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The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 9 3453-3457
Copyright © 2000 by The Endocrine Society

Original Studies

Ontogenetic Pattern of Thyroid Hormone Receptor Expression in the Human Testis

Emmanuele A. Jannini, Anna Crescenzi, Nadia Rucci, Emiliano Screponi, Eleonora Carosa, Anna De Matteis, Enrico Macchia, Giulia d’Amati and Massimino D’Armiento

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We studied the spatiotemporal distribution of thyroid hormone nuclear receptors (TRs) {alpha}1 and {alpha}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 TR{alpha}1 and TR{alpha}2 are both expressed at different levels in fetal and adult testis. At all ages TR{alpha}2 is found at higher levels. Northern analysis showed hybridization signals corresponding to the expression of TR{alpha}2 and TR{alpha}1 in a ratio that increased from 2.6 at 17 weeks of gestation to 12.0 in adulthood. In fact, the expression of TR{alpha}1 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 TR{alpha}1 and TR{alpha}2 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{alpha}, which is localized in Sertoli cells, TRß being always undetectable. Fetal and prepubertal ages represent the period of maximal expression of TR{alpha}1 and TR{alpha}2. The {alpha}2/{alpha}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 TR{alpha}1, and may account for the macroorchidism without virilization occurring when hyposecretion of thyroid hormones occurs before puberty.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THYROID HORMONES play an important role in the growth, development, and metabolism of mammalian tissues, and their excess or deficiency affects many organs and systems. However, controversy exists regarding the impact of iodothyronines on human male reproduction (1). This is due to the apparent clinical irrelevance of signs and symptoms related to male gonadal function during hypo- and hypersecretion of thyroid hormones, to the paucity of well-controlled clinical studies, to the fact that thyroid diseases are more common in females than in males, and, finally, to the demonstration that the adult male gonad of experimental animals is metabolically unresponsive to thyroid hormones (2). Furthermore, the c-erbA{alpha}2 sequence (TR{alpha}2), 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 TR{alpha}1 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 (TR{alpha}1, 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 {alpha}1 and {alpha}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.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Subjects

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. 1Go 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 TR{alpha}1 (corresponding to the last 600 nt of the TR{alpha}1 cDNA) and with the EcoRI-EcoRI fragment specific for TR{alpha}2 (17) (corresponding to the last 600 nt of the TR{alpha}2 gene). Another set of identical filters was hybridized with the full-length sequence of TR{alpha}. 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 TR{alpha}2 and TR{alpha}1.



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  		Figure 1. Schematic representation of the cDNA of hTR{alpha}1, hTR{alpha}2, and hTRß. The thick squares represent the coding region composed by the A/B domain (striped), the DNA binding domain (DBD; white), the hinge region (black), and the ligand-binding domain (LBD; dotted). TR{alpha}1 and TR{alpha}2 have the same sequence until the splicing site (arrows). Oligos used for PCR and size of amplified fragments are indicated. The restriction sites EcoRI and XhoI are used to cut the specific fragments for {alpha}1 and {alpha}2 used in Northern analysis.

 
Semiquantitative RT-PCR analysis

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 TR{alpha}1 (445 bp), TR{alpha}2 (445 bp), and TRß (212 bp) with hß-actin (287 bp) as internal control, using the following primers (see Fig. 1Go for details):

TR{alpha}1: upstream 5'-GCCAAAAAACTGCCCATGTTCTCCGAG-3', downstream 5'- GGCAGGCCCCGATCATGCGGAGGTCAG-3'; TR{alpha}2: 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 TR{alpha}1, TR{alpha}2, 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 TR{alpha}1, TR{alpha}2, and TRß oligos described above and visualized with the in situ hybridization detection kit (DAKO Corp.).


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of TR{alpha} 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 TR{alpha}1 and {alpha}2. As shown in Fig. 2Go (top), TR{alpha}1 and TR{alpha}2 are both expressed in the fetal tissues and decrease in the adult gonad, TR{alpha}2 showing a lower decrease.



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  		Figure 2. Expression of various TR isoform mRNAs in human testis during development and adulthood. Semiquantitative RT-PCR analysis of fetal (17 weeks of gestation) and adult (a pool of mRNAs extracted from testes of patients 38, 42, and 52 yr of age) human testis. The cDNA transcribed from 2 µg total RNA from testes or SK-N-SH cells (positive control) was used as a template for the PCR amplification of TR{alpha}1, TR{alpha}2, TRß, and hß-actin, used as internal control.

