possesses greatest substrate specificity for .5n-2 arachidonoyl-esters of phosphatidylethanolamines.” (h) At least part of the phospholipase AZ activity of the human chorioamnion is contained in lysosomes.’ The results of these investigations have been reviewed recently,‘” and others have confirmed many of these findings. For example, Grieves and Ligginsyg have also demonstrated phospholipase A activity in human amnion tissue, and Keirse and associate? found an increased concentration of arachidonic acid in the am-

nionic fluid of laboring women, which tontinuc’ci to increase with increasing cervical dilation. The findings of the present study are con&tent with the possibility that alteration in the expression (tt thrs enzymatic activities of fetal membrane Ivsoscm~es is an early event in the biochemical process that leads to the initiation of human parturition. Such a process would allow for the expression of lysosomal phospholipase A, activity resulting in accelerated hydrolysis of phosphatidylethanolamine, the release of free arachidonic acid. increased prostaglandin synthesis, and the initiation of‘ human parturition. We wish to express our thanks to Mr. David Dowd for his technical assistance in the performance of this study.

REFERENCES

1. Gustavii, B.: Labour. A delayed menstruation? (Letter), Lancet 2:1149, 1972. 2. Schwarz, B. E., Schultz, F. M., MacDonald, P. C., and Johnston, J. M.: Initiation of human parturition. IV. Demonstration of phospholipase As in the lysosomes of human fetal membranes. AM. J. OBSTET. GYNECOL. 125:1089, 1976. 3. Liggins, G. C., Christine, S. F., Grieves, S. A., and Schwartz. A. L.: Control of oarturition in man. Biol. Rerod. 16:39, 1977. ’ 4. lund, A.: Lysozyme in amniotic fluid during rivanolinduced second trimester abortion, Prostaglandins 17: 149, 1979. 5. Gustavii, B.: Sweeping of the fetal membranes by a physiologic saline solution: Effect on decidual cells, AM. J. OBSTET. GYNECOL. 120:531, 1974. 6. Levvy, G. A., and Conchie, J.: Mammalian glycosi$ases and their inhibition bv aldonolactones. in Neufeld, E. F., and Ginsburg, V., ediiors: Methods in’Enzymology, New York, 1966, vol. 8, Academic Press, Inc., p. 580. 7. Bergmeyer, H. U.: Methods in Enzymatic Analysis, ed. 2, New York, 1974, Academic Press, Inc., p. 574. 8. Snedecor, G. W., and Cochran, W. G.: Shortcut and non-parametric methods, in Statistical Methods, ed. 6, Ames, Iowa, 1967, The Iowa State University Press, p. 130. 9. Movat, H. Z., Uriuhara, T., Macmorine, D. L., and Burke, J. S.: A permeability factor released from leukocytes after phagocytosis of immune complexes and its possible role in the Arthus reaction, Life Sci. 3:1025, 1964. 10. Treadwell, P. E.: Association of the lysosomes of the reticuloendothelial system with mouse anaphylaxis, J. Immunol. 94~692, 1965. 11. Oronsky, A., Ignarro, L., and Perper, R.: Release of cartilage mucopolysaccharide-degrading neutral protease from human leukocytes, J. Exp. Med. 138:461, 1973.

t)

12. MacDonald, P. C., Schultz, F. M., Duenhoelter, J. H., et al.: Initiation of human parturition. I. Mechanism of action of arachidonic acid. Obstet. Gvnecol. 44:629, 1974. 13. Lands, W. E. M., and !&muelsson,‘B.: Phospholiiid precursors of prostaglandins, Biochim. Biophys. Acla 164: 426, 1968. 14. Schwarz, B. E., Schultz, F. M., MacDonald, P. C., and Johnston, J. M.: Initiation of human parturition. III. Fetal membrane content of prostaglandin Es and FPa precursor, Obstet. Gynecol. 40:564, 1975. 15. Okita, J, R., Okazaki, T., MacDonald, P. C., and Johnston, J. M.: Alterations in phospholipid content of human fetal membranes during parturition, Prckeedings of the Twenty-sixth Annual Meeting of the Society for Gynecologic Investigation, San Diego, California, March 21-24, 1979, p. 114. (Abst.) 16. Curbelo, V., Bejar, R., Benirschke, K., and Gluck, L.: Premature labor: Placental arachidonic acid (20: 4), Proceedings of the Twenty-sixth Annual Meeting of the Society for Gynecologic Investigation, San Diego, California, March 21-24, 1979, p. 87. (Abst.) 17. Okazaki, T., Okita, J. R., MacDonald, P. C., and Johnston, J. M.: Initiation of human parturition. X. Substrate specificity of phospholipase Al in human fetal membranes, AM. j. OI~TE;. GY~ECOL. 130~432, 1978. 18. MacDonald, P. C., Porter, J. C., Schwarz, B. E., and Johnston, J. M.: Initiation of parturition in the human female, Semin. Perinatol. 2:273, 1978. 19. Grieves, S. A., and L&gins, G. C.: Phospholipase A activity in human and ovine uterine tissues, Prostaelandins l&229, 1976. 20. Keirse, M. J. N. C., Hicks, B. R., Mitchell, M. D., and Turnbull, A. C.: Increase of the prostaglandin precursor, arachidonic acid, in amniotic fluid during spontaneous labour. Br. J. Obstet. Gynaecol. 84~937, 1977.