Placenta (2004), 25, 705–711doi:10.1016/j.placenta.2004.02.009

Anatomical and Histological Characterization of the Female

Reproductive Organs of Mouse Deer (Tragulidae)

J. Kimuraa,*, M. Sasakib, H. Endoc and K. Fukutad

a Department of Veterinary Medicine, College of Bioresource Science, Nihon University, 1866 Kameino, Fujisawa,Kanagawa 252-8510, Japan; b Department of Veterinary Anatomy, Obihiro University of Agriculture andVeterinary Medicine, Obihiro, Hokkaido, Japan; c Department of Zoology, National Science Museum, Tokyo, Japan;d Laboratory of Animal Morphology and Function, Graduate School of Bioagricultural Science, Nagoya University,Nagoya, Aichi, Japan

Paper accepted 20 February 2004

The Tragulidae may be a type that represents the earliest ruminant families to evolve. The female reproductive organs including

ovary, oviduct, uterus and placenta were anatomically and histologically investigated. The structures of the ovary and oviduct were

very similar to other ruminants. However, the gross structure of the placenta was diffuse and thus noticeably different from other

ruminants which are polycotyledonary. Histologically, the placenta of Tragulidae appears to be epitheliochorial and therefore

similar to other ruminants. Numerous trophoblastic binucleated cells which are characteristic of all other ruminants were

observed. These results suggest that the placenta of Tragulidae is a transitional type between diffuse epitheliochorial and

polycotyledonary synepitheliochorial categories.

Placenta (2004), 25, 705–711 � 2004 Elsevier Ltd. All rights reserved.

INTRODUCTION

The suborder Ruminantia divides into seven families, one of

which is the Tragulidae. Two genera of the Tragulidae are

found in South Africa, India and South-east Asia. The lesser

mouse deer, Tragulus javanicus, is the smallest among living

hoofed animals [1,2], the adult weighing about 1.5 kg and

rarely over 2 kg [3,4]. Although they look like tiny deer, mouse

deer differ in several particulars. They share a number of

characteristics with nonruminants: they lack horns or antlers;

the males have continually growing, tusk-like upper canines;

they have sharp-crowned premolars and four fully developed

toes [5]. The stomach is simpler and (like the camel’s) has

three instead of four effective compartments. The distribution

pattern of gut endocrine cells in the lesser mouse deer is closer

to that in the pig rather than in the domestic ruminants [6].

The phylogenetical relationship of Tragulidae and other artio-

dactyls has been analyzed by means of DNA sequence analysis

of secretory ribonuclease [7] and growth hormone [8] and this

has established the considerable phylogenetical difference be-

tween Tragulidae and the other, more recently evolved, ru-

minants. Recently, Endo et al. [9] investigated the functional

morphology of the mastication muscles in Tragulus javanicusand showed a significant difference in mastication strategy

compared with the grazer such as a developed Bovid species.

* Corresponding author. Tel.: +81-466-84-3629.E-mail address: kimura@brs.nihon-u.ac.jp (J. Kimura).

0143–4004/$–see front matter

These data suggest that the Tragulidae represent the earliest

ruminants to evolve.

Breeding data for this animal are limited, there are few

reports describing the seasonality, period of gestation, the age at

sexual maturity and so on [5,10,11]. Three papers in late 19th

and early 20th centuries observed the placenta of mouse deer

macroscopically and stated that the mouse deer has a diffuse

placenta [12–14]. However, there have been no reports about

mouse deer placenta structure since then. In this study, the

existence of diffuse placenta was verified by the observation of

the mid- and full-term pregnant uterine tissues with the light

microscope. This research is also designed to establish the

characteristics of the female reproductive organs of this animal

as a basis for understanding the reproductive physiology of this

endangered species. Such information is a necessary pre-

requisite for design of effective conservation strategies.

MATERIALS AND METHODS

Nine female mouse deer were used in this study (Table 1).

