CONCOMITANT ADMINISTRATION OF RAPHIA MAMBILLENSES EXTRACT AND
PDE-5 INHIBITORS ON L-NAME INDUCED TESTICULAR DYSFUNCTION IN
WISTAR RAT
BY
OKON SHEMAIAH UDOH
(18/46KA122)
BEING A PROJECT SUBMITTED TO THE DEPARTMENT OF ANATOMY,
FACULTY OF BASIC MEDICAL SCIENCE, COLLEGE OF HEALTH SCIENCE,
UNIVERSITY OF ILORIN, KWARA STATE,NIGERIA.
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE AWARD OF
BACHELOR OF SCIENCE (B.Sc) DEGREE IN ANATOMY
SEPTEMBER, 2023
1
CERTIFICATION
This is to certify that this project was done and presented by OKON SHEMAIAH UDOH with
the matric number 18/46KA122 and has been read and approved as meeting part of the requirement
for the award of Bachelor of Science (B.Sc.) honour degree in Anatomy under the Department of
Anatomy, University of Ilorin, Kwara State, Nigeria.
………………………..
..……………………
PROF. A.O. OYEWOPO
DATE
SUPERVISOR
B.Sc (Ilorin) M.Sc. (Lagos) Ph.D. (Lagos)
………………………..
…………………….
DR. A. IBRAHIM
DATE
LEVEL ADVISER
B.Sc (Maiduguri); M.Sc. (Ilorin) Ph.D. (Cape town)
MPH (Ibadan)
……………………….
………………………
PROF.A.OLAWEOPO.
DATE
HEAD OF DEPARTMENT
MB;BS (Ilorin), M.Sc.(Lagos).
FWACS
………………………….
………………………
EXTERNALEXAMINER
DATE
2
DEDICATION
This work is dedicated to the Department of Anatomy
3
ACKNOWLEDGEMENT
My ultimate thanks for the completion of my Bachelor of science diploma Programme goes to
God.Also my appreciation goes to my project supervisor in person of PROF.A.O. Oyewopo whose
guidance and assistance made this work a success.
I also owe a big thanks to my parents, Mr Okon Udoh and my mum, Mrs Udoh for their constant
support both financially morally during my B.Sc. school years. I pray you shall eat the fruit your
Labor, all my family members are not excluded, I thank you for the love and support you showed
me during my academic race.
My profound gratitude goes to the H.O.D. in person of PROF.A. Olawepo and the entire
lecturers of the department of anatomy. I also appreciate the effort of my colleagues Mary,
muamed, damola and all the anatomy students of 2022/2023 set.
4
TABLE OF CONTENT
Title page
Certification
Dedication
Acknowledgement
Abstract
List of table
Table of figure
CHAPTER ONE : INTRODUCTION
1.0
Background of study
1.1 Statement of research problem
1.2 Experimental design
1.3 Aim of study
1.4 Objectives of study
1.5 Research question
1.6 Hypothesis statement
CHAPTER TWO: REVIEW OF RELATED LITERATURE
LITERATURE REVIEW
2.0 Infertility
2.1 Male infertility
2.1.1. Histology of adult male Wistar rat testes
2.2.2 Morphometry of adult male Wistar rat testes
2.3 Testicular dysfunction
2.4 Causes of testicular dysfunction
2.5. Nitric oxide
5
2.6. The role of nitric oxide in penile erection
2.7 Phosphodiesterase -5 inhibitors
2.70 Mechanism of action
2.71. Safety and adverse effect
2.7.2. PDE -5 inhibition and NO
2.8. L- NAME
2.80. Mechanism through which L- NAME induces testicular dysfunction
2.9. Raphia mambillenses
CHAPTER THREE: RESEARCH METHODOLOGY
3.1. Material and methods
3.2 Experimental animals
3.2.1. Procurement of animals
3.2.2. Animal care
3.2.3 Animal grouping
3.2.4. Animal feed and water measurement
3.3 Procurement and preparation of L-NAME solution
3.4 Procurement and preparation of the aqueous extracts of raphia mambillensis seed
3.5 Preparation and treatment with sildenafil citrate (viagra)
3.6 Animal sacrifice and tissue processing for histological studies
3.7 Blood collection
3.8. Tissue processing for histological studies
3.9. Biochemical analysis
3.10. Ethnical approval
6
CHAPTER FOUR: ANALYSIS OF RESULT
4.0. Results
4.1. Analysis of result
4.2. Histology analysis
CHAPTER FIVE: DISCUSSION
5.0. Discussion
5.1. Histological improvement
5.2. Conclusion
5.3. References
7
LIST OF TABLES
Table.
Title.
3.2.3Grouping of experimental animals
4.1. Biochemical and hormonal assay for experimental animals
8
TABLES OF FIGURES
Figure.
Title.
4.1.1
Graphical Representation of testosterone levels in the experimental groups
4.2. 2
Graphical Representation of nitric oxide (NO) levels in the experimental group
4.1.3
Graphical representation of Luteinizing Hormone (LH) in the experimental groups
4.2.1.
Histology of Control group
4.2.2.
Histology of L-NAME
4.2.3.
Histology of Raph a mambillenses
4.2.4.
Histology of Sildenafil citrate
4.2.5.
Histology of L-NAME +Sildenafil citrate
4.2.6.
Histology of L-NAME +Raphia mambillenses
9
LIST OF ABBREVIATIONS
AANOV.
Analysis of Variance
cGMP
Cyclic guanine monophosphate
DNA
DEOXYRIBONUCLEIC ACID
FSH
Follicle stimulating hormone
HPG
Hypothalamic pituitary gonadal axis
IACUC
Institutional Animal Care and Use Committee
KgBW
Kilogram/body weight
L-NAME
Nω-nitro-L-arginine methyl ester
LH
Luteinizing hormone
NO
Nitric oxide
NOS
Nitric oxide synthase
PDE 5
Phosphodiesterase 5
ROS
Reactive oxygen species
SEM
Standard Error of Mean
\
10
ABSTRACT
Testicular dysfunction is a pressing issue that can significantly impact male reproductive health,
potentially leading to infertility and a host of related physiological and psychological concerns.
This study seeks to unravel the potential therapeutic benefits of concomitant administration of
Raphia mambillenses and PDE-5 inhibitors in mitigating testicular dysfunction induced by LNAME, an established nitric oxide synthase inhibitor, in adult male Wistar rats.
The primary focus is on histomorphometric analysis, which involves the quantitative assessment
of tissue structure, to elucidate the impact of these interventions on testicular health. The rats are
divided into various groups, including those treated with L-NAME to induce testicular
dysfunction, those administered Raphia mambillenses extract, those given PDE-5 inhibitors, and
those subjected to both Raphia mambillenses and PDE-5 inhibitors. The L-NAME group
exihibited significant altercation in testicular histology; the seminiferous tubules and the lumen,
reduced sperm quality ; sperm count, sperm morphology, decreased testosterone levels compared
to the control group. However, the L-NAME + sildenafil group displayed improved testicular
histology, enhanced sperm count and restored testosterone level. Control groups are included for
comparison. In conclusion, the findings of this study suggests that PDE5 inhibitors have a
protective effect on testicular function in L-NAME induced testicular function in adult male Wistar
rat.
.
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CHAPTER ONE
INTRODUCTION
1.0 BACKGROUND OF STUDY
The testis is a vital organ in the male reproductive system. It has two essential roles: synthesis of
steroid hormones and production of sperms (Luaibe et al ., 2017) Several agents have a harmful
influence on the testes, either directly, by acting on the germ cells, or indirectly, by affecting the
somatic Sertoli and interstitial cells. Testicular dysfunction is a multifaceted condition that can
significantly impact male reproductive health. It is characterized by impaired spermatogenesis,
hormonal imbalances, and a range of sexual dysfunction issues. The causes of testicular
dysfunction are diverse, including genetic factors, environmental influences, and pathological
condition (Al-Majed et al., 2006)
Testicular function encompasses a range of processes crucial for male reproductive health. The
testes are responsible for spermatogenesis, the production of sperm, and the synthesis of sex
hormones, primarily testosterone. Both of these functions are vital for male fertility and overall
well-being (parker, 1993)
Spermatogenesis is the complex process through which male germ cells (spermatogonia)
differentiate into mature spermatozoa. It occurs within the seminiferous tubules of the testes and
involves multiple stages, including mitosis, meiosis, and spermiogenesis. The precise regulation
of spermatogenesis is essential for maintaining fertility (Sharpe, 2003)
Testicular function is intricately regulated by hormones, including luteinizing hormone (LH) and
follicle-stimulating hormone (FSH) from the anterior pituitary gland. These hormones stimulate
the Leydig cells to produce testosterone and Sertoli cells to support spermatogenesis.
12
The hypothalamic-pituitary-gonadal (HPG) axis tightly controls these hormonal interactions
(O’Donnell et al ., 1996)
The morphology of the testes, including their size, weight, and cellular composition, can provide
valuable insights into testicular health and function. Changes in testicular morphology may be
indicative of underlying issues affecting spermatogenesis and hormone production (Roosen-Runge
and Anderson, 1976)
1.1 STATEMENT OF RESEARCH PROBLEM
There is a need to investigate the impact of concomitant administration of Raphia mambillenses,
a natural plant extract, and PDE inhibitors on L-NAME induced testicular dysfunction in adult
male Wistar rats. This study aims to assess the histomorphometric changes resulting from this
combined intervention, including effects on spermatogenesis, testicular morphology, oxidative
stress markers, and vascular parameters (Smith et al., 2020)
This research problem encapsulates several essential aspects, including the need to elucidate the
potential synergistic effects of Raphia mambillenses and PDE-5 inhibitors, their influence on
critical histomorphometric parameters related to testicular function, and their potential in
ameliorating oxidative stress and vascular impairments. Understanding these effects is
fundamental to advancing our knowledge of potential treatments for testicular dysfunction and
ultimately improving male reproductive health (Tang et al., 2019)
1.2 EXPERIMENTAL DESIGN:
The study involves a randomized, controlled animal experiment, where adult male Wistar rats
are divided into different groups: control group, L-NAME-induced testicular dysfunction group,
groups receiving PDE-5 inhibitors, groups receiving
13
Raphia mambillenses, and groups receiving a combination of PDE-5 inhibitors and L-NAME.
The rats are treated for a specific duration, and their testicular tissues are then subjected to
histomorphometric analysis.
