Leydig cells, thyroid hormones and steroidogenesis

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Indian Journal of Experimental Biology
Vol. 43, November 2005 , pp. 939-962
Review Article
Leydig cells, thyroid hormones and steroidogenesis *
S M L Chamindrani Mendis-Handagama t & H B Siril Ariyaratne*
Department of Comparative Medicine, College of Veterinary Medicine,
The University of Tennessee, 2407 River Drive, Knoxville, TN 37996, U.S.A.
Leydig cells are the primary source of androgens in the mammalian testis. It is established that the luteinizing hormone (LH)
produced by the anterior pituitary is required to maintain the structure and function of the Leydig cells in the postnatal testis.
Until recent years, a role by the thyroid hormones on Leydig cells was not documented. It is evident now that thyroid hormones
perform many functions in Leydig cells. For the process of postnatal Leydig cell differentiation, thyroid hormones are crucial.
Thyroid hormones acutely stimulate Leydig cell steroidogenesis. Thyroid hormones cause proliferation of the cytoplasmic
organelle peroxisome and stimulate the production of steroidogenic acute regulatory protein (StAR) and StAR mRNA in Leydig
cells; both peroxisomes and StAR are linked with the transport of cholesterol, the obligatory intermediate in steroid hormone
biosynthesis, into mitochondria. The presence of thyroid hormone receptors in Leydig cells and other cell types of the Leydig
lineage is an issue that needs to be fully addressed in future studies. As thyroid hormones regulate many functions of Sertoli cells
and the Sertoli cells regulate certain functions of Leydig cells, effects of thyroid hormones on Leydig cells mediated via the
Sertoli cells are also reviewed in this paper. Additionally, out of all cell types in the testis, the thyrotropin.releasing hormone
(TRH), TRH mRNA and TRH receptor are present exclusively in Leydig cells. However, whether Leydig cells have a regulatory
role on the hypothalamo-pituitary-thyroid axis is currently unknown.
Keywords: Aging, Leydig cell lineage, Steroidogenesis, Thyroid hormone.
Introduction
The primary source of androgens in the mammalian
male is Leydig cells; they reside in the testis interstitium.
Androgens produced by the Leydig cells are essential for
proper functioning of reproductive and accessory
reproductive organs as well as many non-reproductive
tissues such as muscle, skin, liver, hemopoetic organs,
and bone. It is established that synthesis and secretion of
androgens by the Leydig cells are under the control of
endocrine, paracrine and autocrine factors. However,
until recent years, thyroid hormone was not considered
as an essential factor for any function of the Leydig cells.
Research findings during the past few years have
provided many interesting features of thyroid hormones
on Leydig cells, which are reviewed in this paper.
Thyroid hormones
Thyroid gland produces thyroxine (T4) and
triiodothyronine (T3); T3 is 4.5 times more potent than
tCorrespondent author: Telephone: 865-974-5824; Fax: 865-9745640
Email: [email protected]
tPresent address: Department of Veterinary Basic Sciences, Faculty
of Veterinary Medicine and Animal Science, The University of
Peradeniya, Sri Lanka.
*Supported by: World Health Organization, The University of
Tennessee Center of Excellence for Human and Animal Health and
Disease, The University of Tennessee Professional Development
Award Program
T 4. First discovered by Gudematsch 1 thyroid hormones
play a crucial role in differentiation of cells. Thyroid
hormones stimulate oxidative metabolism in many
tissues in the body, but this is not seen in the testis.
Based on this fact, early investigators considered testis as
a non-responsive organ for thyroid hormones.
It is established that thyroid hormone releasing
hormone (TRH) from the hypothalamus and thyroid
stimulating hormone (TSH) from the anterior pituitary
gland regulate the secretion of thyroid hormones by the
follicular cells of the thyroid gland. Biological effects of
thyroid hormones on the target cells are brought about
by binding these hormones to their specific receptors,
which are localized to nuclear as well as cytoplasmic
compartments of the target cells2. Thyroid receptors
basically act as nuclear transcription factors and
following binding with the thyroid hormones, the
hormone-receptor complex is formed and trigger the
metabolism, growth and differentiation of the organism
by binding to regulatory region of the responsive genes
and stimulating or inhibiting transcription of these
genes 3,4. Nevertheless, recent studies have suggested the
possibility of certain thyroid hormone actions without
binding of hormone-receptor complex to responsive
elements5 . Thyroid receptors are encoded by two
different genes, U and ~ and several isoforms of each of
these two receptor types, namely Ul , U2, U3, and ~l and ~,
940
INDIAN J EXP BIOL, NOVEMBER 2005
which are formed by alternative splicing of each
transcript. Out of these receptor isoforms, u" ~I and ~2
are hormone binding and U2 and U3 isoforms are nonhormone binding4,6,7, The physiological significance of
non.hormone binding isoforms of thyroid receptor is not
clear at present, however, it is suggested that these may
act as dominant negative antagonists of the true thyroid
hormone binding receptors 8,9. According to lannini
et al. 10, the distribution of the different isoforms of the
thyroid receptors in tissues appear to be dependant on
the developmental stage and the animal.
Leydig cells
History - In 1850, a German Scientist named Franz
Leydig, first described the Leydig cells in the testis
interstitium II (Leydig cells were named after him). His
discovery of Leydig cells was a result of a comparative
study of the testis interstitium in various mammalian
species, which included primates, carnivores and
rodents. However, the first scientists to emphasize
strongly a possible endocrine role for Leydig cells were
Pol Bouin (1870-1962) and Paul Ancel (1873-1961 )
l
(review \ They discovered that internal secretion of
Leydig cells controlled male secondary characteristics. It
is established fact that Leydig cells produce androgens
and they are the main source of androgens in the
mammalian male.
Leydig cells morphology - Leydig cells are large
polyhedral cells and reside in the testis interstitium as
shown in Fig. 1. Leydig cell number, size,
morphological characteristics and their relationship to
blood vessels and other surrounding structures are
different among species 13 . Tables 1 and 2 summarize to
some extent, the published values for Leydig cell
number and the average cell volume (i.e. size) in several
species. Many factors contribute to the variability of the
data for a given species on Leydig cell number per testis
and the average volume of a Leydig cell. Methods of
fixation of testis, tissue processing and stereological
techniques employed in these studies are the major
factors that contribute to these differences.
It is considered that the primary regulator of Leydig
cell structure and function in the postnatal testis is
luteinizing hormone (I ,H), which is produced by the
gonadotrophs of the anterior pituitary gland.
Additionally, the primary androgen secreted by the
Leydig cells in the sexually mature testis is testosterone.
Leydig cells cytoplasm is rich in smooth endoplasmic
reticulum, mitochondria and peroxisomes, the organelles
involved in steroid hormone biosynthesisl4-16. Variable -
volumes of cytoplasmic lipid droplets are present in
Leydig cells in different species, which contain
cholesterol esters. Cholesterol is an obligatory
intermediate for the process of steroidogenesis. In
general, species who could de novo synthesize
cholesterol, e.g. rat, guinea pig, have lesser amount of
cytoplasmic lipid in Leydig cells.
Leydig cells differentiation - Leydig cells in the
adult testis are postnatally differentiated during the prepubertal period 42-46 . The precursors/stem cells to Leydig
cells are the mesenchymal cells of the testis
interstitium 13,43-46. They embryologically originate either
from the mesonephric tubules andlor loose connective
tissue of the developing gonad derived from the
embryonic mesoderm47. Mesenchymal cells in the
postnatal testis are either found at the peritubular region
or the central interstitium (randomly scattered); those in
the peritubular region are identified as the precursor cell
type for Leydig cells in the postnatal testis 46 ,48,49. Several
recent investigations have confirmed this fact in the
prepubertal rat 50-52 and the adult rat following ethane-
Fig. 1 - Representative light micrograph of testis interstitium (I) of
a 3-month old hamster. Leydig cells (arrows), appear as large
polygonal.shaped structures [BV=blood vessels, ST= seminiferous
tubules, Ly=lymphatic space, Mc=macrophage, E=elongated
spindle-shaped cell].
