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BOR - Papers in Press, published online ahead of print January 8, 2003.
Biol Reprod 2003, 10.1095/biolreprod.102.008466
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BIOLOGY OF REPRODUCTION 68, 1975–1981 (2003)
DOI: 10.1095/biolreprod.102.008466
© 2003 by the Society for the Study of Reproduction, Inc.

Testis

INSL3 Ligand-Receptor System in the Equine Testis1

Thomas Klonisch2,3, Klaus Steger5, Astrid Kehlen4, William R. Allen6, Christine Froehlich3, Johannes Kauffold7, Martin Bergmann5, and Sabine Hombach-Klonisch3

Departments of Anatomy and Cell Biology3 Immunology,4 Martin Luther University Halle-Wittenberg, Faculty of Medicine, Grosse Steinstrasse 52, D-06097 Halle (Saale), Germany Institute of Veterinary Anatomy,5 Justus Liebig University Giessen, D-35392 Giessen, Germany Department of Clinical Veterinary Medicine,6 Equine Fertility Unit, University of Cambridge, Newmarket, Suffolk, CB8 9BH, United Kingdom Large Animal Clinic for Theriogenology and Ambulatory Services,7 Faculty of Veterinary Medicine, University of Leipzig, D-04103 Leipzig, Germany


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We employed molecular and immunological techniques to investigate the expression of INSL3, a member of the insulin-like superfamily, in prepubertal testis, postpubertal testes exhibiting normal and disturbed spermatogenesis, and cryptorchid testes of male horses. In addition, the partial cDNA coding sequences of the equine homologue of the human relaxin/INSL3-receptor Lgr8 were determined. Nonradioactive in-situ hybridization with a cRNA probe for equine Insl3 and immunohistochemistry with a specific rabbit INSL3 antiserum localized Insl3 transcripts and immunoreactive INSL3 ligand to Leydig cells in all types of testes investigated. Quantitative polymerase chain reaction analysis revealed a down-regulation of Insl3 and an up-regulation of the relaxin/INSL3-receptor expression in unilateral cryptorchid versus descended testes. Western blot analysis of protein extracts from adult normal and cryptorchid testes and prepubertal testes showed a single immunoreactive band at 14.5 kDa, which correlates with the predicted size of equine proINSL3. Densitometric analysis of Western blot data of adult normal testes revealed significantly stronger expression of immunoreactive proINSL3 as compared to extracts derived from cryptorchid or prepubertal testes. Thus, decreased expression of immunoreactive INSL3 in cryptorchid and prepubertal equine testis is transcriptionally regulated. The detection of transcripts for equine Lgr8 in the testis has identified the testis as a potential target of INSL3.

Leydig cells, placenta, relaxin, trophoblast


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The heterodimeric peptide hormone INSL3, also known as relaxin-like factor or Leydig-insulin-like peptide, is a member of the insulin-like superfamily [1–3]. The INSL3 is essential for differentiation of the gubernaculum testis. Male mice with a deletion of the Insl3 gene display impaired testicular descent and infertility [4, 5]. In the human, as in animal species, INSL3 is a constitutive marker of testicular Leydig cells in the postpubertal testis [6–9], and the testis is the major source of circulating serum levels determined in men and male rats [10, 11]. The potential physiological role of INSL3 in cryptorchidism is still unknown. Mutations and polymorphism of the Insl3 gene are not a major cause of testicular maldescent, with or without undermasculinization, in men [12–15]. Among endocrine factors potentially involved in cryptorchidism, diethylstilbestrol has been shown to interfere with gubernaculum development by down-regulating Insl3 gene expression [16].

Female INSL3-knockout mice show age-related disturbances of the estrous cycle and follicular development, which have been attributed to an increased rate of follicular apoptosis [5, 17]. In women, increased expression of INSL3 is observed in the glandular endometrial epithelium during the proliferative phase of the menstrual cycle, suggesting that estrogens may affect Insl3 gene activity in the uterus [18].

In the human, the testis expresses relaxin receptors, which were recently shown to belong to a larger family of leucine-rich, G protein-coupled receptors (LGR) [19]. Human LGR7 and LGR8 were demonstrated to specifically bind relaxin at nanomolar concentrations [19]. Deletion of the murine GREAT gene, a homologue to human LGR8, results in a cryptorchid phenotype similar to that seen in INSL3-deficient mice [4, 5, 20]. Indeed, LGR8 has recently been identified as a relaxin/INSL3 receptor that is able to bind both relaxin and INSL3 [21].