Overall, the background of the histomorphometry study aims to explore potential interventions
that may alleviate testicular dysfunction induced by L-NAME in adult male Wistar rats, with a
focus on the effects of concomitant administration of Raphia mambillenses and PDE-5 inhibitors.
It is important to remember that specific details of the study may vary based on the actual research
conducted, and the above outline serves as a general framework for such investigations.
1.3 AIM OF STUDY
The aims and objectives of investigating the histomorphometry study of concomitant
administration of Raphia mambillenses and PDE-5 inhibitors on L-NAME induced testicular
dysfunction in adult male Wistar rats can be summarized as follows::
•
To Evaluate the Effects on Testicular Morphology: The primary aim of the study is to
investigate the histomorphometric changes in the testicular tissues of adult male Wistar rats
induced by L-NAME administration. The study aims to assess any alterations in testicular
structure, including seminiferous tubule diameter, interstitial tissue, and Leydig cell
morphology.
•
To Examine Spermatogenesis: Another aim is to examine the impact of L-NAME-induced
testicular dysfunction on spermatogenesis, which refers to the process of sperm cell
development within the seminiferous tubules. The study aims to assess the quantity and
quality of sperm cells in the testicular tissues.
14
•
To Investigate the Potential Protective Effects of PDE-5 Inhibitors: The study seeks to
examine whether concomitant administration of PDE-5 inhibitors, such as sildenafil,
tadalafil, or vardenafil, can mitigate L-NAME-induced testicular dysfunction. This
investigation includes evaluating whether PDE-5 inhibitors can enhance nitric oxide (NO)
signaling, improve blood flow to the testes, and consequently, ameliorate testicular
function.
•
To Explore the Therapeutic Potential of Raphia mambillenses: The study aims to
investigate the effects of Raphia mambillenses, a plant with potential medicinal properties,
on L-NAME-induced testicular dysfunction. The objective is to determine whether Raphia
mambillenses administration can offer protective effects on testicular health and
reproductive function.
1.4 OBJECTIVES:
•
To Induce Testicular Dysfunction in Adult Male Wistar Rats: The study’s initial objective
is to induce testicular dysfunction in the adult male Wistar rats using L-NAME, a known
nitric oxide synthase inhibitor.
•
To Divide the Rats into Experimental Groups: The rats will be randomly divided into
different experimental groups, including a control group, a group receiving L-NAME,
groups receiving PDE-5 inhibitors, groups receiving Raphia mambillenses, and groups
receiving a combination of PDE-5 inhibitors and Raphia mambillenses.
•
To Administer Interventions: The study aims to administer the respective interventions (LNAME, PDE-5 inhibitors, and Raphia mambillenses) to the designated groups for a
specific duration based on the experimental design.
15
•
To Perform Histomorphometric Analysis: After the treatment period, the testicular tissues
of the rats will be harvested and subjected to histomorphometric analysis to quantify
various parameters related to testicular structure and spermatogenesis.
The overall aims and objectives of this histomorphometry study are to advance our
understanding of the potential therapeutic effects of PDE-5 inhibitors and Raphia
mambillenses in mitigating testicular dysfunction induced by L-NAME in adult male Wistar
rats. The study may provide valuable insights into the mechanisms of testicular dysfunction
and may have implications for further research on potential treatments for testicular
dysfunction in humans.
1.5 RESEARCHQUESTION
•
How does the concomitant administration of Raphia mambillenses and PDE-5 inhibitors
impact the histomorphometry of testicular tissues in adult male Wistar rats with L-NAME
induced testicular dysfunction?
•
What are the specific changes in testicular structure, including seminiferous tubule
diameter, interstitial tissue, and Leydig cell morphology, following the concomitant
treatment of Raphia mambillenses and PDE-5 inhibitors in the L-NAME induced testicular
dysfunction model?
•
Does the concomitant administration of Raphia mambillenses and PDE-5 inhibitors lead to
improvements in spermatogenesis in adult male Wistar rats with L-NAME induced
testicular dysfunction, and if so, to what extent?
•
How does the combined treatment of Raphiamambillenses and PDE-5 inhibitors affect the
hormonal regulation of testosterone, luteinizing hormone (LH), and follicle-stimulating
hormone (FSH) in the context of L-NAME induced testicular dysfunction?
16
These research questions aim to explore the effects of Raphia mambillenses and PDE-5
inhibitors, both individually and in combination, on histomorphometric parameters related to
testicular structure, spermatogenesis, and hormonal regulation in the context of L-NAME
induced testicular dysfunction. By addressing these questions, the study seeks to shed light on
the potential therapeutic benefits and mechanisms of action of these interventions for
improving testicular health and function.
1.6 HYPOTHESISSTATEMENT
The concomitant administration of Raphia mambillenses and PDE-5 inhibitors in adult male
Wistar rats with L-NAME induced testicular dysfunction will lead to significant improvements in
testicular histomorphometry, including increased seminiferous tubule diameter, improved
spermatogenesis, and enhanced hormonal regulation compared to the L-NAME-induced testicular
dysfunction group. Additionally, we expect that the combination treatment will show more
pronounced positive effects on testicular health parameters compared to the individual
administration of Raphia mambillenses or PDE-5 inhibitors alone, indicating potential synergistic
effects of the concomitant therapy on testicular dysfunction in this experimental model.”
17
CHAPTER TWO
REVIEW OF RELATED LITERATURE
2.0. INFERTILITY
Infertility is usually defined as the inability of a couple to conceive even after one year of
unprotected, frequent sexual intercourse (Fertil, 2008) It affects about 15% of all couples in the
United States and at least 180 million worldwide (Thonneau et al ., 1991) Male infertility is defined
as the inability of a male to make a fertile female pregnant, also for a minimum of at least one year
of unprotected intercourse. The male is solely responsible for about 20% and is a contributing
factor in another 30% to 40% of all infertility cases. As male and female causes often co-exist, it
is important that both partners are investigated for infertility and managed together. Overall, the
male factor is substantially contributory in about 50% of all cases of Infertility (Hull et al ., 1985)
There are several reasons for male fertility to occur, including both reversible and irreversible
conditions. Other factors that could influence each of the partners could be their age, medications,
surgical history, exposure to environmental toxins, genetic problems, and systemic diseases. The
key purpose for evaluating a male for infertility is to identify his contributing factors, offer
treatment for those that are reversible, determine if he is a candidate for assisted reproductive
techniques (ART) and offer counseling for irreversible and untreatable conditions (Shih et
al.,2019) In rare cases, male infertility could be a herald to a more serious condition. This is an
additional reason to do a comprehensive evaluation of the male partners of infertile couples; so
that any significant, underlying medical conditions can be identified and treated (Honig et al .,
1994)
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2.1. MALE INFERTILITY
Male infertility refers to a sexually mature male's inability to impregnate a fertile female. In
humans it accounts for 40–50% of infertility (Pandruvada et al., 2021) It affects approximately 7%
of all men. Male infertility is commonly due to deficiencies in the semen, and semen quality is
used as a surrogate measure of male fecundity. More recently, advance sperm analyses that
examine intracellular sperm components are being developed (Turner et al ., 2020)
Etiology
There are multiple causes for male infertility, which can be broadly classified due to their general
underlying etiology. These include endocrine disorders (usually due to hypogonadism) at an
estimated 2% to 5%, sperm transport disorders (such as vasectomy) at 5%, primary testicular
defects (which include abnormal sperm parameters without any identifiable cause) at 65% to 80%
and idiopathic (where an infertile male has normal sperm and semen parameters) at 10% to 20%
(Winters and Walsh, 2014) These are broad estimates only as accurate statistics are unavailable
due to general underreporting, cultural factors, and regional variations. Patients sent to a tertiary
referral center are more likely to have their condition reported, while private patients may never
have their data collected (Winters and Walsh, 2014) A partial summary of specific etiologies is
listed below:
•
Endocrinological cause – congenital GnRH Deficiency (Kallmann syndrome), Prader Willi
syndrome, Laurence – Moon – Beidl syndrome, iron overload syndrome, familial
cerebellar ataxia, head trauma, intracranial radiation, testosterone supplementation, or
hyperthyroidism.
•
Idiopathic – idiopathic male infertility (10% to 20%) where semen parameters are all
normal, but the male remains infertile.
19
•
Genetic causes – mutations of the cystic fibrosis transmembrane conductance regulator
(CFTR) gene, primary ciliary dyskinesia, Kallmann syndrome, Klinefelter’s syndrome,
Young syndrome, Sertoli cell-only syndrome, Kal- 1, Kal -2, FSH, LH, FGFS,
GnRH1/GNRHR PROK2/PROK2R gene deficiencies, chromosomal anomalies, Y
chromosome micro deletion, AR mutations, gr/gr deletion.
•
Congenital urogenital abnormalities – absent, dysfunctional, or obstructed epididymis,
congenital abnormalities of the vas deferens, undescended testes, ejaculatory duct disorders
(cysts).
•
Acquired urogenital abnormalities – bilateral obstruction or ligation of the vas deferens,
bilateral orchiectomy, epididymitis, varicoceles, retrograde ejaculation.
•
Urogenital tract infections – Gonococci, chlamydia, syphilis, tuberculosis, recurrent
urogenital infections, prostatitis, and recurrent prostatovesiculitis.
•
Sexual dysfunction – premature ejaculation, anejaculation, infrequent sexual intercourse,
and erectile dysfunction.
•
Malignancies – sellar masses, pituitary macroadenomas, craniopharyngiomas, and surgical
or radiation treatment to these conditions, testicular tumors, or adrenal tumors leading to
an excess of androgens.
•
Medications or drugs – cannabinoids, opioids, psychotropic drugs can cause inhibition of
GnRH, exogenous testosterone or androgenic steroids supplementation, GnRH analogs and
antagonists used in prostatic carcinoma, chronic glucocorticoid therapy, alkylating agents,
antiandrogens, ketoconazole, cimetidine.
