A morphological and ultrastructural study of the female reproductive
tract and placenta of the lesser Bushbaby (Galago senegalensis)
By
ALLAN NJOGU, B.V.M (NBI)
Department of Veterinary Anatomy
University of Nairobi.
A thesis submitted in part fulfillment for the degree of Master of Science
(Veterinary Anatomy) in the University of Nairobi.
2003.
I
DECLARATION
This thesis is my original work and has not been presented for a degree in
any other university.
-------------------------------ALLAN NJOGU
Department of Veterinary Anatomy
University of Nairobi.
This thesis has been submitted for examination with our approval as
University supervisors.
----------------------------------G.E. OTIANGA-OWITI, BVM, MSc, Ph.D.
Associate professor of Veterinary Anatomy.
Dept. of Vet. Anatomy
----------------------------------D. ODUOR-OKELO, BVSc, DVM, MSc, Ph.D.
Professor of Veterinary Anatomy
Dept. of Vet. Anatomy
II
TABLE OF CONTENTS
CHAPTER 1.0
1.1 General introduction ......................................................................... 1
1.2 Literature review............................................................................... 4
1.2.1 Prosimian classification ................................................................. 4
1.2.2 Ecology and social organisation ..................................................... 7
1.2.3 Characteristics................................................................................ 9
1.2.4 Reproductive behaviour ................................................................10
1.2.5 Reproductive anatomy ..................................................................14
1.2.6 Maternal recognition of pregnancy................................................18
1.2.7 Implantation and early post-implantation development .................19
1.2.8 Yolk sac placenta ..........................................................................22
1.2.9 The amnion ...................................................................................23
1.2.10 The chorion ................................................................................24
1.2.11 The allantois ...............................................................................24
1.2.12 The placental classification and development .............................26
1.3 Aims and objectives .........................................................................37
CHAPTER 2.0 Materials and methods .................................................38
2.1 Animal specimens............................................................................38
2.2 Dissection and fixation. ...................................................................38
2.3 Tissue fixation. ................................................................................39
2.4 Processing for histology...................................................................40
2.5 Processing for electron microscopy .................................................40
CHAPTER 3.0 Results ...........................................................................42
3.1 General remarks ...............................................................................42
III
3.2 The ovaries ......................................................................................42
3.2.1 Non-gravid animals .......................................................................42
3.2.2 Gravid animals ..............................................................................43
3.3 The oviduct ......................................................................................44
3.4 The uterus ........................................................................................45
3.4.1 The non-pregnant uterus ...............................................................45
3.5 The cervix ........................................................................................46
3.6 The vagina .......................................................................................46
3.7 The vestibulum vaginae ...................................................................47
3.8 The clitoris .......................................................................................47
3.9 The pregnant uterus .........................................................................48
3.10 The definitive (chorioallantoic) placenta. .........................................48
3.10.1 Microscopic observations ...........................................................48
3.10.2 Electron microscopy...................................................................48
3.11 Chorionic vesicles............................................................................50
3.12 Glandular epithelium. ......................................................................53
CHAPTER 4.0 Discussion ......................................................................54
4.1 Taxonomy, characteristics and distribution. .....................................54
4.2 Litter size .........................................................................................55
4.3 The ovary.........................................................................................56
4.4 The reproductive tract, placenta and accessory structures ................57
4.5 Chorionic vesicles............................................................................64
4.6 Summary and future research needs. ................................................66
CHAPTER 5.0 References .....................................................................67
IV
Abstract.
The histology of the reproductive tract and the ultrastructure of the
chorioallantoic placenta of the lesser bushbaby (Galago senegalensis) was
studied in four specimens obtained from the field and fixed promptly with
glutaraldehyde. An overview of the main results reveals that implantation is
superficial and the placenta is of the epitheliochorial and diffuse type.
A corpus luteum of pregnancy is absent during the later stages of pregnancy.
The trophoblasts of the chorionic villi other than those lining the chorionic
vesicles are characterized by the presence of many lipid droplets. It was
inferred that the significance of these droplets could be that they serve as a
store of cholesterol, which is the principle substrate for the biosynthesis of
progesterone (in the absence of a corpus luteum of pregnancy).
In the later stages of gestation, the fetal capillaries indented the trophoblastic
epithelium. In addition chorionic vesicles, similar to those reported by King
(1984) in the Galago crassicaudata, are observed to develop with advanced
pregnancy. These structures grossly appeared as placentomes on the
placental surface giving the impression that the placenta is cotyledonary in
nature. The trophoblasts lining the chorionic vesicles were generally
columnar with the apical surfaces bulging outward to form a dome shape.
V
There were deep clefts between these trophoblasts, and these clefts were
occupied by abundant microvilli of adjacent cells. These probably constitute
surface specializations that increase absorptive surface area. Many coated
pits and vesicles were also observed on the apical surface of these cells.
Maternal glandular epithelium consisted mainly of columnar epithelial cells.
These had abundant flattened cisternae of granular endoplasmic reticulum
usually with an apical-basal orientation. Their nuclei had abundant
euchromatin relative to the amount of heterochromatin. They also had a
prominent Golgi apparatus quite characteristic of protein synthesizing cells.
Granulated cells (mast cells) were observed within the maternal connective
tissue. These were identified by their characteristic metachromatic staining
with toluidine blue. Langerhans cells were observed as scattered clear cells
with dark staining nuclei within the non-keratinized stratified squamous
epithelium of the vestibulum. The roles of the mast cells and Langerhans
cells are discussed.
It was concluded that the morphology of the reproductive tract of
Galago senegalensis does not differ from that of other lower primates whose
placentae are also epitheliochorial and diffuse in nature.
VI
Acknowledgments
I am grateful for the financial support given by the University of
Nairobi in the form of a post graduate scholarship.
I wish to express my sincere appreciation to Prof. G. E Otianga-Owiti
and Prof. Oduor-Okelo for their invaluable advice and supervision during
the period of this study.
I also wish to thank those who provided the necessary technical
assistance in histology, electron-microscopy and photography, Messrs. P.K
Kiguru, S. Ochieng and J.M Gachoka of the Department of Veterinary
Anatomy and C. Wells of the International Livestock Research Institute
(ILRI).
My appreciation to the entire Ngoroi family for their moral and
financial support and encouragement.
VII
CHAPTER 1.0
1.1 GENERAL INTRODUCTION
Bush babies belong to the order Primate. This order is divided into
two sub orders namely; Prosimii (lower primates) where the bush baby
belongs and Simii (higher primates) which includes monkeys, baboons,
gorillas chimpanzees and man. In the area of primate taxonomy, studies of
the simian primates are more extensive mainly because they are diurnal
animals living in a sensory world similar to that of man where vision
dominates and most of the species are terrestrial and thus accessible for
observation.
Evolutionary studies show that the simians are the closest kin to man
showing great similarities with respect to morphological and behavioural
pattern (Hill, 1953, Napier and Napier, 1967). As a result of these
similarities, simians have been used as models in various aspects of human
research studies including the understanding of the virology and
immunology of HIV (Human Immunodeficiency Virus) within the genital
tract (Miller and Hu, 1999; Ambrose et al, 2001). Other aspects include
developmental anatomy, nutritional disorders and effects of drugs (Penniston
and Tanumihardjo, 2001; Platt et al., 2001; Amaral, 2002). They are also
1
used extensively as models for research in human reproduction (Wango,
1990).
On the other hand, prosimians are almost exclusively arboreal, usually
nocturnal (Bearder, 1987), and thus their sensory world is different from that
of man. Prosimians are classified as primates because they retain singular
mobility of the limbs that have long spatulate five digit hands and feet with
opposable thumbs. They also retain a clavicle (Kingdon, 1971).
In these primates, olfaction and auditory senses are generally acute. These
are important in the trailing and locating of prey in the night (darkness).
They are less accessible, and thus more difficult to study especially in the
field as most sleep during the day. Phylogenetically they are farther placed
from man and differ greatly in their morphology and behavior and for this
reason, prosimians have received less attention than their simian
counterparts. Studies on primates would however, be incomplete if
prosimians were to be ignored totally. Wilson (1969) stated that all
published studies on experimental teratology in primates have dealt with the
higher or simian forms. But the search for a suitable animal model for the
preclinical testing of drugs need not necessarily be limited to the higher
primates if a prosimian form can be found that parallels human sensitivity in
a way comparable to that already shown in macaques and baboons. The ideal
2
animal would be a small primate that is easy to handle and maintain, has a
short gestation period and produces more than one offspring per year.
Interest in prosimians has been aroused by the hope of tracing the
evolutionary sequence of primates and also in the search for more nonhuman models for use in biomedical research. In order to identify an
appropriate
model, basic
information about their physiology and
morphology is necessary to determine the phylogenetic proximity to man.
Other factors such as availability, ease of handling, adaptability to life in
captivity, size (which determines space requirements and husbandry costs)
and length of gestation also play an important but mostly secondary role to
phylogenetic proximity in selecting an appropriate model.
Available information on the ultrastructure of the female reproductive
system and especially the placenta of the lesser bush baby (Galago
senegalensis) is inadequate and reference is made to other studies on
primates in drawing similarities and comparison. In addition most of it is not
specific to the species. Therefore, the objective of this study was to examine
the histology and the ultrastructure of the placenta and the female
reproductive tract of the lesser bush baby (Galago senegalensis) and
compare with information available on other lower primates and primates in
general.
3
1.2 LITERATURE REVIEW
1.2.1 Prosimian classification
There are numerous systems of primate classification, each with its
own merits. Most primate systematists classify them into lower (prosimians)
and higher (simian) primates (Simpson, 1945; Hill, 1953; Napier and Napier,
1967; Kingdon, 1971). A general classification of the primates is shown in
Chart 1 (modified after Young, 1962)
4
ORDER
SUBORDER
SUPERFAMILY
FAMILY
Pongidae
(Apes)
Hominoidae
(Apes and Man)
Anthropoidea
Hominidae
(Man)
Cercopithecoidea
(Old world monkeys)
Ceboidea
(New world monkeys)
(New
Cercopithecidae
Callithricidae
world
Cebidae
Primates
Tarsoidea
Tarsiidae(Tarsiers)
Galagidae
(Bush babies)
Prosimii
Lorisoidea
Lorisidae
(Lorises)
Daubentoniidae
(Aye Aye)
Lemuroidea
Indriidae (Indri
group)
Lemuridae
(Lemurs)
(Lemurs)
Chart 1. A classification of the order primates (adapted from Young, 1962)
Extinct families, according to Young, have been excluded. [Classification based on muscular and
skeletal systems.]
5
GALAGIDAE
1) Genus; Galago
a) Sub genus; Galago
Species; Galago senegalensis
Galago gallarum
Galago moholi
b) Sub genus; Euoticus
species; Galago elegantulus
Galago matschiei
2) Genus; Galagoides
a) Sub genus; Galagoides
Species; Galagoides demidoffi
Galagoides thomasi
Galagoides zanzibaricus
b) Sub genus; Schiurocheirus
Species; Galagoides alleni
3) Genus; Otolemur
Species; Otolemur crassicaudata
Otolemur garnetti
Chart 2. Members of the family galagidae. (adapted from Olson, 1979,
1986)
6
Olson (1979, 1986) identified 11 species and classified the greater
galagos in a distinct genus, the 'Otolemur'. However, Eaglen and Simons
(1980) argue that species level separation is appropriate and there is no
justification for placing these Galagos in a distinct genus.
The criteria used in distinguishing different species of the genus
Galago vary widely. Zimmerman et al (1988) assert that, in addition to
different geographical habitats, striking divergences between vocalizations
of the Senegal and South African lesser bush babies detected using
spectrograms and oscillograms also provide strong support for the separation
of the two forms into distinct species, Galago senegalensis (G. senegalensis)
and Galago moholi (G. moholi).
1.2.2 Ecology and social organization
Bush babies are distributed exclusively in Africa with upto eleven
phenotypically recognized distinct species (Nash et al., 1989). In Kenya six
species are to be found, four of these being lesser Galagos namely; G.
senegalensis, G. zanzibaricus, G. gallarum and G. thomasi. The other two
species are greater Galagos namely; G. crassicaudata and G. garnetti
(Eaglen and Simons, 1980; Nash et al., 1989).
The G. senegalensis commonly known as the lesser bush baby
occupies a geographic range that stretches across Africa from Senegal to the
7
Gulf of Aden, in the area between the Sahara and the coastal forest in the
west and the Congo basin in central Africa (Hill, 1953; Napier and Napier,
1967; Nash et al., 1989). In East Africa, it is found throughout Kenya,
Uganda and Tanzania where it inhabits the savannas and bush land
(Kingdon, 1971).