Cryptorchidism in the horse is of clinical and economic importance. Therefore, in the present study, we investigated the expression of equine (e) INSL3 in cryptorchid testes, in descended prepubertal testes, and in postpubertal testes exhibiting normal and disturbed spermatogenesis. Finally, we determined the partial cDNA sequences of equine relaxin/INSL3-receptor eLgr8 gene and demonstrated a potentially functional INSL3 ligand-receptor network within the equine testis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tissues

Testicles were collected from 19 adult stallions with normal testes (n = 9), testes with disturbed spermatogenesis (n = 7), and cryptorchid inguinal testes (n = 3). Prepubertal testes were obtained from eight colts showing unilateral cryptorchidism. Both the contralateral descended testis and the abdominally situated (n = 3) and inguinally situated (n = 5) testes were recovered from each animal. Small biopsy specimens of testicular parenchyma were cryopreserved in liquid nitrogen for reverse transcription-polymerase chain reaction (RT-PCR) or fixed in phosphate-buffered formalin or Bouin solution. Testicular tissues were obtained during routine medical procedures, and no animal was operated on specifically for the present study.

Isolation of RNA, cDNA Synthesis, and RT-PCR

Total RNA was extracted using Trizol reagent (Invitrogen, Karlsruhe, Germany) from three snap-frozen testis samples with histologically normal and impaired spermatogenesis, respectively. The amount and purity of the RNA isolated were determined by spectrophotometry at 260 and 280 nm [22]. Five micrograms of total RNA were used to generate first-strand cDNA, employing a Superscript reverse transcriptase kit and 500 ng/ml of oligo d(T) primer (both from Invitrogen). For RT-PCR of cDNA isolated from testicular tissues, specific oligonucleotide primers (Table 1, primers 1 and 2) for eInsl3 (accession no. AB033169) were employed. The Insl3 primers flanked the putative single intron at the N-terminus of the C-domain of eInsl3 to preclude any genomic DNA amplification. A specific amplicon of 540 base pairs (bp) encoding equine cytochrome P45017{alpha}-hydroxylase/17–20 lyase (Cyp17; accession no. D88184) was obtained by RT-PCR with specific primers (Table 1, primers 3 and 4). A nested RT-PCR approach was employed to clone a partial eLgr8 cDNA employing primers 5–8 (Table 1). Primers 5 and 6 were designed according to the human LGR8 sequence, whereas the nested primers 7 and 8 were specific for eLgr8. The partial cDNA sequence of eLgr8 was deposited under GenBank accession number AY196483. All RT-PCR reactions were carried out in 50 µl of solution containing 1 µl of cDNA, 5 µl of 10x Advantage cDNA polymerase mix buffer, 100 µM dNTP, 10 pmol of each primer, and 2.5 U of Taq DNA-polymerase (Clontech, Heidelberg, Germany). The PCR cycles consisted of an initial denaturation for 3 min at 95°C, followed by 40 cycles of denaturation at 95°C and annealing at 65°C (both for 1 min each), and an elongation step for 2 min at 72°C, and a final extension cycle for 10 min at 72°C. For nested RT-PCR, annealing temperatures of 60°C and 65°C were used for the outer and nested Lgr8 primer pairs, respectively. The PCR product was separated on a 1% low-melting point agarose gel, purified by Magic column extraction, and cloned into the pGEM-T vector (both from Promega, Mannheim, Germany). Sequence analysis of the PCR-amplified cDNA clones employed the PRISM dye Deoxy Terminator cycle sequencing kit (Perkin-Elmer, Foster City, CA) and T7 or SP6 sequencing primers.


