JOURNAL OF MORPHOLOGY 258:158 178 (2003)

Morphogenesis of Extraembryonic Membranes and

Placentation in Mabuya mabouya (Squamata, Scincidae)
Adriana Jerez1,2 and Martha Patricia Ramrez-Pinilla1*
1

Laboratorio de Biologa Reproductiva de Vertebrados, Escuela de Biologa, Universidad Industrial de Santander,

Bucaramanga, Colombia
2
Museo de Ciencias Naturales, Universidad Nacional de Salta, Salta, Argentina
ABSTRACT Topological and histological analyses of
Mabuya mabouya embryos at different developmental
stages showed an extraembryonic membrane sequence as
follows: a bilaminar omphalopleure and progressive mesodermal expansion around the whole yolk sac at gastrula
stages; mesodermal split and formation of an exocoelom in
the entire embryonic chamber at neurula stages; beginning of the expansion of the allantois into the exocoelom to
form a chorioallantoic membrane at pharyngula stages;
complete extension of the allantois into the exocoelom
between limb-bud to preparturition stages. Thus, a placental sequence could be enumerated: bilaminar yolk sac
placenta; chorioplacenta; allantoplacenta. All placentas
are highly specialized for nutrient absorption from early
developmental stages. The bistratied extraembryonic ectoderm possesses an external layer with cuboidal cells and
a microvillar surface around the whole yolk sac, which
absorbs uterine secretions during development of the bilaminar yolk sac placenta and chorioplacenta. During gastrulation, with mesodermal expansion a dorsal absorptive
plaque forms above the embryo and several smaller absorptive plaques develop antimesometrially. Both structures are similar histologically and are active in histotrophic transfer from gastrula stages until the end of
development. The dorsal absorptive plaque will constitute
the placentome and paraplacentome during allantoplacental development. At late gastrula early neurula stages
some absorptive plaques form chorionic concavities or chorionic bags that are penetrated by a long uterine fold and
seem to have a specialized histotrophic and/or metabolic
role. The extraembryonic mesoderm does not ingress into
the yolk sac and neither an isolated yolk mass nor a yolk
cleft are formed. This derived pattern of development may
be related to the drastic reduction of the egg size and
obligatory placentotrophy from early developmental
stages. Our results show new specialized placentotrophic
structures and a novel arrangement of extraembryonic
membrane morphogenesis for Squamata. J. Morphol. 258:
158 178, 2003. 2003 Wiley-Liss, Inc.
KEY WORDS: Mabuya mabouya; reptiles; placental development; extraembryonic membranes; matrotrophy

Neotropical species of the genus Mabuya are viviparous and exhibit a substantial degree of matrotrophy, which is attributed to the development of a
specialized type of chorioallantoic placenta designated as type IV allantoplacenta (Blackburn, 1993).
2003 WILEY-LISS, INC.

This type is the most specialized of the squamate

allantoplacentae known today (Blackburn, 1993,
1998). The morphology of the chorioallantoic placenta of these species has been described for two
Brazilian species, M. heathi (Blackburn et al., 1984)
and M. nigropunctata (bistriata, Avila-Pires, 1995)
(Vitt and Blackburn, 1991), for Mexican species
(Villagran-Santacruz et al., 1994), and Colombian
populations of M. mabouya (Jerez and RamrezPinilla, 2001). Although the general arrangement of
the uterine and extraembryonic elements of the placenta in the embryonic chamber are similar for all
the studied species and follows the type IV allantoplacenta of Blackburn (1993), in embryos at the nal
stages of development of Colombian populations ascribed as M. mabouya we found: 1) a complete extension of the allantoic vesicle in the exocoelom of
the embryonic chamber, as observed in M. heathi
and M. nigropunctata; 2) a bistratied chorionic epithelium regionally differentiated for nutritional
transfer and gas exchange; 3) a placentome developed especially in the preparturition stage; 4) a
paraplacentome at the mesometrial pole, delimited
and very active in histotrophic transfer of nutrients;
5) chorionic areolas only observed in the preparturition stage, receiving materials mainly from endometrial glands; and 6) a mixed allantoplacenta at
the abembryonic pole, functionally specialized in nutrient transfer (absorptive plaques) and gas exchange (segments similar to the type I allantoplacenta of Blackburn, 1993). Thus, there are several
placental specializations related to histotrophic
transport: at the mesometrial pole (placentome,
paraplacentome, and chorionic areolas), and at the
antimesometrial pole (absorptive plaques) (Jerez
and Ramrez-Pinilla, 2001).

*Correspondence to: Martha Patricia Ramrez-Pinilla, Escuela de

Biologa, Universidad Industrial de Santander, Bucaramanga, Colombia. E-mail: mpramir@uis.edu.co

DOI: 10.1002/jmor.10138

PLACENTAL DEVELOPMENT IN M. MABOUYA

The ontogeny of the extraembryonic membranes

and placentation is unknown for New World
Mabuya. Because features described for M.
mabouya show interesting specializations for placentotrophic nutrition, the study of their development and comparison with placental development in
related groups is of great importance. The present
study describes the ontogeny of the extraembryonic
membranes and the consequent succession of placental arrangements formed in the embryonic chambers of M. mabouya females, compares it with the
description for other species, and presents new morphological features related to histotrophic transfer
from the earliest embryonic development stages until the formation of the mature allantoplacenta.
MATERIALS AND METHODS
Mabuya mabouya pregnant females were obtained by manual
collection in the Inspeccion de Polica de Guadualito, Municipio
de Yacop, Cundinamarca, Colombia (0537N, 7418W, 840 m
altitude), and by loan of herpetological collections (Museo Colegio
San Jose, Instituto de Ciencias Naturales Universidad Nacional
de Colombia, and Universidad del Valle). The females collected
were killed by cardiac injection of anesthesia. All the females
were dissected and the gravid reproductive tracts were excised.
The incubation chambers were xed in Bouins solution (in the
museum females they were xed in 10% formalin). The tissues
were dehydrated in an ethanol series, embedded in paraplast,
and sectioned serially at 6 m. Sections were mounted on glass
slides coated with albumen and stained with hematoxylin and
eosin, Periodic acid-Schiff (PAS)-Alcian Blue (pH 1 and pH 2.5),
and Masson Trichromic (MT) methods (Luna, 1968).
The Dufaure and Hubert (1961) and Hubert (1985) developmental tables were followed for the determination of embryonic
stages. Staging was based on serial sections in early embryos and
on dissection of incubation chambers when possible. The terminology and placental categories follow Stewart and Blackburn
(1988) and Blackburn (1993). We used embryos from oviductal
eggs recently ovulated (in which no blastomeres were observed) to
stage 34 of development. Detailed histological descriptions were
made on collected specimens. Museum specimens were used to
compare and corroborate the topological descriptions. The photomicrographs were taken using an Olympus microscope. The
morphometric data (i.e., epithelial heights) were obtained with an
ocular micrometer on an Olympus microscope. In order to compare the topological and histological features of the Mabuya
mabouya placental sequence, we use mainly the description of
Blackburn and Callard (1997) for Chalcides chalcides since they
studied a similar series of developmental stages.

RESULTS
Oviductal Egg
Some females had oviductal eggs in which we did
not nd evidence of fertilization or the beginning of
segmentation (Fig. 1). They could correspond to recently ovulated and possibly fertilized eggs. These
oviductal eggs were not considered abortive because
they lacked of signs of degenerative processes. The
egg is surrounded by the shell membrane, a brous
acellular layer that is eosinophilic, stains pale blue
with Masson Trichrome (MT), and is positive for
acid mucoproteins (Fig. 2). It was 3.25 0.46 m
(n 8) thick and bilayered (the external leaf 1.5

