Advances in Bioscience and Biotechnology, 2013, 4, 1-7 ABB
http://dx.doi.org/10.4236/abb.2013.410A4001 Published Online October 2013 (http://www.scirp.org/journal/abb/)
Testicular expression of the TGF-β1 system and the control
of Leydig cell proliferation
Gonzalez Candela Rocio1*, Calandra Ricardo Saul2, Gonzalez-Calvar Silvia Ines2,3
1Research Center of Biomedical Biotechnology, Environmental and Diagnostic Studies, Maimónides University, Buenos Aires, Ar-
gentina
2Institute of Biology and Experimental Medicine, National Council for Scientific and Technical Research, Buenos Aires, Argentina
3School of Medicine, Buenos Aires University, Buenos Aires, Argentina
Received 24 June 2013; revised 25 July 2013; accepted 15 August 2013
Copyright © 2013 Gonzalez Candela Rocio et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Transforming growth factor beta 1 (TGF-β1) is a key
regulator that modulates male reproductive function.
Testicular TGF-β1 modulates the steroidogenesis of
Leydig cells, the organization of peritubular myoid
cells, testis development and spermatogenesis. Several
studies have indicated that TGF-β1 is involved in the
tight balance between proliferative and apoptotic re-
sponses in the Leydig cells. In the present review, we
summarize the direct effects of this cytokine in Ley-
dig cells under normal and pathological conditions.
We analyze the effect of TGF-β1 in Leydig cells de-
pending on the type of receptors involved in the sig-
naling pathway of TGF-β1. Our group has been ana-
lyzing the canonical and non canonical intracellular
signaling pathways of TGF-β1 that are involved in the
expression of proliferative and apoptotic markers in
Leydig cells. On the basis of our studies and from
those of other authors we conclude that the balance
between the expression of TGF-β1 receptors and co
receptors is of relevance in Leydig cell physiology and
pathophisiology.
Keywords: TGF-β1; Leydig; ALK; Endoglin
1. INTRODUCTION
The normal function of the testis has long been recog-
nized to be dependent on gonadotropins hormones. Nu-
merous reports over the past years have clearly indicated
that locally produced factors may play an important role
in the regulation of Leydig cell functions. In this contex t,
many factors produced locally in the testes that act in a
paracrine and/or autocrine manner are able to have plei-
otropic actions on Leydig cell homeostasis. Particularly,
the transforming growth factor beta (TGF-β) family mem-
bers play important roles in Leydig cell differentiation,
proliferation and secretory functions. This review will
focus on two aspects: first, the localization of TGF-β1
system in the testis of different species and second, the
action and signaling pathways of the TGF-β1 involved in
a mechanism that controls Leydig cell proliferation.
1.1. The TGF-β Family
The TGF-β family controls diverse cellular processes in-
cluding cell proliferation, differentiation, migration and
apoptosis [1]. TGF-β family comprises more than 30 pro-
teins that share between 30% to 80% sequence homology
[1]. The best characterized members of this family in-
clude five isoforms of TGF-β (TGF-β1-5), inhibins, ac-
tivins, bone morphogenetic proteins (BMPs), anti-Mul-
lerian Hormone (AMH) and growth and differentiation
factors (GDFs) [2,3]. TGF-β members have an important
role during embryogenesis, organogenesis and mainte-
nance of tissue homeostasis during adult life [4]. In addi-
tion, deregulation of the TGF-β signaling has been im-
plicated in the progression of diseases such as cancer and
autoimmune fibrosis [5].
In reproductive tissues, TGF-β members regulate the
development of the gonads and accessory sex glands,
spermatogenesis, immunoregulation of pregnancy, embr-
yo implantation and placental development [6].
1.2. Localization of TGF-β1 in the Testis
Five isoforms of TGF-β encoded by different genes have
been described [7-9]. TGF-β1, TGF-β2 and TGF-β3 are
expressed in the mammalian testis whereas TGF-β4 and
5 are expressed in birds and toads [6]. Particularly, tes-
ticular TGF-β1 modulates a variety of cellular processes,
*Corresponding a uthor.

G. C. Rocio et al. / Advances in Bioscience and Biotechnology 4 (2013) 1-7 2
comprising the secretory function of Leydig cells, the or-
ganization of peritubular myoid cells, testis development
and spermatogenesis [9-11]. TGF-β has been detected
both in somatic cells (Leydig cells, peritubular myoid cells
and Sertoli cells) and germ cells from different species
including pigs, rats, mice, hamsters and humans [12-15].
It has been also demonstrated that different factors such
as age, photoperiod or pathological conditions modulate
the expression of TGF-β1 in the testis [16-18].