Bush babies are nocturnal arboreal animals although movements
between sleeping sites during the daytime have been reported in response to
heavy rainfall or extremes of temperature and when food is in short supply
(Kingdon, 1971). Their social organization consists of groups of upto six
members of the same polygamous family (Kingdon, 1971). These animals
are omnivorous in the wild feeding on insects, small birds, acacia gum, fruit,
seeds and nectar. They use a specialized tooth-scraper or tooth-comb
(procumbent incisors and canines of the lower jaw) to scrape hardened gum
from the surface of trees (Bearder, 1987). Both eyes and ears are important
in locating prey (Kingdon, 1971). It has been observed that G. senegalensis
will not breed when the diet is deficient in protein or will turn to feeding on
their young. This phenomenon has been suggested for all insectivorous
prosimians (Doyle, 1974).
Communication is by olfactory and auditory signals. Other calls of
social significance such as clucking vocalization during courtship or
8
threatening sounds when aggression is imminent, have also been reported
(Tandy, 1974).
Defense from environmental hazards, including adverse climatic
conditions and predators has a major effect on the behavior of bush babies.
Predation is countered by a combination of camouflage and direct protection
behind a screen of vegetation. If an attack is imminent the animal will either
escape rapidly or defend itself by biting and spiting (Bearder, 1987).
The families of lorisoidea have distinct styles of locomotion. Lorises
are slow moving quadrupedal climbers while bush babies are vertical
clingers and leapers. Bush babies are active and agile with a maximum
horizontal leap of around 5 meters in the most specialized species like G.
senegalensis and G. elegantulus (Charles-Dominique, 1974; Bearder, 1987).
1.2.3 Characteristics
The G. senegalensis has a gray to brownish gray dorsum, the flanks of
its legs are variable but always distinctly yellow in colour (Kingdon, 1971;
Nash et al., 1989). It has a broad head with a short muzzle and relatively
large eyes. The interocular strip is prominent due to the distinctly dark
circumocular rings. Its tail is not bushy and the nails are not pointed (Nash et
al., 1989).
9
Statistics on the body sizes as given by Nash et al., (1989), Bearder (1987)
and Kingdon (1971) are as follows;
Body weight (g)
112-300(mean =206)
Head and body length (mm)
132-210(mean =65)
Tail length (mm)
195-303(mean =261)
Ear height (mm)
21-57(mean =37)
Hind foot length (mm)
52-78(mean =69)
1.2.4 Reproductive behaviour
The Senegal galago is known to have a life span of upto 14 years in
captivity (Kingdon, 1971). Both male and female G. senegalensis reach
sexual maturity well before 1 year of age. Manley (1966b) observed oestrous
changes in the external genitalia of two females at 196 and 209 days of age.
One of these females copulated and conceived during her third oestrous
cycle at 257 days of age.
Seasonal breeding is common to all non-human primates under natural
conditions. Field studies generally support the phenomenon that female of
prosimians experience at least two restricted periods of mating and two
pregnancies per year. The data also suggest that the second conception in
each bimodal season results from postpartum oestrous cycles among females
that had conceived some 4 months earlier in the first period of mating
10
(Doyle, 1974). Ecological conditions such as photo period, rainfall, food
availability and quality influence the onset of ovulation and timing of
conceptions. Within groups, sexual behaviour is generally restricted to
breeding females and a single behaviourally dominant male in non-human
primates (Pope et al., 1987; Ghosh and Sengupta, 1992; Digby, 1999;
Ziegler et al., 2000).
Most prosimians are reported to experience more than one oestrous
period in a year (polyestrous ) under both laboratory and wild conditions.
Studies using G. senegalensis females from Nuba mountains of Sudan have
shown indirect evidence of bimodal seasonality in breeding (Butler, 1967a).
On the other hand, Darney and Franklin (1982) analyzed the oestrous cycle
of laboratory-housed Senegal galago (G. senegalensis) and reported no
seasonal trend in cycling since 5 out of the 11 cycling females in the study
exhibited vaginal estrus during each month. They also reported that cycle
lengths and duration of estrus were consistent for each female but varied
significantly among females. Individuals’ average cycle lengths varied from
29.2 to 39.3 days and duration of oestrus from 4.8 to 6.7 days. Behavioural
oestrus (sexual receptivity), however, persists for only 1-3 days (Butler,
1967a and Doyle et al., 1971)
11
Estimates of cycle length in this species vary according to whatever
facet of the reproductive cycle is being measured. De lowther (1940) used
changes in the external genitalia and sexual behaviour of one female to
measure estrus and estimated the cycle length at 43.5 days with a range of
43-45 days. On the basis of vaginal smears in a single female, PetterRousseaux (1962) reported a mean cycle length of 39.0 days with a range of
36-42 days. Estimates of the cycle length based on changes in the external
genitalia of 4 females studied by Manley (1966a), yielded a mean periodicity
of 31.7 days with a range of 19-31 days. Doyle et al. (1971) reported
subspecies differences in several measures of reproductive activity for G.
senegalensis, but some of the variability in the recorded estimates of cycle
length may well be due to lack of uniformity in the attributes measured.
In general, the Senegal galago follows a typical pattern of the
mammalian estrous cycle but shows a number of special features. During
seasonal periods of sexual quiescence or the diestrous phase of the oestrous
cycle, the vulva of the female G. senegalensis is imperforate. Around the
time of ovulation (which occurs spontaneously), however, the vaginal orifice
opens widely and the labia and clitoris become red and inflated (Butler,
1967b). In G. senegalensis females studied, this swelling persisted for 24-48
12
hours and the epithelial lining of the vagina developed a shiny white
appearance throughout oestrous.
Olfactory signals promote an increased frequency of visits by the
males. The males of G. senegalensis, together with other prosimian genera,
have mating chases in which upto 6 males follow a female at the peak of
oestrus. However the dominant male drives away the subordinate ones.
Ejaculation is accompanied by a loud call by the mating male (Doyle et al.,
1967) and this is sometimes followed by grooming of self or both the female
and male (Bearder, 1987). In G. senegalensis, seasonal breeding occurs with
primary conception peaking between December and March. Post partum
estrus after a gestation period of 120-130 days was a normal feature in this
species and usually occurs between April and August (Hill, 1953; Butler,
1957; Doyle et al., 1967). Post partum oestrous has also been observed in
other members of the lower primate group (Hill, 1953; Ioannou, 1966).
When giving birth the female is generally on her own in a nest
(prepared by herself) or a hollow in a tree. The maximum litter size in the
lesser bush baby is two but more commonly the female gives birth to just
one offspring. The newborn galago weighs about 12 grams and it suckles for
6 weeks and by two months it is able to feed itself. In Galago moholi,
studies on the effect of lactation on inter birth interval have shown that
13
females whose infants died within 3 weeks of birth had significantly shorter
interbirth intervals and post partum anovulatory intervals than did females
who raised their infants until weaning (Izard and Simons, 1987). The
female’s isolation at parturition is of such importance as to prevent
cannibalism of the infant born into social groups. Cannibalism, however
does not seem to be as much of a problem in the G. moholi. Isolation also
affords protection to the infant from predators (Izard and Simons, 1986).
A study done to determine the effect of gravidity status (primigravid
or multigravid) on neonatal mortality and litter size showed that gravidity
status has no effect on the percentage of multiple births in G. senegalensis
(Izard and Simons, 1986). Births generally coincide with warm wet seasons
when food and cover are readily available (Bearder, 1987).
1.2.5 Reproductive anatomy
The gross anatomy of the external genitalia of lesser galago from
newborn to adult has been described in both male and female by Haines et al
(1976). They reported that the penis is perpendicular to the body wall at
birth and by about 8 weeks of age the testes have descended into the scrotum
and remain permanently in the scrotum throughout adult life, the scrotum is
sessile, the penis oriented obliquely rostral, and the genitalia are covered by
luxuriant pelage. In the young female, the clitoris is also perpendicular to the
14
body wall and the labia and vaginal orifice, although not obvious, are
located at its caudal base. In the adult female, the clitoris remains
perpendicular, slender, essentially devoid of fur, and has the urethral
opening in its tip. The labia and vaginal orifice are at its caudal base,
obscured by fur.
The female genitalia of the bush baby is externally represented by a
vulva opening, well developed clitoris and labial folds (Hill, 1953; Haines et
al, 1976). During seasonal periods of sexual quiescence or the diestrous
phase of the ovarian cycle, the vulva of the female G. senegalensis is
imperforate being closed by a thin membrane. Eaton et al., (1973) have also
reported this in the thick tailed bush baby (G. crassicaudata).
Internally, the reproductive tract is represented by a pair of ovaries
(gonads), paired fallopian or uterine tubes, a bicornuate uterus with a small
body, cervix and vagina (Mossman, 1987).
The structural changes in the ovaries, uterus and vagina of the G.
senegalensis at several stages of the oestrous cycle in both wild and captive
animals have been described (Butler, 1967a). The general pattern of these
changes was similar to that seen in other mammals but showed the following
special features: periodic opening and closing of the vaginal orifice; a
prolonged post ovulatory invasion of the uterus and vagina by large numbers
15
of eosinophilic leukocytes; an unusually long life of the corpus luteum of the
non pregnant cycle. Furthermore, the endometrium appears to have a dual
arterial supply like that seen in menstruating higher primates, although the
animals do not menstruate. The coiled elastic endometrial arteries form a
vascular adaptation to permitting rapid dilatation of the uterus during early
pregnancy. The dual endometrial arterial supply consists of straight basal
arteries that supply the glandular zone of the endometrium and the spiral,
elastic arteries in both the myometrium and the endometrium. The
relationship of the spiral, elastic arteries to menstruation in various primates
was investigated and shown to be absent in New World monkeys which do
not menstruate in the accepted sense of the word (Kaiser, 1947a& b). These
species exhibit round cell infiltration of the endometrium, epithelial
disorganization and microscopic haemorrhage. He concluded that the
amount of menstrual flow was proportional to the degree of development of
the spiral arteries.
The urethra perforates the whole length of the peniform clitoris.
However, a peniform clitoris is not peculiar to prosimians as it has also been
observed in lower mammals like rodents where it is referred to as the urinary
papilla (Oduor-Okelo, 1979).
16
Further observations on the general structure and organization of
germinal cords in the ovaries of the lesser bush baby showed that they
occupy a wide area of the ovarian cortex beneath the surface epithelium and
are separated from the medullary region by a zone of definitive, small,
primary follicles (Butler, 1968, 1971). Pope (1982) describes the fine
structure of germinal nests in the adult ovary of the lesser bush baby (G.
senegalensis) and reports that the fine structure of oogonia and oocytes in
the germinal nests parallels that seen during oogenesis in the fetal and
neonatal ovary of human, rhesus monkey and most other mammalian
species. Additionally, pedunculated corpus luteum (CL) in the right ovary of
a cycling lesser bush baby has been described twenty-six days post ovulation
(Butler, 1966).
In early pregnancy the corpus luteum of pregnancy reaches a
maximum diameter of 1.5-2.0 mm but decreases to 1.0 mm by the time the
embryo has grown to a crown-rump (CR) length of 4 mm (Butler, 1960). By
the time the fetus measures 12.0 mm CR length, which is near the end of the
first trimester of pregnancy, the corpus luteum is replaced by a corpus
albicans that lacks luteal cells and is only 0.5 mm in diameter. At 20 mm CR
length, the corpus albicans is still clearly visible. After the first trimester,
17
pregnancy is thought to be maintained by the placenta since no accessory
corpora lutea have been observed at this stage (Butler, 1960).
1.2.6 Maternal recognition of pregnancy
For pregnancy to proceed beyond its very earliest stages, a signal must
be received by the mother from the foetus to ensure that the presence of the
conceptus is recognised. This phenomenon is known as maternal recognition
of pregnancy (Short, 1969; Heap and Perry, 1977; Heap et al., 1981). In
primates, the chorionic gonadotrophin is known to mediate maternal
recognition of pregnancy. The biologically active chorionic gonadotrophin is
secreted by primate embryonic trophoblast cells. This substance prevents the
normal cyclic regression of the corpus luteum and ensures continued
production of progesterone.
(Seshagiri and Hearn, 1993; Lopata et al.,
1995). Progesterone, from ovarian source is the primary determinant of
embryo-endometrial maturation and synchronization for implantation in
primates (Ghosh et al., 1997). A structural similarity exists between
chorionic gonadotrophin molecules in term placentae extracts from man,
apes, prosimian, Old and New World monkeys. This similarity suggests a
function similar to that of human chorionic gonadotrophin (Hobson and
Wide, 1981).
