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Original Studies |
Department of Experimental Medicine (E.A.J., N.R., E.S., E.C.), University of L’Aquila, L’Aquila 67100; Institute of Urology (A.D.M.) and Department of Experimental Medicine and Pathology (G.d.A., M.D.A.), University of Rome "La Sapienza", Rome 00161; Department of Endocrinology (E.M.), University of Pisa, Pisa 56124; and Diagnostic Department (A.C.), "Regina Apostolorum" Hospital, Albano Laziale 00041, Italy
Address correspondence and requests for reprints to: Massimino D’Armiento, M.D., Chair of Endocrinology, Department of Experimental Medicine and Pathology, University of Rome "La Sapienza," Policlinico Umberto I–Viale del Policlinico, 155, 00161 Rome, Italy. E-mail: massimino.darmiento{at}uniroma1.it
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Abstract |
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1 and 2 and ß messenger
RNA (mRNA) levels in normal human testicular tissue during development
and in adulthood. Nonpathological specimens from five aborted fetuses
(17 and 23 weeks of gestation, three and two cases, respectively) and
from four patients undergoing orchiectomy (18 months old and 38-, 42-,
and 52-yr-old, respectively) were analyzed by Northern blot,
semiquantitative RT-PCR amplification using DNA sequences or
specifically designed primers for the TR isoforms, and in
situ hybridization.
By using PCR amplification, we found that
TR1 and TR2 are
both expressed at different levels in fetal and adult testis. At all
ages TR2 is found at higher levels. Northern
analysis showed hybridization signals corresponding to the expression
of TR2 and TR1 in
a ratio that increased from 2.6 at 17 weeks of gestation to 12.0 in
adulthood. In fact, the expression of TR1
dramatically decreased throughout development, being faintly detectable
in the adult testis. Expression of TRß was not detected
at any age studied. This finding was further confirmed by PCR, which
did not amplify TRß either in fetal or in adult testis
mRNAs. In situ hybridization studies showed the absence
of TRß and that TR1 and
TR2 colocalized in Sertoli cells of
prepubertal testis, whereas germ and interstitial cells appeared devoid
of TR mRNA signals.
From these results it can be concluded that the human testis
exclusively expresses TR, which is localized in Sertoli
cells, TRß being always undetectable. Fetal and
prepubertal ages represent the period of maximal expression of
TR1 and TR2. The
2/1 ratio rises dramatically after
development. These results confirm a critical window for the action of
thyroid hormone in human testis, in the period of maximal expression of
T3 binding isoform TR1, and may
account for the macroorchidism without virilization occurring when
hyposecretion of thyroid hormones occurs before puberty.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2
sequence (TR2), which
encodes a thyroid hormone receptor (TR) isoform that does not bind the
hormone (3) but exerts a dominant negative effect on the action of
other TR isoforms (4), has been cloned from a human adult testis
cDNA library (5), suggesting that this tissue is unresponsive to
thyroid hormones. However, in prepubertal rat, we showed the presence
of nuclear-binding activity for thyroid hormones (6), due to the
expression of the TR1
messenger RNA (mRNA) (7), and a growing amount of evidence (8, 9, 10, 11)
demonstrates that prepubertal testis is a target organ for
iodothyronines.
mRNA of other TR forms and isoforms that bind T3
(TR1,
TRß1, and
TRß2) have never been
probed in human testis. We now study the expression pattern of
different TR forms and isoforms in fetal, prepubertal, and adult human
testis, showing the exclusive presence of 1
and 2 isoforms, localized within the
seminiferous epithelium in Sertoli cells, expressed at different levels
during and after development, the ß form being virtually absent at
all studied ages.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human fetal testes were obtained from five prostaglandin abortions at 17 weeks (three cases) and 23 weeks (two cases) of gestation with the permission of the Committee of Clinical Investigation. The ages of the fetuses were estimated from the menstrual history and from the measurements of fetal size, and samples from fetuses of the same gestational age were pooled. Postnatal human testicular tissues from four patients of various ages (18 months old and 38-, 42-, and 52-yr-old, respectively) were obtained at surgery for testis or prostate cancer. The patients had received neither hormonal nor chemotherapeutic medication, nor had they received radiotherapy before the operation; they had no endocrine disease, and none of them had suffered from cryptorchidism. The organs were quickly dissected, macroscopically examined, and frozen at -70 C until RNA isolation. Adult testis RNAs were pooled. The edges were cut off from all samples used in this study and embedded in paraffin for histopathological examination, which showed that all tissues were normal.
