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Immunohistochemical
observation on luteinizing hormone in rat testes before and after testicular
capsulotomy
Da-Nian
QIN1, Mary A.
Lung2
1Department
of Physiology, Shantou University School of Medicine, Shantou 515031,
China
2Department of Physiology, University of Hong Kong, HKSAR,
China
Asian J Androl 2001 Sep; 3: 227-230
Keywords:
testis;
testicular capsulotomy; luteinizing hormone; immunohistochemistry
Abstract
Aim: In the testicular capsulotomized rats, although there was a significant increase in the luteinizing hormone (LH) levels, the secretion of testosterone remained low. In order to clarify the mechanisms of this phenomenon, the binding of endogenous LH to the testes were observed before and after testicular capsulotomy. Methods: Peroxidase-anti-peroxidase (PAP) method was used to detect the binding of LH to the testes in rats. Results: An intense positive staining of LH was found in the Leydig cells of both the normal and sham-operated control testes. However, at 40 d after operation, the LH immunoreactivity was decreased in the Leydig cells of the capsulotomized testis. By d 60, only very weak positive staining could be observed in these cells. Conclusion: A progressive reduction of endogenous LH binding to the testis occurred in the capsulotomized rat.
1
Introduction
It
was found in our previous studies that testicular capsulotomy induced a progressive
degeneration of seminiferous tubules in adult male rats with a progressive
reduction in the testosterone concentration in the testicular venous blood
and a gradual increase in the LH and follicular stimulating hormone (FSH)
levels
in the peripheral blood from approximately 30 d post-capsulotomy. At that
time,
the fertility of capsulotomizd rats was gradually depressed[1-4]. It
was traditionally
believed that level of testosterone was principally controlled by the
amount of LH released from the pituitary gland. However, in the testicular
capsulotomized
rats, although there was a significant increase in LH levels, the secretion
of testosterone remained low[3]. The resent study was designed to
clarify the mechanism of this phenomenon. 2 Materials and methods
2.1 Experimental animals and testicular capsulotomy
Mature (60 d of age) Sprague-Dawley male rats were obtained from the Laboratory Animal Unit of The University of Hong Kong and divided at random into the control, the sham-operated and the capsulotomized groups of 6 rats each. Surgical intervention was performed in the Minimal Disease Operation Theatre of the University. The animals were anaesthetized intraperitoneally with sodium pentobarbitone (Sigma, USA) at a priming dose of 60 mgkg-1and a maintenance dose of 10 mgkg-1h-1. Testicular capsulotomy was carried out as previously described[1]. Briefly, with the aid of a dissecting microscope (Wild M60, Switzerland), the two outer layers of the capsule, i.e., the tunica vaginalis and the tunica albuginea, were carefully incised starting half-way down the rostral half of the testis a long the two lateral borders down to the middle of the caudal half of the testis.2.2 PAP technique for staining LH
The
LH in the testicular sections were localized according to the method of
Adams and Brumlow[5]. Sections of the testis were deparaffinized
with xylene, and washed in Lugol's solution and sodium thiosulphate for
2 min each. The sections were hydrated in a series of ethanol, rinsed
in phosphate buffered saline (PBS)
at pH 7.4, incubated for 30 min in 0.3% hydrogen peroxide (Sigma, USA)
in methanol in order to destroy the endogenous peroxidase activity, rinsed
for 5 min in each of the three changes of PBS, and incubated for 30 min
in 4% normal goat serum (NGS, Sigma, USA) in PBS to block the nonspecific
binding of LH antiserum to the tissue. The NGS was shaken off from the
slides and the sections were incubated
for 24 h in ovine LH (oLH) antiserum (1:100 dilution, Amersham, UK) in
a moist chamber, rinsed for 5 min in each of the three changes of PBS,
incubated for 30 min with the link antibody (goat anti-rabbit IgG,
5 ug/mL; Sigma, USA) in a
moist chamber, rinsed for 5 min in each of the three changes of PBS, incubated
with peroxidase-anti-peroxidase (PAP, 180
ug/mL; Sigma, USA), rinsed for 5 min in
each of the three changes of PBS and stained for 10 min in freshly prepared
diaminobenzidine (DAB) solution at 2
in a shaking apparatus. The sections were then
rinsed for 5 min in each of the two changes of deionized water, dehydrated in
a series of ethanols, cleared in xylene and mounted with coverslips.
The following controls were used: 1) examination of Leydig cells in testicular sections of rats which were either normal or sham-operated for the presence of endogenous LH according to the methods described above, 2) omission of primary antiserum .
The DAB solution was prepared by dissolving 25 mg 3,3-diaminobenzidine (Sigma, USA) in 200 mL 0.05 mol/L Tris-HCL buffer. The solution was then filtered and 2 mL 0.3% freshly prepared hydrogen peroxide was added. This gave a final DAB concent ration of 0.0125% and H2O2 concentration of 0.003%.