•
Environmental toxins – insecticides, fungicides, pesticides, smoking, excess alcohol
(Winters and Walsh, 2014)
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2.1.1 HISTOLOGY OF ADULT MALE WISTAR RAT TESTES
The histology of the adult male Wistar rat testis involves the microscopic examination of its
various components, including the seminiferous tubules, interstitial tissue, and supporting
cells:
•
Sertoli Cells: Sertoli cells are supporting cells found within the seminiferous tubules. They
provide physical and nutritional support to developing germ cells and are essential for
spermatogenesis (Johnson etal., 2015)
•
Epididymus: The testes are partially covered by the epididymus which consists of 3
regions: the caput epididymus (located at the top), the corpus epididymus (running down
one side), and the cauda epididymus (the portion at the bottom of the testes) The caput
epididymus is enveloped in fat and leads into the ductus deferens. The epididymus stores
the sperm that have been manufactured within the testes (Cold and Taylor ,1999)
•
Leydig Cells: Leydig cells, also known as interstitial cells, are located in the interstitial
tissue surrounding the seminiferous tubules. They produce testosterone, which is critical
for the maintenance of male reproductive function (Davidoff et al., 2013).
•
Rete Testis: The rete testis is a network of tubules that connects the seminiferous tubules
to the epididymis. It serves as a conduit for sperm transport (Nistal et al., 2006)
•
Seminiferous Tubules: The bulk of testicular tissue is composed of seminiferous tubules,
coiled structures where sperm production, or spermatogenesis, takes place. These tubules
are lined with germinal epithelium, where spermatogonia develop into mature spermatozoa
(Clermont, 1972).
1.
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2.1.2 MORPHOMETRY OF ADULT MALE WISTAR RAT TESTES
Morphometry of the adult male Wistar rat testis involves the quantitative measurement of various
anatomical and histological parameters within the testicular tissue. Here are morphometric aspect
of a Wistar rat testis:
❖ Leydig Cells: Leydig cells are thought to be the source of most, if not all, the
testosterone produced by the testis. The goal of this study was to obtain quantitative
information about rat Leydig cells and their organelles that might be correlated with
pertinent physiological and biochemical data available either now or in the future.
Morphometric analysis of Leydig cells in mature normal rats was carried out on
tissue fixed by perfusion with buffered glutaraldehyde, and embedded in glycol
methacrylate for light microscopy and in Epon for electron microscopy. The most
prominent ultrastructural Features exhibited by these cells are an abundant Smooth
endoplasmic reticulum (SER) and fairly Numerous mitochondria. Lipid droplets
are common in Leydig cells of some species, although not In those of adult
laboratory rats. The biosynthesis Of testosterone is catalyzed by enzymes located
Predominantly on membranes of the SER and in Adjacent cytoplasm, although few
steps occur on The inner mitochondrial membranes. The total Leydig cell
population in I g of rat testis produces 6.7 ng of testosterone/min in vivo. If we
could Determined the number of Leydig cells in that Amount of testis, it would be
possible to calculate The rate of testosterone production per average Leydig cell
(Free and tillson 1973)
❖ Seminiferous tubules: Histomorphometry of seminiferous tubules in adult male
Wistar rats involves the quantitative analysis of various parameters within these
tubules, providing valuable insights into spermatogenesis and testicular health.
22
•
Seminiferous Tubule Diameter: Measuring the diameter of seminiferous tubules is
a critical histomorphometric parameter. It reflects the structural integrity of the
tubules and can provide information about their developmental stage and health
(Staub et al., 1984).
•
Tubule Length: Quantifying the length of seminiferous tubules allows for the
assessment of the total tubular surface area available for spermatogenesis
(Berndston et al., 2008).
•
Epithelial Height: The height of the germinal epithelium within seminiferous
tubules is an essential histomorphometric parameter. It reflects the degree of
spermatogenic activity and the presence of mature spermatozoa (Griswold, 2016).
•
Lumen Diameter: Measuring the diameter of the tubular lumen provides insights
into the overall structure of the seminiferous tubules and the space available for
spermatozoa (Berndston et al., 2008)
❖ Sertoli Cells: Histomorphometry of Sertoli cells in adult male Wistar rats involves
the quantitative analysis of various parameters related to these critical supporting
cells within the seminiferous tubules.
•
Sertoli Cell Count: Quantifying the number of Sertoli cells within a defined area of
seminiferous tubules provides essential information about their density and
distribution (Johnson et al., 2015)
•
Sertoli Cell Diameter: Measuring the diameter of Sertoli cells can indicate their size
and morphology within the seminiferous tubules (Orth et al., 1988)
•
Sertoli Cell Nuclei: Analyzing the characteristics of Sertoli cell nuclei, such as size
and shape, can offer insights into their structural features and function (Hess et al.,
1993)
23
•
Sertoli Cell Junctions: Assessing the presence and integrity of Sertoli cell junctions,
such as the blood-testis barrier, is crucial for understanding their role in
spermatogenesis (Mruk et al., 2011)
2.2. SPERMATOGENESIS
Spermatogenesis is the process by which haploid spermatozoa develop from germ cells in the
seminiferous tubules of the testis. This process starts with the mitotic division of the stem cells
located close to the basement membrane of the tubules (de Kretser et al ., 1998)
Stages of Spermatogenesis:
Spermatogenesis in Wistar rats, as in many mammals, is a complex and sequential process
occurring within the seminiferous tubules of the testes. It comprises three main phases: mitosis,
meiosis, and spermiogenesis (Skinner, 1991)
MITOSIS:
Spermatogenesis initiates with mitosis, involving the proliferation of spermatogonia, the male
germ stem cells, through a series of divisions. These cells are situated at the basal compartment of
the seminiferous tubules and represent the source of all future germ cells. Some spermatogonia
remain as stem cells, while others differentiate into primary spermatocytes (Russell et al., 1990)
Cell division by mitosis gives rise to genetically identical cells in which the total number of
chromosomes is maintained. Therefore, mitosis is also known as equational division. In general,
mitosis is preceded by S phase of interphase during which DNA replication occurs (Todd and
Isabella, 2019) and is often followed by telophase and cytokinesis; which divides the cytoplasm,
organelles and cell membrane of one cell into two new cells containing roughly equal shares of
these cellular components (Carter, 2014) The different stages of mitosis altogether define the
24
mitotic (M) phase of a cell cycle—the division of the mother cell into two daughter cells
genetically identical to each other (Petra et al ., 2018)
MEIOSIS:
Primary spermatocytes, the result of mitotic divisions, undergo meiosis, a reduction division
process, that produces haploid secondary spermatocytes. These secondary spermatocytes further
divide into haploid round spermatids, marked by a significant reduction in chromosome number.
Meiosis ensures genetic diversity in offspring by recombination and segregation of genetic
material (Russell et al., 1990).
SPERMIOGENESIS
Spermiogenesis is the final stage of spermatogenesis, during which the spermatids develop into
mature spermatozoa. At the beginning of the stage, the spermatid is a more or less circular cell
containing a nucleus, Golgi apparatus, centriole and mitochondria; by the end of the process, it has
radically transformed into an elongated spermatozoon, complete with a head, midpiece, and tail
(O'Donnell et al ., 2011)
•
Golgi phase
The spermatids, which up until now have been mostly radially symmetrical, begin to develop
polarity. The head forms at one end, where the Golgi apparatus creates enzymes that will become
the acrosome. At the other end, it develops a thickened midpiece, where the mitochondria gather
and the distal centriole begins to form an axoneme.
Spermatid DNA also undergoes packaging, becoming highly condensed. The DNA is first
packaged with specific nuclear basic proteins, which are subsequently replaced with protamines
during spermatid elongation. The resultant tightly packed chromatin is transcriptionally inactive
((O'Donnell et al ., 2011)
25
•
Cap/acrosome phase
The Golgi apparatus surrounds the condensed nucleus, becoming the acrosomal cap.
•
Formation of tail
One of the centrioles of the cell elongates to become the tail of the sperm. A temporary
structure called the “manchette” assists in this elongation. During this phase, the developing
spermatozoa orient themselves so that their tails point towards the center of the lumen, away
from the epithelium (Fraser, 1998)
•
Maturation phase
The excess cytoplasm, known as residual body of Regaud, is phagocytized by surrounding
Sertoli cells in the testes (Fraser, 1998)
2.3. TESTICULAR DYSFUNCTION
Testicular failure, also known as primary hypogonadism, is an uncommon condition that is
characterized by the inability of the testicles to produce sperm and the male hormone testosterone.
The are many factors that have been postulated as causes of the condition, which give rise to a
wide array of signs and symptoms. These factors can sometimes make diagnosis as well as
treatment of testicular failure a challenging problem (Yolanda, 2023)
2.4. CAUSES OF TESTICULAR DYSFUNCTION
❖ HORMONAL IMBALANCES:
•
Hypogonadism: Hypogonadism is a condition characterized by insufficient testosterone
production. It can be caused by primary factors (testicular dysfunction) or secondary
factors (disorders of the hypothalamus or pituitary gland). Primary hypogonadism may
result from genetic conditions, aging, or testicular injury (Bhasin et al., 2018)
26
•
Hyperprolactinemia: Elevated levels of the hormone prolactin can inhibit gonadotropinreleasing hormone (GnRH), which in turn suppresses testosterone production. This
hormonal imbalance can stem from pituitary tumors or medications (Melmed et al., 2017)
❖ VARICOCELE
A varicocele is an abnormal enlargement of the pampiniform venous plexus in the male
scrotum; the female equivalent of painful swelling to the embryologically identical
pampiniform venous plexus is called pelvic compression syndrome. This plexus of veins
drains blood from the testicles back to the heart. The vessels originate in the abdomen and
course down through the inguinal canal as part of the spermatic cord on their way to the
testis. Varicoceles occur in around 15% to 20% of all men (White et al ., 2019) The
incidence of varicocele increase with age.
Often the greatest concern with respect to varicocele is its effect on male fertility. The
relationship between varicocele and infertility is unclear. Some men with the condition are
fertile, some have sperm that are normal in shape and move normally but are compromised
in function, and some have sperm with abnormal shapes or that do not move well
(.Eisenberg et al ., 2011) Theories as to how varicocele affects sperm function include
damage via excess heat caused by the blood pooling and oxidative stress on sperm (Dabaja
et al ., 2013)
❖ INFECTION AND INFLAMMATION
•
ORCHITIS
Orchitis is defined as the inflammation of the testicle unilaterally or bilaterally usually caused by
viruses and bacteria. Testes are two oval male reproductive organs situated in the scrotum. They
are responsible for the production of male sex hormones and sperm. The testis is innervated by the
testicular plexus, which contains nerves originating from the renal and aortic plexus.
27
The main arterial supply is by paired testicular artery, arising from the abdominal aorta, which
passes via the inguinal canal inside the spermatic cord. Venous drainage is through the
pampiniform plexus. As testis are originally retroperitoneal organs, lymphatic drainage is through
para-aortic lymph nodes, while superficial inguinal lymph nodes drain the scrotum.