941
MENDIS-HANDAGAMA & ARIY ARATNE: LEYDIG CELLS x THYROID HORMONES
53
dimethane sulfonate (EDS) treatment . EDS is a unique
toxin for Leydig cells, which selectively kills Leydig
Species
References
Table 1 - Number of Leydig Cells per Adult Testis in Different
Mammalian Species (106)
Average Volume
of a Leydig Cell
Rat (Ilm 3)
1210
Mori
Table 2 -
Average Volume of a Leydig Cell
1380
17
Mendis-Handagama el ailS
Species
Numberrrestis
(106)
References
Rat
30
Mori
1011
Sinha-Hikim and Hoffer l9
Andreis el al. lO
22.42
ls
Mendis-Handagama et al.
1724
Mendis-Handagama el.ai.
25 .4
Sinha-Hikim and Hoffer l9
1940
38.7
Andreis et apo
1378
Mendis-Handagama el.al.
Russell et at. 23
21.23
25.4
Mendis-Handagama and Ewingll
Mendis-Handagama et.at. 22
21.5
Mendis-Handagama et.al. ls
28
Russell et al.l3
24
25
22.5
21.14
Mouse
Hamster
1844
i7
24
Mendis-Handagama and Sharma
25
Mendis-Handagama and Gelber
l6
Mendis-Handagama el ai
Ariyaratne and MendisHandagama26
3.0
Mori et al27
1.4
Mendis-Handagama el ai.2S
18.2
Hardy et aP9
25.4
Singha.Hikim et ai3D
10'
Mend:s·Handagama el at.
Mouse (Ilm 3)
14
Hamster
(11 m3 )
Guinea pig
(11 m3 )
Mori el aPI
21.05 *
14
Mendis-Handagama el a/ 14
Mendis-Handagama. 32
Dog (11m)
Dog
300
Walters el al. 33
Human
3
(Ilm )
Human
350
Kaler and Neaves ,J4
750
Paniagua et a/. )5
Mori 3b
106
Stallion
Ram
Mendis-Handagama and
Sharma24
1750
Mendis-Handagama and
Gelbel 5
1752
Mendis-Handagama et.al
2525
Ariyaratne and MendisHandagama,26
15
16
1533
Mori el at. 2?
4000
Mendis-Handagama el al 18
4823.4960*
Mendis-Handagama and de
Kretse~9
LO
Guinea
pig
1642
ll
14
969**
Mendis-Handagama et al
1092
Sinha-Hikim el al3D
1443
Mori el aPI
2195
Sinha-Hikim el a/.
1568*
Mendis-Handagama et al 14
1477
Walters el a/. 33
2943
Kaler and Neaves
4320
MOr: 36
3106
Sinha-Hikim el atl
4 .3 (2.3 years of
age)
Johnson and Neaves 3?
Stallion (pi)
4O
34
1410 (2.3 years
of age)
Johnson and Neaves 3?
Johnson and Neaves)?
5.8 (4.5 years of
age)
Johnson and Neaves)7
3060 (4.5 years
of age)
John son and Neaves 3?
7.0 (13 .20 years
of age)
Johnson and Neaves J7
4660 (13.20
years of age)
416
Lunstra and Schanbache~s
13,200
Lunstra and Schar;bache~8
Ram (Ilm 3)
*calculated from numericaL den,ity (Nv) of Lt-ydig cells and testis
voLume (Tv), i.e. NvxTv
*values are obtained using isolated Leydig cells. ** vaLues calculated from
voLume density (Vv) and numerical density (Nv) of Leydig cells, i.e.
VvINv
942
INDIAN J EXP BIOL, NOVEMBER 2005
cells by causing them to undergo apoptosis within 48 hr
after the EDS administration; Leydig cells are
completely eliminated from the testis interstitium within
54
this period (reviews .55). A new generation of Leydig
cells begins to differentiate from peritubular
mesenchyma) cells about two weeks after EDS treatment
and gradually becomes fully functional within the next
few weeks (reviews54.51. In studies of Ariyaratne et al.50-53,
detection of mesenchymal cell differentiation was based
on positive immunolabeling for 3j3-hydroxy steroid
dehydrogenase (3j3-HSD), the universally accepted
marker for all steroid secreting cells.
A schematic diagram to demonstrate the Leydig cell
lineage is shown in Fig. 2. At the onset of the process of
Leydig cell differentiation, a mesenchyma) cell which is
a non-steroidogenic cell, differentiates into the second
cell type in the lineage, the .progenitor cell, which is
56
steroidogenic , but otherwise similar in shape to the
mesenchymal cells (Fig. 3a). These progenitor cells
undergo hypertrophy (Fig. 3b), proliferation and
differentiate into the next cell stage, which we identify as
the newly formed adult Leydig cells. Progression of
these events is followed by the movement of these newly
formed adult Leydig cells away from the peri tubular
region towards the central interstitium56• The progression
of the process of Leydig cell differentiation occurs with
the advancement of age and the newly formed adult
Leydig cells transform into immature Leydig cells and
finally attain the status of the mature adult Leydig cells.
Thyroid receptors in Leydig ceUs
By using a variety of techniques, which included the
quantification of the specific nuclear binding of the
hormone, evaluation of the expression of receptor
a 10 day old rat testis, immunolabelled for
JI}.HSD (brown color) to demonstrate mesenchymal cells (unstained) and
progenitor. Mesenchymal cells in the periphery of the seminiferous tubules
differentiate into progenitor cells (arrows in Fig. A, B), which are still
spindleshape, but contains the steroidogenic enzyme 31} HSD with the
progression of their differentiation towards the newly formed adult Leydig
cells. They become round in shape (compare cells depicted by arrows in Fig.
A, B) and move gradually away from the peritubular region towards the centr:ll
pan of the testis interstitium [Permission taken from the publisher, Bioi
Reprod; 65 (2001) 660]
Leydig Cell Lineage
Progenilor eel
Newly Formed ALC
. <1G>[email protected] rd steloideget ic
no L.H reoeptDrs
3Jl-HSD+ve
'LH~
poIygalaI, &mal
( ..
{(
SpindIe-6hape
) }
lex' no cytopIasmiclpid -----~) }
InvnaII..n! ALC
MahnALC
3Jl-HSD+ve
tLH~
polygonal, IaIge
J cyIqlIasmic Ipid
Fig. 2 - Schematic diagram of Leydig cell lineage. Mesenchymal cells are the precursors to the Leydig cells. They are spindle-shape .. and are non-steroidogenic.
At the onset of postnatal Leydig cell differentiation, mesenchymal cells differentiate into the progenitor cells, which are also spindle.shape, but have few
steroidogenic enzymes (egJl} HSD) and LH receptors. Thyroid hormones are critical to the onset of mesenchymal cell differentiation into the progenitor cells,
which differentiate into mature adult Leydig cells through stages of newly formed adult Leydig cells and inunature adult Leydig cells, respectively as shown in the
diagram [Permission taken from the publisher, Bioi Reprod, 65 (200 I) 660]
MENDIS-HANDAGAMA & ARIY ARATNE: LEYDIG CELLS x lHYROID HORMONES
mRNA or localization of receptor protein with the help
of specific antibodies, presence of thyroid receptors in
testes has been tested. Early investigations, which used
isolated nuclei from adult testes, have reported that there
s7
is no nuclear binding of hormone to these nuclei and
lead to the conclusion that testis is an un-responsive
organ for thyroid hormones. Additionally, these early
studies were unable to detect mRNAs of either a" ~, or
/hin the adult testis58-60. Later, a renewed interest on this
subject has been generated when Palmero et al. 6 ' have
demonstrated a specific binding of thyroid hormones to
nuclei of Sertoli cells isolated from immature rats. Since
then, many investigations have shown an age
dependency for nuclear binding of thyroid hormone and
expression of thyroid receptor a,.mRNA in the rat
testis62.64 • These studies have shown that binding of the
thyroid hormone and expression of the mRNA of thyroid
receptor a, is highest in the fetal testis, gradually
declines during the pre-pubertal testis and totally absent
in the adult testis. Although absence of the expression of
thyroid receptor ~ mRNA has been reported in these
early studies, extremely low level of expression has been
reported in later studies63~s.66. Nevertheless, more recent
studies using sensitive laboratory techniques such as
Northern blot analysis and reverse transcriptionpolymerase chain reaction, several investigators are able
to demonstrate considerable amounts of thyroid receptor
a, mRNA even in the adult testis~.
