Download Leydig cells, thyroid hormones and steroidogenesis

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>-~-@ 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
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0
:;,11) ...
~§l
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2000
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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
•
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45
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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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.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
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c:j2
3000
2000
"0
4)
OJ
c:
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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.
References
I
Gudernatsch 1 F, Feeding experiments on tadpoles. I. The
influence of specific organs given as food on growth and
differentiation. A contribution to the knowledge of organs wit~
internal secretion. Wilhelm Roux Arch Entwicklungsmech
Organismen, 35 (1912) 457.
2
D' Armiento M, & lannini E A, Thyroid hormone action. in
Thyroid disease: Basic science, pathology, clinical and
laboratory diagnoses, edited by L Troncone, B Shapiro, MA
Satta & F Monaco (CRC Press, Boca Raton, FL) 1992, 145 .
3
Oppenheimer J H, Thyroid hormone action at the molecular
level. in Werner and Ingbar's The thyroid, edition 6, edited by
L E Braverman, & R D Utiger (Lippincott, Philadelphia) 1991 ,
204.
4
Lazar M A, Thyroid hormone receptors: Multiple forms,
mUltiple possibilities. Endocr. Rev, 14 (1993) 184.
5
Shibusawa N, Hashimoto K, Nikrodhanond A A, Liberman M
C, Applebury M L, Liao X H, Robbins J T, Refetoff S, Cohen
R N & Wondisford F E, Thyroid hormone action in the
absence of thyroid hormone receptor DNA binding in vivo. J
Clin Invest, 112 (2003) 588.
6
Evans R M, The steroid and thyroid hormone receptor superfamil y. Science, 240 (1988) 889.
7
Chin W W, Nuclear thyroid hormone receptors. in NucLear·
Hormone Receptors, edited by M G Parker, (Academic Press,
New York) 1991,79.
8
O'Shea P 1 & Williams G R, Insight into the physiologIcal
actions of thyroid hormone receptors from genetically
modified mice J Endocrinol, 175 (2002) 553.
MENDIS-HANDAGAMA & ARIY ARATNE: LEYDIG CELLS x THYROID HORMONES
955
9
Flamant F & Samarut 1. Thyroid hormone receptors: Lessons
from knockout and knockin mutant mice. Trends Endocrinol.
Metab. 14 (2003) 85.
25
Mendis-Handagama S M L C & Gelber S J, Signs of aging are
apparent in the testis interstitium of Sprague Dawley rats at 6
months of age, Tissue Cell, 27 (1995) 689.
10
lannini EA. Ulisse S & D' Armiento M. Thyroid hormone and
male gonadal function Endocrin Rev, 16 (1995) 443.
26
II
Leydig F, Zur Anatomie der mann lichen Geschlechtsorgane
und Analdrusen der Saugetgiere; Z. wiss.Zool. 2 (1850) I.
Ariyaratne H B S & Mendis-Handagama S M L C, Changes in
the testis interstitium of Sprague Dawley rats from birth to
sexual maturity, Bioi Reprod, 62 (2000) 680.
27
Mori H, Hiromoto N, Nakahara M & Shiraishi T, Stereological
analysis of Leydig cell ultrastructure in aged humans, J CUn
Endocrinol Metab , 55 (1982) 634.
28
Mendis-Handagama S M L C, Kerr 1 B & de Kretser D M,
Experimental cryptorchidism in the adult mouse: I Qualitative
and quantitative light microscopic morphology, J Androl, II
(l990b) 539 .
29
Hardy M P, Mendis-Handagama S M L C, Zirkin B R &
Ewing L L, Photoperiodic variation in Leydig cell numbers in
the testis of the golden hamster:A possible mechanism for their
renewal during recrudescence. J Exp Zool, 244 (1987) 269.
30
Sinha Hikim A P, Bartke A & Russell L D, The seasonal
breeding hamster as a model to study structure.function
relationship in the testis, Tissue Cell, 20 (1988) 63.
31
Mori H, Shimizu D, Takeda A, Takioka Y & Fukunishi R,
Stereologic analysis of Leydig cells in normal guinea pig testis,
J Eleclron Microsc, 29 (1980) 8.
32
Mendis-Handagama S M L C, Mitosis in normal guinea pig
Leydig cells, J Androl, 12 (1991) 240.
33
Walters J R Walters, luniewicz P E, Oesterling 1, MendisHandagama S M L C. Zirkin B R & Ewing L L, The effect of
inhibition of aromatase enzyme activity on Leydig cell
structure and function in beagles, Endocrinology, 123 (1988)
2223.
34
Kaler LW & Neaves W B, Attrition of the human Leydig cell
population with advancing age Anal Rec, 192 (1978) 513.
35
Panigua R, Rodriguez M C, Nistal M, Fraile B, Regadera J &
Amat P, Changes in surface area and number of Leydig cells in
relation to the six stages of the cycle of the human
seminiferous epithelium, Anal Embryol, 178 (1988) 423 .
36
Mori H, Testicular interstitial cells (Leydig cells), Cell, 12
(1980) 11.
37
Johnson L & Neaves W B, Age.related changes in the Leydig
cell population, seminiferous tubules and sperm production in
stallions, Bioi Reprod, 24 (1981) 703.
38
Lunstra D D & Schanbacher B D, Testicular function and
Leydig cell ultrastructure In the long-term bilaterally
cryptorchid rams, Bioi Reprod, 38 (1988) 211.
39
Mendis-Handagarua S M L C, Kerr J B & de Kretser D M,
Experimental cryptorchidism in the adult mouse III Qualitative
and quantitative electron microscopic morphology of Leydig
cells, J Androl, 12 (1991) 335.
40
Sinha Hikim A P, Chakraborty J & Ihunjhunwala J S, Effects
of experimental torsion of the spermatic cord on Leydig cell
function In the guinea pig testis: An ultrastructural
stereological analyses, Urol Res, 14 (1986) 253 .
41
Sinha Hikim A P, Chakraborty 1 & Jhunjhunwala J S,
Unilateral (orsion of spermatic cord in men : effect on Leydig
cells, U,-?logy, 29 (1987) 40.
12
13
14
15
Christensen A K, The history of studies on Leydig cells: The
first century; in: The Leydig Cell, edited by AH Payne, MP
Hardy and L D Russell (Vienna, IL: Cache River Press)
1996, I.
Fawcett D W, Neaves W B & Flores M N, Comparative
observations on intertubular lymphatics and the organization
of the interstitial tissue of the mammalian testis, Biol Reprod, 9
(1973) 500.
Mendis-Handagama S L M C , Zirkin B R & Ewing L L,
Comparison of the testis interstitium with testosterone
secretion in hamster, rat and guinea pig testes perfused in vitro,
AmJAnal. 181 (1988) 12.
Mendis-Handagama S M L C, Watkins P A, Gelber S 1 &
Scallen T 1, Leydig cell peroxisomes and sterol carrier
protein.2 in luteinizing hormone. deprived rats, Endocrinology,
131 (1992) 2839.
