Study of Level of Inhibin B and Ultra structure of Sertoli Cells in Contra-Lateral Testis after Unilateral Blunt Testis Trauma in Rat
This study was designed to evaluate the ultrastructure of contra lateral testis tissue and measurement of Serum inhibin B following unilateral blunt testis trauma. Twenty pre-pubertal male wistar albino rats aged 3 weeks were divided into 4 equal groups that each containing five rats. Group I was the control group. Group II was used as a Sham group. Group III had right orchiectomy initially. Group IV was the trauma group in which the right testis was placed on a firm sterile surface and the metal rod weighting 100 g was drooped on to the testis from a height of 5.5 cm. Seven weeks after initial operation 3 mL blood samples were obtained from each rat to determine inhibin B levels and contra lateral orchiectomies were performed in all groups to microscopically investigate electron. Inhibin B levels decreased in groups 3 and 4. The difference between group 3 with groups 1 and 2 was significant (p = 0.003 and 0.02). Also the difference between group 4 with groups 1 and 2 was significant (p = 0.006 and 0.002) but the difference between group 3 and 4 was not significant (p = 0.08). In group III (orchiectomy) TEM showed a normal sperm morphology and normal disruption of different stages of the spermatogonial maturation. Debris and vacuolar changes were seen in sertoli cells. Morphology of leydig cells slightly modified and the dilated cisternae of the Smooth Endoplasmic Reticulum (SER) were observed in group IV( trauma) mitochondria with degenerated cristae and enlarged vacuole were observed.
Received: March 15, 2010;
Accepted: May 04, 2010;
Published: August 18, 2010
Testicular trauma is defined as any injuries sustained by the testicle. Types
of injuries include blunt, penetrating or degloving. Blunt Trauma refers to
injuries sustained from objects applied with any significant force to the scrotum
and testicle. This can occur with various types of activity. Blunt trauma accounts
for approximately 85% of cases (Saleh et al., 2009).
The most common cause of blunt testicular trauma is sport injuries. Most blunt
testicular injuries are unilateral and isolated (i.e., without other associated
injuries) (Luchey et al., 2009). Unilateral testicular
injury is always considered with contralateral testicular damages. Any kind
of testicular injury may result in unilateral and contralateral testicular atrophy
which will impair fertility (Ozkan et al., 2003).
There is evidence that unilateral blunt testicular trauma affects contralateral
testicular germ cell maturation through an autoimmune response (Shaul
et al., 1997). The testicular maturation is not complete until puberty
and any injury to the pre-pubertal testis is likely to affect the spermatogenesis
maturation in later periods (Srinivas et al., 1999a).
Inhibin B is the relevant circulating inhibin form in the human male. Inhibin
B is a dimmer of α- and βB-subunits. It is produced exclusively
by the testis. The Sertoli cell is considered the predominant source of inhibin
B (Srinivas et al., 2003; Meachem
et al., 2001). This notion is currently challenged and confounded
by a mounting number of sometimes contradictory studies, based on immunolocalisation
of inhibin subunits and on the in vitro and/or in vivo production
of inhibin B in various experimental settings. These studies indicate that germ
cells and possibly even Leydig cells would produce inhibin as well (Grootenhuis
et al., 1990; Handelsman et al., 1990).
The human male inhibin subunits seem to be differentially localized and secreted
depending on age and cell type. For example, in the fetal testis and bB subunits,
but not bA were immunolocalised in the sertoli and leydig cells (Risbridger
et al., 1998). Ozkan et al. (2003)
showed that Unilateral Testis Trauma (UTT) has an adverse on the contralateral
testis and decreased levels of inhibin B following unilateral testis trauma
may reflect this contralateral testis damage. This may be consequence of sertoli
cell degeneration or less in the contralateral testis because the serum inhibin
B level in orchioctomy group was normal. Srinivas showed that grade I unilateral
blunt testicular trauma in prepubertal rats significantly affected germ cell
maturation in both ipsilateral and contralateral testis and altered the sex
hormone profile (Andersson et al., 1998; Majdic
et al., 1997). This study was designed to evaluate the ultrastructure
of contra lateral testis tissue following unilateral testis trauma and measurement
of Serum inhibin B.
