FORM AND FUNCTION: THE
INSEPARABLE TWINS IN VETERINARY
MEDICINE
An Inaugural Lecture delivered
at the University of Ibadan
on Thursday, 19h February,' 2009
by
BANKOLE OLUSUI OKE
Professor of Veterinary Anatomy
Faculty dJ Veterinary' Med-t'cine
, University ~f Ibadan,
Ibadan, Nigeria.
UNIVERSITY OF IBADAN
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Ibadan University Press
Publishing House
University of Ibadan
Ibadan, Nigeria.
© University of Ibadan 2009
Ibadan, Nigeria
First Published 2009
All Rights Reserved
ISBN: 978-121- 457- 0
978-978-121-457 -8
Printed by: Ibadan University Printery
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The Vice-Chancellor, Deputy Vice-Chancellor (Administration),
Deputy Vice-Chancellor (Academic), Registrar, Librarian,
Provost of the College of Medicine, Dean of the Faculty of
Veterinary Medicine, Dean of the Postgraduate School, Deans
of other Faculties, and of Students, Directors of Institutes,
Distinguished Ladies and Gentlemen.
Preamble
It is indeed for me a great honour, to be called upon to deliver
this lecture, the 8th in the 2008/2009 series of inaugural lectures
on behalf of the Faculty of Veterinary Medicine, and the 27th to
be delivered by professors of this Faculty since 1976 when
Professor Desmond H. Hill, the founding Dean of the Faculty
delivered the first. This lecture also is the third to be delivered
by my department (Veterinary Anatomy). The first was
delivered by Professor S. F. Amakiri in 1984, followed by
Professor T. A. Aire in 1986. In spite of being the third
inaugural lecturer, I am by no means the third professor to be
produced by the Department. That third chair was occupied by
Professor Oyewale Adeyemo in 1993. I am thus the fourth of
five professors to be produced by the department. Professor S.K.
Onwuka and I are still in the department while the others have
found their ways to other climes. Indeed, the issue of 'turn-
taking' in the delivery of inaugural lectures in the faculty is a
settled tradition as one's turn is determined by one's seniority in
the attainment of the professorial rank. However, Mr. Chairman,
this is the second inaugural lecture from our residential address.
There was no seniority consideration when in December 2005,
Professor Gbemisola Oke gave the first-a case of what men
can do, women can do earl ier.
Inaugural lecturers are expected to give an account of their
contributions to their fields within one hour and in as simplified
a form as possible. Thus, if one's contribution is as varied as
mine is, in a field as challenging as veterinary medicine, the
main task would be to determine what is to be expunged and
what is to remain, and then, to weave a thread to link those that
remam,
During this lecture therefore, I intend to give a glimpse of
my career and highlight the purple patches of my work and in
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the process acknowledge my mentors, those with whom I have
collaborated and those who have assisted me along the way.
Before I go any further in discussing my work, Mr. Vice-
Chancellor, I crave your indulgence to look, no matter how
cursorily or superficially, at the main component of the title of
this lecture, which simply implies that the scope of my research,
which though has its roots in morphology, has wide applications
in veterinary medicine.
Morphology or Anatomy
Morphology is the branch of biology that deals with the form
and structure of organisms without consideration of function.
The term anatomy, which stems from the Greek word
"Anatemnein" meaning "to dissect", "to cut apart" (Budras et a1.
2002), also has similar application but wider dimension in that it
goes beyond dissection or description of form to the
interrelationship between form and function as well as
application to clinical practice. Anatomy is divided into
macroscopic (gross) anatomy, histology (microanatomy), and
developmental anatomy (often called embryology). Anatomy is
reserved for the oldest and most encompassing area called
macroscopic anatomy. In macroscopic' anatomy, gross dissection
assumes a pride of place as the first method of gaining insight
into the organ or tissue.
In our department here at Ibadan, our subjects at the
undergraduate level include gross and microscopic anatomy,
embryology, neuroanatomy, avian anatomy, comparative
aspects of anatomy as well as applied or clinical anatomy. All
these are geared towards a comprehensive understanding of
structure and its relationship to function. Because the surgeon,
however competent, and other health professionals (patholo-
gists, clinicians, e.t.c.) cannot practice their art without a work-
ing knowledge of anatomy, the latter can be described as the
basis for medical practice and the health sciences.
The term histology is derived from the Greek word histos,
meaning tissue (web), and logia (knowledge). The discovery,
development, and use of the light microscope in its various
forms helped scientists to make great advances in the field of
histology and general structure of cells (Heath and Young
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2000). Embedded in the scope of histology is ultrastructure,
where the use of the electron-microscope comes into the study
of cells or tissues. Earlier, before the advent of ultrastructural
studies as enabled by electron microscopy, cell morphology
(histology and cell physiology) and cell biology were treated as
separate entities (King 1966). Ultrastructure constitutes the
bridge between histology and cytology (cell biology). An
ultrastructural study therefore makes cell structure and cell
function interrelated parts of a whole rather than antagonists. An
overview of most experiments and studies involving the use of
electron microscope presents the goal of the experiments and
uses structural profiles to deduce function.
