J. Anat. (2001) 198, pp. 443–453, with 4 figures Printed in the United Kingdom 443

NADPH-diaphorase activity and nitric oxide synthase

isoforms in the trophoblast of Calomys callosus

NECI MORAES1,2, DOUGLAS ZAGO2, SONIA GAGIOTI2, MARA SANDRA HOSHIDA2 AND

ESTELA BEVILACQUA2

"Department of Morphological Sciences, Federal University of Santa Catarina, FlorianoU polis, SC, Brazil, and

#Department of Histology & Embryology, Institute of Biomedical Sciences, University of Sah o Paulo, Sah o Paulo, Brazil

(Accepted 24 October 2000)

The pattern of expression of a variety of placental nitric oxide synthase isoforms has contributed to

elucidating the regulatory mechanisms of nitric oxide (NO) synthesis during gestation. The maintenance of

vascular tone, attenuation of vasoconstriction, prevention of platelet and leukocyte adhesion to the

trophoblast surface, and possible participation in uterine blood flow seem to be the main functions of NO

generated at the fetal-maternal interface in humans and mice. Extending this knowledge to other rodent

species commonly used as laboratory animals, in this study we focus on NADPH-diaphorase activity and

the distribution of nitric oxide synthase isoforms (NOS) in the trophoblast cells of Calomys callosus during

different phases of pregnancy. NADPH-diaphorase activity was evaluated cytochemically and the presence

of NOS isoforms detected by immunohistochemistry. These techniques were performed on pre- and

postimplantation embryos in situ and in vitro, as well as in placentae on d 14 and 18 of pregnancy. Neither

NADPH-diaphorase activity nor inducible or endothelial NOS isoforms were found in pre-implanting

embryos except after culturing for at least 48 h, when some of the embryonic cells were positive for the

diaphorase reaction. On d 6±5 of pregnancy, trophoblast cells showed intense diaphorase activity both in situ

and under in vitro conditions. A positive reaction was also found in the different placental trophoblast cells

on d 14 and 18 of pregnancy. The inducible NOS (iNOS) isoform, but not the endothelial isoform, was

immunodetected in trophoblast cells from the placenta and from postimplantation embryos in situ and

under in vitro conditions. These results strongly suggest the production of NO by the iNOS isoform in the

trophoblast of Calomys callosus after embryo implantation. The data also emphasise a possible role for the

trophoblast in producing and releasing cytotoxic molecules at the fetal-maternal interface.

Key words : Pregnancy; placenta; inducible nitric oxide synthase.

Vasodilatation, relaxation of nonvascular smooth

muscle, inhibition of platelet aggregation, inhibition

of leukocyte adhesion and rolling, neurotransmission,

and mediation of cytotoxic macrophage effects are

important roles attributed to nitric oxide (NO)

(Moncada et al. 1991; McKendrick & Radomski,

1998). Some of these events occur during pregnancy

during which NO also plays a pivotal role, assuring

gestational success (Myatt et al. 1992; Molna! r et al.

1994; Kaufmann & Castellucci, 1997; Zarlingo et al.

Correspondence to Dr Estela Bevilacqua, Depto de Histologia e Embriologia, Instituto de Cie# ncias Biome!dicas, Universidade de Sa4 o Paulo,

Av. Prof Lineu Prestes, 1524, 05508-900 Sa4 o Paulo, SP, Brazil. Fax: ­55-11-818-7307; e-mail : bevilacq!usp.br

1997). In the human placenta, the maintenance of

vascular tone, attenuation of vasoconstriction, and

prevention of platelet and leukocyte adhesion to the

trophoblast surface seem to be the main functions of

NO generated at the fetal-maternal interface (Myatt

et al. 1993; Kaufmann & Castellucci, 1997).

In mice, a role for NO and oxygen-reactive species

released by the trophoblast cells has been associated

with a potent cytotoxic mechanism which appears to

facilitate the phagocytic process during implantation

(Bellavite et al. 1990; Gagioti et al. 1995, 1996, 2000).