MATERIALS AND METHODS
This experimental study was performed in the Physiology Research Center of Ahvaz Jundishapur University of Medical Sciences (AJUMS) from March 2008 to August 2009. Twenty male wistar rats aged 3 weeks were divided into 4 equal groups each containing 5 rats. All the rats were provided with equal amount of commercial pellet feed once a day at a scheduled time and water was made available and libitum. They were housed in cages under standard elaborating conditions with 12 h light and dark cycles. Rats were anesthetized by intraperitoneal ketamine (1 mg 100 g-1 b.wt.). All instruments used for the procedure were sterilized. Abdomens of group II (sham) were sham operated without disturbing either testis. In group III right orchiectomy performed initially. In group IV a transverse lower abdominal incision was made and the right testis was placed on a sterile firm surface and 100 g sterile weight was dropped on to the testis from a height of 5.5 cm. The tunica albuginea was open thereby exposing seminiferous tubules and the tunica vaginalis was then closed with fine absorbable suture. The abdominal incision was closed in layers with polyglactin suture and the wound was cleaned with povidone-iodine. Seven weeks after initial operation 3 mL blood samples were obtained from each rat to determine inhibin B levels and contra lateral orchiectomies were performed in all groups to microscopically investigate electron.
Electron microscopy: For electron microscopy, the testicular specimens were fixed with 2.5% glutaraldehyde in 0.1 M sodium buffer phosphate (pH 7.2) for 3 h at 4°C, washed in the same buffer for 1 h at 4°C and post-fixed with 1% osmium tetroxide in sodium phosphate buffer for 1 h at 4°C. The tissues were then dehydrated in graded series of ethanol, starting at 50% each step for 10 min, after two changes in propylene oxide. The tissue ecimens were embedded in araldite. Ultrathin sections were prepared with Mg-uranyl acetate and lead citrate for the electron microscopic evaluation (Philips CM 10 TEM).
Measurement of inhibin B levels: Serum inhibin B was measured using specific two-site ELISAs.
Statistical analysis: Level of serum inhibin B was reported as the Means±SD. The statistical significance of difference between the control and experimental groups was determined by the unpaired t-test. Differences between the means were considered to be significant when p<0.05 was achieved.
Electron microscopic findings: The EM sections were studied under the
following categories: (1) Seminiferous tubules: Basement Membrane (BM); spermatogenesis
series: various stage of maturation and sperm morphology and sertoli cells:
nuclear membrane and organelles. (2) Extra Cellular Matrix (ECM) and leydig
cells. Features of degeneration were fragmentation of the cell membrane, disorganization
of organelles. Disruptions of the Endoplasmic Reticulum (ER) and rarefaction
of mitochondria. In group I (control) and group II( sham), TEM showed a normal
BM, normal sperm morphology and normal disruption of different stages of the
spermatogonial maturation, normal leydig cells, sertoli cells, normal ECM and
||(a) Electron microscopy of testicular tissue in orchiectomy
group. vacuolar changes → of interstitial tissue and slightly morphological
changes ↔ of leydig cells were observed, (b) electron microscopy of
testicular tissue in trauma group. Nuclear membrane degeneration →
of primary and secondary spermatocytes was observed in this group. Dens
apoptotic body ↔ increase in cells, (c) electron microscopy of testicular
tissue in trauma group. Morphological changes of interstitial cells specially
leydig cells → and vacuolar changes of interstitial tissue ↔ and
(d) Electron microscopy of testicular tissue in trauma group. Mitochondria
with degenerated cristae ↔ and enlarged vacuole → were observed
|| Inhibin B level in all groups
In group III (orchiectomy) TEM showed a normal sperm morphology and normal
disruption of different stages of the spermatogonial maturation. Debris and
vacuolar changes were seen in sertoli cells. Morphology of leydig cells slightly
modified and the dilated cisternae of the Smooth Endoplasmic Reticulum (SER)
were observed (Fig. 1a). In group IV (trauma) mitochondria
with degenerated cristae and enlarged vacuole were observed. Markedly morphological
changes of interstitial cells specially leydig cells and nuclear membrane degeneration
of primary and secondary spermatocytes were observed in this group. Dens apoptotic
body increase in cells (Fig. 1b-d).
Inhibin B levels: The level of inhibin B was 410.3±15.9, 435.5±20.3, 290.5±28.5, 270.8±30.8 pg mL-1 in control (I), sham (II), orchiectomy (III) and trauma (IV) groups, respectively (Table 1). Statistical analysis show that the difference between group 3 with groups I and II was significant (p = 0.003 and 0.02). Also, the difference between group IV with groups I and II was significant (p = 0.006 and 0.002) but the difference between group III and IV was not significant (p = 0.08).
