Effect of Tamsulosin drug (flomax) on fertility in Albino male mice

University of Baghdad
Effect of Tamsulosin drug (flomax)
on fertility in Albino male mice
A Thesis Submitted
By
Dina Khudhair Husain Ali
B.SC in Biology/Baghdad university 2006
To the College of Science, University of Baghdad, in partial
fulfillment of the requirements for degree of master of science
in
Biology /zoology
Supervised by
Prof. Dr. Sabah Naser Alwachi
February
Rabia alawal
2012A.D.
1433A.H.
‫ﺑِﺴْﻢِ ﺍﻟﻠَّﻪِ ﺍﻟﺮَّﺣْﻤَﻦِ ﺍﻟﺮَّﺣِﻴﻢ‬
‫ﻗَﺎﻟُﻮﺍ ﺳُﺒْﺤَﺎﻧَﻚَ ﻟَﺎ ﻋِﻠْﻢَ ﻟَﻨَﺎ ﺇِﻟَّﺎ ﻣَﺎ ﻋَﻠَّﻤْﺘَﻨَﺎ‬
‫ﺇِﻧَّﻚَ ﺃَﻧْﺖَ ﺍﻟْﻌَﻠِﻴﻢُ ﺍﻟْﺤَﻜِﻴﻢُ‬
‫ﺻﺪﻕ ﺍﷲ ﺍﻟﻌﻈﻴﻢ‬
‫ﺳﻮﺭﻩ ﺍﻟﺒﻘﺮﺓ )‬
‫‪(32‬‬
‫‪Declaration‬‬
I declare that this thesis was prepared by Dina Khudhair Husain
Ali , under my supervision at the Department of Biology, College of
Science, University of Baghdad, in partial fulfillment of the
requirements for degree of M.SC of Science in Biology/ Zoology
Signature
Supervisor: Dr. Sabah N. Alwachi
Scientific Degree: Professor
Date:
/
/
In view of the available recommendation . I forward this thesis for
debate by the examination committee.
Signature
Name: Dr. Hayfa H. Hassani
Scientific Degree: Professor
Head of Department Biology
Date:
/
/
Dedication
This work is dedicated to:
My father ……………….who brightened my road to
success
My mother……………………………..…the source of
kindness
My husband………………..for his love and
encouragement
My sisters and brothers……………...,,,,,,,,for their
supports
Dina
Acknowledgements
In the name of the God ,the first who deserve all thanks and
appreciation for granting me with well ,strength and helps with which
this research had been accomplished .
I would also like to express my special thanks to my respectful
supervisor Prof. Dr. Sabah N. Alwachi for his patience , advise and
encouragement throughout the period of the research .
I would like also to express my sincere gratitude & appreciation
to the head of Department Biology , Collage of Science in University
Baghdad and the her staff for their support and cooperation.
I would like to express my special thanks and debts to Dr. Jabar
H. Al-Hilfy, Dr. Hind Husain , Majida H. Mehdy and Sura M. for
their assistants during my research period.
I would also like to thank Dr. Mohammed joied from Pathology
Department in College of Veterinary Medicine for his assistance.
Deep appreciation and thanks for my friends Nusaiba Amir ,
Shaman Nasser , Zainab Khudhair, Noor Jaafer , Israa Salem and
Maha Nidal
from Biology Department for their support and
cooperation.
Finally , I would like to express my deep thanks and appreciation to
my family especially my parents and husband Mohammed for their
love and encouragement throughout my study .
Dina
Summary
Summary
The present study was conducted to investigate the effect of Tamsulosin
hydrochloride on testicular function and fertility in albino male mice (Mus
musculus) . Thirty mature male mice with average body weight of 25-30g and
8-10 weeks of age were randomly divided in to three group (10 mice / group).
The first group was injected intraperitoneally (I.P) with distilled water as a
control group and the other groups (second and third) were also injected I.P
with two concentrations of Tamsulosin hydrochloride (8 , 16 µg /kg.b.wt)
daily for a period of 42 days.
The results showed a significant (p<0.05) decrease in the body weights
and rate of testes weights in male mice after the treatment with Tamsulosin
hydrochloridefor for two concentrations compared with control group.
The treatment with Tamsulosin hydrochloride also showed a significant
(p<0.05) decrease in percentage of sperm motility ,viability and sperm
concentrations in the epididymis at dose 16 µg /kg.b.wt compared with the
control group but there was no significant (p<0.05) decrease in the percentage
of sperm motility and concentrations at dose 8 µg /kg.b.wt ,while there was a
significant (p<0.05) decrease in the percentage of viability of sperm at the
same dose (8 µg /kg.b.wt ) compared with the control group. There was also a
significant increase (p<0.05) in the percentage of morphologically abnormal
sperm for two concentrations (8 , 16 µg /kg.b.wt ) compared with the control
group.
Histological examination of testes indicated that the treatment with the
drug induced a significant (p<0.05) decrease in seminiferous tubules , primary
spermatocytes
,spermatid and decrease in number of
I
the
leydig’s cells
Summary
clusters, while there was a significant (p<0.05) increase in the interstitial space
between tubules comparing with the control group.
The histological examination also revealed an existence of some
changes in the testes such as necrosis in the tubule and oedema in the
interstitial spaces
The treatment with all concentrations also caused a significant (p<0.05)
decrease in the testosterone levels in blood serum comparing with the control
group .
It was concluded from this investigation that Tamsulosin has a negative
effect on testicular function (fertility).
II
List of contents
No.
Contents
Summary
Page No.
I
III
VI
VIII
IX
1
1-1
1.2
List of Contents
List of Figures
List of Tables
List of Abbreviations
Chapter one : Introduction and literatures review
Introduction Literatures review
Introduction
Literature Review
1-2-1
Alpha adrenergic antagonist (alpha blockers)
3
1-2-2
Tamsulosin Hydrochloride
4
1-2-2-1
Tamsulosin hydrochloride description
4
1-2-2-2
Trade names of Tamsulosin hydrochloride
7
1-3
Route of Tamsulosin Hydrochloride within the body
7
1-3- 1
Absorption
7
1-3-2
1-3-3
Distribution
Metabolism
7
8
1-3-4
Interaction
8
1-3-5
1-4
Excretion
Side effect of Tamsulosin hydrochloride
9
9
1-5
Male reproductive system
11
1-6
Characteristics of the sperm
13
1-6-1
Sperm morphology
13
1-6-2
Sperm concentration
15
1-6-3
1-6-4
Sperm viability
Sperm motility
15
16
1-7
Spermatogenesis
17
1-7-1
Spermatocytogenesis (mitosis)
18
1-7-2
Spermatidogenesis (meiosis)
18
1-7-3
Spermiogenesis
18
1
1
3
III
1-8
1-9
1-10
1-10-1
1-13-2
1-13-3
1-13-4
Role of sertoli cell in spermatogenesis
Hormonal control of spermatogenesis
Testosterone hormone
Chemistry and biosynthesis of testosterone
Transport and metabolism of testosterone
Action of testosterone
The role of testosterone in spermatogenesis
Chapter two: Materials and Methods
19
21
23
23
26
27
28
2
Materials and Methods
29
2-1
Materials
29
2-1-1
Equipments and Apparatus
29
2-1-2
Chemicals
31
2-2
Methods
32
2-1-1
Preparation of solutions
Preparation of solutions
32
32
2-2-1
2-2-1-1 Preparation of histological solutions
2-2-1-2
2-2-1-3
32
33
33
2-2-2
2-2-3
2-2-4
2-2-5
2-2-6
Sperm count solutions
Preparation of Tamsulosin hydrochloride (Flomax )
solution
Experimental Animals
Blood collection
Killing of the animals
Collection and Preparation of Sperms
Sperms parameters
2-2-6-1
2-2-6-2
2-2-6-3
2-2-6-4
Percentage of sperm motility
sperm concentration
percentage of sperm viability
Percentage of morphologically abnormal sperm
35
35
35
36
2-2-7
2-2-8
2-2-9
Histological study
Microscopic Examination
Statistical analysis
Chapter three: Results and discussion
Testosterone concentration in serum
Changes in testes and body weights
Sperm parameters
36
38
38
3-1
3-2
3-3
33
34
34
34
35
39
41
43
IV
3-3-1
Sperm concentration in the epididymis
43
3-3-2
Percentage of sperm motility in the epididymis
47
3-3-3
3-3-4
3-4
Percentage of dead sperms in the epididymis
48
Percentage of morphologically abnormal sperms in the 50
epididymis
The Histological studies
52
3-4-1
The diameter of seminiferous tubules Interstitial Space 52
3-4-2
Interstitial Space
54
3-4-3
3-4-4
Diameter of Leydig’s cells
primary spermatocytes
56
58
3-4-5
Spermatid
Conclusions & Recommendations
59
60
References
61
V
List of Figures
No.
Subjects
No
1.1
subjects
Page
Pages
Chemical structure of Tamsulosin hydrochloride (Patel & 5
Patel, 2010).
1.2
3-1
3. 2
The intratesticler steroidogenic pathway for synthesis of 25
testosterone ( Rwerdloff Ronald et al., 2010).
Sperm morphology from mice treated with Tamsulosin 51
Hydrochloride showing (A) normal sperm , (B) abnormal
tail (bent tail) , (C) abnormal head ( hummer head ) (E
stain) 40X.
Section in mouse testes (control group), showing normal 60
structure of seminiferous tubules ,diameter of seminiferous
tubules (ST), interstitial space (IS) ,spermatide and primary
spermatocyte (prim. Sp.) (H and E) X 10 for figure (A)
,40Xfor figure (B).
3. 3
Section in mouce testes (treated group 8µg/KgB.W for A 60
,16 µg/KgB.W for B.) showing abnormal structure of
seminiferous tubules. diameter of seminiferous tubules
(DST), interstitial space (IS), oedema (E) and necrosis (N)
(H and E) X 10.
3. 4
Section in mouse testis (control group) showing normal 61
structure of Leydig’s cell clusters (H & E X 40)
3. 5
Section in mouce testis (treated group with A 8µg /kg.b.wt. 61
, B 16 µg /kg.b.wt) showing abnormal structure of
Leydig’s cell (H & E X 40).
VI
List of Tables
Subjects
NO.
Page
2.1
The equipments and apparatus used .
29
2.2
Chemicals used
31
3.1
Effect of Tamsulosin hydrochloride (8µg /kg.b.wt.
and 39
16µg/kg.b.wt.) on serum Testosterone level (ng/ml) in male
mice
3.2
3.3
Effect of different concentrations of Tamsulosin HCl
43
(8µg/KgB.W and 16µg/Kg B.W )on the rate of testes and
body weights
Effect of Tamsulosin (µg /kg.b.wt. ) on sperms parameters in 46
male mice.
3.4
Effect of Tamsulosin (µg /kg.b.wt. ) on diameter of 55
seminiferous tubules and interstitial space in male mice .
3.5
Effect of Tamsulosin 8µg/KgB.W and 16 µg /kg.b.wt. on
57
diameters of primary spermatocyte ,spermatids .
VII
List of abbreviations
AAG
Primarley Alpha -1-acid Glycoprotein
AR
Adrenoceptor
ABP
Androgen Binding Protein
BPH
Benign Prostate Hyperplasia
CPPS
Chronic Pelvic Pain Syndrome
CP
Chronic Prostatitis
DNA
De-oxy Ribo Nucleic Acid
DHT
Dihydrotestosterone
DHEA
De Hydro Epi Androsterone
E2
Oestradiol
FSH
Follicle Stimulating Hormone
GnRH
Gonadotrophin Releasing Hormone
GBG
Gonadal steroid Binding Globulin
HCl
Hydrochloric Acid
IP
Intraperitonealy
IU
International Unit
ICSH
Interstitial Cell Stimulating Hormone
LH
Luteinizing Hormone
LUTS
Lower Urinary Tract Symptoms
1n
Half chromosome number
PSA
Prostate - Specific Antigen
RE
Retrograde Ejaculation
VIII
RPMI 1640
STAR
Roswell Park Memorial Institute-1640
SPR
Stone Passage Rates
S.T.
Seminiferous Tubules
WHO
World Health Organization
Steroidgenic a cute Regulatory protein
IX
Chapter One
Introduction and Literatures Review
Chapter One
1: Introduction and Literatures Review
1-1: Introduction
Alpha blockers represent one of the
most common therapies for
chronic pelvic pain syndrome / chronic prostatitis (CPPS/CP) . The rationale
is based on presence of varied receptors in the lower genitourinary tract .
Starting in the early 1970 , reports suggested that certain patients might
benefit from treatment with alpha –blocker .Recently alpha blockers had side
effects that severely limited their utility (Lee et al., 2008).
Tamsulosin hydrochloride , an alpha blocker , works by blocking nerve
ending called alpha receptors . This will relaxes the smooth muscles of the
prostate and bladder . Tamsulosin is used also
to treat benign prostate
hyperplasia (BPH) in men which can be recognized by enlargement of
prostate and also known as an enlarged prostate which causes difficulties
with urination (Sonnberg, 2003) . It was discovered by research team led by
Toichi Takenaka in 1980 at first as a potential anti-hypertension drug. Later it
was used to treat several disease cases ,benign prostate hyperplasia (BPH)
,lower urinary tract symptoms associated with BPH and urine retention
(Hara, 2003).
