a) Title Expression of receptors c-kit and androgens in the histological and functional organization in human testicle b) Authors 1 Rodríguez H, Sarabia L, Tamayo C, Sepúlveda M, Inostroza J, 2 Espinoza-Navarro O, 3 Araya JC, 4 Moriguchi K. c) Affiliation 1 Laboratory of Histoembriology. Faculty of Medicine. University of Chile. Santiago-Chile. 2 University of Tarapacá, Arica- Chile. 3 Laboratory of Histopathology Histomed. Viña del Mar- Chile. 4 Department of Anatomy. School of Dentistry. Aichi- Gakuin University. Nagoya-Japan. d) Author for reprint Prof. Dr. Omar Espinoza-Navarro. Laboratory of Biology of Reproduction and Development. University of Tarapacá. Casilla 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 1
37
Embed
Malatión SUMMARY is a ... - Universidad de Chiledocencia.med.uchile.cl/morfologia/histologia/paperAJA2007 OMA… · Web viewKey Word: Human reproduction, testicle, interstitial
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
a) Title
Expression of receptors c-kit and androgens in the histological and functional organization in
human testicle
b) Authors
1Rodríguez H, Sarabia L, Tamayo C, Sepúlveda M, Inostroza J, 2Espinoza-Navarro O, 3Araya
JC, 4Moriguchi K.
c) Affiliation
1Laboratory of Histoembriology. Faculty of Medicine. University of Chile. Santiago-Chile.
2University of Tarapacá, Arica-Chile. 3Laboratory of Histopathology Histomed. Viña del Mar-
Chile. 4Department of Anatomy. School of Dentistry. Aichi-Gakuin University. Nagoya-Japan.
d) Author for reprint
Prof. Dr. Omar Espinoza-Navarro. Laboratory of Biology of Reproduction and Development.
University of Tarapacá. Casilla 7/D, Velásquez 1775, Arica-Chile. Telephone: 56 58 205415,
During the embryonic and fetal development of the testicle, a series of changes occurs in both
cellular distribution and histological organization that lasts until it reaches sexual maturity. This
developmental process ensures the optimal organization to produce masculine gametes and male
hormones [1].
Somatic cells of the testicle are Leydig cells, peritubular myoids cells and Sertoli cells. In these
cells the presence of androgen receptors (AR) has been demonstrated, with variable
immunohistochemical positivity according to the age and state of the cycle of the
spermatogenesis.
The androgens mediate a wide range of physiological responses and are especially important in
male sexual maturation, the maintenance of spermatogenesis, and male gonadotropin regulation
[2].
The effects of androgens are mediated through the androgen receptor (AR), a 110-kDa ligand-
inducible nuclear receptor that regulates the expression of target genes through binding to an
androgen response element. Mutations of the AR may result in male infertility or complete or
partial androgen insensitivity [3].
From the beginning the processes of cellular migration, proliferation and apoptosis, where the
cells expresses a series of proteins with receptor function, are the main biological mechanism
driving testicle organization.
4
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
4
There are many kinds of AR immunoreactive cells in normal rat testis, such as Leydig cells,
endothelial cells, myoid cells, Sertoli cells, as well as spermatogenic cells [4]. In the human
testis, AR immunoexpression was observed in Sertoli cells, peritubular myoid cells, Leydig cells,
and periarteriolar cells, but not in germinal cells. There is no correlation with the intensity of AR
immunoexpression in either Sertoli cells or peritubular myoid cells in regard to spermatogenic
cells. Perhaps an inappropriate expression of the AR is a cause or a consequence of idiopathic
infertility in the human patients [5].
The membrane receptor c-kit, a tyrosine quinase protein type III, directly regulates the
proliferation and apoptosis of stem cells and it is involved in cell migration [6]. In the
embryofetal and immature testicle, the receptor would be expressed by the somatic cells (Sertoli
and Leydig) and germinal cells (spermatogonia types A, Intermediate and B). Some authors even
describe an incomplete c-kit protein in some meiotics stages. Nevertheless, in the adult man
there would take place a reduction in the number and type of cells that express the protein c-kit
(of unknown function), which probably is an important factor in the regulation of the endocrine
and gametogenic functions of the adult testicle. Therefore, the expression of the c-kit receptors
may perhaps constitute one of the regulating factors of the spermatogenesis, by means of
modulating proliferation and apoptosis in the seminiferous epithelium, as well as regulation of
tubular motility.