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  		TABLE 1. List of oligonucleotide primers employed for the specific amplification of the indicated equine target genes

Construction of RNA Standards and Quantitative PCR Analysis

ß2-Microglobulin was used as an endogenous control for the normalization of quantitation. Quantitative PCR (Q-PCR) standard was constructed as described previously [23] employing the ß2-microglobulin primers 9 and 11 (Table 1). The resulting PCR amplicon was gel purified (QIA quick Gel Extraction Kit; Qiagen, Hilden, Germany) and transcripted in vitro with SP6 RNA polymerase, and recombinant RNA was quantified at 260 nm before use as standard in the Q-PCR reaction with primers 10 and 11. For quantification, 1 µl of the reverse transcriptase reaction mixture was added to 25 µl of reaction mixture consisting of 1x Advantage2 reaction buffer, 1.5 U of Taq polymerase (Clontech), 0.2x SYBR Green (Biozym; Hess, Oldendorf, Germany), 200 µM of each dNTP, and 0.5 µM of each specific primer. Primer pairs 1 and 2, 7 and 8, and 10 and 11 (Table 1) were employed for Q-PCR amplification of eInsl3, eLgr8, and ß2-microglobulin transcripts, respectively. A negative control without template was included. Six dilutions of each standard and cDNAs of three different animals were run in separate tubes in duplicate in an RG2000 cycler (LTF, Wasserburg, Germany). Initial denaturation at 95°C for 5 min was followed by 40 cycles with denaturation at 95°C for 15 sec, annealing at 65°C for 30 sec, and elongation at 72°C for 20 sec. The fluorescence intensity of the double-strand specific SYBR Green reflecting the amount of formed amplicon was read after each elongation step at 82°C using the Rotor-Gene software 4.4 (LTF) and the new release version 4.6. Relative quantitation of gene expression of both elnsl3 and eLgr8 was performed employing the comparative cycle threshold (Ct) method ({Delta}{Delta}Ct) with ß2-microglobulin as the endogenous standard. Both quantitation softwares revealed similar results.

Digoxigenin-Labeling of cRNA and In Situ Hybridization

For cRNA synthesis, 5 µg of the pGEM-T plasmid clone containing the cloned insert specific for eInsl3 and a pGEM-T construct with the insert for equine cytochrome P45017{alpha}-hydroxylase/17–20 lyase were digested with the restriction enzymes NcoI (sense cRNA)/NotI (antisense cRNA) and SacI (sense cRNA)/NotI (antisense cRNA), respectively (all enzymes from New England Biolabs, Schwalbach/Taunus, Germany). Digested plasmids were extracted in phenol and precipitated. Employing a cRNA synthesis kit (AMS Biotechnology, Wiesbaden, Germany) and a digoxigenin (DIG)-RNA 10x labeling mix (Boehringer Mannheim, Mannheim, Germany), cRNA synthesis was performed with 1 µg of digested and extracted plasmids. After precipitation of the DIG-labeled cRNA, the pellet was dissolved in 70 µl of diethyl pyrocarbonate water, and the quantity of cRNA was determined by dot-blot analysis of serial dilutions of DIG-labeled cRNA [24]. For nonradioactive in situ hybridization, serial dewaxed sections (thickness, 5 µm) of equine testis and placenta were digested with proteinase K at 30 µg/ml (Boehringer Mannheim), postfixed in 4% paraformaldehyde, and processed as described by Lewis and Wells [25] employing a 1:1000 dilution of an anti-DIG alkaline phosphatase-conjugated Fab-antibody in 1% BSA (Boehringer Mannheim). Specific hybridization signals were visualized using the chromogen combination 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium (both from Sigma, Munich, Germany). Sections were counterstained with hematoxylin, mounted in glycerol gel, and examined under a bright-field microscope.

Immunohistochemistry

Paraffin-embedded serial sections (thickness, 5 µm) of equine testis were dewaxed and rehydrated in PBS-0.1% Tween 20 (PBS-T). Endogenous peroxidase activity was blocked by incubation in 3% H2O2 in methanol for 25 min at room temperature. For the immunodetection of eINSL3, a rabbit polyclonal antiserum generated against a synthetic 15-mer epitope (N'-EKLCGHHFVRALVRV-C'; BioGenes, Berlin, Germany) located within the B-domain of human INSL3 was used. This antiserum had been shown previously to react specifically with a glutathione-S-transferase human INSL3 fusion protein, immunoreactive proINSL3, and a processed INSL3 of 6 kDa in extracts of human placenta and canine testis [18, 26, 27]. For the immunodetection of INSL3 within equine testis, nonspecific-binding sites were first blocked by incubation with 10% nonimmune swine serum in PBS-T containing 3% BSA. Tissue sections were incubated overnight at 4°C with the rabbit polyclonal INSL3-antiserum diluted 1:800 in PBS-T containing 3% BSA. After three washing steps (3 x 10 min in PBS-T), the sections were incubated for 1 h each at room temperature with a swine anti-rabbit immunoglobulin at 1:100, followed by a rabbit peroxidase antiperoxidase antibody at 1:100 in PBS-T. All sections were counterstained with hematoxylin and mounted in glycerol gel.

