CHARACTERIZATION OF OVARIAN FOLLICULAR
STRUCTURES (MEDULLA, CORTEX AND PRIMARY FOLLICLES)
OF WHITE FULANI CATTLE
BY
OYEKUNLE, M. KOLADE
MATRIC NO: 2006/0542
DEPARTMENT OF ANIMAL PHYSIOLOGY
A PROJECT SUBMITTED TO THE COLLEGE OF ANIMAL
SCIENCE & LIVESTOCK PRODUCTION
IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE
AWARD OF BACHELOR OF AGRICULTURE (B. Agric) OF THE
UNIVERSITY OF AGRICULTURE, ABEOKUTA.
OGUN STATE
JUNE 2011
1
CERTIFICATION
I hereby certify that OYEKUNLE MOSHOOD KOLADE with matriculation number
2006/0542 of the Department of Animal Physiology, University of Agriculture, Abeokuta,
under my supervision, carried out the work as reported in this project.
-------------------------------
-----------------------------
Dr. O. F. SMITH
DATE
(SUPERVISOR)
2
DEDICATION
I dedicate this project to God Almighty who in his infinite mercies kept me till this
moment to witness this memorable day.
I also dedicate this project to my wonderful parents, Mr. and Mrs. M. K. Oyekunle who
have been very supportive both prayerfully and financially, and also to my siblings.
3
ACKNOWLEDGEMENT
My special gratitude goes to God Almighty, the author and finisher of my faith, who has
always been the pivot upon which my strength lies.
I wish to express my profound gratitude to my project supervisor, who is also the Acting
H.O.D., Dr O. F. Smith for his creativity, objectivity coupled with timely supervision of
my project. His comments and fatherly advice has really enhanced the quality of this
work and its early completion inspite of his tight engagements. I pray that God Almighty
will continue to be with him and his family.
My sincere appreciation goes to my father. Mr. M. K. Oyekunle for his fatherly love. his
support financially and for his prayers which has contributed to the success of this
project. May the good God Almighty reward him abundantly and grant him his heart
desires.
I also wish to extend my special love to my mother. Mrs. L. I. Oyekunle for her care and
support, and to my siblings, Oluwaseun, Adeboye, Adebayo, Boluwatife, Abiodun, and
Oluwatobi (Oyekunle) for their faithfulness and understanding throughout my stay in
Abeokuta. God bless you all.
Many thanks to Acting HOD, Veterinary Pathology, Dr. S. O. Omotainse and the
laboratory Technologist, Mr. E. O. Anise for their efforts in time and space to make sure
this project was successful.
I will not forget the inestimable impact that Redeemed Christian Fellowship (RCF),
UNAAB had on my life. With the help of God, all the Ex-cos and members of this
4
fellowship from 2005 till date had been a source of blessing to me both spiritually and
physically. I pray the favor of God will never run dry on the altar of RCF, UNAAB.
I also wish to express my appreciation to the lecturers and staff of my Department, Dr.
M.O. Abioja, Dr. I. J. James, Dr J. A. Abiona, Dr. T.J. Williams, Dr. O.A. Ladokun, Dr.
J.O. Daramola, Dr. (Mrs.) O.G. Sodipe and others not mentioned, for their contributions
and moral advice towards the completion of this project.
The unity and unanimity expressed by my and fellow graduating students of Animal
Physiology, especially my project mates (Usamah Simon and Joachim Abraham) has also
contributed enormously to the conclusion of this project. I love you all.
I will like to appreciate Prof. Mrs O.A.T. Sotonade for her mentorship and timely advice
which served as an aid to the execution of this project.