The chemical structure of Tamsulosin hydrochloride is composed of
methoxy - benzenesulfonamide which , differs from other alpha blockers
such as Alfuzosin , Terazosin and Doxazosin which are quinazolin
derivatives (Schulman,1996).
The abnormal ejaculation of semen is a typical but infrequent side
effect of some α1-adrenoceptor antagonists , particularly those with
selectivity for α1A-adrenoceptors such as Silodosin and Tamsulosin . Recent
clinical studies suggested that this represents a relative ejaculation rather
1
Chapter One
Introduction and Literatures Review
than a retrograde ejaculation . The reduced ejaculation and related male
infertility is shown to be caused by an impaired function of the vas deferens
rather than by alterations in sperm formation number or function (Michel,
2007).
Comparing to other α-antagonists , Tamsulosin hydrochloride has
greater specificity for α-1 receptors in the human prostate which do not
affect receptors on blood vessels. It is the most frequently prescribed
medication for the treatment of lower urinary tract symptoms (Bari et al.,
2011).
Tamsulosin has also affected the sexual function in men, which can
cause males to experience retrograde ejaculation. In males, retrograde
ejaculation occurs when the fluid to be ejaculated, which would normally exit
the body via the urethra, is redirected to the urinary bladder. Normally, the
sphincter of the bladder contracts and the ejaculate goes to the urethra, the
area of least pressure. In retrograde ejaculation, this sphincter does not
function properly (Roehrborn et al., 2008)
α1-adrenoceptor blockade has a potent anti fertility effect in male rats,
because libido and mating performance remained essentially uninhibited. On
the other hand, the anti-fertility effect was accompanied by significant
impairment in ejaculatory (Ratensooria and Wadsworth, 2009).
α-receptor blockers eg. phenoxybenzamine , Prazasin and Tamsulosin ,
that interfere with seminal emission , cause sperm retention in the tail of the
epididymis . These drugs can either decrease vaginal sperm numbers and
cause infertility as a results of an ejaculation (Nieschlag and Behre, 2009).
Aims of the study:Studying The physiological and the histological effects of Tamsulosin
hydrochloride on testicular functions of albino male mice (fertility).
2
Chapter One
Introduction and Literatures Review
1-2: Literature Review
1-2-1:Alpha adrenergic antagonist (alpha blockers).
Adrenergic receptors were originally divided in to alpha and beta
subtypes , with alpha further classified in to alpha -1 and alpha-2 receptors.
Currently there are three
subtypes in the alpha1 subfamily: 1a ,1b ,1d.
Differences in receptor amino acid sequence among these subtypes alter the
binding properties of specific agonist and antagonist . In human prostate cell ,
the alpha -1a receptor type is the most prevalent type , making the targeting of
this subtype therapeutically useful (Blanchad and Abu Baker, 2010).
Docherrty (2010) has been pointed that alpha1-Adrenoceptors (AR) can
be divided in to alpha 1a , alpha 1b and alpha 1d adrenoceptors . A fourth
alpha 1-adrenoceptor , the alpha 1L- represents a functional phenotype of the
alpha 1a-adrenoceptor.
Alpha receptors were further grouped in to α1 and α2 subtypes . The α1
receptors are located primarily in the smooth muscle of the prostate gland and
bladder neck but also to the lesser degree in the vascular system (Tewari and
Naryan, 1999) . Molecular studies showed that the α1 subtypes represents
70% of α1receptors in the prostate gland (Chen et al., 2000).
The first generation of alpha adrenergic antagonists (i.e. phenoxy
benzamine) blocked both alpha 1 and alpha 2 receptors in the prostate and
vasculature (Lee, 2003).
Second generation of alpha –adrenergic antagonist have greater affinity
for alpha 1 receptors than for alpha 2 receptors , leading to an improved side
effect profile . These second generation agents include Prazocin , Terazocin,
Doxazocin and Alfuzocin (Debruyne, 2000).
Tamsulosin hydrochloride , the third generation alpha adrenergic
antagonists , was selective adrenergic receptor blocker with high binding
affinity for the alpha 1d receptor (Nobile et al., 1997 and Kuritzky, 2005).
3
Chapter One
Introduction and Literatures Review
Tamsulosin is selective α1- adrenergic receptor blocker with a tenfold
greater affinity for α1a and α1d receptors than for α1b adrenergic receptors .
This distinguishes Tamsulosin from other blockers Terazocin , Doxazocin and
Alfuzocin that all have similar affinity for all three α1-receptor subtypes.
Study has shown that alpha (α1a AR) selective antagonists relieve bladder
based irritability symptoms , in contrast blockade of the alpha 1b- adrenergic
receptors leads to orthostatic hypotension ; a side effect associated with non
select alpha blockers (Zelefsky and Stephen, 2010).
1-2-2:Tamsulosin Hydrochloride
1-2-2-1: Description of Tamsulosin Hydrochloride
Tamsulosin hydrochloride is an antagonists of alpha 1a adrenoceptors
in the prostate . Tamsulosin hydrochloride is (R)-5-[2-[[2-(2-ethoxyphenoxy)
ethyl] amino] propyl]-2-methoxybenzenesulfonamide , monohydrochloride.
Tamsulosin HCl. found as a white crystals that melt with decomposition at
approximately 230°C. it is sparingly soluble in water and in methanol and
practically insoluble in ether (Patel & Patel, 2010).
The empirical formula of Tamsulosin HCl. is C20H28N2O5S.HCl and
the molecular weight it HCl. is 444.98. and its structural formula is:
4
Chapter One
Introduction and Literatures Review
Figure (1-1): Chemical structure of Tamsulosin hydrochloride (Patel & Patel,
2010).
Tamsulosin hydrochloride belongs to a class of alpha -1a receptor
antagonists that have performed selectivity for prostate smooth muscle,
although it does have some binding to vascular smooth muscle receptors as
well. Tamsulosin binding is remarkable for its significantly lower degree of
non specific binding compared to other alpha-receptor antagonists
(Naryan
et al., 2000).
The symptoms associated with BPH are related to bladder outlet
obstruction, which is comprised of two underlying components: static and
dynamic.
The static component is related to an increased in prostate size caused
in part , by proliferation of smooth muscle cells in the prostate stroma . The
dynamic component is a function of an increase in smooth muscle tone in the
prostate and bladder neck leading to constriction of the bladder outlet.
Smooth muscle tone is mediated by the sympathetic nervous stimulation of
alpha-1 adrenocepter , which are abundant in the prostate , prostatic capsule ,
prostatic urethra , and bladder neck (Boheringer Ingelheim, 2009).
5
Chapter One
Introduction and Literatures Review
The combined uses of Longbishu and Tamsulosin hydrochloride is
effective and safe in the treatment of chronic a bacterial prostatitis (Huil and
Hai-qiu, 2007).
Tamsulosin treatment is recommended for patients with the stone
diameter smaller than 8mm because of its feasibility and effectiveness
(MengYuan et al., 2009).
Alpha-1 adrenocepter antagonists were considered the first line of
treatment for managing lower urinary tract symptoms associated with BPH
(Grinwan et al., 2010).
Tamsulosin was superior to placebo in providing symptomatic relief in
men with chronic prostatitis or chronic pelvic pain syndrome particularly in
those with more sever symptoms (Nickel et al., 2004 and Yamanishi et al.,
2009 ).
Tamsulosin therapy for un complicated distal urethral calculi augments
stone passage rates (SPR) shortens passage time and decrease need for
analgesia. Particularly, Tamsulosin shortens the passage time for smaller
stones and augments the passage rate for larger stones (Abdel-Meguid et al.,
2010).
Currently , Sasaki et al., (2008) also found that α1a adrenocepter
antagonists could become useful medication for stone passage in urolithiasis
patients . In fact , Tzortzis et al., (2009) noted that "medical expulsive therapy
using α1-adrenoceptor antagonists such as Tamsulosin , augments the stone
expulsion rate compared to standard therapy for moderately sized distal
urethral stones.
6
Chapter One
Introduction and Literatures Review
1-2-2-2:Trade names of Tamsulosin hydrochloride
Tamsulosin is launched in Japan in august , 1993 under the trade name
of Harnal (Hara, 2003) . Tamsulosin trade names are Alna , Amsulosin
hydrochloride , Expras , Flomax, Omic, Omix , Omnic, Pradif , Scotex and
Urolosin (Miline , 2005).
1-3: Route of Tamsulosin Hydrochloride within the body
1-3- 1: Absorption
Tamsulosin is absorbed in the intestine and almost completely
bioavailable . Both the rate and extent of absorption of Tamsulosin were
reduced when taken within 30 minutes of a meal.
Tamsulosin shows dose proportional plasma exposure after single dose
of it in the feed state, plasma concentration of Tamsulosin peak at around 6
hours and in the steady state , which is reached by day 5 of multiple dosing
(Anonemous, 2010).
After oral administration, more than 90% of Tamsulosin is absorbed.
However, its administration with food decreases bioavailability by 30%
(Brophy et al., 2010).
The absorption of the drug is also variable and the onset of action is 1-2
hours (Bryant and Kathleen, 2011). After oral administration, peak plasma
concentration is reached in 5 days (Narayan et al.,2000).
Absorption of Tamsulosin hydrochloride (Flomax) capsules 0.4 mg is
essentially complete (>90%) following oral administration under fasting
conditions , the apparent half-life of Tamsulosin hydrochloride is
approximately 9 to 13 hours in healthy volunteers and 14 to 15 hours in the un
healthy man (Chong Kim , 2009).
1-3-2:Distribution
After administration of tamsulosin in healthy male adults , it distributed
to extra cellular fluid in the body.
7
Chapter One
Introduction and Literatures Review
In addition , whole body studies in mice , rat and dogs indicate that
Tamsulosin is widely distributed to most tissue including kidney , prostate,
liver , gall bladder , heart , aorta , brown fat and minimally distributed to the
brain , spinal cord and testes.
Tamsulosin is extensively bound to human plasma protein (94% to
99%) , primarily alpha -1-acid glycoprotein (AAG) in human , with linear
binding over a with concentration range (20 to 600 ng/ml) . The results of in
vitro study indicate that the binding of Tamsulosin to human plasma protein is
not affected by amitriptyline and diclofenic (Kim et al., 2001).
1-3-3: Metabolism :
Tamsulosin hydrochloride is extensively metabolized by cytochrome
p450 enzyme in the liver and less than 10% of the dose is excreted in urine
unchanged. Inhibition of hepatic druid –metabolizing enzyme may lead to
increase exposure to Tamsulosin .
The metabolites of Tamsulosin HCl. undergo extensive conjugation to
glucuronide or sulfate prior to renal excretion. However, results of the in vitro
testing of the Tamsulosin HCl. interaction with diclofenac and warfarin were
equivocal (Brophy et al., 2010).
1-3-4: Interaction
Kluwer ( 2011) noticed that there are potential drug interactions with
Tamsulosin HCl.
1) Alpha blockers: additive effect of both drugs.
2) Cimitidin: risk of decreased Tamsulosin clearance.
3) CYP2D6 inhibitors: such as Fluoxetine, Paraxetine and Terbinafine
4) CYP3A4 inhibitors: such as Crythromycin , Ketoconazole
5) Phosphodiestrase inhibitors increased risk of hypotension
8
Chapter One
Introduction and Literatures Review
1-3-5: Excretion
Approximately 10% of the drug is excreted unchanged in the urine
(Brophy et al., 2010). Bryant and Knights (2011) shows that the excretion of
Tamsulosin occurs via urine & feces . Hepatic metabolism with metabolite
excreted in urine (Narayan et al.,2000).
The administration of Tamsulosin hydrochloride to 4 healthy
volunteers showed that 97% of the administered was recorded with urine
(76%) representing , the primary route of excretion compared to feces (21%)
over 168 hours. The elimination half life of Tamsulosin HCl in plasma ranged
from 5 to 7 hours. Because of absorption rate controlled pharmacokinetics
with Flomax capsule (Boehringer Ingelheim, 2009).
1- 4: Side effect of Tamsulosin hydrochloride
A starting point of the mice studies was the observation that α1a, α1b
α1d adrenergic receptor reduced fertility in mice ; experiment with different
combination of male and female wild-type of rat indicated that the impaired
ability to induce pregnancy resulted from male infertility (Michel, 2007) .
In the rat , the cauda epidydimis contain post functional α1-but not α2
adrenoceptor , whears in the guinea pig , the cauda epidydimis contain both
α1 and α2 adrenoceptor (Queriroz et al., 2001).
Studies by Bhathal et al., ( 1974) and Ratensooria & Wadsworth ,
(2009) have shown that the rats epididymis is significally affected by α1a
adrenocepters blockade . In vivo treatment α1a adrenocepters antagonist
induced a decrease in ejaculatory capacity associated with reduction in the
fertilization ability of the sperm , suggesting a role for the sympathetic
nervous system infertility maintenance via α1A adrenocepters (Griwan et al.,
2010) . However, dizziness, rhinitis, general weakness and fatigue have been
reported with Tamsulosin (Lowe, 2005 and Kaplan, 2005).