During the past few decades, especially in human healthcare
delivery, there has been an explosion of new techniques for
imaging anatomy in living patients. Examples range from
endoscopy and laparoscopy to computed tomography (CT) and
Magnetic Resonance Imaging (MRI) together with newly
emerging technology of 3D or three-dimensional visualization.
The other types of investigative methods are used to develop
observations, hypotheses, and conclusions in cell and molecular
biology as well as observe behaviour of cell organelles, and
cellular and molecular structures (Wolfe 1993). Some of these
include centrifugation, gel electrophoresis, blotting techniques,
chromatography, polymerase chain reaction, cell culture,
antibody production, DNA sequencing, DNA fingerprinting,
DNA hybridization, and DNA cloning. As of now, cytology is
the rave-with immunocytochemistry playing on the stage with
loud ovation. I have been part of this rave as will be shown later
in this lecture.
The Beginning
During my pre-clinical years, I discovered my flair for anatomy
and the reason is simple. Every fine artist or architect can be
described as an anatomist of some sort as he/she deals basically
with structures. I have always had a flair for fine art and this was
utilized to advantage during my pre-clinical years. I realized
early in the anatomy class that my best strategy for
understanding and easily recollecting things in the dissecting
room was to do a sketch of the details and label instantly. That
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way, it became permanently engraved in my memory. Dr. Tom
Aire, who obviously was similarly endowed and with the benefit
of having expressed the talent more extensively, would instantly
spot my sketches on his supervisory rounds in our dissection
classes. After a few suggestions here and there, my sketches
often became the standard for the rest of the class. This
unknowingly was the beginning of a special mentoring
relationship between Professor Aire and 1. The mentoring was
suspended as I graduated into the clinical years, only to be
reactivated after my NYSC and a short stint at the Ogun State
Veterinary Services as Veterinary Officer II. Professor Aire
invited me to join the staff of veterinary anatomy department
when a vacancy existed. My good friend, Professor C.A.O.
Adeyefa was the one who sought me out in Abeokuta to deliver
the urgent message. At that time, there was no 'GSM' and every
one was at the mercy of 'NITEL'.
I joined the university service in the Department of
Veterinary Anatomy in March, 1980 and I delved into
teaching-the best way I could-with all the artistic flair and
enthusiasm. Nobody hinted me that the best teaching methods
alone cannot take one far. So, I returned to Professor Aire who
laughed at my ignorance of what it took to progress in
academics and he initiated me into the world of scientific
research. This started from his ongoing research work on the
stages of the seminiferous epithelium of the African giant rat
(Aire 1980). The work was classical, following in the mould of
what had earlier been done by Clermont on the seminiferous
epithelium of the human testis in 1963. I jumped at it and in the
process began to learn techniques of microstereology, accurate
sectioning, serial sectioning, photography, and more
importantly, literature search. I enthusiastically embraced this
nurturing. I was later introduced to areas of further work which
led to my meeting with Professor S.S. Ajayi of the Department
of Wildlife and Fisheries Management. As a pioneer worker on
the African giant rat, he focused on the domestication of the rat
which ordinarily is a wild rodent. My immediate duty was to
meet with him and get as much information as I could. I foupd
him to be very warm and receptive.
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Mr. Vice-Chancellor Sir, before I introduce the African
giant rat, I wish to highlight the aspects of my research which I
intend to discuss in this lecture. They are:
• Breeding pattern of the giant rat as influenced by
season, in aid of its domestication.
• Characterization of the epididymis of the giant rat, also
aimed at complementing the process of domestication.
• Our initial efforts in the study of the female African
giant rat.
• Developing new methodologies in our research that can
replace those that are often difficult to utilize or
purchase.
• The significant outcomes of our studies on gossypol
usage especially in malnutrition and parasitism.
My main focus is related to how structure in each of these
studies has elucidated important functional implications.
The African Giant Rat
The African giant rat (Cricetomys gambianus, Waterhouse)
known in Yoruba as Okete, is a wild rat widely spread in sub-
Saharan Africa (Rosevear 1969). The big size of this rat makes
it one of the most striking of Africa's rodents. They have been
found to be fairly tame and docile in captivity even though
shortly after being captured, they tend to exhibit such escape
reactions as violent struggles and hitting head and tail against
the cage.
Fig. 1: The African giant rat (Cricetomys gambianus, Waterhouse)
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Their ability to adapt to various environmental conditions,
social acceptance in Nigeria (especially southern Nigeria), their
value as a delicacy among rural populations, and considering the
need to meet the increasing demand for animal protein among
other considerations recommended this wild rodent for
domestication.
Since Ajayi pioneered its domestication here in Ibadan
(Ajayi 1975), I decided to study the biology of its reproduction
(Oke 1988) as a means of providing adequate and needed
informati n and baseline data on both the morphology and
physiology of its systems to aid the domestication process. In
addition, we felt that the rat may become a valuable laboratory
animal because of its large size and availability in West Africa,
thus becoming useful in the assessment of some pathological
conditions or the efficacy of various pharmacological substances
on different organ systems both in the study of diseases and
during drug screening programs (Holtz 1972).
Seasonal Studies in the Giant Rat
Attempts to breed this rodent on a large scale were not
successful because detailed knowledge of its reproductive
biology was scanty and sometimes incorrect. For example,
Rosevear (1969) suggested that the rat in the wild is a seasonal
breeder. Ajayi (1975), whose work was based on the rat in
captivity, on the other hand, reported that they reproduced
throughout the year and did not show any seasonal peak in
breeding.