18
In ovine and bovine, there is evidence suggesting that the conceptuses
produce an antiluteolytic agent known as the trophoblast protein-1 for only a
limited time during early pregnancy. This substance prevents the destruction
of the corpus luteum by prostaglandin-F2 released from the non pregnant
uterus (Bazer et al., 1986; Fincher et al., 1986; Knickerbocker et al., 1986)
1.2.7 Implantation and early post-implantation development
Implantation is defined as the stage in early pregnancy during which
the blastocyst looses its free floating status and assumes a fixed position
within the uterus for the purpose of achieving efficient physiologic exchange
to meet the increasing requirements by the foetus (Enders and Schlafke,
1986). The process starts with the apposition of blastocyst to the uterine
surface and is completed when the placenta is formed (Wimsatt, 1975;
Bjorkman, 1976). Three major patterns of implantation are recognized
(Wimsatt, 1975; Dyce et al., 1996) viz.
i) Centric (Superficial): - the blastocyst expands to fill most of the
uterine cavity (e.g. domestic animals, rhesus monkey and baboon).
ii) Eccentric: - the blastocyst implants in a crevice or fold of the uterine
wall (e.g. rat and mouse).
iii) Interstitial: - where the blastocyst penetrates through the uterine
epithelium and becomes completely embedded in the stroma e.g.
19
woman, chimpanzee and hystricomorph rodents (Roberts and Perry,
1974; Oduor-Okelo and Gombe, 1991)
The eccentric and interstitial types of implantation mainly occur in
species in which the blastocyst is small before implantation and either seeks
out a nest in a cleft of the lumen or burrows into the endometrium. The
blastocysts of domestic mammals grow considerably before implantation
and remain centrally within the lumen and thus related to the whole
circumference of the uterus. (Dyce et al., 1996)
Adhesion of trophoblast of the blastocyst to the uterine epithelium, its
penetration, invasion of the endometrial stroma, the dilation of maternal
vessels and establishment of the basic organisation of the placenta all occur
within the first week following the initiation of implantation in the human,
macaques and several other higher primates (Enders, 1995).
Early implantation and development stages of the Olive baboon,
Papio cynocephalus anubis have been studied and the sequence of events
parallel that of the rhesus monkey (Tarara et al., 1987). These events
include; adhesion of a thick trophoblastic plate to a uterine epithelial plaque,
formation of primary villi and later secondary villi by cytotrophoblasts and
lining of large spaces containing maternal blood by syncytiotrophoblasts. In
the Rhesus monkeys, penetration of the uterine epithelium by syncytial
20
trophoblasts occurs only at the margin of the inner cell mass (Enders and
Schlafke, 1986). However in the Rhesus monkey and baboon, implantation
is superficial whereas in the chimpanzee and humans it is interstitial (Enders
and Schlafke, 1986).
During early post-implantation stages in higher primates, the
trophoblast replaces the uterine epithelium and processes of syncytial
trophoblast invade the dilated superficial maternal vessels. In subsequent
lacunar stages there is rapid elevation of the developing conceptus above the
uterine surface as the lacunae enlarge. Cytotrophoblast rapidly enters
maternal vessels and arterioles are partially or completely occluded by
migrating cytotrophoblast (Tarara et al, 1987; Enders and King, 1991;
Blankenship et al, 1993a; 1993b; Enders et al, 1997).
Amoroso (1952) stated that implantation in the lorisoidea is of the
central type with the fetal membranes remaining external to the uterine
tissue. In Galago demidoffi, however, Gerard (1932) described a temporary
decidua capsularis around the early blastocyst, formed presumably as a
result of secondary enclosure and not of true interstitial implantation.
According to Luckett (1977), this condition may be described as `pseudo
interstitial implantation' since the narrow opening of the implantation
chamber is continuous with the main uterine lumen. A similar implantation
21
type has been described in the elephant shrews (Van der Horst, 1950; OduorOkelo, 1979, 1984).
In Galago senegalensis, a unique abembryonic attachment plaque
consisting of large (‘giant’) trophoblast cells has been reported to occur
(Butler, 1967b). The uterine epithelium disappears in the area of the
attachment plaque so that the abembryonic trophoblast cells appear to be
apposed to the basal lamina of the epithelium. At its maximum development,
the plaque occupies about one-fourth to one-half of the circumference of the
blastocyst. It then begins to recede as the uterine epithelium reforms.
1.2.8 Yolk sac placenta
In a survey on the development and structure of the placenta and fetal
membranes of non-human primates, King (1993) noted that most
strepsirhines (prosimii) are characterized by a large yolk sac early in
gestation and a transient choriovitelline placenta. In some haplorhines
(simii), fetal membranes may include a yolk sac, which is unusual because a
secondary yolk sac is formed. The small secondary yolk sac develops in 12to 13-day human and macaque embryos as a result of pinching off of a
portion of the larger primary yolk sac. Development of a secondary yolk
sac in higher primates appears to be related causally to differential rates of
expansion of the blastocyst and primary yolk sac within the simplex uterus.
22
The yolk sac has both synthetic and absorptive functions in early gestation
(Luckett, 1975; King, 1993). The ultrastructure of the yolk sac endoderm
and mesothelium of the African green monkey (Cercopithecus aethiops) has
been shown to be similar to those of comparable stages in other primates
(Owiti et al., 1989).
1.2.9 The amnion
In strepsirhines (prosimii), amniogenesis occurs by folding (Butler
1967b, King, 1993). However in haplorhines (simii) the amnion forms by
cavitation (Luckett, 1975; Enders et al., 1986). At implantation, epiblast
cells begin to show marked evidence of polarity. They form a spherical
aggregate with their basal ends toward the basal lamina and apical ends
toward the interior. This change in polarity of the cells sets the stage for the
formation of an internal space. However, the cytological evidence of
separation of the cells that will form the amniotic epithelium from the rest of
the epiblast is only seen when the cavity begins to form. This cavity appears
within the epiblast and enlarges to become the amniotic cavity (Enders et al.,
1986; Sadler, 1990).
In prosimii the amnion arises by folding of the extra-embryonic
somatopleure to form a sac around the embryo. The double layered
somatopleure, consisting of the ectoderm and somatic mesoderm, is thrown
23
into two cresentic folds. The earliest fold to appear is just in front of the
embryo; later, a second fold arises just behind the embryo. These two folds
advance towards each other over the head and caudal regions. The
concluding step is the fusion of the several layers located at the margin of
the folds. The result is the formation of two separate compound membranes.
The inner membrane is the amnion. It is lined with ectoderm and covered
externally with somatic mesoderm. The amnion fills with fluid transudate
within which the embryo is suspended. It serves to protect the fetus against
drying, allows the fetus to develop unimpeded and also allows for change of
position (Arey, 1974)
1.2.10
The chorion
The outer sac of the somatopleure is the chorion whose component
layers are in reverse order to those of the amnion. The ectoderm is the
covering layer, whereas the mesoderm furnishes the lining. The chorion
covers both the embryo and all other fetal membranes and is separated from
them by the extra embryonic coelom. It goes to constitute the definitive
placenta (Amoroso, 1952; Arey, 1974).
1.2.11
The allantois
The allantois in humans appears at about the 16th day of development.
It however remains as a rudimentary structure but by no means vestigial. Its
24
proximal portion becomes the epithelium of the urachus (Boyd and
Hamilton, 1970; Sadler, 1990).
The allantois in Lorisoidea and Lemuroidea is described as large and
multilobulate, involving the formation of four sacculations of the allantoic
vesicle. This peculiar configuration of the allantois has also been observed in
unrelated species like the hyrax, Procavia capensis (Wislocki and Van der
Westhuysen, 1940; Sturgess, 1948), the cape anteater, Orycteropus
(Mossman, 1957) and the African elephant Loxodonta africana (Amoroso
and Perry, 1964; Perry, 1974). This is thought to be responsible for early
vascularization of the entire chorion by the direct in-growth of the umbilical
vessels following fusion of the primary allantoic lobe with a small localized
area of the chorionic surface to form the chorioallantoic membrane (Hill,
1932).
The allantois arises as an out pouch of the ventral floor of the hind gut
thus it consists of the endoderm and the splanchnic mesoderm. It pushes into
the extraembryonic coelom where it dilates to form the allantoic sac
connected to the hindgut by a narrower allantoic stalk. Fusion of the
allantoic sac with the overlying chorion produces a functionally common
membrane. The allantoic blood vessels ramify, in a process known as
angiogenesis within the combined mesodermal layer of the chorion and the
25
allantois allowing for physiologic exchange between the fetus and its
environment (Amoroso, 1952; Arey, 1974).
Angiogenesis in the placenta refers to the formation of new vascular
beds and is a critical process for normal tissue growth and development
(Reynolds and Redmer, 2001). The mammalian placenta is an organ through
which respiratory gases, nutrients and wastes are exchanged between the
maternal and foetal systems. The rate of transplacental exchange depends
primarily on the rate of placental blood flow. Increased uterine vascular
resistance and reduced uterine blood flow can be used as predictors of highrisk pregnancies and are associated with foetal retardation (Trudinger et al,
1985). The rates of placental blood flow, in turn are dependent on placental
vascularization. Therefore placental angiogenesis is critical for successful
development (Reynolds and Redmer, 2001).
1.2.12
The placental classification and development
Mossman (1937) defined the mammalian placenta as an apposition or
fusion of the fetal membranes to the uterine mucosa for the purpose of
physiological exchange. This definition provides a good working definition
that is broad enough to cover the diverse types of mammalian placentae. The
placenta is a temporary organ found only in eutherian mammals at the site
where the physiologic exchanges between the mother and the fetus occur. It
26
consists of a fetal part and a maternal part (Bjorkman, 1976; Junqueira et al.,
1977). The placenta has enormous structural diversity among different
species. Vascularization of the fetal part of the placenta is effected by the
extra-embryonic splanchnopleure of both the yolk sac and the allantois. This
fetal component can be apposed to the uterine tissue to form the
choriovitelline
and the
chorioallantoic
placentae
respectively.
The
choriovitelline placenta develops early but undergoes rapid or gradual
involution in domestic mammals e.g. horse and carnivores. In some
mammals e.g. the rodents, the yolk sac locally replaces the chorion and thus
forms the outer most embryonic membrane (vitelline placenta). The
chorioallantoic placenta establishes a more widespread vascularization. It is
thus the more efficient organ for mediating physiologic exchange between
parent and offspring (Amoroso, 1952; Bjorkman, 1976; Leiser and
Kaufmann, 1994). Classification, of the chorioallantoic placenta is done
according to several different principles (Amoroso, 1952), namely:
A) Grossly (distribution pattern of the chorionic villi).
On this basis, four types of placental configurations are recognized;
i) Diffuse placenta- villi distributed uniformly on the chorionic sac e.g
sow and mare.
ii) Cotyledonary placenta- isolated tufts of branched chorionic villi
27
(cotyledons) on the chorionic sac. These attach to preformed oval
prominences (caruncles), on the endometrial surface e.g. ruminants.
iii) Zonary placenta- the chorionic villi occupy a gird-like band around the
equator of the sac e.g. carnivores.
iv) Discoid placenta- the chorionic villi are restricted to a disk-shaped area
of the chorion e.g. primates and rodents.
B) The degree to which the fetal membranes are anchored to the
endometrium thus determining the amount of uterine tissue lost at
parturition. On this basis, two types are recognized namely;
i) Non deciduate type- the fetal components are separated from the
maternal components at parturition without loss of the later e.g. horse
and pig.
ii) Deciduate type- the hypertrophied part of the endometrial stroma, the
decidua, is shed with fetal membranes after parturition e.g. human
and guinea pig.
C) Area of contact between the fetal and uterine parts.
An increase in this area enhances the capacity for fetal-maternal
exchange. On this basis, three placental types are recognized namely;
i) Folded placenta- the chorionic and endometrial surfaces interlock with
primary and secondary ridges and corresponding fossae e.g. sow.
28
ii) Villous placenta- branched chorionic villi fit into corresponding
uterine crypts e.g. ruminants and mare, or are freely exposed to
maternal blood e.g. higher primates.
iii) Labyrinthine placenta- chorionic protrusions anastomose to form a
labyrinth e.g. carnivores and rodents.
D) Histologic classification.
The number of tissue layers that separate the fetal and the maternal blood.
Although the fetal component layers remain constant, the number of the
maternal tissue layers varies with the species. On this basis, four
placental types are recognized namely;
i) Epitheliochorial placenta- all the three layers (uterine epithelium,
connective tissue layer and maternal endothelium) are present.
ii) Synepitheliochorial placenta- is similar to the epitheliochorial type but
some trophoblastic cells-called the binucleate giant cells migrate to the
maternal side and fuse with uterine epithelial cells to form hybrid
synplasms or a fetomaternal syncytium at the fetomaternal interface
(Wooding, 1992).
iii) Endotheliochorial placenta- the uterine epithelium and connective
tissue layers are absent but the maternal endothelium is present.
iv) Hemochorial placenta- all the three maternal tissue layers are absent.