9
Chapter One
Introduction and Literatures Review
Tamsulosin does not disturb homodynamic in patient with benign
prostate enlargement (Nieminen et al., 2007) . α1-adernoceptor blockade has
a potent anti fertility effect in male rats (Ratensooria and Wadsworth, 2009 ).
One of the adverse side effect commonly reported with different α1
blockers was sexual dysfunction. This sexual dysfunction has been related to
changes in ejaculation (either retrograde or diminished ejaculation) .
Moreover, α1 blockers differ in their likhood of causing abnormal ejaculation.
In long term open label extension study, 30% of patient treated with
Tamsulosin reported abnormal ejaculation. In contrast, incidence of abnormal
ejaculation non related to the use of nonselective α1-blockers such as
Doxazosin , Terazocin or Alfuzosin generally were lower than 1.5% (Kaplan ,
2009).
In another study , Hellstrom and Sikkasc (2006) examined the effect of
Tamsulosin
and the non selective α-blocker Alfuzosin on ejaculatory
function in healthy volunteers and found that Tamsulosin 0.8 mg /day caused
markedly reduced ejaculate volume in 90% of patients and an ejaculate in
35% of participants. These ejaculatory disorders with Tamsulosin were not
attributed to retrograde ejaculation. In contrast, an ejaculation was not
observed in any subjects in the Alfuzosin or placebo groups.
However, analysis of post climatic urine samples showed no increase in
sperm counts , suggesting that retrograde ejaculation (RE) did not occur . In
addition, others have theorized that α1A-selective blockers may results in
reduced or absent seminal emission via inhibition of smooth muscle
contraction (Michel, 2007). Also postural hypotension may be caused by
Tamsulosin (Naryan et al., 2000).
Tamsulosin has been implicated in decreased contractility of seminal
vesicles, vas deferens as well as acting of central nervous system and
theoretically affecting neurostimulasion of ejaculation (Poch and Sigman,
10
Chapter One
Introduction and Literatures Review
2010) . Also Intraoperative floppy iris syndrome occurred in 67% of the
patients treated with Tamsulosin (Bidaguren et al., 2007).
11
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1-5: Male reproductive system
The human male reproductive system consist of testes, epididymis ,
ductus deferences , urethra , scrotum and penis as well as the accessory
glands: seminal vesicles , prostate
and bulb urethral or cowper’s gland
(Lingappa and Farey, 2000).
The testes are two small avoid organ reside in the scrotum outside the
body. Each testicle is surrounded by three layers of tissues: tunica vaginalis ,
the tunica albuginea and the tunica vasculosa (Wangh and Grant, 2001).
After puberty, testes produce male sex cells called spermatozoa or
sperms and a male sex hormone called testosterone (Brichfored, 2009) .
There are 2000-3000 lobules in each testis , each lobule consists of 1-4
convoluted seminiferous tubules composed of germinal epithelial and
Leydig’s cells that secrete testosterone in to the blood stream (Ganong, 2003;
Alwachi, 2008).
The seminiferous tubule was lined by two types of cells. The first type
is called spermatogenic cell (spermatagonia) which divided by meiosis to
produce immature sperms or spermatides . Each spermatid contain a haploid
number of chromosome while the second type called sertoli cells, which
secrete oestrogen and nutrient which prolong the survival of the sperms
(Aspinall et al., 2009).
Each epididymis is a tightly coiled , thread like tube about 6 meters
long , it emerges from the top of the testis and then courses upward to become
vas deferens (Shier et al., 2000) . The epididymis is an organ in which
sperms undergo final maturation and storage prior to ejaculation (Queiróz et
al., 2002).
The epididymis is divided in to three main regions a head (caput) , a
body (corbus) and long tail (cauda) (Romer et al., 1977 and Seely et al.,
1998) . Spermatozoa are capable of surviving longer in the epididymis than in
any other segment of the reproductive tract and the cauda is the major site of
12
Chapter One
Introduction and Literatures Review
storage spermatozoa in the duct system (La Barbera, 1996). The development
of the epididymis and accessory gland are under the control of androgens
(Kocak et al., 2001).
Each vas deference also , called ductus deferences , is muscular tube
about 45 cm long (Shier et al., 2000) . The enlarge part of vas deference
called ampulla .The ampulla of the ductus deferences with the proximal
region of the seminal vesicle to form the terminal portion of the reproductive
duct system called ejaculatory duct (Mckinley and O’loughlin, 2006).
Accessory gland produce little fluid to transport the sperms from the
testes to the female tract and to aid their survival (Aspinall et al., 2009).
The alveoli of the seminal vesicles are lined with a pseudostratified
epithelium whose cells contain numerous granules and clumps of yellow
pigment. Some of the epithelial cells have flagella. The secretion of the
seminal vesicles is yellow, visicous liquid containg globulin and fructose.
This secretion provides the majority of the ejaculate volume (Linda and
Schust , 2010).
The prostate gland is a complete encapsulated a walnut measuring a
approximately 2 cm by 3 cm . This gland includes submucosal gland that
produce mucin and more than 30 tubuloaleveolar gland that open directly
through numerous duct in to the prostatic urethra (Mckinley and O’loughlin,
2006). The function of it is to store and secrete a slightly alkaline fluid, milky
or white in appearance (Huggins et al., 1942) . Usually, the prostate fluid
contributed 37–44% of the whole ejaculate and the seminal vesicle
contributed 55–61% ( Ndovi et al., 2007).
The bulbuorethral gland (also called cowper's gland) is small, paired
bulb or membranous urethral glands that may be mistaken for prostatic
carcinoma in biopsy specimens. They are composed of closely packed
uniform acine lined by cytologically benign cells with a abundant apical
13
Chapter One
Introduction and Literatures Review
mucinous cytoplasm campers embedded in smooth muscle, mimicking the
infiltrative pattern of prostate cancer (Bostwick and Cheng, 2008).
Each gland produces a clear, viscous secretion. This fluid helps to
lubricate the urethra for spermatozoa to pass through, neutralizing traces of
acidic urine in urethra and helps flush out any residual urine or foreign matter.
It is possible for this fluid to pick up sperm, remaining, in the urethral bulb
from previous ejaculation and carry them out prior to the next ejaculation.
The cowper’s gland also produces some amount of Prostate - Specific
Antigen (PSA) and cowper's tumors may increase PSA level that makes
prostate cancer suspected (Chughtai et al., 2005).
1-6: Characteristics of the sperm
1-6-1:Sperm morphology
Mammalian spermatozoa are highly differentiated cells. Their
particular structural organization results from complex morphogenetic
changes during spermiogenesis. These include:
(I) The formation of the acrosome derived from Golgi vesicles.
(II) The formation of an axoneme from the distal centriole, surrounded by
periaxonemal structures , which together form the flagellum.
(III) The migration and development of mitochondria in a helicoidal sheath
Around the axoneme in the midpiece .
(IV) The formation of an anisotropic sperm head (with a ellipsoid face and a
pear-shaped profile) containing a highly condensed nucleus.
(V) The disappearance of the vast majority of the cytoplasm (Auger, 2010).
The normal sperms head should be oval in shape. In normal sperm there
must be no defect in neck, mide piece and tail and no cytoplasmic droplet ,
more than one –third the size of a normal sperm is the head (Mendeluk et al.,
1997).
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Introduction and Literatures Review
Normal spermatozoa sought in the ejaculate are those that are
biologically selected by reaching endocervical mucus. These spermatozoa
recovered and stained in air dried fixed smears, exhibit no defect of the head
or tail (Garrett et al., 1997).
Sperms have regular oval shaped head with an intact and slender mid
piece and principal piece displaying no breaks. The acrosome is clearly
visible and covers 40-70% of the sperm area. The vacuoles do not exceed
20% of the acrosomal area and not present in the post acrosomal region. The
cytoplasm occupying <30%. The men were considered normal when produce
few potentially fertilizing spermatozoa which bind to zona pellucida (Liue et
al., 2003).
The morphologic examination of spermatozoa is a sensitive indicator of
the quality of spermatogenesis and fertility (Kruger et al., 1988).
The defect in the process of spermatogenesis lead to abnormalities
known as the primary abnormalities that may occur in the sperm head ,
including the massive head (Macrocephalic) , small head (Microcephalic ) ,
double head and round head (WHO, 1992).
1-6-2:Sperm concentration
This term should be used instead of sperm density to a void confusion
with the specific gravity of spermatozoa (Brinsden, 2005).
The concentration or the total number of sperm is a marker of the
integrity of testicular function . If the number of sperms are small by usual
fertility standard, but their motility and morphology are excellent, then
fertility might be normal . But if the number of sperm are large but their
motility and morphology are poor, the person might be infertile (WHO, 1980)
. In general, sperms concentration in normal men ranges from about 20X106 /
ml to 200X 106 / ml. There is a large variation among and within people
(WHO, 1987).
15
Chapter One
Introduction and Literatures Review
Normal sperm concentration have been reported to range between 20-
250 million per milliliter. Azoospermia is a complete absence of sperm.
Sperm count less than normal may be due to chromosomal disorders , ductal
obstruction , drugs , gonadotropin deficiencies maturation arrest , pituitary
gland disorders , radiation , renal failure and hormonal abnormality (Mundt
and Shanahan , 2011).
1-6-3: Sperm viability
Determination whether non motile sperms are viable or non viable is
important in establishing a cause for infertility in males. The membranes of
dead sperm are damaged and can easily take up eosin stain. The membranes
of viable sperm remain intact and do not allow eosin stain to penetrate leaving
the sperm colorless (they will appear white). Eosin stain can be used alone or
in conjugation with nigrosin stain. which provide dark back ground against
which the red colored dead sperm and white colorless sperms can be
visualized. Sperms that are non motile may be a live but may have effects of
the tail piece. However, the proportion of viable sperms, dead sperms can not
demonstrate motility. Normally >75% of sperm are viable (Mundt and
Shanahan, 2011).
Reduced percentage of motility with high percentage of viable sperms
may reflect structural or metabolic abnormalities of sperms that are derived
from abnormalities in testicular function or anti motility factor in the seminal
plasma (Siegel, 1993).
There are two general approaches to viability testing, traditional
staining versus the hypo osmotic swell test. The advantage of this approach is
that the sperms remain viable and can be used for intracytoplasmic sperm
injection (Hsiao and Schlegal, 2011).
16
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Introduction and Literatures Review
1-6-4:Sperm motility
Motility is the amount of flagellar motion and forward motion that a
sperm makes . The most commonly system of motility grades used as follow:
1- zero -0.4 indication no motility
2- One denotes sluggish motion with no forward progression.
3- Two denotes slow forward progression.
4- Three Indicates sperm moving in a straight line at great speed. If motility is
low (<30%) a viability stain should be performed (Hsiao and Schlegal, 2011).
Fertilization of ovum is dependent on the ability of sperm to reach and
unite with it. Motility should be evaluated within one hour of specimen
collection, because motility will decrease over time. The movement of sperm
is evaluated and may be subjectively estimated or counted in to three
categories. These categories may be called high motile, low motile, non
motile (Mundt and Shanahan , 2011).
The presence of progressively motile sperm in the ejaculate is critical to
ensure an adequate sperm transport and fertilization. Sperm motility is
considered as compromised if the percentage of forward progressive sperm
falls bellow 50% within 60 min of sample collection (Agarwal and Said,
2011).
The presence of low sperm motility asthenozoospermia could occur as
a result of prolonged time to processing of collected samples . Samples
container may be toxic to the sperm and sample exposure to extreme
temperature or sun light may result in decreased sperm motility . Long period
of abstinence also proved to be a cause of a asthenozoospermia include sperm
axonemal
deformities,
excessive
leukocyte
and
unknown
factors.
Asthenozoospermia is also most commonly seen with anti sperm antibodies.
The observation of sperm clumping combined with low sperm motility is a
further indication of the presence of anti sperm antibodies (WHO, 1999).
17
Chapter One
Introduction and Literatures Review
Sperm motility is usually rated in two ways : the fraction or percentage
of all sperm that are moving and the quality of sperm movement which
include active motile, low motile and non motile (Turek, 2000).
1-7: Spermatogenesis
Spermatogenesis is a process of generating mature sperms with halve
the number of chromosomes (haploid) produced from germ cell precursors
with full number of chromosomes (diploid) (Wang et al., 2009).
The efficacy of spermatogenesis is being reflected by daily sperm
production in adulthood is known to be determined by the total number of
functional sertoli cell (Sharp , 1994).
The duration of spermatogenesis is considered to be constant and it is
vary considerably in species thus, it is measured about 64 days in man (Heller
and Clermont 1963) and about 35 days in mice (Berne and Levy, 1993).
Spermatogenesis happened within several structures of the male
reproductive system. The initial stages occur within the testes and progress to
the epididymis where the developing gametes mature and are stored until
ejaculation. The seminiferous tubule of the testes is the starting point for this
process (Middendroff et al., 2002).
Spermatogenesis process may be divided in to three phases:
spermatocyto-
-genesis
(mitosis)
, spermatidogenesis
(meiosis) and
spermiogenesis (Schlegel and Chang , 1998).
1-7-1: Spermatocytogenesis (mitosis)
Spermatocytogenesis is the process of which male spermatogonia
become progressively modified and enlarged to form large primary
spermatocytes (Guyton and Hall, 2006).