In present study we observed that the level of inhibin B in two experimental
groups (groups III and IV) significantly decreased in compared to others two
groups. This can consequence of sertoli and leydig cells degeneration in contralateral
testis. Because in these groups TEM showed vacuolar changes in ECM, morphological
changes of leydig cells, dilated cisternae of SER, mitochondria with degenerated
cristae in sertoli cells and degeneration of spermatocyte cells. Earliely, Ozkan
et al. (2003) declared that in peripubertal rats the serum inhibin
B levels in trauma groups in agreement with our result decreased but in contrast
with present study the serum inhibin B level of the orchiectomy group was not
different from the control group. Ozkan et al. (2003)
said that the inhibin B levels of orchiectomy group might have been decreased
but after a 6 week period the remaining testis might have improved its function
by compensating for impaired fertility (II). Present results reinforced the
Ozkan et al. (2003) study. In other study that
performed by Shaul et al. (1997) in postpubertal
rats, the group undergoing orchiectomy following UTT had a similarly high fertility
rate compared to the control group (III). This finding is confirm by this results.
Wolfe et al. (1985) studied the serum quality
of contralateral testicle of bulls after unilateral orchiectomy. They observed
a decreased percentage of normal spermatozoa only on postoperative day 6 but
at the end of the 8 weeks there was no difference in sperm mobility scores and
percentage of normal spermatozoa (Wolfe et al., 1985).
Srinivas et al. (1999b) reported that UBTT affects
the contralateral testis and fertility. Anti Sperm Antibody (ASA) mediate this
damage and orchiectomy performed around 6 h after trauma or short-term cyclosporine
therapy prevents the damage. Experimental models were chosen to study the effect
of UBTT, in which reduced fertility (Slavis et al.,
1990; Srinivas et al., 1999b), antibody
mediated (Srinivas et al., 1999a) and cell-mediated
(Sakamoto et al., 1995, 1998;
Sharma et al., 1999) immune response have been
demonstrated. In these studies, the technique of inflicting injury and documentation
of the injury was not standardized. In this study serum inhibin B levels in
orchiectomy and trauma groups were less than control groups. The difference
between orchiectomy and trauma group was not significant. Also, the electron
microscopic findings in these two group almost similar. Present results indicated
that the injury in prepubertal period was sensitively and repair might need
a long period. Additionally the grade of injury is importance. In the present
study, the ultrastructure details of the contralateral testis were studied using
Transmission Electron Microscopy (TEM). According to our best knowledge out
report represents the first study designed to evaluate the ultrastructure of
contra lateral testis tissue following unilateral testis trauma. In agreement
with our present study, earlier, Nambirajan et al.
(2002) reported the ultrastructure changes observed in the undescended testis
(UDT) were also reflected in the Contralateral Descended Testis (CDT). These
changes in the CDT are significant. In this study electron photomicrographies
of contralateral descended testis showed a irregularly thickened basement membrane,
vacuolation, focal degeneration of spermatocyte and dens apoptotic body. Also,
the size of ER and number of sertoli cells increased (Nambirajan
et al., 2002). Dokmeci et al. (2007)
considered the protective effects of ibuprofen on testicular torsion/detorsion-induced
Ischemia/Reperfusion (I/R) injury in rats. After I/R, slightly dilated cisternae
of SER and markedly swollen mitochondria with degenerated cristae were observed
in sertoli cells in the ipsilateral testis of the late orchiectomy model. After
I/R markedly degenerated cristae of mitochondria and slightly dilated cisternae
of SER were observed in the spermatid cells contralateral testes of the late
Mogilner et al. (2006) concluded that testicular
ischemia in rats led to histological damage in the ipsilateral testis. In the
contralateral testis , minimal damage was observed and testicular ischemia additionary
caused an increase in germ cells apoptosis in the contralateral testis. In present
study on prepubertal rats, histological changes of contralateral testis following
unilateral testis trauma were observed in orchiectomy and trauma groups. Some
changes were similar to histological changes in torsion and undescended testis.
In group III (orchiectomy) TEM showed a normal sperm morphology and normal disruption
of different stages of the spermatogonial maturation. Debris and vacuolar changes
were seen in sertoli cells. Morphology of leydig cells slightly modified and
the dilated cisternae of the Smooth Endoplasmic Reticulum (SER) were observed
in sertoli cells. In group IV (trauma) mitochondria with degenerated cristae
and enlarged vacuole were observed. Markedly morphological changes of interstitial
cells specially leydig cells and nuclear membrane degeneration of primary and
secondary spermatocytes were observed in this group. Dens apoptotic body increase
in cells. These changes could probably be responsible for the decrease in serum
inhibin B levels and decrease in fertility seen in previous studies.
Recent study indicated that UTT affected the contralateral testis and serum inhibin B levels reflect sertoli cell function and spermatogenetic activity.
This project (Grant No. PRC53) was financially supported by the Research Deputy of Ahvaz Jundishapur University of Medical Sciences (AJUMS). Authors would like to express their great appreciation for their support.
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