Orchitis can be acute and symptomatic on the presentation or asymptomatic and chronic
(Pilatz et al ., 2019) Isolated orchitis is rare and is usually accompanied by an infection of
the epididymis. The major route for the spread is blood-borne dissemination for isolated
orchitis (Krieger et al ., 1984) Ascending infections can also involve testis.
Various bacteria and viruses cause orchitis.
•
Orchitis in young patients is usually viral, with mumps and rubella being the most common
causes. Reports exist of cases of orchitis after the measles, mumps, and rubella (MMR)
vaccine (Kanda et al ., 2014)
•
Other viruses include coxsackievirus, varicella, echovirus, and cytomegalovirus.
Bacterial infections of the prostate and urinary tract infection can cause orchitis. Common
causes of bacterial orchitis include Escherichia coli, Klebsiella pneumoniae, Pseudomonas
aeruginosa, and Staphylococcus and Streptococcus species.
•
Bacteria that can cause sexually transmitted infections can also cause orchitis in sexually
active males. Common organisms are Neisseria gonorrhoeae, Chlamydia trachomatis, and
Treponema pallidum
•
Mycobacterium avium complex, Cryptococcus neoformans, Toxoplasma gondii,
Haemophilus parainfluenzae, and Candida albicans have been reported to cause orchitis in
immunocompromised patients
Complications are preventable with accurate diagnosis and efficient management of patients.
28
Most patients recover without sequelae; however, there are reports of the following
complications:
•
Testicular atrophy (up to 60% of cases demonstrate some degree of atrophy)
•
Impaired fertility
•
Sterility (rare)
•
Epididymitis
•
Reactive hydrocele
Rarely in cases of pyogenic orchitis and testicular infarction, an abscess can be formed which
requires surgical consultation for management (Street et al ., 2017)
•
EPIDIDYMITIS
The epididymis is part of the genitourinary tract that includes the testes, the vas deferens,
the prostate, the urethra, and the bladder. Epididymitis is an infection or inflammation of
the epididymis, the tubular structure located on the posterior and superior aspect of the
testis where sperms mature prior to ejaculation. Because of its proximity to the testis, any
infectious or inflammatory process affecting the epididymis may spread to the testis itself,
a condition known as epididymo-orchitis (Louette et al ., 2018)
The majority of cases of epididymitis occur as a result of bacterial infection. The types of
bacterial infection include common urinary pathogens as well as pathogens known to cause
sexually transmitted disease. In most cases of epididymitis, infection occurs either as a
result of the retrograde flow of urine, most commonly seen in elderly males, or as a result
of a sexually transmitted disease, most often encountered in males ages 20 to 40. In males
prior to sexual maturity, the most common cause of epididymitis is inflammation that
occurs as a result of trauma or repetitive activities such as sports.
29
The possibility of a sexually transmitted disease, however, must be considered even in
males prior to sexual maturity due to the possibility of sexual abuse. Other possible causes
of epididymitis include chemical, drug-induced, and viral infections (Agrawal et al ., 2018)
Epididymitis if not treated properly or promptly can lead to the following complications:
•
Infection of the epididymis can lead to the formation of an epididymal abscess
•
Progression of the infection can lead to the involvement of the testicle, causing epididymoorchitis or a testicular abscess
•
Sepsis is a potential consequence of severe infection
•
Bilateral epididymitis may result in sterility due to occlusion of the ductules from
peritubular fibrosis (Agrawal et al ., 2018)
❖ GENETIC FACTORS:
Many genes have been implicated in the disorders of TDS, with genome wide association
studies (GWAS) regularly identifying new gene variants that play a role in abnormal testes
development. Some of these are specific to certain disorders, and some are part of a ‘risk
factor network’ that connect TGCC, hypospadias, cryptorchidism, poor semen quality. The
majority of these genes are involved in fetal gonad development. Mutations in androgen
receptor genes are highly implicated, as these are involved in penile development, testes
descent, and testes development (Skakkebæk et al ., 2001) Testicular germ cell cancer
(TGCC) shows a strong genetic disposition, with the most significant gene variants being
those linked to gonad formation and germ cell function (Niels et al ., 2016)
30
❖ ENVIRONMENTAL FACTORS
Exposure of a male fetus to substances that disrupt hormone systems, particularly
chemicals that inhibit the action of androgens (male sex hormones) during the development
of the reproductive system, has been shown to cause many of the characteristic TDS
disorders. These include environmental estrogens and anti-androgens found in food and
water sources that have been contaminated with synthetic hormones and pesticides used in
agriculture (Meyts, 2006) In historical cases, medicines given to pregnant women, like
diethylstilbestrol (DES), have caused many of the features of TDS in fetuses exposed to
this chemical during gestation (Gill et al ., 1979) The impact of environmental chemicals
is well documented in animal models (Skakkebæk et al ., 2001)
If a substance affects Sertoli and Leydig cell differentiation (a common feature of TDS
disorders) at an early developmental stage, germ cell growth and testosterone production
will be impaired (Niels et al ., 2016) These processes are essential for testes descent and
genitalia development, meaning that genital abnormalities like cryptorchidism or
hypospadias may be present from birth, and fertility problems and TGCC become apparent
during adult life. Severity or number of disorders may therefore be dependent on the timing
of the environmental exposure Environmental factors can act directly, or via epigenetic
mechanisms, and it is likely that a genetic susceptibility augmented by environmental
factors is the primary cause of TDS (Skakkebæk et al ., 2001)
❖ PATHOGENESIS
The TDS hypothesis proposes that testicular dysgenesis, which has various primary causes,
can lead to abnormalities in Sertoli and/or Leydig cell function.
31
This leads to both impaired germ cell development and hormonal changes during male
sexual differentiation. For instance, insufficient production of testosterone can result in
incomplete masculinisation, whilst reduced expression of insulin-like factor 3 can lead to
incomplete testes descent (Sharpe et al ., 2008) The downstream disorders of such
abnormalities
can
include
both
genital
malformations
(e.g.hypospadias
and
cryptorchidism) and delayed reproductive disorders (e.g. testicular cancer and poor semen
quality) which comprise TDS (Bay et al ., 2006)
2.5 NITRIC OXIDE
Nitric oxide (NO) is an unorthodox messenger molecule, which has numerous molecular
target. NO control servoregulatory functions such as neurotransmission (Schuman and
Madison ,1991) or vascular tone (Förstermann et al ., 1986) (by stimulating NOsensitive guanylyl cyclase), regulates gene transcription and mRNA translation (e.g. by
binding to iron-responsive elements) (Liu et al., 2002) and produces post-translational
modifications of proteins (e.g. by ADP ribosylation)
An important mode of inactivation of NO is its reaction with superoxide anion (O2−•)
to form the potent oxidant peroxynitrite (ONOO−). This compound can cause oxidative
damage, nitration, and S-nitrosylation of biomolecules including proteins, lipids, and
DNA. Nitrosative stress by ONOO− has been implicated in DNA single-strand breakage,
followed by poly-ADP-ribose polymerase (PARP) activation. In mammals, NO can be
generated by three different isoforms of the enzyme NO synthase (NOS; l-arginine,
NADPH:oxygen oxidoreductases, NO forming; EC 1.14.13.39). The isozymes are referred
to as neuronal ‘n’NOS (or NOS I), inducible ‘i’NOS (or NOS II), and endothelial ‘e’NOS
[or NOS III] (Lee et al ., 2003)
32
MECHANISM OF NITRIC OXIDE SYNTHESIS
All isoforms of NOS utilize l-arginine as the substrate, and molecular oxygen and
reduced nicotinamide-adenine-dinucleotide phosphate (NADPH) as co-substrates. Flavin
adenine dinucleotide (FAD), flavin mononucleotide (FMN), and (6R-)5,6,7,8-tetrahydrol-biopterin (BH4) are cofactors of all isozymes. All NOS proteins are homodimers. A
functional NOS transfers electrons from NADPH, via the flavins FAD and FMN in the
carboxy-terminal reductase domain, to the haem in the amino-terminal oxygenase domain.
The oxygenase domain also binds the essential cofactor BH4, molecular oxygen, and the
substrate l-arginine (Alderton et al ., 2001) At the haem site, the electrons are used to
reduce and activate O2 and to oxidize l-arginine to l-citrulline and NO. Sequences located
near the cysteine ligand of the haem are also apparently involved in l-arginine and BH4
binding (Nishimura et al ., 1995)
In a second step, NOS oxidizes Nω-hydroxy-l-arginine to l-citrulline and NO. All
isoforms of NOS bind calmodulin. In nNOS and eNOS, calmodulin binding is brought
about by an increase in intracellular Ca2+ (half-maximal activity between 200 and 400
nM). When calmodulin affinity to NOS increases, it facilitates the flow of electrons from
NADPH in the reductase domain to the haem in the oxygenase domain. In inducible NOS
(iNOS), calmodulin already binds at extremely low intracellular Ca2+ concentrations
(below 40 nM) due to a different amino acid structure of the calmodulin-binding site. All
NOS proteins contain a zinc–thiolate cluster formed by a zinc ion that is tetrahedrally
coordinated to two CysXXXXCys motifs (one contributed by each monomer) at the NOS
dimer interface. Zinc in NOS has a structural rather than a catalytic function (Hemmenset
al., 2000)
2.
33
(Research gate, 2021)
2.6THE ROLE OF NITRIC OXIDE IN PENILE ERECTION
The functional state of the penis, flaccid or erect is governed by smooth muscle tone.
Sympathetic contractile factors maintain flaccidity whilst parasympathetic factors induce
smooth muscle relaxation and erection. It is generally accepted that nitric oxide (NO) is the
principal agent responsible for relaxation of penile smooth muscle. NO is derived from two
principal sources: directly from non-adrenergic non-cholinergic parasympathetic nerves
and indirectly from the endothelium lining cavernosal sinusoids and blood vessels in
response to cholinergic stimulation. The generation of NO from L-arginine is catalysed by
nitric oxide synthase (NOS).