16
Mendis-Handagama S M L C, Watkins P A, Gelber S 1 &
Scallen T 1, The effect of chronic luteinizing hormone
treatment on adult rat Leydig cells, Tissue Cell, 30 (1998a) 64.
17
Mori H. Tesicular interstitial cells (Leydig Crlls), Cell. 12
(l980a) 11.
18
Mendis-Handagama S L M C, Risbridger G P & de Kreter D
M, Morphometric analysis of the components of the neonatal
and adult rat testis interstitium. Int J Androl, 10 (1987) 525.
19
Sinha Hikim A P & Hoffer A P, Quantitative Anaysis of germ
cells and Leydig cells in rats made infertile with gossypol,
Contraceptioll, 35 (1987) 395.
20
21
22
23
24
Andreis P G, Cavallini L, Malendowicz G, Belloni A S,
Rebuffat P & Nussdorfer G G, Morphological and functional
responses of rat Leydig cells to a prolonged treatment with
human chorionic gonadotropins, J Submicrosc Cytol Pathol.
21 (1989) 7010.
Mendis-Handagama S M L C & Ewing L L, Sources of error
in the estimation of Leydig cell numbers in control and
atrophied mammalian testes, J Microscop (Oxford), 159 (1990)
73 .
Mendis-Handagama S M L C, Watkins P A, Gelber S 1,
Scallen T 1, Zirkin B R & Ewing L L,Luteinizing hormone
causes rapid and transient changes in rat Leydig cell
peroxisome volume and intraperoxisomal sterol carrier
protein .2 content. Endocrinology, 127 (I 990a) 2947.
Russell L D, Corbin T J. Ren H P, Amador A G, Bartke A &
Ghosh S, Structural changes in rat Leydig cells post
hypophysectomy: A morphometric and endocrine study,
Endocrinology, 131 (1992) 498
Mendis-Handagama S M L C & Sharma 0 P, Effects of
neonatal administration of the reversible goitrogen
propylthiouracil on the testis interstitium in adult rats, J
Reprod Fert, 100 (1994) 85.
956
INDIAN 1 EXP BIOL, NOVEMBER 2005
42
Roosen-Runge, E C & Anderson A D, The development of the
imerstitial cells in the testis of albino rat; Acta Anal, 37 (1959)
125 .
43
Mancini R E, Vilar 0, Lavieri J C, Andrada 1 A & Heinrich 1
J, Development of Leydig cells in the normal human testis; A
cytological, cytochemical and quantitative study; Am J Anat,
58
Murray M B, Zilz N D, McCreary N L, MacDonald M 1 &
Towle H C, Isolation and characterization of rat cDNA clones
for two distinct thyroid hormone receptors, J Bioi Chem. 263
(1988) 12770.
59
Santos A, Freake H C, Rosenburg M E. Schwartz H L &
Oppenheimer 1 H, Triiodothyronine nuclear binding capacity
in rat tissues correlates with a 60 kilobase (kb) and not a
26 kb messenger ribonucleic acid hybridization signal
generated by a human c.erbA probe, Mol Endocrinol, 2 (1988)
992.
60
Barsano C P, Iqbal Z, Pullen G L, Munoz E & Singh S P,
Tissue.specific differences in the compartmentalization of rat
nuclear triiodothyronine receptors, Acta Endocrinol (Copenh).
122 (1990) 181.
61
Palmero S, Maggiani S & Fugassa E, Nuclear triiodothyronine
receptors in rat Sertoli cells, Mol Cell Endocrinol, 58 (1988)
253 .
62
Jannini E A, Olivieri M, Francavilla S, Gulino A, Ziparo E &
D' Armiento
M,
Ontogenesis
of
the
nuclear
3,5,3' .triiodothyronine receptors in the rat testis,
Endocrinology, 126 (1990) 2521.
112 (1963) 203.
44
Baillie A H, Further observations on the growth and
histochemistry of Leydig tissue in the postnatal prepubertal
mouse testis, J Anat Lond, 98 (1964) 403 .
45
Niemi M & Kormano M, Cell renewal in the interstitial tissue
of postnatal prepubertal rat testis, Endocrinology, 74 (1964)
996.
46
Lording D W & de Kretser D M, Comparative ultrastructural
and histochemical studies of the interstitial cells of the rat
testis during fetal and postnatal development, J Reprod Fert,
29 (1972) 261.
47
Byscove A G, Differentiation ofmarnmalian embryonic gonad,
Physiol Rev, 66 (1986) 77.
48
Russell L D, de Franca L R, Hess R & Cooke P,
Characteristics of mitotic cells in developing and adult rat
testes with observations on cell lineage, Tissue Cell, 27 (1995)
105.
63
Chemes H E, Leydig cell development in humans, in The
Leydig Cell, edited by AH Payne, MP Hardy and LD Russell
(Cache River Press, Vienna, IL) 1996 175.
lannini E A, Dolci S, Ulisse S & Nicodem V M,
Developmental regulation of the thyroid hormone receptor a I
mRNA expression in the rat testis, Mol Endocrinol, 8 (1994)
89.
64
Bunick D, Kirby 1, Hess R A & Cooke P S, Developmental
expression of testis messenger ribonucleic acids in the rat
following propylthiouracil.induced neonatal hypothyroidism,
Bioi Reprod, 51 (1994) 706.
65
Palmero S, De Marco P & Fugassa E, Thyroid hormone
receptor beta mRNA expression in Sertoli cells isolated from
prepubertal testis, J Mol Endocrillol, 14 (1995) 131.
66
Buzzard J J, Morrison J R, O'Bryan M K, Song Q & Wreford
N G, Developmental expression pfthyroid hormone receptors
in the rat testis, Bioi Reprod, 62 (2000) 664.
49
50
51
Ariyaratne H B S, Mason JI & Mendis-Handagama S M L C,
Effects of thyroid and luteinizing hormone on the onset of
precursor cell differentiation into Leydig progenitor cells in the
prepubertal rat testis, Bioi Reprod, 63 (2000a) 898.
Ariyaratne H B S, Mendis-Handagama S M L C, Hales D B &
Mason 1 I, Studies on the onset of Leydig precursor cell
differentiation in the prepubertal rat testis, Bioi Reprod, 63
(2000b) 165.
52
Ariyaratne H B S, Mason 1 I & Mendis-Handagama S M L C,
Effects of triiodothyronine on testicular interstitial cells and
androgen secretory capacity of the prepubertal rat, Biol
Reprod, 63 (2000d) 493 .
67
lannini E A, Crescenzi A, Rucci N. Screponi E. Carosa E, de
Matteis A, Macchia E, d' Amati G & D' Armiento M,
Ontogenic pattern of thyroid hormone receptor expression in
the human testis, J Clin Endocrinol Melab, 85 (2000) 3453.