To assure the euthyroidism of the fetuses, free T3, free T4, and TSH values were determined by commercial kits and compared with the normal levels for gestational age (12). Normal levels of the same hormones were found at the time of surgery in postnatal and adult subjects.
RNA preparation from human testes and Northern analysis
Total RNA was isolated by the guanidinum isothiocyanate procedure (13), and poly(A)+ RNA was prepared by oligo(dT)-cellulose column chromatography (Fast Track kit, Celbio, Italy). Total RNA extracted from a confluent culture of human SK-N-SH neurogenic tumor cells (14) was used throughout the experiments as a positive control.
Poly(A)+ RNA (7 µg) samples were denatured,
separated by electrophoresis, blotted on nylon membranes, and probed at
high stringency with the (32)P-labeled human TR cDNAs (see Fig. 1
for details). In particular, the
full-length sequence of human TRß
(15) was used as a probe in a cohybridization
experiment with the 32P-labeled
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (16) sequence used as
internal control. Filters were hybridized with the
XhoI-EcoRI cDNA fragment specific for the
TR1 (corresponding to
the last 600 nt of the
TR1 cDNA) and with the
EcoRI-EcoRI fragment specific for
TR2 (17) (corresponding
to the last 600 nt of the
TR2 gene). Another set
of identical filters was hybridized with the full-length sequence of
TR. GAPDH was again used as control probe.
Autoradiograms were analyzed densitometrically, and the results were
expressed as arbitrary units of optical densities. The expression
pattern was expressed as the ratio between intensities from
TR2 and
TR1.
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First-strand cDNA was made using, for each sample, 2 µg total
RNA in the presence of M-MuLV reverse transcriptase and random primers
provided from the First-strand cDNA Synthesis Kit (Pharmacia Biotech, Cologno Monzese, Italy). The cDNA obtained was used as
a template for the PCR coamplification of
TR1 (445 bp),
TR2 (445 bp), and
TRß (212 bp) with hß-actin (287 bp) as
internal control, using the following primers (see Fig. 1
for
details):
TR1: upstream
5'-GCCAAAAAACTGCCCATGTTCTCCGAG-3', downstream 5'-
GGCAGGCCCCGATCATGCGGAGGTCAG-3';
TR2: upstream
5'-GCCAAAAAACTGCCCATGTTCTCCCAG-3', downstream
5'-TGTACAGAATCGAACTCTGCACTTCTC-3'; TRß:
upstream 5'-CAATTACCAGAGTGGTGGATTTTGCCA-3', downstream
5'-CACCACCCCAAGACCCCCATTTTTCAGCTGGACGC-3'; hß-actin: upstream
5'-AGCGGGAAATCGTGCGTG-3', downstream 5'-CAGGGTACATGGTGGTGCC-3'.
PCR was performed using a DNA thermal cycler (PCR system 9700;
Perkin-Elmer Corp., Rome, Italy) in the presence of
Taq DNA polymerase (2 units per tube) (Perkin-Elmer Corp.) with 15 pmol of both upstream and downstream primers and
2.2 mM MgCl. Coamplifications were carried out at
94 C for 1 min, 55 C for 1 min, and 72 C for 1 min for the first 10
cycles in the presence of
TR1,
TR2, and
TRß primers, and then, for the remaining 25
cycles, hß-actin primers were added to the reactions. The experiments
were repeated on at least three different occasions.
In situ hybridization
The DAKOGenPoint Catalyzed Signal Amplification System
(DAKO Corp. S.p.A., Milan, Italy) was used for in
situ hybridization. Normal testis from a 26-month-old subject
undergoing orchiectomy as treatment for monolateral testis cancer and
human SK-N-SH neurogenic tumor cells were fixed in 10% buffered
formalin for 12–15 h. The patient did not receive hormonal and
chemotherapeutic medication or radiotherapy before the operation.