Western Blot Analysis

Snap-frozen testicular tissues were lysed in 63 mM Tris, 2% SDS, and 10% saccharose. After boiling for 5 min and subsequent centrifugation (5 min, 13 000 rpm), the amount of protein in the supernatant was determined with the protein assay kit (Bio-Rad, Munich, Germany) and then stored at -80°C until used. Protein extracts (20 µg/lane) were separated under nonreducing conditions on a 15% SDS-polyacrylamide gel and blotted onto a nitrocellulose membrane (Amersham, Braunschweig, Germany). After blocking treatment with PBS-T containing 5% milk powder and 1% BSA, membranes were incubated overnight at 4°C with the rabbit polyclonal INSL3 antiserum diluted 1:10 000 in blocking solution. After washing with PBS-T (3 x 10 min), specific binding was detected with a peroxidase-conjugated goat anti-rabbit immunoglobulin (Invitrogen) diluted 1:20 000 in PBS-T and visualized with the enhanced chemiluminescence Western blot detection reagent (Amersham). Blots incubated with rabbit nonimmune serum (DAKO, Hamburg, Germany) at similar dilution as the primary antiserum were used as negative control. Immunoreactive proINSL3 bands were quantified by densitometry. Equal loading of protein was assessed on stripped blots by immunodetection of ß-actin with a mouse monoclonal antibody diluted 1:1000 and a peroxidase-conjugated rabbit anti-mouse immunoglobulin diluted 1:5000 (both from Sigma).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The RT-PCR analysis on total RNA and mRNA derived from postpubertal normal and cryptorchid equine testis revealed single amplification products at 212 bp encoding eInsl3 and an amplicon of 653 bp encoding eLgr8, respectively. The proper phase of translation was obtained by alignment with the corresponding known human cDNA sequences (Fig. 1). Control experiments that employed water instead of cDNA did not yield amplicons (Fig. 1). The Q-PCR analysis revealed significantly reduced expression of eInsl3 mRNA in cryptorchid as compared to descended equine testis with normal spermatogenesis (Fig. 2). The eLgr8 transcripts were up-regulated 2-fold in cryptorchid equine testis (Fig. 2). The eLgr8 partial cDNA sequence encoded a deduced peptide sequence of 154 amino acids (aa), which corresponded to the transmembrane regions 1–6 in human LGR8. Equine LGR8 peptide displayed a 94.8% and 80.5% aa identity with human LGR8 and mouse GREAT protein sequence, respectively, but only a 55.3% aa identity with the corresponding peptide region of the human LGR7 (Fig.3).



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  		FIG. 1. RT-PCR with specific primers for eInsl3 revealed Insl3 amplicons of 212 bp (1 and 2) and eLgr8 amplicons of 653 bp (4 and 5) in cDNA preparations of postpubertal normal (1 and 4) and cryptorchid (2 and 5) equine testis (n = 3 each). Replacing cDNA with water in the PCR negative control reactions did not reveal amplification products (3 and 6). M, Marker



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  		FIG. 2. Q-PCR analysis of the transcriptional activity of the eInsl3 and eLgr8 gene in postpubertal descended and cryptorchid equine testis. The transcriptional rates of the target genes eInsl3 and eLgr8 in descended equine testis with normal spermatogenesis were set at 100% to serve as a reference point. Cryptorchid equine testis revealed a transcriptional down-regulation of eInsl3 by up to 50%, whereas expression of the Insl3 receptor eLgr8 was up-regulated by approximately 2-fold in comparison to descended equine testis



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  		FIG. 3. Nucleic acid sequence (653 bp) and deduced amino acid (aa) sequence of the partial cDNA sequence of equine relaxin/INSL3 receptor, eLgr8, corresponding to the nucleic acids 1271–1923 of the cDNA of human Lgr8. Nested primers employed for the specific amplification of the eLgr8 partial cDNA sequence are marked in italics. Alignment of the deduced partial aa sequence of eLGR8 (217 aa) and the corresponding aa sequences of human LGR8 (aa 425–641) and mouse Great protein (aa 408–624) corresponded to the start of the LGR8 transmembrane region 1 (TM1) to the beginning of TM6. Equine LGR8 protein showed 93.1% and 79.7% identity with human LGR8 and mouse Great protein, respectively