In the peritubular compartment, the myoid cells are androgen dependent, surround the
seminiferous tubules and secrete basal lamina components [7]. These myoid peritubular cells
with contractile properties could be regulating the motility of the seminiferous tubules.
5
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
5
Therefore the contractility of the seminiferous tubules and the displacement of the intratubular
fluid could be regulated through testosterone an specific receptor, and by the activity of the
positive c-kit cells.
In the present work, in the interstitium of the adult human testicle we describe positive c-kit cells
associated to the peritubular contractile cells, probably interrelated and regulated by testosterone.
6
96
97
98
99
100
101
102
6
Materials and methods
Human testicular tissue
Testicular tissue was obtained from prostate cancer patients (n= 10, between 60 to 65 years old),
under procedures of uni or bilateral testicular surgery (orchiectomy) corresponding to an
androgen ablation therapy. The patients are asked to authorize the use of the tissues, after
explaining the aims of the work.
The work fulfills the requirements of the Bioethical Committee, for handling human tissues,
according to the Clinical Hospital Jose Joaquin Aguirre, of the University of Chile.
Fixing of testicular tissue was in 40 g/L buffered formaldehyde pH 7.4 in PBS (phosphate buffer
saline).
All staining procedures for light microscopy were performed on 4 m paraffin-embedded
sections. The sections were deparaffinized in xylene and re-hydrated from graded alcohol to
distilled water. Routine histological examinations were made from testicular sections stained with
hematoxylin-eosin (or Papanicolaou stain).
For detection of c-kit a rabbit antihuman antibody and horseradish peroxidase enzyme-labeled
polymer conjugated to polyclonal rabbit secondary antibodies (LSAB-2; DAKO Corp.) was
utilized. Similar protocols were done with antibodies against specific Actin of smooth muscle and
neurofilaments (NeoMakers ref RB 9010-R7) and the androgen receptor (LabVision CO. Ref RB
9030 - PO), revealed with UltraVision Plus Detection System, Anti polivalente, HRP. For
7
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
7
immunodetection of c-kit (CD117) and AR, antigenic retrieval was made by the microwave-
citrate buffer method. All slides were incubated with the primary antibody for 10 minutes at R.T.,
then incubated with the secondary antibody, a biotinylated goat anti-rabbit for another 10 minutes
at R.T., and finally with Peroxidase-Streptavidin conjugated and with AEC
(Aminoethylcarbazole) or DAB (Diaminobenzydine) as chromogen (DAB) to develop the color
reaction. Endogenous peroxidase was inactivated by 3 % H2O2 for 5 minutes (30 % perhydrol
p.a., Merck). Negative controls were performed by either blocking with appropriate non-immune
serum or by omitting the primary antibody from the protocol. Histological interpretations were
directly made from stained slides as well as from digitalized images.
The results were registered in digitalized images (digital camera Nikon® Coolpix 4500, 4
Megapixeles of resolution), and the quantification was made in a Nikon Microscope Eclipse. For
the c-kit cells, AR, and smooth muscle specific actin, the number of immunopositives cells by
seminiferous tubule was registered, considering a total of 1,200 tubules.
Results
Testicular histology usually considers an organization in compartments: tubular, peritubular and
intertubular or interstitial. The cellularity in each one of them is different, and the functions are
also different, with an intratesticular paracrine regulation, which assures the functional
interdependence between the compartments.
8
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
8
In tables 1, graphics 1 and 2, the quantitative results of the technique of immunohistochemistry of
the testicular cells that express the protein c-kit, the androgen receptor and specific actin of
smooth muscular are shown.
In table 1, the quantification of the c-kit positive cells is observed, in the adult human testicle,
assumed to be pacemarker cells, though scarce in number. They are found in association to
structures that present contractile properties such as arterioles and, in greater amount, around the
seminiferous tubules.
In graphic 1, in the different compartments of the adult human testicle there are different cellular
populations that express the androgen receptor protein. Also the contractile peritubular cells
express in a high percentage the androgen receptor.
In graphic 2, in the peritubule the cells express actin, protein specific of the smooth muscle,
which allows to confirm that these cells are of muscular character and that they could be
participating in the regulation of the motility of the seminiferous tubules in association with the c-
kit positive cells.