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Chapter One
Introduction and Literatures Review
1-7-2: Spermatidogenesis (meiosis)
In this phase , each primary spermatocytes undergoes a reduction
division (first meiotic division ) to yield two haploid secondary spermatocytes
which bear the (1n) version chromosome number (Guyton and Hall, 2006).
Each secondary sprmatocytes then undergo the second meiotic division
to form two haploid rounded spermatids which are located near the lumen of
seminiferous tubules, after that no divisions occur and spermatides enters the
phases of morphological changes (Berne and Levy, 1993).
The special type of nuclear division, which forms haploids gametes, is
termed meiosis. The mitotic phase terminates at the primary spermatocytes ,
which at first resembles the cytological characteristics of spermatogonia from
which they a rise (Gupta , 2005) .
1-7-3: Spermiogenesis
The final phase of differentiation of male germ cells prior to their
release from seminiferous tubule. During this phase the round spermatides
which are relatively less an differentiated haploid germ cell, undergo complex
morphological , biochemical and physiological changes that result in the
formation of a symmetrical flagellated spermatozoa (Gupta, 2005 and de
Rooij, 2001).
During spermiogenesis , the spermatides begin to grow a tail and
develop a thickened mid-piece , where the mitochondria gather and form an
axoneme . Spermatide DNA also undergoes packaging, becoming highly
condensed . The Golgi apparatus surrounded the condensed nucleus,
becoming the acrosome ,one of the centrioles of the cell elongates to become
the tail of the sperm (de Rooij, 2001).
The end result of spermiogenesis phases, two mature spermatozoa per
each transformed spermatide , then released in the lumen of seminiferous
tubules by the process of spermiation which is managed by sertoli cells , that
19
Chapter One
Introduction and Literatures Review
many studies had shown the differentiation of spermatides into spermatozoa
occur within the cytoplasm of these cells (Berne and Levy, 1993).
Maintenance of spermatogenesis requires testosterone androgen
production, in turn , is regulated by the luteinizing hormone (LH) . Follicle
stimulating hormone (FSH) required for development of the seminiferous
epithelium and initiation and maintenance of the mitotic phases of
spermatogenesis (La Barbera, 1996).
1-8: Role of Sertoli cell in spermatogenesis
Sertoli cell has served important roles in spermatogenesis support and
nutrition of the developing germ cells ; compartmentalization
of the
seminiferous tubules by right junction , which provides a protected and
specialized environment for the developing cells ; controlled release of mature
spermatides in to the tubular lumen (spermiation ) ; secretion of fluid , protein
and several grouth factor and phagocytosis of the degenerating cells and
phagocytosis of the excess cytoplasm (residual body ) that remain from
released sperm (Cheng, 2008).
Sertoli cell also mediates the action of FSH and LH stimulated
testosterone production in the testes. FSH induce sertoli cell to produce
nutrient for sperm ,LH induce Leydig cell to produce T which has important
role in maintaining normal spermatogenesis (Sharp, 1994).
Sertoli cells provide physical support for the germ cells is a previous
from their topographic relationship. Sertoli cell show both configurational
relationship and specialized contact with the developing germ cells. The
configurational relationship and contacts suggest that sertoli cell originates
four to five different germ cell generation in one tubule , so that they all
evolve synchronously to form the next stage . They also suggest facilitation of
germ cell movement upward as each layer of cells matures (Barth and Oka,
1989) .
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Chapter One
Introduction and Literatures Review
Although it is suggested that FSH plays a major role initiation,
maintaining quantitatively normal spermatogenesis, whearse FSH play
qualitative role and is not strictly necessary for fertility (Sharp, 1994).
Sertoli cell may control the temporal and special organization of
spermatogenesis these observation on spermatogenesis imply a remarkable
degree of temporal and spatial organization among the spermatogenic cells.
The cytoplasm of an adjacent sertoli cells are in continuity with one another
via extensive gap junctional contact. In addition, each sertoli cell spans the
tubule from per tubular basement membrane to lumen for communication
(Johnson and Everit, 2007).
Sertoli cell works to regulate the proliferation of primary germ cells
primordial (germ cell) and continuity of the spermatogenesis where sertoli
cells processed testicular blood barrier by strong connected junctions and are
thus equipped with a small environment to facilitate the spermatogenesis
(Haywood et al., 2004).
1-9: Hormonal control of spermatogenesis
Spermatogenesis in mammals requires the actions of a complex
assortment of peptide and steroid hormones, each of which plays an important
role in the normal functioning of the seminiferous epithelium. These
hormonal messengers are critical not only for regulation of male germ cell
development but also for the proliferation and function of the somatic cell
types required for proper development of the testes ( Holdcraft and Braun ,
2004).
These include the interstitial steroidogenic leydig’s cells whose primary
function appears to be the production of testosterone. The myoid cells that
surround the seminiferous tubules provide physical support and contractile
motion to these structures and the sertoli cells, whose direct contact with
proliferating and differentiating germ cells, within the seminiferous tubules,
21
Chapter One
Introduction and Literatures Review
makes them essential for providing both physical and nutritional support
for spermatogenesis (Meng et al., 2005).
The spermatogenesis process and all other aspects of male reproductive
function depend on the presence of reproductive hormones produced by the
hypothalamus, anterior pituitary and testes (Tilbrook and Clarke, 2001).
Hormonal regulation of spermatogenesis is organized as a control
circuit with a negative feed-back mechanism involving the hypothalamus,
pituitary gland and testes (Brehm and Stegar, 2005).
Selected neuron in the nucleus of hypothalamus secrete the peptide
gonadotropin releasing hormone (GnRH) in to the pituitary portal blood
stream. The GnRH travels to the anterior pituitary, where specialized cells
termed gonadotrops initiate signaling in response to GnRH binding. In
response to GnRH ,triggered signaling gonadotrops synthesized and secrete
LH and FSH (Lingappa and Farey, 2000).
Baker et al., (2003) pointed that the loss of GnRH hormones in the
mice causes the pool of fatty droplets in the leydig’s cells and secretion of
steroidal hormones.
The spermatogenesis in mammals required an act common to all
peptide and steroid hormones ; each of them has a key role in the normal
functioning of the seminal lining in regulating the growth and development of
germ cells and the proliferation of somatic cells important in the development
of the testes cell such as leydig’s cells , sertoli cells and muscle cells that
surround the seminiferous tubules (Holdcraft and Braun, 2004).
Luteinizing hormone, T and FSH are the prime regulators, which
control spermatogenesis. However, androgens are indispensable for initiation
and maintenance of spermatogenesis , although testosterone feeds back on
both gonadotrophic hormone , an additional feed back loop exists between the
testes and the brain for FSH . Inhibin , activin and follistatin are involved in
22
Chapter One
Introduction and Literatures Review
this regulatory system . While inhibin functions to suppress FSH secretion,
(Gupta, 2005).
Follicle stimulating hormone stimulate the proliferation of the
spermatogonia and formation of the primary spermatocytes , while androgens
are involved in bringing about the meiosis division of the primary
spermatocyte and their final conversion in to the spermatide . Follicle
stimulating hormone is also influences the secretion of sertoli cells, secretion
of androgen binding protein (ABP) is under the control of FSH . However,
LH control the synthesis of androgens by the Leydig cells clusters (Negi,
2009). Follicle stimulating hormone and testosterone act through the sertoli
cell since the receptors for those hormone are located on these cells and not
on the germ cells (Verhoeven et al., 2007).
Follicle stimulating hormone stimulates the production of androgen
binding protein by sertoli cells. ABP is essential to concentrating testosterone
in levels high enough to initiate and maintain spermatogenesis, which can be
20-50 times higher than the concentration found in blood. The hormone
inhibin acts to decrease the levels of FSH. Studies from rodent models
suggest that gonadotropin hormone (LH and FSH) suppresing the
proapoptotic signals and therefore promote spermatogenic cell survival
(Pareek et al., 2007).
Within the testes, LH causes synthesis of testosterone by intertubular
leydig’s cells, which negatively influences hormone release in the
hypothalamus and pituitary by contrast , FSH acts on sertoli cells . It induces
the production of ABP by means of which testosterone can pass the sertoli –
sertoli junction complexes and also inducing the production of activin and
inhibin by sertoli cells which both influence hormone release in the
hypothalamus and pituitary (Brehm and Stegar , 2005).
23
Chapter One
Introduction and Literatures Review
1-10:Testosterone hormone
1-10-1: Chemistry and biosynthesis of testosterone
Testosterone is the major hormone of testes , it is synthesized from
cholesterol and is also formed from androstenedione secreted by the Leydig’s
cells (Negi, 2009).
It transformed to dihydrotestosterone in the cytoplasm of the prostate
gland cells and other accessory sexual gland cells . The interstitial cell of the
testes, known as , leydig’s cell is the main source of testosterone . The
activity of these cells mediated by the interstitial cell stimulating hormone
(ICSH) which is produced by the pituitary gland (Alwachi, 2008).
The first steps of T production and processing occur in mitochondria or
smooth endoplasmic reticulum of leydig’s cells, cholesterol is the main and
started compound in the manufacturing process (Beamer et al., 1983).
As summarized in figure (1.1) Testosterone is synthesized in the
leydig’s cell of the interstitial tissue of the testes by an enzymatic sequence
steps from cholesterol which predominantly formed by synthesis from acetate.
Also contribute the first and rate –limiting step in gonadel and adrenal steroid
–genesis in the transfer of steroidogenic substrate cholesterol from the outer
mitochondrial membrane to the inner membrane mediated by the cholesterol
transport protein steroidgenic a cute regulatory protein (STAR) (Swerdloff et
al., 2010).
After testosterone form from the testes, testosterone is bound by plasma
protein in the circulation about 45% of plasma testosterone . In adult men
bound with high affinity to sex binding globulin (SBG) , 50% is loosing
bound to albumin 9.1-2% to cortisol-binding globulin and less than 4% is free
(not protein bound ) (Winters and Clark, 2003).
24
Chapter One
Introduction and Literatures Review
cholesterol
P4 50 scc
3B-HSD
pregnenolone
Progesterone
P450C17
B450C17
3B-HSD
17 α -oltpregnenolone
17 α -oH-progestrone
P450c17
P450c17
3B-HSD
An endrostenedione
DHEA
17B-HSD
17B-HSD
3B-HSD
Androstenediol
Testosterone
Figure (1.2) The intratestesticler steroidogenic pathway for synthesis of
testosterone (Swerdloff Ronald et al., 2010).
25
Chapter One
Introduction and Literatures Review
1-13-2.Transport and metabolism of testosterone
The normal range of Testesterone is 2.8-8.2 ng /ml testosterone
secretion rate is 4.9% in normal adult males. Small amount of testosterone are
also secreted in females, probably from the ovary, but possibly from the
adrenal as well. ninety eight percent of the testosterone in plasma is bound to
protein; 65% is bound to a B-globulin called gonadal steroid binding globulin
(GBG) or sex steroid binding globulin, and 33 percent to albumin. (Ganong ,
2005) . 70% of testosterone hormone turned to oestradiol hormone through
the process of aromatization, small quantities of it secreted by the adrenal
gland, and about 30% secreted by the testes, as part of it produces by leydig’s
cells ( Ganong,1985).
The testosterone hormone is also metabolized to oestradiole (E2) , this
metabolism occur due to aromatize in the brain , testes and adipose tissue , in
the normal men , the rate between estradiol hormone levels are compared with
the total testosterone is known and between the levels of DHT and
testesterone hormone in the plasma is about the ratios 1:200 and 1:10
respectively (Winters et al., 1999).
Steroid hormone are diffused freely and rapidly a cross cell membrane
as following its synthesis , such as testosterone leave leydig’s cells to enter
the testicular interstitial compartment and diffuse across the capillary
endothelium in to the circulation (Winters and Clark, 2003).
26
Chapter One
Introduction and Literatures Review
1-13-3.Action of testosterone
In addition to their action, during development , testosterone and other
androgens exert an inhibitory feed back effect on pituitary LH secretion ;
develop and maintain the male secondary sex characteristics ; exert an
important protein – anabolic , growth promoting effect and along with FSH ,
maintain spermatogenesis increase the synthesis and decrease the break down
of protein and thus , results in increase in the rate of growth (Ganong, 2005) .
Testosterone contributes to the development of seminal vesicles and
epididymis , while it seems that the development of the testicle and scrotum
and penis stimulates by the dihydrotestosterone (Alwachi, 2008).
Testosterone deficiency in men is manifested typically by symptoms of
hypogonadism, including decreases in erectile function and libido.
Testosterone also has an important role in the regulation of normal growth,
bone metabolism and body composition. Specifically, T deficiency is an
important risk factor for osteoporosis and fractures in men. Men with
testosterone deficiency have significant decreases in bone density, particularly
in the trabecular bone compartment (Laurence, 2000)
Normal levels of testosterone in males are requires to maintain
secondary sexual characteristics , fertility , muscle mass , hair and sexual
function as part of natural male aging . There is generally decrease in
testosterone levels secondary to diminished gonodal function. The effect of
reduced testosterone levels varies among subjects and has become known as
androgen decline in the aging male. In conjucation with testosterone reduction
in men, a decline in erectile function is often seen. This is importante such as
in the metabolic syndrome in which the hypogonodal state is frequently a
ccompained by erectile and sexual dysfunction (Jones, 2009).