1.3. The TGF-β Receptors in the Testis
Five type II and seven type I receptors that bind mem-
bers of the TGF-β family have been identified in mam-
mals [19-21]. These receptors are transmembrane pro-
teins with an intracellular serine/threo nine kinase domain.
TGF-β type II receptors include the receptors for activin
(ActR-IIA, ActR-IIB), BMPs (BMPRII), AMH (AMHR-
II) and TGF-β (TGF-βRII) [3]. TGF-β type I receptors
(TGF-βRI) comprise the activin like receptor kinase
(ALK) 1 - 7, which can be classified in three groups ac-
cording to their sequence. The first group includes ALK-
5, ALK-4, ALK-7 and GDFs receptor. The second group
consists of the two BMP type I receptors, ALK-3 and
ALK-6. Finally, in the third group, are included the ALK-
1 and ALK-2 receptors [22]. The receptors involved in
the TGF-β1 action are the TGF-βRII and TGF-βRI, par-
ticularly, ALK-1 and ALK-5 [9,23-25]. The presence of
TGF-βRII and ALK-5 receptors has been extensively de-
monstrated in Leydig cells, Sertoli cells and germ cells
from mice [14], rats [26], boars [18], pigs [27], hamsters
[15] and humans [13,28]. ALK-1 receptor was recently
observed in Leydig cells from mice [14], hamsters [15]
and humans [28].
Other receptors capable of binding TGF-β members
are TGF-β receptor type III (TGF-βRIII) or betaglycan
[29] and the co-receptor endoglin [30,31]. The presence
of TGF-βRIII has been characterized in the testicular
cells [32] but endoglin receptor was characterized prima-
rily in endothelial cells [33]. Recently, the expression of
the protein and mRNA of endoglin has been reported in
Leydig cells from human and mouse testis [14,28]. The
pattern of expression of the TGF-β receptors in a given
cell leads to different responses exerted by TGF-β1 (see
below).
1.4. Canonical and Non-Canonical TGF-β1
Signal Transduction Pathway
The TGF-β receptors have the ability to associate as ho-
momeric and heteromeric complexes. In this context,
TGF-βRII forms homodimers regardless of TGF-β bind-
ing and is constituti vely autophosphory lat ed [34,35]. TGF-
βRI also forms homodimers independently of ligand and
binding. Both ALK-1 and ALK-5 are not able to effi-
ciently bind to the ligand, except when they are associ-
ated with the TGF-βRII. Therefore, the binding of TGF-
β1 to TGF-βRII induces the recruitment and phosphory-
lation of ALK-1 and/or ALK-5 receptors and triggers the
intracellular signal transduction through the Smad pro-
teins (namely canonical mechanism) [4]. Eight different
Smad proteins are encoded by the human and mouse ge-
nome [4]. Only five Smad proteins (Smad1, Smad2,
Smad3, Smad5 and Smad8) act as a substrate for the
TGF-β receptors and are called R-Smads [5]. In this con-
text, ALK-1 receptor signals via the phosphorylation of
Smads 1/5 and ALK-5 receptor signals via the phospho-
rylation of Smads 2/3 [8]. This process induces the trans-
duction of the extracellular signal of TGF-β1 activating
downstream gene transcription [19,20,36,37].
The presence of Smad proteins in the testis has not
been thoroughly studied. At present, it has been describ-
ed in rats, i.e. the expression of the Smads 1, 2 and 3 in
Leydig cells, Sertoli cells and germ cells [38,39]. In
mouse postnatal testis, Smad1 has been detected in sper-
matocytes and round spermatids [40]. In the prepubertal
mouse testis, the expression of Smad5 has been reported
in spermatogonia cells [41]. In the human testis, Smad 1,
2, 3 and 5 have been localized in Leydig cells and semi-
niferous tubules [2 8,42].
Besides these intracellular mechanisms involving Smad
proteins, TGF-β1 exerts its action via non-smads proteins
(namely non-canonical mechanism), particularly, JNK (Jun
N-terminal kinase), p38 MAPK (mitogen-activated pro-
tein kinase p38) and ERK (extracellular-signal-regulat-
ed kinase) [43-45]. The first evidence of this mechanism
comes from observations in which TGF-β1 can activate
the ERK signalling pathway promoting the activation of
p21 (cell cycle inhibitory factor) in intestine and lung
epithelial cells from rats [46]. At present, the best charac-
terized TGF-β1 signalling pathway that does not involve
Smad proteins is the JNK and p38 MAPK pathway [47].
A direct demonstration of this mechanism is based on re-
sults performed in murine mammary epithelial cells with
a mutation in the TGF-βRI receptor at the binding and
phosporilating Smads sites. It was observed that this mu-
tated receptor is capable to induce an intracellular re-
sponse in the presence of TGF-β1 activating JNK and
p38 MAPK pathways and leading to cell apoptosis [48,
49].