53
Ariyaratne H B S, Mills N, Mason J I & Mendis-Handagama S
M L C, Effects of thyroid hormone on Leydig cell regeneration
in the adult rat following ethane dimethane sulphonate
treatment, Biol Reprod, 63 (2000c) 1115.
68
54
Morris I D, Leydig cell toxicology, in The Leydig Cell, edited
by A H Payne M P Hardy and L D Russell (V;enna, IL: Cache
River Press) 1996, 573
69
55
Teerds K J, Regeneration of Leyc;lig cells after depletion by
EDS: A model for postnatal Leydig cell renewal, in The Leydig
Cell, edited by A H Payne M P Hardy and L D Russell,
(Vienna, IL: Clche River Press) 1996,203
Canale D, Agostini M, Giorgilli G. Caglieresi C, Scartabelli G.
Nordini V, lannini EA, Martino E, Pinchera A & Macchia E,
Thyroid hormone receptors in neonatal, prepubertal and adult
rat testis, J Androl, 22 (2001) 284.
Palmero S, Benahmed M, Morera A M, Trucchi P & Fugass~'
E, Identification of nuclear tri.iodothyronine receptors in
Sertoli cells from immature piglet testes, J Mol Endocrinol, 9
(1992) 55.
56
Mendis-Handagatna S M L C & Ariyaratne H B S,
Differentiation of the adult I .e ydig cell popuLtion in the
postnatal testis, Bioi Reprod, 65'(2001) 660.
57
Oppenheimer J H, Schwartz H L & Surks M I, Tissue
differences in the concentration of triiodothyronine .nuclear
binding sites in the rat: liver, kidney, pituitary, heart, brain,
spleen and testis, Endocrinology, 95 (1974) 897 .
70
71
Lue M, Faure R, Ruel 1 & Dussault J H, A monoclonal
antibody to the rat nuclear triiodothyronine receptor:
production and characterization, Endocrinology, 123 (1988)
180.
Macchia E, Nakai A, Janiga A, Sakurai A, Fisfalen M E,
Gardner P, Soltani K & DeGroot L, Characterization of
site.specific poly-clonal antibodies to c.erbA peptides
recognizing human thyroid hormone receptors ai, a2 and /3
and. native 3,5 ,3' .triiodothyronine receptor and study of
tissue distribution of the antigen, Endocrinology, 126 (1990)
3232.
MENDIS-HANDAGAMA & ARIY ARA TNE: LEYDIG CELLS x THYROID HORMONES
72
Tagami T, Nakamura H, Sasaki S, Mori T, Yoshioka H,
Yoshida H & Imura H, Immunohistochemical localization of
nuclear 3,5,3' .triiodothyronine receptor proteins in rat tissues
studied with anti.serum against c.erbAff3. receptor,
Endocrinology, 127 (1990) 1727.
73 Strait KA , Schwartz H L, Seybold V S, Ling N C &
Oppenheimer J H, Immunofluorescence localization of thyroid
hormone receptor protein beta I and variant alpha 2 in selected
tissues: Cerebellar Purkinje cells as a mode for beta I
receptor.mediated developmental effects of thyroid hormone in
brain, Proc NaIl Acad Sci USA, 88 (1991) 3887.
74 Tagami T, Nakamura H, Sasaki S, Miyoshi Y & Imura H,
Estimation of the protein content of thyroid hormone receptor
alpha I and beta I in rat tissues by western blotting,
Endocrinology, 132 (1993) 275.
75 Hardy M P, Sharma R S, Arambepola N K, Sottas C M,
Ru ssell L D, Bunick D, Hess R A & Cooke P S, Increased
proliferation of Leydig cells induced by neonatal
hypothyroidism in the rat, 1 Androl, 17 (1996) 231.
76 McCoard S A, Wise T H & Ford J J, Endocrine and molecular
influences on testicular development in Meishan and White
Composite boars, 1 Endocrinol, 178 (2003) 405 .
77 Hagenbuch B & Meier P J, Organic anion transporting
polypeptides of the OATP/SLC21 family: Phylogenetic
classification as OA TP/SLCO superfamily, new nomenclature
and molecular/functional properties, Pflugers Arch, 447
(2004) 653 .
78 Fujiwara K, Adachi H, Nishio T, Unno M, Tpkui T, Okabe M,
Onogawa T, Suzuki T, Asano N, Tanemoto M, Seki M, Shiiba
K, Suzuki M, Kondo Y, Nunoki K, Shirnosegawa T, Iinuma K,
Ito S, Matsuno S & Abe T, Identification of thyroid hormone
transporters in humans: Different molecules are involved in a
tissue.specific manner, Endocrinology, 142 (2001) 2005.
79 Jansen J, Friesman E C, Millici C & Visser T J, Thyroid
hormone transporters in health and disease, Thyroid, IS (2005)
757.
80 Pizzagalli F, Hagenbuch B, Stieger B, Klenk U, Folkers G &
Meier P J, Identification of a novel human organic anion
transporting pol peptide as a high affinity thyroxine transporter,
Mol Endocrinol, 16, (2002) 2283.
81
82
83
84
Suzuki S, Mori J I, Kobayashi M, Inagaki T, Komatsu A,
Yamashita K, Takeda T, Miyamoto T, Ichikawa K &
Hashizume K, Presence of functional domains In
NADPH.dependent cytosolic 35'3' .triiodo.L.thyronine (1'3)
.binding protein (p38CTBP) molecule:analyses with deletion
mutants, Horm Melab Res, 35 (2003) 577.
Mendis-Handagarna S M L C, Ariyaratne H B S, Teunissen,
van Manen KR & Haupt RL, Differentiation of adult Leydig
cells in the neoaatal rat testis is arrested by hypothyroidism,
Bioi Reprod, 59 (I 998b J 351.
Teerds K J, de Rooij D G, de jong F H & van Haaster L H,
Development of the adult Leydig cell population in the rat
testis is affected by neonatal thyroid hormone levels, Bioi
Reprod, 59 (1998) 344.
Mendis-Handagama S M L C & Ariyaratne H B S, Prolonged
and transient neonatal hypothyroidism on Leydig cell
differentiation in the postnatal rat testis, Arch Androl, 50
(2004) 347.
957
85
Maran R R, Arunakaran J, Jayaraj D A, Ravichandran K,
Ravisankar B & Aruldhas M M, Transient neonatal,
hypothyroidism alters plasma and testicular sex steroid
concentration in pubertal rats, Endocr Res, 26 (2000a) 411.
86
Langer P, Tajtakova M, Fodor G, Kocan A, Bohov P,
Michalek J & Kreze A, Increased thyroid volume and
prevalence of thyroid disorders in an area heavily polluted by
polychlorinated biphenyls, Eur 1, Endocr, 139 (1998) 402.
87
Nagayarna J, Okamura K, lida T, Hirakawa H, Matsueds T,
Tsuji H, Hasegawa M, Sato K, Ma H Y, Yanagawa T, Igarashi
H, FuIrushige J & Watanabe T, Postnatal exposure to
chlorinated dioxins and related chemicals on thyroid hormone
status in Japanese breastJed infants, Chemosphere, 37 (1998)
1789.