29
The trophoblast is exposed to maternal blood. In this type of placenta
the fetal epithelium may consist of one, two or three layers of
trophoblasts (Enders, 1965). These placentae are referred to as
hemomonochorial (woman, guinea pig, chinchilla), hemodichorial
(rabbit) and hemotrichorial (rat and mouse) respectively.
The histologic system of classification has enjoyed greater popularity since
it seems to define different degrees of placental permeability that is
supposedly less in the 'primitive' epitheliochorial than in the 'advanced'
hemochorial type (Dyce et al, 1996). Grosser (1927), believed that the
barrier separating fetal from maternal blood becomes thinner, simpler and
more efficient both ontogenetically and phylogenetically. It is now known
that species low in the phylogenetic scale possess an 'advanced' placenta e.g.
rodents. Also closely related species can exhibit wide differences in their
placental types for instance carnivores are generally thought to have the
endotheliochorial type of placenta but the hyena has the hemochorial type
(Wynn and Amoroso, 1964; Dempsey 1969; Oduor-Okelo and Neaves,
1982). Grosser's (1927) emphasis upon the thickness of the barrier also
failed to draw attention to the proven active processes of placental transfer
such as those accomplished by phagocytosis by the yolksac/choriovitelline
placentae or by paraplacental regions as exemplified by the marginal
30
hematomas of carnivore placentation (Dempsey, 1969; Leisser and
Kaufmann, 1994).
The growth and differentiation of primate placenta show all variations
between the typical labyrinthine placenta in the lower primates and the
typical villous type seen in the greater apes and man (Amoroso 1952).
The placentae of many of the lorisidae and lemuridae are of the simple
diffuse, indeciduate and epitheliochorial type where the outer membrane of
the embryo (the chorion) is in contact with the whole of the uterine wall and
the maternal and fetal blood streams are separated by a six layer barrier
across which physiologic exchange takes place (Napier and Napier, 1967).
After birth the placenta is stripped off the uterine wall bringing no maternal
layers with it (non-deciduate type). This is quite unlike those of higher
primates, which are discoidal, deciduate, and hemochorial type (Amoroso,
1952; Napier and Napier, 1967). However, Gerard (1932) observed that in
the development of its placenta, Galago demidoffi exhibits certain unusual
and quite unexpected features that distinguish it from all other prosimii so
far investigated. In this animal there is found an area of endotheliochorial
placentation surrounded on all sides by a diffuse epitheliochorial region. In
this area, referred to as 'Zona d' implantation', the uterine epithelium
disappears and is replaced by a layer of trophoblastic cells. Here the
31
maternal capillaries form a rich plexus that is in contact with the
trophoblastic cells thus establishing an endotheliochorial type of placenta.
According to Hill (1932), the relation between fetal and maternal
circulations in the prosimii placenta is brought about by the formation of
vascular trophoblastic villi that fit into crypts lined by a persistent uterine
epithelium. The uterine epithelium persists throughout pregnancy and is said
to be actively secretory in galago (Amoroso, 1952). In higher primates the
placenta remains actively secretory throughout pregnancy (Fazleabas et al.,
1993).
The uterine glands in the galago, open for the most part in groups,
their openings being on depressed areas of the mucosa opposite which are
specially modified absorptive areas of the chorion termed the chorionic
vesicles (Hill, 1932; Amoroso, 1952; King, 1984). The fine structure of the
placental villi and the chorionic vesicles of the Galago crassicaudata
suggests that the placenta is of the diffuse epitheliochorial type and the
chorionic vesicles are invaginations of the chorion opposite the mouths of
the uterine glands (King, 1984). The trophoblast of the placental villi is
engaged in both hemotrophic and histotrophic nourishment of the embryo
and the specialized chorionic vesicles are particularly important in providing
histotrophic nutrients to the embryo, especially secretions of the uterine
32
glands. The mesodermal component of the chorionic vesicle of the G.
crassicaudata includes a capillary network and a layer of smooth muscle
cells (King, 1984).
The chorionic villi are seen in their simplest condition in the lorisidae
where they appear as simple nodular processes separated by extremely thin
walls of the uterine crypts (Amoroso, 1952). However, in the galago they are
finely branched and show a distinct tendency to be polygonal. In lemuridae
they attain a much more extensive development and take the form of large
leaf-like folds. Amoroso (1952) further observed that the mesodermal core
of the villi is very vascular and the capillary vessels immediately underlie
the covering epithelium but nowhere do they indent the trophoblast cells.
However, electron microscopic studies reveal that the fetal capillaries indent
the trophoblastic cells later in gestation, and the trophoblast over the
capillaries correspondingly became thinner with advancing gestation (King,
1984).
The mode of chorionic apposition in galago is such that there is a
separation between the endometrium and the tips of the chorionic villi.
These spaces are filled with histotrophic material derived from the uterine
epithelium (Amoroso, 1952; King, 1984).
33
A comparison of the placenta and fetal membranes of the strepsirhini
(prosimii) and haplorhini (simii), with the exception that of the Tarsius
which is constantly being shifted from one suborder to another, suggests that
the placenta and fetal membranes of the strepsirhini and haplorhini are
radically different (Butler, 1982; King 1993) (see table 1).
34
Table 1 Summary of the major features of the placenta and fetal
membranes of the strepsirhini and haplorhini.
Strepsirhini
Haplorhini
Trophoblast;
a)Non invasive cytotrophoblasts
+
b)Invasive syncytiotrophoblast
+
c) Rauber's layer (Polar trophoblast)
+
Implantation
a) Interstitial
+
b) Superficial
+
Blastocyst attachment
a) Paraembryonic
+
b) Embryonic
+
Amniogenesis
a) Folding
+
35
b) Cavitation
+
Choriovitelline placenta
a) Present
+
b) Absent
+
Allantoic vesicle
a) Large and permanent
+
b) Small and rudimentary
+
Chorioallantoic placenta
Diffuse, epitheliochorial
+
b) Discoidal, hemochorial
+
Note: Some strepsirhini may have small areas of transition invasive
trophoblast [Adapted from Butler, (1982)].
36
1.3 Aims and objectives:
From the literature review it is apparent that the available information
on the morphology of the female reproductive system and especially the
placenta of the G. senegalensis is inadequate and reference is made to other
studies on primates in drawing similarities and comparison. In addition,
most of it is not specific to the species. The objective of this study is
therefore to describe the morphology of the female reproductive tract of the
lesser bush baby with special emphasis on the fine structure of the placenta.
37
CHAPTER 2.0 MATERIALS AND METHODS
2.1 Animal specimens
A total of four female lesser bush babies were used in this study.
These animals were captured at Kilimambogo area of Thika district by use
of baited traps that were set up at night when these nocturnal animals emerge
from the sleeping nests to forage. Acacia gum was used as bait placed in the
trap such that the animal has to squeeze its whole body through a narrow
entrance to get to the bait. Once in, the animal was trapped and later put in a
gunny bag for transportation to the laboratory. In the laboratory, they were
kept separately in wire cages measuring 2 cubic feet for a day and supplied
with fruits (bananas) and fresh drinking water.
2.2 Dissection and fixation.
In preparation for dissection, each animal was deeply anaesthetized by
intramuscular injections of a combination of Ketamine hydrochloride
(15mg/kg body weight) and xylazine hydrochloride (0.5mg/kg body weight).
The animals were weighed, their body lengths measured and their respective
measurements recorded. The animals were laparotomised by use of a scalpel
blade, the abdominal aorta located and cannulated cranial to the ovarian
arteries. Two bush babies were found pregnant on laparotomy. The bush
babies were euthanized with an intravenous overdose of sodium
38
pentobarbitone (200mg/ml). Immediately the caudal vena cava was opened
and perfusion (through the cannulated abdominal aorta ) by gravity of the
lower trunk, genitalia and limbs was done first with 0.85% sodium chloride
(physiological saline) warmed to 35C to clear the tissues of blood. This was
done for 3 minutes or where necessary, to effect.
2.3 Tissue fixation.
Fixation of tissues was done both by perfusion and immersion to
achieve good and thorough fixation especially for the early conceptus and
placenta. Tissues from the two pregnant bush babies were fixed for
transmission electron microscopy while the non-pregnant ones were fixed
for histology. Fixation followed clearing of tissues using warm saline and
the fixatives used were either Bouin's or glutaraldehyde fixative (depending
on whether the tissues were to be processed further for light or electron
microscopy) for 10-15 minutes. 2.5% glutaraldehyde mixture in 0.1M
cacodylate buffer (pH=7.2) was used for electron microscopy whereas other
tissue samples were fixed in Bouin's for routine histology.
After successful fixation, as evidenced by the hardening of the organs,
the whole reproductive tract and placenta (where present) were dissected
out, measured, weighed and immersed in their respective fixatives for later
processing. The various portions of the reproductive tract were identified,
39
cut into smaller pieces and processed accordingly for both light and electron
microscopic studies. These cut sections were further fixed by immersion into
appropriate fixatives.
2.4 Processing for histology
The Bouin's solution fixed tissues were processed for routine
histological sections. This involved dehydration through ascending
concentrations of ethanol (50%, 70%, 90% and 100%), clearing using
methyl benzoate and infiltration and embedding in molten wax. The
embedded tissues were then mounted on wooden blocks and 5mm thick
sections cut with a sliding microtome. The sections obtained were
subsequently stained with haematoxylin and eosin and examined for light
microscopy studies.
2.5 Processing for electron microscopy
Tissues fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer were
subsequently diced, washed in 0.1M cacodylate buffer, post fixed in 2%
osmium tetroxide for 2 hours at room temperature, dehydrated through
ascending concentrations of ethanol (50% for 15 minutes, 2 changes of 70%
for 10 minutes each, 2 changes of 80% each for 15 minutes, 2 changes of
95% each for 15 minutes and finally 2 changes of 100% each for 30
minutes) and embedded in epon resin. Semi-thin sections were cut with glass
40
knives on a Sorvall MT-1 `Porter Blum' microtome and stained with
methylene blue or toluidine blue stain and examined with the light
microscope for the purpose of tissue selection and orientation. Ultra thin
sections were cut with diamond knife (Diatome) using an Ultra Reichert
microtome. These were subsequently mounted on copper grids and double
stained with uranyl acetate followed with lead citrate and examined with a
Zeiss EM microscope.
41
CHAPTER 3.0 Results
3.1 General remarks
The lesser bush babies examined in this study were adult females and
their body weights ranged from 151.08 g to 191.90 g (Table 2), with the
mean body weight being 171.45 g. The body morphology of a typical female
lesser bush baby is shown in figures 1. They have a broad head with a short
muzzle and prominent dark circumocular rings. They have large ears and
large eyes for night vision. The hind legs are longer and more powerful than
the forelegs to aid in jumping (like a kangaroo). The fingers and toes are
flattened at the ends with pads of thick skin to give a better grip on tree
trunks and branches. The back foot index toe has extended claw to assist in
grasping. The animals have a silvery gray to brown woolly fur and the flanks
of their legs are yellow in colour.
Each animal has two ovaries, two oviducts and a bicornuate uterus
connected to the vagina via a cervix simplex (Figs 2, 3 &4).
3.2 The ovaries
3.2.1 Non-gravid animals
3.2.1.1 Macroscopic observations
The ovaries of the non-pregnant animals are small, oval and
flattened. Both ovaries are approximately equal in size in the non42
pregnant animals. The ovaries measured about 4 x 3 x 2 mm and their
shapes varied from round to oval. Each ovary is enclosed by an extensive
ovarian bursa consisting of the mesovarium and mesosalpinx.
3.2.1.2 Microscopic observations
The two zones (i.e. the cortex and the medulla) of the ovary are
clearly distinguishable (Fig. 5). The ovary of the non-pregnant lesser
galago has a single layer of low cuboidal cells forming the surface
(germinal) epithelium. Beneath the surface is a continuous layer of tunica
albuginea (Fig. 6). This is made up of dense regular connective tissue
fibers, running parallel to the surface. The outer zone, the cortex,
contains follicles and interstitial gland masses (Fig 6). The follicles
appear to be in various stages of development. A vesicular follicle shows
inner and outer granulosa. An antrum was present in most of the
secondary and vesicular follicles. Secondary, vesicular and atretic
follicles have distinct granulosa cells and thin thecal cell layers (Fig 7)
3.2.2 Gravid animals
The ovary of the pregnant bush baby was found to be nonlobulated and generally smooth. Histologically the cortex was
characterized by the presence of degenerating corpus albicans and
follicles at varying stages of development. No apparent corpus luteum
43
verum was observed. The medulla was reduced to occupy a small area at
the center of the ovary. (Fig 8).