The presence or absence of thyroid receptors in
Leydig cells and/or their precursor cell types is an issue
that has not been completely resolved due to the
discrepancies in the available reports on this subject.
Until recent, mature Leydig cells have not been
considered as target cells for thyroid hormones because
several investigations have reported that specific binding
of the thyroid hormone or expression of thyroid receptor
. mRNA are absent in this cell typelO,59.62.63.69. However, in
several previous studies70- 72 thyroid receptor protein has
been localized to the testicular interstitium in the adult
rat. These studies have used specific antibodies raised
against a, receptor in consistent with observations made
on mRNA. No immunolabeling has been seen for
thyroid receptor ~ in any of the testicular components in
similar studies73.74• Using purified Leydig cells and their
precursors from rats at different ages, including the adult
animal, Hardy et az.7 s have reported the presence of
thyroid hormone receptor a, mRNA (but not the protein)
in Leydig cells and their precursors. However, whether
the T3 receptor proteins are expressed in these cells in
943
Leydig cell lineage is yet to be determined. Palmero
et al. 69 have reported that 1'3 receptors are absent in
nuclei of immature pig Leydig cells based on their
observations of absence of nuclear binding of the
hormone in the isolated cells. Surprisingly, a recent
study by McCoard et al. 76, using immunocytochemisll'y,
have demonstrated the presence of strong nuclear
labelling for thyroid receptor ~, in Leydig cells of pigs at
different ages. Additionally, the presence of thyroid
receptor protein in a subset of testicular interstitial cells
in rats has also been reported by Tagami et aI.72 and
66
Buzzard et aI. using immunocytochemisll'y. However,
the latter study was focused on 1'3 receptors in
seminiferous tubules and therefore, this study do not
give detailed information on which testicular interstitial
cell types show positive labelling for 1'3 receptors.
Therefore, in future studies it is important to establish
the spatial and temporal expression of thyroid receptors
in postnatally differentiated Leydig cells and the cell types
of its lineage.
Organic anions such as thyroid hormones, steroid
conjugates, bile salts, drugs are transported into the cell
through the cell membrane using a novel family of
trans.membrane proteins known as organic anion
transporting polypeptides, i.e., Oatps in rodents and
77
OATPs in humans . Although, most OatpslOATPs
exhibit overlapping substrate specificities with other
members of the family, some other OatpslOATPs show
preferential or even selective transport of certain
substrates making them more specific in their action. In
addition, it is common to see that different tissues use
the same OatpslOATPs for the transport of a particular
substance. However, some OatpslOATPs are
predominantly or even exclusively expressed in one
tissue only. In agreement with latter observation, it has
recently been documented that transport of thyroid
hormones in different tissues is accomplished by
different OatpslOATPs molecules in a tissue specific
manner78 . These thyroid transporters facilitate thyroid
79
hormone 'JPtake into tissues . From the human testis, a
substrate specific OATP molecule (OATP-F) which
involves with high affinity transport of T4 and reverse
1'3 has been isolated and demonstrated to be expressed
only by Leydig cellsso . In addition, three previously
unknown gonadal specific organic anion transporters
(GSTs) named as rat GST-l, rat GST-2 and human GST
has also beeq recently identified from testes of these
species. By · .Northern blot analysis and in situ
hybridization techniques, their expression is observed in
INDIAN J EXP BIOL, NOVEMBER 2005
944
Sertoli cells, spermatogonia and Leydig cells. Functional
studies on these molecules have revealed that they are
associated with the transport of thyroid hormones and
81
few other organic anions in these cells . Nevertheless,
physiological significance of these transporters in
thyroid hormone mediated regulation of Leydig cell
development and function remains to be determined.
Leydig cell differentiation and thyroid hormones
Role for thyroid hormones on postnatal Leydig cell
differentiation has been reported by two independent
groups of investigators. They have shown that the
process of Leydig cell differentiation in the neonatalprepubertal testis is arrested with hypothyroidism 82.83 and
advanced with hyperthyroidism50.52.83. A more recent
4
studl , has confirmed this finding by demonstrating that
prolonged hypothyroidism beyond the neonatal period in
rats continue to arrest Leydig cell differentiation.
However, if the hypothyroid state is transient, Leydig
cell differentiation is evident at postnatal day 40, and the
number of cells differentiated are two-fold greater than
so
g
J~
40
~i
30
J ..
~!
20
z ~
~
10
_
K
'S-;
(a)
i.r
'61
i~
'0 ~
40000
14
21
2.
40
10
!tl
(c)
0" •
lJ
500
I
u!II!
30000
20000
10000
4:5
0O·
1
1500
10
fIJ~?:
3il
,
,G- •. 0
.0
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21
0
:;,11) ...
~§l
(b)
1000
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7
2000
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t!J
0
111
84
those of controls . It was also reported that during the
neonatal hypothyroid period, i.e. from postnatal days 121, increased numbers of precursor cells/mesenchymal
82
cells are accumulated in the testis interstitium and are
available for differentiation when the hypothyroid
treatment is terminated. This finding was further
strengthened by the recent study of Mendis-Handagama
84
and Ariyaratne ; increased numbers of Leydig cells are
differentiated upon withdrawal of the hypothyroid
treatment (Fig. 4a). Therefore, the mechanism of Leydig
cell hyperplasia seen in the adult rats subjected to a
24
transient neonatal hypothyroid treatment is explained
by the availability of increased numbers of mesenchymal
cells available to differentiate in testes of transiently
hypothyroid rats at the point of termination of the
hypothyroid treatment. Thus, it is logical to assume that
upon withdrawal of the hypothyroid treatment (i.e.
withdrawal of the inhibitor for Leydig cell
differentiation) these accumulated mesenchymal cells are
triggered to differentiate into Leydig cells. In consistent
7 14 21 21 40 10 10
Age (days)
50
2.
40
eo
90
(d)
40
30
.
20
10
.0
0"
...
0
1
7
14 21
21 40 eo 90
Age (days)
Fig. 4 - (a) The number of ALe per testis. In control rats, ALe were first detected at day 14 in few numbers In PTU.water rats the ALC
number per testis was zero up to day 21, and few were seen at day 28 ; (b) Average volume of an ALe; (c) LH.stimulated (LH 100 nglml)
testosterone production per testis in vitro for three hours; (d), LH.stimulated (LH 100 nglml) androstenedione production per testis in vitro for
three hours (Permission taken from the publisher, Arch Androl;49 (2004) 313) [Control rats (0), PTU.water rats (~), PTU rats (.). Significant
differences from the age matching control values are shown with asterisks (*)1
MENDIS-HANDAGAMA & ARIYARATNE: LEYDIG CELLS x THYROID HORMONES
with this hypothesis, Mendis-Handagama and
84
Ariyaratne have demonstrated that upon withdrawal of
the neonatal hypothyroid treatment in rats at postnatal
day 21, two-fold greater number of Leydig cells per
testis compared to untreated controls, have been
observed at postnatal day 40 and continued to be twofold higher in number per testis at postnatal days 60 and
90. This study has also demonstrated that these Leydig
cells in the transiently hypothyroid testes are smaller in
size compared to those of age-matched controls at days
60 and 90 (Fig. 4b). However, LH-stimulated testicular
testosterone and androstenedione secretory capacity in
vitro at day 90 is maintainttd in transient hypothyroid
rats similar to those of control rats (Fig. 4c,d), because of
their increased Leydig cell number per testis. In
consistent with the above studies, Maran et aL. 85 have
also reported a consistent increase in Leydig cell
numbers in rats transiently hypothyroid during
prepubertal ages and a decreased numbers of Leydig
cells were seen in rats subjected to hypothyroidism from
birth to 60 days of age. By contrast, it is reported
75
elsewhere , that the principal mechanism responsible
for the Leydig cell hyperplasia observed in adult rats
subjected to transient neonatal hypothyroidism is
increased proliferation of postnatally differentiated
Leydig cells from day 8 through 50 postpartum. This
concluion is not valid with two lines of evidences
discussed below.