In descended postpubertal equine testis, specific eINSL3 immunostaining was detected exclusively in Leydig cells (Fig. 4A). Similar to the immunolocalization of eINSL3, nonradioactive in situ hybridization with an antisense eInsl3-DIG-labeled cRNA probe revealed an identical hybridization pattern in all testicular tissues investigated (Fig. 4B). Leydig cells in descended testes displaying disturbances in spermatogenesis (Fig. 4C), in cryptorchid testis with abdominal or inguinal localization (Fig. 4D), and in prepubertal equine testis (Fig. 4E) were also immunopositive for eINSL3. Leydig cells in equine testis were further characterized by the detection of specific hybridization signals for cytochrome P45017{alpha}-hydroxylase/17–20 lyase transcripts (Fig. 4, F and G). All the testicular sections that were treated with a nonimmune serum (Fig. 4H) or with the sense cRNA probe (Fig. 4I) were devoid of staining. Paraformaldehyde- and Bouin-fixed testicular tissues revealed an identical hybridization and immunoreactive staining pattern.



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  		FIG. 4. Detection of immunoreactive eINSL3 (A) and eInsl3 transcripts (B) in Leydig cells of paraffin-embedded normal postpubertal equine testis. Similar to hybridization signals obtained by nonradioactive in situ hybridization (data not shown), immunoreactive eINSL3 was also observed in postpubertal descended testis suffering disturbances in spermatogenesis (C), in cryptorchid testis (D), and in prepubertal testis (E). Leydig cells were further characterized by nonradioactive in situ hybridization using an antisense cRNA probe that encoded for equine cytochrome P45017{alpha}-hydroxylase/17–20 lyase (F and G). Representative sections from a prepubertal (F) and a cryptorchid equine testis (G) are shown. Sections treated in immunohistochemistry with nonimmune serum as the primary antiserum (H) and with the corresponding sense cRNA probe in the in situ hybridization (I) were devoid of staining. Original magnification: A–E and G–I, x200; F, x400

Western blot analysis of protein extracts from postpubertal normal, cryptorchid, and prepubertal testes demonstrated an immunoreactive band at 14.5 kDa, which correlated with the predicted size of pro-eINSL3 (Fig. 5). Densitometric analysis revealed significantly reduced amounts of immunoreactive eINSL3 in protein extracts (20 µg/lane) of cryptorchid and prepubertal testes as compared to normal equine testis. For prepubertal testis extracts, a 4-fold higher protein concentration had to be applied to detect a clear immunoreactive eINSL3 band (Fig. 5).



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  		FIG. 5. Densitometric analysis of immunodetection of a 14.5 kDa pro-eINSL3 with a rabbit polyclonal serum against INSL3 in protein extracts (20 µg/lane) of normal (1), cryptorchid (2), and prepubertal testis (3). Employing immunoreactive ß-actin to assess equal protein loading, expression of pro-eINSL3 ligand was strongest in postpubertal normal testes (1) and significantly weaker in cryptorchid testes (2). Prepubertal testes extracts (3) displayed very weak immunoreaction for pro-eINSL3 and 4- to 5-fold higher amounts of protein of up to 100 µg/lane were needed to obtain an immunoreactive signal comparable in intensity to that with the normal equine testis


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In horses, cryptorchidism is an important developmental defect, because the retained testis does not produce fertile spermatozoa yet continues to secrete high concentrations of androgenic hormones that promote stallion-like behavior in the affected animal. The INSL3 is an essential hormone for the development of the gubernaculum testis, which initiates testicular descent [4, 5, 10, 28]. In mice, the level of Insl3 mRNA in the testis was shown to remain constant during the first 3 wk after birth, to begin increasing at the time of the first round of spermiogenesis, and to reach peak levels during adulthood [29]. In the present study, we identified eINSL3 as a marker of Leydig cells in pre- and postpubertal equine testis. The localization of eInsl3 transcripts in Leydig cells was independent of the location of the testis in the animal. By contrast, eInsl3 gene activity in testicular Leydig cells appeared to be affected by cryptorchidism, because we observed reduced expression of eInsl3 transcripts and immunoreactive eINSL3 in cryptorchid testes despite the presence of multiple Leydig cells. In comparison, prepubertal testes, both normal and cryptorchid, contain few and, probably, less differentiated Leydig cells, which may explain the higher protein concentrations necessary to detect immunoreactive eINSL3.