27
Chapter One
Introduction and Literatures Review
1-13-4.The role of testosterone in spermatogenesis
Testosterone leaves the testes through the blood stream and 95% of
circulating testosterone in the adult man is derived from testicular secretion;
the remainder arises from metabolic conversion of precursor steroids.
Predominantly secreted by the adrenal cortex such as dehydroepiandrosterone
DHEA (Ishimaru et al., 1978).
Moreover ,T is essential for spermatogenesis , fertility and maintenance
of the male phenotype (Pakarainen et al., 2005).
Testosterone produced by the leydig’s cells under the stimulation of
LH is not only secreted in the system circulation but diffuse from leydig’s
cells in the interstitial space in to the seminiferous tubule where it enter sertoli
cells . Which can convert testosterone to DHT and leaves sertoli cells to enter
the testicular fluid around the germ cells . Resulting in high intra testicular
levels (Jones and López, 2006 and Swerdloff al., 2010).
leydig’s cells secrete the androgen (T) , especially under contact of
Luteinizing Hormone (LH) that secreted by the pituitary gland which is
known as interstitial cell stimulating hormone (ICSH) in males ;also it may be
helped by Follicle Stimulating Hormone (FSH) to stimulated sertoli cells to
production Androgen Binding Protein(ABP) (Alwachi, 2008).
The testosterone act as a synergistic with FSH to regulate the division
of spermatogonia and the continuation of the spermatogenesis (Singh and
Handelsman , 1996).
It is testosterone and possibly DHT that stimulate certain phases of
spermatogenesis, not LH directly , for example testosterone in the rat
stimulate the first meiotic division during which diploid primary
spermatocytes are converted to haploid secondary spermatocytes . Thus, LH
causes this division only indirectly by causing the secretion of testosterone
from the leydig’s cells (Jones and López, 2006).
28
Chapter Two
Chapter Two
Materials and Methods
2:Materials and Methods
2-1: Materials
2-1-1: Equipments and Apparatus
The equipments and apparatus which were used throught the study are
listed in table 2-1
(Table 2-1): The equipments and apparatus used .
Instruments and Equipments
Suppliers
Eppendorf Centrifuge
Olympus
(Japan)
autoclave
Webeco gmbh
(Germany)
Electrical Oven
Memmert
(Germany)
Eppendorf tubes
Espif
(Germany)
Dissecting microscope
Sartorius
(Germany)
Hot plate
Heidolph
(Germany )
Incubator
Eppendorf
(Germany)
Light microscope
Novex
(Holland)
Mini-VIDAS
Bio meieusvitek
(USA)
Petri dish
Localy made
(plastic)
Digital Camera
Sony- Japan
Rotary microtom
Gallenkamp
( England)
Slides and cover glasses
Memmert
(Germany)
29
Country
Chapter Two
Water bath
Memmert
Electric balance
Sartorius
30
Materials and Methods
(Germany)
(Germany)
Chapter Two
Materials and Methods
2-1-2:Chemicals
Chemicals which were used throught the study are listed in table 2-2 (Table 2-2)
Table (2-2):Chemicals used.
Chemical
Suppliers
Ethanol 70%
Switzerland ,fluka
Physiological saline
Locally prepared
Bouin s fixative
Switzerland
Zylene
Gainal and Chemical, USA
Paraffin wax
fluka, Switzerland
Glycerine
fluka, Switzerland
Hematoxylin Stain
BDH, England
Eosin stain
Reidle, Germany
Nigrosin
BDH, England
Albumin
Canada Balsam
BDH , England
RPMI-1640
BDH , England
Glatial acetic acide
BDH , England
Sodium citrate
BDH , England
Formaldehyde
Fluka- switzerland
31
Chapter Two
Materials and Methods
2-2 :Methods
2-2-1 :Preparation of solutions:
2-2-1-1: Preparation of histological solutions :
- Bouin’s fixative prepared by mixing the following:
1- Saturated alcoholic picric acid solution (75 ml)
2- Formaldehyde 40% (25ml)
3- Glacial acetic acid (5ml) (Galigher & kozloff, 1946).
- Eosin stain: it was prepared by mixing:
1- Eosin pigment (1 gm)
2- Distilled water (30ml)
3- Ethyl alcohol (70 ml) (Luna & Lee, 1968)
- Harris –Hematoxylin Stain: prepared as a mixture of
1- Hematoxylin Stain (1.5 gm)
2- Absolut ethyl alcohol (15 ml)
3- Potassium Alum (30 gm)
4- Red mercury oxide (1,5 gm)
5- Glacial acetic acid (12ml)
6- Distilled water (25ml) (Luna and Lee, 1968).
Mayer's Adhesive: It was prepared by mixing:
1- Albumin (50 ml)
2- Glycerol (50 ml)
32
Chapter Two
Materials and Methods
2-2-1-2 :Sperm count solutions :
Eosin –Nigrosin stain: It was prepared from:
1- (1 gm) of Eosin stain soluble in (100 ml) of 3% sodium citrate.
2 - (5gm) of Nigrosin stain soluble in (100 ml) of 3% sodium citrate (Hancook, 1951)
2-2-1-3: Preparation of Tamsulosin hydrochloride (Flomax ) solution
Tamsulosin hydrochloride
(Flomax) was obtained from (Astellas pharma
Europe B.V. Leiderdorp, The Netherlands) at concentration (o.4mg) , The Tamsulosin
hydrochloride doses (8 ,16µg /kg.b.wt) was prepared by dissolving one capsule of the
drug (400µg/ml) in 100ml of distilled water to prepare a (8µg /kg.b.wt) .
Same procedure was followed using one capsule of the drug and dissolved in
200 ml of distilled water to prepare the second dose (16µg /kg.b.wt). (Patel and
Patel., 2010).
2-2-2: Experimental Animals:
Thirty albino Swiss male mice (Mus musculs) aged 8-10 weeks were obtained
from the animal house of Al-kindy company for vaccines.
The weight range was 35-30gm . The animals were housed in plastic caged.
The caged were embedded with wooden shelves, under natural 10 hr light and 12 hr
dark, The animals were caged at lab temperature of 23 – 25°C, and the animals were
feed ad libitum .They were divided into three groups , 10 animals ⁄ group.
Group 1 : as control , animals were treated with (o.1ml ) of distilled water.
Group 2 : Animal were given 8 µg /kg.b.wt /day of Flomax for each mouse .
Group 3: Animal were given 16 µg /kg.b.wt/day (0.4 µg) of Flomax for each mouse.
The doses were daily injected intraperitonially for 42 successive days
(Tambaro. et al., 2005)
33
Chapter Two
Materials and Methods
2-2-3 :Blood collection
Animals weight has been recorded before and after the injection by using
electrical balance , Blood samples (0.5-1.0 ml) were obtained (immediately after
sacrificing) by heart puncture and placed into eppendrof tubes and allowed to clot .
Serum was separated by centrifugation for 10 minutes at 3000 rpm. The isolated
serum was kept at 4C˚ bellow zero to determine the testosterone hormone level. Mini
VIDAS radioimmunoassay for serum testosterone was carried out with a testosterone
kit.
2-2-4: Killing of the animals
Animals were killed by cervical dislocation, Immediately after killing the
abdominal cavity was opened in overturned (T) shape, and then the male reproductive
organs were extirpated (right testes & right testes epididymis). The adipose tissues
were removed. The right testes were placed on a filter paper to be weighted with an
electrical balance. The tail of the Epididymis was utilized to study sperm parameters.
The testes were fixed in Bouin’s fixative for 24hr then transferred to 70% ethyl
alcohol for storage, and then it was submitted to microscopic examination for
histological study.
2-2-5: Collection and Preparation of sperms
Soon after killing and dissection, the caudal of right epididymis of were isolated
and placed in Petri dish containing 1ml of RPMI-1640 Medium which is norishing
media and composed of
Fetal bovine serum (10g), Penicillin (1000000 IU),
Streptomycin (1g) , Heps (4g) and Sodium bicarbonate (1%). Which maintained at
37˚C to prevent cold shocks, and minced by using microsurgical scissor and forceps to
release sperm from the tail of the epididymis ( Nara and McCulloch , 1985 ).
34
Chapter Two
Materials and Methods
2-2-6: Sperms parameters:
2-2-6-1: Percentage of sperm motility:
Sperm motility represents the quantitative parameter of motile sperms expressed
as a percentage (Silverage and Turner, 2001). A drop of semen was placed on a
warmed 37˚C slide and examined under the power of 400X. Active sperm motility
percentage was assessed according to the following equation
No. of motile sperm
Motility (%) = ________________________________ X 100
Total N. of sperms
2-2-6-2: sperm concentration:
A drop of spermatozoa was placed on a slide and covered with a cover slide.
Concentration of spermatozoa (sperm/ml) was calculated from the mean number of
sperm in five high powers microscopically fields under magnification of 400X .This
number multiplied by a factor of one million (x106/ml) (Smith and Mayer, 1955).
Sperm concentration =No. of spermatozoa X multiplication factor
2-2-6-3:percentage of sperm viability:
A drop of sperm suspension was mixed with a drop of eosin stain (1%) and two
drops of nigrosin stain. Then a thin smear of semen –eosin - nigrosin mixture was
made using other slide which used to make a thin smear in a third slide and the third
slide left to dry at room temperature , the slides was examined under light microscope
at (400x). The dead sperms appear pink colour while the normal ones show a blue
colour. also the morphology abnormal sperm were determined by this stain.
35
Chapter Two
Materials and Methods
The sperm viability were estimated according to the following equation (Hafez,
1987)
No. of dead sperm
Percentage of dead sperm % =_____________________
X 100
Total sperm No.
2-2-6-4: Percentage of morphologically abnormal sperm:
Spermatozoa were examined for normal and abnormal morphology of head or
tail or both. To calculate the abnormality of sperm by using the same slide which was
used for calculating live and dead sperm according to Dale and Edler, 1997.
No. of abnormal sperms
Abnormality % = _______________________ X100
Total N. of sperms
2-2-7 Histological study:
Testes were kept in Bouin’s solution for 24 hr then they are washed by 70%
ethyl alcohol for several times until the yellow colour was removad. Testes were kept
in ethyl alcohol 70% of until use.
Dehydration, clearing, infiltration, embedding, sectioning, staining and
mounting were respectively done to prepare the slides for histological examination
according to Bancroft and Stevens (1982).
36
Chapter Two
Materials and Methods
1) Dehydration
Water was withdrawn from
the tissue by passing specimens in a series of
progressive concentrations of ethanol (80%, 90%, 100%, and 100%) and for 45 min
for each concentration.
2) Clearing
The specimens were placed in xylene for 45 min. for clearance
3) Infiltration
Samples were set with a mixture of xylene and melted paraffin wax (58-5˚C) in
(1:1) ratio for 30 minutes in an electric oven, and then the samples were transferred to
a new melted wax for only three phase and one hour for each phase.
4) Embedding
The specimens were placed in a metal template filled with melted paraffin and
left until wax is solidified. The paraffin blocks were removed from the template and
submerged in cold water for 2-3 hours.
5) Sectioning
Sectioning was done by a microtome. At a thickness of 5µm, and the sections
were placed on clean slide put in water bath (45-50°C) for two min there after they
were fixed on the slide by using Mayer s̉ albumin.
6) de waxing
The section were putted in xylene at two phases , for 30 minutes each phase.
7) Staining
The slide passed in descending serial concentration of ethyl alcohol: 100%,
95%, 90%, 80%, 70% for two minutes in each concentration , then washed with
37
Chapter Two
Materials and Methods
distilled water for two minutes. Sections were stained with Haematoxilin Harris for
two minutes and then washed with tap water. Then the slides were stained with
alcoholic eosin for two minutes and then passed ascending concentration of ethanol is:
70% , 80% ,90% ,95% , 100% , about twice for each concentration and then the
slides were transferred to xylene for clearing in two stages in a period of 15 minutes
8) Mounting
Sections were mounted with canada balsam then covered with cover slide.
2-2-8 :Microscopic Examination:
Compound light microscope was used to study the histological changes in
seminiferous tubules , interstitial spaces , spermatid and leydig’s cells clusters.
Diameter were assessed in each testes using previously calibrated micrometer (Ocular
micrometer , stage micrometer) .The diameter of 20 seminiferous tubules was
measured in four fields(5 seminiferous tubules per field) . In same way diameter of
spermatids , and spermatocyte were measured in four fields and the mean value of
each was calculated .The interstitial space measured between two consecutive
seminiferous tubules by using the ocular micrometer . Photos were taken to visualize
some of results using a light microscope supplied with Sony camera
2-2-9: Statistical analysis
Data were statistically analyzed by 1-way analysis of variance with ANOVAtest. Data are presented as means ± SD. The level of significance of P < 0.05 was used
for analysis of variance test (ANOVA) (Al-Mohammed et al., 1986).
