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Testis |
a Cattedra di Endocrinologia, Dipartimento di Medicina Interna, Università "Tor Vergata," 00133 Rome, Italy
b Cattedra di Andrologia, Dipartimento di Fisiopatologia Medica, Università "La Sapienza," 00161 Rome, Italy
c Cattedra di Medicina Interna, Dipartimento di Fisiopatologia Medica, Università di Roma "La Sapienza," 00161 Rome, Italy
d Dipartimento di Medicina Sperimentale, Laboratorio di Istologia ed Embriologia, Seconda Università di Napoli, 80138 Naples, Italy
e Dipartimento di Tossicologia, Università di Cagliari, 09124 Cagliari, Italy
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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leptin, leptin receptor, Leydig cells, testis, testosterone
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It is known that animal [17] and human [18] models of leptin resistance and deficiency show a severe impairment of the reproductive function. Homozygote mutations of leptin receptor (db/db mutants) have low levels of circulating testosterone, fail to undergo normal sexual maturation, and are infertile. These alterations are due to the absence of leptin stimulatory actions on GnRH neurons in the hypothalamus and a consequent hypogonadotropic hypogonadism [19]. In men, it has been shown that leptin levels rise by 
50% before the onset of puberty and decrease to baseline after its initiation [20], whereas in male monkeys, nocturnal leptin levels rise about 10–20 days before the prepubertal increase in pulsatile LH release [21]. These findings follow previous negative studies [22] and suggest that maturation of the HPG axis requires the integrity of the leptin-leptin receptor system. It is likely that, in the hypothalamus, leptin concentrations above a minimal threshold are necessary to trigger sexual maturation and maintain reproductive function [19].
In the present study, we investigated the developmental pattern of Ob-R gene expression and protein localization in rat testis from embryonic life to adulthood to gain more insight into the delicate cross talk between adipose tissue and testicular function during the entire life span. Our aim was to study whether critical events such as Leydig cell differentiation, birth, sexual maturation, and aging may affect leptin receptor expression and lead to significant modifications in the interaction between leptin and Leydig cell function. In order to better characterize Leydig cell specificity and differentiation status, we used an anti-relaxin like factor (RLF) antibody as a control in the immunohistochemical studies because RLF is considered to be a specific marker for the mature fetal as well as the adult Leydig cells [23, 24]. Finally, the effects of leptin on hCG-stimulated testosterone and intracellular cAMP production by Leydig cells isolated from prepubertal and adult rat testes were studied.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Male Sprague-Dawley rats (at least three per age group) (Charles River Laboratories, Wilmington, MA) of different embryonic (E14.5 and E19.5) and postnatal ages (7-, 14-, 21-, 35-, 60-, 90-day-old rats) were used. The animals were provided with a standard laboratory diet and normal light hours (14L:10D) and temperature (21–22°C) and were killed by oxygen deprivation. At early and late gestational ages (see Results), pregnant rats were killed and fetuses were removed. For the determination of the age of the embryos, the morning after vaginal plug formation was considered as Day 0.5 of embryonic development. Testes were rapidly removed and fixed overnight in Bouin solution for immunohistochemical experiments, frozen in liquid nitrogen for mRNA extraction, or placed in ice-cold PBS and decapsulated for collagenase digestion and subsequent Leydig cells purification and extraction. In separate experiments, sexually immature juvenile (21-day-old) and adult (60-day-old) male rats [25] were anesthetized with chloral hydrate and perfused transcardially with PBS followed by 4% paraformaldehyde in PBS, and brains were extracted after decapitation.
Immunohistochemistry
Bouin-fixed testes were washed twice in PBS, dehydrated, treated with xylene, embedded in paraffin, and sectioned at a thickness of 5 µm. Sections were dewaxed, hydrated, and rinsed with PBS. Endogenous peroxidases were blocked by incubation with 3% hydrogen peroxide in distilled water for 20 min at room temperature. Leydig cells were detected using a polyclonal anti-rat RLF antibody raised in rabbit (generously provided by Professor Richard Ivell, Institute of Hormone and Fertility Research, Hamburg, Germany) at 1:1000 dilution. The primary antibody against leptin receptor (rabbit anti-human Ob-R polyclonal antibody [H-300; Santa Cruz Biotechnology, Santa Cruz, CA]), specific for all the isoforms of the receptor, was used at 1:150 dilution, and sections were incubated overnight at 4°C. The following steps were performed according to the manufacturer's instructions (Histostain-Plus kit; Zymed Laboratories). The avidin-biotin immunoperoxidase system with 3-amino-9-ethylcarbazole as chromogen was used to visualize bound antibodies. The preparations were counterstained with hematoxylin, dehydrated, cleared, and mounted, as previously described [26].
