3 Correspondence: Division of Endocrinology, Central Drug Research Institute, Lucknow-226 001, India, Tel: 212411-418 EPABX/4246 Extn., Fax: 91-0522-223405/223938, Email: purshottam123@rediffmail.com

Acknowledgement: We are thankful to Dr. N.M. Khanna, former scientist for generous supply of compound 95/588. Technical assistance was ably provided by Mrs. R. Pant and S. Kitchlu and Mr. H.K. Checker did excellent typing. The study was financed by University Grants Commission (to SF) and supported by the Ministry of Health and Family Welfare, Government of India, New Delhi

The study reports alterations in the level of two nucleic acid catabolizing enzymes, adenosine deaminase (ADA) and 5’-nucleo- tidase (5’-NT) in normal implantation swellings (with and without embryo), inter-implantation area (IA) of the uterus and spontaneous/drug-induced resorbed (SR/DR) tissues in hamster during early to mid gestation [normal-days 7-9 and day-12 post-coitum (p.c.); resorbed day-8 and 12 p.c.]. In normal implantation swellings there was a progressive rise in ADA concentration, being maximum on day 9 and 12 p.c. in all the three tissues. In the resorbed tissues (both SR/DR), on the contrary, the enzyme concentration was remarkably very low on day 8 p.c. On day 12 p.c. it remained low in SR but remarkably increased in DR, even higher to the normal implantations of same day. The enzyme 5’NT, on the contrary, showed decreasing trend from day 7-9 p.c. in implantation swellings (with and without embryo both) but remained virtually equal in IA on all the three days. On day 12 p.c. the level of the enzyme was more or less equal to that of day 8 p.c. However, in SR and DR there was no consistency in the enzyme level on both day 8 and 12 p.c. The results indicate that only ADA participates in tissue proliferation/regeneration. Its low activity may be due to the resorption of implantations, irrespective of the gestation day; while its higher activity in DR on day 12 p.c. may be due to tissue regeneration that occurs on cessation of drug treatment.

In peri and immediate post-implantation, the trophectodermal cells of blastocyst begin proliferation and multiplication in the uterine endometrium after invading it. These cells also stimulate stromal cells to undergo cytodifferentiation, a process known as deciduoma formation. To perform these functions a battery of biochemical indices are alleged to participate. Among these are the two nucleic acid enzymes, 5’-nucleotidase (5’-NT; EC 3.1.3.5) and adenosine deaminase (ADA; EC 3.5.4.4), reported to be associated with growth and development of preplacental and decidual tissues (Knudsen et al., 1991; Jenuth et al., 1996). While the former enzyme is involved in the formation of adenosine and 2-deoxyadenosine, the latter mediates the breakdown of these products into inosine and deoxyinosine, respectively. Since adenosine is a regulatory nucleoside that has a catalytic effect on the vascular development of the uterus, its excess may trigger the release of biochemical signals which coordinate early post-implantation events including apoptosis (Adair et al., 1989). This is destined to undergo as the consequence of invasion by the proliferating trophoblasts (Blackburn et. al., 1992). The 2-deoxyadenosine, on the other hand, is an embryotoxic metabolite, that primarily activates the apoptosis (Gao et al., 1994). The adequate level of these enzymes thus appears to be essential for cell/tissue growth and proliferation during pregnancy.

The enzyme ADA is reported to be initially expressed antimesometrially in the primary decidua and then shifts to the mesometrial side, mainly to the basal zone of the developing placenta in which it accounts for 95% activity at the foeto-placental site (Hong et al., 1991; Witte et al., 1991). The evidence for expression of ADA in early post-implantation development has been provided by the administration of R-deoxycoformycin (DCF), an inhibitor of this enzyme, which increases the incidence of embryo resorptions in mouse. The drug, however, was given on the gestation day 7 or 8; treatment earlier or later to these days did not show any effect. The site of DCF action was mainly on the antimesometrial decidua, prospective neural plate and the primary mesenchyme of trilaminar disc, the arrest of whose growth might have affected the embryonic development at early somite stage (Knudsen et al., 1987, 1989). Since the morphogenesis of ectoplacental cone (EPC), a preplacental tissue highly rich in trophoblasts (Kirby, 1971), begins on the gestation days 7 and 8 in this species, the development of EPC might have also been affected.