The differentiation of two species, T. napu and T. javanicus has

not been established, so in this paper all animals used were

classed as Tragulidae. Animals were killed by intraperitoneal

injection of an overdose of ketamine hydrochloride. Female

reproductive organs including ovary, oviduct and uterus were

excised and fixed in 10% formalin or Bouin’s solution. After

the gross observation of these organs, they were dissected and

samples were dehydrated, embedded in paraffin, sectioned and

� 2004 Elsevier Ltd. All rights reserved.

706 Placenta (2004), Vol. 25

Table 1. Biological data for the mouse deers used in the study

Number 3 4 5 6 8 9 11 12 13Date of collection Sept. 2001 Sept. 2001 Sept. 2001 March 2002 March 2002 Sept. 2002 Sept. 2002 Sept. 2002 Sept. 2002Body weight (g) 140 280 250 180 200 200 345 260 410Body length (mm) 417 512 498 500 460 492 570 523 600Tail length (mm) NA NA NA NA NA 77 82 73 89Hind footlength (mm)

110 130 130 120 120 135 140 136 151

Ear length (mm) 35 36 37 38 40 44 42 37 39Number of fetus 0 0 1 in R 1 in R 1 in R 0 1 in R 0 1 in RVolume of ovary(right) (mm3)

591 157 733 290 828 424 302 350 NA

Volume of ovary(left) (mm3)

509 469 838 1175 1175 321 1175 283 NA

Existence of large CL R No R L and R L L L No NAWidth of uterinehorn (right) (mm)

8.5 7.3 NA NA NA 6 NA 7.4 NA

Width of uterinehorn (left) (mm)

4.5 3.7 NA NA NA 3.5 NA 4 NA

Volume of ovary was calculated as follows: V ¼ 4=3pabc (a: long axis; b: short axis; c: width of ovary).R: right; L: left.

stained with HE, AB ( pH 1.0), AB ( pH 2.5), PAS and Masson’s

Trichrome stain. Some sections of ovary and placenta were

stained immunohistochemically using the avidin–biotin–

peroxidase complex method [15] with the antisera to human

cytokeratin (DAKO-AE1/AE3, Dako, Carpinteria, CA, USA,

code M3515) and rat cytochrome P450 side chain cleavage en-

zyme (SCC) (Chemicon, Tamecula, CA, USA, code AB1244).

Briefly, after an overnight incubation at 4 (C with the primary

antiserum (!200 dilution), sections were incubated with bio-

tinylated antimouse or antirabbit immunoglobulins (Nichirei,

Tokyo, Japan) for 30 min. Following this step, the avidin–

biotin–peroxidase complex was applied to the sections for 5 min.

A brown reaction product was obtained by subsequently

incubating the sections in diaminobenzidine–H2O2 solution.

Staining controls included the omission of the primary anti-

serum. All stained sections were observed by light microscope.

The size and the weight of ovary, uterus and the fetus were

measured after the fixation process. In case of an obvious preg-

nancy, the age of the fetus was estimated using the hind foot

length instead of crown rump length (HFL), because the

fixation makes the measurement of crown rump length

inaccurate [16].

RESULTS

Biological data for each individual are listed in Table 1.

Ovary

As in other ruminant ovaries, the narrow medulla and broad

cortex were clearly differentiated. Primordial follicles are

evenly distributed mainly in the outer cortex which is common

in ruminants and the sow, whereas in carnivora the follicles

occur in clusters. In pregnant mouse deer, the ovary is at-

tached directly to the uterine wall and there is no mesovarium

(Figure 1). This observation was first reported by Mossman

and Duke [17] and verified in this study. This is the only

exception to the rule reported so far that the mammalian ovary

is suspended by the mesovarium.

The epithelium of follicles consisted of follicular epithelial

cells ( granulosa cells) and two layers of thecal cells, internal

and external thecal cells, which were identified by HE stain

and SCC immunohistochemistry which clearly differentiated

the internal thecal cells with a positive reaction from the ex-

ternal thecal cells with none. Follicles normally contained one

oocyte, very occasionally two oocytes. This is like in cats and

dogs rather than in ruminants which show only one oocyte per

follicle. Large corpora lutea were observed in the ovaries of

pregnant individuals and consisted of large and small cells.