The presence of cytotoxic molecules in the trophoblast

invasion pathway appears to influence the fate of the

surrounding maternal cells, allowing or even trigger-

ing their internalisation by the trophoblast. Vascular

rearrangement, neovascularisation, and vasodilata-

tion, which take place in the endometrium sur-

rounding the embryo (Welsh & Enders, 1987) con-

comitant with the implantation process also may

result from the action of NO derived from an

endothelial nitric oxide synthase (eNOS) isoform,

released by the trophoblast, functioning together with

other components of maternal origin (McLaughlin et

al. 1978; Benirschke & Kaufmann, 1995; Holcberg et

al. 1995).

Extending knowledge of this specific feature of

pregnancy to other species is of considerable im-

portance in comprehending how trophoblast cells

perform their particular functions and contribute to

gestational success. In this context, we focus our study

on Calomys callosus (Rodentia: Cricetidae). This

eminently South American species is a reservoir for

various pathogens and may represent an important

vector for the spread of diseases (Justines & Johnson,

1969, 1970; Johnson et al. 1975). Over the last few

decades, C. callosus has been kept under laboratory

conditions where various congenital infections in

which the trophoblast may play a relevant role can be

investigated (Ferro et al. 1999). From the reproductive

point of view, C. callosus is a polyoestrus rodent

whose oestrus cycles last around 6±6 d; puberty

frequently occurs at 40±1 d (³7±6) in females and

19±6 d (³6±6) in males. The adult animal is 12 cm

long, weighs 30³8 g and produces litters of 5³3 pups

(Mello, 1978). C. callosus is also considered to be an

important laboratory animal as it may be a reservoir

for Trypanosoma cruzi, the aetiological agent of

Chagas’ disease (Ribeiro, 1973), and for Argentine

haemorrhagic fever (Justines & Johnson, 1969).

These considerations led us to examine the pro-

duction of NO by the trophoblast which may

participate in the dynamics of maternal circulation,

acting to maintain adequate blood flow during

pregnancy, and mediating the phagocytic activity of

trophoblast cells in the placental barrier. We invest-

igated possible sites of NOS activity (NADPH-

diaphorase enzyme complex) and the expression of

endothelial and macrophage isoforms of NOS in the

trophoblast of Calomys callosus. The ample struc-

tural, cellular and molecular similarities found be-

tween trophoblast giant cell populations in vivo and

in vitro in various rodents (Mehrotra, 1988; Iguchi et

al. 1993; Albieri et al. 1999; Hoshida, personal

communication) allowed parallel studies under both

conditions.

Materials

Fetal calf serum (FCS) was purchased from Culti-

lab, Campinas, SP, Brazil. Tris(hydroxymethyl-

aminomethan) and sodium chloride were obtained

from Merck, Darmstadt, Germany. MEM nonessen-

tial amino acids, insulin, uridine, penicillin and

streptomycin were purchased from Gibco BRL,

Grand Island, NY, USA. Glycol methacrylate resin

was obtained from LKB, Sweden. Polyclonal en-

dothelial NOS (eNOS) antibody and polyclonal

macrophage NOS (iNOS) antibody were obtained

from Santa Cruz Biotechnology, Santa Cruz, CA,

USA. Biotinylated goat antirabbit IgG, streptoavidin-

alkaline phosphatase complex kit, Fast Red TR}naph-

thol AS-MX developer kit, β-NADPH, PBS, BSA,

Dulbecco’s modified Eagles medium (D-MEM), L

(­) lactic acid, pyruvic acid, adenosine, guanosine,

thymidine, nitro blue tetrazolium (NBT), paraform-

aldehyde, poly--lysine solution, paraplast and Triton

were obtained from Sigma, St Louis, MO, USA.

Animals

Virgin female Calomys callosus aged 3–4 mo were

caged overnight with males, and successful mating

was checked on the following morning. The presence

of a vaginal plug established the first half-day of

pregnancy (dop). Food and water were available ad

libitum. The photoperiod was 12L:12D.

Pregnant females were killed by cervical dislocation

and their uteri immediately dissected in PBS con-

taining 0±3% BSA. All procedures were performed in

accordance with the ethical principles for animal

research adopted by the Brazilian College of Animal

Experimentation.