To everyone who has in one way or the other contributed to the success of this project, I
love you all. GOD bless you
5
TABLE OF CONTENTS
Contents
Page
Title
i
Certification
ii
Dedication
iii
Acknowledgement
iv-v
Table of contents
vi-vii
Abstract
viii
CHAPTER ONE
1.0
INTRODUCTION
1-3
1.1
Justification
3
1.2
Broad objective
3
1.3
Specific objectives
4
CHAPTER TWO
2.0
LITERATURE REVIEW
5
2.1 Histology and structure of the ovary
5-6
2.2 Ovarian follicle
6-7
2.3 Histology and staining of tissue
7
2.4 Stages of follicle development
8-10
CHAPTER THREE
3.0
MATERIALS AND METHOD
11
3.1
Study area
11
3.2
Materials
11
3.3
Sample collection and Experimental site
11-12
6
3.4
Tissue processing, sectioning, and staining
12
CHAPETR FOUR
4.0
RESULT
13
4.1
Medulla and Cortex
13-14
4.2
Primary follicle
14-15
4.3
Discussion
16
CHAPTER FIVE
5.0
CONCLISION AND RECOMMENDATION
REFERENCES
17
18-20
7
ABSTRACT
A total of 12 ovaries of white Fulani cows were collected from the abattoir and used for
this study. The ovaries were collected and fixed in 10% formalin solution which served
as preservative for the samples thus preventing it from being denatured and still retaining
its cellular components before being transported to the laboratory for examination.
Histological study on the ovaries of these cows revealed the presence of follicles at
different stages. Subsequent to a successful, trimming, processing and staining technique,
the slide (containing the stained ovary sample) was observed under the light microscope
at various stages of development. The medulla and the cortex on which the follicles are
located were clearly seen under a light microscope. On the cortex is found the primary
follicle, while the medulla contains other follicles such as the secondary, tertiary and
ovulated follicles. The primary follicle consists of a primary oocyte with a single layer of
follicular cells. It is smaller than other follicles and also few in number in the ovaries
obtained from the abattoir. Due to the increasing demand on beef, knowledge of the
pattern of follicle development is increasingly becoming critical in order to improve and
develop methods that can manipulate the fertility and productivity of animals.
8
CHAPTER ONE
1.0
INTRODUCTION
Cattles are not only important agricultural species but their ovarian follicle dynamics
makes them ideal models for humans (Adams and Pierson, 1995), including the changes
that occur on ageing at menopause (Malhi et al., 2005). Ovarian follicles are the basic
unit of female reproductive biology, each of which is composed of roughly spherical
aggregations of cells found in the ovary. The adult ovary contains a reserve of inactive
primordial follicles. Each contains a small non-growing oocyte and a layer of nondividing pregranulosa cells encapsulated by the follicular basal lamina. These structures
are periodically initiated to grow and develop, culminating in ovulation of usually a
single competent oocyte. The cells of the ovarian follicle are the oocyte, grunulosa cells
and the cells of the internal and external theca layers. Following ovulation, the granulosa
cell and theca cells differentiate into the large and small luteal cells of the corpus luteum,
and the vascular supply of the corpus luteum is derived from the capillaries of the theca
interna. All non ovulating follicles undergo atresia (which is the death of follicles) and
regression.
9
Fig. 1; Diagram of structures that can be identified in a cross section of an ovary of a reproductively active
cattle.
A follicle is an anatomical structure in which the primary oocyte develops. The primary
role of the follicle is oocyte support. In biology, folliculogenesis is the maturation of
ovarian follicle, a densely packed shell of somatic cells that contain an immature oocyte.
Folliculogenesis describes the progression of a number of small primordial follicles into a
large, preovulatory follicle that enter the menstrual cycle (Fortune et al, 2000). The
process of folliculogenesis begins continuously; meaning that at any time the ovary
contains follicles in all stages of development and ends when a mature oocyte departs
from the preovulatory follicle in a process called ‘ovulation’.An ovary is subdivided into
cortical (ovarian cortex) and medullary compartments (ovarian medulla). The cortex
usually also contains the remains of degenerated follicles called atretic follicles which
may arise at any stage of follicular development.
10
The medulla is composed of loose areolar connective tissue containing numerous elastic
and reticular fibers, large blood vessels, nerves and lymphatics.