34
There has been controversy over the relative prevalence of endothelial or neuronal NOS
within the penis of different animal species. This review examines the role of NO in the
penis in detail. Established and new treatments for erectile dysfunction whose effects are
mediated via manipulation of the NO pathway are also described (J Cartledge et al., 2001)
2.7 PHOSPHODIESTERASE -5 INHIBITORS
During sexual arousal, nitric oxide (NO) is released from nerve terminals and endothelial
cells in the corpus cavernosum. NO activates guanylate cyclase to convert guanosine
triphosphate (GTP) into cyclic guanosine monophosphate (cGMP), triggering a cGMPdependent cascade of events. The accumulation of cGMP leads to smooth-muscle
relaxation in the corpus cavernosum and increased blood flow to the penis.
PDE5 is an enzyme found primarily in the smooth muscle of the corpus cavernosum that
selectively cleaves and degrades cGMP to 5′-GMP. PDE5 inhibitors are similar in structure
to cGMP; they competitively bind to PDE5 and inhibit cGMP hydrolysis, thus enhancing
the effects of NO. This increase in cGMP in the smooth muscle cells is responsible for
prolonging an erection.
PDE5 inhibitors lack a direct effect on corpus cavernosum smooth-muscle relaxation.
Therefore, after administration, adequate sexual stimulation is necessary for an erection to
occur (Limin et al.,2010)
Among the phosphodiesterase inhibitors, sildenafil is the more common agent acting on
PDE-5 and is FDA-approved in 1998 for erectile dysfunction . Sildenafil use is not limited
to the treatment of erectile dysfunction(ED) and obtained FDA approval in 2005 to treat
pulmonary arterial hypertension (PAH) in adults.
35
The use of Sildenafil in pediatric populations for pulmonary arterial hypertension(PAH)
has not received approval and is considered off-label use. Vardenafil and avanafil are other
PDE-5 inhibitors, also approved for erectile dysfunction(ED). Tadalafil is a PDE-5
inhibitor
approved
to
treat
benign
prostatic
hyperplasia(BPH)
and
erectile
dysfunction(ED). As both conditions may coincide in the later durations of life, tadalafil
may be used as monotherapy for its therapeutic use to treat both conditions (Mónica et
al.,2019)
MECHANISMOFACTION
Cyclic adenosine monophosphate(cAMP) and cyclic guanosine monophosphate
(cGMP) are intracellular second messenger molecules degraded and inactivated by the
enzyme phosphodiesterase [PDE] (Pasmanter et al.,2022) Phosphodiesterase inhibitors
exert their effects on their targeted phosphodiesterase enzymes(PDE-3, PDE-4, PDE-5),
preventing cGMP or cAMP degradation, further increasing their levels in smooth muscle
cells, causing relaxation and vasodilatory effect in target cells. PDE-3 inhibitors exert their
effects by increasing cAMP levels in the myocardium, peripheral vasculature, and platelets.
This further causes positive inotropic effects by increasing ionized calcium in the
myocardium, vasodilation of the peripheral vessels, and preventing platelet aggregation
and can be used to treat decompensated cardiac failure and peripheral arterial
disease(PAD).
PDE-4 is an enzyme found in cells of the lungs, and PDE-4 inhibitors inhibit the
degradation of intracellular cyclic adenosine monophosphate(cAMP) and increase cAMP
levels in target cells, further causing bronchial muscle relaxation. Along with its effects on
the lungs, it also decreases pro-inflammatory mediators (Zebda et al., 2018)
36
These agents are useful in treating COPD, psoriatic arthritis(PA), and atopic
dermatitis(AD). Crisaborole is a PDE-4 inhibitor indicated for mild to moderate atopic
dermatitis, and its mechanism of action its therapeutic effect is not fully understood.
PDE-5 inhibitors carry out their action by preventing the breakdown of cyclic guanosine
monophosphate (cGMP), further increasing cGMP levels in target cells. The endothelial
cells of the penile smooth muscle corpus cavernosum release nitric oxide(NO), which
initiates the enzyme guanylate cyclase, further enhancing the synthesis of cyclic guanosine
monophosphate (cGMP). The prevention of cGMP breakdown further prolongs an erection
and is used therapeutically to treat erectile dysfunction(ED) (Dhaliwal, 2023) PDE-5
inhibitors also cause pulmonary vasodilation and may be useful for treating pulmonary
arterial hypertension(PAH) as PAH may be correlated with the weakened release of nitric
oxide in the pulmonary vasculature, resulting in decreased cGMP levels. Nonspecific
inhibitors(PDE-3,4,5) exert their action, increasing cAMP levels in the pulmonary
vasculature, resulting in bronchial relaxation and further decreasing pro-inflammatory
mediators (Bergstrand, 1980)
2.7.1SAFETY AND ADVERSE EFFECT
PDE-5 inhibitors are generally well tolerated for the treatment of ED. The most common
adverse drug reactions reported include headache, flushing, nasal congestion,
nasopharyngitis, and dyspepsia. Rare but serious reports of prolonged erections lasting
more than 4 hours and priapism (painful erections lasting more than 6 hours) have been
reported with PDE5 inhibitors. Patients should be advised to seek immediate medical
attention if they experience these events. Cases of priapism that are not immediately treated
can lead to permanent penile tissue damage (Omori, 2007)
37
Visual abnormalities have been reported with PDE5 inhibitors. In July 2005, an FDA
alert recommended that men discontinue all such agents and seek immediate medical
attention if they experienced a sudden loss of vision .Cases of non-arteritic anterior
ischemic optic neuropathy (NAION) were reported during postmarketing experience with
PDE5 inhibitors (Pomeranz and Bhavsar, 2005) In this condition, blood flow to the optic
nerve is blocked. Although evidence of a cause-and-effect relationship is insufficient,
caution should be used in prescribing PDE5 inhibitors, particularly for men with preexisting risk factors for the development of NAION, such as hypertension, diabetes, and
hyperlipidemia.
Sudden hearing loss was also reported in postmarketing studies. In October 2007, the
FDA requested that this potential risk be displayed more prominently on the labels for
PDE5 inhibitors. At the time of the announcement, 29 cases of hearing loss had been
reported in postmarketing analyses. Additional cases were identified in a retrospective
analysis of the clinical trials. Although the direct association of hearing loss with PDE5
inhibitor use has not been established, patients were advised to discontinue the use of all
PDE5 inhibitors and to seek medical attention if they experienced a sudden decrease in or
a loss of hearing (Cialis, 2011)
Back pain and myalgia have been reported with tadalafil. In general, the pain was
reported as mild to moderate in severity, occurring 12 to 24 hours after administration and
typically resolving within 48 hours without medical treatment. When treatment was
necessary, acetaminophen and nonsteroidal anti-inflammatory drugs (NSAIDs) were
generally effective in alleviating the reported pain (Cialis, 2011)
38
2.7.2 PDE-5 INHIBITON AND NO
NO promotes penile vasodilation and blood flow by diffusing across the smooth muscle
membrane and activating sGC to produce cGMP, resulting in an enzymatic cascade that
inhibits calcium influx, lowers cytosolic calcium concentrations, and thus induces
relaxation of cavernosal smooth muscle. PDE‐5 catalyzes the degradation of cGMP,
facilitating smooth muscle contraction (Friebe, 2003) PDE‐5 is the most important of the
PDEs in the corpora cavernosa (Corbin et al.,2002) By selectively blocking the PDE‐5
enzyme, PDE‐5 inhibitors thus preserve and sustain the NO‐triggered increase in cGMP
that promotes cavernosal trabecular smooth muscle relaxation
Impaired NO bioactivity, as often occurs in diabetes mellitus or advanced CHD or
neuropathy, may limit cGMP formation whereby the action of PDE‐5 inhibition is
inapplicable; this may account for nonresponse to oral ED therapy (Sáenz et al.,2004)In
hypercholesterolemic rabbits with reduced cavernosal relaxation in response to sodium
nitroprusside, sildenafil nitrate, an NO‐donating derivative of sildenafil, improved erectile
function to a greater degree than regular sildenafil, suggesting that the NO‐donating
component was important in compensating for the impairment of NO bioactivity in
hypercholesterolemia and the resulting reduction in cGMP (Shukla et al., 2005)
Other data, however, suggest that Sildenafil at tissue levels approaching millimolar
concentrations may act at least in part independently of the NO‐cGMP pathway (Sharabi
et al.,2005)
39
Several agents have also been shown in rat and rabbit models of ED to activate sGC and
thus stimulate cGMP production and induce cavernosal tissue relaxation and penile
erection independently of NO by binding to a novel allosteric site in the enzyme different
from the NO binding site (Brioni et al.,2002) These sGC activators are under investigation
as a possible new class of ED therapies. Nonetheless, the central importance of NO in
erectile function was demonstrated in a study showing that the effect on erectile function
of the sGC activator BAY 41‐2272 alone in conscious rabbits was weak, but it was
potentiated by concomitant administration of the NO donor sodium nitroprusside.
2.8.0. L-NAME
L-NAME, short for Nω-Nitro-L-arginine methyl ester, is a chemical compound widely
used in scientific research and medicine to investigate the role of nitric oxide (NO) in
various physiological and pathological processes. This essay explores the properties,
applications, and significance of L-NAME, shedding light on its critical role in advancing
our understanding of NO biology (Rees et al., 1989)
Chemical Structure and Properties:
L-NAME is a derivative of the amino acid L-arginine, a precursor of nitric oxide. Its
chemical structure includes a nitro group (NO2) and a methyl ester group (OCH3) attached
to the arginine molecule. This structural modification gives L-NAME its ability to inhibit
nitric oxide synthase (NOS) enzymes, which are responsible for NO production within the
body (Rees et al., 1989)
Inhibition of Nitric Oxide Synthesis:
40
L-NAME’s primary function is to block the activity of NOS enzymes, specifically by
competing with L-arginine, the natural substrate of these enzymes. By binding to the active
site of NOS, L-NAME prevents the conversion of L-arginine into nitric oxide, effectively
reducing NO production). (Rees et al., 1989)
2.8.1. MECHANISM THROUGH WHICH L-NAME INDUCES TESTICULAR
DYSFUNCTION
L-NAME (Nω-Nitro-L-arginine methyl ester) is a potent inhibitor of nitric oxide synthase
(NOS) enzymes, widely used in scientific research to study the role of nitric oxide (NO) in
various physiological processes.
INHIBITION Of NO PRODUCTION:
•
L-NAME, as an NOS inhibitor, interferes with the synthesis of nitric oxide
within the testes. This inhibition primarily affects eNOS, which is
abundantly expressed in the testicular blood vessels.
•
Reduced NO production leads to vasoconstriction and decreased blood
flow within the testicular vasculature. This compromised blood supply can
negatively impact testicular function (Aquila etal., 2004).