Pre-implantation blastocyst collection and culture

On d 3±5 of pregnancy, pre-implantation embryos

were flushed from the uterine horns with PBS-10%

FCS. Late and early blastocysts were selected in a

stereomicroscope and cultured in a 12-well multidish

culture plate containing removable 18-mm diameter

glass coverslips. The embryos were cultured at 36±5 °Cin a humid atmosphere of 5% CO

#in air, in D-MEM

medium supplemented with 10% FCS, 100 U}ml

penicillin, 100 µg}ml streptomycin, 1% MEM non-

essential amino acids, 0±4 µg}ml insulin, 1% nucleo-

sides (adenosine, thymidine, guanosine, uridine),

520 µg}ml lactic acid, 56 µg}ml pyruvic acid and

4 mg}ml BSA.

444 N. Moraes and others

Freshly collected, and 24 and 48 h cultured

blastocysts were used for NOS evaluation.

Ectoplacental cone collection and culture

Embryos were surgically dissected under a stereo-

microscope from the uteri of females killed by cervical

dislocation on d 6±5 of pregnancy. The ectoplacental

cones (ECs) were then carefully separated from the

surrounding embryonic tissues and cultured as pre-

viously described for blastocysts. Three to 5 ECs were

introduced into each well of a 12-well multidish

culture plate covered with removable 18 mm diameter

glass coverslips.

Implantation sites on d 3±5 and 6±5 of pregnancy and

placentae on d 14 and 18

The uteri of females on d 3±5 and 6±5 of pregnancy

were collected immediately after death. The implan-

tation chambers were removed and thinly sliced into

transverse fragments in TBS (Tris 0±02 sodium

chloride 0±85%)-BSA 3%. Some slices were processed

using morphological and immunohistochemical tech-

niques and the remainder were used for cytochemical

assays.

The uteri of females on d 14 and 18 of pregnancy

were dissected and the placentae removed, embedded

in TBS-BSA and carefully dissected from the amniotic

membranes and decidual tissues.

Cytochemical assay for NADPH-diaphorase activity

This assay was performed in tissues fixed at different

stages of pregnancy. The specimens were fixed in 4%

paraformaldehyde in PBS for 1 h at room tem-

perature.

The enzyme complex activity was investigated in

freshly collected, flushed blastocysts, in 24 and 48 h

cultured blastocysts, in implantation sites on d 3±5and 6±5 of pregnancy, in 24 and 48 h cultured ECs,

and in mature placenta.

After fixing and rinsing in PBS, the specimens were

incubated for 30 min at room temperature in 1±0 m

β-NADPH and 0±25 m NBT in PBS, pH 7±4 (Conrad

et al. 1993). The specificity of the reaction was

controlled by omitting the β-NADPH from the

incubation solution. Following incubation, all sam-

ples were postfixed in 4% paraformaldehyde in 0±1

PBS. Noncultured specimens were prepared for

embedding in glycol-methacrylate resin according to

the manufacturer’s instructions and examined in

stained or unstained 5 µm sections. Cultured ECs and

blastocysts which had adhered to the glass coverslips

were rinsed, counterstained with neutral red or left

unstained, dried and mounted in PBS}glycerol (1 :9)

and directly observed by conventional light micro-

scopy.

For ultrastructural studies, the specimens were

postfixed in 4% paraformaldehyde-2% glutaralde-

hyde in 0±1 PBS for 20 min followed by 2% osmium

tetroxide in 0±1 PBS for an additional 20 min at

4 °C.

After fixing, the formazan salts and osmium yield a

weakly soluble complex which appears as electron-

dense areas at the ultrastructural level. The specimens

were then embedded in Spurr’s resin and observed as

stained or unstained thin sections using a JEOL-

CMX-II transmission electron microscope.

Immunohistochemistry

Freshly collected and cultured embryos from d 3±5and 6±5 of pregnancy adhering to glass coverslips were

immunoassayed after rapid fixation (10 min) in a

solution containing 60% methanol, 30% chloroform,

and 10% acetic acid.

Placentae and implantation sites (days of pregnancy

3±5 and 6±5) were investigated in 5 µm thick paraplast

sections. The material was fixed by immersion in 4%

paraformaldehyde in TBS, pH 7±4, for 24 h, followed

by dehydration in ethanol and embedding.