159

0.5 m and the internal 1.8 0.28 m thick). The

ooplasm contains a lax brillar material and a few
small (3.75 0.88 m in diameter) pale yellow granules (with H&E stain). Both materials are dispersed
peripherally near the plasmatic membrane. The center of the ooplasm appears to be translucent. Yolk
platelets or yolk granules were not observed (Figs. 1,
2).
The uterus is slightly distended and the endometrium is folded. The folds are high at the antimesometrial and low at the mesometrial regions of the
incubation chamber. Laterally, there are no folds
(Fig. 1). The luminal uterine epithelium is columnar
(17.9 3.1 m, n 10). Epithelial cells have a
round and prominent nucleus, nondiscernible nucleolus, and a homogeneous eosinophilic cytoplasm. A
few cells possess short cilia. The lamina propria is
well vascularized and contains several open glands
with tubular channels toward the uterine lumen.
Some of the channels contain amorphous material
that stains pale eosinophilic and positive for acid
mucosubstances. The glandular lumen is empty.
Seemingly, the glands had secreted previously. The
myometrium is uniformly thick (28 m).
Early Gastrula Stage
The extraembryonic ectoderm and endoderm have
been developed and completely surround the egg.
Dorsally, this ectoderm is formed by rounded to
cuboidal cells (Fig. 3), while laterally and ventrally
the cells are squamous. The endodermal cells are
large, rounded, with large nuclei and two nucleoli
(Figs. 4, 5). Their cytoplasm presents abundant yellow irregular and heterogeneous granules (2 4 m
diameter, H&E stain). Within the yolk sac there is
nely granular material and a few granules identical to those in the cytoplasm of the endodermal cells.
Both are located peripherally near the cells of the
omphalopleure. The center of the sac appears to be
empty.
The egg is enclosed by a shell membrane that is
continuous laterally and ventrally, whereas at the
embryonic pole, immediately above the embryonic
disk, it has begun a breakup process evidenced by
the presence of isolated and broken bers or its
absence above the embryonic disk (Figs. 35).
The uterine mucosa possesses deep, strongly vascularized folds toward the mesometrial side in front
of the embryonic disc, while toward the abembryonic
pole the folds are very low or absent (Figs. 3 6). The
mucosa is glandular, with obliterated glands in the
embryonic pole, whereas in the ventral and lateral
sides of the chamber the glands have eosinophilic
material in their lumen. The mucosa has subepithelial blood vessels that are observed especially in the
folds of the embryonic pole. The uterine luminal
epithelium in the embryonic chambers is high columnar (25.8 4.08 m, n 5) with an irregular
apex (Fig. 5). The basophilic nuclei are located at

Fig. 1. Oviductal egg of Mabuya mabouya. e, egg; o, ovoplasm; s, shell membrane; u, uterus. Scale bar 140 m.
Fig. 2. Oviductal egg of Mabuya mabouya, detail of egg periphery. e, egg content; eu, uterine epithelium; l, uterine lumen; s,
bilayered shell membrane. Scale bar 7 m.
Fig. 3. Mesometrial hemisphere at early gastrulation of Mabuya mabouya. ec, ectoderm; em, embryo; en, endodermal cells; l,
uterine lumen; ue, uterine epithelium. Scale bar 40 m.
Fig. 4. Abembryonic hemisphere of Mabuya mabouya embryos at early gastrula stage. en, endodermal layer; l, uterine lumen; s,
shell membrane; sq, squamous ectoderm; ue, uterine epithelium. Scale bar 10 m.

Fig. 5. Abembryonic hemisphere of Mabuya mabouya embryos at early gastrula stage. ec, squamous ectoderm; en, endodermal
cells; l, uterine lumen; s, shell membrane; ue, uterine epithelium. Scale bar 7 m.
Fig. 6. Diagram of the embryonic chamber of Mabuya mabouya at early gastrula stage of development. A bilaminar omphalopleure
(bo) extends around the entire egg. em, embryo; l, uterine lumen; u, uterus; ys, yolk sac.
Fig. 7. Embryonic hemisphere of embryos of Mabuya mabouya at gastrulation. em, embryo; dap, dorsal absorptive plaque; ms,
extraembryonic mesoderm. Scale bar 30 m.
Fig. 8. Embryonic hemisphere of Mabuya mabouya embryos at gastrulation. Detail of the dorsal absorptive plaque. c, binucleated
columnar cells; i, interstitial cell; ms, mesoderm; s, basal squamous ectodermal layer. Scale bar 10 m.

Fig. 9. Abembryonic hemisphere of Mabuya mabouya embryos at gastrula stage. bec, bilayered ectoderm; c, cuboidal cells of
bilayered ectoderm; e, endodermal cells; eu, uterine epithelium; g, cytoplasmic granules of the endodermal cells; l, uterine lumen. a:
General view. b: Detail. Scale bar in a 25 m, b 7 m.
Fig. 10. Absorptive plaque in the abembryonic hemisphere of Mabuya mabouya embryos at gastrula stages. bc, binucleated cell; c,
columnar cells; e, endodermal cells; i, internal squamous layer and mesoderm; ic, interstitial cell; l, uterine lumen. a: At early gastrula
stages the columnar cells are mainly mononucleated and few interstitial cells are observed. Scale bar 12 m. b: At advanced gastrula
stages the columnar cells are binucleated and the interstitial cells are evident. Scale bar 15 m.
Fig. 11. Diagram of Mabuya mabouya embryonic chamber at gastrula stage. A bilaminar omphalopleure is formed at the
abembryonic hemisphere and the mesoderm is expanding at the embryonic hemisphere. ap, absorptive plaque; bo, bilaminar
omphalopleura; dap, dorsal absorptive plaque; em, embryo; l, uterine lumen; m, mesoderm at embryonic hemisphere; u, uterus; ys,
yolk sac content.

PLACENTAL DEVELOPMENT IN M. MABOUYA

different levels of the epithelium and contain two

nucleoli. The uterine lumen at the mother/embryo
interface contains material of the same nature as
that of the cellular apex of the luminal epithelium.
In this stage a bilaminar yolk sac placenta is
found, formed by the apposition of the bilaminar
omphalopleure, consisting of extraembryonic ectoderm and endoderm, and the uterine epithelium
(Figs. 5, 6).

Gastrula Stage
The extraembryonic ectoderm is bilayered around
the whole egg. A thin internal layer of squamous
cells is separated from the endodermal cells by a
thin basal lamina, because the layers of the bilaminar omphalopleure are initially in intimate apposition. The external layer, which faces the uterine
epithelium, differs at the embryonic and abembryonic poles. Dorsal to the embryo, this layer forms a
long zone of hypertrophied cells, the dorsal absorptive plaque (Fig. 7) that is composed of two types of
cells. High columnar cells (33.8 4.1 m, n 10)
with one or two nuclei have lightly eosinophilic cytoplasm and short microvilli (Fig. 8). In one embryo,
the microvilli were not evident because the apex of
these cells was irregular and an abundance of eosinophilic material adhered to the cells. A second type
of cells, interstitial cells, are high columnar and very
thin, with ovoid nuclei, and their cytoplasm is almost imperceptible (Fig. 8). Some of these cells are
binucleate. Laterally and ventrally, at the abembryonic pole, the cells of the ectodermal external layer
are cuboidal to squamous with large round and basophilic nuclei and a single prominent nucleolus
(Fig. 9). Some of these cells have microvillar
apexes. In some regions these cells penetrate
among the uterine folds and have an irregular
form. In some embryos the formation of a rst
abembryonic absorptive plaque appears as a short
zone with eosinophilic columnar cells (14 1.54
m, n 6), bearing microvilli (Fig. 10a), some of
them binucleated with basophilic nuclei and a notable basophilic nucleolus. Interstitial cells are
dispersed among them. At the embryonic pole the
mesoderm is a very thin layer of avascular mesenchyme, composed of cells with small ovoid nuclei. In some gastrulas it is not extended beyond or
lateral to the embryonic disk (Fig. 7), whereas in
more advanced gastrulas it has extended laterally
toward the abembryonic pole, where it participates in formation of the absorptive plaques (Fig.
10b). In the latter embryos the dorsal mesoderm
begins to delaminate. A very thin layer adheres to
the extraembryonic ectoderm (Fig. 8) and a similar layer adheres to the yolk sac endoderm. Laterally and ventrally the mesoderm is not delaminated and it does not extend near to the ventral
pole.