Both large and small luteal cells showed a positive reaction

with the SCC antibody. Mature follicles were present in the

ovary of all pregnant animals even in the late gestational period

Figure 1. Uterus of pregnant mouse deer (No. 11). Arrow head: left ovary.

Kimura et al.: Female Reproductive Organs of Mouse Deer 707

(HFL 72.5 mm). No development of interstitial cells was ob-

served which is not significantly different from other ruminant

animals. In the medulla, a gland-like structure was observed

(i.e. epithelial cells surrounding a central lumen) (Figures 2

and 3). Some of these glands were located near the blood vessels.

These gland cells reacted with anti-SCC antibody (Figure 4)

indicating a level of this enzyme equivalent to that shown in the

follicular epithelial cells and the corpus luteum cells.

Oviduct

Ampulla, isthmus and fimbriae were clearly differentiated.

The epithelium of ampullar portion consisted of simple cuboi

dal cells and ciliated cells. Rugal folds were prominent in the

isthmus. The apical cell surface of some of the epithelial cells

in both regions, which protrude into the lumen, reacted with

PAS and AB (pH 2.5) stain. The isthmus has a thicker

muscular tunica and thinner mucosa than the ampulla.

Uterus and fetus

The uterus consists of bilateral horns (cornua) arising from

a single uterine body (Figure 5). On the inner surface of

Figure 2. Glandular structure (arrow) in the ovarian medulla of nonpregnantmouse deer (No. 9). PAS–hematoxylin. Scale 0.3 mm.

Figure 3. High magnification of the glandular structure in the ovarianmedulla (No. 9). Columnar cells line the luminal spaces. PAS–hematoxylin.Scale 0.05 mm.

nonpregnant uterine horn of the mouse deer, there were longi

tudinal folds but caruncles could not be identified. The uterine

epithelium consists of columnar cells. Well-developed coiled

uterine glands were located throughout the lamina propria

mucosa (Figure 6). Uterine glands consist of columnar cells

and reacted with cytokeratin antibody.

Among nine females observed, five individuals had a suf

ficiently large fetus which could be recognized as a swelling in

the uterine horn. All of the fetuses were found in the right

uterine horn (Table 1). The other four individuals also showed

a larger uterine horn on the right side although no conceptus

was found within any (Table 1).

The characteristics of fetus are listed in Table 2. As shown

in this table, the eruption of incisor, the hair growth and the

initiation of darkness of hoof and muzzle were first observed at

60.5 mm (HFL).

Placenta

In appearance, the placenta of mouse deer presents a smooth,

white or yellowish allantoic inner surface. The interaction of

Figure 4. (a) Control section. (b) Glandular structure (arrow) and thefollicular epithelial cells (*); which reacted with SCC antibody. Nonpregnantmouse deer (No. 12). Immunostain. Scale 0.1 mm.

Figure 5. Uterus (U), oviduct (arrow) and ovaries (O) of individual (No. 4)with no fetus. This sample was fixed with Bouin’s solution. Note the larger sizeof right uterine horn (*). Scale indicates 5 mm.

708 Placenta (2004), Vol. 25

maternal and fetal placenta surfaces takes place over virtually

the entire sac, and so these placentas can be described as dif-

fuse with no evidence of caruncular or placentomal differen-

tiation (Figures 7 and 8). In the mid-term pregnancy (HFL:

21 mm), many rugal folds were observed on the surface of

the fetal placenta (Figure 7). In full-term pregnancy (HFL:

725 mm), these folds had disappeared and smooth surface was

observed (Figure 8).

Histologically, endometrial tissue and trophoblastic layers,

including maternal and fetal vessels, were intimately associated

(Figure 9). The placenta consists of enmeshed maternal and

fetal fingerlike villi with no obvious branching. Binucleated

cells (BNC) are dispersed within uninucleate trophoblast cells

which stained with cytokeratin in the trophectodermal villi.

BNC stained positively with PAS (Figures 10–12).