The specimens were first incubated in either TBS-

0±3% Triton added to 1% BSA or normal rabbit

serum in TBS-Triton for 1 h, at room temperature,

followed by incubation for 15 min in 15% acetic acid

in water, to block endogenous alkaline phosphatase

activity. They were then incubated overnight at 4 °Cin polyclonal eNOS antibody (1:25) and in polyclonal

iNOS antibody (1:50).

Antibody binding was detected using biotinylated

goat antirabbit IgG (1:100) in TBS and streptoavidin-

alkaline phosphatase complex (1:40). Alkaline phos-

phatase was revealed using a Fast Red kit. Sections

were counterstained with Mayer’s haematoxylin for

5 min, and washed in distilled water. The slides and

glass coverslips containing the material were then

dried and mounted in PBS}glycerol (1 :9) and ob-

served in a light microscope.

Immunohistochemical controls consisted of sub-

stituting the primary antibody or the biotinylated

antirabbit serum by TBS. Positive controls used

histological preparations of lung tissue. Negative

controls were performed using a nonrelevant antibody

(polyclonal goat antirabbit IgG against Toxoplasma

gondii ).

NADPH-diaphorase and NOS in the trophoblast 445

Fig. 1. Day 3±5 of pregnancy. (a, inset) Histochemical staining for NADPH-diaphorase activity shows no reactivity in the blastocysts (arrows).

The star indicates the blastocoele. (b–d ) After 48 h of culture, embryo outgrowths show giant cells (*) at the periphery and small cells in the

central region (b). Cells reactive for diaphorase are shown in c ; in d, no reaction is seen in the absence of the enzymatic substrate. (e–f ) The

blastocyst (arrow) lying in the uterine lumen is not immunoreactive for iNOS (e, Gomori’s trichrome stain; f, Mayer’s haematoxylin

counterstaining). a–b, ¬200; c–f, inset, ¬160.

446 N. Moraes and others

Fig. 2. Day 6±5 of pregnancy. (a) HE-stained paraplast section of an implantation site in which the ectoplacental cone (Ec) and giant mural

trophoblast (g) can be easily identified. (b–d ) NADPH-diaphorase reactivity (arrows) was found in giant cells (g) of cultured ectoplacental

cones (b), cells of the mural region (c), and polar trophoblast of embryos in situ (d ). ( f ) Immunoreactivity for iNOS is seen in the trophoblast

cells (arrows). (e) Negative control. D, decidua. a, ¬64; b–c, neutral red counterstaining, glycol methacrylate resin; b–d, ¬420; e–f, Mayer’s

haematoxylin counterstaining; e, ¬210; f, ¬420.

NADPH-diaphorase and NOS in the trophoblast 447

NADPH-diaphorase activity

With the exception of pre-implantation embryos,

NADPH-diaphorase activity was found exclusively in

the trophoblast cell populations, in all periods of

pregnancy studied.

Fig. 3. Day 14 of pregnancy. The placenta of Calomys callosus shows 3 distinct regions: the giant cell network (G), the spongiotrophoblast

(S) and the labyrinth (L). Giant cells (a) and cells from the labyrinth (b) are strongly reactive for NADPH-diaphorase (arrows). (c)

Immunolocalisation of iNOS is also present in trophoblast cells of the spongiotrophoblast but mainly in the labyrinth cells. (d ) Negative

control. (e) Positive control : the arrow indicates reactivity in lung macrophages. a–b, neutral red counterstaining, glycol methacrylate resin,

¬320; c–e, Mayer’s haematoxylin counterstaining, c, e, ¬320; d, ¬160.

Isolated blastocyst-stage embryos, enveloped by the

zona pellucida (Fig. 1a), did not show NADPH-

diaphorase activity (Fig. 1a, inset). After 48 h of

culturing, the hatched blastocysts lost their initial

shape and features ; peripheral giant cells were found

surrounding groups of small central cells (Fig. 1b).

Enzymatic activity was present in these embryos,

448 N. Moraes and others

Fig. 4. Day 14 of pregnancy. Cytochemical localisation of NADPH-diaphorase reactivity (arrows). (a) Note the presence of reaction deposits

in vesicles and tubular-like structures present in the cytoplasm of the trophoblast cells. (b) Positive plaques were also found in close proximity

to intermediate filaments (*). Electron micrographs, magnification ¬20000.

mainly in the peripheral giant cells (Fig. 1c). No

reactivity was found in the absence of the enzymatic

substrate NADPH (Fig. 1d).