The growing follicle passes through the following distinct stages that are defined by their
structural characteristics. The stages include; Primordial, Primary, Secondary, Tertiary
and Preovulatory stages.Up till the preovulatory stage, the follicles contain a primary
oocyte that is arrested in prophase of meiosis 1. During the late preovulatory stage, the
oocyte continues meiosis and becomes a secondary oocyte, arrested in metaphase II.
Primordial follicles are indiscernible to the naked eye, however, these eventually
develops into primary, secondary and tertiary vesicular follicles. Tertiary vesicular
follicles (also called mature or ripe vesicular follicles) are sometimes called ‘Graafian
follicles’. After rupturing, the follicle is turned into a corpus luteum.
1.1 JUSTIFICATION

As more and more demands are placed on beef, knowledge of the pattern of
follicle development is becoming increasingly critical in order to improve and
develop methods that manipulate the fertility and productivity of different
animals.

Ovarian follicular study especially in the area of characterization has majorly
been done on foreign breeds, hence the need to carry out study on our local breeds
such as White Fulani cattle.
1.2 BROAD OBJECTIVE

Characterization of ovarian follicular structures (medulla, cortex and primary
follicles) of White Fulani cattle.
11
1.3 SPECIFIC OBJECTIVE

To describe the cortex, medulla and primary follicles of White Fulani cows.
12
CHAPTER TWO
2.0
LITERATURE REVIEW
2.1 HISTOLOGY AND STRUCTURE OF THE OVARY
The ovaries are magnificent glands which are part of the female reproductive system. The
ovaries are about the size and shape of an almond and sit just above the fallopian tubes,
one ovary on each side of the uterus. Every month during ovulation, either the right or
left ovary produces a single mature egg for fertilization. The ovaries lie one each side of
the peritoneal cavity, just above the brim of the pelvis. Each is close to the open, fringed
end of a Fallopian tube, leading to the uterus which lies between and below them. Unlike
the testes, where there is continuous production of sperm after puberty and throughout
life, the ovaries do not produce any more eggs and women are born with all the eggs they
will ever have. The adult ovary contains a reserve of inactive primordial follicles. Each
contains a small non-growing oocyte and a layer of non-dividing pregranulosa cells
encapsulated by the follicular basal lamina. Every day a number of primordial follicles
become active, and the oocyte commences growing while the granulosa cells begin to
divide (Malhi et al., 2005). The ovary though not vital to individual’s survival is vital to
perpetuation of the species. The function of the ovary is to produce the female germ cells
or ova and in some species, to elaborate hormones that assist in regulating the
reproductive cycle (Moscow, 1973). The ovary of all vertebrates’ functions in essentially
the same manner, however, ovarian histology of the various groups differs considerably.
The ovary of a new-born heifer may contain up to 100,000 primordial follicles (Erickson,
1966). However, only a few of these mature and release an ovum. From birth to shortly
before puberty the primordial follicles are in a state of arrested development (dictyotene;
13
the resting stage). Shortly before puberty, many primordial follicles start to grow and
develop in response to hormone (gonadotrophin) stimulation. The presence of developing
follicles indicates active gametogenesis and steroidogenesis. During each oestrous cycle,
several follicles may develop to the Graafian stage, but usually only one reaches full
maturity and ruptures to release the ripe ovum (ovulation): the others become atretic (also
known as degenerating, luteinising or anovulatory follicles) (McDonald, 1980).
2.2 OVARIAN FOLLICLE
Ovarian follicles are the basic units of female reproductive biology, each of which is
composed of roughly spherical aggregations of cells found in the ovary. They contain a
single oocyte (immature ovum or egg). These structures are periodically initiated to grow
and develop, culminating in ovulation of usually a single competent oocyte in humans
(McGee and Hsueh, 2000)).The primary role of the follicle is oocyte support. From birth,
the ovaries of the cow contain a number of immature, primordial follicles. These follicles
contain a similarly immature primary oocyte. A clutch of follicles begins folliculogenesis,
entering a growth pattern that will end in death or in ovulation (the process where the
oocyte leaves the follicle), (Fortune et al., 2000). Ovarian follicles are spherical cell
aggregations within the ovary containing an ovum. During ovulation, an egg is released
from the ovarian follicle. The ovarian follicle is made up of several parts. These parts
include an external fibro-vascular coat, an internal coat of nucleated cells, and a
transparent, albuminous fluid in which the ovum is suspended.