3.3 The oviduct
The oviduct was a thin tortuous tube attached to the body wall by an
extensive delicate and translucent mesosalpinx. In the non-pregnant animals
the oviduct is more convoluted than in pregnant ones. Histologically the
oviduct could be distinguished into the usual four parts: the infundibulum,
the ampulla, the isthmus and the intra uterine part. The initial portion of the
oviduct wall (infundibulum and its fimbriae) approximates the ovary and is
attached to the body wall by a translucent mesosalpinx. The infundibulum
and its fimbriae is made up of an inner mucosal layer thrown into numerous
folds and a thin outer connective tissue covering (Fig. 9). The ampulla wall
consists of a mucosal layer and a muscular layer with a thin inner circular
and a thin outer longitudinal layer. A layer containing blood vessels (stratum
vasculare) is observed between the circular and the longitudinal muscle
layers at the point of attachment of the mesosalpinx to the oviduct. The outer
layer (tunica serosa) consists of loose connective tissue lined by a simple
squamous epithelium (Fig 10). The mucosal layer exhibits primary and
secondary longitudinal folds formed by the epithelial lining and a lamina
propria core. These folds are lower and their number less than those
44
observed in the infundibulum. The epithelium is of the simple columnar type
(Fig. 11). The muscle layer is best developed in the isthmus with a thick
inner circular layer surrounding a mucosal layer (Fig. 12). The mucosal layer
is thrown into simple folds, with no secondary branching, that are lined by a
simple columnar epithelium (Fig. 13).
3.4 Uterus
3.4.1 The non-pregnant uterus
3.4.1.1 Macroscopic observations
The non-pregnant uterus of the lesser galago is V-shaped with two
uterine horns continuing from a small uterine body of about 1-cm in length.
The uterus is suspended on either side by an extensive and transparent
mesometrium.
3.4.1.2 Light microscopy
The uterine wall is composed of an endometrium with numerous
longitudinal folds, a myometrium and a perimetrium (Fig. 14). The structural
appearance of the endometrium depends on the stage of the estrous cycle
and pregnancy. The folds carried a pseudo stratified type of epithelium (Fig.
15). The myometrium is principally composed of two smooth muscle layers
(inner circular and outer longitudinal layers) of near equal thickness (Fig.
16). The perimetrium contains collagenous fibers covered by a simple
squamous epithelium.
45
3.5 The cervix
The cervix is a thick walled structure of about 0.5-cm in length and
width. It has round internal (ostium uteri internum) and external (ostium
uteri externum) cervical orifices. The mucosa of the cervical canal is
elaborated into a series of fairly parallel longitudinal folds that together with
the mucus plug tightly seals the cervix during pregnancy. The cervical wall
consists of a muscular layer that is constituted of fairly thick and distinct
inner circular muscle layer with a thin less distinct outer longitudinal muscle
layer (Fig 17) which is continuous with that of the vagina.
The propria-submucosa contains very dense irregular connective tissue and
glands that open at the base of the mucosal folds (Fig 18). The cervical
lining has a non-keratinized stratified squamous epithelium (Fig. 19).
3.6 The vagina
The vagina is a relatively long and firm tubular structure. In the
pregnant state the vagina is large and patent. It is thrown into a series of
fairly high longitudinal folds that gave its inner surface a corrugated iron
sheet-like appearance (Fig. 20). The propria-submucosa contains dense
regular connective tissue. The tunica muscularis is composed of two smooth
muscle layers, a thin distinct inner circular and a distorted outer longitudinal
layer (Fig 21). The mucosa-submucosa occupies about half of the inner wall
46
thickness, with the muscularis occupying the outer half of the vaginal
thickness covered by a thin tunica adventitia (Fig 21). The vagina is lined by
a non-keratinized stratified squamous epithelium made up of three to five
layers of cells (Fig 22). Externally a loose connective tissue layer (tunica
adventitia) covers the muscular layer.
3.7 The vestibulum vaginae
The vestibulum vaginae is closely related to the rectum dorsally and
urethra ventrally. The epithelium of the vestibulum is stratified squamous of
about 5-6 cell layers thickness but terminally it is continuous with the skin.
Langerhans cells are observed as scattered clear cells with dark staining
nuclei within the epithelium (Fig. 23)
3.8 The clitoris
The clitoris appears as a long peniform structure that has the urethra
perforating it along its length so as to open at the tip (Fig. 24).
Histologically, an outer keratinised stratified squamous epithelium (Fig. 25)
and a corpus cavernosum clitoridis surrounded by a tunica albuginea (Fig.
26) characterizes the clitoris.
47
3.9 The pregnant uterus
The pregnant uterine horn is recognizable by a single locular swelling
(Figs. 4& 27). Blood vessels formed a complex network on the mesometrial
side of the pregnant horn.
The earliest stage of development obtained is represented by bush
baby B. the blastocyst is centrally implanted and the depth of implantation is
superficial with the foetal membrane remaining in the uterine lumen. The
trophoblasts form a simple cuboidal to columnar epithelium (Fig. 28).
3.10 The definitive (chorioallantoic) placenta.
3.10.1
Microscopic observations
The interhemal membrane of the chorioallantoic placenta consists of
the fetal and maternal components. The fetal component is constituted by the
trophoblast, fetal mesenchyme and fetal capillaries and the maternal
component is made up of endometrial epithelium, endometrial loose
connective tissue and endometrial capillary endothelium (Figs. 29 a& b).
There is no erosion of the maternal tissue.
3.10.2
Electron microscopy
3.10.2.1
Fetal component.
This is constituted by the trophoblast epithelium, a basal lamina and
the fetal capillary endothelium. The layer of trophoblasts is cellular (i.e.
48
cytotrophoblasts) layer rather than syncytial (i.e. syncytiotrophoblast) (Fig.
30). They have a microvillous border that interdigitates closely with
processes on the maternal epithelial cells to form an elaborate junctional
zone (Fig. 31). Small vesicles and tubules, as well as numerous
mitochondria are present in the apical cytoplasm. The Golgi apparatus is
perinuclear in position and small amounts of endoplasmic reticulum (ER)
are present throughout the cells. Apparent tight junctions and occasional
desmosomes joined the cells. Droplets with a homogenous content were
particularly abundant in the basal cytoplasm. These are interpreted to be fat
droplets due to their characteristic dark staining with osmium tetroxide (Fig.
32).
In the later stage of gestation, the fetal capillaries indented the
trophoblastic epithelium and these epithelial cells became thinned over the
capillaries (Fig 29 a& b). The capillaries are surrounded by a well-developed
basal lamina (Fig. 33).
3.10.2.2
Maternal component
This is constituted by the uterine epithelium, basal lamina and
maternal capillary endothelium. The uterine epithelial cells are low cuboidal.
The apical border of the epithelial cells have numerous microvilli
interdigitating with microvilli of the trophoblastic cells. The apical
49
cytoplasm contained vesicular bodies and electron dense granules. The
cytoplasm also contains aggregates of smooth and rough endoplasmic
reticulum (Fig. 34).
Uterine capillaries indent the epithelial cells. The capillaries are of the
continuous type and the endothelial cells had numerous cisternae of rough
endoplasmic reticulum (R.E.R). A thin basal lamina surrounded the
endothelium (Fig 34).
Granulated cells (mast cells) are observed within the maternal
connective tissue (Fig. 29a& b, 35, 36). These are identified by their
characteristic metachromatic staining with toluidine blue (i.e. property of
changing color of blue dyes to purple due to their content of heparin). Their
nuclei appear round in shape and their cytoplasm is filled with many darkstaining granules (Fig. 36).
3.11
Chorionic vesicles
These structures grossly appear placentome-like on the placental
surface (Fig. 37) giving the impression that the placenta is cotyledonary in
nature. Light microscopic of the vesicles reveals a layer of vacuolated
columnar chorionic epithelial cells covering the villi that project into the
lumen of the vesicle. A rich capillary bed was present in the mesodermal
cores of the villi, but the capillaries do not indent the epithelium. Fetal
50
mesenchyme around the vesicles, contained blood and lymphatic vessels
embedded within its loose connective tissue. The other component of the
vesicle wall was a layer of fusiform cells (smooth muscle cells) that did not
extend into the villous cores (Figs 38a & b). A structure that appears as an
extension of the amniotic space and its contents is also observed running
across these pictures (Figs. 38a &b).
The ultrastructure of the trophoblastic epithelium of the vesicle villi
appeared quite different from that of trophoblast elsewhere in the placenta.
The cells are generally columnar with the apical surfaces bulging outward
to form a dome shape (Fig. 39 & 40). There are deep clefts between most of
the cells, and this region was occupied by abundant microvilli of
adjacent cells. At the bases of the clefts, tight junctions are present (Fig.
40).
The lumen of the chorionic vesicle often contained electron dense
material. This material could often be observed in the intercellular clefts
extending to the tight junction, the lateral cell membranes are closely
apposed (Fig. 40).
The apical cytoplasm of the trophoblastic cells contained numerous
vesicles and vacuoles of varying sizes. Often these structures
contained moderate amounts of electron-dense material similar to that
51
present in the lumen. In addition to the numerous intracellular vesicles, the
apical and supranuclear cytoplasm also contain granular and agranular
endoplasmic reticulum and mitochondria (Fig. 41). Deeper in the cytoplasm,
electron-dense granules of varying sizes, and containing finely particulate
material, are observed. The cytoplasm in this region also contains some lipid
droplets, profiles of granular and agranular endoplasmic reticulum, and
numerous mitochondria. The nucleus had more abundant euchromatin
relative to the amount of heterochromatin. The later is organized in clumps
mostly attached to the nuclear membrane. The epithelium rested on a basal
lamina (Fig 40 & 41). Many coated pits and vesicles are observed (Fig. 42).
Connective tissue and its accompanying capillaries occupy the core of the
villi, but the capillaries do not indent the epithelium (Fig. 39). The
endothelium was mainly of the continuous type and had a thin basal lamina.
Another prominent component of the wall of the chorionic vesicle was
several layers of fusiform cells that do not extend up into the cores of the
villi. These cells are clearly identified as smooth muscle by their numerous
cytoplasmic filaments, surface invaginations, and the presence of an external
lamina. The layers numbered between ten and fifteen depending on the site
of count and cells in adjacent layers were joined by cytoplasmic processes
(Fig. 43).
52
3.12 Glandular epithelium.
Tubular maternal glands appear in cross section on semi thin sections
(Fig. 44a & b). Maternal glandular epithelium consists of columnar
epithelial cells. These have abundant flattened cisternae of granular
endoplasmic reticulum usually with an apical-basal orientation (Fig. 45a&
b). Their nuclei have abundant euchromatin relative to the amount of
heterochromatin. They also have a prominent Golgi apparatus (Fig 46),
moderate numbers of secretory granules and multivesicular bodies. Both
elongate and circular mitochondria are observed interspersed between long
cisternae of granular endoplasmic reticulum (Fig 47). Their apical
membranes are modified into numerous small microvilli. Tight junctions are
present between the cells just below the surface. Below the tight junctions,
the lateral cell membranes are closely apposed.
The epithelium rests on a basal lamina. Smooth muscle cells are also
part of this wall and are found interspersed within the connective tissue
below the basal lamina (Fig. 48). The basal surface of the uterine gland cells
have numerous infoldings that in effect increase its surface area.
53
CHAPTER 4.0 DISCUSSION
4.1 Taxonomy, characteristics and distribution.
Bush babies (Galago) are confined to Africa, where there are two
large (> 700 g) and six (6) to nine (9) smaller species (< 400 g) with as many
as three species sharing the same habitat. The confusion regarding the
taxonomy of these primates is largely a consequence of their secretive
nocturnal habitats. Separate species may look superficially similar, but their
behaviour is usually distinctly different and such differences are now being
used to help refine the taxonomy. G. moholi and G. senegalensis for
example, were considered a single species due to their shared habitat,
moderate body size and relatively large eyes. They exhibit important
differences in their contact vocalizations, locomotion, litter size and method
of infant carriage as well as Karyotype (Kingdon 1971; Nash et al., 1989).
The division of living primates into prosimians and simians is a
reflection of major differences in their sensory anatomy and physiology. The
anthropoid primates (simians) have occupied diurnal niches since early
stages of their evolution and their sense organs and perceptual abilities are
adapted accordingly. In contrast, all the prosimians, including those lemurs
that are now diurnal, show the hallmarks of a long history of adaptation to
nocturnal conditions; they have relatively large eyes, sensitive nocturnal
54
visions, large independently movable ears, elaborate tactile hairs (vibrissae)
and a well-developed sense of smell. These sensory specializations are
accompanied by differences in organization of the brain (i.e. areas of the
cerebral cortex representing sensory and motor functions) and by a marked
contrast in social relations and systems of communication compared to
anthropoid primates (Charles-Dominique, 1978; Carlson and Nystrom,
1994). Thus the primate order includes an unusually wide array of social
systems, both diurnal and nocturnal. This study shows details of the placenta
and its accessories of the lesser bush baby confirming this premise that the
primate order includes species with varied placental structures.