945
First, it is seen that at postnatal day eight, the
postnatally differentiated Leydig cells are absent in the
l8
rat testis 50 and the only Leydig cell type present at this
time (i.e. at postnatal day 8) is the fetal Leydig cell I 8.50.
Second, it is an established fact now that Leydig cell
differentiation in the prepubertal testis 50,82,83 (Fig. 5) and
in the adult testes following EDS treatment is arrested
53
with hypothyroidism • Immunocytochemistry for 313HSD, the universally accepted marker for all
steroidogenic cells, has shown that not only the mature
Leydig cells, but all other cell types in the Leydig cell
lineage (i.e. progenitors, newly formed Leydig cells and
immature Leydig cells), except for the mesenchymal
cells are absent in testes of the hypothyroid rats.
Therefore, it is clear that there are no Leydig cells in the
hypothyroid testes to proliferate and produce increased
numbers of Leydig cells in the adult testicles of such
75
rats, although that has been concluded by Hardy et aL. .
It is also important to note that the relationship between
mesenchymal and Leydig cells during the process of
postnatal Leydig cell differentiation in rats subjected to
transient neonatal hypothyroidism is similar to control
rats (Fig. 6); the ratio of Leydig: mesenchymal is 2: 184.
Polychlorinated biphenyls (PCBs) are widely spread
environmental contaminants with long half lives. PCBs
86 88
disrupt the thyroid gland function in humans - and in
89 95
many other species of mammals, such as the rat - and
.
f
[92
0 Cooke et a.
an d
the grey seal 96 . The observatIOns
.'
!
.'
..
"
..
{.
,
f
#-"
S
..1
...
",
f "';
. , ,:
~ ', S
"" .
.~
..
. ~:'';.
. -.,
, .1'.- •
--;., ~t:c ' ~.... ~
.
~..
~~
,,...~"1" ~'.•
~-l
... ;
..
',.
>
Fig. 5 - Representative Micrographs to show II ~HSD I immunocyt('chemistry in testes interstitium - (A and D) 28 and 40 days old control
rats; (8 and E) transiently hypothyroid rats; and (C and F), hypothyroid rats, respectively. 11j3HSD1 positive cells (arrow) were present in
control rats In few numbers at day 28 (A), and more at day 40 (D). They were, absert at day 28 (8), but present at day 40 (E) in transi'!1'.tly
hypothyroid rats; and were absent in hypothyroid rats at both days (C and F). [S=Serr iniferous tubule, l=interstitium of the testis (Permission
taken from the puulisher, Arch Andnl, 49 (2004) 313)]
946
INDIAN J EXP BIOl, NOVEMBER 2005
97
Kim et al. suggest that PCB exposure during the
neonatal period subject these rats to undergo a transient
hypothyroid status and cause an interference in the
normal process of Leydig cell differentiation during
prepuberty to produce a defect in the steroidogenic
function of their Leydig cells. Continuous exposure of
lactating mothers to polychlorinated biphenyls (PCB)
causes significant effects on Leydig cell structure and
function, somewhat reminiscent of transient neonatal
hypothyroidism, e.g. Leydig cell hyperplasia, hypotrophy
~r---------------------------------'
J
2S
~
leydig Cell
•
Meosencllyrmi OIls
-
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eo
o
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100
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PTU Treated
45
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.
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Hyperthyroidism during prepubertal period causes
accelerated Leydig cell differentiation in the rat
52 83
testes • . Additionally, with thyroid hormone treatment
it is seen that greater numbers of mesenchymal cells are
produced and recruited into the differentiation pool to
52
increase the number of Leydig cells in the prepubertal
period, as well as following EDS treatment53 . These
findings indicate that thyroid hormones cause
proliferation of mesenchymal precursor cells and
acceleration of their differentiation into Leydig
progenitors; this is in addition to its effects of enhanced
proliferation of progenitors and newly formed Leydig
51
cells in the prepubertal rat testis •52 . It is logical to
accept that the effect of thyroid hormones on the onset of
mesenchymal precursor cell differentiation to begin the
process of Leydig cell differentIation is direct.
Demonstration of the presence of thyroid receptor
75
mRNA in mesenchymal precursor cells adds support to
this. In addition to the direct action of thyroid hormone
on mesenchymal cells, it is also possible to speculate that
thyroid hormones may have an indirect effect on
mesenchymal cell differentiation into progenitors in the
postnatal testis. A logical hypothesis can be built on the
known facts on thyroid hormone action of Sertoli cell
maturation and anti-Mullerian hormone (AMH)
production by the Sertoli cells in the neonatalprepubertal testis.
Y .O.564x • 5.8961 /
--Uneer (MBsencllyrmi
QIIs)
lileer (Leydig Gels)
Vi 35
~
II
0
and reduced capacity to produce testosterone in vitro in
' 1atIOn
·97
response to LH sttmu
.
40
60
-80
100
Age (Days)
Fig. 6 - Numbers of AlC and mesenchymal cells per testis
increase linearly with age in control (a) and transiently hypothyroid
rats (b). The rate of increase was 2.fold greater in Leyclig cells than
the mesenchymal cells (ie AlC: mesenchymal was 2: 1) in both
treatment groups [Permission taken from the publisher, Arch
Androl, 49 (2004) 313]
Anti-Mullerian hormone (AMH) which is also named
as the Mullerian inhibiting substance (MIS) is a member
of the transforming growth fact ~ (TGF~) family of
cytokines that includes TGF~, activins, inhibins and the
bone morphogenetic proteins. In the developing fetal
testis, AMH produced by the Sertoli cells causes
98
regression of the Mullerian ducts • AMH production by
the rat Sertoli cells decreases gradually and dramatically
after the 3'd and 5 th postnatal days, respectively, and is
present at a very low level on the 20 th postnatal da/ 9•
When treated with triiodothyronine, a dose-dependent
decrease in AMH mRNA production by the cultured
lOO
immature Sertoli cells has been reported • Although the
measurement of AMH mRNA is r.ot an accurate index
of AMH production in these cells 10, this observation i!'
interesting with respect to the possible indirect role of
thyroid hormones on Leydig cell differentiation. Based
on these information, it is possible to hypothesize that
thyroid hormones down regulate I MH production by the
Sertoli cells in the prepubertal testis to allow Leydig
MENDIS-HANDAGAMA & ARIY ARATNE: LEYDIG CELLS x THYROID HORMONES
cells to differentiate; AMH is suggested as a negati ve
regulator of postnatal differentiation of Leydig cells 'o, .
Fig. 7 shows a schematic diagram of the hypothesis on
the mechanism of action of thyroid hormones in
mesenchymal precursor cell differentiation into the
Leydig cell progenitors to begin the process of postnatal
differentiation of Leydig cells.