In an attempt to resolve this, I investigated the reproductive
organs of the male during the two major seasons (wet and dry)
of Nigeria using morphometric methods (Oke 1985). My
findings showed that values for the dry season (November -
February) were consistently higher than values obtained for the
wet season in all cases except the epididymal epithelial height,
bulbo-urethral-gland weight, and prostate-gland weight (tables
1, 2, and 3), even though there were no significant differences
between the seasons in most of the values obtained.
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Table 1: Mean Testicular Weight, Volume and Tubular Diameter of
Giant Rats within Seasons
Weight of Volumeof
Live weights No_of testis' testis" Tubular
of animals (g) Season animals (g) (g) diameter (urn)
1165±51-15 Rainy season 26 ro-9±O-78 9-60±O-75 212-S5±8-32
(1430l()27) (Mar-{)ct) (l3-3(}-873) (14-00-720) (242 160)
1158±60-10 Dry season 26 12-[1 ± 1-10 11-74±1-09 228-64 ±6-23
(1286-828) (Nov-Fcb) ( 13-5(}-9-06) (15-20 8-70) (248-1'12)
Significance of
seasonal ditfcrence P>0-05 P>0-05 P>O-05
'Figures are for both left and right testes,
All values are mean j Sf (range).
Table 2: Mean Epididymal Weight, Epididymal Height, and
Epididymal Tubular Diameter of Giant Rats within Seasons
No_of Epididymal Epididymal epithelial Epididymal tubular
Season animals weight" (g) height (urn) diameter (urn)
Rainy Season 26 2-26±0-18 51-40± 1-68 216-45±6-28
(Mar OCI) (3-48-1-12) (6041) (268-184)
Dry season 26 2-SI±0-18 42-30±330 242-82:t 10-80
(Nov-Feb) (3-54-1-52) (6(}-30) (300-205)
Significance of
Seasonal difference P>O-05 P>O-05 P>0-05
-Figures are for both left and right epididymides,
Values are mean ±SE (range).
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Table 3: Mean Weights of Accessory Sex Glands of African Giant
Rats within Seasons
No. of Weight of seminal Weigl:! of bulbo- Weight of prostate
Season Animals vesicles' (g) urethral glands_' (g)_ __glands' (g)----------------_. __ .. .__ . ._.-
Rainy season 26 IO·97±[·64 O'29±O'O6 l'O6±O'[8
(Mar-Oct) ([7'7-12·43) (0,92-0,[0) (2, I (H) [4)
Dry season 16 1l·72±0·93 O·27±0·05 O'89±0'2[
(Nav-Fob) (1580·-8·90) (048-0,06) (2,30-0'15)
Significance of
seasonal difference p>O·OS P<O'05 P>O'05
'"'Figures are for all components or organs.
Values are mean±SE (range).
These findings which corroborated Ajayi (1975), showed that
the male giant rat does not appear to be a seasonal breeder.
Characterization of the Giant Rat Epididymis
In furtherance of the need to contribute to the domestication and
breeding of the giant rat, we also sought to characterize the
epididymis (Oke et al. 1988, 1989; Oke and Aire 1990). The
epididymis, one of the major paths of a male gamete to its
ultimate role in breeding, is important in the thrust towards
improving the breeding capacity of animals as its complex
nature also presents numerous challenges to investigators, like
cytologists, biochemists, pathologists, clinicians, andmorpholo-
gists.
We found the epididymis of the giant rat to be similar to that
of the laboratory rat in both shape and relative sizes of the caput,
corpus, and cauda regions (fig. 2), although there are species
variations in cell specialization along the epididymal duct.
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EO
r
CAPUTI
I
L
oo"'"{
00
CAUDA
Fig. 2: Regions and zones of the epididymis of the giant rat
Five distinct zones of the epididymis, based on epithelial height,
cell types, and structure were observed. These zones, apart from
the efferent ductules are zone 1 (initial segment), zones II, III,
and IV (middle segment), and zone V (terminal segment). Zone
V has two major subzones, proximal (Va) and distal (Vb).
The lining epithelium of the· entire epididymis is a
pseudo stratified columnar ciliated epithelium. It is characterized
by four main cell types which are present in various
combinations in the different zones. These are the principal cell,
the basal cell, the intra-epithelial lymphocytes, and the clear
cell. Apical cells, as seen in the rat, mouse, and guinea-pig
(Hoffer and Greenberg 1978) are absent in the giant rat.
The Principal Cell .
The principal cell is present in all segments and zones and
extends from the basal lamina to the lumen of the duct, into
which its stereocilia project. Principal cells constitute the tallest
of the epithelium and are the basis for the epithelium of the
caput being very tall, thereby reducing the luminal diameter. It
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exhibits clumps of mitochondria (fig. 3) in zone I, numerous
vacuoles and large numbers of dense bodies (fig. 4) in zone II,
and one or two large vacuoles in the supra nuclear region of
zone V. The mitochondrial clumps located close to an extensive
Golgi region are unusual and peculiar to this animal.
Fig. 3: Photomicrograph of the epithelium of the initial segment of the epididymis
(Zone 1). P (Principal cell); B, basal cell. Note the clumps
Mitochondria (Mt).x1280.
