Cytokines in the oviparity/viviparity transition: evidence of the

interleukin-1 system in a species with reproductive bimodality,

the lizard Lacerta vivipara

Luana Paulesu,a,� Elisa Bigliardi,b Eugenio Paccagnini,b Francesca Ietta,a Chiara Cateni,a Claude PierreGuillaume,c and Benoit Heulind

aDepartment of Physiology, Division of Immunoendocrinology and Reproductive Physiology, University of Siena, 53100

Siena, ItalybDepartment of Evolutionary Biology, University of Siena, 53100 Siena, ItalycLaboratoire de biogeograhie et ecologie des Vertebres, EPHE, Universite de Montpellier II, Place Eugene Bataillon, F-34095

Montpellier, FrancedCNRS-UMR6553, Station Biologique, F-35380 Paimpont, France�Author for correspondence (email: paulesu@unisi.it)

SUMMARY Placental viviparity is a reproductive strategyusually attributed to mammals. However, it is also present inother vertebrate species, e.g. in Squamate reptiles. Althoughthe immunological mechanisms that allow the survival of thesemi-allogenic embryo in maternal tissues are still largelyunknown, cytokines seem to play an important role inmammalian reproduction. Previous studies in our laboratoryshowed that interleukin-1 (IL-1), a cytokine associated withimplantation in mice, is also expressed at the materno-fetalinterface of placental viviparous Squamates. In this study,we used the model of Lacerta vivipara, which exhibits re-productive bimodality, that is, the coexistence of oviparousand viviparous populations. By means of immunohisto-chemistry and anti-human antibodies, we showed that

uterine tissues of L. vivipara (seven oviparous and sixviviparous animals) expressed the two IL-1 isoforms, IL-1aand IL-1b, and the type I IL-1 receptor (IL-1R tI) both at thepre-ovulatory stage and during gestation, with no significantdifference between oviparous and viviparous females. In L.vivipara, as in most oviparous Squamates, an important phaseof embryonic development takes place in the mother’s oviduct,before egg-laying. Moreover, although thinner than in ovi-parous females, an eggshell membrane persists throughoutgestation in viviparous females also, which develop a verysimple type of placenta. The data suggest that immunologicalmechanisms that allow the survival of the semi-allogenicembryo in maternal tissues are independent of the timing orintimacy of contact between maternal and fetal tissues.

INTRODUCTION

Viviparity is a widespread reproductive mode among verte-

brates, involving retention of the embryo within the female

reproductive tract. Although viviparity is sometimes viewed

as a mammalian phenomenon, many other vertebrate groups

also contain live-bearing species (exceptions are the Agna-

thans, Birds, Crocodilians, and Chelonians). Viviparity may

involve the formation of a placenta, a structure formed by the

apposition of extra-embryonic membranes (chorion, allantois,

yolk sac) and the maternal uterus (Amoroso 1952). Although

viviparity represents an evolutionary advantage for the de-

veloping embryo, it is also a risk, as the embryo could be

rejected and thus not reach complete maturation (Medawar

1953). Since the embryo bears antigens of paternal origin,

viviparity can be considered a complex conflict between the

selfish genes of the mother and those of her partner. In the

last 20 years, many studies have shown the presence of

cytokines (peptides or glycopeptides with autocrine/paracrine

action) in the placenta of mammals independent of their type

of placenta (Paulesu 1997; Saito 2001; Schafer-Somi 2003).

Previous studies in our laboratory showed that interleukin-1

(IL-1), a pro-inflammatory cytokine associated with implan-

tation in mice, is also expressed by the placenta of nonmam-

malian vertebrates (Paulesu and Cateni 2002). These include

the chorioallantoic placenta of a Squamate reptile, the three-

toed skink Chalcides chalcides (Paulesu et al. 1995), and the

yolk sac placenta of a shark Mustelus canis (Cateni et al.