The peri-implantation surge of adenosine in the pregnant uterus also coincides with the expression of 5’-NT, the enzyme that catalyzes irreversible dephosphorylation of 5’-AMP to adenosine. The level of this enzyme is reported to attain peak in mouse embryo-decidual unit (EDU) on day 5 of gestation, but later declines through day 9 (Blackburn et al., 1992). In EDU the activity is mainly confined to the central stroma, being localized on stromal cell borders. 5’-NT activity has also been detected on giant trophoblast cell surface between day 7 and 13 in mouse (Thompson et al., 1990). However, the details about its tissue specificity corresponding to gestation day are lacking in mouse and no study is available in hamster either.

The present study has been carried out in hamster to determine the activity of the two enzymes in decidual swellings (with and without embryo; D+E and D-E) and in interimplantation area (IA) of the uterus on different days (7-9 and 12 post-coitum; p.c.). It is aimed to analyse the extent of involvement of uterine and embryonic tissues in the expression of the two enzymes. The tissue IA has been studied to see if the enzyme activity is uniformly distributed in the uterus or it is restricted to the implantation swellings only. Moreover, as the role of these enzymes in foetal resorption (quite common in hamster) is not known, their level was determined in the tissue undergoing resorption, natural (spontaneous) or induced by a pregnancy interceptive agent (compound 95/588). The compound, a plant product, belongs to nitrogen containing heterocycles and is undergoing contraceptive efficacy studies in lower animals. It has demonstrated cent-percent interceptive efficacy by oral route in hamster, rat and guinea-pig at 5,30 and 2 mg/kg on days 4-7, 5-9 and 6-10 p.c., respectively. The compound is effective by intravaginal route also (unpublished). This has also shown trophoblastolytic activity on human trophoblasts under going cytodifferentiation in vitro (Jaggi et al., 1999).

1. Animal experimentation
Adult (80-100 g) female Syrian hamsters (Mesocricetus auratus) of Institute’s animal colony, maintained in an air-conditioned room (21±2°C) and maintained on a photoperiod of 14 h light and 10 h darkness were used. They were fed pelleted food (Amrut Seeds Pvt. Ltd., Pune, India) and water ad-libitum. All the experiments were performed under the guidelines of animal ethics committee of the institute.

Virgin females at proestrus were cohabited overnight with healthy adult males (100-150 g) of proven fertility in the ratio of 2:1 in sterile plastic cages. The vaginal smear was examined for spermatozoa next day that was considered as day 1 post-coitum (p.c.). The animals were autopsied under mild inhalation of anesthetic ether (Hydroquinone : Kabra, India) by cervical dislocation on days 7 to 9 and 12 p.c. in case of normal pregnancy, but on days 8 and 12 p.c. only in the case of spontaneous and compound 95/588-induced resorptions.

The compound 95/588 was dissolved in physiological saline and administered (s.c.) to mated females on days 4-7 p.c. at 5 mg/kg body weight. The dose and treatment schedule was decided as per protocol routinely followed for testing peri and immediate post-implantation efficacy in these species. Minimum six animals were used for each data point and each implant was considered as a separate replicate.