163

The extraembryonic endodermal cells are large,

globoid, with basophilic round nuclei, two nucleoli,
and a cytoplasm with yellow pale granules of different sizes (Fig. 9a,b). The endoderm is peripheral to
the yolk sac, whose content seems empty. Only a
few, pale yellow, very small granules can be observed dispersed peripherally.
The endometrium is folded. Dorsally the folds are
long, whereas laterally and ventrally they are short
or are absent. The underlying connective tissue is
highly vascularized and contains glands with
opened tubular channels. Some glands have eosinophilic material in their lumen. The muscular layers
are thick dorsally, whereas laterally and ventrally
they are thin. The luminal uterine epithelium is
columnar (19.25 2.8 m, n 8), with notable
round and basophilic nuclei located in the mid to
basal cellular zone. Some cells are binucleate. Cellular apexes are irregular and the cells appear to
secrete eosinophilic material in the form of conspicuous vesicles and a nely granular matrix into the
uterine lumen, where abundant, nely granular, eosinophilic material of a condensed and vesicular appearance was observed. The shell membrane is only
represented in some embryos by a few amorphous
residues toward the ventral side of the abembryonic
pole.
At this stage we observed a nonvascularized trilaminar omphalopleure that extends from the mesometrial to the lateral and ventral zones of the
embryonic chamber. The formation of the chorioplacenta in the dorsal region above the embryo is evident because the split of the mesoderm has been
initiated (Fig. 11). A bilaminar yolk sac placenta is
present in the ventral hemisphere where the mesoderm has not extended.
Late Gastrula StageEarly Neurula Stage
The mesodermal delamination progressively
forms the chorion. The external leaf of the ectoderm
in the dorsal absorptive plaque is higher (99.7
4.59 m, n 10), with columnar and interstitial
cells (Fig. 12). Lateral and ventral to it the chorion
varies, forming several absorptive plaques of different sizes (Fig. 13). The epithelium of these regions
contains the same two types of epithelial cells that
occur in the dorsal absorptive plaque, but the height
of the epithelium is lower (68.9 10.94 m, n 11,
Fig. 16). The columnar cells are binucleated with
globoid, slightly basophilic nuclei and a strongly basophilic nucleolus. The cytoplasm is slightly eosinophilic and the cellular apices have conspicuous microvilli. The very thin interstitial cells are dispersed
among the columnar cells.
The chorion separating the absorptive plaques
forms large segments in which the external leaf of
the ectoderm is cuboidal to squamous. The cuboidal
cells of this layer have an eosinophilic cytoplasm,
basophilic nuclei, and a prominent nucleolus. Abun-

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A. JEREZ AND M.P. RAMIREZ-PINILLA

Figures 1215

PLACENTAL DEVELOPMENT IN M. MABOUYA

dant material adhering to the cellular apex prevents

the detection of microvilli in these cells. Some cells
show yellow cytoplasmic granules that confer a
swollen appearance to the cell.
Several large absorptive plaques (12 per incubatory chamber), lateral and ventrolateral to the embryonic pole, are unusual in forming invaginations
terminating in large concavities (165 m internal
wide and 130 m deep) that in some cases have
closed their folds to form bags (Fig. 14). The concavities have columnar cells that are deformed by the
presence of cytoplasmic crystalloid yellow granules
and very ne yellow material is present in the cavity
lumen. The bags (475 m wide, 162.5 m deep) have
an external membrane (formed by the closed chorionic folds) that faces the uterus and an internal
membrane (the bottom of the bag) that faces the
exocoelom. Both are continuous with the rest of the
chorion, have a bistratied ectoderm, and a mesoderm continuous with the somatopleure of the entire
embryonic chamber (Fig. 14). The ectodermal external layer that faces the endometrium has cuboidal
cells (Fig. 15), whereas in the cavity of the bag
(internal layer) the epithelium consists of columnar
cells, mainly binucleated, with basophilic nuclei and
a strongly basophilic nucleolus. These cells have an
eosinophilic cytoplasm, but many of them are deformed by the presence of large, yellow cytoplasmic
granules. The interstitial cells are rarely observed.
There is also a nely granular yellow material in the
lumen of the bags. Both concavities and bags are
apposed to a long uterine fold. In the bags the long
uterine fold penetrates into its lumen and the ectodermal cells appear to be strongly deformed. Although the cytoplasmic yellow granules are mostly
observed inside the ectodermal cells of the chorionic

165

bags, they can also be seen dispersed in the rest of

the chorionic cells of the embryonic chamber.
The extraembryonic mesoderm is developed
around the whole embryonic chamber without penetrating the yolk sac. The mesoderm is delaminated
in the dorsal and lateral regions, while the ventral
pole is covered by a nonvascularized trilaminar
omphalopleure (Fig. 17). A small vessel that penetrates into the yolk sac is present next to the embryo. In some embryos the mesoderm associated
with the yolk sac contains a small vessel (Fig. 12).
When the mesodermal split is completed the yolk
sac is splanchnopleuric (Figs. 16, 17). The endodermal cells and the content of the yolk sac are similar
to previous stages.
The uterine mucosa has folds dorsally, whereas
lateral and ventrally they are shorter; however, the
endometrium forms a long fold where it is apposed
to each chorionic concavity or chorionic bag. The
luminal epithelium is columnar (27 7.07 m, n
5). The cells have slightly basophilic, round nuclei
with a small nucleolus and an irregular apex with
adhered eosinophilic material. A few cells contain
yellow granules in their cytoplasm. The lamina propria is vascularized with open glands containing
eosinophilic material (Fig. 13). In the uterine lumen,
a few yellow granules and nely granular eosinophilic material were observed. No remnants of the
shell membrane were observed.
A chorioplacenta is formed by the apposition of the
uterine mucose to the chorion. This chorioplacenta is
highly specialized, forming absorptive structures in
both hemispheres (dorsal absorptive plaque at the
embryonic hemisphere and absorptive plaques, chorionic concavities, and chorionic bags at the abembryonic hemisphere).
Late Neurula Stage

Fig. 12. Dorsal absorptive plaque during late gastrula early

neurula stages of Mabuya mabouya. dap, dorsal absorptive
plaque; l, luminal uterine material; ue, uterine epithelium; v,
blood vessel of vascularized mesoderm at the embryonic hemisphere. Scale bar 40 m.
Fig. 13. Absorptive plaque at the abembryonic hemisphere
during late gastrula early neurula stages of Mabuya mabouya.
ap, absorptive plaque; c, binucleated columnar cells; i, interstitial
cells; l, uterine lumen; ue, uterine epithelium, ug, uterine gland.
Scale bar 50 m.
Fig. 14. Chorionic concavity and chorionic bag at the abembryonic pole during late gastrula early neurula stages of Mabuya
mabouya. ap, absorptive plaque; b, chorionic bag; cc, chorionic
concavity; ch, chorion; ex, exocoelom; l, uterine lumen; u, uterus.
The inset is shown in Figure 15. Scale bar 90 m.
Fig. 15. Detail of Figure 14 showing the uterine and chorionic
epithelia in the chorionic bag at the abembryonic pole during late
gastrula early neurula stages of Mabuya mabouya. c, disorganized cuboidal chorionic cells of the bag external fold; ue, uterine
epithelium; the arrow points out crystalloid material transferred
between the epithelia. Scale bar 10 m.

The exocoelom is continuous at both poles because

the extraembryonic mesoderm is delaminated completely to form the chorion, by association between
the extraembryonic somatic mesoderm and the ectoderm, and the yolk sac splachnopleure, by association between the extraembryonic splanchnic mesoderm and the yolk sac endoderm (Figs. 18, 19).
The chorionic bistratied ectoderm has the same
general features as in the previous stage. The dorsal
absorptive plaque, above the embryo, is wide and
the high columnar cells show dense microvillar surfaces. In the abembryonic hemisphere the chorion
forms several absorptive plaques with the same cellular composition of the dorsal absorptive plaque
(Fig. 18). In a sagittal section of the incubation
chamber we observed between 8 and 10 of these
plaques. The chorionic segments among the absorptive plaques have a squamous to cuboidal epithelium. The chorion as a rule exhibits amorphous material adhered to the cellular apex. Some of the
chorionic cells of the external leaf phagocytose lumi-

Fig. 16. Absorptive plaque at the abembryonic pole during late gastrula early neurula stages of Mabuya mabouya. ap, absorptive
plaque; en, vascularized endoderm; m, delaminating mesoderm; v, endoderm vascularization; ys, yolk sac. Scale bar 15 m.
Fig. 17. Short zone at the abembryonic pole where the mesodermal delamination is complete during late gastrula early neurula
stages of Mabuya mabouya. ap, absorptive plaque; ch, chorion; en, yolk sac endoderm; l, uterine lumen; ms, delaminating mesoderm;
u, uterus; ys, yolk sac. Scale bar 85 m.
Fig. 18. Diagram of an embryonic chamber at late neurula stage of Mabuya mabouya. Chorioplacenta. The extraembryonic
mesoderm is delaminated completely, forming the splanchnopleure and the somatopleure. ap, absorptive plaque; dap, dorsal absorptive plaque; em, embryo; ex, exocoelom; l, uterine lumen; spv, splanchnopleuric blood vessel; u, uterus; ys, yolk sac.

