BIOLOGY OF REPRODUCTION 64, 634643 (2001)

Developmental and Hormonal Regulation of Leptin Receptor (Ob-R) Messenger Ribonucleic Acid Expression in Rat Testis1
Manuel Tena-Sempere,2,3 Leonor Pinilla,3 Fu-Ping Zhang,4 Lucas C. Gonza lez,3 Ilpo Huhtaniemi,4 Felipe F. Casanueva,6 Carlos Dieguez,5 and Enrique Aguilar3
Department of Physiology,3 University of Co rdoba, 14004 Co rdoba, Spain Department of Physiology,4 University of Turku, 20520 Turku, Finland Departments of Physiology5 and Medicine,6 University of Santiago de Compostela, 15705 Santiago de Compostela, Spain
ABSTRACT
In target tissues, leptin receptor (Ob-R) gene expression results in an array of alternatively spliced isoforms (Ob-Ra to ObRf) with different functional features. Recent evidence has pointed to a direct role of leptin in the control of testicular function. However, complete elucidation of the pattern of Ob-R gene expression in the male gonad is still pending. The focus of this study was to characterize in detail the developmental pattern of expression and hormonal regulation of Ob-R gene in rat testis. To this end, the overall expression of Ob-R mRNA was compared to that of the fully functional, long Ob-Rb isoform in different experimental settings, using semiquantitative reverse transcription-polymerase chain reaction. Expression of Ob-R mRNA was detected in testes from 15-, 30-, 45-, and 75-day-old rats at rather constant relative levels. In contrast, testicular expression of Ob-Rb mRNA was higher in pubertal testes (15- to 30-day-old rats) and declined in adulthood. In testes from 30-day-old animals, analysis of isoform distribution revealed that, in addition to abundant Ob-Rb mRNA levels, expression of Ob-Ra, Ob-Rf, and, to a lesser extent, Ob-Rc and Ob-Re messages is detected. Testicular Ob-R mRNA expression appeared sensitive to neonatal imprinting as neonatal treatment with estradiol benzoate (500 g/rat; Day 1 postpartum) resulted in a persistent increase (P 0.01) in the relative expression level of Ob-R mRNA, a phenomenon only partially mimicked by neonatal suppression of serum gonadotropins by means of LHRH-antagonist administration. In addition, neonatal estrogenization differentially altered the pattern of expression of Ob-R isoforms in adult rat testis, as expression of Ob-Rb mRNA was decreased to undetectable levels, whereas that of Ob-Rc remained unaltered, and Ob-Ra, Ob-Rf, and, to a lesser extent, Ob-Re mRNA levels were signicantly increased (P 0.01) by neonatal exposure to estrogen. Finally, down-regulation of testicular Ob-R gene expression by homologous and heterologous signals was demonstrated as relative levels of Ob-R and Ob-Rb mRNAs were signicantly decreased (P 0.01), in a coordinate manner, in rat testis after exposure to human recombinant leptin in vitro, and after stimulation with hCG and FSH in vivo. In conclusion, our results indicate that testicular Ob-R gene expression is developmentally regulated, imprinted by the neonatal endocrine milieu, and sen1 This work was supported by grant PM98-0163 from DGICYT (Ministerio de Educacio n y Cultura, Spain) and project 1FD97-0696-02 (FEDER). 2 Correspondence: Manuel Tena-Sempere, Department of Physiology, Faculty of Medicine, University of Co rdoba, Avda Mene ndez Pidal s/n, 14004 Co rdoba, Spain. FAX: 34 957 218288; e-mail: 1tesem@lucano.uco.es

sitive to regulation by leptin and gonadotropins. The ability of pivotal signals in testicular function to regulate Ob-R gene expression further supports the contention of a direct role of leptin in functional control of the rat testis.

hormone action, leptin receptor, testes

INTRODUCTION

Received: 13 June 2000. First decision: 7 July 2000. Accepted: 25 September 2000. 2001 by the Society for the Study of Reproduction, Inc. ISSN: 0006-3363. http://www.biolreprod.org