2. Processing of tissues 
Each implant was separated from the uterus on the respective pregnancy day in phosphate buffer saline (PBS; pH 7.2). Serosa and myometrium were removed by fine tweezers and the resultant decidual swellings (D) containing embryo (E) and extraembryonic membranes inside (D+E) were transferred to ice cold buffer. However, the embryos were removed (under stereobinocular) from some of the implants by making an incision on the swellings through a sharp edged knife and the embryos were scrapped off from inside by a probe. The resultant tissue, without embryo (D-E), was cleaned in PBS and processed in the same manner as D+E. IAs of the same uterine horn having serosa and myometrium intact (as removal of these layers was not possible in this tissue) were sagitally cut and processed similarly. On day 12 p.c., in the case of normal tissue, the embryonic, placental and decidual components were taken out (after removing serosa and myometrium), cut into small pieces and washed several times in buffer prior to processing for enzymes estimation. In the case of resorbed tissues (spontaneous and compound-induced; day 8 and 12 p.c.) the entire resorption nodes (removal of serosa and myometrium was not possible in this tissue also) were dissected out from the uterus, washed in buffer and used for enzymes estimation.

3. Estimation of enzymes activity
To estimate ADA activity the respective tissues were homogenized in motor driven Ultra Turrax homogenizer in 1.0 ml ice cold PBS. The homogenate was centrifuged at 20,000g for 20 min at 4°C and the supernatant was used for the enzyme assay as per method of Giusti (1974). Degradation of adenosine and 2’-deoxyadenosine to inosine and 2’-deoxyinosine, respectively takes place with simultaneous release of NH₃, which is considered as the index of enzyme activity. In brief, aliquots containing appropriate amount of supernatant (0.2 µg protein for D+E, D-E and DR and 4-5 µg protein for IA and SR) were incubated at 37°C with 0.21mM adenosine for 60 min. The reaction was terminated by adding colour reagent containing sodium nitroprusside and HOCl. The reference was simultaneously made in which a parallel aliquot of the supernatant was added following the colour reagent. Absorbance was determined at 625 nm using ammonium sulphate as the standard.

Enzyme 5’-NT activity was measured according to Heppel and Hilmoe, (1956). The pellet obtained by centrifugation of tissue homogenate was sonicated in 1.15% KCl and the aliquot (50-100 ug protein) was incubated with Tris-HCl buffer (pH 8.5) containing 0.1 M MgCl₂ and 3 µmoles of 5’-AMP. The reaction was allowed to proceed at 37°C for 60 min., following which 5% trichloroacetic acid was added. The supernatant was analysed for inorganic phosphate (Pi) (Sumner, 1949). Each sample was analysed in triplicate and the protein content was determined by Lowry et al., (1951), using bovine serum albumin as a standard.

It may be mentioned that drawing a protein vs. activity curve separately for each tissue determines the minimum amount of protein required for both the enzymes.

For statistical calculations one way analysis of variance was done for each group (D+E, D-E, IA, SR and DR) and multiple comparison of means was performed by Newman Keuls Test (Zar, 1974).

4. Histochemical localization of enzyme ADA
The individual implants and resorption nodes were excised from the uterus and fixed overnight in cold phosphate buffered gluteraldehyde (1.5%) containing 30% sucrose. Specimens were frozen on dry ice and sectioned (12 µm). The cryostat sections were collected on 0.1% poly-L-lysine coated slide, air dried for 1h. rinsed in fresh fixative and then in 25mM sodium phosphate (pH 7.3). The sections were incubated at 37^oC for 20 min with a cocktail consisting of adenosine (7.0 mM), nucleoside phosphorylase (0.2 units/ml), xanthine oxidase (0.1unit /ml), phenazine methosulfate (0.2 mM) and nitro blue tetrazolium (0.8 mM) (all Sigma Chemical Co., USA). Finally these were rinsed, dehydrated in graded series of ethanol, cleared in xylene, mounted and examined. The blue formazon color reaction was photographed using 590 nm filter.