PLACENTAL DEVELOPMENT IN M. MABOUYA

167

Fig. 19. Embryonic pole of Mabuya mabouya embryos at late neurula stage. bv, blood lacunae in the yolk sac splanchnopleure; dap,
dorsal absorptive plaque; em, embryo; ex, exocoelom; l, uterine lumen; sm, somatic mesoderm; spm, splanchnopleuric mesoderm; u,
uterus; ys, yolk sac. Scale bar 100 m.
Fig. 20. Abembryonic pole of Mabuya mabouya embryos at late neurula stage. ap, absorptive plaque; ex, exocoelom; l, uterine
lumen; sp, splanchnopleure; u, uterus; ys, yolk sac. Scale bar 90 m.
Fig. 21. Diagram of the embryonic chamber at pharyngula stage of Mabuya mabouya. The allantois is expanding into the exocoelom
at the embryonic hemisphere. a, allantois; ap, absorptive plaque; dap, dorsal absorptive plaque; em, embryo; ex, exocoelom; l, uterine
lumen; u, uterus; ys, yolk sac.

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A. JEREZ AND M.P. RAMIREZ-PINILLA

Figures 2225

PLACENTAL DEVELOPMENT IN M. MABOUYA

nal uterine granules and other cells exhibit granules

within their cytoplasm. Macroscopically, the dorsal
absorptive plaque is a white broad zone of thick
appearance, while the rest of the embryonic chamber is translucent and presents many dispersed
white patches of different sizes and forms. These
patches correspond to the absorptive plaques, and
the translucent areas of the embryonic chamber, in
which the patches are dispersed, correspond to the
chorionic attened luminal epithelial segments described above.
The vascularized extraembryonic splanchnic mesoderm is a thin membrane that surrounds the yolk
sac completely (Fig. 19). The mesoderm contains a
great number of blood vessels that in some regions
form wide lacunae containing blood cells. We did not
observe any type of isolated yolk mass, yolk cleft, or
intravitelline cells. Instead, the vascularized
splanchnic mesoderm is continuous around the yolk
sac (Fig. 20). The endodermal cells are observed
peripherally and they have the same morphology as
those of the previous stages. However, some
endodermal cells show eosinophilic borders and cytoplasm of lamentous appearance and a few yellow
granules. The interior of the yolk sac is almost empty; it exhibits dispersed granules and amorphous
material near the endodermal cells among a nely
granular matrix.
The endometrium possesses some long vascularized folds that are at at the apex. The lamina
propria is well vascularized and contains many large
active glands in the region of transition between the
interembryonic spaces and the embryonic chamber.
The luminal epithelium is low columnar. The cells
have an apical eosinophilic cytoplasm and basally
located basophilic round nuclei. Some cells contain
apical granules. The apical border of these cells is

Fig. 22. Allantoic vesicle in Mabuya mabouya embryos at

pharyngula stage. a, allantois; ap, absorptive plaques; dap, dorsal
absorptive plaque; em, embryo; ex, exocoelom; ys, yolk sac; u,
uterus. Scale bar 220 m.
Fig. 23. Chorioallantois at the embryonic hemisphere during
pharyngula stages of Mabuya mabouya. a, allantois; av, allantoic
blood vessels; dap, dorsal absorptive plaque; l, uterine lumen; u,
uterus. Scale bar 80 m.
Fig. 24. Embryonic hemisphere during early pharyngula
stage of Mabuya mabouya. a, allantois; dap, dorsal absorptive
plaque; cc, chorionic concavity; uf, uterine fold penetrating the
cavity. Scale bar 65 m.
Fig. 25. Endodermal cells of Mabuya mabouya embryos. bv.
splanchnopleuric blood vessel; en, endodermal cells; spm,
splanchnopleuric mesoderm; ys, yolk sac. a: Endodermal cells
with lightly cytoplasm containing eosinophilic granules and dispersed amorphous yellow granules. Scale bar 10 m. b:
Endodermal cells with empty apical cytoplasm during pharyngula stage of Mabuya mabouya. Scale bar 7 m.

169

irregular and the cells appear to be secreting eosinophilic material into the uterine lumen. This material is pale blue and red when observed in stained
MT slides. The muscular layer and the conjunctive
are quite thinned. The conjunctive is vascularized
and presents a greater quantity of glands at the
abembryonic pole. However, the glands are active
and are widely distributed, and the lumen of some of
these contains amorphous eosinophilic and nely
granular materials. With MT stain they are red and
light blue, respectively.
Eosinophilic granules of different sizes are
present in the uterine lumen. They are of two types,
exhibiting nely granular or brous consistencies.
The nely granular material is red with MT stain
and is observed protruding in the form of large vesicles from the luminal uterine cells. Other small
granules are light blue. Fibrillar amorphous material is mainly observed adhered to the chorionic
cells. This material is more abundant in the transition zone to the interembryonic space.
At these developmental stages, an active chorioplacenta is maintained. It is formed by the chorion
that is regionally specialized in absorptive processes, in apposition to the secretory uterine mucosa.

Pharyngula Stage
The amnion is a very thin membrane surrounding
the embryo. It is composed of at cells with round to
ovoid nuclei. The allantois has invaded the dorsal
and lateral exocoelom under the chorionic tissue,
vascularizing it (Figs. 2123). In early pharyngulas,
the allantois extends from the embryo like a bag of
at cells containing blood cells. Toward the end of
these stages the allantoic vesicle extends dorsally
and laterally. The external allantoic membrane adheres to the chorionic mesoderm, vascularizing it, to
form the chorioallantois (Figs. 2123). Dorsally, the
external membrane is a wide net (Fig. 23), while
laterally it is a very thin membrane. The internal
allantoic membrane is also thin. It adheres to the
amnion in the embryonic hemisphere surrounding
the embryo and covers the dorsal region of the yolk
sac (Fig. 22). The allantoic vesicle does not occupy
the entire exocoelom because there is a ventrolateral
zone without vascularization in the abembryonic
hemisphere.
The chorion in the dorsal absorptive plaque is
composed of the same cells described for the previous stage (Figs. 22, 23). The columnar cells are very
high (89.11 10.37 m, n 9). This dorsal absorptive plaque, which originated in the gastrula stage,
corresponds to the chorionic contribution of the future placentome found in late developmental stages
and is peripherally delimited by a short fold in
which the external chorionic cells are polyhedral. In

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A. JEREZ AND M.P. RAMIREZ-PINILLA

front of this chorionic fold, the uterine mucosa has

low folds. A chorionic concavity was observed near
the dorsal absorptive plaque in one embryo (Fig. 24).
Laterally and ventrally, the external layer of the
ectoderm exhibits squamous to cuboidal cells intercalated with the chorionic absorptive plaques (epithelial height 56.22 14.39 m, n 9), which
structurally are similar to the dorsal chorionic epithelium although of smaller size.
The yolk sac is highly vascularized by the splanchnic mesoderm; it presents small invaginations and
small lobules. The endodermal cells have similar
features to previous stages and contain a very ne
brillar eosinophilic cytoplasm (pale red with MT
stain) and small yellow granules (Fig. 25a). Some of
these cells appear to be swollen by the abundant
eosinophilic material toward the lumen of the yolk
sac, and others seem to have emptied their secretory
content (Fig. 25b). There is material similar to that
found in the cytoplasm of the endodermal cells scattered in the cavity of the sac.
The uterine mucosa presents small folds toward
the dorsal side, while laterally it has large folds.
This latter region connects with the adjacent interembryonic spaces. Ventrally, the embryonic
chamber has very short folds. The vascularized conjunctive has active glands with eosinophilic (pale
blue and red with MT stain) nely granular and
compact amorphous material in their lumen. Some
glands are open but without luminal or cellular secretions. The uterine epithelium is low columnar
(height 18.5 3.62 m, n 10). The epithelial cells
have an irregular apex and appear to be secretory,
sending eosinophilic (red to purple with MT stain)
nely granular material into the uterine lumen. In
the embryonic polar zone there is a short region
where the epithelial cells are lower than in the rest
of the epithelium. This zone exhibits conspicuous
microvilli and the lateral membranes between cells
are indistinguishable. Large, round, and faint nuclei
with a strongly basophilic nucleolus are observed
inside a lightly eosinophilic cytoplasm (Fig. 26). This
region has low folds and is located in front of the
dorsal absorptive plaque. The chorionic plaque is
longer than this syncytial uterine zone.
There is an abundance of amorphous material and
granules of different sizes in the uterine lumen. This
material is similar to that observed in the uterine
luminal epithelial cells and occurs freely in the uterine lumen or adhered to the chorionic cells.
At this stage of development there is a chorioallantoic placenta, characterized by the formation
of the chorioallantoic membrane in apposition to the
uterine epithelium. The chorioallantois is extended
mesometrially and toward the lateral parts of the
embryo. Dorsally, the future placentome begins to
be formed from the dorsal absorptive plaque. Ventrally, the chorioplacenta is maintained.