2. INVOLVEMENT OF TGF-β1 IN
LEYDIG CELL PROLIFERATION
TGF-β1 exerts a crucial role in cell proliferation, differ-
entiation and apoptosis. Although the mechanism of TGF-
β1 signalling has been studied in different experimental
conditions, the action of this cytokine in testis h omeosta-
sis has been poorly an alyzed. It is well known that TGF-
β1 inhibits the growth of most cell types, including epi-

G. C. Rocio et al. / Advances in Bioscience and Biotechnology 4 (2013) 1-7 3
thelial cells, endothelial cells, fibroblasts and hematopoi-
etic cells [50]. On the other hand, it has been observed
that TGF-β1 is able to stimulate cell proliferation in skin
fibroblasts and endothelial cells [50]. In this context, it
has been shown the activation of the TGF-βRII and sub-
sequently of ALK-5 and Smad2/3 by TGF-β1 leads to a
regulation of the gene promoters for the cyclin dependent
kinases (CDK) to suppress their transcription [50 ]. Smad
proteins are responsible for the transcriptional activation
of the two major cyclin inhibitors: p15 and p21. Both are
responsible for inhibiting the activity of CDK proteins
that allow the progression from G1 to S phase of the cell
cycle, leading to cell cycle arrest in G1 phase [4,50]. In
human prostate cells, gastric cells and astrocytes it was
observed that the incubation with TGF-β1 promotes apo-
ptosis by enhancing the levels of p15 and p21 [51-54]. In
ovarian tumor cells, the treatment with TGF-β leads to an
increase in p15 levels and phosphorylation of Smad2
[55]. In the testis, it has been described that the incuba-
tion of ha mster Leydig cells with TGF-β1 stimulated p15
expression in a mechanism that involves the participation
of ALK-5 receptor and activation of p38 MAPK [15]. In
line with this, Wu et al. [45] observed that TGF-β1 acti-
vates the p38 pathway leading to cell ap optosis in murine
podocytes. There is no direct evidence that TGF-β1 via
p38 MAPK induces apoptosis of Leydig cells, although
exists different reports that propose the participation of
this MAPK in the apoptosis of germ cells in rodents [56].
However, studies reported by Gonzalez et al. [15], indi-
cated that the Leydig cells from hamsters with maximal
testicular photoperiodic regression are quiescent and ex-
press a marked increased of p15 which leads to an arrest
of cellular cycle and not necessarily leads the cells to
apoptosis.
It is important to point out, that TGF-β1 exerts bifunc-
tional effects on cell proliferation, a mechanism highly
described in endothelial cells [57]. In this experimental
model, Lebrin et al. [57] showed that TGF-β1 is able to
induce or inhibit cell proliferation. In these “in vitro”
studies, low doses of TGF-β1 stimulated endothelial cell
proliferation, whereas high doses of TGF-β1 inhibited
this respon se [33,57]. The resu lts obtained in endothelial
cells, showed that the bifunctional effect of TGF-β1 is
the result of the balance between the expression of ALK-
5 and ALK-1 receptors. Whereas ALK-5 is responsible
for the inhibition of proliferation, ALK-1 has the oppo-
site effect [57]. In line with this, the co-receptor endoglin
plays a fundamental role. Extracellular and cytoplasmic
domains of endoglin interact with ALK-1 receptor en-
hancing its signal transduction via the Smad1/5 protein
pathway and interfering with ALK-5-Smad2/3 signaling
[31,57-59]. Experiments performed on endothelial cells
lacking the co-receptor endoglin, showed that these cells
were not able to proliferate because the signalling path-
way of TGF-β1 that involves the activation of ALK-1
receptor is reduced, whereas the ALK-5 receptor signal-
ling pathway is stimulated [57]. Then, expression of the
coreceptor endoglin is essential to generate a prolifera-
tive cell response in the presence of TGF-β1. It has been
recently described that low doses of TGF-β1 enhanced
the expression of the early growth response f actor-1 (egr-
1) and Krüppel-like factor 14 (KLF14) in TM3 Leydig
cells [60]. Both KLF14 and egr-1 are transcription fac-
tors responsible for the regulation of the transcription of
various genes. Scanning of the promoter region of en-
doglin gene, one site for egr-1 and eight sites for KLF
family members were detected [60]. Egr-1 is involved in
cell growth, differentiation, and survival [61]. In this con-
text, Hou et al. [61] have reported that TGF-β1 is able to
induce a rapid and transient accumulation of egr-1 pro-
tein and mRNA in human skin fibroblasts. On the other
hand, it has been described that KLF14 is a protein up-
regulated by TGF-β1 that represses TGF-βRII [62]. The
effect of TGF-β1 on the induction of KLF14 and egr-1,
promotes the expression of endoglin, which leads TGF-
β1 to promote the signaling pathway of proliferation
[60].