3
Results
Figure
1. Light micrograohs of testis from a normal rat at the age of 60
d. (A) Positive LH immunoreactivity in the Leydig cells (small arrows).
(B) Negative LH immunoreactivity
in the control (omit primary antiserum) (big arrows).
PAP method, 250.
Figure 2. Light
micrographs of testis at d 5 post-operation. (A) Positive LH immunoreactivity
in the Leydig cells of a sham-operated rat (small arrows). (B) Positive
LH immunoreactivity in the Leydig cells of a capsulotomized rat (big arrows).
PAP method, 630.
Figure 3. Light
micrographs of the testis
(A) LH immunoreactivity was weak in the Leydig cells (small arrows)
at 40 d post-capsulotomy. (B) LH immunoreactivity was
very weak in the Leydig cells (big arrows) at 60 d postcapsulotomy.
PAP method, 630.
4
Discussion
The
positive staining reaction of the Leydig cells by the PAP technique proved
that the procedure in use was sensitive enough to demonstrate endogenous
LH binding in the testis. An intense positive staining of endogenous LH
was found in the Leydig cells of both normal and sham-operated control
testes. After capsulotomy, no obvious change
in LH immunoreactivity was noted in the Leydig cells at 5 d post-operation.
However, the positive staining reaction in the Leydig cells became weakened
at 40 d after capsulotomy and at 60 d, the positive staining reaction
was further decreased. The results suggested that there was a progressive
reduction in the LH
binding to the testis in the capsulotomized rat. What caused this reduction
in the capsulotomized testis?
In
recent years, there is accumulating evidences indicating that the action
of LH on the Leydig
cells is dependent on the local environment, which is created through
interactions between the seminiferous tubules and the interstitial cells;
paracrine factors are believed to be the major means of communication
involved[6]. The paracrine control of Leydig cells by factors
released from the seminiferous tubules has been the subject of numerous
investigations[7]. It was important to
note that a loss of LH binding in Leydig cells was found not only in bilaterally
cryptorchid rats[8,9], but also in unilateral cryptorchid rats[10],
indicating that local factor(s) was also responsible for the loss of LH
binding. Since a progressive degeneration of seminiferous tubules was
induced by testicular capsulotomy, the reduction in LH binding in the
capsulotomized testis might be related to an upset of the local paracrine
control induced by degeneration of the seminiferous tubules.
The progressive reduction of endogenous LH binding to the testis occurred in the capsulotomized rat may explain why the secretion of testosterone remained at low levels, even though there was a notable increase of LH levels after testicular capsulotomy.
Acknowledgements
We
wish to thank Mr. K.K Tsang (The University of Hong Kong) for his skillful
technical assistance.
References
[1]
Qin DN, Lung MA. Studies on the relationship between testicular capsule
and sperm transport in the rat testis. Asian J Androl 2000; 2: 191-8.
[2] Qin DN, Lung MA. Effect of testicular capsulotomy on lipid droplets
in the seminiferous tubules of rats. Asian J Androl 2001; 3: 121-4.
[3] Qin DN, Lung MA. Effect of testicular capsulotomy on secretion of
testosterone and gonadotrophins
in rats. Asian J Androl 2000; 2:
257-61.
[4] Qin DN, Lung MA. Effect of testicular capsulotomy on fertility of
rats. Asian J Androl 2001; 21-5.
[5] Adams CS, Brumlow WB. Immunocytochemical detection of luteinizing
hormone in epididymis of mature mouse. Histochemistry 1989; 91: 485-94.
[6] Saez JM, PerrardSapari MH, Chatelain PG, Tabone E, Rivarola MA.
Paracrine regulation of testicular function. J Steroid Biochem
1987; 27: 317-29.
[7] Bergh H. Local differences in Leydig cell morphology in the adult
rat testis: evidence for a local control of Leydig cell by adjacent seminiferous
tubules. Int J Androl 1982; 52:
325-30.
[8] de Krester DM, Sharp RM, Swanston IA. Alterations in steroidogenesis
and human chorionic
gonadotropin binding in the cryptorchid rat testis. Endocrinolgy
1979; 105: 135-8.
[9] Schanbacher BD. Androgen secretion and characteristics of testicular
hCG binding in cryptorchid
rats. J Reprod Fertil 1980; 59: 145-50.
[10] Risbridger GP, Kerr JB, de Krester DM. An assessment of Leydig cell
function after bilateral or unilateral efferent duct ligation:
further evidence for local control
of Leydig cell function. Endocrinology 1981; 109: 1234-41.
[11] Hall PF. Testicular steroid synthesis:
organization and regulation.
In: Knobil
E, Neill JD. editors. The physiology of reproduction. New York:
Raven Press;1988.
p 975-98.
Correspondence
to: Dr.
Da-Nian QIN, Shantou University School of Medicine, Shantou
515031, China.
Tel:
+86-754-856 6776 Fax: +86-754-855 7562
E-mail: lqchen@mailserv.stu.edu.cn
Received 2001-08-27 Accepted
2001-09-10