Limb-Bud Development Stage (Stages 30 to

34 D&H)
The allantois is extended ventrally within the exocoelom, although it is not joined to the chorion in the
ventral region, leaving a small medial zone without
vascularization (Fig. 27). The external membrane of
the allantois is broadened greatly toward the embryonic pole, while laterally it is a thin membrane (as is
the internal membrane) (Fig. 27). The allantoic vesicle contains amorphous material in its interior.
The chorioallantoic membrane is extended from
the embryonic to the abembryonic hemisphere but
not completely in the polar region. In the mesometrial pole the chorioallantois of the future placentome forms small invaginations. The dorsal absorptive plaque is sharply delimited by a short, wide fold
formed by the binucleated cells that are amorphous
and strongly reduced in size in this zone (Fig. 28). In
the dorsal absorptive plaque the external layer of
the ectodermal epithelium is constituted by the columnar and interstitial cells previously described.
The epithelial height is slightly lower than in preceding stages (70.83 7.06 m, n 12). Laterally
and ventrally, the chorionic cells of the external
layer vary, forming alternating segments of squamous to cuboidal cells, intercalated with shorter segments of columnar and interstitial cells (height
52.30 4.96 m, n 10). These latter structures
constitute the absorptive plaques (Fig. 27).
The lobulated yolk sac is thoroughly vascularized
by the splanchnopleure (Fig. 27). The endodermal
cells contain cytoplasmic amorphous material and
eosinophilic granules of a similar nature to the few
observed in the periphery of the sac. Some cells
exhibit supercial material toward the lumen of the
yolk sac, as if they were secreting or absorbing these
materials to or from the interior of the sac. Other
cells at the ventral region of the yolk sac contain
yellow granules similar to those present in earlier
stages.
The uterine mucosa presents some lower folds at
the dorsal pole. The myometrium is thinned by the
growing embryo. The conjunctive possesses active
glands throughout and the glandular lumen contains eosinophilic material similar to that found in
the uterine lumen. The luminal epithelium is syncytial in a greater zone of the dorsal pole than in the
previous stage. Here, the uterine mucosa forms
small folds (Fig. 29). The epithelial height in the
syncytial region is low; the rounded nuclei are large
and have strongly basophilic nucleoli (Fig. 29).
These cells in front of the dorsal absorptive plaque
have a distinctive microvillar surface. Laterally, at
the embryonic hemisphere, the luminal epithelium
is low columnar (20.54 3.35 m, n 11). At the
lateral and abembryonic regions the epithelium varies. It is low columnar (17.54 4.63 m, n 11),
forming the absorptive plaques, and cuboidal to
squamous (epithelial height 8.6 1.34 m, n 10)

Fig. 26. Embryonic hemisphere of embryos at pharyngula stages. av, allantoic vessel; dap, dorsal absorptive plaque; l, uterine
lumen; ue, uterine epithelium forming a syncytium at the dorsal pole. Scale bar 25 m.
Fig. 27. The allantois occupies the exocoelom during the limb-bud stage of Mabuya mabouya. a, allantois; ap, absorptive plaques;
ex, exocoelom; l, uterine lumen; u, uterus; v, blood vessels in the splanchnopleure; ys, yolk sac. Scale bar 100 m.
Fig. 28. Embryonic hemisphere of embryos at limb-bud stage of Mabuya mabouya. a, allantois; dap, dorsal absorptive plaque; chf,
chorionic fold delimiting the chorionic paraplacentomal zone from the rest of the embryonic chamber; l, uterine lumen; u, uterus. Scale
bar 100 m.
Fig. 29. Uterine luminal epithelium at the embryonic polar zone observed in limb-bud stage embryos. a, allantois; c, binucleated
columnar cells of the dorsal absorptive plaque; s, syncytial region of the luminal uterine epithelium; uv, uterine vascularization. Scale
bar 35 m.

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A. JEREZ AND M.P. RAMIREZ-PINILLA

in the segments among them. The luminal surface of

the epithelial cells of the entire incubation chamber
shows irregular apexes and appears to be secreting
to the uterine lumen, where amorphous and granular material of different sizes has accumulated. This
material stains mainly eosinophilic (red-purple with
MT).
At this stage the chorioallantoic placenta is
formed by the apposition of the chorioallantois to the
uterine epithelium. The chorioallantoic placenta is
extended almost entirely in the embryonic chamber.
There is a gap ventrally where the lateral extensions
of the allantois have not converged and where they
do not vascularize the chorion completely. Toward
the embryonic side the placentome and the paraplacentome are forming from the dorsal absorptive
plaque.
DISCUSSION
The ontogeny of the extraembryonic membranes
determines the sequence of placental development
and both the spatial characteristics of placentation
at specic developmental stages, as well as the nal
topology of placentation (Stewart and Thompson,
2000). In Mabuya mabouya we found an extraembryonic membrane development that determines the
following sequence of placental arrangements: 1) bilaminar yolk sac placenta in early gastrulas; 2) trilaminar nonvascularized omphalopleure at the embryonic hemisphere and bilaminar yolk sac placenta
at the abembryonic hemisphere, during gastrulation; 3) a brief, transitory vascularized trilaminar
omphalopleure that appears almost simultaneously
with mesodermal delamination; 4) chorioplacenta,
from early neurula to pharyngula stages; and 5)
allantoplacenta, from pharyngula to parturition. All
placentas are highly specialized for nutrient absorption from early developmental stages. Uterine secretions come from the endometrial tubular glands and
from the luminal epithelium, both from the incubatory chamber and from interembryonic uterine segments. During bilaminar yolk sac placenta and chorioplacenta arrangements, the uterine luminal
epithelium is secretory and not regionally differentiated, whereas it shows regional differentiation late
in development, during allantoplacenta development.
Shell Membrane
The shell membrane of Mabuya mabouya is thin
(3.25 m), double, and composed of highly sulfated
acid mucosubstances and brous proteins. The presence of a shell membrane has been observed in viviparous squamates as a brous acellular leaf of
variable thickness composed of two or three thin
distinct layers (Blackburn, 1998). For example, it
was observed in several species by Weekes (1929,
1930, 1935); in Xantusia vigilis (Yaron, 1977), in

Elgaria coerulea (Stewart, 1985), in Sceloporus aeneus (a simple membrane average of 6.2 m thick,
Guillette and Jones, 1985), in Chalcides chalcides
(4 10 m thick, Blackburn and Callard, 1997), and
in Chalcides ocellatus tiligugu (an inner shell membrane 4 m thick and an outer brous layer 11 m
thick, Corso et al., 2000). In the snake Thamnophis
sirtalis, Hoffman (1970) found that it is composed of
keratin with a low percentage of sulfur and is bilayered (12 m thick). The eggshell of Chalcides ocellatus tiligugu (Corso et al., 2000) does not have an
outer calcareous layer and is composed of protein
bers (collagen-like material) secreted by the uterine glands, immersed in an interbrillar matrix of
acidic glycoproteins and an innermost amorphous
layer of sulfated glycosaminoglycans, both secreted
by two different types of epithelial cells of the uterine luminal epithelium.
The shell membrane of Mabuya mabouya is secreted during segmentation by the oviductal glands
and is lost in the gastrula stages, rst in the embryonic hemisphere, and nally in the abembryonic
hemisphere, where a few remnants persist in late
gastrulas. By neurula stages, remnants are not
found in either of the two hemispheres. The length
of the time in which the shell membrane surrounds
the embryo varies in viviparous lizards. Stewart and
Thompson (1994) found that in Niveoscincus metallicus remnants are observed until stage 30 32,
while in N. coventryi by stage 30 it was not detected
(Stewart and Thompson, 1998). In Pseudomoia spenceri in stage 30 embryos, a shell membrane is visible
about the perimeter of the egg, and at stage 33, a
remnant of it is observed at the abembryonic pole
(Stewart and Thompson, 1998). Remnants of the
shell membrane of Chalcides ocellatus tiligugu
(Corso et al., 1994) occur in the embryonic hemisphere of embryos at stages 3536, whereas in C.
chalcides it is lost rst in the embryonic hemisphere
toward the late gastrula stage, but remnants of it
are maintained in the abembryonic pole until stage
30 (Blackburn and Callard, 1997). In Eumecia anchietae there is no evidence of a shell membrane at
stage 28 (Flemming and Branch, 2001) and in M.
nigropuctata and M. heathi it is lost by midgestation (Blackburn et al., 1984; Vitt and Blackburn, 1991). Thus, for New World Mabuya and C.
chalcides the shell membrane is entirely lost at the
mesometrial pole very early in development (Blackburn, 1998). The shell degradation process appears
to be faster in these species and in E. anchietae than
in the other matrotrophic species. Possibly this accelerated process in M. mabuya is related to the
beginning of the hypertrophy of the extraembryonic
ectoderm in the dorsal pole during early gastrulation because the shell membrane begins to degenerate just above this zone. We hypothesize that the
hypertrophied extraembryonic ectoderm cells could
play an inductor role in the degradation of the shell,
facilitating the beginning of the histotrophic nutri-

PLACENTAL DEVELOPMENT IN M. MABOUYA

ent transfer from these early stages of development.