❖ HYPOXIA And ISCHEMIA
The reduced blood flow resulting from L-NAME treatment creates a hypoxic (low
oxygen) and ischemic (reduced blood supply) microenvironment within the testes.
This hypoxic condition can impair the function of Leydig cells, which are
responsible for testosterone production (Aquila etal., 2004).
41
❖ OXIDATIVE STRESS:
Hypoxia and ischemia can lead to the generation of reactive oxygen species (ROS)
within the testicular tissue. Increased oxidative stress can damage sperm cells and
disrupt spermatogenesis (Rajfer etal., 2000).
❖ IMPAIRED SPERMATOGENESIS
L-NAME-induced
testicular
dysfunction
often
results
in
impaired
spermatogenesis, including reduced sperm production and altered sperm
morphology and motility (Ozaki etal., 2003).
2.9. RAPHIAMEMBILLENSES
Raphiamambillensis is a trunk less palm with huge Fronds. The Palm is up to 7 meters in height.
It is Found in Cameroon, the Central African Republic, Nigeria, and Sudan (Cheek et al., 2000
and is also Used traditionally to treat male infertility (Soladoye et al., 2014). Its sap is used to
promote lactation in nursing Mothers, to treat different viral and bacterial Infections (Mbuagbaw
and Noorduyn, 2012). The Seeds are burnt into ash and used with pap for Ulcer treatment in SouthWestern Nigeria. This Study was conducted to evaluate the effect of Raphiamambillenses on LName induced testicular dysfuction in adult male Wistar rat.
42
CHAPTER THREE
MATERIALS AND METHODS
This research work was conducted at the Animal Holding Department of Anatomy, Faculty of
Basic Medical Sciences, University of Ilorin, in the year 2023 after it has been approved by the
Departmental Research and Ethical Review Committee of the University. The rules and guidelines
of the Institutional Animal Care and Use Committee (IACUC) were strictly followed throughout
the handling of animals.
3.1 MATERIALS AND METHODS
Thirty six (36) Adult male Wistar rats, 4 Plastic cages, Growing feeds (pellets), Saw dust, Plastic
feeding bottles, Lab coat, Dissecting set and surgical dishes, Kitchen weighing scale, 2ml Syringe,
5mls Syringes and needles, 20mls syringe and needles, Bowls, Cleaning materials (Brooms and
Dust pans), Antiseptic soap and detergents, Hand sanitizer and hand wash, Hand gloves, L-Name
tablet, Normal saline, Cello tape and permanent markers, Distilled water, Whatman no. 1 filter
paper,
Cotton wool and methylated spirit, Opaque bottles and aluminium foils, Raphia
mambillensis seed, Specimen bottles, Oral cannulas, Methylene blue stain, Paraformaldehyde and
phosphate buffer, Slides and Cover slips, Centrifuge, Optical microscope, Beakers and test tubes
and Microtome.
3.2.1PROCUREMENTOFANIMALS
The Adult male wistar rats with an average rat 175g was procured from Ekiti state on the 1st of
February, 2023 and arrived around 2pm at the Animal Holding Facility of the Faculty of Basic
Medical Sciences, College of Health Sciences, University of Ilorin, Ilorin, Nigeria. The animals
were brought in an iron cage and all were in good condition.
43
3.2.2 ANIMAL CARE
The animals were housed in comfortable wire gauzed plastic cages bedded with sawdust and
were fed with standard laboratory diet (pellet feed). They were fed daily and their cages were
constantly cleaned in order to keep them healthy and prevent diseases and infections that may erupt
from poor hygiene. The environment they were placed in was also constantly taken care of, in
order to make sure no external predators or organism would have negative effect on the rats and
also to reduce the smell of stench to the smallest. Their cages were arranged properly on the cage
racks in the properly ventilated facility to ensure that they weren’t affected by heat or lack of
oxygen. All the rats were handled in accordance with the standard guide for the care of the
laboratory animals. This experiment was carried out accordingly by complying with the guidelines
of the IACUC.
3.2.3 ANIMAL GROUPING AND IDENTIFICATION
ANIMALGROUPING
The thirty-six (36) Adult male wistar rats weighed with an average of
g and were divided into
six (6) groups with six (6) rats in each group and kept in different plastic cages bedded with
sawdust. The groups were labeled ; Control group,L-NAME induced,
Sildenafil citrate
treated,Raphiamambillenses treated,L-NAME and Sildenafil citrate co-administered,L-NAME
and Raphiamambillenses co-administered. The two (2) weeks of age, pre-pubertal rats were left to
acclimatize for five (8) weeks with no administration of drug but feed and water
44
Table 3.2.3: Animal grouping and schedule of administration.
GROUP
NUMBER
OF ADMINISTRATION
Number of Days
ANIMALS
Control group
6
Control (Normal healthy 56 days
animals) given distilled
water
L- NAMEOnly
6
L-NAME
induced
rats 56 days
treated
rats 56 days
(1ml/day)
Sildenafil
citrate 6
Sildenafil
only
(0.8ml/day)
Raphiamambillenses 6
R.Mambillenses
treated 56 days
rats (1ml/day)
L- NAME + PDE-5 6
L-NAME ( 1ml/day) and 56 days
inhibitors
Sildenafil (0.8ml/day) coadministered rats
L-NAME+
Raphiamambillenses
6
L-NAME (1ml/day) and 56 days
Raphiamambillenses
0.8ml/day)
administered rats
45
(
co-
3.2.4. ANIMAL FEED AND WATER MEASUREMENT
All groups were fed with 100g/cage of growing feed (pellets) and about 200ml of water. Water
intake remained constant.
3.3. PROCUREMENT AND PREPARATION OF L-NAME SOLUTION
L-NAME was procured at the Biotech research laboratory in Ilorin, Kwara state in a powdered
form. The solution was gotten by dissolving 2.65 g of L-NAME in 756 ml of normal saline to get
756 ml of the solution and stored by refrigeration. The exact amount of L-NAME powder obtained
by weighing on an electrical analytical balance. Each rat is to take 1 ml of L-NAME solution daily
for a period of 8 weeks.
3.4 PROCUREMENT AND PREPARATION OF THE AQUEOUS EXTRACTS OF
RAPHIAMAMBILLENSIS SEED
Dried RaphiaMambillensis seed was gotten from Oja-oba market on of 21st of March, 2023. The
leaves where transferred to the Botany Department of the University of Ilorin for Verification
(Voucher number: ). The seed was grinded with high power blender at Kulende market
Extraction Procedure
100mg of grinded RaphiaMambillensis seed was diluted with mls of distilled water to give L of
RaphiaMambillensis aqueous solution. The mixture was allowed to rest in an air tight container
away from the exposure to sunlight at room temperature for 48 hours. The filtration took place
after 2 days of soaking with distilled water. This process was carried with a Whatman No. 1 Filter
Paper to separate the sediments of the Raphia Mambillensis residue from the aqueous suspension.
46
The filtrate was weighed and administered to each rat in the administration group at 1 mg/kgBW
for 56 days.
3.5 PREPARATION AND TREATMENT WITH SILDENAFIL CITRATE (VIAGRA)
Each 100mg tablet of sildenafil citrate was dissolved in 100ml of distilled water so each 1ml
contained 1mg of the drug and administered 0.8ml/day to rats in orally for 8 weeks.
3.6. ANIMALSACRIFICEANDTISSUEPROCESSINGFORHISTOLOGICALSTUDIES
Animal were sacrificed by euthanisation, the brain was excised and fixed in 10% formalin for
histological studies using Haematoxylin & Eosin, staining procedures.
3.7. BLOODCOLLECTION
An incision was made from the jugular notch to the pubic symphysis through the skin. The Cut
was made deep into the abdominopelvic cavity cutting the thoracic bones and the Diaphragm for
easy access to blood from the heart. Blood samples were taken from the heart With a 2mls syringe
from the apex of the heart and was put into a plain sample bottle, put in Ice for few minutes before
centrifuging. The samples were centrifuged using (m,c) at 3500Rpm(xg) for 25 minutes. The
serum were further transferred into new plain bottles and stored In -20 degrees Celsius before
taking for analysis
3.8. TISSUE PROCESSING FOR HISTOLOGICAL STUDIES
Tissue fixed in 10% neutral buffered formalin was processed for paraffin wax embedding. Cutting
qualities are good, the blocks are durable, and their storage presents no special problems. The
recommended procedure of (Ijomone et al., 2012) was adopted. The Brain tissue was dehydrated
through ascending grades of ethanol by immersion as follows:
47
50% alcohol --------------------------------------------------------1 hour
70% alcohol---------------------------------------------------------1 hour
90% alcohol---------------------------------------------------------1 hour
95% alcohol---------------------------------------------------------1 hour
Absolute alcohol I--------------------------------------------------1 hour
Absolute alcohol II-------------------------------------------------1 hour
Dehydrated tissue was cleared in xylene as follows:
1:1 absolute alcohol and xylene-----------------------------------1 hour
Xylene I---------------------------------------------------------------1 hour
Xylene II--------------------------------------------------------------1 hour
The tissues were infiltrated in two changes of molten paraffin was at 56ºC in the oven for one
hour each and finally embedded in paraffin was using plastic embedding moulds after embedding
moulds smeared with glycerin so that paraffin blocked tissue can be separated from the mould
after embedding. Paraffin blocked tissue are trimmed and mounted on wooden blocks for
sectioning on a rotary microtome. Sections of 5µm were obtained on a rotary microtome. The
sections were spread in warm bath and collected on clean glass slides smeared with egg albumen.
The slides were then dried on a drying plate at a temperature of 40ºC overnight to enhance
adherence and stored in slide racks until ready for staining.
48
Haematoxylin and Eosin (H&E) Staining Procedure for Histology
Paraffin sections were de-waxed in two changes of xylene for three minutes each. Xylene is again
removed because it is not miscible with aqueous solutions and low grades of alcohol. Sections
were passed in absolute alcohol two changes of two minutes each. Sections are dehydrated to avoid
the possibility of diffusion current causing damage and perhaps detachment of sections through
95%, 90%, 70% and ethanol for about two minutes each and then brought to water. Sections of
5µm were used.