2003). These results suggested that IL-1 is an integral part

of maternal–fetal immunity in various classes of viviparous

EVOLUTION & DEVELOPMENT 7:4, 282–288 (2005)

& BLACKWELL PUBLISHING, INC.282

placental vertebrates. Therefore, we hypothesized that the ev-

olution from oviparous to viviparous reproduction was pro-

moted by the secretion of cytokines, e.g. IL-1, in reproductive

tissues. In the present study, we investigated the presence of

the two IL-1 isoforms, IL-1a and IL-1b, and the type I IL-1

receptor (IL-1R tI) in reproductive tissues of another Squa-

mate reptile, the lizard Lacerta vivipara. This species is of

particular interest because it exhibits reproductive bimodality,

that is, the coexistence of oviparous and viviparous popula-

tions with distinct geographic distributions (Heulin et al. 1997;

Surget-Groba et al. 2001). Oviparous females of L. vivipara

lay eggs with relatively thick eggshells (35–65mm), containing

incompletely developed embryos, at stages 30–35 in the ter-

minology of Dufaure and Hubert (1961). Viviparous females

of this species lay fully formed newborns (final stage 40),

which, however, are still encased in a very thin eggshell mem-

brane (less than 10mm) (Panigel 1956; Heulin 1990; Brana

et al. 1991; Heulin et al. 1991, 2002). During the pregnancy of

viviparous females, the nutrition of the developing embryo is

lecithotrophic (from the yolk contained in the ova), and a very

simple chorioallantoic placenta, type I in the terminology of

Blackburn (1993), only allows respiratory, water, and limited

mineral exchanges between the mother and her embryos

through the thin eggshell membrane (Panigel 1956). This

lecithotrophic viviparity is clearly distinct from the placento-

trophic viviparity (placental transfer of nutrients from the

mother to the embryo) of C. chalcides, in which the embry-

onic (chorioallantoic) and maternal (uterine) tissues are very

closely apposite and are not separated by an eggshell mem-

brane (Blackburn 1992, 1993). Because of its reproductive

bimodality, L. vivipara appears to be an ideal model to in-

vestigate the possible role of cytokines in the evolution from

oviparity to viviparity. In addition, it provides us with the

opportunity to examine a type of viviparity (lecithotrophic

and with persistence of an eggshell membrane) for which the

presence of IL-1 cytokines in uterine tissues has not yet been

tested.

MATERIALS AND METHODS

Animals and collection of samplesL. vivipara is a small (adults 45–75mm in snout-vent length)

ground-dwelling lacertid that generally lives in moist habitats. De-

tailed information on the reproductive cycle, life history, and ge-

ographic distribution of its oviparous and viviparous populations

has been published elsewhere (Heulin et al. 1991, 1997). In this

study, we used lizards from the oviparous population of Louvie

(431 060N, 01230W, Alt. 370m) in south-western France and from

the viviparous populations of Paimpont (481N, 21W, Alt. 150m) in

north-western France. The lizards reared in our laboratory were

placed in individual terraria (30 � 20 � 20 cm), each equipped with

a shelter, dishes of food and water, and a 40-W bulb that provided

heat for 6 h/day. Some females were caught in the natural pop-

ulations during autumn, and hibernated in our laboratory (4

months at 41C) under conditions allowing normal vitellogenesis

during the month following hibernation (Gavaud 1983). This sam-

ple was used to examine the uterine tissues of post-lethargic non-

vitellogenetic females (three oviparous and two viviparous animals,

sacrificed at the end of hibernation) and vitellogenetic pre-ovula-

tory females (two oviparous and two viviparous animals, sacrificed

3–4 weeks after the end of hibernation). A few females were also

caught at the end of May. Four females were sacrificed during their

period of intra-uterine egg retention (two oviparous and two vi-

viparous females), either in early June or in late June (embryonic

stages 32 and 34 in oviparous females and stages 31 and 39 in

viviparous females). The females were killed with chloroform and

dissected. Their oviducts were excised, fixed for 24h in 10% buff-

ered formalin, washed, and kept in 75% ethanol until processed.

For females having oviductal eggs, we dissected one egg to deter-

mine the stage of embryonic development according to the no-

menclature of Dufaure and Hubert (1961). Tissues were embedded

in paraffin wax and sectioned at 5mm. Some sections were stained

with hematoxylin and eosin. Only histologically normal sections

were processed for immunohistochemistry. The experiments and

animal captures were performed with the approval of institutional

committees: French Ministry of the Environment (99/27/AUT),

DIREN Aquitaine (DCLE3-190702).