88
Cheek A 0, Kow K, Chen J & McLachlan J A, Potential
mechanisms of thyroid disruption in humans: interaction of
organochlorine compounds with thyroid receptor,
transthyretin, and thyroid biding globulin, Environ Health
Perspect, \07 (1999) 273.
89
Collins W T, Fine structural lesions and hormonal alterations
in thyroid glands of perinatal rats exposed in utero and by the
milk to polychlorinated biphenyls, Am J Palhol, 99 (1980)
125.
90
Saeed A & Hansen L G, Morphometric changes in the
prepUbertal female rat thyroid gland following acute exposure
to 2,2',4,4'-tetrachlorobiphenyl and ArocJor 1242, J Toxieol
Environ Hlth, 51 (1985) 503.
91
Ness D K, Schantz S L, Moshtaghian J & Hansen L G, Effects
of perinatal exposure to specific PCB congeners on thyroid
hormone concentrations and thyroid histology in the rat,
Toxicol Lett, 68 (1993) 311.
92
Cooke P S, Zhao Y D & Hansen L G, Neonatal
polycholrinated biphenyl treatment increases adult testis size
and sperm production in the rat. Toxieol Appl Phormacol, 136
(1996) 112.
93
Kato Y, HaraguchiK, Shibahara T, Masuda Y & Kimura R,
Reduction of thyroid hormone levels by methylsulfonyl
metabolites of polychlorinated biphenyl congeners in rats,
Arch Toxicol, 72 (1998) 54\.
94
Kato Y, Haraguchi K, Shibahara T, Yumoto S, Masuda Y &
Kimura R, Reduction of thyroid hormone levels by
methyl sulfonyl metabolites of tetra. and pentachlorinated
biphenyls in male Sprague Dawley rats, Toxicol Sci, 48 (1999)
5\.
95
Desaulniers D, Leingartner K, Wade M, Finte1man E,
Yagminas A & Foster WG, Effects of acute exposure to PCBs
126 and 153 on anterior pituitary and thyroid hormones and
FSH isoforms in adult Sprague Dawley male rats, Toxicol Sci,
47 (1999) 158.
96
Wolstad S & Jensen B M, Thyroid hormones in grey seal pups
(Halichoerus grypus), Comp Biochem Physiol A Mol
Integr.Physiol, 122 (1999) 157.
97
Kim I, Ariyaratne H B S & Mendis-Handagama S M L C,
Effects of continuous and intermittent exposure of lactating
motners to Aroclor 1242 on testicular steroidogenic function in
the adult offspring, Tissue Cell, 33 (2000) 169.
958
INDIAN J EXP BIOL, NOVEMBER 2005
98
Josso N, Anti.Mullerian hormone: New perspective for sexist
molecule, Endocr Rev, (1986) 421 .
99
Zhou B, Satts L M & Hutson J M, Germ cell development in
neonatal mouse testes in utero requires Mullerian inhibiting
substance, J Urol, 150 (1993) 613 .
100 Kuroda T, Lee M M, Haqq C M, Powell D M, Manganaro T F
& Donohoe P K, Mullerian inhibiting substance ontogeny and
its modulation by follicle. stimulating hormone in the rat testes,
Endocrinology, 127 (1990) 1825.
101 Racine C, Rey R, Forrest M, Louis F, Ferre A, Huhtaniemi I,
Josso N & Clementi N, Receptors for anti.Mullerian hormone
on Leydig cells are responsible for its effects on
steroidogenesis and cell differentiation, Proc Natl Acad Sci
USA, 95 (1998) 594.
102 Sasaki S, Nakamura H, Tagami T, Osamura Y & Imura H,
Demonstration of nuclear 3,5,3 '.triiodothyronine receptor
proteins in gonadotrophs and corticotrophs in rat nterior
pituitary by double immunohistochemical staining,
Endocrinology, 129 (1991) 511.
103 Marshall W A & Tanner J M, Variations in the pattern of
pubertal changes in boys, Arch Dis Child, 45 (1970) 13.
104 Barnes N D, Hayles A B & Ryan R J, Sexual maturation in
juvenile hypothyroidism Mayo, Clin Proc, 48 (1973) 849.
105 Castro-Magana M, Angulo M, Canas A, Sharp A & Fuentes B,
Hypothalamic.pituitary gonadal ax is in boys with primary
hypothyroidism and macroorchidism, J Pediatr, 112 (1988)
397.
106 Franks R C & Stempfel R S jr, Juvenile hypothyroidism and
precocious testicularmaturation, J Clin Endocrinol, 23 ( 1963)
805.
107 Laron Z, Karp M & Dolberg L, Juvenile hypothyroidism with
testicular enlargement, Acta Paediatr Scan, 59 (1970) 317.
108 Hoffman W H, Kovacs K T, Gala R R, Keel B A, Jarrell T S,
Ellegood JO & Burec CL, Macroorchidism and testicular
fibrosis associated with autoimmune thyroiditis, J Endocrinol
Invest, 14 (1991) 609.
109 Cristovao F C, Bisi H, Mendonca B B, Bianco A C & B10ise
W, Severe and mUd neonatal hypothyroidism mediate opposite
effects on Leydig cells of rats, Thyroid, 12 (2002) 13.
liD Chandrasekhar Y, D'Occhio MJ & Setchell B P, Reproductive
hormone secretion and spermatogenic function in
thyrodectomized rams receiving graded doses of exogenous
thyroxin, J Endocrinol, III (1986) 245.
111 Wortsman J, Rosner W & Du f:lu M I, Abnormal testicular
function in men with primary hypothyroidism, Am J Med, 82
(1987) 207.
112 Antony F F, Aruldhas M M, Udhayakumar R C, Maran R R &
Govindarajulu P, Inhibition of Leydig cell activity in. vivo and
in. vitro in hypothyroid rats, J Endocrinol. 144 (1995) 293.
113 Tahmaz L, Gokalp A, Kibar Y, Kocak I, Yalcin 0 & Ozercan
y , Effect of hypothyroidism on the testes in mature rats and
treatment with levothyroxine and zinc, Andrologia, 32, (2000)
85.
114 Foldes J, Banos C, Feher T, Bodrogi L, Szalay F, Borvendeg J,
Csillag J & Tako J, Functional relationships of the
hypothalamic-pituitary-testicular system in Grave's disease
Act, Med Acad Sci Hung, 39 (1982) 109.
115 Monson J P, Barge A, Chowns J, Broadbent M, Noonan K,
Temple R C & Houghton B J, Changes in sex steroid and
gonadotrophin concentrations during treatment of Graves'
thyrotoxicosis in males, Ann Clin Biochem, 25 (1988) 330.