Tissue and cell preparations were pretreated with 0.2–0.8%
pepsin/0.2N HCl at room temperature. Specimens were hybridized with the
biotinylated TR1,
TR2, and
TRß oligos described above and visualized with
the in situ hybridization detection kit (DAKO Corp.).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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isoforms in human testis
To evaluate the expression pattern of TRs in the human testis, we
first performed a semiquantitative RT-PCR analysis on fetal and adult
testicular tissues. mRNAs from fetal (17 weeks of gestation) and adult
testes were analyzed using specific primers for
TR1 and
2. As shown in Fig. 2 (top),
TR1 and
TR2 are both expressed
in the fetal tissues and decrease in the adult gonad,
TR2 showing a lower
decrease.
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1 sequence was first
used to probe the testicular mRNAs obtained from fetuses of 17 and 23
weeks of gestation, from 18-month-old patients, or pooled samples from
three subjects (38-, 42-, and 52-yr-old; Fig. 3, top, A, B, C, and D,
respectively). The 6-kb band signal corresponding to
1 was more intense in the 17 weeks of
gestation testis and it dramatically decreased throughout development,
being faintly detectable in the adult testis, as observed by
RT-PCR.
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2
sequence, the same Northern membrane was rehybridized to study the
expression pattern of the
TR2 isoform (Fig. 3, middle). The 3.2-kb band signal corresponding to
2 followed the expression pattern of the
1 isoform, showing maximal expression at 17
weeks of gestation and a sharp decrease during development and after
birth.
To make a direct comparison of the expression of both the isoforms, a
full-length cDNA probe corresponding to the complete
TR sequence was used. A ratio between the
intensity of the signal from
TR2 and
TR1 was obtained,
normalizing for the GAPDH signal (Fig. 4). The expression pattern from this
experiment further confirms the results obtained by using the specific
1 and 2 probes and
underlines the expression trend of both the isoforms. When evaluating
the ratios between the intensities from 2 and
1, it is interesting to observe that at 17 and
23 weeks of gestation the ratio is 2.6 and 3.2, respectively, and it
further increases after birth, being 4.2 at 18 months and 12.0 at
adulthood.
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To investigate TRß expression, a
preliminary RT-PCR analysis was performed on RNAs extracted from fetal
testes of 17 weeks of gestation and from pooled samples from three
subjects (38-, 42-, and 52-yr-old). As reported in Fig. 2
(bottom), TRß expression was absent
at both ages. Because it was possible that this receptor could have
been expressed late in fetal life or during prepubertal age, the same
mRNAs used for expression were hybridized using a full-length cDNA
probe specific for the TRß sequence. As shown
in Fig. 3 (third panel) no signals were obtained at any age
studied, compared to the SK-N-SH cell line (positive control), which
gave a 10-kb signal corresponding to TRß.
Localization of TR genes during development in human testis
To investigate the regional expression of TR genes in human testis
during development, we used in situ hybridization. A
hybridization signal with specific
TR1 (Fig. 5) and
TR2 (data not shown),
was apparent in prepubertal testis obtained from a subject of 26 months
and localized in the seminiferous epithelium but not in interstitium.
In particular, focal cytoplasmatic precipitates were detected in
Sertoli cells, but not in germ cells of seminiferous cords. In
contrast, the TRß specific oligo did not
hybridize to any region of the prepubertal testis, compared to the
hybridization signal found in the SKN-N-SH cells, used as positive
control (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,
but not TRß, from fetal life to adulthood. The
expression of TR is confined to Sertoli cells
and is ontogenetically regulated.
The presence of two distinct genes encoding TRs suggests that both are
important for the T3-signaling pathway. However,
the and ß forms are expressed in a distinct, but often
overlapping pattern, suggesting that they may mediate both individual
and common functions (18). In fact, there are few tissues in which only
one TR form is expressed (19, 20).
