I'lamto (1992), 13, 223-229

Placental Localization of 15 -Hydroxy- Prostaglandin Dehydrogenase in Early and Term Human Pregnancy

J. J. H. M. ERWICH & M. J. N. C. KEIRSE”

Department of Obstetrics, Leiden University Hospital, R&s- burgerweg IO, 2333 AA Leiden, The *Netherlands ’ To mhom correspondence should be addressed at: Department of Obstetrics, Academisch Ziekenhuis, P.O. Box 9600, 2300 RC Leiden, The Netherlands Paper accepted 16. IO. 1991

SUMMARY

The human placenta possesses a large capacity for inactivation ofprostanoids. This is due to thepresence oflarge quantities ofthe MAD+ -linked 15-hydroxy-prostaglandin dehydrogenase (PGDH type I; EC 1.1.1.141). In order to investigate whether a spec$r localization of PGDH is at the origin of the increasing placental PGDH activi?y during early pregnancy, PGDH activity was localized histochemical!y in placental tissue obtained in early pregnan y and at term. Intracellular PGDHactizQ mas present in three distinct compartments in the placenta. First, the syngltiotropho- blast and second, the underlying ytotrophoblast layer of placental terminal villi contained PGDH. Third, scattered throughout the sections, we,fiund chorion-like cells which showed strong staining for PGDH activity. The intensity of staining in samples before 12 weeks ofgestation was much less than in samples efier 12 weeks oj gestation, which were comparable to those at term. Thus, the fetal cell lajjers at the boundary between the fetal tissues and the maternal blood have a great potential to inactivate prostanoids suggesting that the fetus protects itse!f against prostanoids of maternal origin.

INTRODUCTION

The human placenta possesses a large capacity for inactivation of prostanoids. This is due to the presence of large quantities of the NAD+-linked 15-hydroxy-prostaglandin dehydro- genase (PGDH type I; EC 1.1.1.141) (K eirse and Turnbull, 197.5; Keirse, Hicks and Tumbull, 1976; Willman and Collins, 1978; Jarabak, 1980; Nagai, Tsuruta and Mori, 1987). -411 prostanoids, except PGDZ, are substrates in vitro for PGDH, which oxidizes the 15- hydroxyl-group and is the main enzyme responsible for biological inactivation of prosta- glandins (Hansen, 1976).

0143-4004/92/030223 + 07 $0.5.00/O 0 1992 BailliPre Tindall l,td

224 Placenta (1992), Cbl. 13

In earlier work, we have shown that PGDH activity in the human placenta develops early in pregnancy, increases throughout the first trimester of pregnancy (Keirse, Erwich and Klok, 1985), and is controlled predominantly by the genetic constitution of the fetus (Erwich and Keirse, 1988). Since placental tissue is made up of several cell-types, with their own specific localization, it is useful to know whether there are differences in PGDH activity among these cells. In addition to a further understanding of the importance of placental PGDH, evidence on the localization of the enzyme may provide clues for the function of this important catabolic enzyme.

Histochemical localization of PGDH activity has been performed in several tissues (Nissen and Andersen, 1968; Niimura and Ishida, 1978; Marani et al, 1980; Wohlrab and Essbach, 1984; Chart, Chan and Cook, 1984; De Reeder et al, 1989). Localization of PGDH in human intrauterine tissues obtained from term pregnancies was studied by Duefias et al (1984) and Cheung et al (1990). Duefias et al (1984) showed PGDH activity to be located in the cytoplasma of cells. The highest activity was observed in the fetal membranes, which is consistent with biochemical data (Keirse and Turnbull, 1975), in the cytotrophoblast cells in the basal plate and in the intravascular trophoblast cells. Less intense, but clearly present, PGDH activity was located in the syncytiotrophoblast layer of the placental villi. Using a polyclonal primary antibody to PGDH, Cheung et al (1990) showed the presence of PGDH in invading trophoblast cells in the decidua, in syncytiotrophoblast, and intermediate type trophoblast.

In order to verify published data, extend them to early pregnancy, and to investigate whether a specific localization of PGDH is responsible for the increase in placental PGDH activity in early pregnancy, we studied the histochemical localization of PGDH in placental tissue obtained in early pregnancy and at term.

MATERIALS AND METHODS

Twenty-five samples of human placental tissue from 5 to 19 weeks of gestation (calculated from the day of the last menstrual period) were obtained after informed consent from women undergoing elective termination of pregnancy for social reasons. Only pregnancies with certain duration were included in the study. All terminantions were performed by aspiro- tomy, a one-step vaginal procedure under local anaesthesia (Beekhuizen et al, 1982). Products were collected and rinsed in ice cold saline 0.9 per cent. Pieces of villous trophoblast tissue were identified, blotted, immediately frozen in liquid nitrogen, and stored at -80°C until analysis. In addition, samples were collected from placentae at term after spontaneous vaginal delivery. Frozen tissue was submerged in Tissue tek (Miles Labora- tories, Naperville, IL) and sections (6pm) were cut in a cryostat at - 12°C. Activity of PDGH was demonstrated using a modification of the technique of Nissen and Andersen (1968) as described by De Reeder et al (1989). Frozen sections of placental samples from different gestational ages were put on one slide (five per slide) and incubated for 60 min at 37°C in a medium (50 PuVsection) containing 1 rnM NAD (Sigma), 5 mM Nitro-Blue Tetrazolium salt (Sigma), 4 mM Sodiumazide (Sigma) and 2.8 mM (1 mg/ml) PGEz (Upjohn, Kalamazoo, MI) dissolved in 0.033 M sodium phosphate buffer pH 8.0. After incubation, sections were rinsed in tepid tap water, air dried, fixed in buffered formaldehyde and mounted in glycerine jelly. Negative controls were obtained by omitting PGEz as a substrate on adjacent sections from the same samples.