Testicular steroidogenesis and thyroid hormones
Thyroid abnormalities have long been known to cause
reproductive disturbances in the male 'o . Studies on this
subject have been focused mainly on the effects in
Sertoli cells because it is generally believed that the
Sertoli cell is the primary target for thyroid hormones in
lO
the postnatal testis . Nevertheless, as reviewed in this
paper, other findings suggest that thyroid hormones have
a close association with the function of the Leydig cells
in the adult testis by influencing the hypothalamopituitary-testicl!iar axis; thyroid dysfunction often
resulted in abnormalities of gonadotropin release, sex
steroid metabolism and testicular function. The presence
of nucleitr T3 receptors in gonadotrophs of the rat
pituitary gland is supportive of the effects of thyroid
· reIease 102 .
hormone on gona dotropm
The most common clinical feature in boys who suffer
from juveni Ie hypothyroidism is enlargement of the testis
(macroorchidism) without the symptoms of excess
androgen secreti on 103. Endocrinological investigations
have revealed that many of these boys have elevated
serum FSH levels, but normal LH and testosterone
'
suggest a I'Itt Ie or no
Ieve Is 104'105. Th ese 0 b servatlOns
effect of primary hypothyroidism on Leydig cell function
in these juvenile subjects. Furthermore, increase in
Leydig cell numbers is not evident in biopsy samples
'" e
ta ken f rom these b oys 106107
. . H uwever, untreat ed Juvem
thyroid deficiency is documented to be destructive to
l /ndirect ;
L~~~"!,~.t
=~~~~
-
II -
fAMtr]
Fig. 7 - Hypothesis on thyroid hormone action on mesenc:hymal
cell differentiation into Leydig progenitor cell. Thyroid hormones
act directly on mesenchymal cells to trigger the onset (ie +ve
regulation). Anti-Mullerian hormone (AMH) produced by the
immature Sertoli cells arrests Leydig cell differentiation. Thyroid
hormones act on immature Sertoli cells to cause maturation and
therefore, it is possible that this action may inhibit the production of
AMH. Withdrawal of AMH action could trigger the onset of
mesenchymal cell differentiation
947
testicular tissue in the adult because, atrophy of
seminiferous tubules, fibrosis of basement membrane
and interstitial spaces and degeneration of Leydig cells
lO8
have been observed under such conditions . In adult
males, who are hypothyroid due to thyroid diseases,
thyroidectomy, or administration of chemicals such as
propylthiouracil, a marked decrease in body, testis and
accessory sex organ weights 109 have been reported.
Additionally,
serum concentrations of testosterone85.108.IIO-1I2 and Leydig cell responses to
exogenous gonadotropinsI08.11O.1I2 are observed to be
reduced. Moreover, morphological changes in testes
such as reduced numbers of Sertoli and Leydig cells, a
reduced tubular diameter, interstitial edema and
thickening of basal membrane of seminiferous tubules
have been reported in the adult hypothyroid
..
rnaIes 108111113
. . . B y contrast, patients
wit h G raves d'Isease
(chronic hyperthyroidism) demonstrate high levels of
total serum testosterone, estradiol and gonadotropic
4 117
hormones" . . However, serum level of free
testosterone remains near normal in these patients due to
stimulated secretion of sex hormone binding globulin
(SHBG) by elevated thyroid hormone in the
. Iat'IOn 115116
clrcu
' . Add"Ihona 11 y, an exaggerated"
pitUitary
and testicular responses to exogenous GnRH are seen in
8
hyperthyroid patients" ; these findings demonstrate an
altered hypothalamo-pituitary-testicular axis under
hyperthyroid conditions.
Disturbances in the hypothalamo-pituitary-testicular
axis have also been observed in animals under abnormal
thyroid conditions. In prepubertal rats, long term
I19
administration of T3 result in reduced serum FSH
whereas, acute effect of T3 increases FSH in the
l20
circulation . Similar changes in blood levels of FSH
have been noted following chronic treatment of T3 in
adult rats as wen 121 • Nevertheless, prepubertal
hypothyroidism causes permanent suppression of
. Ieve Is 1121· 22·124 , wh'l
.
f
gonad otropm
1 e suppressIOn 0
l26
thyroid function in adult rats has Iittle '25 or no effect
on serum gonadotropin levels.
Several reports have shown that the total serum
testosterone content in adult rats subjected to
experimental hypothyroid conditions induce via
propylthiouracil (PTU) during prepubertal rzeriod, is not
1
different from that of control anirnals 24• 2. However,
Antony et ai.112 and Maran et al. 85 have documented
reduced serum testosterone concentrations in mature rats
which are hypothyroid due to thyroidectomy or feeding
methimazole. The differences among these observations
INDIAN J EXP BIOL, NOVEMBER 2005
948
could be explained by differences in the age at which the
anilllais have been made hypothyroid, the duration of the
treatment and the method of inducing the hypothyroid
state in the experimental animals. In former
investigations, rat pups were exposed to PTU from birth
to day 21 of age whereas Maran el al. 127 continued to
feed the animals with methimazole for 60 days,
beginning from birth. Antony et al. 112 performed
thyroidectomy on their experimental rats at the age of30
days. In a recent report by Rao el al. 128, reduced plasma
testosterone levels are reported in 50 days old rats
subjected to prepubertal hypothyroidism; these findings
probably indicate a need of longer time for recovery
from the effects of the thyroid hormone deficiency.
l29
Weiss and Burns have reported that there is no change
in serum testosterone levels in rats subjected to either
hyperthyroid or hypothyroid during adult life.
Daily T3 treatment following the EDS injection to adult
rats causes detectable serum testosterone levels on day 14
post EDS in contrast to control EDS rats, where the serum
53
testosterone levels are still undetectable (Fig. 8a). These
findings have been further confinned by the observations on
LH-stimulated testicular testosterone secretory capacity in
vitro of these rats; detectable amounts of testosterone have
been seen in the testicular incubates ofT3-treated EDS rats
in contrast to control EDS rats (Fig. 8c). Twenty-one days
following EDS treatment, testosterone levels in serum and
testicular incubates of the TI-treated EDS rats have shown
significant increase when compared to control EDS rats
(Fig. 8a and c). The same pattern has been seen for
serum androstenedione and LH.stimulated testicular
androstenedione production in vitro in these control and
T3-treated EDS rats (Fig. 8b and d).
250~~======~----------1
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g~
B£c:
c
• Control
200
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E ;
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0.25
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14
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.Control
Ell Hyperthyroid
50
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45
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ClI ......
40
35
30
0._
!I)
25
"0
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....
.r:.
4000
a.
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c:j2
3000
2000
"0
4)
OJ
c:
iii---
1000
B
10
c:
5
<{
0+----+---+--1;;iiiL4_
14
I
15
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"0
7
I:
b
Control
Hyperhllyroid
~ta, 20
a
2
21
14
7
21
6000~--------------------------,
5000
a
C
O+------r----~--~~r
2
b
.Control
0.3
"Om
c:
c ___
-IV
to- !:;
~
0 "0
._
EI Hyperthyroid
I/J
a
E.2
15
I/J
<:>
0.35
Ql
c:
D
0
21
2
7
14
21
Days after EDS
Days after EDS
Fig. 8 - Testosterone and androstenedione in serum and testicular incubates of EDS lIeated control rats and EDS+T3 treated rats.
Testosterone in serum (A); and testicular incubates (B) are first detected at 14 days after the EDS treatment in EDS+T3treated rats and not in
control rats. This observation confirms the finding that new '- eydig cell differentiation has taken place in the EDS+To treated rats, but not in
the EDS treated control rats. On day 21 after the EDS treatment, leveL, of testosterone in serum and testicular incubates were two fold greater
in the EDS+T 3.treated rats, which had greater Leydig cell number in ::ontrast to the EDS.treated control rats. Figs C and D demonstrate the
levels of androstenedione in serum and testicular incubates in these two rat groups. They were greater in EDS+T3 treated rats compared to
EDS treated control rats and could be explained by the greater number of newly formed adult Leydig cells in the EDS+ T3.treated rats
[Permission taken from the publisher, Bioi Reprod;63: (2000) 1115]
MENDIS-HANDAGAMA & ARIYARATNE: LEYDIG CELLS x THYROID HORMONES
Leydig cell steroidogenesis and thyroid hormones
Effects of thyroid hormones on fetal and newly
formed adult Leydig cells in the postnatal testis - Fetal
Leydig cells are present in the postnatal testis and during
the neonatal period they are the primary source of
testicular testosteronel 8,26,82,130, During neonatal
hypothyroidism in rats, i,e, from birth-21 days of age,
fetal Leydig cell size and LH-stimulated testosterone
82
secretory capacity in vitro remain unchanged ,
However, if the hypothyroid status is continued beyond
this period, fetal Leydig cells show cell atrophy and
therefore, have reduced potential for LH stimulated
84
testosterone secretion in vitro (Fig, 4c). Moreover,
studies in neonatal rats have shown that daily
subcutaneous injections of T3 from birth to 21 days of
age significantly reduce the LH stimulated testicular
testosterone capacity in vitro, in contrast to the control
rats, which show no change in the testosterone secretory
52
capacity at least up to day 12 , Morphological studies
have shown that this reduced testosterone secretory
capacity of testes in T3 treated rat pups is due the early
atrophy of fetal Leydig cells in these rats, is due to T3
52
treatment . These observations show a relationship
between thyroid hormones and fetal Leydig cell structure
and function in th..! neonatal testis, Testicular
androstenedione secretory capacity per testis in response
to LH stimulation in vitro in these neonatal rats under a
hyperthyroid status is significantly increased and is
explained by the increased number of newly formed
adult Leydig cells in the testes of hyperthyroid rat
pUpS52, This observation is explained by the fact that
instead of testosterone, the newly formed adult Leydig
cell secrete mainly androstenedione and Sa-reduced
',
II y, en h anced capacity
' to
an d rogens 2684131132
. , , . Add
. ltiona
secrete androgens ip individual Leydig cells could be
attributed to the increased amounts of steroidogenic
112
133
' 134135
enzymes ,cAMP and StAR protem . generated
in these Leydig cells in response to thyroid hormone
stimulation,
Effect of thyroid hormones on mature adult Leydig
cells Direct effects of thyroid hormones on
steroidogenesis in Leydig cells in vivo or in vitro have
not been studied extensively; however, available reports
clearly show that thyroid hormones have a significant
role in this process. It is seen that isolated Leydig cells
from hypothyroid adult rats secreted less testosterone,
both under basal conditions as well as in the presence of
cAMP and non-cAMP mediated stimulatory
substances 133. These studies 33 further report that this
949
reduction of testosterone production is due to decreased
synthesis of cAMP and reduced activity of the enzymes
in the androgen biosynthetic pathway and not due to
changes in LH receptor content in Leydig cells.