Among the features that stand out in the supranuclear region of
the principal cell in the caput region especially in zone II are
large Golgi complexes and numerous cisternae of endoplasmic
reticulum studded with ribosomes and arranged in parallel
lamellae or whorls (fig. 5). These features led us to suggest the
occurrence of protein secretion in the giant rat caput epididymis,
especially in zone II, consistent with reports available in many
species (Nicander and Malmquist 1977) and other cells (Allison
and Davies 1974).
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Fig. 4: Zone II epithelium showing P, principal cell; T, truncated cell, with
dense bodies and vacuoles V); B, Basal cell; M, macrophage-like cell,
x1280.
Fig. 5: EM of rough endoplasmic reticulum occurring in parallel lamellae and in
whorls. x7,248.
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The ultrastructural evidence for secretion in different zones
of the caput epididymis was provided by Oke and Aire (1990)
using the giant rat. Absorption of fluid and particulate matter has
been attributed to the efferent ductules and epididymal
· epithelium, especially in the first part of the epididymis where
Crabo (1965) estimated that over 90% of the fluid leaving the
testis in the bull or boar is reabsorbed. I have been able to
provide morphological con-dation for these notable functions in
the giant rat (Oke 1988).
The Basal Cell
This cell type is present but few in all segments. It is generally
small in size, but smaller in zone III, having large nuclei and
· lucent cytoplasm (fig. 3). They lie on the basal lamina in close
association with intra-epithelial lymphocytes and macrophage-
like cells. In the terminal segment (zones Va and Vb) the basal
cells become progressively flattened cranio-caudally. Yeung et
at. (1994) reported close proximity and morphological similarity
.between basal cells and peri tubular macrophages in man. They
also showed that these basal cells expressed a tissue-fixed,
macrophage-specific antigen. This is consistent with the
hypothesis that links the basal cell with the intra-epithelial
macrophage in relation to the ultimate fate of the basal cell in
the epididymal epithelium.
The Intra-epithelial Lymphocyte (IEL) and Macrophage-like
Cell (MLC)
This cell type is also present in all segments and zones of the
epididymis. IELs with characteristic pale cytoplasm and deeply
heterochromatic nuclei are found in large numbers (fig. 4). The
number of IELs and MLCs increases remarkably in the middle
· segment (zones II, III, and IV). They are mostly basal in
location while some migrate upward (fig. 6) in the epithelium.
IELs and heavily laden MCLs occur in the terminal segment in
large numbers.
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Fig. 6: Epithelium of zone III showing a few subapical vacuoles (arrowhead) and
dense globules (D) in the principal cells. B, basal cell; M, macrophage-like _.
cells; L = IELs. x 1280. - .-
It is interesting that the giant rat epididymal epithelium contains
a large number of intra-epithelial lymphocytesand macrophage-
like cells. Sinowatz et at. (1979) have reported the occurrence of
similar cells in the monkey, man, and bovine epididymides.
Their role in the epididymal epithelium is linked to phagocytosis
of spermatozoa or fragments of spermatozoa (spermiophagy).
They have also been thought to provide a first line of defence
against local, mainly viral, antigen (Beagly and Husband 1998)
and the segregation of sperm antigens from the general
circulation (Dym and RornreII 1975). In intestinal epithelial
cells, cytotoxic IELS have been implicated in epithelial cell
turnover, bringing about apoptosis (Beagly and Husband 1998).
The Clear Cell
The clear cell is present only in the terminal segment (zone V),
where it appears as the dominant feature. The clear cell is
slender in the proximal portion (zone Va) (fig. 7) but very broad,
often flask-shaped in the most distal portion of the terminal
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segment (fig. 8). This cell is broader and lighter-staining than
the principal cell. It contains numerous vacuoles of varying sizes
and numerous dense bodies which indent the nucleus.
Fig. 7: Subzone I in the proximal aspect of the terminal segment of the
epididymis. Principal cell (P), clear cell (c), lymphocyte (L), basal cell (B),
macrophage-like cell (M). Note the presence of long numerous mitochondria
'.- (arrowhead). x1280.
Fig. 8: Subione 6 of the terminal segment of the epididymis. Note the presence of
large vacuoles (V) in the principal cells. The clear cell (c) becomes very
broad; the entire epithelial height is low. G, Golgi zone. x1280.
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The giant rat is similar to the rat and hamster in possessing
large numbers of clear cells in the terminal segment. The clear
cells, which seem to occur only in a few mammals is thought to
be the source of glycerylphosphoryl choline (GPC) which
together with sialoproteins may account for the hyperosmotic
. epididy-mal plasma and sperm maturation (Rajalakshmi 1985).
Apart from secretion, phagocytosis, and the movement of
spermatozoa from the testis to the exterior, acquisition of
fertilizing ability by the sperm cells is also accomplished in the
epididymis through changes in size, electric charge, shape,
metabolic properties. and capacity for motility. Motility of
spermatozoa is acquired as the germ cells traverse the duct, a
phenomenon believed to be facilitated by an increase in intra-
sperm content of cyclic AMP during epididymal transit
(Hoskins et al. 1978) combined with the building of a specific
forward-motility protein.