The growth of the ovarian follicle is initiated by the follicle stimulating hormone (FSH).
The ovarian follicle is an important part of the cattle’s reproductive system.
14
The ovarian follicle, consisting of an oocyte surrounded by granulosa and theca cells,
represents the basic functional unit of the ovary. Over the course of roughly a year, the
primordial follicle undergoes a series of critical changes in character, both histologically
and hormonally (Van den Hurk et al., 2005). They become dependent on hormones
emanating from the host body, causing a substantial increase in growth rate.
2.3 HISTOLOGY AND STAINING OF TISSUE
This is the study of the microscopic anatomy of cells and tissues of plants and animals. It
is performed by examining a thin slice (section) of tissue under a light
microscope or electron microscope. The ability to visualize or differentially identify
microscopic structures is frequently enhanced through the use of histological stains.
Histology is an essential tool of biology and medicine (Stedman’s Medical Dictionaries,
2005).
Biological tissue has little inherent contrast in either the light or electron microscope.
Staining is employed to give both contrast to the tissue as well as highlighting particular
features of interest. Where the underlying mechanistic chemistry of staining is understood,
the term histochemistry is used. Hematoxylin and eosin (H&E stain) is the most
commonly used light microscopical stain in histology and histopathology. Hematoxylin,
a basic dye, stains nuclei blue due to an affinity to nucleic acids in the cell nucleus; eosin,
an acidic dye, stains the cytoplasm pink. Uranyl acetate and lead citrate are commonly
used to impart contrast to tissue in the electron microscope.
15
2.4 STAGES OF FOLLICLE DEVELOPMENT
Primodial follicle
The primordial follicle consists of a primary oocyte and a single layer of flattened
follicular cells. As the follicle develops, alterations occur in the primary oocyte and the
surrounding follicular cells. The primary oocyte produces yolk granules and the
follicular cells change from flattened to cuboidal or columnar. These primordial follicles
contain immature oocytes surrounded by flat, squamous granulosa cells (the support cells)
that are segregated from the oocyte's environment by the basal lamina. Because
primordial follicles can be dormant for up to 50 years in the human, the length of the
ovarian cycle does not include this time (Fortune et al., 2000). The primordial follicles
always form the majority of the follicles in the ovary.

Primary follicle
In the transition of the primordial follicles into primary follicles the follicular epithelium
that surrounds the oocyte becomes iso- to highly prismatic. The primary follicle consists
of a primary oocyte with a single layer of cuboidal/columnar follicular cells. As
development proceeds, the number of follicular cells increases by mitosis forming several
layers around the primary oocyte. As these cells enlarge they release steroid hormones
called estrogens of which estradiol is the dominant one prior to ovulation.
During each cycle, a few primary follicles will continue to develop into secondary
follicles (Oklahoma State University 2000).
The granulosa cells of these primordial follicles change from a flat to a cuboidal structure,
marking the beginning of the primary follicle (Fortune et al., 2000). The oocyte genome
16
is activated and genes become transcribed. Rudimentary paracrine signaling pathways
that are vital for communication between the follicle and oocyte are formed.
Primary follicles develop receptors to follicle stimulating hormone (FSH) at this time, but
they are gonadotropin-independent up until the antral stage. Research has shown,
however, that the presence of FSH accelerates follicle growth.