 
To analyze the expression pattern of both TR isoforms in more detail, Northern blot experiments were performed on testis mRNAs obtained at different ages. An XhoI-EcoRI cDNA fragment corresponding to the last 600 nt of the TR{alpha}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. 3Go, top, A, B, C, and D, respectively). The 6-kb band signal corresponding to {alpha}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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  		Figure 3. Expression of various TR isoform mRNAs in human testis during development and adulthood. Representative Northern blot of 10 µg of poly(A)+ RNA extracted from fetal (lane A, 17 weeks of gestation; lane B, 23 weeks of gestation) and postnatal human testis (lane C, 18 months after birth; lane D, a pool of testes from patients 38, 42, and 52 yr of age). Samples were separated on a denaturing gel, blotted on nylon filters, and hybridized with the cDNA fragments corresponding to the indicated TR or with a human GAPDH probe to assess their integrity and concentration. Ten micrograms of human neuroblastoma SK-N-SH cells Poly(A)+ RNA have been used as positive control. The size of the different mRNAs are also shown. The figure is representative of three separate experiments.

 
Using an EcoRI- EcoRI cDNA corresponding to the last 600 nt of the TR{alpha}2 sequence, the same Northern membrane was rehybridized to study the expression pattern of the TR{alpha}2 isoform (Fig. 3Go, middle). The 3.2-kb band signal corresponding to {alpha}2 followed the expression pattern of the {alpha}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{alpha} sequence was used. A ratio between the intensity of the signal from TR{alpha}2 and TR{alpha}1 was obtained, normalizing for the GAPDH signal (Fig. 4Go). The expression pattern from this experiment further confirms the results obtained by using the specific {alpha}1 and {alpha}2 probes and underlines the expression trend of both the isoforms. When evaluating the ratios between the intensities from {alpha}2 and {alpha}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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  		Figure 4. TR{alpha}1 and TR{alpha}2 expression and ratio during and after development. Expression of TR{alpha}1 (•) and TR{alpha}2 ({blacksquare}) 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{alpha} cDNA have been scanned, and densitometric values of specific bands corresponding to the TR{alpha}1 and TR{alpha}2 mRNAs were normalized to that of GAPDH control probe. The TR{alpha}2/TR{alpha}1 ratio ({blacktriangleup}) was also plotted.

 
Expression of TRß in human testis

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. 2Go (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 {alpha} expression were hybridized using a full-length cDNA probe specific for the TRß sequence. As shown in Fig. 3Go (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 TR{alpha}1 (Fig. 5Go) and TR{alpha}2 (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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  		Figure 5. Localization of TR{alpha} mRNAs by in situ hybridization. The biotinylated oligos used in Fig. 2Go have been hybridized with testis sections from a patient of 26 months. SC, Sertoli cells; GC, germ cells; I, interstitium. Original magnification, x100.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We demonstrated for the first time that human testis, a tissue previously considered virtually devoid of TRs and unresponsive to thyroid hormones, expresses exclusively TR{alpha}, but not TRß, from fetal life to adulthood. The expression of TR{alpha} 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 {alpha} 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{alpha} 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 TR{alpha}1 and TR{alpha}2 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{alpha}, because of the absence of other TR forms.

The ontogenetic pattern of TR{alpha} 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 TR{alpha}1.

Our study demonstrates that human testis expresses TR{alpha}1 even in adulthood, although at low levels, when the {alpha}2/{alpha}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 TR{alpha}2 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 TR{alpha}2 is necessary for male fertility, because only when {alpha}2(26), but not {alpha}1 (24, 25), is absent, mice were sterile. Considering what we have herein demonstrated, that TR{alpha}2 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 {alpha}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 TR{alpha}1 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 TR{alpha}1 and TR{alpha}2 forms, localized in Sertoli cells, TRß being always undetectable. The {alpha}2/{alpha}1 ratio increases progressively from fetal life to adulthood. As in experimental animals, TR{alpha}1 is probably required for normal testis differentiation and development.


   Acknowledgments
 
We are grateful to Dr. Susanna Dolci for critical reading of the manuscript and to Dr. Rosaria Caruso for adapting her English expertise to our needs.

Received February 21, 2000.

Revised June 2, 2000.

Accepted June 9, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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