4.2 Litter size
The pregnant specimen of the G. senegalensis collected for this study
had a single loculus of one embryo/fetus. This is consistent with other
findings on the same species (Cooper, 1966). However the incidence of
multiple births varies among the lesser bush babies. In captive G.
senegalensis, Cooper (1966) reported predominantly single births with only
rare exceptions. Twins were reportedly uncommon in the Senegal galago
caught in the Sudan (Butler, 1966) and in Uganda (Haddow and Ellice,
1964). This is in contrast to Hill's report (1953), that twinning is the rule in
55
the Galagidae. However, Doyle et al., (1971) recorded 16 sets of twins and
one of triplets in 29 pregnancies among 12 captive G. moholi.
4.3 The ovary
The macroscopic and microscopic structure of the lesser bush baby
ovary does not show any striking deviation from that of other mammals.
Both the ovaries of the pregnant and the non-pregnant lesser bush babies
were enclosed in a voluminous ovarian bursa. They have a distinct tunica
albuginea and showed a number of graafian follicles with antra and solid
follicles with granulosa cells. These findings are consistent with those of
Butler (1967a). A corpus albicans is observed in the ovary of the female
bush baby with the advanced pregnancy. A corpus luteum of pregnancy was
absent having regressed into a corpus albicans and no corpora lutea were
observed. This finding is consistent with that of Butler (1960) who reported
that the corpus luteum is replaced by a corpus albicans at the end of the first
trimester. It has also been reported that in women, monkeys, mares, sheep
and guinea pigs, the corpora lutea, while important in the early stages of
gestation are not essential for its completion (Amoroso and Perry, 1977;
Porter and Amoroso, 1977)
56
4.4 The reproductive tract, placenta and accessory structures
The gross anatomical features of the lesser Galago’s reproductive tract
were similar to those seen in other lesser primates (prosimians). The
reproductive tract differs from that of higher primates in that there is
a long peniform clitoris with the urethra perforating the whole length of the
clitoris. This feature makes sexing quite difficult in live animals as it appears
like a penis.
The oviduct (uterine tube) could not be grossly distinguished into the
four segments that characterize it. This was achieved through microscopy.
The infundibulum appeared as a large opening whose lining was thrown into
numerous tall, branching and intercalating folds. The ampulla appeared as a
thin-walled segment caudal to the infundibulum with lower mucosal folds
and a thin muscular layer. A thick muscle layer and a lining membrane that
had simple and very low folds characterized the isthmus. These findings are
typical to those found in other primates. Cell types of the epithelium could
not be distinguished under light microscopy.
The non pregnant uterus is bicornuate with a small uterine body of
about 1 centimeter in length this is unlike that of higher primates which have
a uterus simplex. Histologically the uterus exhibits the typical 3 layered
structure (i.e. the endometrium, the myometrium and the perimetrium) that is
57
characteristic of other mammals (Bloom and Fawcett, 1986). The
endometrium in the non-pregnant uterus has atrophied uterine glands, some
of which have collapsed lumen. However in the pregnant uterus these glands
hypertrophy and are found opposite foetal membrane structures known as
chorionic vesicles. The ultrastructural characteristics of the uterine glands
suggest active synthesis, packaging and secretory activity due to the
presence of extensive rough endoplasmic reticular system and a well
developed Golgi apparatus. Presence of smooth muscle cells in the wall of
the glands suggests active expulsion of secretion from the lumen of the
glands. Their location opposite the chorionic vesicles is strategic in that the
two structures combine to form an efficient and direct route through which
the developing fetus gets adequate nutrients from the mother. Provision of
adequate nutrition to a fetus is the key to a successful pregnancy. The uterine
glands are an important source of nutrients to the human fetus during
organogenesis, when metabolism is essentially anaerobic (Burton et al.,
2002).
The cervix forms the wall of the cervical canal. Its mucosa is thrown
into longitudinal folds that alternate between low and high folds. Cervical
glands open at the bases of the crypts between these folds. The vagina is
devoid of any glands and has a thin outer adventitial layer, a relatively thick
58
muscular layer that is divided into an inner circular and an outer longitudinal
and a mucosa that is highly folded. The vestibulum vaginae continues the
vagina posteriorly and is characterized by a thicker non-keratinized stratified
squamous epithelium. Langerhans cells were observed within the epithelium
as scattered clear cells with dark staining nucleii. Langerhans cells are
dendritic cells found in the upper layers of the stratum spinosum. There
slender processes extend into the intercellular spaces among the cells of the
stratum spinosum and appear to form an almost continuous network in the
epithelium. They contain numerous small vesicles and multivesicular bodies.
Their most distinguishing characteristic is the presence of peculiar
membrane-bounded rod shaped granules known as the Langerhans cells
granules (Bloom and Fawcett, 1986). Langerhans cells were also observed in
the vestibular epithelium. These cells have been shown to participate in the
body's immune response (Parr et al., 1991; Szabo & Short, 2000). The lower
reproductive tract is an important site of contact with pathogenic
microorganisms. Langerhans cells in the vestibular epithelium are well
positioned to sample antigen in the lumen of the reproductive tract, travel to
the draining lymph node, present the antigen to T lymphocytes and initiate
an immune response. Most cases of primary HIV infection are thought to
involve HIV binding initially to the CD4 and CCR5 receptors found on
59
antigen presenting cells-which include macrophages, Langerhans' cells and
dendritic cells- in the genital and rectal mucosa (Szabo and Short, 2000).
The most widely accepted model for sexual transmission of HIV is that of
non-human
simians
such
as
the
rhesus
macaques
with
simian
immunodeficiency virus. However, there is a possibility that such studies
can be done using prosimians such as the G. Senegalensis as alternatives to
the non-human simians that are higher placed phyllogenetically.
This study has shown that the placenta of Galago senegalensis is of
the epitheliochorial type confirming earlier light microscopic studies in this
and other galago species (Butler and Adam, 1964; King, 1984). The single
layer of cytotrophoblast (cellular trophoblast) is in close contact with uterine
epithelium and a dark line, as seen under a light microscope, marks the line
of contact. Electron microscopic examination of this contact line in this
study and that by King (1984) on the Galago crassicaudata shows that at
this
fetal-maternal contact zone, the
uterine epithelial cells and
cytotrophoblasts are intimately apposed by the interdigitating microvilli of
their apical surfaces. The area of the giant cell cytotrophoblast, as reported
by Butler (1967b), was not observed in this study. This, however, does not
contradict his finding because of the reported transitory nature of this
feature. This area was observed by Butler (1967b) in early embryos that
60
measure 4.0 to 5.0 mm. in diameter. The embryos show centrally implanted
bilaminar blastocysts attached temporarily to the surface of the endometrium
denuded of its epithelium. An area of giant trophoblast cells situated at the
abembryonic pole mediates this attachment. By the time the primitive streak
appears nearly all giant trophoblast cells have degenerated and the uterine
epithelium reconstituted.
One notable observation previously reported in G. crassicaudata by
King (1984) was the indentation of the trophoblast by fetal capillaries.
Indentation was reportedly absent in G. senegalensis (Butler and Adam,
1964) and an additionally unnamed species of galago (Amoroso, 1952). In
this study indentation was evident in sections obtained from the late stage of
placenta. These differences probably relate to gestational stages examined
rather than to species differences. Presence of numerous indenting fetal
capillaries in this species accounts for one of the several similarities between
its placenta and that of other species with epitheliochorial placentae such as
the pig (Bjorkman, 1965, 1973) and horse (Samuel et al., 1976).
In pigs (Friess et al., 1980) and the mare (Samuel et al., 1976), cells
contributing to the interhemal barrier have been observed to have a reduced
number of organelles in the area near the capillaries. This has been said to
contribute to the effective transfer of nutrients with reduced uptake by the
61
intervening cytoplasm. This situation was observed in the present study
study.
Presence of large amounts of lipid droplets in the trophoblasts of the
G. senegalensis placenta confirms the previous observations of Butler and
Adam (1964). King (1984) also reported a similar feature in the greater bush
baby (G. crassicaudata). The significance of these droplets could be that
they serve as a store of low-density lipoprotein cholesterol, which is the
principle substrate for the biosynthesis of progesterone. In the humans, the
principal source of progesterone during pregnancy is the placenta. However
the corpus luteum is the major source during the first six to eight weeks of
gestation (Tulchinsky and Hobel, 1973). During this time, progesterone is
important for the development of a secretory endometrium to receive and
implant a blastocyst. The developing trophoblast takes over as the principal
source of progesterone by eight weeks, since the removal of the corpus
luteum before this time, but not after, leads to abortion (Csapo et al., 1973).
Although the placenta produces a large amount of progesterone, it normally
has very limited capacity to synthesize precursor cholesterol from acetate.
Therefore maternal cholesterol in the form of low-density lipoprotein (LDL)
cholesterol, is the principal substrate for the biosynthesis of progesterone
(Winkel et al., 1980; Simpson and MacDonald, 1981). LDL cholesterol
62
attaches to its receptors on the trophoblast and is taken up and degraded to
free cholesterol which is then converted to progesterone and secreted. In the
absence of a corpus luteum of pregnancy as is seen in this study and also as
was reported by Butler (1960), the placenta could serve as a source of extraovarian progesterone. The physiologic role of progesterone includes binding
to receptors in uterine smooth muscle to inhibit contractility and thus ensure
myometrial quiescence. It also inhibits prostaglandin formation, which is
important in inducing parturition. Cytochrome P-450 enzymes are
responsible for conversion from cholesterol to progesterone. These enzymes
are hydrophobic hemoproteins and are therefore located in the lipophilic
membranes of the smooth endoplasmic reticulum and the mitochondrial
cristae (Bruce, 1990).
The fetal capillaries in the placental villi of G. senegalensis are of the
continuous type, similar in most respects to those described in non-primate
epitheliochorial placentas (King, 1984).
Another feature of the G. senegalensis placenta observed in this study
was the presence of mast cells in the maternal connective tissue. Mast cells
have not been reported before in the placentae of this species. Decidual mast
cells have been observed sparsely distributed in placentae of women who
have carried normal pregnancy to term (Marx et al., 1999). Mast cells have
63
also been observed in the endometrium of mares post partum (Welle et al.,
1997). Mast cells have been associated with stress triggered abortions in
mice being actively involved (as reflected by their degranulation) and acting
as the cellular link between stress and resulting abortions (Markert et al.,
1997). Marx et al., (1999) also suggested that decidual mast cells in women
might play an important role in the onset of abortion due to the production of
cytokines such as tumor necrosis factor-alpha (TNF-alpha). In this study, the
mast cells however do not appear to be taking part in an immune reaction to
reject the fetus as an allograft because they are not degranulated.
4.5 Chorionic vesicles.
These structures were observed opposite openings of uterine glands.
The trophoblastic cells of the chorionic vesicles are characterized by the
presence of vesicles and coated pits. By their location and structure, the
chorionic vesicles appear to be involved in maternal-fetal exchange through
absorption of uterine glandular secretion. King (1984) reported a similar
situation in the greater galago (G. crassicaudata). Ferrous iron has also been
localized in the trophoblastic cells lining the chorionic vesicles and the
uterine glands of the lesser bush baby (Bulter and Adam, 1964) suggesting
the involvement of the chorionic vesicle in absorption of nutrients from the
histiotroph by the fetus.
64
A comparison between the structure of the chorionic vesicles in the
greater galago to that of the areola in the pig suggests that the two structures
are similar in many characteristics except the presence of a layer of fusiform
cells in the wall of the chorionic vesicles of the greater galago (King, 1984).
In pigs it has been shown that proteins in the uterine gland secretions, such
as uteroferrin (involved in iron transport), are almost exclusively absorbed
by trophoblasts of the areolar (Palludan et al., 1969). Friess et al., (1981),
demonstrated electron dense material is absorbed via coated vesicles by the
trophoblasts. In this study, involvement of coated pits and vesicles in the
absorption of uterine content has also been demonstrated. It is therefore
possible that uptake of certain macromolecules from the uterine gland
secretion occurs via this active process. Butler and Adam (1964) localized
iron in the chorionic vesicles and uterine glands of the G. senegalensis.
These lend credence to the notion that the chorionic vesicles may play an
important role in transfer of nutrients such as iron from the mother to the
fetus.
Observations in this study show the presence of a layer of fusiform
cells in the wall of the chorionic vesicle and these characteristically appear
as smooth muscle cells. They may be derived from the fetal mesenchyme
and their location and functional significance is still not clearly understood.