The development of sperm motility, and indeed the normal
structure and function of the epididymis is dependent on
hormones, specifically androgens-which reach the epididymis
in the rat testis fluids and the systemic blood circulation.
Androgens bind to androgen binding protein (ABP) of which the
Sertoli cell is the source (Hagenas et al. 1975). In attempting to
determine the relationship between the giant rat and the
laboratory rat, Adeyemo and Oke (1990) analysed testicular and
epididymal proteins by gel electrophoresis (SDS-PAGE). Our
findings (fig. 9) showed that the giant rat epididymis
demonstrated copious bands suggested as ABP. However the
non-recognition of giant rat ABP by anti-rat ABP antibody
indicated that fundamental genetic differences could exist
between the laboratory rat and the giant rat, especially in this
regard.
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:·5
) ,-,
'- i,:
--- t"
Fig. 9: Cytosolic fraction of the African giant rat epididymis analysed by the 2-
DG electrophoresis and stained with Coomasie blue. M = albumin; P =
protein suspected to be GR ABP; H and K = proteins characteristically found
in this tissue
From all of the above, it is quite clear that the specialization of
the epididymal duct along its length is not only morphological
but also biochemical and physiological. This is why those who
perform biochemical studies on materials from the epididymal
duct or pathologists who may be inclined to examining only one
portion of this duct need to be aware of these differences-
which can no longer be regarded as trivial matters of interest to
morphologists only. In addition, its intimate involvement in
spermatozoa acquiring motility and fertilizing ability also offers
ideal sites for action of male antifertility agents.
Initial Studies on the Female African Giant Rat
Vice-Chancellor Sir, studies so far described have focused on
the aspects of the male giant rat in reproduction and hence in
breeding. In the course of the research, the need to investigate
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the female aspects became increasingly apparent-if the vision
of domesticating this otherwise wild rodent was to be realized.
Fortuitously, my first female masters student pro\.,'J; to be so
outstanding in her performance that she was assignecrf'lilework
towards her M.Sc degree. Most interesting however is the fact
that at the commencement of the research, she was Dr. (Miss)
Bankole; by the time she was completing it, she had become Dr.
(Mrs) Oke-by marrying my nephew, Engineer Dapo Oke. The
worth of her work is such that it formed the framework for
another generation of brilliant scholarly pursuit by Dr. A.K.
Akinloye whose Ph.D thesis is on more in-depth investigation of
the female giant rat reproductive system.
Our investigation involved the characterization of the
oestrus cycle in the female using vaginal smears (Oke and Oke
1999). It revealed that the oestrus cycle length is between 4 and
5 days. In addition to the four stages of the oestrus cycle
(proestrus, estrus, metestrus, and diestrus), we also identified
three intermediate stages (late diestrus/early proestrus, late
proestrus/early estrus,-and la¢te metestrus:/early10). ~diestru.s) (fig.
LI at•e-Proestr.'us/ 1 / Me.Estrus
Early-Estrus V
D
Mid-Prosstrus Mid-Metestrus
Late Diesttrusl
E9riy Proestrus Lete-Metestrus/Early-Diestrus
Mi~Di"'".,:
Fig. 10: A chart showing different stages of oestrus cycle in the female African
giant rat
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The vaginal smears obtained during proestrus were mucoid
and contained mainly nucleated epithelial cells while those
obtained during estrus were gritty and contained mainly
superficial cells. Metestrus smears were characteristically dry
and contained leucocytes and superficial cells while those
obtained during diestrus were pus-like with a lot of mucus and
numerous neutrophils. The totality of the findings established
that vaginal exfoliative cytology can also be used to determine
the estrous or heat period to facilitate productive mating in the
giant rat as in other animals.
This particular finding, along with others from the male
research efforts were translated into practical application at
breeding, first from our colony in the Faculty and then on a few
farm facilities outside. It is however disappointing to note that
such proven success in the laboratory could not be replicated nor
sustained on the field because of a vital missing link-input of
extension workers. Even though the crucial role of this cadre of
workers has been long recognized in agricultural practice, its
equivalent is required in almost every sphere of science and
technology as many research findings never make their way into
the real world due to this missing link. Whilst advocating for
government to encourage manpower development in this area,
attention of entrepreneurs should also be drawn to this
potentially lucrative source of opportunities.
Developing New Research Methodologies
For reproducibility of research results, researchers are taught to
adhere very closely to standard research protocols. There are,
however, occasions when the prevailing situation dictates a
departure. It is frequently impracticable to follow some standard
practice because of the peculiarity of the Nigerian circumstances
viz-temperature, electricity failure, and lack of certain
chemicals and reagents. This situation called for being
innovati ve in research design and laboratory protocols-in
tissue studies. As a consequence, we had to develop novel
alternative tissue processing methods? two of which are
described here.
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Use of "Adi-agbon" (Coconut oil) as Clearing Agent in Neural
Histology
As a way of providing a useful substitute to the relatively
expensive and imported clearing agents used in histological
tissue processing, we investigated the use of adi-agbon, an oily
extract of the endosperm of the coconut fruit (Cocoa nucifera.
L) as a clearing agent for embryonic soft tissues (Shokunbi et al.