Weekes (1927) also suggested that the ectoderm
cells would have this function for viviparous lizards.
The degradation of the shell membrane also has
been related to a possible mechanical effect for the
increasing size of the egg (Weekes, 1927; Blackburn
and Callard, 1997). For C. chalcides, Blackburn and
Callard (1997) suggested that differential degradation (rst in the embryonic hemisphere) possibly is a
consequence of a differential action of uterine proteases. Blackburn (1998) summarizes the possible
mechanisms by which the shell membrane may degenerate: 1) swelling of the oviductal egg; 2) the
chorion or the local uterine epithelium assists in the
breakdown of the shell membrane in the embryonic
pole; and/or 3) action of the myometrium, in association with extrusion of the eggs down the oviduct.
There are no data to support any of these hypotheses.
Yolk Sac
The development of the yolk sac proceeds in four
stages in reptiles. The bilaminar omphalopleureforms rst, followed by the nonvascularized trilaminar omphalopleure, vascularized trilaminar
omphalopleure, and nally the yolk sac splachnopleure (Stewart, 1997; Stewart and Thompson,
2000). Similarly, in Mabuya mabouya the yolk sac
follows this sequence of stages. The bilaminar
omphalopleure is transient (early gastrula early
neurula stages). The nonvascularized trilaminar
omphalopleure develops from late gastrula to early
neurula stages and the yolk sac splachnopleure is
formed during neurulation and persists until parturition. All squamates present a yolk sac splachnopleure until the end of gestation. In M. mabouya,
like in the Brazilian species of Mabuya studied
(Blackburn et al., 1984; Vitt and Blackburn, 1991;
Blackburn, 1993), the yolk sac is not associated with
the formation of an omphaloplacenta or an omphalallantoic placenta, as has been observed in other
oviparous and viviparous squamates (Stewart, 1993;
Stewart, 1997; Stewart and Blackburn, 1988; Stewart and Thompson, 2000), including the highly matrotrophic Eumecia anchietae, although in this species the formation of a yolk cleft and isolated yolk
mass could not unequivocally be conrmed (Flemming and Branch, 2001). Recently, Flemming and
Blackburn (2001) mentioned that in this latter species and in Mabuya ivensii, two African species with
microlecithal eggs, the yolk sac placentas are also
atypical for squamates, because no yolk cleft or isolated yolk mass is apparent by mid-development.
Several other reproductive specializations of these
lizards are also shared with the South American
Mabuya. Thus, they suggested that according to
phylogenetic information, placentotrophy in these
African and South American skinks was retained
from a common viviparous ancestor.

173

In the oviductal egg (diameter 1.5 mm), the egg

content is an aqueous substance that contains dispersed amorphous material composed mainly of a
few peripheral small granules. We could not detect
yolk granules or yolk platelets in preovulatory (Gomez and Ramrez-Pinilla, pers. obs.) or postovulatory eggs of Mabuya mabouya. The oviductal eggs
appear to contain some translucent lipid droplets
observed as empty vesicles because they are lost
during sample preparation. Lipids constitute one of
the principal elements of the yolk in lecithotrophic
(Hoffman, 1970; Stewart, 1989; Stewart and
Castillo, 1984; Speake and Thompson, 2000) and
most placentotrophic reptiles (Stewart 1989; Blackburn and Callard, 1997; Thompson et al., 1999a,b,
2000; Speake and Thompson, 2000). However, they
constitute complex and prominent granules or platelets that stain distinctly in normal histological preparations. Speake and Thompson (2000) found that
the eggs of oviparous and viviparous species with a
simple placenta (type I) had different lipid composition than the eggs of viviparous species with complex (types II and III) placentas. They also found
that in placentotrophic species the relative reduction in the lecithotrophic lipid contribution is
brought about largely by a relative reduction in egg
size. However, species with a type IV placenta were
not studied. In Pseudemoia species (type III placenta), Thompson et al. (1999b) found that more than
59% of the total lipid supplied to the embryo is
derived from placental transport. We suppose that
in the microlecithal egg of species with type IV placenta, besides the substantial reduction in total egg
lipid content, the lipid prole is different from that
in the other placentotrophic species, related to their
higher placental complexity. Possibly, a high proportion of the lipids necessary for embryo development
is contributed by the mother through the placenta.
However, the chemical nature of ooplasmic constituents, and the composition and physiological mechanism of transfer of substances through the placentae, are still to be determined for these species.
In early and late gastrulas there is little material
within the yolk sac. Endodermal cells may function
in the absorption of water and nutrients when they
form the bilaminar omphalopleure or through absorptive plaques of the nonvascular trilaminar
omphalopleure. Blackburn et al. (1984) observed
that in Mabuya heathi the oviductal egg grows 3 4
times, possibly by water uptake, during early gestation when the yolk sac placenta is formed. During
late neurulation, pharyngula, and limb-bud stages
the yolk sac grows. It has a thicker wall and develops deeper lobules that are highly vascularized by
the splanchnopleuric mesoderm that surrounds it.
Endodermal cells appear to be very active in secretory (and/or absorptive) processes. From pharyngula
stages, the interior of the yolk sac contains material
like that present in the endodermal cells. This material could pass throughout the endothelium that

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A. JEREZ AND M.P. RAMIREZ-PINILLA

vascularizes the yolk sac to the blood stream. Blackburn et al. (1984) also observed, in late embryos of
M. heathi, that the yolk sac is thick-walled and well
vascularized. The lumen of the yolk sac of matrotrophic Eumecia anchietae (Flemming and Branch,
2001) contains only some liqueed material and very
few yolk granules, and the endodermal cells
(vitelline cells) are vacuolated and proliferate into
the lumen with accompanying vasculature. The development of the yolk sac in the highly matrotrophic
Mabuya and in E. anchietae suggests that the yolk
sac endodermal cells play an important metabolic
role. However, the precise function of these cells is
unknown.
Bilaminar Yolk Sac Placenta
Extraembryonic endoderm and ectoderm are extended completely over the egg by the early gastrula
stage. These membranes form the bilaminar omphalopleure and, in apposition to the uterine epithelium, constitute the rst placental structure. Histologically, it begins as a simple cuboidal (above the
embryonic disc) to squamous membrane during
early gastrulation. Later, the extraembryonic ectoderm becomes bistratied and the external layer
forms a dorsal zone of cellular hypertrophy with
features of absorptive cells. At the abembryonic
hemisphere the external leaf of the extraembryonic
ectoderm changes initially from squamous to cuboidal cells with prominent microvillar surfaces. Microvilli can increase the surface area for exchange
between mother and fetus, suggesting that active
absorption of nutrients begins very early in development, during gastrulation, through both the dorsal
absorptive plaque and the external leaf of the extraembryonic ectoderm. The uterine luminal epithelium and the endometrial glands show secretory activity in correspondence with the absorptive
ectodermal epithelium. A bilaminar extraembryonic
ectoderm is also present in Chalcides chalcides (Ghiara et al., 1987) from the late neurula stage (Blackburn and Callard, 1997). The bistratication of this
layer is accelerated in Mabuya mabouya in comparison to C. chalcides. The bilaminar omphalopleure
typically contributes to an omphaloplacenta or
omphalallantoic placenta in squamates and these
placentas have been considered likely sites for the
uptake of water (Weekes, 1935; Stewart and Thompson, 1996) or nutrients in lecithotrophic (Yaron,
1977), incipient or facultatively matrotrophic
(Swain and Jones, 1997; Thompson 1999a,b), or substantially matrotrophic species (Stewart and
Thompson, 1996). The bilaminar omphalopleure of
Eumecia anchietae, a highly matrotrophic species,
also has characteristics indicative of nutritional
transport (Flemming and Branch, 2001).
The cells of the external layer of extraembryonic
ectoderm in the omphaloplacenta of Chalcides chalcides change from squamous to columnar in some