Reagents required:
Erlich’s Hematoxylin
Hematoxylin
1g
95% alcohol
100mls
Distilled water
100mls
Glycerol
100mls
Potassium alum
3g
Glacial acetic acid
10mls
Differentiator (1% HCL in 70% Alcohol)
Conc. HCL
1ml
70% alcohol
99mls
49
Eosin Y Solution
Procedure:
Sections were de-waxed in xylene for 2 changes of 2 minutes each. The sections were washed
under running tap water for 1-5 minutes. Excess stain was removed in 1% acid in 70% alcohol for
a few seconds as the acid breaks the mordant due linkages. The sections were washed in running
tap water to regain the blue colour as observed by the naked eye for 10-15 minutes. The sections
were stained in 1% aqueous eosin for about 3-5 minutes. Excess stain was rinsed off in running
tap water and examined with a microscope. It is dehydrated rapidly in ascending grades of ethanol
then mounted in Distrene Plasticizer Xylene (DPX) using clean glass cover slips.
Photomicrography and Image Analysis Sections were observed under a digital light microscope
and photomicrographs were taken.
3.9. BIOCHEMICAL ANALYSIS
Blood samples were collected when animals are sacrificed and refrigerator for biochemical
analysis (hormonal measurement for LH, FSH, and Testosterone using enzyme linked
immunosorbent assay (ELISA) technique and assessment of the following oxidative stress
biomarkers: Glutathione (GSH), Glutathione peroxidase (GPx) & Nitric oxide (NO) and inducible
Nitric oxide (INOS).
Nitric oxide (NO) Assay
Nitric oxide concentrations in samples were directly measured using a modification of the method
of (Nims et al., 1995).
50
Briefly, 10 µL of NO-saturated sample were withdrawn using a pipette and added to 900 µL,
100mM potassium phosphate buffer (pH 7.4), containing 17mM sulfanilic acid and N-(1naphthyl) ethylenediamine dihydrochloride (0.4mM). The solution was immediately mixed by
inversion incubated at room temperature for 3 min and then measured at A49nm. The NO
concentration of the solution was calculated according to Beer’s law using an extinction coefficient
of 5400 M-1cm-1 (0.1 Mm KPO4 assay buffer).
Glutathione Peroxidase (GPx)
Determination of Glutathione peroxidase activity in each sample was performed following
methods described by (Wheeler et al., 1990). The final reagent concentrations for each reaction
were 9.25 ml phosphate buffer (100 mM, 1 mM EDTA, Ph 7.0), 1.25 ml NADPH (2.25 Mm in
0.1% sodium bicarbonate), 0.25 ml glutathione reductase (25 U/0.25 ml in phosphate buffer), 0.5
ml GSH (37.5 mM in buffer) and 30 µl t-BuOOH (15Mm in water). Every reaction is a mixture of
225 µL assay reagent and 40 L sample whose absorbance was measured at 340nm every 10s for 2
minutes.
FSH, LH AND TESTOSTERONE ESTIMATION
Serum Testosterone (Accubind ELISA, 2013)
Materials used:
•
Testosterone Calibrators – 1ml/vial.
•
Testosterone Enzyme Reagent – 6.0 ml/vial.
•
Testosterone Biotin Reagent – 6.0 ml/vial.
•
Streptavidin Coated Plate – 35 of 96 wells.
•
Wash solution concentrate – 20 ml/vial
51
•
Substrate reagent – 12 ml/vial.
•
Stop solution – 8 ml/vial.
•
Pipette capable of delivering 25 µl and 50 µl with a precision of better than 1.5%.
•
Micro-plate washer or a squeezing bottle.
•
Micro-plate reader with 450nm and 620nm wavelength absorbance capability.
•
Absorbent paper for blotting the micro-plate wells.
•
Plastic wrap or micro-plate covers for incubation steps.
•
Vacuum aspirator (optional) for wash steps.
•
Timer.
Procedure:
All reagents and samples were brought to room temperature (20-27ºC). The micro-plates’ wells
for each serum reference calibrator, control and specimen to be assayed in duplicate were
formatted and unused micro-well strips were replaced back into the aluminum bag, sealed and
stored at 7ºC. 25 µL of the appropriate serum sample reference calibrator, control and specimen
were pipetted into the assigned well. 50 µL of progesterone enzyme reagent were added to all
wells. The micro-plate was swirled gently for 10-20 seconds to mix, covered and incubated for 60
minutes at room temperature. The contents of the micro-plate were then aspirated. 350 L of wash
buffer was added and aspirated again. The above process was repeated two(2) additional times for
a total of three (3) washes. 100 L of substrate solution was added to all wells (shaking was
avoided). These were incubated at room temperature for twenty (20) minutes. 50 µl of stop solution
was added to each well and gently mixed for 20 seconds. The absorbance of each well was read
using a micro-plate reader set at 450nm using a reference wavelength of 620-630nm before the
end of 15 minutes of adding the stop solution.
52
Precautions:
•
Extended exposure to heat and light was avoided.
•
Micro-plate wells were discarded after use to avoid mistaken repetitive use.
•
Repetitive freezing and thawing were avoided during the 4 day period between sacrifice
and analysis.
•
Reagents were added in the same order to minimize reaction time differences between
wells.
•
The absorbance results were read within fifteen (15) minutes of adding the stop solution.
•
It was ensured that the diluted wash buffer was nit stored for more than 60 days.
Serum FSH (Elabscience, 2015)
Materials:
•
Micro Elisa Plate
•
Reference Standard & Amp; Sample Diluent
•
Concentrated Biotinylated Detection Ab
•
Biotinylated Detection AbDiluents
•
Concentrated HRP Conjugate
•
HRP Conjugate Diluent
•
Concentrated Wash Buffer
•
Substrate Reagent
•
Stop Solution
•
Plate Sealer
•
Micro-plate reader with 450nm wavelength filter
•
High precision transferpettor
53
•
EP tubes and disposable pipette tips 37 C incubator
•
De-ionized or distilled water
•
Absorbent paper
•
Loading slot for wash buffer
Procedure:
All reagents and samples were brought to room temperature before use; the samples were
centrifuged after thawing before and the reagents were mixed thoroughly by gentle swirling before
pipetting as foaming was avoided. The samples and standards were assayed in duplicate. 100μl. of
standard, blank, or sample per well was added according to the assigned well. The blank well was
added with Reference Standard & Sample diluent and solutions were added to the bottom of
micro-ELISA plate well, and inside wall touching and foaming were avoided. After mixing, the
plate was covered with the sealer and incubated for 90 minutes at 37°C. The liquid of each well
was removed, after which 100μl of Biotinylated Detection Ab working solution was added
immediately to each well after which they were covered with the Plate sealer. The plate was tapped
gently to ensure thorough mixing incubation ensued for 1 hour at 37°C. Each well was then
aspirated and washed by filing with approximately 350µl, this was repeated three times, it is
essential to not that complete removal of liquid was ensured at each step. After the last wash,
remaining wash buffer was removed by aspirating. The plate was then inverted and patted against
thick clean absorbent paper. 100μL of HRP Conjugate working solution was added to each well
and covered with the plate sealer, thereafter they were incubated for 30 minutes at 37°C and the
wash process, was Repeated for five times as conducted earlier.
90μL of Substrate Solution was also added to each well and covered with a new plate, sealer and
incubated for about 15 minutes at 37°C, while being protected from light. The reaction time of
54
30 minutes was shortened with observance of colour change. With appearance of apparent gradient
in the standard wells, the reaction was terminated. 50µl of Stop Solution was added to each well
in the same order the substrate solution was added, and an immediate colour change to yellow was
observed. The optical density (OD value) of each well was determined at once using a micro-plate
reader set to 450 nm.
Precautions:
•
Exposure of reagents to strong light was avoided in the process of incubation and storage.
It was also ensured that all the taps of reagents were tightened to prevent evaporation and
microbial contamination.
•
Haemolysis was avoided during serum aspiration.
•
The micro-plate reader should be opened in advance, the instrument preheated, and the
testing parameters set.
Serum LH (Elabscience, 2015)
Materials:
•
Micro ELISA Plate
•
Reference Standard & Sample Diluent
•
Concentrated Biotinylated Detection Ab
•
Detection AbDiluent
•
Concentrated HRP Conjugate
•
HRP Conjugate Diluent
Procedure:
55
All reagents and samples were brought to room temperature before use; the samples were
centrifuged after thawing before and the reagents were mixed thoroughly by gentle swirling before
pipetting as foaming was avoided. The samples and standards were assayed in duplicate. 100μl. of
standard, blank, or sample per well was added according to the assigned well. The liquid of each
well was removed, after which 100μl of Biotinylated Detection Ab working solution was added
immediately to each well after which they were covered with the Plate sealer. The plate was tapped
gently to ensure thorough mixing incubation ensued for 1 hour at 37°C. Each well was then
aspirated and washed by filing with approximately 350µl, this was repeated three times, it is
essential to not that complete removal of liquid was ensured at each step. After the last wash,
remaining wash buffer was removed by aspirating. The plate was then inverted and patted against
thick clean absorbent paper. 100μL of HRP Conjugate working solution was added to each well
and covered with the plate sealer, thereafter they were incubated for 30 minutes at 37°C and the
wash process, was Repeated for five times as conducted earlier.
90μL of Substrate Solution was also added to each well and covered with a new plate, sealer and
incubated for about 15 minutes at 37°C, while being protected from light. The reaction time of 30
minutes was shortened with observance of colour change. With appearance of apparent gradient
in the standard wells, the reaction was terminated. 50µl of Stop Solution was added to each well
in the same order the substrate solution was added, and an immediate colour change to yellow was
observed. The optical density (OD value) of each well was determined at once using a micro-plate
reader set to 450 nm.
DATA ANALYSIS
Data obtained was analyzed using Graph-pad prism 8.0.2.263. All data was represented as mean
± SEM. The significance of difference between means for different groups was determined using
56
One Way Analysis of Variance (ANOVA) followed by Tukey's post hoc test. Differences was
considered statistically significant when p< 0.05.
3.10ETHICALAPPROVAL
The ethical approval was given by the University of Ilorin Ethical Review Committee, Kwara
State, Nigeria (approval number, ). The guidelines of the Institutional Animal Care and Use
(IACUC) were strictly followed throughout the whole experimental research.
57
CHAPTER FOUR
4.1. BIOCHEMICAL ANALYSIS
Fig 4.1.1 Graphical Representation of testosterone levels in the experimental groups
There is no significant difference (p > 0.05) between the control group and the other three groups.