65
Perhaps they regulate the volume of the vesicle as a result of accumulation
of glandular secretions.
4.6 Summary and future research needs.
This study reveals morphological findings that support the
classification of the G. senegalensis placenta as villous and epitheliochorial.
It also reveals the presence of cells such as mast cells within the connective
tissue of the maternal part of the placenta and Langerhans cells in the
epithelial lining of the vestibulum. These cells have also been observed in
other primates, however further studies would be required to ascertain the
exact roles that these cells play vis-a-vis the functions of similar cells
reported in these sites in other primates and mammals in general. In addition
biochemical studies on the placenta would be required to determine if it is a
source (as suspected) of the extra-ovarian progesterone that is required to
carry the pregnancy to term.
66
CHAPTER 5.0
REFERENCES
Amaral D.G (2002). The primate amygdala and the neurobiology of social
behavior: implications for understanding social anxiety. Biol.
Psychiatry 51(1): 11-17.
Ambrose Z, Larsen K, Thompson J, Stevens Y, Finn E, Hu S.I and Bosch
M.L (2001). Evidence of early local viral replication and local
production of antiviral immunity upon mucosal Simian –human
immunodeficiency virus SHIV (89.6) infection in Macaca nemestrina
J. Virol., 75(18): 8589-8596.
Amoroso E.C (1952) Placentation. In: ‘Marshall’s Physiology of
Reproduction Vol 1(2) chapter 15, Parkes A.S, ed, pp. 185-188.
Longmans Green, London.
Amoroso E.C and Perry J.S (1964) The foetal membranes and placenta of
the African Elephant (Loxodonta africana). Phil. Trans. Roy. Soc.
Lond. Ser. B. 248: 1-35.
Amoroso, E.C and Perry, J.S (1977). Ovarian activity during gestation. In:
67
The Ovary, Vol. 2 Lord Zuckerman and Weir B.J, eds. pp. 315-398.
Academic press, London.
Arey L.B (1974) Developmental anatomy; A textbook and Laboratory
manual of embryology. Seventh edition. Saunders company,
Philadelphia.
Bazer F.W, Vallet J.L, Roberts R.M, Sharp D.C and Thatcher W.W (1986).
Role of conceptus secretory products in establishment of pregnancy.
J. Reprod. Fertil. 76: 841-850
Bearder S. K (1987); Lorises, bush babies and tarsiers; Diverse societies in
solitary foragers. In: ‘Primate societies’. Smuts B. B, Cheney D. L,
Seyforth R. M, Wrangham R W, Struhsaker. eds. pp. 11-24,
University of Chicago press, Chicago
Bjorkman N (1965). On the fine structure of the porcine placental barrier.
Acta Anat., 62: 334-342.
Bjorkman N (1973). Fine structure of the fetal-maternal area of exchange in
68
the epitheliochorial and endotheliochorial types of placentation. Acta
Anat. (Basel), 86 [Suppl. 1]: 1-22.
Bjorkman N (1976). Placentation. In: Textbook of Veterinary Histology.
Dellman H. D and Brown E.M eds. Lea and Febiger, Philadelphia.
Blankenship T. N, Enders A. C and King B. F (1993a). Trophoblastic
invasion and the development of uteroplacental arteries in the
macaque: Immunohistochemical localization of cytokeratins, desmin,
type IV collagen, laminin and fibronectin. Cell tissue Res. 272(2):
227-236.
Blankenship T. N, Enders A. C and King B. F (1993b). Trophoblastic
invasion and modification of uterine veins during placental
development in macaques. Cell tissue Res. 274(1): 135-144.
Bloom W and Fawcett D.W (1986) A textbook of Histology. Saunders,
Philadelphia.
Boyd J D and Hamilton W J (1970). The Human Placenta. The Macmillan
69
press Limited, London.
Bruce R.C, (1990). The maternal-fetal placental unit in: Principles and
practice of endocrinology and metabolism. Kenneth L.B, Bilezikian
J.P, Loriaux D.L, Bremner J.W, Rebar W.R, Hung W, Robertson G.L,
Kahn C.R, Wartofsky L. Eds. chapter 111 pp. 887-898. Lippincot co.
Philadelphia.
Burton G.J, Watson A.L, Hempstock J, Skepper J.N and Jauniaux E (2002).
Uterine glands provide histiotrophic nutrition for the human fetus
during the first trimester of pregnancy. J. Clin. Endocrinol. Metab.
87(6): 2954-2959.
Butler H (1957). The breeding cycle of the Senegal galago (Galago
senegalensis senegalensis). Proc. Zoo. Soc. Lond., 129: 147-149
Butler H (1960). Some notes on the breeding cycle of the Senegal galago
(Galago senegalensis senegalensis) in the Sudan. Proc. Zool. Soc.
Lond., 135: 423-430
70
Butler H (1966). Seasonal breeding of the Senegal galago (Galago
senegalensis senegalensis) in the Nuba Mountains, Republic of
Sudan. Folia Primat., 4: 416-423.
Butler H (1967a). The Oestrus cycle of the Senegal bush baby (Galago
senegalensis senegalensis) in the Sudan. J. Zool. Lond., 151: 143-162.
Butler H (1967b). The Giant cell trophoblast of the Senegal Galago (Galago
senegalensis senegalensis) and it’s bearing on the evolution of the
Primate placenta. J. Zool., 152: 195-207.
Butler H (1968). Post puberal oogenesis in prosimiae. In: Int. Cong.
Primates, Vol. 2 Hofer H. O, ed., pp. 15-21.S Krager, Basel.
Butler H (1971). Oogenesis and folliculogenesis. In Comparative
Reproduction of Non human Primates, Hufez E.S, ed., Charles C
Thomas, springfield Illinois.
Butler H (1982). The placenta and fetal membranes of the strepsirhini and
71
Haplorhini. In: ‘The lesser bush baby as an Animal model’ Haines
D.K ed. chapter 10 pp. 183-197 CRC press, Boca Raton, Florida.
Butler H and Adam K.R (1964). The structure of the allantoic placenta of
the Senegal bush baby (Galago senegalensis senegalensis). Folia
Primatol., 2:22-49.
Carlson M and Nystrom P (1994). Tactile discrimination capacity in relation
to size and organisation of somatic sensory cortex in primates: I oldworld prosimian, galago; II new-world anthropoids, saimiri and cebus.
J. Neurosci. 14(3 pt 2): 1516-1541.
Charles-Dominique P (1974). Ecology and feeding behavior of five
sympatric Lorisids in Gabon. In: ‘Prosimian biology’ Doyle G A,
Martin R D and Walker A C eds. Academic press, Lond.
Charles-Dominique, P (1978). Solitary and gregarious prosimians: Evolution
of social structures in primates. In: Recent Advances in Primatology,
Vol. 3: Evolution, Chivers D. J and Joysey K. A eds. Academic press.
London.
72
Cooper R. W (1966). Fourth annual report, Institute of comparative
Biology, Zool. Soc., San Diego, Calif., pp. 5-8.
Csapo A.I, Pulkkinen M.O and Wiest W.G (1973) Effects of luteectomy and
progesterone replacement in early pregnant patients. Am J Obstet.
Gynecol. 115: 759
Darney K. J. Jr. and Franklin L. E (1982). Analysis of the estrous cycle of
the laboratory housed Senegal galago (Galago senegalensis
senegalensis): Natural and induced cycles. Folia Primatol. (Basel),
37(1-2): 106-126.
De lowther, F. L (1940). A study of the activities of a pair of Galago
senegalensis moholi in captivity including the birth and postnatal
development of twins. Zoologica, N.Y., 25: 433-462.
Dempsey E.W. (1969) Comparative aspects of placentae of certain African
mammals. J. Reprod. Fert. Suppl. 6: 189-192.
73
Digby L. J (1999). Sexual behavior and extragroup copulations in a wild
population of common marmosets (Callithrix jacchus). Folia primatol.
(Basel)., 70(3): 136-145.
Doyle G. A (1974) Behavior of prosimians . In ‘Behaviour of non human
primates: Modern Research trends’ Schrier and Stolintz eds. 5: 155353, Academic Press, Lond.
Doyle G. A, Pelletier A and Bekker T (1967). Courtship, mating and
parturition in the lesser bush baby (Galago senegalensis moholi)
under semi-natural conditions. Folia Primat. 7: 169-197.
Doyle G. A, Andersson A. and Bearder S. K (1971). Reproduction in the
lesser bush baby (Galago senegalensis moholi) under semi-natural
conditions. Folia Primatologica (Basel), 14: 15-22.
Dyce K.M, Sack W.O and Wensing C. J. G (1996) A textbook of Veterinary
Anatomy. Second Edition. Saunders Company, Philadelphia.
Eaglen R. H and Simons E. L (1980). Notes on the breeding biology of the
74
thick tailed and silvery Galagos in captivity. J. mammology 61: 534537.
Eaton G. G, Slob A and Resko J. A (1973). Cycles of mating behaviour,
oestrogen and progesterone in the thick-tailed bushbaby (Galago
crassicaudatus) under laboratory conditions. Anim. Behav., 21: 309315.
Enders A.C (1965). Comparative study of the fine structure of the
trophoblast in several hemochorial placentas. Amer. J. Anat. 116: 2967.
Enders A. C (1995). Transition from lacunar to villous stage of implantation
in the macaques, including establishment of the trophoblastic shell.
Acta Anat. (Basel)., 152(3): 151-169.
Enders A. C and King B. F (1991). Early stages of trophoblastic invasion of
the maternal vascular system during implantation in the macaque and
baboon. Am. J. Anat. 192(4): 329-346.
75
Enders A. C and Schlafke S (1986). Implantation in Nonhuman Primates and
in the human. In: Comparative Primate Biology, 3: Reproduction and
Development, 291-310. (Alan R. Liss, Inc. Publ.)
Enders A. C, Schlafke S and Hendrickx A. G (1986). Differentiation of the
embryonic disc, amnion and yolk sac in the rhesus monkey. Am. J.
Anat., 177(2): 161-185.
Enders A. C, Lantz K. C, Peterson P. E and Hendrickx A. G (1997). From
blastocyst to placenta: the morphology of implantation in the baboon.
Human Reprod. Update., 3(6): 561-573.
Fazleabas A. T, Donelly K. M, Mavrogianis P. A and Verhage H. G (1993).
Secretory and morphological changes in the baboon (Papio anubis)
uterus and placenta during early pregnancy. Biol. Reprod., 49(4): 695704.
Fincher K.B, Bazer F.W, Hansen P.J, Thatcher W.W and Roberts R.M
(1986). Ovine conceptus secretory proteins suppress induction of
76
uterine prostaglandin-F2 release by estradiol and oxytocin. J.
Reprod. Fertil. 76: 425-433.
Friess A. E., Sinowatz F., Skolek-Winnisch R and Trautner W (1980). The
placenta of the pig. I. Fine structural changes of the placental barrier
during pregnancy. Anat. Embryol.,158: 179-191.
Friess A. E., Sinowatz F., Skolek-Winnisch R and Trautner W (1981). The
placenta of the pig. II. The ultrastructure of the areolae. Anat.
Embryol. (Berl.), 163: 43-53.
Gerard P (1932) Etudes sur L’ovogenese et L’ontogenese chez les
Lemuriens du genre Galago. Arch. Biol., Paris, 43: 93.
Ghosh D. and Sengupta J. (1992). Patterns of ovulation, conception and preimplantation embryo development during the breeding season in
rhesus monkeys kept under semi-natural conditions. Acta endocrinol.
(Copenh.) 127(2): 168-173.
Ghosh D, Nayak N. R, Kumar P. G, Dhara S and Sengupta J (1997).
77
Hormonal requirement for blastocyst implantation and a new
approach for anti-implantation strategy. Indian J. Physiol. Pharmacol.,
41(2): 101-108.
Grosser O (1927). Fruhentwicklung, Eihautbildung und Placentation des
Menschen und der Saugetiere. Bergmann, Munchen.
Haddow A. J and Ellice J. M (1964). Studies on the bush babies (Galago
spp.) with special reference to the epidemiology of yellow fever.
Trans. R. Soc. Trop. Med. Hyg., 58: 521-538.
Haines D. K, Holmes K. R and Carmichael S. W (1976). Sex determination
of the lesser bush baby, (Galago Senegalensis). Lab. Anim. Sci.
26(3): 430-435.
Heap R.B and Perry J.S (1997). Maternal recognition of pregnancy. In:
Contemporary obstetrics and gynaecology. Chamberlain G.V.P, ed.
pp. 3-7. Northwood publications, London.
Heap R.B, Flint A.P.F and Gadsby J.E (1981). Embryonic signals and
78
maternal recognition. In: Cellular and molecular aspects of
implantation. Glasser R.S and Bullock D.W eds. chapter 19 pp. 311326. Plenum press, New york.