The unique expression of TR in human testis,
its localization in Sertoli cells, and its progressive decrease from
fetal to adulthood parallels that of rat gonad, a tissue in which we
demonstrated the localization of
TR1 and
TR2 in prenatal,
perinatal, and prepubertal Sertoli cells and the absence of
TRß (6, 21). Thus, the
growing number of results obtained with testicular tissues of hypo- and
hyperthyroid rodents may be of value for humans. Furthermore, human
testis, like prepubertal rat testis, can be considered an excellent
model for studying the role of TR, because of
the absence of other TR forms.
The ontogenetic pattern of TR expression in
human testis herein described may explain the abnormalities of the male
reproductive function accompanying the hyposecretion of thyroid
hormones in prepubertal age. In fact, several well-controlled studies
on boys have demonstrated that juvenile hypothyroidism can be
associated with precocious sexual development (for review, see Ref. 1).
This precocity is characterized by enlargement of the testes, in the
absence of activation of the hypotalamus-pituitary-gonadal axis,
without autonomous androgen hypersecretion and virilization that, for
this reason, has been termed "pseudopseudo precocious puberty"
(22). Up to 59 cases of prepubertal hypothyroidism have been reported
in boys (1, 21), and more than 75% had testicular size measurement
compatible with macroorchidism. Testicular biopsies showed a
predominance of the tubular compartment, characterized by an early
onset of spermatogenesis. This could be due to the high levels of FSH
found in this condition (23, 24) and/or to the lack of thyroid hormone
action during the period of maximal expression of
TR1.
Our study demonstrates that human testis expresses
TR1 even in adulthood,
although at low levels, when the
2/1 ratio reaches its
maximum. Additional studies on the spatiotemporal distribution of TR
coactivators and corepressors (25) in human testis are needed to
elucidate whether these levels are sufficient to mediate an action of
thyroid hormones in the adult gonadal tissue. However, clinical reports
suggest that this is not the case. In fact, when reviewing clinical
literature, it is clear that the male reproductive function is not
substantially affected when hyper- or hyposecretion of thyroid hormone
occurs after puberty (1).
The role of non-T3-binding isoform
TR2 in many tissues and
in the testis, both in humans and rodents, is not clear, but from the
knock-out data it seems that the presence of
TR2 is necessary for
male fertility, because only when 2(26), but not 1
(24, 25), is absent, mice were sterile. Considering what we have herein
demonstrated, that TR2
expression spans throughout life also in humans, it can be suggested
that its expression is required for human fertility. Alterations of
this gene may account for some cases of "unexplained" male
infertility. In this respect, it would be interesting to study the
knock-out of 2 isoform alone.
Finally, we demonstrated that TRß, similarly in
animals (6), is undetectable in human testis. This is in agreement with
the fact that in the inherited syndrome of generalized resistance to
thyroid hormones, characterized by the exclusive mutation of
TRß form, abnormalities of gonadal functions
have been observed only in 1 of 146 males reviewed by Refetoff et
al. (27). Furthermore, fertility as well as testicular macro and
micromorphology of
TRß-/- male mice are
apparently normal (19, 24). These observations confirm that
TR1 is the unique TR
expressed in testis and that TRß is not
involved in the altered reproductive functions associated with juvenile
hypothyroidism.
In conclusion, human testis exclusively expresses
TR1 and
TR2 forms, localized in
Sertoli cells, TRß being always undetectable.
The 2/1 ratio
increases progressively from fetal life to adulthood. As in
experimental animals,
TR1 is probably required
for normal testis differentiation and development.
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Acknowledgments |
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Received February 21, 2000.
Revised June 2, 2000.
Accepted June 9, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 mRNA
expression in rat testis. Mol Endocrinol. 8:89–96.[Abstract]
- and ß-thyroid hormone receptor genes
in rat brain and pituitary. Proc Natl Acad Sci USA. 86:7250–7254. and ß in brain development indicated by differential
expression of receptor genes. EMBO J. 10:269–275.[Medline]
gene encoding a thyroid hormone receptor is essential for
post-natal development and thyroid hormone production. EMBO J. 16:4412–4420.[CrossRef][Medline]
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) mRNAs in fetal (17 and 23 weeks
after coitum, a.c., respectively), postnatal (18 months
after birth, a.b.), and adult human testis (a pool of
testes from patients 38, 42, and 52 yr of age). Autoradiographs of
Northern blots performed with the full-length TR
) was also plotted.