Rat brains were postfixed in the same solution of perfusion and cut coronally on a vibratome (40 µm) 2 days later. Free-floating sections were washed in 0.1 M glycine in PBS, washed twice in PBS, and treated with 0.5% H2O2 in PBS, washed three times in PBS, and preincubated in PBS containing 10% normal goat serum and 0.25% Triton X-100 for 1 h at 4°C. Free-floating hypothalamic sections were incubated for 48 h with the primary Ob-R antibody (H-300; Santa Cruz Biotechnology) at a dilution of 1:500. The reaction was visualized using biotinylated secondary antisera and by standard avidin-biotin-horseradish peroxidase technique as described by Dragunow et al. [27]. After stopping the reaction by several washes in PBS, sections were mounted on chrome-alume gelatin-coated slides, dehydrated, and coverslipped with mounting medium. Slides were analyzed with a Zeiss Axioskope 2 light microscope (Oberkochen, Germany) equipped with high-resolution digital camera.
In both testes and brain experiments, negative control sections were processed in the absence of the primary Ob-R antibody and after preincubation of the Ob-R antibody with oversaturating concentrations of Ob-R (541–840) peptide (Santa Cruz Biotechnology) overnight at 4°C.
RNA Extraction and Reverse Transcription-Polymerase Chain Reaction Analysis
Tissue mRNAs were extracted by using a commercial kit (Micro-Fast-Track Kit; Invitrogen, San Diego, CA). First-strand cDNA was synthesized from 0.5 µg of mRNA using an annealing temperature of 65°C in a final volume of 25 µl containing 250 mM Tris-HCl, 375 mM KCl, 15 mM MgCl2, 50 mM dithiothreitol, 0.5 mM dNTPs, 0.5 µg random hexamer oligonucleotide, 200 U M-MLV-RT, 26 U ribonuclease inhibitor (Promega, Madison, WI). ß-Actin was used as a constitutive expressed gene product for comparison of Ob-R abundances in different samples. A 0.5-µl volume of the reverse transcription (RT) products was amplified with 2.5 units of Taq DNA polymerase (Promega) and 20 µM specific rat ß-actin primers (5'-ATTGGCAATGAGCGGTTCCGC-3' [sense, nucleotides 2413–2437] and 5'-CTCCTGCTTGCTGATCCACATC-3' [antisense, nucleotides 2749–2727], GenBank accession no. J00691) in 50 µl of reaction mix containing 50 mM KCl, 10 mM Tris-HCl, 1.5 mM MgCl2, and 10 mM each of the dNTPs. Thermocycling conditions were 1 min of denaturation at 94°C, 1 min of annealing at 58°C, and 1 min of extension at 72°C. To ensure amplification in the exponential phase of the polymerase chain reaction (PCR), reactions were temporarily halted at 20, 25, 30, 35, and 40 cycles and 12 µl of reaction mix were removed from each tube. All products were analyzed by 1.5% agarose gel electrophoresis (data not shown), and 25 cycles was chosen for further analysis. Quantitation of the signals was performed by densitometric analysis using densitometric computer software (Kodak Digital Sciences ID Image Analysis software; Eastman Kodak, Rochester, NY). Dilution of RT products was made when necessary and the amplification procedure was repeated until all samples were standardized for ß-actin content. After standardization, PCR was performed using appropriately diluted RT products in 50 µl of the reaction mix by utilizing 20 µM of each rat Ob-R primer (5'-ATGCTGTGCAGTCACTCAGTG-3' [sense, nucleotides 2284–2303] and 5'-CAACTCCTTCCATAAATACTGGG-3' [antisense, nucleotides 2526–2503], GenBank accession no. U52966). The used set of primers generated a 242-base pair (bp) PCR product corresponding to the extracellular domain common to all Ob-R isoforms. In order to study the developmental pattern of the different isoforms of leptin receptor, we used two more sets of primers (5'-GATATTTGGTCCTCTTCTTCTGG-3' [sense, nucleotides 2786–2809] and 5'AGTTGTGGTGAAATCACATTGG-3' [antisense, nucleotides 3223–3201], GenBank accession no. U52966), generating a 437-bp PCR product corresponding to the intracellular domain of Ob-R, specific