Leptin, the product of the ob gene, has recently emerged as a pivotal signal in body weight homeostasis, neuroendocrine control, and fertility [13]. Such a wide range of biological actions is carried out through interaction with specic cell-surface receptors. Structurally, the leptin receptor (Ob-R) shows high homology to the class I cytokine receptor family, containing a single membrane-spanning domain [4]. Upon ligand-receptor interaction, different intracellular signaling systems are likely recruited, including janus tyrosine kinase-signal transducer and activation of transcription and mitogen-activated protein kinase pathways, although conicting results have been obtained in vivo and in vitro [47]. Worthy to note, expression of the Ob-R gene results in several alternatively spliced isoforms (Ob-Ra to Ob-Rf) that share the extracellular domain but differ in the length of their transmembrane and cytoplasmic coding regions [4, 8 10]. In this context, it is well established that the long ObRb subtype is the functional, signal-transducing isoform in the hypothalamus [3, 4]. However, the functional relevance of the shorter Ob-R subtypes remains to be fully elucidated. The predominant short form of the receptor (Ob-Ra) possesses some signaling capacity [11], and it has been implicated in the modulation of Ob-Rb activity [12, 13] and leptin transport across the blood-brain barrier [8, 14]. In addition, a role for the Ob-Re variant, devoid of transmembrane domain, has been proposed as a soluble leptin-binding protein [4]. On the contrary, no clear function has been assigned to other Ob-R variants. Nevertheless, although conclusive evidence for the precise roles of the array of Ob-R subtypes cloned is pending, it appears likely that leptin action upon target tissues might depend, at least partially, on the balance of expression of different Ob-R isoforms. Compelling evidence indicates that the hypothalamus is the primary target for most of the metabolic and neuroendocrine actions of leptin [3, 15]. However, based on the characterization of leptin receptor distribution and leptin effects on in vitro systems, additional sites for leptin action have been suggested, including the male gonad [1618]. Indeed, recent evidence from our group indicates that leptin is able to inhibit testosterone secretion acting at the testicular level [17, 19]. A similar nding was reported recently using isolated rodent Leydig cells [18]. Moreover, testicular 634

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expression of the Ob-R gene [16] as well as specic detection of the Ob-Rb subtype mRNA in rat Leydig cells [18] provided the potential basis for leptin action on testicular steroidogenesis. However, whether leptin has any additional roles in the regulation of testicular function throughout the life span and whether regulatory mechanisms operate to modulate leptin action upon the male gonad remain to be solved. In the present study, we report the developmental pattern of Ob-R gene expression as well as its hormonal regulation in the rat testis. It was our major goal to gain further insight into the mode of action of leptin on testicular function and to obtain new leads into the regulatory mechanisms and as yet unknown effects of leptin in the rat testis. To this end, overall expression of Ob-R mRNA was compared to that of Ob-Rb in different developmental stages and experimental settings, and expression of the mRNAs encoding different Ob-R isoforms was assessed, using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). It is noteworthy that both the imprinting effects and the acute actions of key hormonal regulators of testicular development and function were studied. The former involved evaluation of the impact of neonatal exposure to estrogen, given its imprinting actions on pivotal events in testicular maturation [2022]. The latter included analysis of the effects of acute testicular exposure to recombinant leptin, hCG, and FSH.
MATERIALS AND METHODS

Animals and Drugs

Male Wistar rats bred in the vivarium of our Institution were used. The day the litters were born was considered Day 1 of life. At this time, the litter size was adjusted to eight rats per dam. The animals were maintained under controlled conditions of light (14 h of light; lights-on at 0700 h) and temperature (22C). The animals were weaned at 21 days of age and housed thereafter in groups of ve rats per cage, with free access to pelleted food and tap water. Human recombinant leptin was produced in Saccharomyces cereviseae as described elsewhere [23] and kindly donated by Eli Lilly (Indianapolis, IN). Highly puried hCG (Profasi HP500) was purchased from Serono (Madrid, Spain). Human recombinant FSH was kindly provided by Prof. J.A.F. Tresguerres (Gonal-F; Serono). Estradiol benzoate (EB) was obtained from Sigma (St. Louis, MO), and the LHRH antagonist Org 30276 (Ac-D-pClPhe-D-pClPheD-Trp-Ser-Tyr-D-Arg-Leu-Arg-Pro-D-Ala-NH2CH3COOH) was generously supplied by Organon (Oss, Netherlands).
Experimental Designs

In experiment 1, overall Ob-R and isoform-specic ObRb mRNA expression at different stages of postnatal testicular development, from early puberty to adulthood, were monitored. To this end, groups of animals (n 5) were sequentially killed and testicular samples obtained from 15-, 30-, 45-, and 75-day-old rats. In addition, analysis of expression of mRNAs for the putative Ob-R isoforms was carried out in testes from 30-day-old rats, an age-point when prominent expression of the mRNA encoding the long Ob-Rb subtype was demonstrated by preliminary assays. In experiment 2, we assessed the effects of neonatal exposure to estrogen on the pattern of Ob-R and Ob-Rb mRNA expression during postnatal testicular develop-