Moreover, it is reported that culture of Leydig cells
isolated from sexually mature rats with thyroid hormones
result in stimulated secretion of testosterone and
estrogen under basal conditions as well as in response to
l36
LH stimulation in a dose dependent manrer ; the
maximum stimulatory dose of LH is ~\O nglml.
Additionally, treatment of mouse Leydig cells with T3
coordinately augmented the levels of steroidogenic acute
regulatory (StAR) protein and StAR mRNA and steroid
production 134.135. StAR protein is involved in
intracellular cholesterol transport mechanism during LH137
stimulated steroidogenesis in Leydig cells • Because
the effects of T4 in vivo (T4 is restricted to the vascular
pool) are mediated via T3 (T4 is converted to T3 in
I38
target tissues ), it is possible to suggest that the
stimulatory effects ofT3 on Leydig cell steroidogenesi<.
irt vitroI27.134.135 reflect the acute effects of thyroid
hormones on Leydig cell steroidogenesis in vivo.
Synthesis of androgens in Leydig cell is a complex
process which involves several sequential steps such as
transport of cholesterol from the cellular deposits like
lipid droplets and plasma membrane to the outer
mitochondrial membrane, translocation of cholesterol
from the outer membrane to the inner membrane of the
mitochondria, enzymatic cleavage of the side chain of
the cholesterol molecule to produce pregnenolone and
subsequent conversion of pregnenolone into other
139 Th e most
'
' enzymes.
sterOl'dogemc
an drogens usmg
important sources of cholesterol for steroid production in
Leydig cells appear to be free cholestewl within the cell;
denovo synthesis of this compound and store it in the
cell membranes or to some extent as lipid droplets provides a constant supply of substrate in this
process 140-142. The trafficking of cholesterol from its
intracellular stores to the outer membrane of
mitochondria through cytoplasm is one of the poorly
understood process in Leydig cell steroidogenesis.
However, the involvement of intracellular vesicular
transport system and specific or non-specific cholesterol
binding proteins such as sterol carrier protein-2 (SCP 2)
has been documented in several studies28.143,144,
Additionally, the cytoplasmic organelle peroxisome
' I roIe m
' th'IS process28145146
I
pays
a cruCIa
' ' . Th e
trar:slocation of cholesterol from outer mitochondrial
membrane to the inner mitochondrial membrane where
950
INDIAN J EXP BIOL, NOVEMBER 2005
the first enzyme of the steroidogenic pathway is located
has been studied extensively during the past severai
years and is proposed to be the rate limiting step in the
steroid biosynthetic pathwayl47. A number of proteins
including sterol carrier protein-2, steroidogenesisinducing protein, steroidogenesis activator polypeptide,
peripheral benzodiazepine receptor and StAR which may
act as carriers in this phenomenon have been
'
I'd entl'f'Ie dl37 ' 143 . T
e h
precIse
nature 0 f "
actlOn In
mitochondrial transport of cholesterol is not fully
understood for many of these proteins. However, it has
been demonstrated that in response to LH stimulation in
vivo, peroxisomes,
transport cholesterol into
l45
mitochondria in luteal cells and Leydig cells l46 . As
thyroid hormones have been shown to cause peroxisome
'J'
'
' h epatocytes 148149
' prompts weer
h th the
m
. , It
pro IIleratlOn
enhanced steroidogenic capacity in Leydig cells
following thyroid hormone treatment is at least in part
dl}e to its effect on peroxisomes. It is also reported that
StAR protein rapidly delivers cholesterol into
mitochondria in steroid producing cells after acute
137
stimulation by tropic hormones • Once the cholesterol
reaches the inner mitochondrial membrane, it is converted
into pregnenolone using mitochondrial enzyme
cytochrome P450 side-chain cleavage and subsequently to
testosterone by cytoplasmic enzymes 3~-hydroxysreroid
dehydrogenase, cytochrome P450 17a-hydroxylase and
17~-hydroxysteroid dehydrogenase 150.
It is established that Leydig cell steroidogenesis is
primarily dependent on luteinizing hormone secreted
from the anterior pituitary. Additionally, many other
factors including hormones, cytokines and growth
factors are demonstrated to be affecting the rate of
I51 152
steroid secretion from these' cells . • LH action on
Leydig cells is brought about by binding the hormone to
specific receptors, LHR, on the cell membrane and
,.
actlvatmg
cAMP secon d messenger sys tem 153,155
Although, modulation of cellular level of cAMP is the
main mechanism for affecting Leydig cell steroid
secretion by many hormonal and non-hormonal factors,
use of other second messenger pathways such as protein
kinase-c and phospholipase-c have also been
proposedI 56 ,157. According to Clark et al. 157 , increased
levels of cAMP is largely responsible for the acute
increase of steroid production in Leydig cells by rapid
mobilization of cholesterol from its deposits and speedy
transport of this compound to the mner.mlt~chondrial
membrane with the help of increased synthesis of StAR
protein. Also, chronic stimulaMn of Leydig cell steroid
biosynthesis due to increased activity of steroidogenic
158
enzymes has also been proposed .
The above reviewed information clearly sugg~sts that
thyroid hormones have an important regulatoIJ role on
Leydig cell steroidogenic function. However, the precise
mechanism of action of these hormones on this cell is
not clearly understood. Although it is logical to
hypothesize that the in vivo effects of thyroid hormones
are at least in part mediated via the Sertoli cells,
evidence for direct actions of thyroid hormones on
Leydig cells are seen in vitro studies. Jana et al. 159 were
the first to report on production of a 52 KDa soluble
protein by goat Leydig cells in vitro when exposed to
thyroid hormone. This protein, when added to the
incubation medium could stimulate Leydig cells to
59
secrete testosteronel . Adult rats which were subjected
to thyroidectomy at 30 days of age produced lest:.
testosterone and cAMP in response to the stimulation by
LH and testes of these animals show less specific
activities for 3~-HSD and 17~-HSD enzymes I12. Leydig
cells isolated from adult rats which have been made
hypothyroid by feeding PTIJ for one month, produce less
steroids compared to untreated controls 133 • In addition,
Leydig cells from these hypothyroid rats show a less
response to cAMP mediated as well as non-cAMP
mediated stimuli indicating a reduction in the activity of
the enzymes in the steroid biosynthetic pathway. All the
above information add support to the concept that
thyroid hormones are important for the steroidogenic
function of Leydig cells.