for the long isoform of the receptor (Ob-Rb); and (5'-ATGCTGTGCAGTCACTCAGTG-3' [sense, nucleotides 2284–2303] and 5'-ACTTCAAAGAGTGTCCGCTCT-3' [antisense, nucleotides 2668–2689], GenBank accession no. D84125), chosen to generate a fragment of 479 bp, specific for the short Ob-Ra isoform. For each gene examined, all primers were derived from separate exons and spanned at least one intron of genomic sequence, thus excluding the possibility of genomic DNA contamination. No PCR product was obtained with any of the set of primers in the absence of cDNA template (data not shown). Thermocycling conditions were 30 sec of denaturation at 94°C, 30 sec of annealing at 55°C, and 60 sec of extension at 72°C, with an additional 2-min extension step at 72°C. Again, reactions were temporarily halted at 25, 30, 33, and 35 cycles to ensure amplification in the exponential phase of PCR; 30 cycles was chosen for densitometric analysis of Ob-R and 33 cycles was chosen for Ob-Ra and Ob-Rb because levels of PCR products increased in a linear fashion for up to 35 cycles. The identities of the generated PCR products were confirmed by cycle sequencing (Thermo Sequenase Radiolabeled Terminator Cycle Sequencing Kit; Amersham Italia, Milan, Italy), and the size was compared with molecular weight markers (100-bp ladder, Promega, Madison, WI). Quantitations of Ob-R, Ob-Ra, and Ob-Rb mRNA were evaluated by densitometric analysis as previously described after normalization with ß-actin.
Leydig Cells Studies
Immature and adult Leydig cells were isolated from Sprague-Dawley rats that were, respectively, 21 (10 rats per experiment) and 60 days old (6 rats per experiments) on the day of isolation. Leydig cells were obtained by collagenase digestion of decapsulated testes as already described [28]. Crude cell suspension was washed and then pelleted at 200 x g for 10 min. The cell pellet was then resuspended in regular Medium 199 (Whittaker M.A. Bioproducts, Walkerville, MD) with Hanks salts and L-glutamine containing 1.4 g/L NaHCO3, 0.5% BSA, 1 mM EDTA, 50 U/ml heparin, 12.5 mg/L deoxyribonuclease, 50 mg/L gentamicin, pH 7.4) and then centrifuged in a discontinuous Percoll density gradient at 800 x g for 30 min at 20°C without rotor brake. Bands between Percoll 68% and 43% were carefully taken, washed twice, and then pelleted and resuspended in Medium 199 containing 0.1% BSA, 50 mg/L gentamicin, and 0.125 mM 3-methylisobutyl xanthine (Aldrich Chemical, Milwaukee, WI). Nitro-blue-tetrazolium staining showed a purity of approximately 90% for immature Leydig cell and 95% for adult Leydig cell fractions. The purified immature and adult rat Leydig cells were plated in 24-well cell culture dishes (0.75 x 106 cells/well) and then incubated at 37°C under 95% O2 and 5% CO2 for 30 and 90 min in the presence or absence of saturating doses of hCG (1 ng/ml; CR 121; preparation kindly provided by the center for Population Research, NICHD, Bethesda, MD) with or without recombinant murine leptin (R & D Systems, Minneapolis, MN) at a final concentration of 150 ng/ml, which in other studies has already been shown to determine the maximal inhibitory effect on hCG-induced testosterone production by adult Leydig cells in culture [14]. At the end of incubation, media were removed and centrifuged at 250 x g for 10 min, and the supernatants were saved for the assay of extracellular testosterone (T). All plated cells were also processed for the analysis of intracellular cAMP, as described elsewhere [14, 29]. The measurements of testosterone and cAMP were performed by RIA, as previously described [14, 30, 31]. For cAMP, the sensitivity of the assay was 1 fmol/ml; the inter- and intraassay coefficients of variation were 6.4% and 4%, respectively. For T, the sensitivity of the method was 10 pg/assay tube; the mean inter- and intraassay coefficients of variation were 10% and 8%, respectively.