DISCUSSION

This paper presents the first published details of the female

reproductive tissues and the placental structure of the mouse

deer. There are no major differences of basic structure of the

ovary compared with other ruminants. The gland-like struc

tures which were consistently observed in the medulla are

probably rete ovarii tubules. Mossman and Duke [17] have

described similar structures in ruminants and many other

Figure 6. Transverse section of left uterine horn of nonpregnant mouse deer(No. 12). Note the uterine glands around the whole uterine circumference.HE. Scale 0.3 mm.

mammals. However, this is the first time they have been shown

to contain SCC activity as do the inner thecal and luteal cells.

In this study, a large and small size of luteal cells was

observed. Both cells, especially small cells, showed a positive

SCC localization. The presence of SCC indicates the potential

for steroid production. There are differences among ruminants

in the source of production of progesterone during the gesta

tional period. For example, in the sheep, but not in the goat,

the placenta has the capability to take over the progesterone-

secreting function of the corpus luteum [18,19]. It is not yet

known whether the corpus luteum of mouse deer secretes pro

gesterone throughout the gestational period or whether there is

a transfer of the production site of steroid hormone to the

placenta later in pregnancy.

There were no significant differences in the structure of the

oviduct of Tragulidae compared with other ruminants such as

cow and goat [20,21].

It is worth emphasizing that each of the five fetuses was

found in the larger right uterine horn. However, three of the

pregnant animals had a mature corpus luteum in the left ovary

and none in the right. This indicates that internal migration of

embryos easily occurs between the two uterine horns. Trans

uterine migration is common in sheep, but it seldom occurs in

cattle. However, in some other ungulate species, like the

impala and the common duiker, pregnancy almost always

occurs in the right uterine horn although they ovulate from

Figure 7. The fetus of pregnant mouse deer (No. 11). Note the many rugalfolds observed on the surface of the fetal placenta.

Table 2. Characteristics of fetus

Number Fetal sex Fetal weight (g) HFL (mm) Hair Incisor Hoof Muzzle

13 F 13.6 16.2 Not developed Not erupted Not pigmented Not pigmented11 M 15.2 21 Not developed Not erupted Not pigmented Not pigmented8 M 16.8 21.9 Not developed Not erupted Not pigmented Not pigmented6 F 19.2 24.8 Not developed Not erupted Not pigmented Not pigmentedUPM-94 F 92.2 60.5 Developed Erupted Half pigmented Half pigmented5 F 170 72.5 Developed Erupted Pigmented Pigmented

UPM-94: this individual is not listed in Table 1, however, only fetus was supplied from University Putra Malaysia. F: female, M: male.

Kimura et al.: Female Reproductive Organs of Mouse Deer 709

either ovary [22]. On the other hand, nearly all pregnancies

occurred in the left horn in all camelidae although, again, the

corpus luteum’s location is equally distributed between the left

and right ovary [23]. This demonstrates the possibility of the

migration of embryos to either uterine horn. The mouse deer

is unique in having a large right uterine horn. This asymmetry

in the size of the left and right uterus of all female mouse deer

observed must be genetically based.

This evidence and the existence of mature follicles in the

ovary in the gestational period confirm the possibility of three

consecutive pregnancies within one year and the occurrence of

the postpartum estrus two days after parturition which were

reported by Kudo et al. [11].

Placentas can be classified by the gross surface structure

into four types: diffuse, cotyledonary, zonary and discoidal. In

ruminants, cotyledonary villus development only occurs at

specialized nonglandular and circular areas of the endome-

trium known as caruncles. They are rich in fibrocytes and have

an extensive blood supply. Caruncles are the site of initial

Figure 8. Well-developed fetus and the placenta of mouse deer (No. 5). Notethat the folds which could be observed on the mid-term fetal placenta surface(Figure 7) are no longer visible and a smooth surface is observed. Thisspecimen was fixed in 10% formalin solution.