regions (Blackburn and Callard, 1997). The bilaminar omphalopleure in the other studied species has
been described as a component of the omphaloplacenta in embryos at approximately stage 30. A stratied ectoderm with hypertrophied external cells has
been observed in other species as well (Weekes,
1927; Hoffman, 1970; Yaron, 1985; Stewart and
Thompson, 1996). In general, the ectodermal cells of
the omphaloplacenta can vary from cuboidal to columnar (Weekes, 1927, 1929, 1930; Yaron, 1977;
Stewart 1985; Villagran-Santa Cruz, 1989; Stewart
and Thompson, 1994, 1996, 1998; Corso et al., 2000;
Flemming and Branch, 2001). A histotrophic nutrient transfer function has been suggested for these
cells based on the development of cuboidal/columnar
epithelium and microvillar surfaces (Stewart and
Thompson, 1996).
Stewart (1993) described the bilaminar omphalopleure as a transitory structure in many amniotes
that is maintained during squamate development as
a structural component of the omphaloplacenta and
omphalallantoic placenta. It is also maintained during development of some marsupials and eutherian
mammals (Mossman, 1987; Stewart, 1993). In Chalcides chalcides the bilaminar omphalopleure is also
the rst placental arrangement, observed until late
stage neurulas, when it constitutes the omphaloplacenta (Blackburn and Callard, 1997). In Mabuya
mabouya, however, the bilaminar yolk sac placenta
forms earlier in development and changes rapidly
due to the rapid mesodermal extension during gastrulation and to a chorioplacenta in early neurulas.
Thus, contrary to that observed in other squamates
during morphogenesis of the extraembryonic membranes, the bilaminar omphalopleure of M. mabouya
is present only during early developmental stages.
Choriovitelline Placenta
This transitory placenta is formed when the vascularized mesoderm invades the bilaminar omphalopleure to form the trilaminar omphalopleure, in
apposition with the uterine wall (Stewart and Blackburn, 1988). In Chelonia, Aves, and Crocodylia the
nonvascularized trilaminar omphalopleure precedes
the sinus terminalis and extends to the base of the
yolk mass. Vascularization of the mesoderm occurs
later (Stewart, 1993). The same pattern was observed in Mabuya mabouya. In squamate reptiles
the trilaminar omphalopleure initially forms in the
same manner, but its development is limited to the
embryonic hemisphere (Stewart, 1993). In M.
mabouya the trilaminar omphalopleure is a very
transitory structure that occurs during gastrulation,
almost simultaneously with mesodermal delamination, to form the chorion and the splanchnopleure.
The trilaminar omphalopleure has been observed
during late neurula to early pharyngula (stages 20
27) in Chalcides chalcides (Blackburn and Callard,
1997), nearly to stage 26 (although embryos were

PLACENTAL DEVELOPMENT IN M. MABOUYA

not staged, we infer this stage according to the description) in Elgaria coerulea (Stewart, 1985), and
not until at least stage 30 in Pseudemoia entrecasteuxii (Stewart and Thompson, 1996), P. spenceri
(Stewart and Thompson, 1998), Niveoscincus metallicus (Stewart and Thompson, 1994), and N. coventryi (Stewart and Thompson, 1998).
The suggestion that the choriovitelline placenta
has the potential role of gas exchange in early development (Blackburn and Callard, 1997) may be
realized if the vascularized trilaminar omphalopleure is maintained during a developmental phase
and over a large area dorsal to the sinus terminalis
(as occurs in Chalcides chalcides). However, in
Mabuya mabouya the transitory character of this
structure, the small area covered by the vascularized trilaminar omphalopleure, and the absorptive
features of the ectodermal cells suggest that any role
of a functional placenta in gas exchange is very
limited.
During gastrulation, new ectodermal structures,
the absorptive plaques, appear at the embryonic and
abembryonic hemispheres. Their development coincides with extension of the extraembryonic mesoderm and may be induced by this process. The dorsal
absorptive plaque will form the placentome in later
developmental stages. The smaller absorptive
plaques develop at lateral and antimesometrial
sides of the embryonic chamber. Cellular features of
these structures are similar and they appear to be
specialized for histotrophic nutrient absorption. The
absorptive plaques originate during development of
the nonvascularized trilaminar omphalopeure and
are maintained until the end of development (Jerez
and Ramirez-Pinilla, 2001).
Chorioplacenta
With the mesodermal split and the consequent
formation of the exocoelom, the apposition between
the chorion and the uterine mucosa constitutes the
chorioplacenta. It is present only at the embryonic
hemisphere in squamates that develop an omphaloplacenta or omphalallantoic placenta (Stewart and
Blackburn, 1988). However, few descriptions of this
placenta can be found. Blackburn and Callard
(1997) describe it for Chalcides chalcides by the
early neurula early pharyngula stage at the embryonic hemisphere. The chorionic surface epithelium
was described as cuboidal to columnar and they
assumed the possibility of histotrophic transfer. In
Mabuya mabouya the chorioplacenta is present during neurulation, but it covers the entire embryonic
chamber because the exocoelom is continuous in
both hemispheres from late neurula stages.
The chorion is regionally specialized, forming hypertrophied cells and structures that indicate the
potential for histotrophic transfer. The absorptive
plaque at the embryonic pole, the lateral absorptive
plaques, and the chorionic concavities and chorionic

175

bags at the abembryonic hemisphere all consist of

columnar cells and interstitial cells. These cells can
actively phagocytize and absorb nutrients from the
luminal uterine epithelium and from the uterine
glands that are very active in secretion processes.
Cuboidal to squamous cells of the chorionic surface
epithelium in the remainder of the embryonic chamber at the abembryonic hemisphere also could function in histotrophic transfer, possibly with a different specic function, because they maintain a
striated border and abundant secretory material is
present over their apical surfaces.
The chorionic concavities and the chorionic bags
are two placental structures that appear at the
abembryonic hemisphere during the chorioplacenta
stages. They are wide and deep invaginations of the
chorion closely related with a high uterine fold that
can be observed in some preparations to penetrate
the bags. They are very transient and only were
observed in late gastrula early neurula stage embryos. The external leaf of the chorionic epithelium
is hypertrophied, similar to the absorptive plaques,
but deformed by the presence of large yellow crystalloid granules. The features of the chorionic cells,
the relation with uterine folds, and the nature of the
cytoplasmic inclusions suggest that they may have a
specialized histotrophic and/or metabolic role. However, this should be investigated. In contrast to observations in other squamates, the chorioplacenta of
Mabuya mabouya is a very important placenta specialized for active histotrophic transfer of nutrients.
Allantoplacenta
The allantoplacenta is formed by the association
between the allantoic vesicle and the chorion. The
chorioallantois is formed at the embryonic hemisphere by pharyngula stages and progressively extends to the abembryonic hemisphere. By the limbbud stage the allantoic vesicle has extended almost
completely into the exocoelom, leaving only a small
area at the abembryonic pole without vascularization. Finally, at stages 35 40 the allantoic vesicle is
continuous in both hemispheres (Jerez and
Ramrez-Pinilla, 2001). For Mabuya heathi, Blackburn et al. (1984) observed that the allantoic vesicle
is expanded into the exocoelom between the fth to
seventh month of gestation.
The allantoplacenta is restricted to the embryonic
hemisphere in species that develop an omphaloplacenta and extends into the yolk cleft in species with
an omphalallantoic placenta (Blackburn, 1993;
Blackburn and Callard, 1997; Stewart, 1993, 1997;
Stewart and Thompson, 1996, 1988). For other matrotrophic species such as Chalcides chalcides
(Blackburn and Callard, 1997), Pseudomoia entrecasteauxii, P. pagenstecheri, and P. spenceri
(Stewart and Thompson, 1996, 1998), the chorioallantois occurs primarily on the embryonic side
of the egg. In Eumecia anchietae at the limb-bud