116 Ford H C, Cooke R R, Keightley E A & Feek C M, Serum
levels of free and bound testosterone in hyperthyroidism, Clin
Endocrinol, 36 (1992) 187.
I 17 Kumar P N, Aruldhas M M & Juneja S C, Influence of
hyperthyroidism induced at pUberty on the epididymal lipids,
number and motility of spermatozoa in rats, Reprod Fertile
Dev, 8 (1996) 373.
S,
Berg
A
&
Kallner
G,
118 Rojdmark
Hypothalamic.pituitary.testicular axis in patients with
hyperthyroidism, Horm Res, 29 (1988) 185.
119 Van Haaster L H, De Jong F H, Docter R & De Rooij D G,
High neonatal triiodothyronine levels reduce the period of
Sertoli cell proliferation and accelerate tubular lumen
formation in the rat testis and increase serum inhibin levels,
Endocrinology, 133 (1993) 755.
120 lannini E A, Ulisse S, Piersanti D, Carosa E, Muzi P, Lazar J
& D' Armiento M, Early thyroid hormone treatment in rats
increases testis size and germ cell number, Endocrinology, 132
(1993) 2726.
121 Schneider G, Kopach K, Ohanian H, Bonnefond V, Mittler 1 C
& Ertel N H The hypothalamic.pituitary.gonadal axis during
hyperthyroidism in the rat, Endocrinology, 105 (1979) 674.
122 Ruiz M, Diego A M, Reyes A, Alonso A & Morell M
Influence of thyroidectomy on serum and pituitary FSH in
control and orchidectomized rats, Res Exp Med. 189 (1989)
85 .
123 Kirby J D, Jetton A E, Cooke P S, Hess R A, Bunick D,
Ackland J F, Turek F W & Schartz N B Developmental
hormonal
profiles
accompanying
the
neonatal
hypothyroidism.induced increase in adult testicular size and
sperm production in the rat, Endocrinology, 131 (1992) 559.
124 Van Haaster L H, De long F H, Docter R & De Rooij D G The
effect of hypothyroidism on Sertoli cell proliferation and
differentiation and hormone levels during testicular
development in the rat, Endocrinology, 131 (1992) 1574.
125 Wong C C, Dohler K D & von zur Munten A, Effects of
triiodothyronine, thyroxine and isopropyl-di-iodothyronine on
thyroid.stimulating hormone in serum and pituitary gland and
on pituitary concentrations of prolactin, growth hormone,
luteinizing hormone and follicle stimulating hormone in
hypothyroid rats, J Endocrinol, 87, (1980) 255.
126 Kalland G A, Vera A, Peterson M & Swerdloff R S
Reproductive hormonal axis of the male rat in experimental
hypothyroidism, Endocrinology, 102 (1978) 476.
127 Maran R R, Ravichandran K, Arunakaran I & Aruldhas M M,
Impact of neonatal hypothyroidism on Leydig cell number,
plasma and testicular interstitial fluid sex steroids
concentration, Endocr Res, 27 (2001) 119.
128 Rao J N, Liang 1 Y, Chakraborti P & Feng P, Effect of thy rod
hormone on the development and gene expression of hormone
receptG1's in rat testes in vive, J Endocrinol Invest, 26 (2003)
435 ..
129 Weiss S R & Burns J M, The effect of acute treatment with
two goitrogens on plasma thyroid hormones, testosterone and
MENDIS-HANDAGAMA & ARIY ARATNE: LEYDIG CELLS x THYROID HORMONES
959
Comp
pregnenolone production, Biochel7l Biophys Res Commun , 154
( 1988) 60.
130 O'Shaughnessy P J, Willerton L & Baker P l , Changes in
Leydig cell gene expression during development in the mouse,
Bioi Reprod, 66 (2002) 966.
145 Mendis-Handagama S M L C, Aten R F, Watkins P A, Scallen
T J & Berhman H R, Peroxisomes and sterol carrier protein.2
in luteal cell steroidogenesis: A possible role in .cholesterol
transport from lipid droplets to mitochondria, Tissue Cell, 27
(1995) 483 .
testicular morphology in adult
BiochemPhysiol A, 90 (1988) 449.
male
rats,
131 Tapanainen J, Kuopio T, Pelliniemi L J & Huhtaniemi I, Rat
testicular endogenous steroids and number of Leydig cells
between the fetal period and sexual maturity, Bioi Reprod, 31
(1984) 1027.
132 Chubb C & Ewing L L, Steroid secretion by sexually immature
rat and rabbit testes perfused in vitro, Endocrinology, 109
(1981) 1999.
133 Valenty S, Guido R, Fazzuoli L, Barreca A, Giusti M &
Giordano G, Decreased steroidogenesis and cAMP production
in. vitro by Leydig cells isolated from rats made hypothyroid
during adulthood, Int J Androl, 20 (1997) 279.
134 Manna P R, Roy P, Clark B J, Stocco D M & Huhtaniemi IT,
Interaction of thyroid hormone and steroidogenic acute
regulatory (StAR) protein in the regulation of murine Leydig
cell steroidogenesis, J Steroid Biochem Mol Bioi, 76 (200 I a)
167.
135 Manna P R, Kero J, Tena-Sempere M, Pakarinan P, Stocco D
M & Huhtaniemi I T, Assessment of mechanisms of thyroid
hormone action in mouse Leydig cells: Regulation of the
steroidogenic acute regulatory protein, steroidogenesis and
luteinizing hormone receptor function, Endocrinology, 142
(200Ib) 319.
136 Maran R R, Arunakaran J & Aruldhas M M, T3 directly
stimulates basal and modulates LH induced testosterone and
oestradiol production by rat Leydig cells in. vitro, Endocr J, 47
(2000b) 417.
146 Mendis-Handagama S M L C, Peroxisomes and intracellular
cholesterol trafficking in Leydig cells following luteinizing
hormone stimulation, Tissue Cell, 32 (2000) 103 .
147 Privalle C T, Crivello J F & Jefcoate C R, Regulation of
intramitochondrial cholesterol transfer to side.chain cleavage
cytochrome P.450 in rat adrenal gland, Proc Natl Acad Sci
USA, 80 (1983) 702.
148 Fringes B & Reith A, Time course of peroxisome biogenesis
during adaptation to mild hyperthyroidism in rat liver: A
morphometric/stereologic study with electron microscopy, Lab
Invest , 47 (1982) 19.
149 Just W W, Hartle F U & Schimassek H, Rat liver peroxisomes
I New peroxisome population induced by thyroid hormones in
the liver of male rats, Eur J Cell Bioi, 26 (1982) 249.
150 Payne A H, Clarke T R & Bain P A, The murine 3 betahydroxysteroid dehydrogenase multi gene family: structure,
function and tissue.specific expression, J Steroid Biochem.
Mol Bioi, 53, (1995) Ill.
151 Mullaney B P & Skinner M K, Growth factors as mediators of
testicular cell-cell interactions, Bail/ieres Clin Endocrinol
Metab,5 (1991) 771.