Every treatment was run in triplicate and every experiments was performed at least three times. Results are the mean ± SEM unless otherwise specified. Statistical significance was determined by ANOVA test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Figure 3 shows the immunolocalization of Ob-R in the hypothalamus of rats at different pubertal status (14 days old versus 60 days old). Different from what was observed in the testis, Ob-R immunopositivity was clearly present both before (Fig. 3, A–C) and after (Fig. 3, D–F) sexual maturation with a similar intensity, and it was mainly localized in the choroid plexus and arcuate nucleus (see arrows), indicating a different regulatory mechanism of Ob-R expression in the testis and brain and suggesting that sexual maturation does not alter the intensity and the pattern of Ob-R expression in the hypothalamus. In both testis and brain experiments, control sections processed in the absence of primary Ob-R antibody and after preincubation of the Ob-R antibody with oversaturating concentrations of Ob-R protein showed no staining.
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Figures 4 and 5 show the semiquantitative RT-PCR analysis of mRNA extracted from rat testes at different ages by using a set of primers specific for the common extracellular domain (Fig. 4) or the short (Ob-Ra) and long (Ob-Rb) isoforms (Fig. 5) of leptin receptor. Ob-R mRNA expression was present in testes from each age group, but semiquantitative analysis showed a 3.5-fold higher transcript concentration in late gestational (E19.5) versus early gestational (E14.5) testes. After birth (7- and 14-day-old rats), Ob-R mRNA was present at lower levels, similar to those expressed on Day E14.5, with a 2-fold increase on Day 21 and significantly higher mRNA concentrations in sexually mature testes, with gradual increases from younger to older ages (35- [3.5-fold], 60- [4.4-fold], and 90-day-old rats [6.7-fold] versus E14.5). The developmental pattern of Ob-Ra mRNA expression was similar to that of Ob-R: at Day E19.5, mRNA concentrations were three times higher than at Day E14.5; at Days 7 and 14, they were relatively low, similar to those present at E14.5, with a subsequent gradual increase from younger to older postnatal ages (21- [2.8-fold], 35- [3.1-fold], 60- [3.5-fold], and 90-day-old [5.15-fold] rats versus E14.5). In line with this pattern of expression, also Ob-Rb mRNA concentrations were relatively higher at E19.5 (4.2-fold versus E14.5) and at adult ages (60- [6.26-fold] and 90-day-old rats [9.5-fold versus E14.5]), whereas they were less abundant at 7 (2.4), 14 (2.6), 21 (3.3), and 35 (3.2-fold versus E14.5) days old.
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In order to establish whether leptin receptor has a functional role in rat prepubertal testis, in vitro studies were performed using isolated Leydig cells. Figure 6, A and B, shows basal and hCG-stimulated (1 ng/ml) T secretion by cultured Leydig cells, isolated from 21-day-old rats (sexually immature) and 60-day-old rats (mature adults), respectively, after 30 min of incubation with leptin (150 ng/ml). As already found in previous studies [14, 32], leptin significantly inhibited hCG-stimulated T production after 30 min of incubation in adult rat Leydig cells in culture (5.2 ± 0.46 ng/ml versus 9.6 ± 0.36 ng/ml, 46% reduction; P < 0.01), whereas it had no effect on hCG-stimulated T production in prepubertal Leydig cells. In both examined systems, leptin did not modify T production in the absence of hCG stimulus. In Figure 6, C and D, are shown basal and hCG-stimulated intracellular cAMP production from the same experiment as in Figure 6, A and B, after 90 min of incubation with leptin (150 ng/ml). In line with previously published results [14], leptin significantly increased intracellular cAMP production in adult Leydig cells (21.49 ± 1.68 fmol/ml versus 14.73 ± 0.99 fmol/L, 46% increase; P < 0.05), whereas it did not affect cAMP production in immature Leydig cells. Again, leptin alone did not affect cAMP production in both 21- and 60-day-old rat Leydig cells. These results indicate that, in rat Leydig cells, the leptin receptor is not functional before sexual maturation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In our study at any studied age, Ob-R immunoreaction was exclusively confined to Leydig cells, whereas no signal was detected in the tubules, in Sertoli cells, or in germ cells. Others, by using in situ hybridization techniques, showed that Ob-R mRNA is expressed in Sertoli cells of adult rats [33]. Thus, the lack of immunoreactivity inside the tubules suggests that Ob-R protein is not translated in Sertoli cells enough to be detectable by immunohistochemistry. Interestingly, in contrast with rat testis, in neonatal and adult mouse testis, it has been found that Ob-R immunoreaction is mainly present in the tubules and is localized to germ cells [34]. In that study, it was found that, in mouse testis, germ cells express leptin receptor in a stage and age-dependent manner, with maximal expression in the spermatocytes of adult animals, and that treatment of isolated seminiferous tubules with leptin leads to STAT-3 phosphorylation; the authors concluded that, in mice, leptin may have a direct role in proliferation and differentiation of germ cells [34]. In contrast, in the present study performed in rat testis, when Ob-R was present, it was exclusively confined to Leydig cells, while no signal was observed within the tubules. These discrepancies might indicate that, as already reported for other testicular peptides [35], the pattern of Ob-R expression in the testis is species specific. It must be noted that, in our studies, the Ob-R antibody used was targeted against a portion of Ob-R (aa 541–840) that is common to all different isoforms of the receptor. Thus, the possibility for differences in antibody selectivity targeting variant isoforms of leptin receptor differentially expressed between the interstitial and tubular compartment is unlikely.