ment. In this setting, 1-day-old male rats were injected s.c. with a single dose of EB (500 g/rat). This regimen is the most effective to induce complete estrogenization in the male rat, which is characterized by alterations in terms of maturation of testicular cell populations and endocrine and spermatogenic functions, but it is devoid of major systemic toxicity [22, 24, 25]. Vehicle (oil)-injected rats served as controls. Groups of animals (n 5) were sequentially killed on Days 15, 30, 45, and 75 of age. In addition, the impact of neonatal estrogenization upon testicular Ob-R isoform mRNA expression was evaluated in adult rat testis. Finally, the potential mechanism(s) involved in the effects of neonatal estrogenization on testicular Ob-R gene expression was explored. Because the effects of neonatal exposure to estrogen on the developing testis could be direct or mediated by postnatal suppression of gonadotropins [26], expression levels of Ob-R and Ob-Rb mRNA were assessed in testes of rats treated neonatally with a potent LHRH antagonist (ANT) and compared to those of neonatally estrogenized males. Male rats were injected s.c. with LHRH-ANT (5 mg/kg body weight) on Days 1, 4, 7, 10, 13, and 15 of age, as described previously [27]. For comparative analysis, three age points were selected for tissue sampling: Day 15 (4 h after the last LHRH-ANT injection), Day 30, and Day 75. In experiment 3, the regulation of testicular Ob-R mRNA expression by its cognate ligand in vitro was studied. As experimental setting, slices of testicular tissue, of approximately 2.0 mm thickness, were prepared from 30day-old rats and incubated for 180 min in the presence of increasing doses (109107 M) of human recombinant leptin, as described elsewhere [17, 19]. Thirty-day-old rats were selected for analysis based on our previous observations of prominent Ob-Rb mRNA expression at this age point. At the end of the incubation period, testicular samples were frozen in liquid nitrogen and stored at 70C until used for analysis of Ob-R and Ob-Rb expression. In experiment 4, the regulation of testicular Ob-R mRNA expression by gonadotropins was studied in vivo. Groups of 30-day-old (n 5) male rats were s.c. injected (1000 h) with vehicle (0.9% NaCl), highly puried hCG (50 IU/rat), or recombinant FSH (12.5 IU). The animals were sequentially killed 2, 4, 8, and 24 h after drug administration. In all in vivo experiments, the testes were immediately removed following decapitation of the animals, frozen in liquid nitrogen, and stored at 70C until used for RNA analyses. When considered relevant, serum samples obtained from experimental animals were assayed for LH, FSH, and testosterone levels, as described previously [28]. All the experimental procedures were approved by the Co rdoba University Committee on Laboratory Animal Care and were conducted in accordance with the European Union Normative for care and use of experimental animals.
Analysis of RNA by Semiquantitative RT-PCR

The RT-PCR, optimized for semiquantitative detection (see below), was used to analyze overall expression levels of Ob-R, as well as isoform-specic Ob-Rb, Ob-Ra, ObRc, Ob-Re, and Ob-Rf mRNAs in the different experimental groups. Total RNA was isolated from testicular samples using the single-step, acid guanidinium thiocyanate-phenol-chloroform extraction method, as described previously [29]. For amplication of the targets, the prim-

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FIG. 2. Representative ethidium bromide-stained gel electrophoresis (out of three independent assays) of Ob-R, isoform-specic Ob-Rb, and L19 cDNA fragments amplied by RT-PCR, using conditions selected for semiquantitative analysis. Assessment of the level of expression of the target mRNAs was carried out from testicular total RNA samples of 15-, 30-, 45-, and 75-day-old rats. The sizes of the generated products were calculated by comparison with the mobility of size markers (M) and are indicated in the right side.

er pairs indicated in Table 1 were used. It is noteworthy that analysis of overall Ob-R mRNA expression was carried out using a primer pair designed to amplify a 418base pair (bp) fragment of rat Ob-R cDNA encoding a region of the extracellular domain of the receptor, common for all cloned isoforms. The reported sets of primers were selected based on a previous reference [30] and synthesized according to the published cDNA sequences of the cloned Ob-R isoforms [810]. In addition, to provide an appropriate internal control, coamplication of a 290-bp
FIG. 1. Optimization of RT-PCR conditions for semiquantitative determination of target mRNAs. For amplication in the exponential phase of PCR, different numbers of cycles were tested for each message. Quantitative analysis of the cycle-dependency for the generated PCR signals revealed a strong linear relationship between cycles 28 to 38 in the case of Ob-R (correlation coefcient r2 0.997) and Ob-Rb (r2 0.9639), and cycles 14 to 23 in the case of L19 ribosomal protein (r2 0.9980). Optimization of RT-PCR conditions was carried out using total RNA from control, 30-day-old rat testis. Values are given as mean SD of two independent determinations. A representative ethidium bromidestained gel electrophoresis of the DNA products generated for each target (Ob-R, Ob-Rb, L19 ribosomal protein) is presented in the insets.

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FIG. 3. Assessment of the level of expression of the mRNAs encoding Ob-Ra, Ob-Rb, Ob-Rc, Ob-Re, and Ob-Rf subtypes in 30-day-old rat testis. A representative ethidium bromide-stained gel electrophoresis (out of three independent assays) of isoform-specic Ob-R cDNA fragments, amplied by semiquantitative RT-PCR, is presented. Reliability of RT-PCR conditions for semiquantitative determination was conrmed by the even amplication pattern of L19 transcripts, as an internal control, in the experimental samples (data not shown). The sizes of the generated products were calculated by comparison with the mobility of a 50-bp DNA step ladder (M); the bp length of representative markers are indicated on the left side.