Postimplantation embryos from d 6±5 of pregnancy

showed strong NADPH-diaphorase activity. This

reactivity was distributed exclusively in the tropho-

blast cells located around the entire embryoblastic

region (EC and mural region, Fig. 2a). The specific

localisation of the enzymatic activity in the tropho-

blast could be seen clearly in methacrylate resin

sections (Fig. 2b, c). Among the trophoblast cell types

present at this point of pregnancy, the giant peripheral

cells appear to be particularly involved in NADPH-

diaphorase staining.

Detection of the enzymatic complex in cultured

ECs also showed strongly positive trophoblast cells in

the first 48 h of conditions in vitro (Fig. 2d). Removal

of β-NADPH from the incubation medium (control)

resulted in a significant loss of NADPH-diaphorase

staining (data not shown).

As seen in other rodents, the Calomys callosus

placenta shows a distinct, regional pattern, which

includes the labyrinth, the spongiotrophoblast and the

zone formed by a network of giant cells. The NADPH

diaphorase reaction was found in the different types of

trophoblast cell present in each of these regions, and

was particularly evident in the labyrinth and in the

giant cells (Fig. 3a, b). There was a strict correlation

between the NADPH-diaphorase activities seen at the

light and electron microscope levels. Of the several

placental cell subpopulations, only the trophoblast

cells showed subcellular NADPH-diaphorase reaction

products, seen as electrondense deposits in vesicles, in

a tubular system of membranes (Fig. 4a), and

associated with intermediate filaments (Fig. 4b). In

the specimens in which the NADPH-diaphorase was

omitted from the incubation medium, no activity was

seen in the trophoblast cells.

Immunohistochemistry

The eNOS reaction was not observed in trophoblast

cells at any stage of development, either in situ or in

vitro. However, evidence for iNOS was found at

different periods of pregnancy from the postimplanta-

tion stages onwards.

On d 3±5 of pregnancy, the blastocysts were lodged

in the antimesometrial region of the uterine crypts but

NADPH-diaphorase and NOS in the trophoblast 449

usually had not yet initiated the implantation process

(Fig. 1e). Neither these blastocysts (Fig. 1 f ) nor the

24 or 48 h cultured embryos showed immunostaining

for iNOS.

Embryos on d 6±5 of pregnancy, lodged at the

implantation site in situ (Fig. 2 f ) or under in vitro

conditions, showed intense immunostaining. There

was no evidence of reactivity in the negative controls

in any specimen evaluated or in those in which the

nonrelevant antibody was used (Fig. 2e).

Inducible activity also appeared in several tropho-

blast cell types in the mature placenta. However, the

reaction was stronger in the cells of the labyrinth zone

than in those of other regions (Fig. 3c). Negative

controls where the primary antibody was omitted

showed no immunolabelling (Fig. 3d).

The iNOS reactivity was confirmed by the strong

positivity seen in the lung macrophages (Fig. 3e).

Normal implantation and placentation are complex

processes requiring the concomitant maternal and

embryonic events, which together contribute to the

success of pregnancy. The vasodilatation that takes

place in the endometrium surrounding the embryo}fetus, the intense phagocytosis exhibited by the

trophoblast during the implantation, and the physio-

logical maintenance of the placental barrier are

examples of such events.

Recent findings suggest a role for NO in many of

these events. Inhibition of NO synthesis can reduce

the number of implanted rodent embryos, empha-

sising the crucial role of NO during this process

(Novaro et al. 1997). In rodents, particularly the

participation of different maternal cells and tissues

and trophoblast subpopulations in NO production

during pregnancy has also been suggested (Natuzzi et

al. 1993; Moorhead et al. 1995; Hunt et al. 1997;

Purcell et al. 1997, 1999; Baylis et al. 1999; Gagioti et

al. 2000).