Figures 1 to the 5 show the cellularity of the different compartments of the testicle, and the
identification of specific cellular types according to the primary antibodies used in the
immunohistochemical techniques.
9
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
9
In figure 1, the testicular histology with Papanicolau stain is observed. The staining of the nuclei
of the cells of Sertoli and the line of the spermatogenesis of the tubular compartment is
outstanding. The peritubular cells are observed of blue color with extended and basophilic nuclei.
Figure 2, shows the cells of the peritubular compartment using the technique of
immunohistochemestry with specific antibodies for smooth muscular actin. The revealed with
AEC allows to observe the positive cells with intense red colour.
Figure 3, shows positive nervous fibers for specific neurofilaments of the nervous system, which
are observed of red color (revealed with AEC) and located towards the vascular tunic of the
testicle.
Figure 4, shows the cellular populations of the testicle that express the androgen receptor. Cells
of Sertoli are observed positive presence of androgen receptor, whereas the germinal line is
negative. In the peritubular compartment the positive cells are arranged circularly and in several
layers. In the interstitial compartment the positive cells are distributed at random.
Figure 5 shows the specific immunohistochemical reaction to identify c-kit cells in cross sections
of seminiferous tubules. Interstitial cells of Cajal (ICC), located in the interstitium, exhibit a
strong immune reaction to the c-kit (*) in their cytoplasm and distributed in the peri-nuclear area.
The nucleus of ICC is clear and is surrounded by a clear reddish halo. The ICC are distributed
close to the periphery of the seminiferous tubules, in contact with the peritubular cells and
separated from the groups of Leydig cells. It must be stated that the tubular and peritubular areas
are negative to the c-kit reaction.
10
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
10
Discussion
This is the first report that describes ICC (c-kit positive cells), in the interstitial compartment of
the testicle (table 1, figure 5) and their relationship with the other cells of the testicle, that
altogether could be regulating the motility of the seminiferous tubules and spermatogenesis.
The seminiferous tubules in human are constituted by the seminiferous epithelium, a complex
stratified epithelial tissue formed by Sertoli cells and spermatogenic cells. The spermatogenesis
includes all the changes that experience the germinal cells: mitosis, meiosis, and cellular
differentiation [8].
In the seminiferous tubules different cellular associations are identified and represent different
stages from differentiation of the germinal cells (figure 1). Therefore, in the cross sections of the
testicle the adjacent seminiferous tubules display different cellular aspects. This complex cells
associations may well be regulated by c-kit cells, as it has been found in the intestinal tract. In
this case, motility, secretion and cell proliferation are under c-kit regulation. This may also be the
case for mitotic activity, which is present only in some stages of the spermatogenic cycle and it
could be controlled by para and autocrine signals, specific for each stage.
In the majority of mammals, the Leydig cells are distributed in the intertubular spaces and are
similar in both ultrastructure and hormonal activity, showing characteristic patterns of growth and
development during the fetal and puberty phases, and also secrete variable amounts of
testosterone.
11
188
189
190
191
192
193
194
195
196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
11
At this time the new molecular concepts revolutionize the medical and clinical practice, with key
molecular findings in the pathogenesis and therapeutics of some diseases. A clear example of it
corresponds to the cells that display the c-kit membrane receptors. The c-kit cells are originated
from mesenchymal "stem" cells that give rise to so called ICC [9], also known as pacemaker in
the gastrointestinal tract, where they regulate the tubular motility [10].
In the gastrointestinal tract the ICC are located near the mesenteric plexus or distributed
separately in the interstice, displaying multiple cytoplasm processes, an developed endoplasmic
reticulum, mitochondria, Golgi apparatus, caveolaes and intermediate filaments [11]. This is in
agreement with our findings, where the c-kit cells are distributed in the interstitial compartment
(figure 5), near the peritubule (muscle cells, figure 2), of the adult human testicle. It is possible
that the cytoplasm processes of the ICC may allow the communication between the cells of the
tubular compartments and the peritubular cells with the interstitial cells of Leydig.
In mesenchymatic tumors the mutations of c-kit cells take place with high frequency, which
implies a constitutive activation of c-kit in absence of the natural stem cell factor (SCF). About a
70% of gastrointestinal mesenchimatic tumors corresponds to mutations in c-kit. These tumors
respond very well to therapy with c-kit inhibitors drugs [12]. Some of these specific drugs could
be useful for future studies of the function of c-kit in the testicle, although the exact relation of
expression of the receptor of androgen and c-kit in the human testicle is not yet clear.