1992). An ideal clearing agent for paraffin processing of
embryonic tissue should satisfactorily de-alcoholize the tissues,
be miscible with molten paraffin wax, and should not cause
excessive hardening and shrinkage of the specimen. Such a
compound should be non-toxic and readily available at modest
cost. However, none of .the conventional clearing agents for
paraffin processing fulfilled all these criteria.
We were able to establish that adi-agbon adequately de-
alcoholizes embryonic soft tissues, permitting paraffin wax
infiltration and easy sectioning. There was some tissue
shrinkage, which was not apparent on gross examination due to
the compactness of the tissues and reduction in extra-cellular
spaces (fig. 11).
Fig. 11: Section of the cerebral cortex of an IS-day mouse embryo cleared in (a)
Adi-agbon (b) xylene.
Note the relative looseness of the neuropil and the prominence of the extra
cellular space in (b). xI600.
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Of the tissues examined, the brain appeared to be most
susceptible to this effect of adi-agbon. However, adi-agbon did
not shrink adult soft tissues (Caxton-Martins et al. 1987). In
spite of these minor short-comings, the use of adi-agbon is still
encouraged because it is available locally and it could serve as a
useful substitute for the other relatively .expensive clearing
agents-especially for routine histological work-where tissue
identification is simply all that is required.
Improved Immunocytochemical Protocol that Employs
Polyester Wax
I was privileged to participate, in conjunction with Dr. Carlos A.
Suarez-Quian at the Department of Cell Biology, Georgetown
University School of Medicine, USA, in developing an
improved protocol for immunocytochemistry of the testicular
tissues. Immunocytochemistry is an ideal tool for determining
both tissue and cellular distribution of proteins. In the testis,
successful immunostaining has been obtained through use of
either frozen or paraffin sections, although both techniques have
their limitations. With freezing, tissue preservation is not
optimal whereas with paraffin embedding, antigenicity is often
destroyed. These limitations have led to numerous ambiguous
results and diagnostic problems.
In order to overcome these limitations, we attempted to
immunocytochemically localize various proteins in the testis.
Our trials (Oke and Suarez-Quian 1993) combined perfusion
fixation with Bouin's fluid embedding of tissue in polyester
wax. The general morphology of the polyester wax-embedded
testis demonstrated excellent preservation of tissue which
compares very well with similar tissues embedded in paraffin
wax (fig. 12). Seminiferous tubules cut in cross section appear
to retain normal morphology when treated with polyester wax
with no major fixation or embedding artifacts.
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Fig. 12: General morphology of testis embedded in polyester wax. S = Sertoli cell
nuclei, P = Pachytene spermatocyte, Z = Zygotene spermatocyte, '" =
interstitial cell. Arrows = mitotic figures, Arrowhead = peri tubular cell
x680.
Fig. 13: Sertoli cell (S) immunostained for ABP (high power). Reaction product
extends from base to lumen (L). Pachytene (P) and Zygotene (Z)
spermatocytes not labeled. x625.
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Using a specific antiserum probe for Sertoli cells (anti-
ABP), a characteristic Sertoli cell immunostaining pattern was
observed (fig. 13). The reaction product was observed to extend
from the base of the tubule to the lumen. There is a general
difficulty in obtaining good and consistent immunolocalization
of ABP in Sertoli cells because of its bi-directional secretion
(apical and basal) and its differential staining pattern as a
function of the cycle of the seminiferous epithelium (Suarez-
Quian and Niklinski 1990). However, we were able to show,
through this method, that intense ABP immunostaining,
spanning the height of the sertoli cell is present in all stages of
the cycle of the seminiferous epithelium.
Specific irnmunostaining of populations of germ cells was
also accomplished in the seminiferous epithelium using a
monoclonal antibody that has been observed to immunostain
nuclear lamins of certain cells in vitro (Suarez-Quian 1988). It
specifically stained spermatogonia and pachytene spermatocytes
(fig. 14).
Fig. 14: Specific localization of reaction product only in the nuclei of pachytene
spermatocyte. Sertoli cells (S) and Round spermtids (R) not immunostained.
x625.
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Fig. 15: Immunostaining for Leydig cells (*). Z and D = Zygotene and Diplotene
spermatocytes. x625
The identity of the immunostained cells was ascertained readily
because of the general preservation of the tissue made possible
by this embedding protocol. This antibody probe has thus been
suggested for identifying populations of these germ cells in
vitro. Immunostaining of interstitial (Leydig) cells was also
accomplished using the rabbit antiserum to the peripheral type
benzodiazepam receptor (PBR) (fig. 15). Neither lymphatic nor
capillary endothelial cells of the interstitium stained with this
antiserum.
Immunolocalization of cytoskeletal proteins (vinculin,
tubulin, and smooth muscle actin) also revealed the specific
efficacy of this method. Vinculin was detected principally at
ectoplasmic specializations between Sertoli cells and elongated
spermatids, at junctional complexes between Sertoli cells and at
kinetochores of spermatocytes at metaphase (fig. 16). Tubulin
immunostaining was present in the cytoplasmic trunk of Sertoli
cells and in the flagella of elongated spermatids (fig. 17).
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Fig. 16: Imrnunostaining for Vinculin in ectoplasmic specializations (short
arrows) and at kinetochores of spermatocytes at metaphase (curved arrows)
x625.