ImmunohistochemistryImmunohistochemical staining of the formalin-fixed 5-mm sections

was performed with mouse antisera recognizing human IL-1 and

StreptABComplex/AP (DAKO, Milan, Italy). After deparaffina-

tion in Bioclear (BioOptica, Milan, Italy) and rehydration in serial

dilutions of ethanol, the histological sections were washed in Tris-

buffered saline (TBS) pH 7.6 and pre-incubated with normal rabbit

serum to prevent nonspecific binding. The slides were first incu-

bated with anti-human IL-1a, IL-1b, and IL-1R tI polyclonal

antibodies (R&D Systems, Abingdon, UK), then with rabbit anti-

goat immunoglobulins (DAKO) diluted 1:500 in TBS, and finally

with streptavidin complex (DAKO) diluted 1:300. Each incubation

was performed for 30min at room temperature and was followed

by three washes in TBS. The alkaline phosphatase reaction was

revealed using naphthol and new fuchsin as a substrate. Endog-

enous alkaline phosphatase was blocked by adding 1mM levami-

sole to the substrate solution. Sections were then washed for 5min

in running tap water and mounted with an aqueous mounting

medium. Negative controls were performed for each tissue by sub-

stituting the primary antibody with the pre-immune serum or TBS.

The specificity of the staining reaction was confirmed by inhibiting

the anti-human IL-1a/IL-1b antibody by incubation (overnight at

41C) with the specific antigen IL-1a, IL-1b (R&D Systems) at a

molar ratio of 1:1 before using it for tissue staining.

RESULTS

Pre-ovulatory stage

Our sample of the pre-ovulatory stage includes the time just

after lethargy and the vitellogenic phase. The following

distinct layers are recognizable in the uterine wall in both

IL-1system in the oviparity and viviparity 283Paulesu et al.

oviparous and viviparous females of L. vivipara: an external

myometrium, formed by outer longitudinal and inner circular

smooth muscle layers, and an endometrial layer formed by

luminal epithelium and a lamina propria of connective tissue

with blood vessels and glands (Figs. 1, A and 2). The luminal

epithelium consists of cuboidal or low columnar cells con-

taining both ciliated and secretory cells. The alveolar glands

become hypertrophic during vitellogenesis (Fig. 1A), and al-

most completely occupy the entire thickness of the lamina

propria, which is well vascularized with numerous blood ves-

sels at the interface between lamina propria and luminal ep-

ithelium. There is strong immunoreactivity for IL-1a and IL-

1b within the glands, with no detectable difference between

oviparous (Fig. 2, A and B) and viviparous (Fig. 2, E and F)

females, whereas only a few cells of the luminal epithelium

appear positive. Immunoreactivity for IL-1R tI is also strong

in the luminal epithelium but with a more spotted localization

in the vitellogenic stage (Fig. 2, C and G). No significant

differences in IL-1 or IL-1R tI expression were observed

among the stages. Negative controls show no immunoreac-

tivity (Fig. 2, D and H).

Gestation

Gestation times corresponding to embryonic stages 32 and 34

in oviparous females and stages 31 and 39 in viviparous fe-

males were examined. In both the oviparous and viviparous

females, the uterus distends owing to the presence of the eggs

and enlarges to form the incubatory chambers. Four to six

embryos are normally present in each female. The uterine wall

becomes thinner, and only flattened secretory cells are present

in the epithelium. Glands are still present in the lamina prop-

ria, but are less dominant and form a thinner layer than in the

vitellogenic stage (Figs. 1, B and 3). The eggshell is much

thicker (36.4� 8.5mm) in the oviparous females (Fig. 3, A–

D) than in the viviparous ones (8.7� 3.9mm) (Figs. 1, B and

3, E–H). Immunoreactivity for IL-1a and IL-1b is mainly

localized in the glandular cells of the lamina propria (Fig. 3,

A, B, E and F). Only a few epithelial cells are immunoreactive

in both oviparous (Fig. 3, A and B) and viviparous (Fig. 3, E

and F) females. IL-1R tI is also widely diffuse in most

epithelial cells in both oviparous (Fig. 3C) and viviparous

(Fig. 3G) females as well as in the endoderm of the allanto-

chorion. No significant differences in IL-1 or IL-1R tI were

observed among the stages. Negative controls show no

immunoreactivity (Fig. 3, D and H).