Hill J. P (1932). Croonian Lecture. The developmental history of the
primates. Philos. Trans. Ser. B., 221: 45-178.
Hill W.C.O (1953). Primates: Comparative anatomy and Taxonomy Vol. 1
University of Edinburgh Press. Edinburgh.
Hobson B M and Wide L (1981). The similarity of chorionic gonadotrophin
and its subunits in term placentae from man, apes, Old and New
World monkeys and a prosimian. Folia Primatol (Basel), 35(1): 51
Ioannou J. M (1966) The oestrus cycle of the potto. J. Reprod. Fert. 11:455457.
Izard M. K and Simons E. L (1986). Infant survival and litter size in
primigravid and multigravid Galagos. J. Med. Primatol., 15(1): 27-35.
79
Izard M. K and Simons E. L (1987). Lactation and interbirth interval in the
Senegal galago (Galago senegalensis moholi). J. Med. Primatol.
16(5): 323-332.
Junqueira L.C, Carneiro J and Contopoulos A (1977). Basic Histology.
Lange Medical Publications, Los Altos, California.
Kaiser I. H (1947a). Histological appearance of coiled arterioles in the
endometrium of rhesus monkey, baboon, chimpanzee and gibon.
Anat. Rec. 99: 199-213.
Kaiser I. H (1947b). Absence of coiled arterioles in the endometrium of
menstruating New world monkeys. Anat. Rec. 99: 353-363.
King B. F (1984). The fine structure of the placenta and chorionic vesicles of
the bush baby, Galago crassicaudata. Am. J. Anat., 169(1): 101-116.
King B. F (1993) Development and structure of the placenta and fetal
membranes of non-human primates. J. Exp. Zool., 226(6): 528-540.
80
Kingdon J (1971) East African mammals: An atlas of evolution in Africa
Vol. 1: 273-328. Academic press, London.
Knickerbocker J.J, Thatcher W.W, Bazer F.W, Drost M, Barron D.H,
Fincher K.B and Roberts R.M (1986). Proteins secreted by Day-16 to18 bovine conceptuses extend corpus luteum function in cows.
J. Reprod. Fertil., 77: 381-91.
Leiser R and Kaufmann P (1994). Placental structure: In a comparative
aspect. Exp. Clinical Endocrinol. 102: 122-134.
Lopata A, Berka J, Simula A, Norman R and Otani T (1995). Differential
distribution of mRNA for the alpha- and beta-subunits of chorionic
gonadotrophin in the implantation stage blastocyst of the marmoset
monkey. Placenta, 16(4): 335-346.
Luckett W. P (1975). The development of primordial and definitive amniotic
cavities in early rhesus monkey and human embryos. Am. J. Anat.,
144(2):149-167.
81
Luckett W. P (1977). Early yolk sac development in the dwarf galago
(Galago demidoffi); A model for early human development. Anat.
Rec., 189: 548
Manley G. H (1966a). Reproduction in Lorisoid primates, Symp. Zool. Soc.
London, 15: 493-509.
Manley G. H (1966b). Prosimians as laboratory animals, Symp. Zool. Soc.
London, 17: 11-39.
Markert U.R, Arck P.C, McBey B.A, Manuel J, Croy B.A, Marshall J.S,
Chaouat G and Clark D.A (1997). Stress triggered abortions are
associated with alterations of granulated cells into the decidua. Am. J.
Reprod. Immunol., 37(1): 94-100.
Marx L, Arck P, Kieslich C, Mitterlechner S, Kapp M and Dietl J (1999).
Decidual mast cells might be involved in the onset of human first
trimester abortion. Am. J. Reprod. Immunol., 41(1): 34-40.
Miller C.J and Hu J (1999) T cell-tropic simian immunodeficiency virus
82
(SIV) and simian-human immunodeficiency viruses are readily
transmitted by vaginal inoculation of rhesus macaques and
langerhans’ cells of the female genital tract are infected with SIV. J.
Infect. Dis. 1999: 179 suppl. 3: 5413-5417.
Mossman H. W (1937). Comparative morphogenesis of the fetal membranes
and accessory uterine structures. Contrib. Embryol. Carnegie Inst. 26:
129-246.
Mossman H. W (1957). The fetal membranes of the Aardvark. Mitt. Natf.
Ges., Bern (N.F.), 14: 119-127.
Mossman H. W (1987) Chapt. 28 & 29. Strepsirhini and Tarsidae. In:
Vertebrate Fetal membranes- Comparative Ontogeny and
Morphology; Evolution; Phylogenic significance; Basic Functions;
Research opportunities. pp. 198-207.
Napier J. R and Napier P. H (1967) A handbook of living primates,
Academic Press, New York.
83
Nash L. T, Bearder S. K and Olson T. R (1989). Synopsis of Galago species
characteristics. Int. J. Primatol. 10(1): 57-80
Oduor-Okelo, D (1979). A study of the fetal membranes and placenta of the
African cane rat (Thryonomys swinderianus Temminck) with some
observations on the placentation in the elephant shrews-Family
Macroscelididae. Ph. D. Thesis, University of Nairobi, 1979.
Oduor-Okelo, D (1984). Histology of the chorioallantoic placenta of the
golden-rumped elephant shrew (Rhynchocyon chrysopygus
Gunther, 1881). Anat. Anz., Jena, 157: 395-407.
Oduor-Okelo, D and Gombe, S (1991). Development of the foetal
membranes in the cane rat (Thryonomys swinderianus): a reinterpretation. Atr. J. Ecol. 29: 157-167.
Oduor-Okelo, D and Neaves W. B (1982) The chorioallantoic placenta of
the spotted Hyaena (Crocuta crocuta Erxleben): An electron
microscopic study. Anat. Rec. 204: 215-222.
84
Olson T. R (1979). Studies on aspects of the morphology and systematics of
the genus Otolemur (Primates: Galagidae), Ph. D thesis, University of
London, London.
Olson T. R (1986). Species diversity and zoogeography in the Galagidae.
Primate rep. 14: 213
Owiti G. E, Tarara R. P and Hendrickx A. G (1989). Fetal membranes and
placenta of the African green monkey (Cercopithecus aethiops). Anat.
Embryol. (Berl.), 179(6): 591-604.
Palludan B, Wegger I and Moustgaard J (1969). Placental transfer of iron. R.
Vet. Agricult. Univ. Yearbook 1970, Kopenhagen, pp. 62-91.
Parr M.B, Kepple L and Parr E.L (1991). Antigen recognition in the female
reproductive tract. II. Endocytosis of horseradish peroxidase by
langerhans cells in murine vaginal epithelium. Biol. Reprod., 45(2):
261-265.
Penniston K.L and Tanumihardjo S.A (2001). Subtoxic hepatic vitamin A
85
concentrations in captive rhesus monkeys (Macaca mulatta). J. Nutr.
131(11): 2904-2909
Petter-Rousseaux A (1962). Recherches sur la biologie de la reproduction
des primates inferieurs. Mammalia 26 suppl. No. 1: 1-87
Perry J. S (1974). Implantation, fetal membranes and early placentation of
the African elephant (Loxodonta africana). Phil. Trans. Roy. Soc.
(London), 269: 109-135.
Platt D.M, Rowlett J.K and Spealman R.D (2001). Discriminative stimulus
effects of intravenous heroin and its metabolites in rhesus monkeys:
opiod and dopaminergic mechanisms. J. Pharmacol. Exp. Ther.,
299(2): 760-767.
Pope R. S (1982) Fine structure of germinal nests in the adult ovary of the
lesser bush baby (Galago senegalensis). In: The lesser bush baby as
an animal model. Haines D.K ed. Chapter 14 pp. 247-267 CRC press,
Boca Raton, Florida
86
Pope N. S, Wilson M. E and Gordon TP (1987). The effect of season on the
induction of sexual behavior by estradiol in female rhesus monkeys.
Biol. Reprod., 36(4): 1047-54.
Porter D. G and Amoroso E. C (1977). The endocrine function of the
placenta, In: Scientific foundations of Obstetrics and Gynaecology.
Philipp E. E, Barnes J and Newton M, Eds. pp 675-712, Heinemann,
London.
Reynolds L P and Redmer D A (2001). Angiogenesis in the placenta.
Biology of Reproduction 64: 1033-1040
Roberts C M and Perry J S (1974). Hystricomorph Embryology. In: Symp.
Zool. Soc. Lond. 34: 333-360 (Rowlands I.W and Weir B.J, Eds.)
Academic press, London.
Sadler T W (1990). Langman's Medical Embryology. Sixth edition.
Williams and Wilkins, Baltimore.
Samuel C. A, Allen W. R and Steven D. H (1976). Studies on the equine
87
placenta. II. Ultrastructure of the placental barrier. J. Reprod. Fertil.,
48: 257-264.
Seshagiri P. B and Hearn J. P (1993). In-vitro development of in-vivo
produced rhesus monkey morulae and blastocysts to hatched ,
attached and post-attached blastocyst stages: morphology and early
secretion of chorionic gonadotrophin. Human Reprod. 8(2): 279-287.
Short R. V (1969). Maternal recognition of pregnancy. In: Fetal Anatomy
Wolstenhome G. E. W and O’Connor M eds. pp 2-26. Churchill,
London.
Simpson G. G (1945). The principles of classification and a classification of
mammals. Bull. Of Amer. Mus. Nat. Hist. 85. 1-350
Simpson E R and McDonald P C (1981). Endocrine physiology of the
placenta. Annu. Rev. Physiol. 43: 163
Sturgess I (1948). The early embryology and placentation of Procavia
capensis. Acta. Zool.29: 393-479.
88
Szabo R and Short R.V (2000). How does male circumsion protect against
HIV infection? Brit. Med. J. 320: 1592-1594.
Tandy J. M (1974) Behaviour and social structure of a laboratory colony of
Galago crassicaudata. In ‘Prosimian Behavior’ Doyle G. A, Martin
R. D, Walker A. C (Eds.) pp. 245-259 Academic press, Lond.
Tarara R, Enders A. C, Hendrickx A. G, Gulam Hussein N, Hodges J. K,
Hearn J. P, Eley R. B and Else J. G (1987). Early implantation and
embryonic development of the baboon stages 5,6 and 7. Anat.
Embryol. (Berl.) 176(3): 267-275.
Trudinger B J, Giles W B and Cook C M (1985). Uteroplacental blood flow
velocity-time waveforms in normal and complicated pregnancy. Br. J.
Obstet. Gynecol. 92: 39-45.
Tulchinsky D and Hobel C J (1973). Plasma human chorionic gonadotropin,
estrone, estradiol, estriol, progesterone and 17-hydroxyprogesterone
in human pregnancy III: early normal pregnancy. Am. J. Obstet.
89
Gynecol. 117: 884
Van der Horst, C. J. (1950) The placentation of Elephantulus. Trans. royal
Soc. S. Afr., Cape Town 32: 435-629.
Wango E.O (1990). Non-human Primates as models for research in human
reproduction. Discovery and innovation 2: 33-36.
Welle M.M, Audige L and Belz J.P (1997). The equine endometrial mast
cell during the puerperal period: Evaluation of mast cell numbers and
types in comparison to other inflammatory changes. Vet. Pathol.
34(1): 23-30.
Wilson J. G (1969). Teratological and reproductive studies in non-human
Primates. In: Methods for Teratological Studies in Experimental
Animals and Man. Nishimura H and Miller J. R., Eds. pp 16-33. Igaka
shorin, Tokyo.
Wimsatt W.A (1975). Some comparative aspects of implantation. Biol.
Reprod., 12:1-40.
90
Wislocki G.B and Van der Westhuysen O.P (1940). The placentation of
Procavian capensis, with a discussion of the placental affinities of the
Hyracoidea. Contrib. Embryol. 28: 65-88. Carnegie Inst. Washington
D.C.
Wooding F B P (1992). Current Topic: The synepitheliochorial placenta of
ruminants: Binucleate cell fusion and hormone production. Placenta
13: 101-113.
Wynn R M and Amoroso E C (1964). Placentation in the spotted hyaena
(Crocuta crocuta Erxleben) with particular reference to circulation.
Amer. J. Anat. 115: 327-362.
Young J. Z (1962) The life of vertebrates; Primates pp 602-651. Oxford
university press, London.
Ziegler T, Hodges K, Winkler P and Heistermann M, (2000). Hormonal
correlates of reproductive seasonality in wild female hanuman langurs
(Presbytis entellus). Am. J. Primatol., 51(2):119-134.
91
Zimmermann E, Bearder S. K, Doyle G. A, Andersson A. B (1988).
Variations in vocal patterns of Senegal and South African lesser bush
babies and their implications for taxonomic relationships. Folia
Primatol. (Basel)., 51(2-3):87-105.
92