38
Chapter three
Results and Discussion
Chapter Three
Results and Discussion
3-1: Testosterone concentration in serum:
The obtained results demonstrated that there was a significant
(p<0.05) decrease in the T. level in the serum of the groups administered
with 8µg /kg.b.wt. and 16µg/kg.b.wt. of Tamsulosin hydrochloride .There
was also a significant decrease (p<0.05) in the T level between treatment
Table 3-1.
Table (3-1): Effect of Tamsulosin hydrochloride (8µg /kg.b.wt. and
16µg/kg.b.wt.) on serum Testosterone level (ng/ml) in male mice .
Testosterone ng/ml
(mean±SD)
Treatments
A
2.86+0.23
Control
B
1.61+0.12
Tamsulosin 8µg/KgB.W
C
0.91+0.09
Tamsulosin 16µg/KgB.W
· A, B, C representing a significant(P<0.05) differences in comparison to each
other .
39
Chapter three
Results and Discussion
These results were in agreement with that of Mocktary et al., (2007)
who noticed that the level of testosterone decreases through either the
effect on steroidogenesis enzymes in testes , or its inactivation properties
on adrenergic systems involved in steroidogenesis.
This effect can be explained by the anti androgenic action as well as
the stimulation of steroidal anti androgen to the negative feedback
inhibition of the hypothalamus which resulted in lowering the
concentration of plasma testosterone . However, it is well known that in
non steroidal anti androgens , the androgenic actions were blocked in
both hypothalamus and target tissues . Therefore , negative feedback
signals were inhibited and production of testosterone was increased in the
testes (Akakura et al., 1998) . There is a great number of steroids like
testosterone which metabolized by CYP3A enzyme . This enzyme
increases these steroids clearance from plasma. Also, CYP3A induction
would produce some metabolites which have endocrine disrupting
function (Guillette ,2006).
These decreases in testosterone level can also be explained by
necrosis induced in the seminiferous tubule as noticed by Hibi et
al.,(1995) .The significant decrease in interstitial cells number lead to
decreased testosterone secretion from interstitial cells .
Serova et al., (1994) pointed that Adrenoceptor blocking drugs
such as phentolamine (alpha receptor blocker) or obsidan (beta blocker)
caused a decrease in testosterone levels in mice.
40
Chapter three
Results and Discussion
3-2: Changes in testes and body weights
There was a significant (p<0.05) decrease in the rate of testes
weights of animals treated with Tamsulosin hydrochloride at the
concentration 8µg /kg.b.wt. and 16µg/kg.b.wt. compared to control
group . There was also a significant (p<0.05) difference in testes weights
between treatments (group I and group II) Table 3-2.
The effects of the drug on testes weight was in consistence with
Mocktary et al., (2007) who noticed that alpha
blockers
caused
reduction in testicular weight . This resulted from the large reduction
of spermatogenic cell number as evidenced by the examination of stained
testes sections (Giuliano, 2006) . Moreover the reduction in testes weight
may be attributed to an impairment of spermatogenesis process that
normally stimulated by FSH and testosterone (Haywood et al., 2003).
The physiologic concentrations of T, LH and FSH play an
important role in spermatogenesis (Zitzmann, 2008), so a significant
decrease of these hormones decreases the number and function of
germinal cells of testes followed by testes weight reduction.
Mhaouty-Kodja et al., (2007) concluded from their work that
alpha1 blocker can cause infertility with reduction in testicular weight.
The results also showed a significant decrease (p<0.05) in body
weight after treatment with concentrations of 8µg /kg.b.wt. and
16µg/kg.b.wt. of the drug compared to control group. There was also a
significant (p<0.05) decrease in body weights between treatment Table
3-2.
This decrease in body weight may be attributed to many factors
such as ; the deficiency in testosterone level , as suggested by Rolf et al.,
(2002) . Testosterone plays a key role in the development of male
41
Chapter three
Results and Discussion
reproductive tissues such as the testes and prostate as well as promoting
secondary sexual characteristics such as increased muscle, bone mass and
growth of body-hair . ( Alwachi, 2008 ; Sherwood ,1991 ; Mooradian et
al.,1987) . In addition, testosterone is essential for health and wellbeing ( Bassil et al., 2009) as well as the prevention of osteoporosis
(Tuck and Francis , 2009).
Many studies have proven that testosterone has a direct effect on
protein synthesis in all tissues and organs of the body and can increase
bone and muscle mass in males (Wade et al .,1968 ) .So the decreased
testosterone could lead to body weight loss via decreasing protein
synthesis and bone /muscle mass .
Tamsulosin hydrochloride also found to cause many disorders such
as asthenia , dizziness , drowsiness , headache , insomnia , syncope ,
vertigo , chest pain orthostatic hypotension , pharyngitis , diarrhea,
general weakness and nausea (Kluwer, 2011) . All of these may be the
cause of decrease in body weights of treated animal.
42
Chapter three
Results and Discussion
Table ( 3-2): Effect of different concentrations of Tamsulosin HCl
(8µg/KgB.W and 16µg/Kg B.W )on the rate of testes and body weights.
Treatments
Control
Tamsulosin
8µg/KgB.W
Tamsulosin
16µg/KgB.W
Final Body
Weight (gm)
(mean±SD)
Testes weight
(gm)/100gm
(mean±SD)
A
32.50+1.30
A
0.20+0.01
B
28.200+0.90
B
0.18+0.02
C
25.20+0.90
C
0.14+0.01
A, B, C representing a significant (P<0.05) differences in comparison to each
other.
3-3: Sperm parameters:
3-3-1: sperm concentration in the epididymis
The results showed that there was a significant (p<0.05) decrease
in sperm concentration of the group administered with Tamsulosin HCl at
a concentration 16µg/kg.b.wt. of Tamsulosin compared with the control
group but there was no significant (p<0.05) decrease in sperm
concentration of the group administered with Tamsulosin hydrochloride
in concentration 8µg /kg.b.wt. of the drug and there was a significant
(p<0.05) difference between treatments Table 3-3).
43
Chapter three
Results and Discussion
Erin et al., (2006) found that Tamsulosin decreases sperm
concentration and total sperm count , semen volume , motility
and
normal morphology .
The clinical findings studied by Michel (2007) suggested that the
drug Tamsulosin have an effect on semen formation , transport and the
cause of the abnormal ejaculation. Moreover, The abnormal ejaculation
of semen is a typical but infrequent side effect of some 1-adrenoceptor
antagonists, particularly those with selectivity for 1A-adrenoceptors such
as Silodosin and Tamsulosin . Recent clinical studies suggest that this
represents a relative an ejaculation rather than a retrograde ejaculation
(Van Dijk et al., 2006 ; Giuliano , 2006 and Michel, 2007) .
Other researchers proposed that the nature of the ejaculation caused
by short-term (5-day) Tamsulosin treatment was reduced the volume of
ejaculation rather than retrograde ejaculation. This could be caused by a
decrease in the contractions of the seminal vesicle, a decrease in the
production of seminal fluid , or both (Hellstrom and Sikka, 2007).
It was found that 0.8 mg Tamsulosin can cause a marked decreased
in ejaculate volume in almost 90% of men and an ejaculation in
approximately 35% of men ( Hellstrom and Sikka , 2007) .
α1A-adrenoceptors was also found to be widely distributed in all
the organs participating in the emission phase (epididymis, vas deferens,
seminal vesicle, prostate gland, prostatic urethra and bladder neck) . This
means that α1A-adrenoceptors plays a role in the emission phase of
ejaculation . Tamsulosin is the only α1-adrenoceptor blocker showing
some α1A selectivity and may affect the first phase of ejaculation
(Giuliano et al., 2004) . However, the decreased capacity of contraction
of the seminal vesicles is proposed as the cause of the ejaculatory
disorder induced by alpha-1 blockers (Hisasue et al., 2006).
44
Chapter three
Results and Discussion
The treatment with Tamsulosin was also found to induce retrograde
ejaculation but not other ejaculatory disorder due to abnormal sperm
progression (Grasso et al, 2006) . The impaired transportation of sperm
from the testes to the vas deferens was found to be the
potential
mechanism of ejaculation dysfunction in mice after treatment with
Tamsulosin (Sanbe, 2009) . Also The administration of Tamsulosin (3
μg/kg i.v.) also caused significant reduction in the contractions evoked by
electrical pulses in the epididymal portion , (Tambaro et al. , 2004) .
It is shown that the decreasing LH, FSH, and testosterone level
could be effective in decreasing spermatogenesis and the number of testes
germinal cells (Shariati et al., 2008) . The process of spermatogenesis is
highly sensitive to fluctuations in the environment, particularly hormones
and temperature. Testosterone is required in large local concentrations to
maintain the process , which is achieved via the binding of testosterone
by androgen binding protein present in the seminiferous tubules
(Harrison and Weiner, 1949).
It is well accepted that the low sperm production was in relation to
the reduced size of the seminiferous tubules (Ratensooria & Wadsworth
,1994) and this study confirm this result . All of These reasons may
explained the reduction in the concentration of sperm after treatment.
45
Chapter three
Results and Discussion
Table 3.3: Effect of Tamsulosin (µg /kg.b.wt. ) on sperms parameters
in male mice.
Treatments
Control
Tamsulosin
Motility of
Sperm
sperms (%)
Concentration (mean±SD)
(×106/ml)
Dead
Abnormalities of
sperms
sperms (%)
(%)
(mean±SD)
(mean±S
D)
A
A
A
A
30.61+3.43 76.40+5.56 18.90+1.7
14.70+1.60
3
0
A
26.94+4.22
A
B
70.00+5.77 24.70+3.2
4
0
B
20.90+1.30
B
21.34+4.81
B
C
57.90+4.88 30.70+1.8
0
C
29.70+2.90
8µg/KgB.W
Tamsulosin
16µg/KgB.W
A, B, C representing a significant (P<0.05) differences in comparison to each
other.
46
Chapter three
Results and Discussion
3-3-2: Percentage of sperm motility in the epididymis
The statistical analysis showed a significant (p<0.05) decrease in
sperm motility of the group administered with Tamsulosin 16µg/kg.b.wt.
compared with control group while the results of other treated group,
8µg/kg.b.wt, demonstrated that there was no (p<0.05)
significant
decrease (p<0.05) in sperm motility compared with control group and
there was a significant difference (p<0.05) in sperm motility between
treatment Table 3-3.
The low percentage of motile sperm or sperm forward progression ,
, or both , may be due to spermatozoa structure defect (Sigman and
Howards, 1998) . On the other hand the treatment with Tamsulosin may
induce retrograde ejaculation but not other ejaculatory disorder due to
abnormal sperm progression (Grasso et al, 2006).
The function of seminal vesicle is important for fertility parameters
as sperm motility, sperm chromatin stability, and immune-protection and
may be changed in case of its hypo function (Gonzales, 2001) . The
prostatic secretion makes spermatozoa motile and helps to neutralize
vaginal acidity (Mann et al., 2006) . The seminal vesicles and the
accessory sex glands secrete fructose, which acts as a donor of energy to
the spermatozoa. It is the major carbohydrate found in seminal plasma,
and appears essential for normal sperm motility (Ahmed et al., 2010) .
47
Chapter three
Results and Discussion
3-3-3: Percentage of dead sperms in the epididymis
Determination of the percentage of dead sperm is one of the
important criteria in the assessment of sperm functions. The statistical
analysis revealed that there was a significant increase in the percentage of
dead sperms of both treated groups 8µg / kg.b.wt. and 16µg/kg.b.wt.
compared with control group. There was a significant difference (p<0.05)
in dead sperm between treatments. Table 3-3.
This may be attributed to effect of the drug on leydig’s cells as well
as , on the T hormone as core hormone in the spermatogenesis and
maturity of sperm . Decline in T is causing the small number of living
sperm as well as affect the function of the epididymis and its tissues
that lead to mature sperm and thus the effect leads to a negative impact
on the percentage of living sperm . (Joshi & Ambaye, 1968) .
Sertoli cells comprise the main structural component of the
seminiferous tubules. They are responsible for the structural support for
germ cell development (Vogl et al., 2000). So, any negative response of
sertoli cells leads to the production of few sperm and had lost its vitality
as it reach dead to the epididymis , because the essential function of
sertoli cell is nutrition and support to the cells responsible for the
formation of sperm and then the negative impact of these cells would
have an effect on sperm (Joshi & Ambaye, 1968).
Other researchers proposed that sertoli cell facilitate germ cell
movement and mature germ cell release (Mruk and Cheng , 2004) and
secretion of diverse functional glycoprotein and peptides to nourish germ
cells (Skinner, 1993) as well as , maintenance of the blood-testis barrier,
and secretion of seminiferous tubular fluid (Waites and Gladwell, 1982).
48
Chapter three
Results and Discussion
It is well accepted that the glycoprotein hormones, FSH and LH,
are involved in the process of spermatogenesis (Sharpe,1994) . However,
sertoli cells express functional receptors for FSH ( Krishnamurthy et al
.,2000).
49
Chapter three
Results and Discussion
3-3-4: Percentage of abnormal morphology of sperms in the
epididymis
Table 3-3 show that there was a significant(p<0.05) increase in
the percentage of morphologically abnormal sperm in animals
administered
with Tamsulosin 8µg /kg.b.wt. and 16µg/kg.b.wt.
compared with control groups . There was also a significant (p<0.05)
difference (p<0.05) in sperm abnormalities between treatments . This
may be explained by many proposals demonstrated by many other
workers.