Biochemical
A progressive and significant increase was noticed in ADA activity of tissue D+E as compared to the tissues D-E and IA from day 7 to 9 p.c. (Table 1). On day 12 p.c. the enzyme level, however, showed a decline from the level of day 9 (Tables 2 and 3). Virtually the increasing pattern was noticed in tissue D-E also except that the difference between day 7 and 8 p.c. was not as significant as between day 7 and 9 p.c. Whereas in tissue IA, despite of showing similar day-wise increasing trend, the enzyme activity was considerably low. On the other hand, in spontaneously resorbed (SR) tissue taken on day 8 or 12 p.c. the enzyme activity was very low as compared in those of normal D+E and D-E tissues of days 7-9 p.c. (Table 2), but was comparable to the enzyme level of the tissue IA of day 7 p.c. In DR, the enzyme level on day 8 p.c. though was higher to that of day 8 p.c. SR, but was still lower to those of normal D+E and D-E tissues of the same day. However, when compared with tissue IA, the enzyme activity was significantly higher to those of day 7 and 8 p.c. but was lower to that of day 9 p.c. On the contrary, in day 12 p.c. DR tissue, the enzyme activity was increased several folds, not only over the DR/SR tissues of day 8 p.c. but even to those of the normal D+E and D-E tissues of days 7-9 and D+E of day 12 p.c. as well.

The results of 5’-NT activity in D+E, D-E, IA and in SR/DR on respective days are presented in Tables 1, 2 and 3 respectively. The enzyme activity was found to decrease from day 7 to 9 p.c. in tissues D+E and D-E but not in tissue IA in which there was no significant difference between day 7-9 p.c. In tissues SR and DR also no remarkable difference was noticed in the enzyme activity between day 8 and 12 p.c. and neither between the two tissues, except that on day 8 p.c. the enzyme activity was more in tissue SR over to DR. On day 12 p.c. exactly the reverse pattern was observed (Tables 2 and 3).

Histochemical
Examination of the sections of normal embryo-attachment site in the uterus revealed a mild staining in endothelial, mesometrial and decidual cells. But a pronounced staining was noticed in giant trophoblasts and placental tissue. The chorioallantoic plate and yolk sac had, however, very mild staining. On gestation day 7 (plug day as day 1) the staining was confined to antimesometrial decidua and the periphery of ectoplacental cavity (Figures 1 and 2). On gestation day 8, the activity shifted to mesometrial decidua, with higher intensity (Figure 3). Mild to moderate staining was also noticed in extraembryonic fold. On gestation day 9 the staining was noticed in the area of neural fold and along the myometrium (Figure 4). The uterine tissue of compound 95/588-treated animals, which contained mostly degenerated endometrial cell mass, did not show any specific reactivity. In the uterine sections of day 8 SR, a cavity was noticed in between the endometrium and myometrium (Figure 5). Whereas, in the sections of day 8 DR no such cavity was noticed between the two tissues (Figure 6). The enzyme reactivity was seen only in the outer region of the endometrium.

The early post-implantation period is heralded by certain essential morphogenetic changes in endometrium as well as extra embryonic membranes of the supporting placenta. This is required for the development of fetoplacental complex and to support these phenomena there is a surge of adenosine in peri-implantation phase uterus. Later developments, however, lead to marked elevation of ADA, the adenosine degrading enzyme. This is well demonstrated in rat and mouse (Knudsen et al., 1988 & 1989; Hong et al., 1991). On analysis of the cellular localization, this enzyme has been found to be initially expressed on the antimesometrial side of the uterus on day 6 (plug day as day 0), but later shifts to the mesometrial decidua and the basal zone of the placenta (day 7 and 8 p.c.), reaching to maximum on day 9 p.c. (Witte et al., 1991).

Our results in hamster, in which the decidual swellings (with and without embryo; D+E and D-E) have been used to estimate enzyme activity, show that the ADA level increases on day 7 p.c. (plug day as day 1) and then reaches maximum on day 9 p.c. in tissues D+E and D-E both. We have not estimated the enzyme activity prior to day 7 p.c., as it was difficult to separate endometrium from the myometrium and serosa. Moreover, the removal of embryo was also not possible on this day. Since our aim was to analyse the extent of contributions made by the embryonic and endometrial tissues per se in raising the enzyme level, we used the decidual swellings (without myometrium and serosa). The enzyme activity was, however, determined (not reported here) in these two layers to check whether they show any activity. It was very negligible in both the layers. We did not estimate the enzyme activity beyond day 9 except day 12 p.c. on which it was required for comparison with tissues SR and DR, the reason is that the ectoplacental cone (a preplacental tissue highly rich in trophoblasts) is fully developed on this day in hamster (Ward, 1948).

