A morphological and ultrastructural study of the female reproductive tract and placenta of the ( 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 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 ...... 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 , 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 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 . 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 non- human 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 Hominidae (Apes and Man) (Man)

Anthropoidea Cercopithecoidea Cercopithecidae (Old world monkeys)

Ceboidea (New world monkeys) Callithricidae (New world Cebidae Primates

Tarsiidae(Tarsiers) Tarsoidea

Galagidae (Bush babies)

Prosimii Lorisoidea Lorisidae ()

Daubentoniidae (Aye Aye) Lemuroidea Indriidae (Indri group)

Lemuridae () (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 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 , 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, Petter-

Rousseaux (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 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 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; Oduor-

Okelo, 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 12- to 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 high- risk 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 non- 42 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 non- lobulated 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 dark- staining 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 extra- ovarian 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 (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 old-

world 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: 155-

353, 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: 534-

537.

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: 309-

315.

Enders A.C (1965). Comparative study of the fine structure of the

trophoblast in several hemochorial placentas. Amer. J. Anat. 116: 29-

67.

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): 695-

704.

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 pre-

implantation 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. 311-

326. 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 . J. Reprod. Fert. 11:455-

457.

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 to-

18 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 re-

interpretation. 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