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,

Fax: 56 58 205484. E-mail: oespinoz@uta.cl

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Summary

Aims: We sought to identify the presence of interstitial cells of Cajal, muscle cells, nerves and

androgen receptor positive cells in adult human testicle, using immunohistochemical detection

with the antibodies for c-kit/CD-117, actin smooth muscle (ASMS) specific, neurofilament (N)

and androgen receptor (RA), respectively. Methods: Sections each 4 m thickness were

obtained, according to the bioethical norms of the University of Chile, from patients (n= 10) with

diagnosis of prostate cancer, submitted to orchiectomy of testicular biopsies. These sections were

processed by routine techniques of histology and for immunohistochemistry using specific

antibodies against c-Kit/CD-117, smooth muscle actin specific, neurofilament and androgen

receptor. Results: We show the presence of cells c-kit/CD-117, with diverse degrees of

positivity, distributed mainly in the interstitial peritubular area of the human testicle. The

peritubular myoides cells were positive to the presence of the actin smooth muscle and, this same

cell type showed positivity to androgen receptor. The neurofilaments elements (+) only were

observed in the vascular tunic. Conclusion: the specific immunohistochemistry directed to

detect proteins c-kit/CD-117, muscle cells, neurofilament and androgen dependence peritubular

cells in the testis has allowed to describe the presence of the interstitial cells of Cajal in human

testicular interstitium, opening a new perspective for the functional interpretation of the testicular

cellularity and tubular motility. Possibly associated functionally to peribubulars cells of smooth

muscle to regulate the mobility of the seminiferous tubules, whose integration and function

would be androgen dependent. With respect to the cells that express the c-kit receptor, these were

exclusively in the interstitial compartment. This cellular type in addition of the muscular cells of

peritubules, allows to develop the analogy with the regulation of tubular mobility, as it happens

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in the gastrointestinal apparatus. Added to the absence of nervous fibers to the interior of the

testicle, they are limited his disposition until the vascular tunic.

Short running title: Interstitial Cajal Cells / Human Testicle.

Key Word: Human reproduction, testicle, interstitial cajal cells, ICC, tubular motility, AR.

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Introduction

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.

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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.

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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.

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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

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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.

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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.

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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.

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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.

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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

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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

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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

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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].

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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.

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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.

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TABLES

Table 1. Quantification and distribution c-kit + cells in the testis: immunocytochemistry reaction.

Cells Distribution (%)Cell/ 0.25mm2 Peritubular Perivascular

c-kit (+) Cells 0.4 ± 0.002 82 18

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Graph 1 Table 2. Quantification and distribution of the cells of the compartments of the adult

human testicle that express the androgen receptor (a,b: p≤ 0.05).

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Graph 2 Table 3. Cells of human adult testicle that express specific actin of smooth muscle.

Seminiferous tubules and vascular system.

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GRAPHICS, FIGURES AND TABLES

Table 1: The quantification was made by count each cells immunocytochemistry positive by area

(40x), with registration the position near seminiferous tubules or vascular area.

Graph 1: The quantification was made by the count of immunopositive cells present in the

different compartments of the testicle.

Graph 2: The quantification was made by count each cells immunocytochemistry positive by

tubules and arterioles of the adult human testicle (40x). The results are expressed in percentage of

immunopositive cells in the walls of the seminiferous tubules and the arterioles.

Figure 1: Histological organization of the adult human testicle with stain of Papanicolau.

Peritubular compartment with blue dyeing and the nuclei of the different cellular populations

from the tubular compartment, germinal cells and Sertoli strongly basofiles 1) interstitial

Compartment, 2) Peritubular compartment, 3) Cells of Sertoli, 4) cells of the germinal line

(100x).

Figure 2. Transversal section of adult human testicle with cells immunopositives for specific

smooth muscular actin. The arrows indicate the positive cells of the peritubular

compartment(40x).

Figure 3. Transversal section of adult human testicle with immunopositives cells for

neurofilaments. The arrows indicate positive nervous fibers, those that are arranged exclusively

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from the vascular tunic outwards of the testicle (20x). ST: seminiferous tubules; VT: vascular

tunic, AT: albuginean tunic, BV: blood vessel. (↑) nervous Fibers: neurofilaments (+).

Figure 4. Transversal section of adult human testicle with immunopositives cells for the

androgen receptor (AR). The arrows indicate the nucleus of the immunopositives cells (40x). 1)

tubular compartment; 2) Peritubular compartment; 3) interstitial compartment.

Figure 5. It represent cross sections of human adult testis with positive immuno-

histochemistochemical reaction for the interstitial cajal cells. (↑) immuno-histochemistry positive

for the c-kit receptor in the interstitial cajal cells, (1) tubular compartment, (2) peritubular

compartment, and (3) interstitial compartment (40x).

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