Our next aim was to see whether the proliferating trophoblasts per se augment the enzyme activity or work together with the differentiating endometrial cells (known as decidual cells) during embryogenesis in this species. To get answer to this, the enzyme activity was determined in trophoblast and decidual cells ,isolated from day 8 p.c. embryo and decidual swellings (Farheen & Mehrotra, 2000), respectively. It was found more in the former cells. Further, to validate the biochemical findings, histochemistry of day 7 to 9 p.c. implantation swellings was done in normal as well as SR/DR tissues on day 8 and 12 p.c., respectively. While on day 7 p.c. the intensity of staining for ADA was noticed on antimesometrial side only, it spread to the entire decidua on day 9 p.c. In tissue SR, on the other hand, the staining intensity was very low on both the days, but in tissue DR it was remarkably high on day 12 p.c. as compared to that on day 8 p.c. The histochemical findings thus support the biochemical findings with regard to the very high activity of the enzyme on day 12 p.c. in the DR tissue. The obvious reasons for this could be the regeneration of uterine decidua after cessation of drug treatment. The results, thus clearly indicate that enzyme ADA plays a definite role in tissue proliferation and/or regeneration, irrespective of the physiological status i.e. whether it is the early post-implantation days in normal pregnancy or after the withdrawal of drug treatment.

The extremely low level of ADA in SR seems to be due to the augmentation of process of necrosis, which begins during resorption (Knudsen et.al., 1987; Gao et al., 1994).

Higher level of ADA in normal implantation swellings or in regenerating uterine tissue after the withdrawal of drug treatment reflects that the tissue proliferation and/or regeneration is dependant on this enzyme (Knudsen et al., 1988, 1991; Hong et al., 1991; Witte et al., 1991; Blackburn et. al., 1992). The maximum level of enzyme in the regenerating tissue thus seems to be the representation of its speedy recovery. Possibly, due to this reason the ADA level increases in sites undergoing regeneration on day 12 p.c. (i.e. four days after the cessation of drug treatment) (Table 3). The findings thus clearly indicate that a pronounced ADA activity is essential for promoting the tissue growth. The importance of the enzyme ADA during fetal stages of development has also been shown by Wakemiya et al.(1995), who noticed that the ADA deficient fetuses, that also lacked ADA in trophoblasts of their adjoining placenta, died perinatally in association with profound purine metabolic disturbances and hepatocellular impairment.

In case of SR the available information states that it is associated with the metabolic formation of 2-deoxyadenosine, the level of which increases as the consequence of DNA degradation. The tissue sites of cell death (during resorption) may become the natural focal points for 2-deoxyadenosine formation, while those of the healthy sites are dependent on elevated ADA activity for protection (Henderson and Smith, 1981 Doherty et al, 1991). Such dependency, besides in pregnant female reproductive tract (Knudsen et al., 1992), has also been demonstrated in thymus (Ratech et al., 1981), cardiac and skeletal muscles (Rubio et.al., 1973) and heart (Schuntz et.al., 1981).

Regression of uterine epithelium during implantation and of sub-apical stroma during early placentation in rodents takes place as the consequence of programmed cell death (apoptosis) (Welsh and Enders, 1985). This is a natural phenomenon. But in case of resorptions (spontaneous or drug-induced) the early embryo faces in utero challenge of excess 2-deoxyadenosine, made available in the absence of enzyme ADA, which may become the causative factor for resorptions.

Among the several reasons responsible for SR in rodent species the genetic disorders may be one of them, due to which the pregnancy fails to sustain. Enzyme ADA is the manifestations of this and its activity remaining unchanged from day 8 p.c. to day 12 p.c. may be because of this defect.