Erich. Keirse: Plucental Localization of PGDH 225

RESULTS

PGDH activity was demonstrated clearly by intense staining in placental sections from both early and term pregnancies. Control sections obtained by omitting the substrate, showed a very low level of background staining, which consisted mainly of specific small granular deposits.

Presence of PGDH activity was located within the cytotrophoblast and syncytiotrophoblast layer of placental terminal villi. Differences in staining intensity within the same sections indicate higher PGDH activity in the syncytium than in the cytotrophoblast cells of the villi. .\mong the villi, fields of larger cells were present, sometimes showing a syncytial covering. Often, these fields are attached to villi. These cell groups showed a very intense staining reaction, indicating the presence of relatively large quantities of PGDH. Only the cytoplas- matic areas of the cells were stained (Figures 1 and 2, with adjacent plates).

Estimation of differences in intensity of staining between sections is only semi-quantitative and should, therefore, be considered with caution. When sections of increasing gestational age processed on the same slide were compared, samples earlier than 12 weeks of gestation showed substantially less staining than those of over 12 weeks of gestation; staining in the latter was comparable to that seen at term. No distinct differences in localization of PGDH activiv could be demonstrated between samples of early and of more advanced gestational ages. However, in some samples obtained before 10 weeks the syncytiotrophoblast appeared to be more intensely stained than the underlying cytotrophoblast layer when compared with sections of more advanced pregnancies, indicating that the syncytial localization of PGDH is more predominant in early pregnancy.

DISCUSSION

intracellular PGDH activity was present in three distinct localizations within the placenta. First, the syncytiotrophoblast and second, the underlying cytotrophoblast layer of the placental villi contained PGDH. Third, scattered throughout the sections, groups of cells were identified which stained strongly for PGDH activity.

Indications for the identity of the latter cell groups comes from Boyd and Hamilton (1970). They described the presence of such cells in the human placenta throughout pregnancy and defined them as cytotrophoblastic cell islands, attached to an anchoringvillus or to a placental septum and of cytotrophoblastic origin. They concluded that these islands are part of the cytotrophoblastic shell, which originally develops from the blastocystic trophoblast.

Our data on placental villous tissue at term are in accordance with the findings of Dueiias et al (1984), who also found PGDH activity in terminal villi to be located mainly in the syncytiotrophoblast layer. The localization of PGDH activity in this study agrees with the distribution of immunoreactive PGDH as described by Cheung et al (1990). However, the staining for PGDH activity in cytotrophoblast obtained from term placentae, albeit relatively low, is a new finding. Cheung et al (1990) found no detectable PGDH in cytotrophoblast at term. This suggests that our method of localizing PGDH activity is more sensitive than the method employed by Cheung et al (1990).

Although the histochemical method used in this study had been validated and used on several other tissues (see Introduction), in general results obtained by histochemical methods are difficult to quantitate; thus, large differences in staining intensity are required to indicate

Placeeta (1992), Vol. 13

(b)

Figure 1. Positive but low reaction of PGDH activity of placental villus tissue of 8 weeks and 4 days of pregnancy (64x). S, syncytiotrophoblast; C, cytotrophoblast; I, intense staining cell islands.

A

gure 2. Positive reaction of PGDH activity of placental villus tissue of 16 weeks of pregnancy (64x syncytiotrophoblast; C, cptotrophoblast; I, intense staining cell islands.

228 Placenta (1992), Vol. 13

true differences in enzyme activity. Notwithstanding this, our results show an increase in staining intensity and thus in PGDH activity with gestational age, which is commensurate with data from biochemical measurements (Keirse et al, 1985).

As to the questions of where PGDH activity is located in early pregnancy and where the increase with gestational age takes place, our findings indicate that the syncytiotrophoblast lining of the villi and the cytotrophoblastic shell are the primary locations of PGDH activity and that PGDH activity increases simultaneously in both locations with increasing ges- tational age.

The observation that prostacyclin is also a substrate for PGDH in vitro (McGuire and Sun, 1978; Jarabak and Fried, 1979; Jarabak, Luncsford and Berkowitz, 1983) makes the development of placental PGDH activity in early pregnancy even more interesting. Data from Jarabak, Watkins and Lindheimer (1987) indicate an elevated PGDH activity in placental tissue obtained from term pregnancies complicated by pre-eclampsia.

Little is known thus far about the role of placental PGDH in early gestation. The fact that the fetal tissues at the utmost boundary between maternal blood and the developing fetus contain such high levels of PGDH activity, suggests that the main function of the enzyme may be to protect the fetal-placental unit from unwanted biological effects of maternal prostanoids.

ACKNOWLEDGEMENTS

The research was supported by the Praeventiefonds, The Hague (grant 28-1118). We thank Conny van Munsteren and Dr Ernst de Reeder, Department of Anatomy, Leiden University, for their assistance.

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