Figure 9. Placenta of mouse deer (No. 8). The placenta consists of enmeshedmaternal (lower) and fetal (upper) fingerlike villi. PAS–hematoxylin. Scale0.3 mm.

attachment of the fetal trophoblast to the maternal uterine

epithelium. The subsequently developed enmeshed maternal

and fetal villi form the placentomes [24,25]. Even though the

number of placentomes in the suborder Ruminantia is vari-

able (e.g., two to six in deer and more than 100 in giraffe),

all ruminants so far examined show a cotyledonary placenta.

The statements that musk deer have a diffuse placenta by

Amoroso [26] and Mossman [27] have recently been denied

by Benirschke [28] although none of these reports include

illustration to verify their claims. No placentomes were observed

in Tragulus in this study. So, Tragulus is the only animal

belonging to the suborder Ruminantia examined so far which

does not possess a cotyledonary placenta.

Mammalian placentas have also been classified by the man-

ner of interaction between fetal trophoblast and uterine

epithelium into four categories: epitheliochorial, endothelio-

chorial, syndesmochorial and hemochorial placentas [26]. The

mature ruminant placenta has more recently been shown to be

neither entirely syndesmochorial, defined by Ludwig [29],

Figure 10. Placenta of mouse deer (No. 11). Many binucleated cells wereobserved (arrows) in the transverse section of maternal and fetal villi. PAS–hematoxylin. Scale 0.2 mm.

Figure 11. Placenta of mouse deer (No. 11). Many binucleated cells wereobserved (arrows) in the longitudinal section of maternal and fetal villi. PAS–hematoxylin. Scale 0.2 mm.

710 Placenta (2004), Vol. 25

Steven [30,31] and Ramsey [32], with no uterine epithelium,

nor epitheliochorial with two apposed cell layers whose only

anatomical interaction is interdigitated microvilli. Wooding

[33] has redefined the ruminant placenta as a ‘‘synepithelio-

chorial’’ placenta. This term accommodates the variety and

reflects the cellular origins of the ruminant placenta.

PAS positive BNC were found in the trophectodermal villi

of the Tragulidae in the present study. In the last decade,

evidence has accumulated to show that a characteristic feature

of the ruminant placenta is the population of fetal chorionic

BNC which migrates throughout pregnancy through the

chorionic tight junction to fuse with uterine epithelial cells or

their derivatives. Ruminant BNC are directly involved in the

modification of the uterine epithelium, beginning at implan-

tation and continuing until term [24,34–36]. BNC are known to

produce hormones such as placental lactogens [37–39]. Pla-

cental lactogens have been shown to be characteristic molecules

in the ruminant placenta, released from the BNC into both

maternal and fetal blood throughout pregnancy. We have been

unable to demonstrate the location and existence of placental

lactogen in the mouse deer by immunohistochemistry using

a bovine placental lactogen antibody, possibly because of a lack

of reactivity of the bovine antibody we used with a different

species (data not shown). It will be important to use molecular,

biological and protein purification methods to establish

whether Tragulus does produce a placental lactogen.

A diffuse placenta is found in Bunodonta, Perissodactyla,

Cetacea, Manidae, Lemuroidea, Scalopus aquaticus [26] and all

Camelidae [40]. The basis of the classification between diffuse

and cotyledonous is whether proliferation of maternal and fetal

tissue to form villi occurs over all the available surface of the

chorionic sac or whether it is restricted to specialized regions.

The diffuse placenta seems to be the primitive form. The

present histological data do not allow an unequivocal identi-

fication of the exact nature and origin of the epithelium apposed

to the trophectoderm of Tragulidae. The presence of the

characteristic ruminant BNC in the trophectoderm suggests

Figure 12. Placenta of mouse deer (No. 11). Many binucleated cells wereobserved (arrows) in the transverse section of maternal and fetal villi. Thecytoplasm of these cells reacted strongly with PAS (arrows). Note thetrophectoderm (T) and uterine (U) epithelium. PAS–hematoxylin. Scale0.05 mm.

that the placenta is synepitheliochorial, but an electron micro-

scope study is required to establish the detail. Since Tragulidae

is the most primitive ruminant judged by molecular and gross

structural consideration, detailed placental cellular structure

may provide important clues as to how the polycotyledonary

type developed.

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