DISCUSSION

Squamate reptiles (i.e. lizards, snakes, and amphisbaenians)

are of particular interest for the study of the evolutionary

transition from oviparous to viviparous reproduction, since

viviparity has evolved far more often in Squamates (100

times) than in other vertebrate lineages (34 times) (Blackburn

1992, 1995). Moreover, Squamate reptiles offer several ideal

models for comparative studies, that is, very closely related

taxa (and even conspecific populations) with different repro-

ductive modes. Such models, including L. vivipara, improve

our understanding of the oviparous/viviparous transition be-

cause they minimize the confounding effect of phylogenetic

differences (Heulin et al. 1991; Qualls et al. 1995; Smith and

Shine 1997; Lobo and Espinoza 1999). This study used the

model of L. vivipara to investigate the role of cytokines,

namely IL-1, in the oviparity/viviparity transition. More spe-

cifically, the expression of the two IL-1 isoforms, IL-1a and

IL-1b, and their functional membrane receptor, IL-1R tI, was

investigated in uterine tissues from oviparous and viviparous

females of L. vivipara. The tissues were examined by immuno-

histochemistry using anti-human IL-1a, IL-1b, and IL-1R tI

antibodies at predetermined stages, including the time after

lethargy, vitellogenesis, and early and late gestation. We

showed that immunoreactivity for IL-1a, IL-1b, and IL-1R tI

was expressed in uterine tissues of both oviparous and vivi-

parous females of L. vivipara. Whereas IL-1a and IL-1b were

mainly distributed within the glands of the lamina propria,

IL-1R tI was also widely expressed by the luminal epithelial

cells. No significant differences were observed among the

stages nor between oviparous and viviparous females.

Fig. 1. Uterine tissues of Lacertavivipara stained with hematoxylinand eosin. (A): Oviparous femaleduring vitellogenesis. (B): Vivipa-rous female during gestation. m,myometrium; lamina propria withblood vessels (arrows) and glands(arrowheads); e, luminal epitheli-um; es, eggshell.

284 EVOLUTION & DEVELOPMENT Vol. 7, No. 4, July^August 2005

Previous studies have demonstrated the presence of the

IL-1 system in mammals with well-developed placental

structures, either invasive (of the hemochorial type) as in hu-

mans and mice (De et al. 1993; Simon et al. 1994) or non-

invasive (of the epitheliochorial type) as in pigs (Ross et al.

2003). The presence of the IL-1 system in reproductive tissues

of Squamates has also been documented for a lizard of the

family Scincidae, the three-toed skink C. chalcides (Paulesu

et al. 1995; Romagnoli et al. 2003), which exhibits a well-

developed epitheliochorial chorioallantoic placenta allowing

placentotrophy (Ghiara et al. 1987). The present study of L.

vivipara is the first evidence of the presence of the IL-1 system

not only in a less sophisticated type of viviparity (lecithotro-

phy) but also in oviparity. This suggests that uterine expres-

sion of the IL-1 system is not related to the complexity of

placental structures, nor even to the presence or absence of a

placenta. Indeed, it is worth noting that, in most oviparous

Squamates (including L. vivipara), an important phase of

embryonic development takes place in the mother’s oviduct,

before egg-laying (Shine 1983; Brana et al. 1991; Blackburn

Fig. 2. Interleukin-1 (IL-1) immu-noreactivity in uterine tissues ofoviparous and viviparous femalesof Lacerta vivipara at the pre-ovulatory stage. (A–D), time afterlethargy. IL-1a (A) and IL-1b (B)in oviparous females; type I IL-1receptor (IL-1R tI) (C) in vivipa-rous females. Strong immunoreac-tivity for IL-1a and IL-1bis present in the alveolar glands,whereas only a few cells of theluminal epithelium are positive.Immunoreactivity for IL-1R tI ispresent in glandular cells, as wellas being diffuse along the lumi-nal epithelium. (E–H): vitellogenicstage. IL-1a (E), IL-1b (F) in vi-viparous females; IL-1R tI (G) inoviparous females. Immunoreac-tivity for IL-1a, IL-1b, and IL-1RtI is present in the hypertrophicalveolar glands, which almostcompletely occupy the entirethickness of the lamina propria.Immunoreactivity for IL-1R tI isalso widely diffuse along the lumi-nal epithelium. Negative controls(D, H) do not show any staining.m, myometrium; g, glands; e, lumi-nal epithelium.

IL-1system in the oviparity and viviparity 285Paulesu et al.