In the present study, we investigate the presence of

NADPH-diaphorase activity and NOS isoforms as a

condition for NO production, in the trophoblast of

Calomys callosus. The NADPH-diaphorase activity

and the presence of eNOS}iNOS were cytochemically

and immunohistochemically evaluated in trophoblast

cells obtained from 3±5- and 6±5-d-old Calomys

callosus embryos, and from mature placentae on d 14

and 18 of pregnancy. In an effort to establish a model

for further molecular studies, we also estimated NOS

expression by cultured trophoblast cells obtained

from 3±5 and 6±5 d-old Calomys callosus embryos.

In our material, the NADPH-diaphorase staining

was obtained after fixation, as shown in other studies

specifically detecting components of the NOS complex

(Hope & Vincent, 1989). According to Matsumoto et

al. (1993), fixation apparently blocks the NO-forming

ability of NOS, but not its ability to transfer electrons

from NADPH to reduce NBT (Matejevic et al. 1996).

Pre-implanting blastocysts showed no diaphorase

activity, except after 48 h of culture, when peripheral

giant cells were reactive. Immunostaining for en-

dothelial and macrophage isoforms of NOS, however,

was not detected in flushed embryos or in cultured

blastocysts, in contrast to the expected colocalisation

of sites of NADPH-diaphorase activity and NOS

immunoreactivity (Bredt et al. 1991a, b).

Possible explanations for this apparent discrepancy

in the experimental data, may be that either the

enzymatic activity seen under culture conditions is

associated with other dehydrogenases not specifically

related to the NOS isoforms, or that the presence of

cells expressing the enzyme in cultured embryos occurs

at a specific stage of trophoblast differentiation,

randomly found during the blastocyst outgrowth in

vitro.

Also, the total amount of NOS protein per

blastocyst may be insufficient for detection by

immunohistochemical methods. Zarlingo et al. (1997)

found no NOS immunoreactivity in the rat but

localised the protein by Western blotting. The small

number of cells and low total amount of protein per

blastocyst are limiting factors for many of the current

experimental approaches that are necessary to confirm

the presence or absence of NOS activity at this

embryonic stage.

Thus we cannot assume that pre-implanting, blasto-

cyst stage embryos are not involved in NO production,

although the expression of NOS in the blastocysts of

other species also has not been demonstrated (Baylis

et al. 1999; Gagioti et al. 2000).

The localisation of NADPH diaphorase activity in

embryos on d 6±5 of pregnancy, however, shows

intense diaphorase reactivity in the trophoblast cells.

The reaction products were restricted exclusively to

the trophoblast population, and predominate in the

trophoblast giant cells both under in situ and in vitro

conditions. The pattern of diaphorase staining was

similar in situ and in vitro, although in culture the

reactive cells were not so numerous. These findings are

consistent with those for giant trophoblast cells

obtained from mouse embryos on d 7–8 of pregnancy

(Moorhead et al. 1995; Gagioti et al. 2000).

Immunodetection of the inducible NOS isoform in

postimplantation trophoblast cells on d 6±5 of preg-

450 N. Moraes and others

nancy supports the observations on NADPH-dia-

phorase activity. This NOS isoform occurred as

patches in the cytoplasm of 24 and 48 h EC cultures,

in trophoblast giant cells in situ.

The expression of NADPH-diaphorase activity and

the immunolocalisation of iNOS in the trophoblast

giant cells of implanting embryos suggest that NOS

activation and NO production may be controlled

events within the trophoblast giant cell differentiation

programme. By maintaining their NO-related char-

acteristics under in vitro conditions, trophoblast giant

cells may be excellent candidates for further studies of

NO-specific metabolic pathway in vitro (Eis et al.

1995).

We also localised NADPH-diaphorase activity and

iNOS in subpopulations of placental trophoblast

cells. As in other stages of trophoblast differentiation,

reactivity was demonstrated as small patches distribu-

ted in the cytoplasm of different trophoblast cell

types. The reason for such patchiness is not clear, but

may reflect the presence of cytoplasmic areas (either

membrane-bound or not) in the trophoblast cells

related to inducible NO formation. Our electron-

microscopic findings are consistent with this possi-

bility. In all cases, reactivity for NADPH-diaphorase

was only seen in trophoblastic components of the

placenta and present either in the peripheral cyto-

plasm of these cells, in close proximity to intermediate

filament bundles, or in specific structures such as

vesicles and tubular, endosome-like components.

Similar results have also been reported by Wolf et al.