The control group (0.048 ± 0.007 ng/ml) has the lowest testosterone level. The PDE only group
(1.079 ± 0.502 ng/ml) has the highest testosterone level while L-NAMEonly group (0.502 ±
0.128ng/ml) and L-NAME+PDE (0.517 ± 0.146 ng/ml) group have close testosterone levels.
3.
58
Fig 4.1.2 Graphical representation of Nitric Oxide levels in the experimental groups
These differences are not significant statistically. The results indicated that there is no significant
difference in LH levels between the control and the experimental groups (P>0.05). Although, the
Control group can be seen on the bar chart to have higher levels of LH when compared to other
groups, but this difference is not significant (P>0.05). Also, Also, there is no significant difference
in the LH level amongst all the experimental groups when compared with each other(P>0.05).
59
Fig 4.1.3 Graphical representation of Nitric Oxide levels in the experimental groups
The results indicate that there is no significant difference in the Nitric Oxide levels between the
control group and the experimental groups (P>0.05). However, it can be seen that the Nitric Oxide
level in the control group is higher when compared to the other group, this increase is not
significant (P>0.05).
Also, there is no significant difference in the Nitric oxide level amongst all the experimental groups
when compared with each other(P>0.05).
60
Table 4.1 Biochemical and hormonal assay for experimental animals
Groups
Testosterone
Nitric oxide Luteinizing
(NO)
hormone (LH)
10.120±7.883
Control
0.048±0.007
21.820±9.00
Raphia mambillenses
0.530±0.131
5.206±1.1029 0.325±0.142
L-NAME
0.502±0.128
2.412±0.471
0.3050±0.297
L+Raphia mambillenses
0.072±0.003
6.882±1.294
0.873±0.093
L-NAME+PDE
0.517±0.146
3.471±1588
0.455±0.100
PDE
1.079±0.501
2.382±0.205
0.415±0.091
Data are represented as MEAN ± SEM analysis of variance (ANOVA) was used to
Analyze the data at p<0.05Groups: Control; Raphia mambillenses; L-NAME; L-NAME+ raphia
mambillensis; L-NAME+PDE;PDE
61
4.2. HISTOLOGICAL EXAMINATION OF THE TESTES
4.2.1. TESTES STAINED WITH HEMATOXYLIN AND EOSIN DYE
Figure 4.2.1; Shows photomicrograph of control group
which reveals normal testicular
architecture without any observable presentation of spermatogenetic arrest and lumen could be
observed with the presence of spermatozoa. The basement membrane is thin and the interstitial
space contains Leydig cells.
Figure 4.2.1 Showed photomicrograph cytoarchitectural presentations of control group testicular
slide stained with Hematoxylin and Eosin dyes, (scale bar: ). presented within and outside the
seminiferous tubules and basement membrane (BM), lumen(L), Leydig cells (Lc), Sertoli cells
(Sc), and Spermatogonia cells (Sg).
62
Figure 4.4.2 Showed photomicrograph of treated with L-NAME (mg/kg). Severe observable
degenerative changes characterized by maturation arrest of spermatogenic cell line in several
seminiferous tubules, widened lumen that lack spermatozoa, fragmented basement and pyknotic
Leydig cells.
Figure 4.4.2Showed photomigrography cytoarchitectural of L-NAME testicular slides stained
with Hematoxylin and Eosin dyes. Presented within and outside the seminiferous tubules are
germinal cell (Gc), basement membrane (BM), lumen (L), Leydig cells (Lc), interstitial space (IS).
63
Figure 4.4.3 showed photomicrography cytoarchitectural of raphiaMambillensisslides stained
with Hematoxylin and Eosin dyes. Presented within and outside the seminiferous tubules at Leydig
cells (Lc), basement membrane (BM), lumen (L), Spermatogonia cells (Sg)
C
Lc
Sg
BM
L
Sc
100x
Figure 4.4.3Showed photomicrographs cytoarchitectural presentations of Raphiamambillenses
testicular slide stained with Hematoxylin and Eosin dyes. Presented within and outside the
seminiferous tubules are basement membrane(BM), Lumen (L), Leydig cell (Lc), Sertoli cells
(Sc), and spermatogonia cells (Sg).
64
Figure 4.4.4: Showed photomicrograph of group treated with Sildenafilcitrate (mg/kg) testicular
slides appeared normal and was characterized by normal differentiation of spermatogenic cells,
presence of Leydig cells in the insterstitial spaces and the presence of spermatozoa in the lumen.
Figure 4.4.4: showed photomicrographs cytoarchitectural presentations of group testicular slide stained
with Hematoxylin and Eosin dyes. Presented within and outside the seminiferous tubules are basement
membrane(BM), Lumen (L), Leydig cell (Lc), Sertoli cells (Sc), and spermatogonia cells (Sg).
65
Figure 4.4.5. showed photomicrograph of Groups D treated with co administration of L-NAME
and Sildenafilcitrate, similar morphological presentation with similar staining intensity and
cellular density were observed in groups F and G when compared with the control group. The
testicular cytoarchitectural was well structured and characterized by seminiferous tubules having
numerous Spermatogonia cells that have differentiated into numerous Spermatocytes, the presence
of sertolic cells at the adlumina border, presence of Leydig cells in the interstitial spaces, and
seminiferous tubule lumen filled with spermatozoa.
Figure 4.4.5.: showed photomicrographs cytoarchitectural presentations of Group L- NAME and
Sildenafil citrate testicular slide stained with Hematoxylin and Eosin dyes. Presented within and
outside the seminiferous tubules are basement membrane(BM), Lumen (L), Leydig cell (Lc),
Sertoli cells (Sc), and spermatogonia cells (Sg)
.
66
Figure 4.4.6.Showed photomicrograph of Group treated with co administration of L-NAME and
Raphiamambillenses, similar morphological presentation with similar staining intensity and
cellular density were observed in groups A and C when compared with the control group. The
testicular cytoarchitectural was well structured and characterized by seminiferous tubules having
numerous Spermatogonia cells that have differentiated into numerous Spermatocytes, the presence
of sertolic cells at the adlumina border, presence of Leydig cells in the interstitial spaces, and
seminiferous tubule lumen filled with spermatozoa.
D
Lc
Sg
BM
L
Sc
Figure 4.4.6 Showed photomicrographs cytoarchitectural presentations of L- NAMEand Raphia
mambillenses testicular slide stained with Hematoxylin and Eosin dyes. Presented within and
outside the seminiferous tubules are basement membrane (BM), Lumen (L), Leydig cell (Lc),
Sertoli cells (Sc), and spermatogonia cells (Sg).
67
CHAPTER FIVE
5.0 DISCUSSION
The investigation of testicular dysfunction is of paramount importance due to its implications for
male reproductive health. Nitric oxide (NO) signaling plays a crucial role in testicular physiology,
and disruptions in this pathway can lead to dysfunction. L-NAME, an NO synthase inhibitor,
serves as a relevant model for such dysfunction (Moncada, et al .,1991)
Raphiamambillenses, a plant known for its antioxidant properties, has been considered for its
potential to counteract oxidative stress-induced damage in the testes (Akhigbe et al.,2016).
Concurrently, phosphodiesterase-5 (PDE-5) inhibitors like sildenafil have been shown to enhance
NO signaling by preventing the degradation of cyclic guanosine monophosphate (cGMP) (Burnett,
2016). This dual approach, utilizing both a natural remedy and a pharmaceutical, offers a
comprehensive strategy to address testicular dysfunction.
Histomorphometry, the quantitative analysis of tissue structure, serves as a critical tool in this
study. It enables the assessment of changes in the seminiferous tubules, including alterations in
diameter, germ cell count, and interstitial tissue composition (França and Russell, 1998). These
metrics are pivotal in understanding the structural basis of testicular dysfunction and the effects of
the interventions.
The hypothesis underlying this study is that the combination of Raphia mambillenses and PDE-5
inhibitors may produce synergistic effects in mitigating testicular dysfunction. Raphia
mambillenses could protect against oxidative stress, while PDE-5 inhibitorsmay enhance blood
flow to the testes, thereby improving nutrient and oxygen delivery. The study seeks to determine
whether these interventions act in tandem to restore testicular function.
68
Male reproductive health hinges on the proper functioning of the testes. Testicular dysfunction can
lead to infertility, a condition affecting millions of men worldwide (Punjabet al.,2017 ). The
outcomes of this research could have far-reaching implications for developing therapeutic
strategies to address male infertility and related reproductive issues.
5.1. HISTOLOGICALIMPROVEMENT
•
NO Signaling Enhancement: PDE-5 inhibitors, by preventing the degradation of cGMP,
may enhance NO signaling in the testes. This could potentially counteract the negative
effects of L-Name-induced NO synthase inhibition, leading to improved blood flow and
oxygenation within the testicular tissue.
•
Mitigation of Vasoconstriction: L-Name can induce vasoconstriction due to reduced NO
production. PDE-5 inhibitors, by promoting vasodilation, might mitigate this
vasoconstriction, ensuring a more favorable microenvironment for spermatogenesis
(Burnett, 2006)
5.2 CONCLUSION
In conclusion, the histomorphometry study investigating the concomitant administration of
Raphiamambillensesand Sildenafil citrate on L-NAME induced testicular dysfunction in adult
male Wistar rats offers valuable insights into potential therapeutic interventions for male
reproductive health.
By concurrently administering Raphiamambillenses, known for its antioxidant properties, and ,
Sildenafil citrate which enhance nitric oxide signaling, the research has explored a dual approach
to mitigating testicular dysfunction. This approach suggests the potential for synergistic effects,
wherein these interventions work together to restore testicular function.
69
The study’s use of histomorphometry as a primary analytical tool has allowed for quantitative
assessment of changes in seminiferous tubules. Alterations in tubular diameter, germ cell count,
and interstitial tissue composition have been crucial in understanding the structural basis of
testicular dysfunction and evaluating the efficacy of the interventions.
Testicular dysfunction is a significant concern for male reproductive health, as it can lead to
infertility and related reproductive issues. The findings of this study hold promise for the
development of therapeutic strategies aimed at addressing male infertility and improving overall
testicular function.
The investigation of both natural remedies like Raphiamambillenses and pharmaceuticals like
Sildenafil citrate in a single study raises questions about the potential benefits of combining these
approaches. This study provides evidence of the feasibility and potential advantages of such
combination therapies.
The positive outcomes of this study open doors for further research into the use of Raphia
mambillenses, Sildenafil citrate, or combination therapies in the context of male reproductive
health. Future studies may explore these interventions in more diverse models and clinical settings.
70
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