In men, as in many animal species, INSL3 has previously been shown to be a constitutive Leydig cell marker in postpubertal normal testis [6, 7]. Furthermore, bovine testicular Leydig cells have been estimated to produce large amounts of INSL3 [30]. Measurable concentrations of INSL3 in peripheral serum have also been reported in men and adult male rats [10, 11]. Given the large number of Leydig cells within the adult equine testis, eINSL3 may be a major secretion product of the descended testis that contributes significantly to serum levels of eINSL3 in the stallion. In contrast to the Leydig cell products in equine testis that change concurrently with spermatogenesis, including testosterone [31], meiosis-activating sterol [32], aromatase [33], or alpha inhibin [34], an apparent lack of regulation of eINSL3 expression in Leydig cells by the seminiferous epithelial cell cycle has previously been reported in men and dogs [7, 26]. In the equine testis, we also detected eINSL3 expression despite marked differences in the functional state of the seminiferous epithelial cell cycle. However, the quantitative differences we observed in postpubertal normal as compared to cryptorchid and prepubertal testes suggest regulatory mechanisms to affect INSL3 production in testicular Leydig cells. Little information is currently available regarding factors that regulate INSL3 expression in Leydig cells in the descended or cryptorchid testis. In azoospermic hpg-mice suffering a deletion in the hypothalamically expressed gene for GnRH, which results in a defective hypothalamo-pituitary-gonadal axis, Leydig cells appear to be arrested in a prepubertal state of differentiation, and they lack testicular INSL3 expression [35]. Treatment with LH or hCG initiates INSL3 gene activation, thereby indicating that gonadotrophs may influence INSL3 expression by inducing Leydig cell differentiation [35]. The nuclear orphan-receptor steroidogenic factor-1 (SF-1) plays an essential role in transcriptional activation of the Insl3 gene [16]. In the mouse, a defined nucleotide sequence of 153 bp within the Insl3 promoter immediately before the Insl3 coding region has been shown to contain three consensus binding sites for SF-1, which mediate transactivation of the Insl3 promoter in the Leydig cell line MA-10 [36]. Employed in the present study to identify testicular Leydig cells, the gene for cytochrome P45017{alpha}-hydroxylase/17–20 lyase, a critical branchpoint enzyme for steroid biosynthesis, is also transcriptionally activated by SF-1 [37]. The effect of cryptorchidism on one or more of these controlling mechanisms involved in Insl3 gene regulation requires further investigation.

The equine testis expressed eLgr8 transcripts and was identified as a target tissue for the actions of eINSL3. Distinct from the endocrine actions of INSL3 on LGR8-expressing, INSL3-negative gubernaculum tissue during testicular descent [16, 38], the intratesticular INSL3-LGR8 ligand-receptor system appears to reflect a novel function of INSL3 hormonal activity. Within the unilateral cryptorchid as compared to the descended equine testis, a down-regulation of eINSL3 ligand and a 2-fold up-regulation of eLgr8 transcripts was observed. Although speculative, the transcriptional differences of the INSL3-LGR8 ligand-receptor system may reflect altered differentiation of the testicular cell populations expressing INSL3 and LGR8 in the descended and cryptorchid testis. With the generation of specific LGR8 antisera, future experiments will attempt to identify the cellular source of LGR8 in normal and cryptorchid testis.

In conclusion, we have identified the equine testis as a source and target tissue of INSL3, suggesting a functional intratesticular INSL3-LGR8 endocrine network within the equine testis.


   ACKNOWLEDGMENTS
 
The authors would like to thank Mrs. E. Schlueter for excellent technical assistance.


   FOOTNOTES
 
1 Funded in part by Fonds der Chemischen Industrie (FCI) and by the Deutsche Forschungsgemeinschaft (KL 1249/5-1 and 5-2; HO 2319/3-1). Back

2 Correspondence: Thomas Klonisch, Department of Anatomy and Cell Biology, Medical Faculty of the Martin Luther University Halle-Wittenberg, Grosse Steinstrasse 52, D-06097 Halle/Saale, Germany. FAX: 49 0 345 557 1700; thomas.klonisch{at}medizin.uni-halle.de Back

Received: 14 June 2002.

First decision: 1 July 2002.

Accepted: 26 December 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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