(1995) in neuronal cells in which the NADPH-

diaphorase reaction was found in association with

cytoskeleton elements and the intracellular system of

membranes.

Based on these experimental approaches, we believe

that active NO-generation takes place in the tropho-

blast cells of Calomys callosus. The participation of

the trophoblast in the production of NO has been

demonstrated in previous studies in humans, in the

late stages of placental maturation (Conrad et al.

1993; Myatt et al. 1993; Buttery et al. 1994; Eis et al.

1995), and in rodents such as guinea pigs (Nanaev et

al. 1995), rats (Purcell et al. 1997; Zarlingo et al. 1997)

and mice (Moorhead et al. 1995; Matejevic et al.

1996; Baylis et al. 1999; Purcell et al. 1999; Gagioti et

al. 2000).

Most of these previous studies demonstrated eNOS

expression among other possible isoforms, by tropho-

blast cells ; however in C. callosus this isoform was not

revealed by immunological techniques. At least 2

reasons for this discrepancy may justify our findings:

(1) nonoptimal, decreased amounts of eNOS protein,

but not of iNOS protein led us to misinterpret our

findings, or (2) eNOS is not expressed by the

trophoblast of C. callosus.

The main physiological role attributed to eNOS in

the placenta is the production of NO, which inhibits

platelet aggregation at the trophoblast surface and

acts as an endothelium-relaxing factor. The lack of

participation by the Calomys callosus trophoblast in

these events may reflect intrinsic species-specific

differences, compensated by mechanisms exhibited by

maternal cells and tissues. In other rodents, oestrogen

produced by the ovaries at the time of blastocyst

implantation seems to induce NOS activation in the

endothelial cells lining the uterine arteries, which leads

to the production of a significant amount of NO

(Chaves et al. 1993; Weiner et al. 1994; Novaro et al.

1997; Biswas et al. 1998). A role for leukocytes in NO

production by the pregnant uterus also has been

suggested in rats and mice (Huang et al. 1995; Hunt

et al. 1997) and in decidual cells (Haddad et al. 1995;

Moorhead et al. 1995).

The presence of iNOS, as a means of releasing NO

at the maternoplacental interface in Calomys callosus,

however, may be of specific physiological significance.

It is largely assumed that trophoblast cells are involved

in the active phagocytosis of maternal elements.

However, the expression of iNOS by inflammatory

cells has been proposed as an event in the phagocytosis

process since the NO produced and released shows a

cytostatic}cytotoxic effect (Moncada et al. 1991;

Radomski, 1995; McKendrick & Radomski, 1998).

Thus this finding may correlate with the intense

phagocytic activity of the rodent trophoblast cells

during the first half of the pregnancy. Maternal cells

interposed in the trophoblast invasion pathway during

embryo implantation are actively phagocytosed

(Bevilacqua & Abrahamsohn, 1989) and this event

may assure the success of the implantation process.

Studies undertaken in our laboratory have demon-

strated the ability of the mouse trophoblast to produce

oxygen-reactive species with cytotoxic properties at

this specific period after implantation (Gagioti et al.

1995, 1996). Coincidentally, at this time, the tropho-

blast is intensely involved in gaining space, expanding

into the maternal stroma.

The expression in Calomys callosus of iNOS as a

source of NO in the placental cells at the maternal

interface also may be involved in the phagocytic

events occurring during pregnancy. The phagocytic

activity of placental cells may be restored under

special conditions during maternal immune challenge

(Myatt et al. 1997) or in the presence of maternal

pathogens at the trophoblast-endometrium interface

NADPH-diaphorase and NOS in the trophoblast 451

(Amarante & Bevilacqua, 1999). Thus, in this context,

the activation of iNOS may be related to the protective

and fetal defence mechanisms exhibited by the

trophoblast.

In conclusion, our data on NADPH-diaphorase

activity and iNOS immunolocalisation support the

possible participation of the trophoblast in events of

fundamental importance to the successful implan-

tation process and in defence mechanisms triggered

during pregnancy. The data also suggest the in-

volvement of NO in the maintenance of appropriate

conditions for phagocytosis at the placental barrier.

We express our gratitude to Rosangela de Oliveira for

technical assistance, and to Dr John McNamara and

Mr John Norman for revision of the English manu-

script.

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