fragment of the L19 ribosomal protein mRNA was carried out in each sample using the primer pair included in Table 1, generated according to the rat L19 ribosomal protein cDNA [31]. For amplication of the targets, RT and PCR were run in two separate steps. Furthermore, to enable appropriate amplication in the exponential phase for each target, PCR amplication of common and isoform-specic Ob-R and L19 ribosomal protein transcripts was carried out in separate reactions with a different number of cycles (see below) but using similar amounts of the corresponding cDNA templates, generated in single RT reactions, as described elsewhere [28, 32]. Briey, equal amounts of total testicular RNA (4 g) were heat denatured and reverse transcribed by incubation at 42C for 90 min with 12.5 U avian myeloblastosis virus (AMV) RT (Promega, Madison, WI), 20 U ribonuclease inhibitor RNasin (Promega), 200 nM deoxy-NTP mixture, and 1 nM specic and L19 antisense primers, in a nal volume of 30 l of 1 AMVRT buffer. The reactions were terminated by heating at 97C for 5 min and cooling on ice, followed by dilution of the RT cDNA samples with nuclease-free H2O (nal

volume 60 l). For semiquantitative PCR, 10-l aliquots of the cDNA samples (equivalent to 650 ng total RNA input) were amplied in 50 l of 1 PCR buffer in the presence of 2.5 U Taq-DNA polymerase (Promega), 200 nM deoxy-NTP mixture, and the appropriate primer pairs (1 nM of each primer; see above). The PCR reactions consisted of an initial denaturing cycle at 97C for 5 min, followed by a variable number of cycles of amplication dened by denaturation at 96C for 1.5 min, annealing at 55C for 1.5 min, and extension at 72C for 3 min. A nal extension cycle of 72C for 15 min was included. The number of cycles was optimized to ensure amplication in the exponential phase of PCR. Different numbers of cycles were tested for Ob-R and Ob-R isoforms (ranging between 25 and 45) and L19 ribosomal protein (ranging between 14 and 30) and 33 and 20 cycles, respectively, were chosen for further analysis (see Results). The generated cDNA fragments were resolved in 1.5% agarose gels containing ethidium bromide (0.1 g/ml) and visualized using a digital imaging system (Gelprinter Super software; TDI Ltd., Madrid, Spain), their molecular sizes being determined by comparison with size markers run together with the cDNA products (PCR 50-bp Step Ladder; Promega). Specicity of PCR products was conrmed by Southern hybridization, using radiolabeled nested oligo-primers, and/or digestion with specic restriction enzymes (data not shown), as described elsewhere [30, 33]. For quantitative evaluation, absolute optical densities (OD) of RT-PCR signals were obtained by densitometric scanning using an image analysis system (1-D Manager; TDI Ltd.). The values for the specic targets were normalized according to those of L19 ribosomal protein to express arbitrary units of relative abundance of the specic messages (i.e., relative expression). To ensure that equal inputs of RNA were added to RT-PCR reactions, only samples yielding roughly similar OD values for L19 bands were considered for further analysis. In addition, to minimize potential RT-PCR artifacts due to inherent reaction variability, all data points were repeated, for each target, at least three times using independent RNA samples. Finally, in all assays, liquid controls and reactions without RT were included, yielding negative amplication (data not shown).
Presentation of Data and Statistics When appropriate, semiquantitative data are presented as mean SEM from at least three independent obser-

TABLE 1. Oligo-primer pairs used for RT-PCR amplication of Ob-R-related and L19 ribosomal protein transcripts.a Target Ob-R Ob-Ra Ob-Rb Ob-Rc Ob-Re Ob-Rf L19
a

Oligo-primers Ob-R sense Ob-R as Ob-Ra sense Ob-Ra as Ob-Rb sense Ob-Rb as Ob-Rc sense Ob-Rc as Ob-Re sense Ob-Re as Ob-Rf sense Ob-Rf as L19-sense L19-as (5-CTCCGCACTCACAGGCAACA-3) (5-TGGATCGGGCTTCACAACAA-3) (5-CCTATCGAGAAATATCAGTTTA-3) (5-TCAAAGAGTGTCCGCTCTCT-3) (5-TGGCCCATGAGTAAAGTGAAT-3) (5-CCAGAAGAAGAGGACCAAATA-3) (5-GTGTCCTGCTGCTCGGAACACTGT-3) (5-AAAGATCTCTATGTAATAGAGTAT-3) (5-GCAGAATCAGCACACACTGTT-3) (5-GTAAAAGCACAGTACACATACC-3) (5-AGAGGATATATAGTGGATGC-3) (5-CACAAATGAGCCATCTTCAAA-3) (5-GAAATCGCCAATGCCAACTC-3) (5-ACCTTCAGGTACAGGCTGTG-3)

Expected size (bp) 418 285 386 188 301 411 290

Linear range 2838 (33) 2838 (33) 2838 (33) 3038 (33) 3238 (33) 2838 (33) 1423 (20)

For each target, the primer pair used for amplication is included. In addition, the expected size of the generated cDNA products and the range of number cycles that resulted in a linear amplication during optimization assays is presented. In parentheses, the number of cycles selected for semiquantitative RT-PCR analysis is indicated for each target.