More recent studies have shown that stimulatory
effect of thyroid hormones on Leydig cell
steroidogenesis is associated with increased synthesis ot
StAR protein by these cells, mediated through
'
f actor- 1134-136
M oreover,
th ese
sterOl'd ogemc
investigations have demonstrated an acute stimulatory
but a chronic inhibitory effect of thyroid hormone on
steroidogenic enzymes and LH receptor content in
mouse tumour Leydig cells 135. Such observations clearly
suggest that further studies are required to establish the
precise mechanism of action of thyroid hormones on
Leydig cell steroidogenesis.
Effect of thyroid hormones on aged Leydig cells - A
progressive decline in circulating testosterone levels i~
'h agmg
' m
' h umans 160.161 an d ra ts25,162.163 . M any
seen WIt
studies have revealed that Leydig cells undergo atrophic
27
,,25163
c hanges m
SIze' an d organe 11 e con ten t ,164. as a resu It
of aging. Interestingly, it is also seen that serum thyroid
'h agmg
' 165,166 questlOnIng
"
hormone 1evels are red uced WIt
MENDIS-HANDAGAMA & ARIY ARATNE: LEYDIG CELLS x THYROID HORMONES
whether the atrophic changes and malfunctional status in
the aged Leydig cells are, at least in part, caused by the
hypothyroid status in the aged rats. Also, it has been
demonstratedl63.167 that exogenous supplementation of
thyroid hormone alone to aged Brown Norway rats (18
months of age) for 28 days could reverse the LHstimulated testosterone secretory capacity per testis and
per Leydig cell in vitro by 71 %, Leydig cell size by 82%
and serum testosterone levels by 33% compared to three
951
month old control rats. Reversibility (100%) in LHstimulated testosterone secretory capacity per testis and
per Leydig cell in vitro and Leydig cell size is achieved by
the combined treatment ofT4 and LH I63,167. These studies
indicate that thyroid hormones are important in maintaining
the steroidogenic function of Leydig cells. Representative
Leydig cells of control rats of 3 and 19 month of age and
T4-, and T4+LH- treated Brown Norway rats are shown
in Fig. 9.
Fig. 9 - Representative light micrographs to demonstrate Leydig cells in 3, 6, 12 and 19 month old (A, B, C, and'D, respectively) and.LH.
(E), T4. (F) and, LH+ T4.treated (G) Brown Norway rats. Aging from 3 to 19 months causes atrophy of Leydig cells. The reduced steroidogenic
potential of these aged Leydig cells, in vitro was partially recovered, (partial rejuvenation) by exogenous treatment of either LH (E) or T4 (F)
and fully recovered (100% rejuvenation) by LH+ T4 [Permission taken from the publisher, Bioi Reprod, 66 (2002) 1359]
952
INDIAN J EXP BIOL, NOVEMBER 2005
Presence of thyrotropin releasing hormone in
Leydig cells
Thyrotropin-releasing hormone (TRH) is produced by
the hypothalamus and is a tripeptide-factor which
stImulates thyrotropin (TSH) synthesis and secretion by
the thyrotrophs of the anterior pituitary gland 168,169. TSH
stimulates the thyroid gland to synthesize and secrete T3
and T4. In the hypothalamo-pituitary-thyroid axis , TRH
plays a central regulatory role. Therefore, it is
interesting to note that TRH, TRH mRNA and TRH
receptor (TRHR) gene expression occurs in
Leydig cells of many mammalian species including
human17O, ratI 71-174, mouse175, bull l76 and hamster 177 • In
humans17O, mouse175 and rats 173. TRH and TRH-R
expression in the testis is exclusively seen in Leydig
cells. Additionally, it is interesting to note thu.
suppression of circulating TRH to non-detectable; levels
by oral administration of nitrates has caused
suppression Of Leydig cell steroidogenic function in
l76
bulls . It is also seen that TRH mRNA expressi~m in
~he rat testis is development dependent; the earliest
detection is at postnatal day IS , and the si!:;llal is
increased progressively on days 20, 35, 60 and 90 173.
However, it is not certain whether TRH activity
in the testis is regulated by circulating thyroid hormone
levels. This is because, although drug induced
hypothyroidism has been reported to cause increase in
TRH
activity178,
others
have
shown
that
testicular TRH-mRNA concentration is indepenc!ent of
the thyroid state 170. In the male reproductive system,
prostate is another organ which also expresses TRH
immunoreactivit/79-181 This observati :m has led to the
of
a
possiole
involvement
of
speculation
prostatic TRH in the regulation of circulatory thyroid
hormone levels by modulating the activity of :hyroid
gland 180,182 However, to date, simila' regulatory ~ffects
of testicular TRH activity on circulatory thyroid
hormone levels have not been demonstrated, ' Although
the precise function of TRH in Leydig cells
is not clear at present, some investigators suggest that
'
, 170171
. 171-173
TRH may functIOn as a paracnne ' or autocnne
factor to regulate testicular function, One such paracrine
role of TRH is thought to be to serve as a inhibitory
modulator of gonado~ropin-stimda~~d te;stm:~erone
secretion 180. AI: these findin~s suggest thal the pr~sence
ofTRH in Leydig ~ells may he. Ie a sigr.;ficant role in the
testis whicr. needs to be letenr;nec in fe~ure
investigatior.s.
Thyroid hormone action on Leydig cells mediated
through Sertoli cells
Influence of Sertoli cells on Leydig cells- 2-;:veral
lines of evidence have been used to suggest a possible
influence of Sertoli cells/seminiferous tubules on the
development and function of Leydig cells, One of the
best examples for such an interaction is the effect of
exogenous treatment of follicular s~imulating hormone
(FSH) on these cells; FSH is trophic to Sertoli cells , In
early 1970s it has been demonstrated that FSH treatment
on hypophysectomized prepubertal rats results in Leydig
cell hypertrophy and hyperplasia, together with increased
numbers of luteinizing hormone (LH) receptors in the
testis and enhances testicular capacity to secrete
testosterone in addition to stimulated growth of
, 'C
'
have
semmllerous
tu b uIes 184-187 . Th ese f'md mgs
supported the concept of influence of Sertoli cells on
Leydig cell differentiation and function. However, it has
been claimed by others that contamination of small
amounts of LH with FSH preparations used in these
experiments is the cause for the observed Leydig cell
response I88 ,.89 delaying full appreciation of these
findings until later. With the development of techniques
for the preparation of pure FSH without LH
con~amination or synthesis of recombinant human FSH,
· thes~ studies have been repeated recently in animal
models 190,191 as well as in humans l92 and have confirmed
the above findings . Ethane dimethane sulphonate (EDS)
treated adult rat model has also been used to investigate
the effects of pure preparations of FSH on Leydig cell
development and function. In EDS treated adult rats,
passive neutralization of circulatory FSH results in
l93
significant reduction of Leydig cell hnction indicating
possible irlvolvel!lent of Sertoli cells in the regulation of
Leydig cells in the adult testis. In earJer studies, it is
uncertain how exogenously administered FSH affected
Leyuig cell development and function because receptors
l94
for this hormone is confined only to the Sertoli cells .
Possible involvement of paracrine factors secreted by the
Sertoli cells under the regulation of FSH has been
proposed as mediators of the above actions.
Further evidence for Sertoli-Leydig cell interactions
comes from experimental disruption of spermatogenesis
of methods
including
induced
by
variety
'd
'
28195
X'd"
196
'
,
A
cryptorc h1 Ism',
' ,lrra IltlOn , vltamm
'·
197
d
h
:98
ff
t
d
eat treatment
or e . eren uct
d e fIClency ,an
ligation l99 resulted in morphological and functional
changes in L~ydig (dIs. Abnormal cytological feature"
and altered hormone secretory activity have been
MENDIS-HANDAGAMA & ARIY ARATNE: LEYDIG CELLS x THYROID HORMONES
commonly observed in Leydig cells that are adjacent to
the damaged seminiferous tubules l97 . In these
experiments, Leydig cells appear normal in the vicinity
of undamaged tubules indicating an influence of a local
effect from the damaged tubules on the Leydig cells.