The lowering of the enzyme activity in DR caused by the compound. on day 8 p.c. is transitory because withdrawal of the treatment brings ADA activity back to normal level or even enhances it further (day 12 p.c.). This may possibly be due to the fact that the tissue becomes highly proliferative in order to get itself regenerated. However, in case of normal pregnancy the enzyme by preventing local accumulation of 2-deoxyadenosine and adenosine at the implantation sites, promotes embryonic development. Possibly due to this reason the embryo is especially sensitive to these bioactive purines (Gao et al., 1994).

The activity of 5’-NT, on the other hand, shows a trend inversely proportional to ADA during the progress of pregnancy in all the three tissue (D+E, D-E and IA) (Table 2). The enzyme level remains equally low in SR as well as DR uterine sites of foetal resorptions on both day 8 and 12 p.c. (Table 3).

Two metabolic pathways are considered as potential source of adenosine formation in mammalian tissue; 1) involvement of 5’-NT, an ectozyme that yields adenosine through enzymatic dephosphorylation of cyclic 5’-AMP, and 2) augmentation of cellular transmethylation that produces adenosine through enzymatic hydrolysis of S-adenosylhomocysteine (SAH) (Rubio et al., 1973; Schuntz et al., 1981). Since no SAH hydrolase activity is reported in embryo-decidual unit (Blackburn et al., 1992), the level of adenosine appears to be regulated through 5’-NT. Possibly due to this reason, a very low level of 5’-NT is detected in the proliferating tissue. But the low level of the enzyme in resorbed tissue, SR or DR, indicates that possibly the 5’-NT has no direct role in uterine necrosis as well. However, a decline in the level of 5’-NT along with its mRNA has been accounted for regression of primary decidua (representing antimesometrial side) between day 6 and 9 of gestation in rat (Welsh and Enders, 1987; Parr and Parr 1989). Since we have studied the entire decidual swelling that includes both decidua basalis and capsularis, the decreased 5’-NT activity may also be an outcome of the consequence resulting in regression of the entire decidual tissue.

The inference drawn from the present findings thus indicates that the enzyme ADA is linked with the normal growth of uterine tissue during early embryonic development as well as with the regenerating tissue. While the remarkably high activity of the enzyme in these tissues reflects both the processes, lowering of activity (day 8 and 12 p.c. SR tissue and day 8 p.c. DR tissue) reflects the process of necrosis. The regenerative process in SR fails to occur possibly due to some irreversible changes in the purine metabolism that might have taken place in the embryonic and/or decidual tissue, resulting in resorption of the fetuses with no chance of reversal. Since no evidence is available in literature on this phenomenon the stated inference seems to be relevant. Moreover, the possibility of linking this with genetic disorder or immunoincompetency of the foetus vis-a-vis the decidual tissue, can not be ruled out as ADA is linked with ‘T’ lymphocytes also (Bulmer, 1996), whose number is reported to go down remarkably in recurrent spontaneous abortions in human subjects (resorption is not known in human) (Hill et al., 1995).

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Table 1

Table 2

Table 3

Figure 1

Section of gestation day 7 hamster decidual swelling showing embryo inside the implantation chamber. Intense enzyme staining on antimesometrial side may be noted.

Figure 2 

Showing magnified area of antimesometrial side (Fig.1) denoting intense staining (asterisk).

Figure 3

Section of gestation day 8 hamster decidual swelling intense staining in mesometrial area (arrow). Mild staining may also noted in extraembryonic fold (arrow head).

Figure 4

Section of gestation day 9 hamster decidual swelling. Enzyme staining is seen on neural plate (arrow) and some part of the myometrium.

Figure 5

Uterine section of spontaneous resorption (day 8) showing mild staining in outer region of resorbed endometrium.

Figure 6

Uterine section of compound 95/588 induced resorption (day 8). Mild staining in periphery only may be noted.

Activity of enzyme ADA and 5'-NT in hamster
(n moles/min/mg protein ; n=5)