Secondary follicle
The acquisition of a second layer of granulosa cells marks the graduation of the primary
follicle to the secondary follicle. By this point, follicle mitotic activity is high and it isn't
long before more and more layers of granulosa cells are formed. The secondary follicle
consists of several layers of cuboidal/columnar follicular cells, now collectively called
the membrana granulosa which begin to secrete follicular fluid. The late-term secondary
follicle is also known as the preantral follicle. Histologically, the preantral follicle is
marked by a fully grown oocyte surrounded by a zona pellucida, approximately nine
layers of granulosa cells, a basal lamina, a theca interna, a capillary net, and a theca
externa (Van den Hurk., 2005).
Tertiary follicle and ovulation
If the secondary follicles survive, tertiary follicles are engendered. Their identifying
characteristic is a fluid-filled cavity, the antral follicle. Tertiary follicles can attain a
tremendous size that is hampered only by the availability of FSH, which it is now
dependent on. A fully developed tertiary follicle eventually ruptures, a process
known as ovulation. The ruptured follicle will undergo a dramatic transformation
into the corpus luteum, a steroidiogenic cluster of cells that maintains the
17
endometrium of the uterus by the secretion of large amounts of progesterone.
The oocyte is now competent to undergo fertilization and will travel down the
fallopian tubes to eventually become implanted in the uterus.

Cortex and medulla
The ovary is composed of the medulla and its outer shell, the cortex. The medulla is
composed primary of blood vessel, nerves, and connective tissue. The medulla is
composed of loose areolar connective tissue containing numerous elastic and
reticular fibers, large blood vessels, nerves and lymphatics.
The cortex contains those cell and tissue layers associated with ovum and hormone
production. The outermost layer of the cortex of the ovary is the surface epithelium.
The surface epithelium because it was believed to be the origin of female germ cells
(oogonia). It is now known that germ cells do not arise from this epithelial layer.
They arise from embryonic gut tissue and then migrate to the cortex of the
embryonic gonad. Just beneath the sureace epithelium is a thin,dense layer of the
connective tissue,the tunica albuginea ovarii. Below the tunica albuginea ovarii is
the parenchyma, known as the functional because it contains ovarian follicles and
the cells which produce ovarian hormones. In the cortical compartment the oocytes
are present within the various follicle stages. Both blood and lymph vessels are
found in the loose connective tissue of the ovarian medulla.
18
CHAPTER THREE
3.0
MATERIALS AND METHODS
3.1 STUDY AREA
The study was conducted on the ovaries of cows slaughtered at Lafenwa abattoir
Abeokuta, Ogun state. The areas surrounding the abattoir were characterized by
moderate rainfall and sparse vegetation.
3.2 MATERIALS
1) Ovary samples
2) 10% formalin
3) Grades of alcohol (70%, 90%, 100%)
4) Xylene
5) Paraffin wax
6) Frosted N glass slides
7) Microtome
8) Furnace
9) Hematoxilin & Eosin (H & E stains)
10) Leukart moulds
11) Tissue basket
12) Distilled Water
3.3 SAMPLE COLLECTION AND EXPERIMENTAL SITE
The breed of cattle used in this study is White Fulani. A total of 12 ovaries were
collected and fixed into 10% formalin solution which serves as a preservative that
prevented the sample collected from being denatured and still retaining its cellular
19
components before being transported to the laboratory for examination. The
experiment was carried out at the Veterinary Pathology laboratory, College of
Veterinary Medicine, University of Agriculture, Abeokuta, Ogun State.
3.4 TISSUE PROCESSING, SECTIONING AND STAINING
The ovary samples were trimmed to about 3-5nm in thickness and fixed in 10% formalsaline. The tissue was processed by placing in bouins fluid for couple of hours after
which it was transferred into a tissue basket for dehydration. The dehydration process
was done by placing the tissue basket with the tissue through series of grades of alcohol
(70%, 90% & 100% respectively), then into a xylene-alcohol mixture, and finally into
xylene at 2⅟2 hours intervals each.