In addition, other factors involving hormonal regula-
tion can modulate the expression of endoglin. It has been
described in mouse Leydig cells, that progesterone is
able to induce the expression of the co receptor endoglin
[63] and, that the effect of low concentrations of TGF-β1
plus progesterone showed an increased in the proliferat-
ing cell nuclear antigen (PCNA) expression and a de-
crease in Bax (pro-apoptotic gene) expression [63]. In
the same work, the authors reported a clear increase in
Smad1/5 phosporylation after mouse Leydig cell incuba-
tion with TGF-β1 and progesterone [63]. In the testis, the
control of the Leydig cell proliferation exerted by TGF-
β1 was confirmed using the non-tumoral mouse Leydig
cell line TM3. In this model, the gene expression of p15
was induced by low doses TG F-β1 [60]. The presence of
progesterone in the incubation media, induced the ex-
pression of endoglin and the proliferative marker PCNA
and abolished the stimulatory effect of TGF-β1 on p15
expression [60]. In this regard, it has been described that
Smad proteins activate the transcription of p15, which
inhibits CDK-6 and CDK-4 proteins, influencing the cel-
lular cycle and leading to a quiescent state of the cells
[53,54,64]. Therefore, the effects of TGF-β1 on cell pro-
liferation and cell cycle arrest would depend on the bal-
ance between ALK-1 and ALK-5 receptors and the ex-
pression of endoglin in the cell.
3. EXPRESSION OF THE TGF-β1
SYSTEM IN THE HYPERPLASIA OF
LEYDIG CELLS
It has been described in a variety of tissues that TGF-β1

G. C. Rocio et al. / Advances in Bioscience and Biotechnology 4 (2013) 1-7 4
and its receptors are altered in pathological conditions
such as cancer and hyperplasia [65]. The identificatio n of
factors involved in the progression of testicular patholo-
gies such as Leydig cell hyperplasia (LCH) is crucial to
understand this patho logy.
Studies performed in experimental animals have shown
that Leydig cell hyperplasia (LCH) may be induced by
hormones and alterations in the production of paracrine
growth factors [66]. It is known that LH, androgens or
estrogens stimulate the proliferation of Leydig cells [67,
68]. Concerning this issue, it has been reported that Ley-
dig cells of transgenic mice over-expressing TGF-β1 are
prominent [69]. In a model of transgenic mice over ex-
pressing hCG which show hyperplasia and hyperthtropy
of Leydig cells, the expression of TGF-β1, ALK-1 an d co
receptor endoglin was increased respect to control ani-
mals [14]. Moreover, morphometric studies revealed that
the “in vivo” intratesticular injection of normal mouse
testis with TGFG-β1 plus progesterone caused an in-
crease in the volume of Leydig cells [63].
In patients with certain testicular pathologies, the his-
tological patterns found are hypospermatogenesis, Ger-
minal Arrest an d Serto li cells on ly Syndro me (SCO) with
and without focus of spermatogenesis [70]. It has been
reported that in some of these patients, Leydig cells show
hyperplasia, characterized by the presence of small nod-
ules, often bilateral, thus being a benign pathology for
the patient [71]. Dobashi et al. [72] have described that
the serum levels of TGF-β1 were higher in patients with
SCO than in normal controls. Gonzalez et al. [28] have
shown that in patients that present LCH associated with
SCO or hypospermatogenesis, the expression of TGF-β1
protein and mRNA was higher than in patients with SCO
or hipospermatogenesis alone.
Although the expression of TGF-β1, TGF-βRII and
ALK-5 receptors has long been described [13,72], scarce
information is known about the localization of ALK-1
and co receptor endoglin in human testis both in normal
and pathological conditions. Recently, it has been de-
scribe the presence of ALK-1 receptor and co receptor
endoglin in Leydig cells from normal and pathological
testicular biopsies [28]. Moreover, an increased in the ex-
pression of these two receptors has been detected in pa-
tients with LCH [28].
4. CONCLUSION
In conclusion, the effect that TGF-β1 exerts on the pro-
liferation of Leydig cells does not primarily depend on
factors such as the concentration of this cytokine in the
cell environment, but more importantly, depends on the
type of receptors and co receptors present in the Leydig
cells. The description of the action of TGF-β1 in the tes-
tis contributes to enlarging our knowledge on Leyd ig cell
regulation and might be a clue to study signaling path-
ways to key outcomes in male reproductive health.
5. ACKNOWLEDGEMENTS
This study was supported by Grants from Consejo Nacional de Inves-
tigaciones Científicas y Técnicas (CONI-CET), Agencia Nacional de
Promoción Científica y Técnica (ANPCyT) and Facultad de Medicina-
Universidad de Buenos Aires, Argentina.
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