Two overlapping generations of Leydig cells populate the rat testis. The first (i.e., fetal Leydig cells) develops during testicular embryogenesis, produces the T required for male sexual differentiation, and persists in the postnatal testis before degenerating after 2 wk of age. The second derives from mesenchymal-like undifferentiated cells, develops into mature adult Leydig cells throughout puberty, predominates in the adult rat testis, and supplies the testosterone production required for the onset of spermatogenesis and maintenance of male reproductive function [36]. In order to better characterize the specificity and the differential status of Leydig cells throughout development, immunohistochemical studies were performed by using an antibody against RLF, which is a specific marker of rodent mature fetal and adult-type Leydig cells [23, 24]. A very similar pattern of expression of RLF and Ob-R was observed. In the rat gonad, RLF and Ob-R expression was not observed in fetal Leydig cells at early gestational age (Figs. 1A and 2A), whereas it became evident in testes at late gestational age (Figs. 1B and 2B). After birth, concomitantly with the known decline in testosterone production, Leydig cells did not express Ob-R until sexual maturation (Fig. 1, C–E), while the anti-RLF staining identified persistent mature fetal Leydig cells in the interstitium (Fig. 2C; see arrows). During sexual maturation and in adult age, concomitantly with the differentiation and the development of the adult population of Leydig cells, Ob-R immunoreaction was again clearly present and abundantly expressed in the interstitium (Fig. 1, F–H); at the same studied ages, the intense anti-RLF positivity marked the mature generation of Leydig cells, with a characteristic increase in intensity from early pubertal to postpubertal periods (Fig. 2, F–H). Altogether, these findings indicate that Ob-R immunopositivity is characteristic of mature Leydig cells, both fetal and adult type, and has a characteristic pattern of expression during testicular development: It is absent after birth, when testosterone production by fetal Leydig cells declines, and it is switched on during and after sexual maturation, when the adult population of Leydig cells differentiates to mature type and becomes capable of full T production.
The explanation of the peculiar pattern of Ob-R in the rat testis during development is not known. It is possible that, in the testis, Ob-R expression is positively regulated by androgens because it is characteristic of mature androgen-secreting Leydig cells both in gestational and in postnatal life. Interestingly, an imperfect estrogen-responsive element and two SP-1 sites have been identified close to the most frequently used transcriptional start site of the rat Ob-R gene [37]. Leydig cells are the major site of estrogen synthesis in rat testis because of aromatase activity, which increases during cell maturation [38]. Thus, the possibility that androgens produced by Leydig cells could regulate testicular Ob-R expression indirectly through locally aromatase-induced estrogens cannot be excluded. Moreover, it has recently been shown that neonatal estrogenization up-regulates the overall Ob-R mRNA expression in rat testis [39]. Different from RLF and in accordance with our proposed model, Ob-R immunostaining was absent in the residual mature fetal Leydig cells in postnatal testis before sexual maturation concomitantly with the decline in T production commonly observed after birth. Gonadotropins are also possible candidates in stimulating the expression of Ob-R in Leydig cells during the peripubertal period. However, recent in vivo studies showed that, in 30-day-old rats, hCG treatment down-regulates testicular Ob-R mRNA expression [39]. The regulation of Ob-R expression in Leydig cells by gonadal steroids needs further study. Also, how changes in Ob-R expression fit with normal developmental pattern of T secretion is not known at present. The finding of a lack of expression of a functional leptin receptor in a period of low T secretion, i.e., early fetal life and prepuberty, further indicates that leptin has mainly a regulatory role on gonadotropin control of androgen production [14], the same as has been found for many other regulators of testicular function [35].