The tissue was now taken through infiltration process for embedding in a water bath for
another 1⅟2 hours interval and afterward it was infused in paraffin wax. The paraffin wax
was melted with the aid of a furnace and was poured in a leukart mould for blocking and
the furnace was set at 800 C temperature reading.
The next step involved dewaxing, then rehydrating by placing the tissue in xylene, 100%
alcohol, 90% alcohol, 70% alcohol respectively, and then washed in running water.

Trimming of tissue with the aid of a disposable blade

Sectioning

Mounting on glass slides

Staining with the H & E

Using cover slip to cover the tissue.
The prepared slides were observed under the microscope and pictures of the follicles
were then taken.
20
CHAPTER FOUR
4.0
RESULTS
Subsequent to a successful processing and staining technique, the slide (containing the
stained ovary sample) was observed under the light microscope at various stages of
development.
4.1 MEDULLA AND CORTEX
The medulla and the cortex were clearly seen, on which the follicles are located (Figs. 2
and 3). On the cortex, is found the primary follicle and in the inner medulla are other
follicles such as secondary, tertiary etc
(Magnification 20x)
Fig 2: Histological section of the medulla of a reproductively active cattle
21
(Magnification 20x)
Fig 3: Histological section of the cortex of white Fulani cattle
4.2 PRIMARY FOLLICLE
The granulosa cells of these primordial follicles change from a flat to a cuboidal
structure, marking the beginning of the primary follicle. The primary follicle (Fig. 3)
consists of a primary oocyte with a single layer of cuboidal/columnar follicular cells. It is
very small and scanty which made it very difficult to observe. More of the tertiary and
graafian follicles were seen. Ovulated follicles and corpus luteum were also observed
22
(Magnification 20x)
Fig 4: Histological section of the ovary (primary follicle of white Fulani cattle).
23
4.3
DISCUSSION
The success of ovarian follicle observation for the purpose of description depends largely
on the age of the cow. This is because the older the cow, the lesser the number of follicles
present. At any point in time the follicles of the ovary appear at various stages of
development which continue to ovulate at every estrous cycle as the cow grows older,
thereby reducing the number of follicles present (a state where all the follicles had
ovulated, and fertility can no longer be achieved).
Many studies have probed the characteristics of ovarian follicles since they were first
described by de Graaf in 1672. It now seems that many follicles develop from the
primordial resting pool in all species during all reproductive states. However, the number and
growth pattern of follicles, once they become gonadotrophin-dependent (at the time of
emergence or recruitment), is variable among species.
Among domestic species pigs are an exception as they do not have follicle waves during
oestrous cycles. This may be due to suppressed FSH secretion (that drives follicle waves)
or to an overriding mechanism that ensures many follicles develop to give the high
ovulation rate typical in pigs. Similar is the case with chickens that continuously develop
selected follicles, ensuring that a large healthy follicle is available for ovulation
approximately every day (Bossis et al. 2000). As more and more demands are placed on
beef, knowledge of the pattern of follicle development is becoming increasingly critical
in order to improve and develop methods that manipulate the fertility and productivity of
different animals.
24
CHAPTER FIVE
5.0
CONCLUSION AND RECOMMENDATION
Since the study of ovarian follicles in the cow requires slaughtering the entire cow which
is very expensive especially in this part of the country. The alternative is to collect
ovaries from the slaughtered cows at the abattoir. About 98 percent of the total
population of cows slaughtered daily in Nigeria are cows that are over-aged after they
have been used for the production of either milk or work for several years and had
become less productive, therefore, less information are available for the purpose of
characterization. The use of ultrasonogrphy of cows, carried out at least daily, is highly
recommended for identifying follicles for description (Irving-Rodgers et al. 2009).
Though expensive, it is a very reliable method for observing larger number of heifers
without injury or eventual sacrificing of the animal, which made it initially, expensive,
but eventually saves cost and time especially if large numbers of animals are involved.
25
REFERENCES
Adams, G.P. and Pierson, R.A. (1995). Bovine model for study of ovarian follicular
dynamics in humans. Theriogenology 43; 113–120.