152 Saez J M, Avallet 0, Naville D, Perrard.Sapori M H &
Chatelain P G, Sertoli.Leydig cell communications, Ann NY
Acad Sci, 564 (1989) 210.
137 Stocco D M, Acute regulation of Leydig cell steroidogenesis,
in The Leydig Cell, edited by Payne AH, Hardy MP and
Russell LD (Cache River Press, Vienna, IL) 1996, 241 .
153 Ascoli M & Segaloff D L, On the structure of the luteinizing
hormone/chorionic gonadotropin receptor, Endocr Rev, 10
(1989) 27.
138 Norman A W & Litwick G L, Thyroid Hormones in
Hormones, edited by AW Norman, GL Litwick (Academic
Press, San Diego) 1997, 169.
154 Saez M, Leydig cells: Endocrine, paracrine and autocrine
regulation, Endocr Rev, 15 (1994) 574.
139 Payne A H & Youngblood G L, Regulation of expression of
steroidogenic enzymes in Leydig cells, Bioi Reprod, 52,
(1995) 217.
140 Charreau E H, Calvo J C, Nozu K, Pignataro 0 , Call K &
Dufau M L, Hormonal modulation of 3-hydroxy-3rnethylglutaryl coenzyme A reductase acttvlty in
gonadotropin.stimulated and -desensitized testicular Leydig
cells, J Bioi Chem, 256 (1981) 1271.
141 Freeman D A & Ascoli M, Studies on the source of cholesterol
l·.sed for steroid biosynthesis in cultured Leydig tumor cells, J
Bioi Chern, 257 (1982) 14231.
142 Hou J W, COllins D C & Schleicher R L, Sources of
cholesterol for testosterone biosynthesis in murine Leydig
cells, Endocrinology, 127 (1990) 2047.
143 Scallen T l, Pastuszyn A, Noland B J, Chanderbhan R,
Kharroubi A & Vahouny G V, Sterol carrier, and lipid transfer
proteins, Chem Phys Lipids, 38 (1985) 239.
144 Van Noort M, Romrnerts F F, Van Amerogen A & Wirtz K W,
intracellular redistribution of SCP2 in Leydig cells after
hormonal stimulation may contribute to increased
155 Cooke B A, Signal transduction involving cyclic
Amp.dependent and cyclic AMP-independent mechanisms in
the control of steroidogenesis, MolCell Elldocrinol, 151 (1999)
25 .
156 Cooke B A, Is cyclic AMP an obi igatory second messenger for
luteinizing hormone? Mol Cell Endocrinol, 69 (1990) C II .
157 Clark B J, Soo S C, Caron K M, Ikeda Y, Parker K L & Stocco
D M, Hormonal and developmental regulation of the
steroidogenic acute regulatory protein, Mol Endocrinol, 9
(1995) 1346.
158 O'Shaughnessy P J & Payne A H, Differential effects of single
and repeated administration of gonadotropins on testosterone
production and steroidogenic enzymes in Leydig cell
popUlations, J Bioi Chem, 257 (1982) 11503.
159 lana N R, Halder S & Bhattacharya S, Thyroid hormone
induces a 52 kDa soluble protein in goat testis Leydig cell
which stimulates androgen release, Biochim Biophys Acta,
1292 (1996) 209 .
160
Holl~nder N & Hollander V P, The microdetermination ot
testosterone in human spermatic vein blood, J CUrt
Endocrinol, 18 (1958) 966.
960
INDIAN J EXP BIOL, NOVEMBER 2005
161 Vermulen A, Leydig cell function in old age in Hypothalamus.
Pituitary and Aging, edited by AV Everille and JA Burgess
(Charles C Thomas, Springfield, IL) 1976,458.
176 Zraly Z, Bendova J, Svecova D, Faldikova L, V ~~nik Z &
Zajicova A, Effects of oral intake of nitrates on reproductive
function of bulls. Vet Med (Praha), 42 (1997) 345.
162 Bethea C L & Walker R F, Age.related changes in
reproductive hormones and in Leydig cell responsivity in the
male Fisher 344 rat I, Gerontology, 34 (1979) 21.
177 Rao J N, Debeljuk L, Bartke A, Gao Y p, Wilber J F & Feng
P, The detection ofthyrotropin.re1easing hormone (TRH) and
TRH receptor gene expression in Siberian hamster testis,
Peptides, 18 (1997) 1217.
163 Kim I, Ariyaratne H B S & Mendis-Handagama S M L C,
Changes in the testis interstitium of Brown Norway rats with
aging and effects of luteinizing and thyroid hormones on the
aged testes in enhancing the steroidogenic potential, Bioi
Reprod, 66 (2002) 1359
164 Ichihara I, Kawamura H & Pelliniemi L J, Ultrastructure and
morphometry of testicular Leydig cell:; and the interstitial
components correlated with testosterone in aging rats, Cell
Tissue Res, 271 (1993)241.
165 Cizza G, Brady L S, Calogero A E, Bagdy G, Lynn A B, Kling
M A, Blackman M R, Chrousos G P & Gold P W, Central
hypothyroidism is associated with advanced age in male
Fischer 344IN rats: in vivo and in vitro studies, Endocrinology,
13I (1992) 2672.
166 Cizza G, Brady L S, Pacak K, Blackman M R, Gold P W &
Chrousos G P, Stress.induced inhibition of the
hypothalamic.pituitary.thyroid axis is allenuated in the aged
Fischer 3441N male rat, Neuroendocrinology, 62 (1995) 506.
167 Mendis-Handagama S M L C, Kim I & Ariyaratne H B S,
Rejuvenation of aged Leydig cells is possible in a rodent
species, in Andrology in the 21" century: Proceedings of the
Vlllh International Congress of Andrology, edited by B
Robaire, H Chemes C Morales, (Medimont Publications,
Englewood, NJ) 2001, 745.
168 Burgus R, Dunn T F, Desiderio D, Ward D N, Vale W &
Guillemin R, Characterization of ovine hypothalamic
hypophysiotropic TSH.releasing factor, Nature, 226 (1970)
321.
169 Schall)' A Y, Redding T W, Boweres C Y & Barrett J F,
Isolation of properties of porcine thyrotropin.releasing
hormone, J Bioi Chem, 244 (1969) 4077.
170 Wilber J F & Xu A H, The thyrotropin releasing hormone gene
1998: Cloning, characterization and transcriptional regulation
in the central nervous system, heart and testis, Thyroid, 8
(1998) 897.
171 Feng P, Gu J, Kim U J, Carnell N E & Wilber J F,
Identification, localization and developmental studies of rat
thyrotropin.releasing hormone mRNA in the testis,
Neuropeptides, 24 (1993) 63.
172 Satoh T, Feng P, Kim U J & \ 'ilber J F, Identification of
thyrotropin. releasing hormone l ' · .~ ptor in the rat testis,
Neuropeplides 27 (1994) 195.