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FIG. 4. On the left side, representative ethidium bromide-stained gel electrophoresis (out of three independent assays) of Ob-R, isoform-specic Ob-Rb, and L19 cDNA fragments amplied by semiquantitative RT-PCR, from testicular total RNA samples of control (C) and neonatally estrogenized (EB) rats at different developmental stages: 15-, 30-, 45-, and 75-day-old animals. The sizes of the generated products are indicated. On the right side, a compilation of semiquantitative data on the steady-state levels of Ob-R and Ob-Rb mRNAs in the above experimental groups is presented. Relative levels of expression were obtained, in each sample, by normalization of absolute ODs of each target (Ob-R and Ob-Rb) to that of L19 signals. Values are given as mean SEM of at least three independent determinations. ** P 0.01 versus values from corresponding controls (ANOVA followed by Tukeys test).

vations. Statistically signicant differences between groups were determined by analysis of variance (ANOVA), followed by Tukey test. P 0.05 was considered statistically signicant.
RESULTS

FIG. 5. Expression levels of the mRNAs encoding Ob-Ra, Ob-Rb, ObRc, Ob-Re, and Ob-Rf subtypes in testes from adult (75-day-old) control (C) and neonatally estrogenized (EB) rats. A representative ethidium bromide-stained gel electrophoresis (out of three independent assays) of isoform-specic Ob-R cDNA fragments, amplied by semiquantitative RT-PCR, is presented. In addition, as internal control, the corresponding amplication pattern using L19 primers is included. The sizes of the generated products were calculated by comparison with the mobility of a 50-bp DNA step ladder (M); the bp length of representative markers are indicated on the left side.

Optimization of Semiquantitative RT-PCR Assays The RT-PCR assays were optimized for semiquantitative analysis of expression levels of common and isoformspecic Ob-R as well as L19 ribosomal protein mRNAs. Such a method was selected due to its ability to detect alternately spliced isoforms with high sensitivity, in contrast with conventional hybridization-based assays such as Northern hybridizations. To obtain optimal conditions for amplication, i.e., in the exponential phase of PCR, different numbers of PCR cycles were tested for each message (see Table 1). This procedure was carried out in detail for Ob-R, Ob-Rb, and L19 transcripts. As shown in Figure 1, plotting of intensity of PCR signals (as expressed by absolute OD values) against the number of amplication cycles revealed a strong linear relationship between cycles 28 to 38 in case of Ob-R (correlation coefcient r2 0.997) and Ob-Rb (r2 0.9639), and cycles 14 to 23 in case of L19 ribosomal protein (r2 0.9980). Thus, PCR amplication of Ob-R-related and L19 ribosomal protein transcripts was carried out in separate reactions, using 33 and 20 amplication cycles, respectively (Table 1). The validity of the above RT-PCR assays for semiquantitative evaluation is supported by 1) the selection, for each target,

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FIG. 6. Representative ethidium bromide-stained gel electrophoresis (out of three independent assays) of Ob-R, isoform-specic Ob-Rb, and L19 cDNA fragments amplied by RT-PCR, from total testicular RNA samples of controls (C), neonatally estrogenized (EB) rats, and rats treated neonatally with a potent LHRH-ANT (A). For comparison, three age points are presented: Day 15 (4 h after the last LHRH-ANT injection), Day 30, and Day 75 of age. The sizes of the generated products are indicated. On the right side, a compilation of semiquantitative data on the steady-state levels of Ob-R and Ob-Rb mRNAs in the above experimental groups is presented. Relative levels of expression were obtained, in each sample, by normalization of absolute ODs of each target (Ob-R and Ob-Rb) to that of L19 signals. Values are given as mean SEM of at least three independent determinations. **P 0.01 versus values from corresponding controls; aP 0.01 versus values from corresponding EB-injected groups (ANOVA followed by Tukeys test).

of amplication conditions in the exponential phase of PCR (see above), 2) the repetitive observation of results within experimental groups (at least three assays per data point using independent RNA samples), and 3) the use of an appropriate internal control (L19 ribosomal protein).
Expression of the Ob-R Gene in Rat Testis at Different Stages of Postnatal Development

Relative expression levels of overall Ob-R and isoformspecic Ob-Rb mRNAs were evaluated by semiquantitative RT-PCR in rat testis during postnatal development. In detail, based in previous reports [34], the following age points were selected for analysis: 15, 30, 45, and 75 days postpartum, i.e., from early pubertal to adult stage of testicular development. The RT-PCR analysis demonstrated testicular expression of Ob-R mRNA at all age points studies, at rather constant relative levels. However, assessment of testicular Ob-Rb mRNA expression indicated clear-cut changes in the relative levels of this message that were signicantly higher (P 0.01) during the pubertal period of testicular development, with maximum values in 30-day-old rat testis, and declining thereafter (Fig. 2).
Expression of mRNAs Encoding Putative Ob-R Isoforms in 30-Day-Old Rat Testis Detection of the highest expression levels of the mRNA encoding the functional, long Ob-R isoform (Ob-Rb) in