Bossis, I., Wettemann, R.P., Welty, S.D., Vizcarra, J. and Spicer, L.J. (2000).
Nutritionally induced an ovulation in beef heifers: ovarian and endocrine
function during realimentation and resumption of ovulation. Biology of
Reproduction. 62; 1436–1444.
Caglar, G., Asimakopoulos, B., Nikolettos, N., Diedrich, K. and Al-Hasani, S. (2005).
"Recombinant LH in ovarian stimulation.". Reprod Biomed Online 10 (6); 774-85.
Craig, J., Orisaka, M., Wang, H., Orisaka, S., Thompson, W., Zhu, C., Kotsuji, F. and
Tsang, B.K. (2000). Gonadotropin and intra-ovarian signals regulating follicle
development and atresia: the delicate balance between life and death. Endocr
Rev. 142; 2184-93.
Erickson, B.H.(1966). Development and senescence of the post natal bovine ovary. J
Anim Sci 25; 800–805.
Fortune, J., Cushman, R., Wahl, C., Kito, S. (2000). "The primordial to primary follicle
transition." Mol Cell Endocrinol 163 (1-2); 53-60
Fortune, J.E., Rivera, G.M., Evans, A.C. and Turzillo, A.M. (2001). Differentiation of
dominant versus subordinate follicles in cattle. Biology of Reproduction 65; 648–
654.
Fortune, J.E. (2007). The early stages of follicular development: activation of primordial
folliclesand growth of preantral follicles. Anim Reprod Sci 2003 , 78;135-63.
Front Biosci 12;3628-39.
26
Gougeon, A. and Chainy, G.B. (1987). Morphometric studies of small follicles in ovaries
of women at different ages. Journal of Reproduction and Fertility 81; 433–442.
Halpin, D.M., Charlton, H.M. and Faddy, M.J. (1986). Effects of gonadotrophin
deficiency on follicular development in hypogonadal (hpg) mice. J Reprod Fertil,
78;119-25.
Irving-Rodgers HF, Harland ML, Sullivan TR & Rodgers RJ (2009). Studies of granulosa
cell maturation in dominant and subordinate bovine follicles: novel extracellular
matrix focimatrix is co-ordinately regulated with cholesterol side-chain cleavage
CYP11A1. Reproduction 137; 825–834.
Isachenko, V., Lapidus, I. and Isachenko, E. (2009). "Human ovarian tissue vitrification
versus conventional freezing: morphological, endocrinological, and molecular
biological evaluation.". Reproduction 138; 319–27.
Malhi, P.S., Adams, G.P. and Singh, J. (2005). Bovine model for the study of
reproductive aging in women: follicular, luteal, and endocrine characteristics.
Biology of Reproduction.73; 45–53
Mcdonald, L. E. (1980). Veterinary endocrinology and reproduction. Lea and Febiger,
Philadalphia. Pp 274-398.
McGee, E. A., and Hsueh, A. J. (2000). Initial and cyclic recruitment of ovarian follicles.
Endocrine Reviews 21; 200-14.
27
McNatty, K.P., Reader, K., Smith, P., Heath, D.A. and Juengel J.L. (2006). Control of
ovarian follicular development to the gonadotrophin-dependent phase: Soc
Reprod Fertil Suppl .64;55-68.
Oktay K, Oktem O (November 2008). "Ovarian cryopreservation and transplantation for
fertility preservation for medical indications: report of an ongoing experience".
Fertil. Steril.. Doi:10.1016/j.fertnstert.2008.10.006.
Van den Hurk, R and Zhao J. (2005). "Formation of mammalian oocytes and their
growth, differentiation and maturation within ovarian follicles." Theriogenology
63 (6); 1717-51.
Van Wezel, I.L. and Rodgers, R.J. (1996). Morphological characterization of bovine
primordial follicles and their environment in vivo. Biology of Reproduction.vol.
55(1); Pp1003–1011
28