Fig. 17: Tubulin immunostaining 111 Sertoli cells apical cytoplasm (arrowhead)
x605.
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Thus, we have shown that antibodies to tubulin can serve as
probes to indicate the presence of the supranuclear region, trunk
region, and apical cytoplasm of Sertoli cells, which is consistent
with previous reports on microtubules within Sertoli cell
cytoplasm (Vogi 1988).
Generally therefore, this procedure for routine immunocyto-
chemical localization of proteins in the testis has several
advantages which are as follows:
(i) The low melting temperature (380 C) of polyester wax
reduces loss of antigenicity.
(ii) Elimination of harmful solvents like xylene enhances
antigen preservation.
(iii) The protocol does not require learning new techniques,
is relatively quick and inexpensive.
(iv) Significantly, the excellent histological detail permits
accurate detection of immunoreactivity.
(v) The results are permanent, allowing prolonged
observations of even low immunostaining signals.
Gossypol Studies and Relevant Comments
Mr. Vice-Chancellor Sir, a number of our research activiues
have implications way beyond veterinary anatomy and, as a
matter of fact, some of our research efforts can easily be
extrapolated to the human population. A good example is our
study on gossypol.
Gossypol is a polyphenolic substance that occurs naturally
in cotton plants. It is a common constituent of livestock feeds. In
addition, it has been evaluated extensively as a potential male
contraceptive. It has been reported to reduce spermatogenesis in
the male and to be a safe intravaginal contraceptive in the
female. In addition to its antifertility effects, it exhibits
antiproliferative, antimetastatic, and parasiticidal properties
being very effective against trypanosomoses, a blood parasitosis
of man and livestock, which causes debility and reduced
productivity.
We sought to understand the antifertility effect of gossypol,
its toxicity, and antitrypanosomal efficacy in a Third-World
environment where several variables interplay simultaneously.
Not fewer than three Ph.D theses have emanated from the series
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of research hypotheses posed and for which answers have been
provided for the 'knowledge bank'. One notable study that
attracted unexpected international approbation and recognition
was derived from the thesis of Dr. Benson Akingbemi
(Akingbemi et at. 1996), in which we investigated reproductive
parameters in gossypol-treated male rats that had been infected
experimentally with Trypanosoma brucei and experimentally
malnourished. This was with a view to generating information
which may not only be useful to livestock farmers in
trypanosome-endemic zones but, might also influence the design
of clinical investigations concerning potential contraceptive
agents, especially in the Third World where blood parasitoses,
protein malnutrition, and rapid population growth are
widespread.
The findings showed that most of the parameters studied
(testicular size and diameter and length of seminiferous tubules)
were lowest in the protein-malnourished-gossypol-treated-
trypanosome-infected animals-when compared to those
obtained from corresponding animals that were only gossypol-
treated or trypanosome-infected, suggesting that reproductive
capacity could be impaired in protein-malnourished,
trypanosome-infected animals fed on gossypol-containing
products even when there are no obvious clinical signs of
disease. Therefore, we suggested screening for haemoparasitism
prior to evaluation and/or use of gossypol in human subjects,
especially in communities that are confronted with severe food
shortages.
Mr. Vice-Chancellor Sir, the gossypol acetic acid used in
this work was kindly donated by the WHO Special Programme
of Research Development and Training in Human Reproduction,
while the financial support came from University of Ibadan
Senate Research Grant and University of Zimbabwe Research
Board. Some parts of the work were done at the Institute of
Veterinary Anatomy in Berlin, when Dr. Akingbemi was on a
DAAD-funded study visit and at the University of Liverpool by
Professor Aire while on a British Council-supported visit. It was
eventually published by the International Journal of Andrology
in 1996 and to our surprise, won the second prize of the
European Academy of Andrology which was accompanied with
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a cash award in 1997. Of great significance was an uncommon
special editor's note, written at the end of the article as follows:
Some readers may be puzzled by the paper by
Akingbemi et al., and may consider that the journal
should not be publishing studies in which so many
experimental manipulations are inflicted in different
combinations on laboratory rats. When so many
variables are at play, how can we then learn
anything of value? This was precisely my own
reaction when I first received and read this paper.
However, if you ignore your immediate response
and read the rationale for the study design, you will
appreciate that it is both logical and relevant to the
evaluation and application of methods of fertility
regulation in man and domestic animals in much of
the world ....
He went further to query the use of gossypol in modem day
scientific investigation, but quickly rationalized even this, based
on the argument of the authors. The end-part of the comment
reads:
But most importantly, the quality of the scientific
thought and practice in this paper is to be
commended, not least because the circumstances
under which this research was performed are
probably a good deal worse than those which most
of us take for granted.
In the light of these comments we must re-examine the current
state of basic science research in our country and make concrete
and determined efforts to advance scientific knowledge and
improve the quality of life of both humans and animals.
Basic science research is pivotal in the development of new
technologies and is the fastest route to a career in industry and
technology. As today's high-tech industry is based on
yesterday's basic research, so is our industrial and technological
future based on the results of today's basic research. To attain
sustainable development in science therefore, we need high
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quality education-especially at the university level and cutting-
edge research in our institutes. In a world where basic research
is now being carried out at the biotechnological, molecular
biology, genomic, proteomic, and metabolomic levels, the
current situation in our university (or universities) leaves much
to be desired. The lack of requisite research equipment and
facilities in our laboratories, incessant power outages, and poor
water supply militate greatly against rational and intelligent
reasoning, which are keys to high productivity in basic research.