The detection of c-kit, also known as membrane antigen CD117, in some cases altogether
12
212
213
214
215
216
217
218
219
220
221
222
223
224
225
226
227
228
229
230
231
232
233
234
12
with histological criteria defines the diagnosis of specific pathologies, as the gastrointestinal
stromal tumors.
Originally the c-kit cell was described in immature hematopoyetic cells. Nevertheless, at the
moment they have been described in several cellular types during the embryonic development
and the adult life [13], including the germinal cells and of some specific stroma where they are
known as ICC.
In mammalian embryos the cell lines of male germinal cells evolve to be the gonocytes of
seminiferous cords, precursors of spermatogonias. The development of the normal testicle
depends on the proliferation of primordial germinal cells and their aggregation with the Sertoli
cells precursors. All these events are associated to the expression of proto-oncogene c-kit that
codifies the protein kinase III [14].
Albanesi et al. [15], describe that type A spermatogonia express the c-kit receptor in the testicle
after birth, and that its transcription stops during meiosis, where the proto-oncogene would be
active for a very short periods at the end of spermatogenesis.
C-kit plays an important role in tyrosine phosphorylation of the protein substrate, with the
subsequent intracellular signaling cascade activation that controls cellular proliferation,
apoptosis, migration and differentiation.
The c-kit protein has been well described in a variety of normal human cells, like mastocytes,
melanocytes, and germinal cells. In contrast, less information has appeared for c-kit expression in
13
235
236
237
238
239
240
241
242
243
244
245
246
247
248
249
250
251
252
253
254
255
256
257
258
13
cells of the adult testicle [16]. The deregulation of c-kit has been involved in the genesis of a
variety of tumoral pathologies; its role in the pathogenesis of tumors is still not clear.
In the present work the c-kit cells are present only in the interstitial compartment of the adult
human testicle. Hence they could be regulating the motility of the seminiferous tubules through
the peritubular contractile cells (graphic 2, figure 2). Moreover, this relation with the testicular
tubular motility could be regulated and mediated by testosterone through the activation of the
androgen receptor on the cells of the peritubular compartment (graphic 1, figure 4).
According to the observed results, the innervation of the human testicle is represented by the
presence of nervous fiber that reaches the vascular tunic of the testicle, without entering the
parenchyme (figure 3). According to this, the presence of the c-kit cells in the testicle and their
distribution in the vicinities of contractile cells of the peritubule, their would represent the
formation of true testicular pacemarkers for the regulation of the motility of the seminiferous
tubules and presumably the fertility of the adult man.
All these facts would allow to postulate that the c-kit (+) cells in the adult human testicle could
exert a function of local pacemarkers, similar to the intestine. C-kit cells and its function would
be androgen dependent.
Finally, it is important to highlight that during spermatogenesis a delicate endo, para and
autocrine regulation exists that supports events of cellular division and differentiation such as
mitosis, meiosis and production of potentially fertile spermatozoa. Nevertheless, available
information in the adult is very scarce for the presence of c-kit cells in the adult testicle, and
14
259
260
261
262
263
264
265
266
267
268
269
270
271
272
273
274
275
276
277
278
279
280
281
282
14
nothing is known about its relationship with Androgen Receptors, myoid cells neither its
interaction with the hypothalamic-hypophyseal-testicular axis.
Until today never before the possibility of the presence of stem-progenitors cells (ICC) in the
adult testicle has been considered. ICC in the intertubular spaces could be regulated by the
hypothalamic-hypophyseal-testicular axis. At present, ICC may be confused with Leydig cells.
In adult liver the presence of ICC cells is described in the stroma and they are called oval cells ,
identified by the expression of the c-kit receptors [17].
In the context of the testicular pathologies, the obstruction of the excurrent ducts is a persistent
cause of infertility diagnosis. In these circumstances it is possible that the c-kit cells appear
increased in number in the testicular interstice, similarly to the situation described in the liver in
cases of biliary obstruction [17].
All these immunohistochemical aspects here analysed must be considered in a testis integral
analysis, in human, and other animal models [18-19].