30-day-old rat testis moved us to evaluate the pattern of relative expression of the different Ob-R subtypes at this age point. The RT-PCR analysis using isoform-specic primer pairs conrmed that Ob-Rb mRNA is abundantly expressed in the pubertal rat testis. In addition, testicular expression of the mRNAs encoding Ob-Ra, Ob-Rf, and Ob-Rc isoforms was also detected, whereas almost negligible signals for Ob-Re were amplied in 30-day-old animals (Fig. 3). This pattern of Ob-R isoform expression was analogous to that found in the adult (75-day-old) rat testis (data not shown).
Expression of the Ob-R Gene in Rat Testis after Neonatal Estrogenization: Mechanism(s) of Action

Expression of overall Ob-R as well as isoform-specic Ob-Rb mRNAs was monitored in testes from 15-, 30-, 45-, and 75-day-old rats after exposure to a single dose of estradiol benzoate (500 g/rat) on Day 1 postpartum. The RT-PCR analysis revealed that neonatal estrogenization induced a signicant increase (P 0.01) in relative expression levels of testicular Ob-R mRNA at all age points studied. In striking contrast, Ob-Rb expression levels in rat testes from neonatally estrogenized rats remained unchanged in 15-day-old animals, whereas a decline to undetectable levels was observed from Day 30 postpartum onward (Fig. 4). The divergent response to neonatal estrogenization in

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FIG. 7. Assessment of the level of expression of Ob-R and isoform-specic Ob-Rb mRNAs in 30-day-old rat testis after exposure in vitro to increasing concentrations (10910 7 M) of recombinant leptin. Testicular samples challenged with medium (Dulbeccos modied Eagles medium) alone served as controls. On the left side, a representative ethidium bromide-stained gel electrophoresis (out of three independent assays) of target cDNA fragments, amplied by semiquantitative RT-PCR in the experimental samples, is presented. On the right side, a compilation of semiquantitative data on the steady-state levels of Ob-R and Ob-Rb mRNAs in the above experimental groups is depicted. Relative levels of expression were obtained, in each sample, by normalization of absolute ODs of each target (Ob-R and Ob-Rb) to that of L19 signals. Values are given as mean SEM of at least three independent determinations. Data with different superscript letters are signicantly different ( P 0.01; ANOVA followed by Tukeys test).

terms of testicular expression of Ob-R and Ob-Rb mRNAs prompted us to evaluate the impact of this experimental manipulation upon the pattern of Ob-R isoform expression in adult rat testis. The RT-PCR analysis using isoformspecic primer pairs conrmed and extended our previous observations: expression of Ob-Rb mRNA was decreased to undetectable levels, whereas that of Ob-Rc remained unchanged and Ob-Ra, Ob-Rf, and, to a lesser extent, ObRe mRNA levels were signicantly increased (P 0.01) by neonatal exposure to estrogen (Fig. 5). In addition, expression levels of Ob-R and Ob-Rb mRNAs were assessed in testes of rats treated neonatally with a potent LHRH-ANT and compared to those observed after neonatal estrogenization. This setting was selected to differentiate between direct and indirect (i.e., those mediated by estrogen-induced suppression of gonadotropins) effects of neonatal estrogen exposure upon testicular Ob-R gene expression. The effectiveness of neonatal blockade of endogenous LHRH actions in our experimental setting was demonstrated by the signicant (P 0.01) suppression of serum LH, FSH, and testosterone (T) levels at the end of the 15-day period of treatment with LHRH-ANT (LH, 0.021 0.002 g/L vs. 0.216 0.08 g/L in paired controls; FSH, 0.497 0.078 g/L vs. 0.855 0.062 g/L in paired controls; T, 136.5 53.5 ng/L vs. 542.5 94.5 ng/L in paired controls). These responses were similar in magnitude to those induced by neonatal estrogenization in pair-aged animals (LH, 0.025

0.007 g/L; FSH, 0.376 0.049 g/L; T, 105.5 41.5 ng/L). Comparative analysis indicated that Ob-R and ObRb mRNA expression in testes from 15-day-old rats were similarly affected by neonatal treatment with estrogen or LHRH-ANT. However, from Day 30 onward, divergent responses were obtained between the experimental models as neonatal estrogenization, but not LHRH-ANT treatment, was able to persistently elevate the relative expression levels of Ob-R message, whereas signicant differences (P 0.01) were detected in the magnitude of suppression of Ob-Rb mRNA levels between neonatally estrogenized and LHRH-ANT-treated rats (Fig. 6).