The pattern of expression of Ob-R in the testis was different from that observed in rat hypothalamus, where a marked Ob-R immunoreaction was evident both before and after sexual maturation. Ob-R was mainly localized in the arcuate nucleus and choroid plexus (Fig. 3; see arrows), with no significant difference in the intensity of Ob-R immunostaining before and after sexual maturation. These observations are in line with previous studies showing that Ob-R immunopositivity is present in the paraventricular nucleus at the late embryonic age, in the newborn, and during the suckling period, even if at a weak level compared with the adult age [40]. Therefore, it appears that different mechanisms control Ob-R expression in the testis and in the hypothalamus during development, probably through a tissue-specific different promoter [41] and/or a different local hormonal milieu [35].
Semiquantitative RT-PCR results obtained on testes from rats of different ages were in line with the immunohistochemical data (Figs. 4 and 5): Ob-R mRNA expression was significantly higher at late gestational (E19.5) versus early gestational (E14.5) age. After birth (7 and 14 days old), Ob-R mRNA levels were similar to those found on day E14.5, showed a slight increase on Day 21, and were much higher in mature testes, with a gradual increase from younger to older ages. Also, the developmental pattern of mRNA concentrations of the short (Ob-Ra) and long (Ob-Rb) isoforms of Ob-R was very similar to that of overall testicular Ob-R splice variants. Ob-R protein was markedly switched on at the time of sexual maturation, and absolutely no protein signal was detected at all studied prepubertal ages. These observations suggest that Ob-R expression is transcriptionally regulated, even if a posttranscriptional level of regulation cannot be excluded.
These latter data are partially in contrast with evidence from other authors [39], who detected Ob-R mRNA in testes from 15-, 30-, 45-, and 75-day-old rats at rather constant relative levels, while the Ob-Rb isoform mRNA concentration was found to be higher in pubertal testes (30 days old), with a subsequent decline in adulthood (75 days old). We cannot explain the discrepancies between these and our findings. However, in contrast with the report by Tena-Sempere et al. [39], we performed all RT-PCR studies on messenger RNA rather than total RNA, which allows a more specific and accurate quantification of transcripts. In addition, we used Sprague-Dawley rats, while the others used Wistar rats. Finally, in our study, we found a valuable consistency between immunohistochemical data, mRNA expression analysis, and functional findings. In order to establish whether immunohistochemical and RT-PCR results had functional relevance, studies on Leydig cells isolated before and after sexual maturation were performed. We recently showed that leptin is able to suppress hCG-induced T production and increase cAMP production by adult isolated Leydig cells in vitro [14]. Also, leptin levels in obese human subjects are the best hormonal predictor of the obesity-related reduction in androgen response to hCG tests in vivo [42]. In line with the morphological data and RT-PCR results, leptin did not modify either hCG-stimulated T or intracellular cAMP production by cultured prepubertal Leydig cells (Fig. 6), where Ob-R immunostaining was negative and the relative abundance of Ob-R mRNA was low. Similar results were reported by other authors using slices of prepubertal testicular tissue incubated in the presence of hCG and leptin [32]. Taken together, these results indicate that, in rat Leydig cells, the Ob 
Ob-R system is not functional before sexual maturation.
In conclusion our results underline how complex are the leptin-leptin receptor interactions at different anatomic levels, and indicate that this system may regulate Leydig cell function in a concentration and age-dependent manner. It appears that the Ob Ob-R system in the testis may serve to negatively regulate T production by Leydig cells during adult but not prepubertal life and that it could have a role during testicular embryogenesis and Leydig cell maturation in prenatal life.
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FOOTNOTES |
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2 Correspondence: Andrea Fabbri, Endocrinology Unit, Department of Internal Medicine, University Tor Vergata, Via di Tor Vergata 135, 00133 Rome, Italy. FAX: 39 06 72596663; a_fabbri{at}hotmail.com
Received: 28 May 2002.
First decision: 14 June 2002.
Accepted: 16 October 2002.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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