15
283
284
285
286
287
288
289
290
291
292
293
294
295
296
297
298
15
References
1.- Rodriguez H, Rios A, Sarabia L, Araya JC. Immunohistochemistry of vimentin and desmin intermediate filaments and enolase enzyme in seminiferous tubules from senile individuals. Rev Int Androl 2004; 2; 9-14.
2.- Carreau S, Silandre D, Bourguiba S, Hamden K, Said L, Lambard S, Galeraud-Denis I, Delalande C. Estrogens and male reproduction: a new concept. Braz J Med Biol Res 2007. 40(6)761-768.
3.- Brockschmidt F, Nothen M, Hillmer A. The two most common alleles of the coding GGN repeat in the androgen receptor gene cause differences in protein function. J Mol Endocrinol 2007. 39(1)1-8.
4.- Zhang C, Yeh S, Chen Y, Wu C, Chuang K, Lin H, Wang R, Chang Y, Mendis-Handagama C, Hu L, Lardy H, Chang C. Oligozoospermia with normal fertility in male mice lacking the androgen receptor in testis peritubular myoid cells. Proc Natl Acad Sci U S A. 2006. 103(47)17718-23.
5.- Loukil L, Boudawara T, Ayadi I, Bahloul A, Jlidi R, Ayadi H, keskes L. High androgen receptor immunoexpression in human "Sertoli cell only" testis. Arch Inst Pasteur Tunis 2005. 82(1-4)47-51. 6.- Ullrich A, Schlessinger J. Signal transduction by receptors with tyrosine kinase activity. Cell 1990; 61; 203-212.
7.- Jeanes A, Wilhelm D, Wilson M, Bowles J, McClive P, Sinclair A, Koopman P. Evaluation of candidate markers for the peritubular myoid cell lineage in the developing mouse testis.Reproduction. 2005. 130(4)509-516.
8.- Tourtellotte W, Nagarajan R, Auyeung A, Mueller C, Milbrandt J. Infertility associated with incomplete spermatogenic arrest and oligozoospermia in Egr4-deficient mice. Development 1999; 126; 5061-5071.
9.- Yorke R, Chirala M, Younes M. c-Kit Proto-Oncogene Product Is Rarely Detected in Colorectal Adenocarcinoma. J Clin Oncol 2003; 21; 3885-3886.
10.- Hagger R, Finlayson C, Jeffrey L, Kumar D. Role of the interstitial cells of Cajal in the control of gut motility. Br J Surg 1997; 84; 445-450.
11- Park S, Kim M, Kim H, Song B,. Chi J. Ultrastructural Studies of Gastrointestinal Stromal Tumors. J Korean Med Sci 2004; 19; 234-244.
12.- Joensuu H, Fletcher C, Dimitrijevic S, et al. Management of malignant gastrointestinal stromal tumours. Lancet Oncol 2002; 3; 655-664.
13.- Potti A, Hille R, Koch M. Immunohistochemical determination of HER-2/neu over expression in malignant melanoma reveals no prognostic value, while c-Kit (CD117) over expression exhibits potential therapeutic implications. J Carcinogen 2003; 2; 8-7.
14.- Dym M, Jia M, Dirami G, et al. Expression of the c-kit receptor and its autophosphorylation in immature rat type A spermatogonia. Biol Reprod 1995; 52; 8-19.
15.- Albanesi C, Geremia R, Giorgio M, et al. A cell- and development stage-specific promoter drives the expression of a truncated c-kit protein during mouse spermatid elongation. Development 1996; 122; 1291-1302.
16.- Nocka K, Majumder S, Chabot B, et al. Expression c-kit gene products is known cellular targets of W mutations in normal and W mutant mice-evidence for an impaired c-kit kinase in mutant mice. Gene Dev 1989; 3; 816-826.
17.- Qin A, Zhou X, Zhang W, Yu H, Xie Q. Characterization and enrichment of hepatic progenitor cells in adult rat liver. World J Gastroenterol 2004; 10; 1480-1486.
18.- Espinoza-Navarro O, Bustos-Obregón E. Effect of malathion on the male reproductive
organs of earthworms, Eisenia foetida. Asian J Andrology 2005; 7(1)97-101.
19.- Rodriguez H, Guzman M, Espinoza O. Parathion effects on proteins synthesis in the
seminiferous tubules of mice. Ecotoxicol Environ Safety 65:129-133.