1995; Andrews and Mathies 2000). This may explain why

immune mechanisms, including IL-1, are expressed in mater-

nal tissues in both oviparous and viviparous Squamates. It

should also be noted that the evolution of viviparity in Squa-

mates is always associated with a thinning (or loss in some

species) of the eggshell, thus facilitating the exchange of sub-

stances between the mother and the embryo (at least respi-

ratory gas and water and sometimes nutrients) (Blackburn

1993; Guillette 1993). For example, an eggshell membrane per-

sists throughout gestation in viviparous females of L. vivipara,

although it is thinner than in oviparous females of the same

species (Panigel 1956; Heulin 1990). Therefore, we can spec-

ulate that expression of the IL-1 system in uterine tissues of

oviparous and viviparous populations of L. vivipara is a sign

of the maternal immune response to fetal antigens independ-

ent of the timing or intimacy of contact between the maternal

and fetal tissues. It is known that IL-1 is heavily involved in

blastocyst adhesion and implantation in mice and humans.

This cytokine is expressed by the murine and human end-

ometrium during the peri-implantation period (De et al. 1993;

Fig. 3. Interleukin-1 (IL-1) immu-noreactivity in uterine tissues ofoviparous and viviparous femalesof Lacerta vivipara during gesta-tion. (A–D): Oviparous females atembryonic stage 32. (E, F): Vivi-parous females at embryonic stage31. (G): Viviparous female at em-bryonic stage 39. Immunoreactiv-ity for IL-1a, IL-1b (A, B, E, F),and type I IL-1 receptor (IL-1R tI)(C, G) is clearly present in theglandular cells of the lamina prop-ria; IL-1R tI is also strongly ex-pressed in the luminal epithelialcells as well as in the endoderm ofthe allantochorion. Negative con-trols (D, H) show no reactivity. m,myometrium; g, glands; e, luminalepithelium; es, eggshell; ca, chor-ioallantoic membrane.

286 EVOLUTION & DEVELOPMENT Vol. 7, No. 4, July^August 2005

Simon et al. 1993) and acts directly on the murine endo-

metrium (Simon et al. 1998). The IL-1 molecule and the IL-1

signalling system are evolutionarily conservative. In agree-

ment with our previous reports on C. chalcides (Romagnoli

et al. 2003), the present study demonstrates expression of the

IL-1 system since the pre-ovulatory stage. Together, these

findings indicate a role of IL-1 in the immunoregulatory

processes allowing establishment and maintenance of preg-

nancy even in species exhibiting embryo retention with or

without formation of a placenta. Expression of IL-1 has been

documented throughout gestation (Paulesu et al. 1995; Tuo et

al. 1996) as well as in term and preterm parturition (Steinborn

et al. 1996). On these bases, the present findings of expression

of IL-1 in L.vivipara at different times of gestation as well as

at the peri-ovulatory stage support that IL-1 is a mediator in

the cytokine network at the materno–fetal interface, playing

different roles throughout gestation.

The IL-1b gene has been cloned and sequenced in various

classes of vertebrates, including cartilaginous fishes, bony

fishes, amphibians, birds, and several species of mammals

(Bird et al. 2002). Unfortunately, the gene has not yet been

cloned in reptiles. However, the sequences obtained in various

species suggest the presence of an IL-1b gene in the ancestor

of all vertebrates (Bird et al. 2002). Moreover, several studies

show that IL-1b is secreted and acts in Squamate reptiles

(Dunlap and Church 1996; Mondal and Rai 2001, 2002).

Thus far, no sequence data are available for the IL-1a gene

except in mammals, whereas homologues of the mammalian

IL-1 type I receptor gene have been cloned in chicken (Guida

et al. 1992) and salmon (Subramaniam et al. 2002). IL-1

receptors show significant sequence similarities in their cyto-

plasmic domain to toll-like receptors that mediate dorso-ven-

tral polarity in Drosophila (O’Neill and Greene 1998; Akira

et al. 2001). This has led to the definition of a receptor su-

perfamily, the IL-1R/toll-like receptor. Further studies on

cloning and sequencing of IL-1 system genes in reptiles could

provide a definitive response concerning the role of IL-1 in the

evolution of viviparity.

AcknowledgmentsWe would like to thank Dr. Peter Christie for careful revision ofEnglish. This work was supported by research grants from theUniversity of Siena (PAR Projects 2003).

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