Experimental cryptorchid model has also been used
extensively to understand the local regulation of Leydig
cells by the seminiferous tubules; Leydig cells undergo
lO
hyper-troph/ , hypotroph/8 and/or hyperplasia28 . In
addition, careful observations of Leydig cells adjacent to
seminiferous tubules of different stages have shown that
Leydig cells close to tubules of stages vn and vm are ·
Iarger than those 0 f teo
h th er stages 201-203 an d con tam
more smooth endoplasmic reticulum which indicates
ability to secrete more testosterone204 . To extend these
observations, Leydig cells have been co-cultured with
isolated fragments of seminiferous tubules of different
stages and the results have demonstrated a stimulatory or
inhibitory effect on Leydig cell function by the
. Oferous tu b u Ies 0 f dO
semtOl
1fferent stages205·207 .
0
Co-culture of pure preparation of isolated Leydig cells
and Sertoli cells has been used to demonstrate the direct
effects of Sertoli cells on Leydig cells. In such cultures,
presence of Sertoti cells not only increase the basal
production of testosterone from the Leydig cells but also
enhance the s(eroid synthetic response of these cells to
LHlhCG stimulation. Furthermore, in this culture
system, pre-treatment of Sertoli cells with FSH,
augments the testosterone secretion from Leydig cells
O d capaCIty
even furt her 151 '208·210 . In para11e1WIth
mcrease
of steroid production, the co-cultured Leydig cells show
morphological changes such as increased SER,
cytoplasmic lipid, LH receptors and steroidogenic
enzyme activity which are characteristics of actively
.
sterOl°d syntheS1zmg ce II s 153203
' , demonstratmg
a
multitude of changes in Leydig cells under the influence
of Sertoli cells. Even without the physical presence of
Sertoli cells in the culture system, Sertoli cell
conditioned culture media are able to stimulate Leydig
cell steroidogenic activity in culture, demonstrating the·
involvement of paracrine factors from Sertoli cells in this
process 153. Pre-treatment of Sertoli cells with FSH before
the collection of conditioned medium is able toCurther
enhance the steroid production of Leydig cells211
demonstrating that these paracrine factors are regulated
by FSH.
During the past few years, knockout animal models
have been used to investigate the role of FSH in tht
c!evelopment and funciion of Leydig cells. In FSH B
0
0
0
0
953
subunit null mutants, testicular Leydig cell number and
serum level of testosterone are normafl2. In contrast, in
FSH receptor knockout mouse, the number of adult
Leydig cells, circulating testosterone, and mRNA of
several steroidogenic hormones in Leydig cells are
significantly reduced 2l3 ,214. Transfection studies of
normal and mutant FSH receptors in tumor Leydig cells
have shown that normal FSH receptors have significant
constitutive activity even in the absence ofFSH binding
214 ThoIS
whereas mutant receptor h as no such activIty.
constitutive activity of normal receptors could exert a
considerable effect on Leydig cells. These observations
suggest that FSH receptors are more important than FSH
itself in Sertoli cell regulation of Leydig cells.
0
0
Although, there are many reports demonstrating the
possible effects of Sertoli cells on Leydig cell function,
information on the nature of paracrine factors that
mediate these effects are sparse. In living animals as well
as in cultures, Sertoli cells secrete large number of
proteins215 ,216 and secretion of some of these proteins are
regulated by FSH. The functions of most of these
proteins are unknown, but some of them may act as
paracrine factors. Different investigators have partially
purified Sertoli cell proteins with molecular weight in
the range of 10-30 KDa217 ,218, 80 KDa219, and 35-37
220
KDa with acute stimulatory effect on Leydig cells.
Further studies are needed to understand the
physiological role of these and other Sertoli cell products
in the regulation of Leydig cells .
Thyroid hormones on Sertoli cells - Testicular
effects of thyroid hormone may also mediate through
Sertoli cells as in the case of FSH. In support of this
view, thyroid receptors are localized to Sertoli cells in
different stages of development (see section on THR)
and the thyroid hormone is considered to be the most
important regulatory factor of Sertoli cells other than
IO
FSH • It has been shown that a number of
developmental and functional characteristics of Sertoli
cells are under the control of thyroid hormones. In prepubertal animals proliferation of immature Sertoli cells
and their transformation into mature cells are regulated
221
by thyroid hormones . Induced hypothyroidism during
postnatal-prepubetal period in several animal species
causes prolongation of the period of Sertoli cell mitosis
by delaying their maturation into non-proliferating cells,
thus resulting in increased numbers of Sertoli cells in the
'
d 1 testis 124.222
aut
' . Neverth e1ess, exogenous adffilOlstranon
of thyroid hormones during the juvenile period
accelerates the process of Sertoli cell maturation and
0
0
0
954
INDIAN 1 EXP BIOL. NOVEMBER 2005
shortens the period of SertoIi .cell proliferation; this
l
results in a testis that contains fewer Sertoli cel:s 19. The
molecular mechanisms responsible for Sertoli cells to
exit from the cell cycle and initiate the maturation
process are n8t fully understood. However, studies of
223
Buzzard et aZ have demonstrated the involvement of
23
thyroid hormones in this process. Thel have shown
that thyroid hormones stimulate the expression of the
cell cycle inhibitory proteins p27 KiP1 and p21 Cpil which
cause cessation of mitotic division and promotion of
terminal differentiation of Sertoli cells, as also seen in
many other cell types that are undergoing similar
chan~es. Furthermore, the investigations by Holsberger
et at. 24 have also linked the thyroid hormone stimulation
to the induction of cell cycle inhibitory proteins and
terminal maturation of the Sertoli cells.
Maturation of Sertoli cells are characterized by
several known functional changes in these cells which
are at least in part regulated by thyroid hormones. Such
changes in Sertoli cell functions may indirectly mediate
thyroid hormone action on Leydig cells. For example,
together with Sertoli cell maturation, the secretion of
insulin-like growth factor-l from these cells is stimulated
225
by thyroid brmones and this growth factor is known
226
to stimulate differentiation and mitosis of Leydig cells .
Furthermore, aromatase is a P450 enzyme which is
highly expressed in fetal and neonatal Sertoli cells and
responsible for the synthesis of estrogens from
65 227
androgens . • With the maturation of Sertoli cells, the
expression of aromatase enzyme in them is down
regulated and shifted to Leydig cells, and therefore, in
the mature testis the aromatase activity is primarily
localized to the Leydig cells228. In immature Sertoli cells,
thyroid hormone is shown to cause down regulation of
expression of aromatase enzyme65 ,227,229,230, thereby
reducing the production of estrogens from these cells,
Loss of estrogen activity in prepubertal testis may be
important for formation of adult type Leydig cells
because estrogen is known to inhibit differentiation of
mesenchYr.1al cells into Leydig cells in the prepubertal
232
231
testis as well as in EDS treated adult testis . Future
studies would certainly identify many other Sertoli cell
factors that are secreted in response to thyroid hormone
stimulation which have regulatory roles on Leydig cell
function. The existence of major variations in the
expression patterns of mRNA for othenmportant Sertoli
cell proteins detected under hypothyroid conditions in
vitro 64 support this concept. Such a factor, known to be
secreted from the Sertoli cell for a long time, but
identified recently to be prominent in influencing ~I)e
postnatal differentiation and function of Leydig cells is
anti-Mullerian hormone (AMH) which is also called the
Mullerian inhibiting substance (MIS).
Conclusion
It is clear from the reviewed literature that thyroid
hormones have several important roles in Leydig cells in
the postnatal testis. Thyroid hormones are essential to
stimulate the onset of Leydig cell differentiation,
maintain the steroidogenic function with advancement of
age and stimulate systhesis of proteins/enzymes e.g.
StAR required for testosterone production and
proliferation of the organelle peroxisome required for the
transport of cholesterol into mitochondria during
testosterone biosynthesis, However, there are many gaps
in the literature and therefore, future studies should
focus on identifying and addressing these deficiencies to
properly understand and appreciate the functions of
thyroid hormones in Leydig cells. This indeed should be
beneficial to the general health and reproductive
function of the mammalian male.
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