173 Liu XP, Li Z, Zhang Y Q & Xu R J, Expression of
thyrotropin.releasing hormone recept:>r mRNA in the rat testi~
development, Shi Yan Sheng Wu Xue Bao, 34 (2001) 109.
174 Li Z, Zhang Y, Liu X & Xu R, Correlation of expression of
preprothyrotropin.releasing hormone and receptor with rat
testis development, Chin MedJ (Engl), 115 (2002) 12,
175 Zhang S H, Zhang Y Q & Vacca.Galloway L L, Identification
of thyrotropin.releasing hormone recepto' mRNA in the
Leydig cells of the mouse testis, Neuropeptides, 29 (1995)
309.
178 Pekary A E, Bhasin S, Smith V, Sugawara M, Swerdloff R S
& Hershman J M, Thyroid hormone modulation of
thyrotrophin,releasing hormone (TRH) and TRH-Gly levels in
the male rat reproductive system, J Endocrinol, 114, (1987)
271.
179 Cockle S M, Aitken A, Beg F & Smyth D G, A novel peptide,
pyroglutamylproline amide, in the rabbit prostate complex,
structurally related to thyrotrophin. releasing hormone, J Bioi
Chern, 264 (1989) 7788.
180 Bilek R, Gkonos P J, Ravianini M A, Smyth D G & Roos B A,
The thyrotrophin. releasing hormone (TRH) .like peptides in rat
prostate are not formed by expression of the TRH gene but are
suppressed by thyroid hormone, J Endocrinol Hypothyroidism,
Bioi Reprod, 51 (1992) 706.
181 Gkonos P J, Kwok C K, Block N L& Roos B A, Identification
of the human seminal TRH-like peptide pGlu.Phe-Pro-NH2 in
normal human prostate, Pep tides, 15 (1994) 1281.
182 Maran R R, Aruldhas M M, Udhayakumar R C, Subramanian
S, Rajendiran G, Antony F F, Arunakaran J & Govindarajulu
P, Impact of altered thyroid hormone status on pros~atic
glycosidases, Int J Androl, 21 (1998) 121.
183 Gerendai I, Sziebert P, Gorcs T, Csaba Z & Csemus V, Effect
of intratesticular administration of TRH, or anti.TRH
antiserum on function of ratleslis, Life Sci, 67 (2000) 269.
184 Odell W D, SwerdloffR S, Jacobs H S & Hescox M A, FSH,
induction of sensitivity to LH: One cause of sexual maturation
in the male rat, Endocrinology, 92 (1973) 160.
185 EI.Safoury S & Bartke A, Effects of follicle slimulating
hormone and luteinizing hormone on plasma testosterone
levels in hypophysectomized and in intact immature and aduh
male rats, J Endocrinol, 61 (1974) 193 .
186 Chen Y I, Payne A H & Kelch R P, FSH, stimulation of
Leydig cell function in the hypophysectomized immature rat,
Proc Soc Exp Bioi Med, 153 (1976) 473 .
187 Kerr J B & Sharpe R M, Follicle.stimulating hormone
induction of Leydig. cell maturation, Endocrinology, 116
(1985) 2592.
188 Purvis K, Clausen 0 P & Hansson V, LH contamination may
explain FSH effects on rat Leydig cells, J Reprod Fertil, 56,
(1979) 657.
189 Moger W H & Murphy P R, Re-evaluation of the effect of
tollicle.stimulaling hormone on the steroidogenic capacity ot
the testis: The effects of r:~uraminidase treated FSH
preparations, Biol Reprod, 26 (1982) 422.
190 Teerds K J, Rommerts F F, van de Kant H J & de Rooij D G,
Leydig ::ell number and function in the adult cynomolgus
monkr::y (Macaca fascicularis) is increased by daily hCG
treatment but not by daily FSH treatment, J Reprod Fertii, 87
(1989) 141.
MENDIS-HANDAGAMA & ARIYARATNE: LEYDIG CELLS x THYROID HORMONES
191 Russell L D, Corbin T J, Borg K E, Franca L R, Grasso P &
Bartke A, Recombinant human follicle .stimulating hormone is
capable of exerting a biological effect in the adult
hypophysectomized rat by reducing the numbers of
degenerating germ cells, Endocrinology, 133 (1993) 2062.
192 Levalle 0, Zylbersztein C, Aszpis S, Aquilano D, Terradas C,
Colombani M, Aranda C & Scaglia H, Recombinant human
follicle.stimulating hormone administration increases
testosterone production in men, possibly by a Sertoli
cell.secreted nonsteroid factor, 1 c/in Endocrinol Metab, 83
(1998) 3973.
193 Sri raman V, Sairam M R & Rao A J, Evaluation of relative
roles of LH and FSH in regulation of differentiation of Leydig
cells using an ethane 1,2-dimethylsulfonate-treated adult rat
model, 176 (2003) 151.
194 Means A R, Fakunding J L, Huckins C, Tindall D J & Vitale
R, Follicle-stimulating hormone, the Sertoli cell, and
spermatogenesis, Recent Prog Harm Res, 32 (1976) 477 .
195 Risbridger G P, Kerr j B, Peake R, Rich K A & de Kretser D
M, Temporal changes in rat Leydig cell function after the
induction of bilateral cryptorchidism, 1 Reprod Fertil, 63
(1981) 415 .
196 Dohle G R, Smit M & Weber R F, Androgen and male fertility
Worl 1 Ural 21 (2003) 341.
197 Rich K A, Kerr J B & de Kretser D M, Evidence for Leydig
cell dysfunction in rats with seminiferous tubule damage, Mol
Cell Endocrinol, 13 (1979) 123.
198 Jegou B, Laws A 0 & de Kretser D M, Changes in testicular
function induced by short.term exposure of the rat testis to
heat: further evidence for interaction of germ cells, Sertoli cells
and Leydig cells, lilt 1 Androl, 7 (1984) 244.
199 Au C L, Robertson D M & de Kretser D M, Relationship
between testicular inhibin content and serum FSH
concentrations in rats after bilateral efferent duct ligation, 1
Reprod Fertil , 72, (1984) 351.
200 Kerr J B, Rich K A & de Kretser D M, Alterations of the fine
structure and androgen secretion of interstitial cells in the
experimentally cryptorchid rat testis, Bioi Reprod, 20 (1979)
409.
201 Bergh A, Local differences in Leydig cell morphology in the
adult rat testis: Evidence for a local control of Leydig cells by
adjacent seminiferous tubules, Inl 1 Androl, 5 (1982) 325.
961
207 Vihko K K & Huhtaniemi I, A rat seminiferous epithelial
factor that inhibits Leydig cell camp and testosterone
production : mechanism of action, stage specific secretion, and
partial characterization, Mol Cell Endocrillol, 65 (1989) 119.
208 Tabone E, Benahmed M, Reventos J & Saez J M, Interactions
between immature porcine Leydig and Sertoli cells in vitro An
ultrastructural and biochemical study, Cell Tissue Res, 237
(1984) 357.