Regulation of Ob-R and Isoform-Specic Ob-Rb mRNA Expression in Rat Testis by Leptin and Gonadotropins

The ability of different hormonal signals to modulate testicular expression of Ob-R and Ob-Rb mRNAs was assessed in 30-day-old animals. Challenge of testicular tissue in vitro for 180 min with increasing doses of human recombinant leptin (109107 M) induced a signicant (P 0.01) coordinate, dose-dependent decrease in the steady-state levels of overall Ob-R as well as isoformspecic Ob-Rb mRNAs (Fig. 7). In addition, the effects of exposure to hCG (as a superagonist of LH) and FSH in vivo were explored. Time-course analysis along a 24-h period revealed that exposure to 50 IU of highly puried hCG induced a signicant decrease (P 0.01) in Ob-R

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FIG. 8. Relative expression levels of Ob-R and isoform-specic Ob-Rb mRNAs in 30-day-old rat testis at different times (224 h) after exposure to 50 IU hCG or 12.5 IU human recombinant (h) FSH in vivo. On the left side, a representative ethidium bromide-stained gel electrophoresis (out of three independent assays) of target cDNA fragments, amplied by semiquantitative RT-PCR in the experimental samples, is presented. On the right side, a compilation of semiquantitative data on the steady-state levels of Ob-R and Ob-Rb mRNAs in the above experimental groups is depicted. Relative levels of expression were obtained, in each sample, by normalization of absolute ODs of each target (Ob-R and Ob-Rb) to that of L19 signals. Values are given as mean SEM of at least three independent determinations. Data with different superscript letters are signicantly different (P 0.01; ANOVA followed by Tukeys test). ND, Nondetectable signal.

and Ob-Rb mRNAs. However, subtle differences in this pattern of response were detected between targets. Overall expression of Ob-R mRNA was already decreased 2 h after hCG administration, reached the lowest levels at 4 h, and increased thereafter, with complete recovery by 24 h. In contrast, Ob-Rb mRNA levels remained unaffected 2 h after hCG and declined thereafter, with minimum levels at 8 h and incomplete recovery at 24 h after hCG exposure. In a similar setting, administration of 12.5 IU human recombinant FSH induced a decrease in Ob-R and Ob-Rb mRNAs to barely detectable levels at all time points (2 24 h) studied (Fig. 8).
DISCUSSION

The biological actions of leptin on body weight homeostasis, neuroendocrine function, and fertility are carried out through interaction with its specic receptor (ObR) in different target tissues. As anticipated on the basis of previous observations [1619], adult rat testis expresses the Ob-R gene, and testicular expression of the long ObRb isoform, i.e., the functional, signal-transducing Ob-R subtype in the hypothalamus, was demonstrated. Interestingly, Ob-R mRNA expression in rat testis was detected at rather constant relative levels along postnatal development. On the contrary, isoform-specic Ob-Rb mRNA expression was signicantly higher during the pubertal period of testicular development (1530 days postpartum) and declined in the adult. Considering that the ability of leptin to suppress testosterone secretion directly at the testicular level is restricted to the adult period [17], our present results provide the molecular basis for additional, yet undened, effects of leptin on rat testicular development and function, as suggested very recently in the mouse

[35]. In keeping with this view, it is apparent from our data that the pattern of alternative splicing of the Ob-R gene in rat testis is differentially modulated throughout the life-span. Assessment of Ob-R isoform expression in rat testis has not been previously published. The RT-PCR analysis using isoform-specic primer pairs indicated that, in addition to abundant Ob-Rb isoform mRNA expression, other Ob-R subtype messages are expressed in rat testis, with moderate levels for Ob-Ra, Ob-Rf, and, to a lesser extent, ObRc isoforms, and almost negligible signals for Ob-Re. Worthy to note, however, is that direct quantitative comparison of signal intensity for the different Ob-R variants can be hampered by differences in the amplication efciency of the cDNA targets using different primer pairs. The reported pattern of Ob-R isoform expression is analogous to that of the adult rat testis, although subtle differences in the relative abundance of Ob-Rb message, which was higher in 30-day-old rat testis, were noticed. Our obvious next step will be the identication of the pattern of cellular distribution of Ob-R isoforms within the rat testis. However, novel data on expression levels of the mRNAs encoding different Ob-R variants are relevant considering the divergent functional capacities of Ob-R subtypes [4, 814], as well as the proposed interaction between Ob-R isoforms in signaling the biological effects of leptin [12, 13]. Recently, a physiological role for estrogen in the regulation of testicular development and function has become evident [21]. Indeed, key events in testicular maturation, as Sertoli cell development [20] and expression of estrogen receptor (ER) and ER genes [28], are likely imprinted by neonatal estrogen. Moreover, estrogen is in-