The dearth of research grants, poor funding, and non-
availability of personnel (trained in biotechnology and
molecular biology techniques) are also serious obstacles. Mr.
Vice-Chancellor, at this juncture, mention must be made of the
efforts by the current university administration to reverse the
trend and point our university to the path of excellence. This is
evidenced by the visible transformation of the campus and the
remarkable achievement fostered by the MacArthur Foundation
Grants which have assisted us a lot in the areas of Information
Communication Technology (ICT) , staff-training overseas, and
the provision of a central multi-disciplinary research laboratory.
Indeed, when the history of this university is written, certain
names would stand out clearly in gold.
However, to attain and maintain world-class status to which
this institution aspires, a lot more needs to be accomplished. In
order to keep up with the ever advancing frontiers of scientific
research, our university's academic exchange programme should
be strengthened. Short- and medium-term training in
international laboratories should continue as has been done in
the last three years or so, while staff should be encouraged and
sponsored to attend conferences and workshops both locally and
internationally and to be members of specialized learned
societies. The University as a matter of urgency needs to train a
new crop of technologists particularly in the basic sciences.
At the risk of being too insistent on this issue of electric
power and water generation, I think the university should seek to
explore other alternatives for the provision of these basic
amenities. While all kinds of suggestions have been proffered, I
continue to wonder if dredging of our own Awba dam and the
streams that supply it would not help in generating power and
good drinking water, and in the process increase our internally
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generated revenue. This can be based on a public-private
partnership for the establishment of an independent power
project that will benefit not only the university community, but
also the immediate or surrounding communities. Yes, the big
and usual question is: "Where is the money?"
Summary
I have, in the last forty minutes or so, shown how structural
studies have been utilized in forming useful opinions on the
functions of cells, tissues, and organs and how these can be
utilized to advantage.
1. Unlike previously held opinion that the African giant
rat is a seasonal breeder, our results show that the male
giant rat can breed throughout the year. Therefore,
seasonal changes may not affect a successful breeding
of the rat. We have been able to elucidate this through
morphological means.
2. Prior to our studies, the cell types in the epididymis of
the giant rat had not been described. Our efforts have
led to a detailed description of structure which is
correlated with their functions, among which are
protein secretion, absorption, local antigenic defense,
and sperm maturation.
3. Our pioneering efforts in the studies of the female giant
rat are bound to lead to a fuller understanding of the
reproductive functions as evidenced by their structural
peculiarities. It is our hope that these efforts will be
translated to practical application at improving breeding
in the giant rat especially with the aid of extension
workers.
4. I have shown in this lecture that alternative materials
and methods are available which can be exploited to
replace expensive ones especially in testicular
immunocytochemistry.
5. It has also been mentioned that feeding animals with
gossypol-containing products especially when they are
malnourished and trypanosome-infected could lead to
impairment of reproduction even when there are no
other signs of disease.
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Acknowledgements
I wish to express sincere thanks to my parents: Papa, Simeon
Emiola Oke and Mama, Irene Titilola Oke (nee Olukoga) both
of blessed memory. Working together, they ensured that we,
their children, all got quality education. Indeed, when I was yet
a little boy, Papa was fond of calling me 'professor' because,
according to him, I like reading all available literature, and my
diction was way beyond my age. They both died before I could
attain this position and to them, I dedicate this precious moment
of my career.
I remember very vividly the very strong contribution of my
principal at the Ijebu-Ode Grammar School, Rev. N. E. Ade
Osisanya. I cannot fail to mention my mentor, Professor Tom
Aire. I thank the late Dr. Charles Oluseyi Osunfisan, former
CVO, Director of Veterinary Services and later Permanent
Secretary in the Ogun State Civil Service; Drs Martin Dym,
Carlos Suarez-Quian, Vasilli Papadopoulos and Mr. William
Vornberger (all of the Anatomy Department of Georgetown
University Washington D.C., USA), and the Rockefeller
Foundation.
I will also like to thank my colleagues in the Faculty, for
their immense love and cooperation, during my stint as Dean of
the Faculty. In this connection, I must mention the past Deans
especially my predecessor, Professor Arowolo, who nudged me
into contesting for that office when I was not even thinking of it.
I also thank the current Dean, Professor G.A.T. Ogundipe and
all members of the Faculty especially those who worked with
me during our modest effort in the Dean's Office at that time.
My department has always been, for me, another home. It is
such a wonderful place with wonderful people. I thank everyone
for the wonderful working relationship, and pray it continues.
I want to acknowledge all members of the Oke and Olukoga
families as well as my in-laws, the Aderinokuns, for their love
and fraternity. May God continue to raise you all up. Amen. I
certainly will not forget to publicly thank my wife, Professor
Gbemisola Oke, my three children, Damilola, Babajide, and
Temitope for their loving care, devotion, and understanding all
these years. Finally, I thank God for all He has been to me over
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the years and for making it possible for me to be alive today to
give this lecture.
Mr. Vice-Chancellor, ladies and gentlemen, I thank you for
your attention.
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