209 Benahmed M, Grenot C, Tabone E, Sanchez P & Morera A M,
FSH regulates cultured Leydig cell function via Sertoli cell
proteins : an in vitro study, Biochem Biophys Res Commun,
132 (1985) 729 .
210 Perrard.Sapori M H, Chatelain P, Vallier P & Saez J M, In
vitro interactions between Sertoli cells and steroidogenic cells,
Biochem BiophysRes Commun, 134, (1986) 957 .
211 Perrard.Sapori M H. Chatelain P C, Rogemond N & Saez J M,
Modulation of Leydig cell function by culture with Sertoli
cells or with Sertoli cell-conditioned medium: Effect of
insulin, somatomedin C and FSH , Mol Cell Endocrinol, 50,
(1987) 193.
212 Wreford N G, Rajendrakumar T, Matzuk M M & de Kretser D
M 200 I Analysis of the testicular phenotype of the folliclestimulating hormone beta. subunit knockout and the activin
type II receptor knockout mice by stereo logical analyses,
Endocrinology, 142 (2001) 2916.
213 Krishnamurty H, Kats R, Danilovich N. Javeshghani D &
Sairam M R, Intercellular communication between Sertoli and
Leydig cells in the absence of follicle-stimulating hormonereceptor signalling, Bioi Reprod, 65 (2001) 1201.
214 Baker PJ , Pakarinen p, Huhtaniemi I T, Abel M H, Charlton H
M, Kumar T R & O'Shaughnessy P J, Failure of normal
Leydig cell development in follicle .stimulating hormone (FSH)
receptor.deficient mice, but not FSH beta-deficient mice: Role
for constitutive FSH receptor activity, Endocrinology, 144
(2003) 138.
215 Wright W W, Musto N A, Mather J P & Bardin C W, Sertoli
cells secrete both testis.specific and serum proteins , Proc Natl
Acad Sci USA ,78 (1981) 7565.
216 Perrard M H, Saez J M & Dazord A, FSH stimulation of
cytosolic protein synthesis in cultured pig Sertoli cells, 1
Steroid Biochem, 22 (1985) 281.
202 Bergh A, Paracrine regulation of Leydig cells by seminiferous
tubules, Int 1 Androl, 6 (1983) 57.
217 Verhoeven G & Cailleau 1, A facto r in spent media from
Sertoli.cell-enriched cultures that stimulates steroidogenesis in
Leydig cells, Mol Cell Endocrinol, 40 (1985) 57.
203 Bergh A, Development of stage-specific paracrine regulation
of Leydig cells by the seminiferous tubules, lnt 1 Androl, 8
(1985) 80.
218 Verhoeven G & Cailleau J, A Leydig stimulatory factor
produced by human testicular tubules, Mol Cell Endocrinol, 49
(1987) 137.
204 Fouquet J P, Ultrastructural analysis of a local regulation of
Leydig cells in the adult monkey (Macaca fascicularis) and rat,
1 Reprod Fertil, 79 (1987) 49.
219 Papadopoulos V, Identification and purification of a human
Sertoli cell secreted protein (Hscsp.80) stimulating Leydig cell
steroid biosynthesis, 1 Clin Endocrin Metab, 72 (1991) 1332.
205 Parvinen M, Nikula H & Huhtaniemi I, Influence of rat
seminiferous tubules on Leydig cell testosterone production in
vitro, Mol Cell Endocrinol, 37, (1984) 331.
220 Cheng C Y, Morris I & Bardin, C W, Testins are structurally
related to the mouse cysteine protein base precursor but devoid
of any protease/anti .protease activity, Biochem Biophys Res
Commun, 191 (1993) 224.
206 Syed V, Khan S A & Ritzen E M, Stage-specific inhibition of
interstitial cell testosterone secretion by rat semini ferous
tubules in vitro, Mol Cell Endocrinol, 40 (1985) 257.
221 Sharpe R M, McKinnell C, Kivlin C & Fisher J S,
Proliferation and functional maturation of Sertoli cells, and
962
INDIAN J EXP BIOL, NOVEMBER 2005
their relevance to disorders of testis function in adulthood,
Reproduction , 125 (2003) 769.
222 Hess R A, Cooke P S, Bunick D & Kirby J D, Adult testicular
enlargement induced by neonatal hypothyroidism is
accompanied by increased Sertoli and germ cell numbers.
Endocrinology, 132 (1993) 2607.
223 Buzzard J J, Wreford N G & Morrison J R, Thyroid hormone,
retinoic acid and testosterone suppress proliferation and induce
markers of differentiation in cultured rat Sertoli cells, Bioi
Reprod, 144 (2003) 3722.
224 Holsberger D R, lirawatnotai S, Kiyokawa H & Cooke P S,
Thyroid hormone regulates the cell cycle inhibitor p27Kip 1 in
postnatal murine Sertoli cells, Endocrinology, 144 (2003)
3719.
225 Palmero S, Prati M, Barreca A, Minuto F, Giordano G &
Fugassa E, Thyroid hormone stimulates the production of
insulin.Iike growth factor I (IGF. I) by immature rat Sertoli
cells, Mol Cell Endocrinol, 68 (1990) 61 .
226 Lin T, Wang D, Hu J & Stocco D M, Upregulation of human
chorionic gonadotrophin-induced steroidogenic acute
regulatory protein by insulin-like growth factor.! in rat Leydig
cells, Endocrine, 8, (1998) 73.
227 Pan no M L, Salerno M, Lanzino M, De Luca G, Maggiol ini
M, Straface S' V, Prati M, Palmero S, Bolla E & Fugassa E,
Follow-up study on the effects of thyroid hormone
administration on androgen metabolism of peripubeltal rat
Sertoli cells, Eur J Endocrinol, 13:t, (1995) 236.
228 Carreau S~ Genissel C, Bilinska B & Levallet J, Sources of
oestrogen in the testis and reproductive tract of the male, Int J
Androl, 22 (1999) 211.
229 Ulisse S, Jannini E A, Carosa E, Piersanti D, Graziano F M &
D' Armiento M, Inhibition of aromatase activity in rat Sertoli
cells by thyroid hormone, J Endocrinol, 140 (1994) 431.
230 Catalano S, Pezzi V, Chimento A, Giordano C, Carpino A,
Young M & McPhaul M J, Triiodothyronine decrease the
activity of the proximal promoter (PH) of the aromatase gene
in the mouse Sertoli cell line, TM4, Mol Endocrinol, 17
(2003) 923 .
231 Dhar J D & Setty B S, Epididymal response to exogenous
testosterone in rats sterilized neonatally by estrogen,
Endokrinologie, 68 (1976) 14.
232 Abney T 0 & Myers R B, 17beta-estradiol inhibition of
Leydig cell regeneration in the ethane dirnethysulfonate-treated
mature rat, J Androl, 12 (1991) 295