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volved in the regulation of differentiation and function of testicular Leydig cells [36], as well as modulation of luminal uid resorption in the epididymis [37]. On this basis, we aimed at elucidating whether testicular expression of the Ob-R gene is sensitive to neonatal exposure to estrogen. Our results indicated that the relative expression levels of Ob-R mRNA are persistently up-regulated in testes from neonatally estrogenized rats throughout postnatal development. This response, however, did not involve a coordinated increase in expression of the mRNAs encoding the different putative Ob-R isoforms, as testicular ObRb mRNA levels were decreased to undetectable levels, whereas those of the Ob-Rc variant remained unchanged in adult, neonatally estrogenized rats; the suppression in terms of Ob-Rb expression being detected from Day 30 postpartum onward. On the contrary, a rise in Ob-Ra, ObRf, and, to a lesser extent, Ob-Re mRNA levels likely accounted for the observed increase in overall testicular Ob-R expression after neonatal exposure to estrogen. Therefore, it is apparent that divergent responses in terms of Ob-R isoform expression are elicited by neonatal estrogenization in rat testis that resulted in blunted expression of the message encoding the long, fully functional Ob-Rb isoform, but enhanced mRNA expression of several shorter Ob-R variants. Considering that the functional capacities of the long and short Ob-R isoforms are strikingly different [4, 6, 814], it is tempting to postulate that net leptin signaling is altered in the neonatally estrogenized rat testis. The contribution of this phenomenon to the plethora of functional defects in the male gonad induced by neonatal exposure to supraphysiological doses of estrogen remains to be elucidated. The effects of neonatal exposure to estrogen on the developing testis could be direct or mediated by postnatal suppression of gonadotropins [26]. Thus, the mechanism(s) whereby estrogen permanently imprints the pattern of testicular expression of Ob-R gene was approached by comparing its effects to those induced by the blockade of gonadotropin secretion via administration of a potent LHRH ANT during the neonatal period. Partially divergent responses detected between these two models suggest that estrogen-induced suppression of serum gonadotropin levels during the neonatal period cannot solely account for the above effects and open up the possibility of a direct effect of estrogen upon the developing testis in the modulation of Ob-R gene expression. Worthy of note is that selective suppression of Ob-Rb mRNA could be attributable, at least partially, to impaired development of testicular cell types expressing this message after neonatal estrogenization [25, 38, 39], as overall Ob-R mRNA expression in rodent testis shows a scattered pattern of cellular distribution [40], and altered maturation of germ, Sertoli, and Leydig cells has been demonstrated in this experimental model [25, 38, 39]. However, it is unlikely that the observed increase in testicular expression of the mRNAs encoding several short Ob-R subtypes could be explained through alterations in the cellular composition of the testis. In this context, it becomes relevant to identify the pattern of cellular expression of the different Ob-R isoforms in the neonatally estrogenized rat testis. The events involved in the modulation of the biological actions of leptin in target tissues likely include regulation of Ob-R expression. However, despite the proven expression of Ob-R gene in rat testis [16, 18], and the involvement of leptin in the control testicular steroidogenesis [1719], to our knowledge no attempt has been made ei-

ther to assess the ability of gonadotropins, the pivotal signals in the hormonal regulation of testicular function, to modulate Ob-R mRNA expression, or to elucidate whether testicular Ob-R mRNA expression is regulated by its cognate ligand. In our experiments, down-regulation of testicular Ob-R gene expression by homologous and heterologous signals was demonstrated as relative levels of ObR and Ob-Rb mRNAs were coordinately decreased in rat testis by exposure to human recombinant leptin in vitro, and after stimulation with hCG and FSH in vivo. In this sense, it was demonstrated recently that ligand-induced decrease in the number of cell-surface Ob-Rs is a mechanism whereby leptin desensitizes its own response [41, 42]. Our present data extend those previous observations, indicating that, in addition, ligand-induced down-regulation of Ob-R in rat testis may involve an actual decrease in Ob-R mRNA levels. A similar mechanism has been proposed for desensitization of other receptor systems in rat testis [43]. Moreover, if this model applies to other target tissues, it may explain the reported decrease in hypothalamic Ob-Rb mRNA expression during pregnancyinduced hyperleptinemia [30]. Furthermore, our analysis demonstrated that, in addition to its cognate ligand, Ob-R mRNA expression is under the control of heterologous signals, i.e., LH and FSH. This may be, at least partially, the basis for the sharp decline in testicular Ob-Rb mRNA levels following pubertal development, a period when high serum gonadotropin levels are achieved [28]. From a general standpoint, an analogous phenomenon has been observed by our group in rat adrenal, where leptin inhibits corticosterone secretion [44, 45], and Ob-R mRNA expression is down-regulated by heterologous (ACTH) as well as homologous factors [46]. Thus, it is tempting to suggest that leptin action upon the testis is controlled, at least partially, by ligand- and gonadotropin-induced downregulation of Ob-R gene expression. An analogous response pattern may apply to other steroidogenic tissues. In conclusion, our results indicate that testicular Ob-R gene expression is developmentally regulated, imprinted by the neonatal endocrine milieu, and sensitive to regulation by homologous and heterologous signals. Overall, data presented herein provide evidence for a novel regulatory step at the level of Ob-R mRNA expression whereby biological actions of leptin upon rat testis are modulated by gonadotropins and leptin itself.
ACKNOWLEDGMENTS
Leptin was a generous gift from Eli Lilly (Indianapolis, IN). Recombinant FSH was donated by Serono (Madrid, Spain). The authors are indebted to Roc o Campo n and Inmaculada Aguilar for their excellent technical assistance.

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