Lenzi et al., (1998) demonstrated that , altered sperm morphology
might reflect disturbances during spermiogenesis , spermiation and sperm
passage through epididymis . It is accepted that sperm morphology is a
sensitive indicator of overall testicular health because the sperm
morphologic characteristics are determined during spermatogenesis
(Turk, 2000).
It was also found , that any defect in spermatogenesis process may
affect the sperms and lead to the production of abnormal or deformed
sperm. This can also happen during the passage of sperm in the
epididymis ( Alwachi, 2008) .
The other reason may be the decrease in the level of testosterone.
However, T is necessary for normal sperm development and promote
differentiation of spermatogonia (Mehta et al., 2008) . So this may
explain the sperm abnormalities in treated mice Fig 3-1
50
Chapter three
Results and Discussion
A
B
C
Figure (3-1): Sperm morphology from mice treated with Tamsulosin
Hydrochloride showing (A) normal sperm , (B) abnormal tail (bent tail) ,
(C) abnormal head ( hummer head ) (E stain) 40X.
51
Chapter three
Results and Discussion
3-4: The Histological studies
3-4-1: The diameter of Seminiferous tubules
Tissue examination of normal testes of mice showed that the
seminiferous tubule are spherical or ovoid shape , lined by germinal
epithelium consisting primarly of spermatogenic cells which are in
progressive stages of spermatogenesis (primary spermatocytes ,
secondary spermatocytes , spermatide and sperms in the lumen of
seminiferous tubule , between seminiferous tubule there are interstitial
space with group of steroid producing interstitial leydig’s cells (Fig 3-2) .
The results of seminiferous tubule showed there was a significant
(p<0.05) decrease in the diameter of the seminiferous tubule in the treated
groups 8µg /kg.b.wt. and 16µg/kg.b.wt. compared to control group. There
was also a significant difference (p<0.05) in diameter of the seminiferous
tubule between treatment.
The histological examination in this study showed decrease in the
diameter of seminiferous tubule and thickness of epithelium. Also an
increase in the diameter of interstitial spaces with oedema in interstitial
tissues accompanied with low testosterone level Fig 3-3
The reduction in the diameter of seminiferous tubules could be
referred to the reduction in testosterone hormone level, since testosterone
has an important role in the development and growth of male
reproductive ducts and epithelial cells of epididymal tubules (Umezu et
al., 2004) .
Other researchers proposed that these reduction may be due to the
destruction of gap junctions (between sertoli and germ cells) in mice
which may impact on both spermatogenesis and T production because the
important role of these junctions in the regulation of cells growth and
52
Chapter three
Results and Discussion
differentiation by controlling the transport of small molecules, including
secondary messengers between adjacent cells and a cross the epithelium
toward the lumen (St-Pierre et al., 2003) . However, the reduction in
tubular diameter may be due to severe germ-cell impairment, although
the distribution is irregular (Guarch, 1992).
It is well accepted that testosterone stimulates the epithelium of the
seminiferous tubule to produce sperm and control the process of
spermatogenesis (Alwachi, 2008) .
This observation is in consistency to that reported by Back et al.,
(1977) and Mausle et al.,( 1982) and who found that the significant
decrease in the diameter of seminiferous tubule
caused by different
antiandrogens may be referred to the changes that happens in
spermatogenesis . Also Mhaouty- Kodja (2007) pointed that these
changes mainly resulted from the large reduction of spermatogenic cell
number .
The reason of degeneration and necrosis changes in seminiferous
tubules in mice group treated with Tamsulosin may be due to the reduced
number of
leydig’s cells which lead to the reduction in level of T
hormone which are responsible of active division of spermatogenic cells
in spermatogenesis therefore, the reduction in level of T hormone leads
to the damage of these cells and then damage in the seminiferous tubules
(Gyton & Hill, 2000 and Sigman and Howards ,1998) Fig 3-3
53
Chapter three
Results and Discussion
3-4-2: Interstitial Space
The histological examination also proved that the treated mice
groups ( 8µg /kg.b.wt. and 16µg/kg.b.wt.) showed a significant (p<0.05)
increase in the interstitial space compared to the control groups. There
was also a significant difference (p<0.05) in interstitial space between
treatment Table-3.4
In the present study the presence of edema in interstitial tissues
may be due to accumulation of water in the tissue of testes because of an
increase in the spaces between seminiferous tubule resulting from low
number in leydig’s cells and this lead to increase diffusion of fluid inside
interstitial tissues (Luis et al., 1986).
Therefore as the seminiferous tubules were reduced in diameter,
the interstitial spaces were increased . Fig 3-3
54
Chapter three
Results and Discussion
Table( 3.4): Effect of Tamsulosin (µg /kg.b.wt. ) on diameter of
seminiferous tubules and interstitial space in male mice .
Treatments
Diameter of
seminiferous
tubules (μm)
(mean±SD)
Interstitial space
(μm)
(mean±SD)
Control
A
208.43+2.88
A
19.15+1.80
Tamsulosin
8µg/KgB.W
B
183.00+2.94
B
28.29+1.78
Tamsulosin
16µg/KgB.W
C
169.43+4.43
C
38.29+2.10
A, B, C representing a significant (P<0.05) differences in comparison to each
other.
55
Chapter three
Results and Discussion
3-4-3: Leydig’s cells clusters
The result obtained from histological studies demonstrated that
there was a significant(p<0.05) decrease in number of leydig’s cells
clusters of animal groups treated with Tamsulosin (8µg /kg.b.wt. ,
16µg/kg.b.wt.)
compared to control group. There was a significant
difference (p<0.05) in the number of leydig’s cells clusters between
treatment Table-3.4.
The reason for the decreases in diameter of leydig’s cells clusters
refer to damage in the leydig’s cells leading to low level of T. hormone
responsible for effective on the cells divisions during spermatogenesis .
Damaging of leydig’s cells and reduce in its number has a direct impact
on the level of the testosterone hormone (Baille et al., 1966). Fig 3-5
The leydig’s cells drive spermatogenesis via the secretion of
testosterone which acts on the sertoli cells to create an environment
which enables normal progression of germ cells through stage VII of the
spermatogenic cycle. In addition, testosterone is involved in the control of
the vasculature, and hence the formation of testicular interstitial fluid,
presumably again via effects on the sertoli when leydig’s cells regenerate
and mature, it can be shown that both the rate and the location of
regenerating leydig’s cells is determined by an interplay between
endocrine (LH and perhaps FSH) and paracrine factors that secrete these
hormones (Sharp et al ., 1990).
56
Chapter three
Results and Discussion
.
Table 3.5: Effect of Tamsulosin 8µg/KgB.W and 16 µg /kg.b.wt. on
diameters of primary spermatocyte ,spermatids .
Treatments
Primary
spermatocytes(μm)
(mean±SD)
Spermatids(μm)
(mean±SD)
Control
A
6.74+0.37
A
4.35+0.50
Tamsulosin
8µg/KgB.W
B
4.561+ 0.29
B
3.36+0.38
Tamsulosin
16µg/KgB.W
B
4.59+0.45
C
2.60+0.33
A, B, C representing a significant (P<0.05) differences in comparison to each
other.
57
Chapter three
Results and Discussion
3-4-4:The diameter of Primary spermatocytes
The histological examination of testes proved that the treated mice
group with Tamsulosin showed a significant (p<0.05) decrease in the
diameter of primary spermatocytes after 42 days of mice exposure to
8µg /kg.b.wt. and 16µg/kg.b.wt. compared to control group Table 3-5.
It is well known that T or its metabolites such as DHT is essential
for maintenance of spermatogenesis by stimulation of leydig’s cells with
pituitary hormone LH , which binds to specific high affinity receptors on
the surface of leydig’s cells, resulting in increased productions of cyclic
AMP (Hold craft and Braun, 2004)
FSH also stimulates sperm production and it is essential to initiate
spermatogenesis, while T maintains spermatogenesis (O’Riordan et al.,
1988).
Uhler et al., (2003) reported that serum FSH levels has been used
as a marker of spermatogenesis in the clinical evaluation of male
infertility.
Ganong (1995) proved that conversion of primary spermatocyte
into secondary spermatocyte (Meiosis I ) are dependent on T hormone
and the final step of maturation of spermatids are dependent on FSH.
However, decreases FSH level lead to inhibiting the production of ABP
from sertoli cells, and then decreasing T hormone level in testes which
adversely affect the spermatogenesis (Yin et al., 2007).
Anderson (1993) pointed that pituitary secretion of LH stimulate
the leydig’s cells of the testes to produce T. Some of the T produced is
delivered to the seminiferous tubules, where it acts on sertoli cells to
stimulate spermatogenesis and some enters the blood stream .
58
Chapter three
Results and Discussion
3-4-4: The diameter of Spermatid
The results showed that there was a significant (p<0.05) decrease
(p<0.05) in the diameter of spermatids after treatment with Tamsulosin
8µg /kg.b.wt. and 16µg/kg.b.wt. compared with the control group . There
was also a significant difference (p<0.05) in diameter of the spermatid
between treatment Table 3-5.
Research showed that the meiosis of the primary spermatocyte and
its conversion to a secondary spermatocyte , requires essentially the
presence of the testosterone and follicle-stimulating hormone which is
essential for the development of spermatide and its conversion to mature
sperm (Alwachi , 2008) .
Testosterone has been shown to be essential for normal
spermatogenesis , because it stimulates the conversion of round
spermatids to elongated spermatids (Maureen Isoken, 2010) . Also the
decrease in testosterone hormone led to decrease in spermatogenesis
(Hammani et al., 2010), While Tamsulosin hydrochloride decreased the
level of testosterone hormone (Mokhatary , 2007) and the secretion of
testosterone stimulate sperms production and control of spermatogenesis
(AL-Alwachi, 2008) . So changes in diameter of spermatide occur after
treatment with Tamsulosin hydrochloride
Anthony et al., (1989) reported that, spermatogenic activity
requires sufficient T concentration and they indicated that ,T level could
be critical for the final steps of spermatogenesis.
59
Chapter three
Results and Discussion
A
B
I.S
Prim. Sp
.
spermatid
DST
Fig(3. 2): Section in mouce testis (control group), showing normal
structure of seminiferous tubules ,diameter of seminiferous tubules (ST),
interstitial space (IS) ,spermatid and primary spermatocyte (prim. Sp.) (H
and E) X 10 for figure (A) ,40X for figure (B).
N
N
IS
DST
DST
E
E
IS
B
A
Fig(3. 3):Section in mouse testis (treated group 8µg/KgB.W for A and 16
µg/KgB.W for B.) showing abnormal structure of seminiferous tubules.
diameter of seminiferous tubules (DST), interstitial space (IS), oedema
(E) and necrosis (N) (H and E) X 10.
60
Chapter three
Results and Discussion
Fig(3. 4):Section in mouse testis (control group) showing normal
structure and normal number of Leydig’s cell clusters (H & E X 40).
A
B
Fig(3. 5): Section in mouse testis (treated group with A 8µg /kg.b.wt. , B
16 µg /kg.b.wt) showing abnormal structure and abnormal number of
Leydig’s cell clusters (H & E X 40).
61
Conclusions and Recommendations
Conclusions
From the results of the present study it could be concluded that :
Tamsulosin hydrochloride has negative effect on fertility in male albino mice
especially prolonged uses can be important cause of infertility.
Recommendations
The following suggestions may be recommended for further studies:
1- Study the effect of Tamsulosin hydrochloride on accessory gland of male
reproductive system such as prostate gland , seminal vesicle.
2- Study of other types of alpha blockers used in benign prostate hyperplasia
therapy and make a comparisons between them.
3- Conduct electron microscope study to clarify the exact effects caused by the
drug which block the growth of sperm in the seminiferous tubule and vitality in
the epididymis.
4-Study the effect of Tamsulosin on other hormones LH ,FSH .