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A. JEREZ AND M.P. RAMIREZ-PINILLA

stage the chorioallantois occurs throughout the incubation chamber, enveloping the entire embryo,
including the yolk sac (Fleeming and Branch, 2001).
The allantoplacenta of Mabuya mabouya forms
the placentome and paraplacentome in the mesometrial hemisphere and absorptive plaques and segments of gas exchange in the antimesometrial hemisphere (see detailed description and a comparative
analysis in Jerez and Ramrez-Pinilla, 2001). The
placentome is formed from gastrula stages as the
dorsal absorptive plaque and is folded and interdigitates with specialized uterine folds at the nal developmental stages (stages 35 40). The placentome
in Chalcides chalcides, Pseudomoia entrecasteauxii,
P. pagenstecheri, and P. spenceri (Stewart and
Thompson, 1996, 1998; Blackburn and Callard,
1997) is formed during the limb-bud stage and the
morphology is different from the New World
Mabuya. However, in C. chalcides during late
neurula early pharyngula stages the ectoderm of
the chorioplacenta is bistratied and has columnar
external cells, showing an early specialization of the
mesometrial zone with histotrophic functions
(Blackburn and Callard, 1997).
Morphogenesis of Extraembryonic
Membranes
The ontogeny of the extraembryonic membranes
in squamate reptiles has been studied in very few
viviparous (Weekes, 1927, 1929, 1930; Hoffman,
1970; Stewart, 1985, 1989; Villagran-Santa Cruz,
1989; Stewart and Thompson, 1994, 1996, 1998;
Blackburn and Callard, 1997; Flemming and
Branch, 2001) and oviparous species (Stewart, 1985;
Stewart and Thompson, 1996; Stewart and Florian,
2000). A similar pattern of extraembryonic membrane development has been observed in Squamata,
different from that of the nonsquamate reptiles
(Chelonia, Archosauria). The mesoderm penetrates
the yolk sac, forming an intravitelline mesoderm,
and a yolk cleft is formed. The outer compartment of
yolk, the isolated yolk mass, is lined externally by
the bilaminar omphalopleure (Stewart, 1993, 1997).
Stewart (1997) considered that the isolated yolk
mass is a synapomorphy for Squamata, or possibly
Lepidosauria, because the development of the yolk
sac has not been studied in Sphenodon. Blackburn
and Callard (1997) and Flemming and Branch
(2001), respectively, tested the hypothesis that the
highly matrotrophic Chalcides chalcides and Eumecia anchietae, evolved a novel pattern of extraembryonic membrane morphogenesis, in association
with yolk reduction and placentotrophy. However,
they found that these species follow the squamate
pattern of development and concluded that substantial matrotrophy may evolve in squamates without
major modications in the morphogenetic pattern.
However, recently Flemming and Blackburn (2001)
observed that in the highly matrotrophic African

skinks E. anchietae and Mabuya ivensii, as in Neotropical Mabuya, an atypical yolk sac arrangement
is developed. Interestingly, Greer et al. (2000) considered that the South American species of Mabuya
seem to be a monophyletic group distinguished by
three characters, one of which is the substantial
degree of matrotrophy. Also, the author suggested
that the sister taxa to South American viviparous
species are the African viviparous species M. capensis, M. ivensii, and M. vittata. Thus, as suggested by
Flemming and Blackburn (2001), morphological features related with placentotrophy could be retained
from a common viviparous ancestor.
In Mabuya mabouya we did not nd the characteristic squamate pattern of extraembryonic membrane development. Thus, this species exhibits a
similar pattern to that of Archosauria and Chelonia,
where the mesoderm does not ingress into the yolk
sac and consequently an isolated yolk mass or a yolk
cleft are not formed. This feature (extraembryonic
membrane morphogenesis similar to nonsquamate
reptiles) constitutes a novel arrangement of extraembryonic membrane morphogenesis for squamates and would be related to the drastic reduction
of the egg size and obligatory placentotrophy from
early developmental stages, as previously proposed
by Weekes (1935) and Blackburn and Callard
(1997).
This pattern observed in Mabuya mabouya would
constitute a reversion. If this pattern is shared with
other South American species of Mabuya (as it appears to be according to the observations of Blackburn, 1993; Blackburn et al., 1984; Vitt and Blackburn, 1991; Villagran-Santa Cruz, pers. commun.)
and the matrotrophic species of the African lineage,
and if both groups form a monophyletic taxon, it
would dene a homologous reversion originated before the separation between Africa and America.
However, there are several species that have not
been studied. To test this hypothesis the relationship between structure and function, together with
the morphogenetic pattern of extraembryonic membranes in other New World species of Mabuya and in
the African lineages, should be studied.
ACKNOWLEDGMENTS
We thank the authorities and people of the Inspeccion de Polica de Guadualito for their hospitality
during our collection trips, especially Profesora Elsa
de Alvarez and her family; the students and teachers at the elementary school of Guadualito; Don
Rodolfo Alvarez and his family; several other students of the Escuela de Biologa, UIS for help in the
capture of the animals; Prof. Rosa Aura Gavilan,
Escuela de Biologa UIS, for the use of their photomicroscopes; Prof. Maricela Villagran-Santacruz,
Facultad de Ciencias, Universidad Autonoma de
Mexico, for showing us the results of her work with
different American species of Mabuya; Biologist Pa-

PLACENTAL DEVELOPMENT IN M. MABOUYA

ola Sanchez for technical assistance with histology,

microphotography, and editing; Prof. Dr. Daniel R.
Miranda for support and helpful suggestions; Prof.
Olga Castano, Instituto de Ciencias Naturales, Universidad Nacional de Colombia, Dr. Fernando Castro, Departamento de Biologa de la Universidad del
Valle, and the Hno. Luis Alberto Zamudio Director
del Museo del Colegio San Jose, Medelln, who facilitated study of the museum specimens; and Dr.
James Stewart for helpful comments on the manuscript.

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APPENDIX
Acronyms: CJS Colegio San Jose, Medelln; ICN Instituto
de Ciencias Naturales, Universidad Nacional de Colombia,
Bogota; UVC Universidad del Valle; UIS-R Coleccion Herpetologica, Museo de Historia Natural, Universidad Industrial de
Santander.
Museum specimens: CSJ 079 (Medelln), CSJ 080 (Medelln),
CSJ 249 (Maicao), ICN 4307(Sucre), ICN 4324 (Meta), ICN 7876
(Guaina), UVC 5183 (Cauca), UVC 5185 (Valle del Cauca).
Collected specimens: UIS-R-0259, (3/2) number of embryos in
left/right oviduct, late neurulas, collected 22 February 1998; UIS-

R-0261, (3/4), early gastrulas, 17 February 1998; UIS-R-0264,

(3/4) pharyngulas, 20 February 1998; UIS-R-0265, (5/2), late neurulas, 22 February 1998; UIS-R-0266, (3/3) early gastrulas, 23
February 1998; UIS-R-0269, (5/3) gastrulas, 24 February 1998;
UIS-R-0270, (2/2) oviductal eggs, 22 February 1998; UIS-R-0289,
(4/3) limb-bud stage embryos, 8 April 1998; UIS-R-0294, (4/4)
gastrulas, 9 April 1998; UIS-R-0292, (3/1) gastrulas, 6 April 1998;
UIS-R-0312, (3/2) late gastrula-early neurulas; 12 June 1998;
UIS-R-0315, (5/4) pharyngulas, 9 June 1998; UIS-R-0316, (5/2)
late gastrula-early neurulas, 7 June 1998; UIS-R-0317, (3/5) late
gastrula-early neurulas, 9 June 1998; UIS-R-0320, (3/2) limb-bud
stage embryos, 10 June 1998; UIS-R-0341, (4/2) pharyngulas, 10
August 1998; UIS-R-0342, (3/2) late gastrula-early neurulas, 10
August 1998; UIS-R-0343, (2/3) late gastrula-early neurulas, 10
August 1998; UIS-R-0344, (2/2) late neurulas, 10 August 1998;
UIS-R-0349, (3/1) pharyngulas, 10 August 1998; UIS-R-0351,
(3/2) limb-bud stage embryos, 10 August 1998; UIS-R-0353, (2/3)
late gastrula-early neurulas, 10 August 1998; UIS-R-0360, (3/3)
oviductal egg, 4 October 1998; UIS-R-0363, (3/2) gastrulas, 5
October 1998; UIS-R-0366, (2/3) pharyngulas, 6 October 1998;
UIS-R-0398, (4/4) gastrulas, 8 December 1998; UIS-R-0438, (2/2)
early gastrula, 15 February 1999.