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‫‪Summary‬‬
‫ﺍﻟﺨﻼﺻﺔ‬
‫ﺻﻤﻤﺖ ﻫﺬﻩ ﺍﻟﺪﺭﺍﺱﻩ ﻟﻠﺘﺤﺮﻱ ﻋﻦ ﺗﺄﺛﻴﺮﻋﻘﺎﺭ ﻫﻴﺪﺭﻭﻛﻠﻮﺭﻳﺪ ﺍﻟﺘﺎﻣﺴﻮﻟﻮﺳﻴﻦ ﻋﻠﻰ ﻭﻇﻴﻔﺔ ﺍﻟﺨﺼﻴﺔ‬
‫ﻭﺍﻹﺧﺼﺎﺏ ﻓﻲ ﺫﻛﻮﺭ ﺍﻟﻔﺌﺮﺍﻥ ﺍﻟﺒﻴﺾ ‪ .‬ﺍﺳﺘﺨﺪﺍﻡ ﺛﻼﺛﻮﻥ ﺫﻛﺮ ﺑﺎﻟﻎ ﺗﺮﺍﻭﺡ ﻣﻌﺪﻝ ﺃﻭﺯﺍﻧﻬﺎ‬
‫‪ 30-25‬ﻏﻢ‬
‫ﻭﺃﻋﻤﺎﺭﻫﺎ ‪ 10-8‬ﺃﺳﺒﻮﻉ ﻭﻗﺴﻤﺖ ﻋﺸﻮﺍﺋﻴﺎ ﺇﻟﻰ ﺛﻼﺙ ﻣﺠﺎﻣﻴﻊ )‪ 10‬ﺫﻛﻮﺭ ﻟﻠﻤﺠﻤﻮﻋﺔ (‪.‬‬
‫ﺣﻘﻨﺖ ﺍﻟﻤﺠﻤﻮﻋﺔ ﺍﻷﻭﻟﻰ ﺑﺎﻟﻤﺎء ﺍﻟﻤﻘﻄﺮ ﺗﺤﺖ ﺍﻟﺒﺮﻳﺘﻮﻥ ﻭﺍﻋﺘﺒﺮﺕ ﻣﺠﻤﻮﻋﻪ ﺳﻴﻄﺮﺓ‬
‫ﻭ ﺍﻟﻤﺠﺎﻣﻴﻊ‬
‫ﺍﻷﺧﺮﻯ )ﺍﻟﺜﺎﻧﻴﻪ ﻭﺍﻟﺜﺎﻟﺜﻪ( ﺣﻘﻨﺖ ﺗﺤﺖ ﺍﻟﺒﺮﻳﺘﻮﻥ ﺑﺘﺮﺍﻛﻴﺰ ﻣﻘﺪﺍﺭﻫﺎ ‪ 16 ، 8‬ﻣﺎﻳﻜﺮﻭﻏﺮﺍﻡ‪/‬ﻛﻐﻢ ﻣﻦ ﻭﺯﻥ‬
‫ﺍﻟﺠﺴﻢ ﻳﻮﻣﻴﺎ ﻟﻔﺘﺮﺓ ‪ 42‬ﻳﻮﻡ‪.‬‬
‫ﺃﻇﻬﺮﺕ ﺍ ﻟﻨﺘﺎﺋﺞ ﺍﻧﺨﻔﺎﺽ ﻣﻌﻨﻮﻱ ﻓﻲ ﺍﻭﺯﺍﻥ ﺍﻟﺠﺴﻢ ﻭﻣﻌﺪﻝ ﺍﻭﺯﺍﻥ ﺍﻟﺨﺼﻰ ﻟﺬﻛﻮﺭ ﺍﻟﻔﺌﺮﺍﻥ ﺑﻌﺪ‬
‫ﺍﻟﻤﻌﺎﻣﻠﺔ ﺏﻋﻘﺎﺭ ﻫﻴﺪﺭﻭﻛﻠﻮﺍﻳﺪ ﺍﻟﺘﺎﻣﺴﻮﻟﻮﺳﻴﻦ ﻟﻠﺘﺮﻛﻴﺰﻳﻦ ﻣﻘﺎﺭﻧﻪ ﺑﻤﺠﻤﻮﻋﻪ ﺍﻟﺴﻴﻄﺮﻩ‪.‬‬
‫ﻛﻤﺎ ﺃﻇﻬﺮﺕ ﺍﻟﻤﻌﺎﻣﻠﺔ ﺑﻌﻘﺎﺭ ﺍﻟﺘﺎﻣﺴﻮﻟﻮﺳﻴﻦ ﺍﻧﺨﻔﺎﺽ ﻣﻌﻨﻮﻱ ﻓﻲ ﺍﻟﻨﺴﺒﺔ ﺍﻟﻤﺌﻮﻳﺔ ﻟﺤﺮﻛﻪ ﺍﻟﻨﻄﻒ‬
‫ﻭﺍﻟﻨﺴﺒﺔ ﺍﻟﻤﺌﻮﻳﺔ ﻟﻠﻨﻄﻒ ﺍﻟﺤﻴﺔ ﻭﺗﺮﻛﻴﺰ ﺍﻟﻨﻄﻒ ﻓﻲ ﺫﻳﻞ ﺍﻟﺒﺮﺑﺦ ﻓﻲ ﺍﻝﺟﺮﻋﻪ ‪ 16‬ﻣﺎﻳﻜﺮﻭﻏﺮﺍﻡ‪/‬ﻛﻐﻢ ﻣﻦ ﻭﺯﻥ‬
‫ﺍﻟﺠﺴﻢ ﻟﻜﻦ ﻟﻴﺲ ﻫﻨﺎﻙ ﺍﻧﺨﻔﺎﺽ ﻣﻌﻨﻮﻱ ﻓﻲ ﺍﻟﻨﺴﺒﺔ ﺍﻟﻤﺌﻮﻳﺔ ﻟﺤﺮﻛﻪ ﺍﻟﻨﻄﻒ ﻭﺗﺮﻛﻴﺰ ﺍﻟﻨﻄﻒ ﻓﻲ ﺍﻝﺟﺮﻋﻪ ‪8‬‬
‫ﻣﺎﻳﻜﺮﻭﻏﺮﺍﻡ ‪/‬ﻛﻐﻢ ﻣﻦ ﻭﺯﻥ ﺍﻟﺠﺴﻢ ‪ ،‬ﺑﻴﻨﻤﺎ ﻫﻨﺎﻙ ﺍﻧﺨﻔﺎﺽ ﻣﻌﻨﻮﻱ ﻓﻲ ﺍﻟﻨﺴﺒﺔ ﺍﻟﻤﺌﻮﻳﺔ ﻟﻠﻨﻄﻒ ﺍﻟﺤﻴﺔ ﻟﻨﻔﺲ‬
‫ﺍﻟﺠﺮﻋﺔ ﻣﻘﺎﺭﻧﻪ ﻣﻊ ﻣﺠﻤﻮﻋﻪ ﺍﻟﺴﻴﻄﺮﺓ‬
‫‪ ،‬ﻭﻫﻨﺎﻙ ﺍﺭﺗﻔﺎﻉ ﻣﻌﻨﻮﻱ ﻓﻲ ﺍﻟﻨﺴﺒﺔ ﺍﻟﻤﺌﻮﻳﺔ ﻟﻠﺘﺸﻮﻫﺎﺕ‬
‫ﺍﻟﻤﻮﺭﻓﻮﻟﻮﺟﻴﻪ ﻓﻲ ﺍﻟﻨﻄﻒ ﻟﻠﺘﺮﻛﻴﺰﻳﻦ ﻣﻘﺎﺭﻧﻪ ﺑﻤﺠﻤﻮﻋﻪ ﺍﻟﺴﻴﻄﺮﺓ‪.‬‬
‫ﺑﻴﻨﺖ ﺍﻟﻔﺤﻮﺻﺎﺕ ﺍﻟﻤﺠﻬﺮﻳﻪ ﻻﻧﺴﺠﺔ ﺍﻟﺨﺼﻰ ﻟﻠﻤﺠﺎﻣﻴﻊ ﺍﻟﻤﻌﺎﻣﻠﺔ ﺑﻬﺬﺍ ﺍﻟﻌﻘﺎﺭ ﺣﺪﻭﺙ ﺍﻧﺨﻔﺎﺽ‬
‫ﻣﻌﻨﻮﻱ ﻓﻲ ﻣﻌﺪﻝ ﺃﻗﻄﺎﺭ ﺍﻟﻨﺒﻴﺒﺎﺕ ﺍﻟﻤﻨﻮﻳﺔ ‪ ،‬ﺍﻟﺨﻼﻳﺎ ﺍﻟﻤﻮﻟﺪﺓ ﻟﻠﻨﻄﻒ ‪،‬ﻃﻼﺋﻊ ﺍﻟﻨﻄﻒ ﻭﺍﻧﺨﻔﺎﺽ ﻓﻲ ﺍﻋﺪﺍﺩ‬
‫ﺧﻼﻳﺎ ﻻﻳﺪﻙ‪ ،‬ﺑﻴﻨﻤﺎ ﺣﺼﻞ ﺍﺭﺗﻔﺎﻉ ﻣﻌﻨﻮﻱ ﻓﻲ ﺍﻟﻤﺴﺎﻓﺎﺕ ﺍﻟﺒﻴﻨﻴﺔ ﻣﻘﺎﺭﻧﻪ ﺑﻤﺠﻤﻮﻋﻪ ﺍﻟﺴﻴﻄﺮﻩ ‪.‬‬
‫ﺍﻇﻬﺮﺕ ﺍﻟﻔﺤﻮﺻﺎﺕ ﺍﻟﻤﺠﻬﺮﻳﻪ ﺃﻳﻀﺎ ﻭﺟﻮﺩ ﺑﻌﺾ ﺍﻟﺘﻐﻴﺮﺍﺕ ﻓﻲ ﻧﺴﻴﺞ ﺍﻟﺨﺼﻴﺔ ﺗﻀﻤﻨﺖ ﺗﻨﺨﺮ ﻓﻲ‬
‫ﻧﺴﻴﺞ ﺍﻟﻨﺒﻴﺒﺎﺕ ﺍﻟﻤﻨﻮﻳﻪ ﻭﺣﺪﻭﺙ ﺍﻟﻮﺫﻣﻪ ﻓﻲ ﺍﻟﻔﺴﺢ ﺍﻟﺒﻴﻨﻴﻪ ‪.‬‬
‫ﺑﻴﻨﻤﺎ ﺃﻇﻬﺮﺕ ﺍﻟﻤﻌﺎﻣﻠﺔ ﻭﻟﺠﻤﻴﻊ ﺍﻟﺘﺮﺍﻛﻴﺰ ﺍﻟﻤﺴﺘﺨﺪﻣﺔ ﺍﻧﺨﻔﺎﺽ ﻣﻌﻨﻮﻱ‬
‫ﻓﻲ ﻣﺴﺘﻮﻯ ﻫﺮﻣﻮﻥ‬
‫ﺍﻟﺘﺴﺘﺴﺘﻴﺮﻭﻥ ﻓﻲ ﺑﻼﺯﻣﺎ ﺍﻟﺪﻡ ﻟﻠﺤﻴﻮﺍﻧﺎﺕ ﺍﻟﻤﻌﺎﻣﻠﺔ ﻣﻘﺎﺭﻧﻪ ﺑﻤﺠﻤﻮﻋﻪ ﺍﻟﺴﻴﻄﺮﺓ‪.‬‬
‫ﻧﺴﺘﻨﺘﺞ ﻣﻦ ﻫﺬﻩ ﺍﻟﺪﺭﺍﺳﺔ ﺃﻥ ﻟﻌﻘﺎﺭ ﺍﻟﺘﺎﻣﺴﻮﻟﻮﺳﻴﻦ ﺗﺄﺛﻴﺮ ﺳﻠﺒﻲ ﻓﻲ ﻭﺿﻴﻔﻪ ﺍﻟﺨﺼﻴﺔ )ﺍﻟﺨﺼﻮﺑﺔ(‪.‬‬
‫ﺟﺎﻣﻌﺔ ﺑﻐﺪﺍﺩ‬
‫ﺗﺄﺛﻴﺮ ﻋﻘﺎﺭ ﺍﻟﺘﺎﻣﺴﻮﻟﻮﺳﻴﻦ )ﺍﻟﻔﻠﻮﻣﺎﻛﺲ( ﻋﻠﻰ ﺍﻟﺨﺼﻮﺑﺔ ﻓﻲ‬
‫ﺫﻛﻮﺭ ﺍﻟﻔﺌﺮﺍﻥ ﺍﻟﺒﻴﺾ‬
‫ﺭﺳﺎﻟﺔ ﻣﻘﺪﻣﺔ‬
‫ﻣﻦ ﻗﺒﻞ‬
‫ﺩﻳﻨﺎ ﺧﻀﻴﺮ ﺣﺴﻴﻦ ﻋﻠﻲ‬
‫ﺑﻜﺎﻟﻮﺭﻳﻮﺱ ‪/‬ﻋﻠﻮﻡ ﺣﻴﺎﺓ ‪ /‬ﺟﺎﻣﻌﻪ ﺑﻐﺪﺍﺩ ‪2006‬‬
‫ﺇﻟﻰ ﻛﻠﻴﺔ ﺍﻟﻌﻠﻮﻡ ﻓﻲ ﺟﺎﻣﻌﺔ ﺑﻐﺪﺍﺩ ﻭﻫﻲ ﺟﺰء ﻣﻦ ﻣﺘﻄﻠﺒﺎﺕ ﻧﻴﻞ‬
‫ﺩﺭﺟﺔ ﺍﻟﻤﺎﺟﺴﺘﻴﺮ ﻓﻲ ﻋﻠﻮﻡ ﺍﻟﺤﻴﺎﺓ ‪/‬ﻋﻠﻢ ﺍﻟﺤﻴﻮﺍﻥ‬
‫ﺑﺄﺷﺮﺍﻑ‬
‫ﺃ‪.‬ﺩ‪ .‬ﺻﺒﺎﺡ ﻧﺎﺻﺮ ﺍﻟﻌﻠﻮﭼﻲ‬
‫ﺭﺑﻴﻊ ﺍﻟﺜﺎﻧﻲ‬
‫ﺷﺒﺎﻁ‬
‫‪1433‬ﻫ‬
‫‪ 2012‬ﻡ‬
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