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BOR - Papers in Press, published online ahead of print May 19, 2004.
Biol Reprod 2004, 10.1095/biolreprod.103.024752
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BIOLOGY OF REPRODUCTION 71, 1079–1087 (2004)
DOI: 10.1095/biolreprod.103.024752
© 2004 by the Society for the Study of Reproduction, Inc.

Testis

Seasonal Expression of INSL3 and Lgr8/Insl3 Receptor Transcripts Indicates Variable Differentiation of Leydig Cells in the Roe Deer Testis

Sabine Hombach-Klonisch1,2, Jennifer Schön4, Astrid Kehlen3, Steffen Blottner4, and Thomas Klonisch1,2

Departments of Anatomy and Cell Biology2 Immunology,3 Martin Luther University, Faculty of Medicine, Halle/Saale, Germany D-06097 Institute for Zoo and Wildlife Research,4 Berlin, Germany D-10252


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Roe deer are seasonal breeders and show cyclic variation in testicular volume and cellular differentiation within the tubular and interstitial testis compartment. We have employed the roe deer as a model to elucidate the expression of the postpubertal Leydig cell marker INSL3 during seasonal changes in Leydig cell differentiation. Roe deer testis and serum samples were collected bimonthly throughout the complete reproductive cycle. Peak levels of testicular Insl3 mRNA and INSL3 immunoprotein were detected well before the onset of rut in April and coincided with the highest percentage of INSL3-positive cell number/square millimeter of testicular interstitial area. During the winter (December, February), roe deer INSL3 was exclusively detected in a subpopulation of alpha-actin-negative, spindle-shaped peritubular cells. Concordant with the increase in INSL3 production in April and 1 mo after the reported LH peak, a sharp increase in serum testosterone concentrations was observed. High serum testosterone concentrations coincided with the presence of detectable 17alpha-hydroxylase, mRNA and protein, in Leydig cells. Upregulation of INSL3 production in spring appeared to reflect LH-dependent differentiation of Leydig cells. The considerable changes in percentage of INSL3 immunopositive cells within the numerically constant interstitial cell population indicated cyclic seasonal de- and redifferentiation of Leydig cells. A complex functional role of the INSL3/LGR8 ligand-receptor system in the roe deer testis was suggested by the detection of specific hybridization signals for roe deer Lgr8 transcripts in Sertoli cells of the roe deer testis.

INSL3, Leydig cells, Lgr8, male reproductive tract, relaxin, roe deer, seasonal reproduction, testis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mature males of mammals with seasonal reproduction show circannual cycles of testicular growth and involution (for review, see [1, 2]). Such seasonally regulated breeding is associated with considerable changes in testis mass, structure, and function throughout the year and includes variations in the proportion and differentiation of cells of both the tubular and interstitial testicular compartment. During these photoperiodically induced transitions between the active and inactive state of the testes, Leydig cells vary in size, ultrastructure, and androgen production and, in some species, also in total cell number [3, 4]. Regulation and kinetics of this circannual process of Leydig cell differentiation in adult males of seasonally reproducing species are not well understood, and investigations of these cellular events require the establishment of an intact relationship between Leydig cells and other testicular cell types [5].

Seasonally reproducing animals represent ideal models to study basic mechanisms of spermatogenesis and steroidogenesis and the impact of controlling factors. Many cervid species of northern temperate origin show such cyclic changes between completely arrested and highly activated spermatogenesis synchronized by the hypothalamic-pituitary-gonadal (hpg) axis [6–8]. As the seasonal breeding hamster was introduced as a model to study structure-function relationships in rodent testis [9], we have employed the roe deer (Capreolus capreolus) as an established animal model to investigate the continuous transition between active and inactive state in the testis of adult ruminants. Roe deer is characterized by drastically reduced testicular weight (to ~20%) and testis function during the winter months as well as high spermatogenic activity and testosterone production during the short rutting season from mid-July to mid-August [10–13]. Already in September, shortly after the rut, declining serum levels of testosterone, a main secretion product of testicular Leydig cells, signal a substantial reduction in Leydig cell activity. As in inactive periods of other seasonally breeding mammals [for reviews, see 3, 4], basal testosterone serum levels are observed in roe deer from October until February [11, 14–17]. Testis involution is already initiated during the late rut in August and involves both the tubular and interstitial testis compartment [10, 18–20]. Shortly after the rut, a sharp decline in mitotic and meiotic cell figures within the seminiferous tubules and a profound morphological change of Leydig cells is observed [10, 11, 19]. Leydig cell involution continues from October until early spring the following year and involves a marked reduction in cytoplasmic volume and testosterone production [10].

The INSL3 peptide hormone, also known as relaxin-like factor or Leydig cell-derived insulin-like factor, is regarded as a marker for Leydig cells of the postpubertal testis in various species, including men [21, 22]. In rodents, Insl3 mRNA expression is developmentally regulated, with fetal Leydig cells secreting INSL3 and testosterone in a gonadotropin-independent manner [23]. Homozygous Insl3-deficient male mice exhibit bilateral cryptorchidism, reflecting an essential role of INSL3 during the first phase of testicular descent [24, 25]. With the onset of puberty and the differentiation of adult-type Leydig cells, Insl3 gene activity is strongly upregulated [26, 27] and mature Leydig cells produce INSL3 in a constitutive manner during adult life [22, 23, 27]. A potential role of a functional hypothalamic-pituitary-testicular axis in Leydig cell differentiation and concomitant INSL3 expression is evidenced by the fact that the hypogonadal (hpg) mouse with a defect in the hypothalamic gene encoding for GnRH contains testes that remain in a prepubertal state. Exposure of immature testicular Leydig cells of hpg mice with the LH-analogue human chorionic gonadotropin (hCG) results in the development of adult-type testicular Leydig cells expressing INSL3, indicating that, at least in rodents, postnatal initiation of INSL3 production by testicular Leydig cells is LH dependent [26].

Currently, information is lacking on the expression of INSL3 and its G-protein-coupled receptor LGR8 [28–30] in testicular Leydig cells undergoing physiological differentiation processes during the seasonal reproductive cycle. We have employed the roe deer to analyze the expression of INSL3, mRNA and protein, and the transcriptional activity of the INSL3 receptor Lgr8 in testes collected during the complete annual reproductive cycle. We tested the hypothesis that the INSL3/LGR8 ligand-receptor system in seasonal breeders is subject to changes corresponding to the seasonal alterations in steroidogenic and spermatogenic activity.


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

A total of 18 adult male roe bucks (Capreolus capreolus) were included between August 1998 and June 2000. Bucks were kept under semifree ranging conditions in large, outside enclosures. Animals were treated in accordance with the National Animal Welfare Legislation. All procedures were approved by the local Office for the Preservation of Environment and Nature, Eberswalde, Germany. Every 2 mo during a complete seasonal cycle (February, April, June, August, October, and December), three individual bucks were castrated as described previously [13]. The three bucks studied per month were primary castrates to avoid interference of measurements with compensatory endocrine actions known to occur in the remaining testis after removal of one testis. Briefly, bucks were immobilized by i.m. administration of xylazin (2 mg/kg body mass) in combination with ketamin (4 mg/kg) using a blow pipe. Anesthesia was maintained by application of an isofluran-oxygen mixture (2–3 vol% Isofluran, 1.0 L O2-flow rate) via endotracheal tube. After the operation procedure, the anesthesia was antagonized with antipamezolhydrochlorid (200 µg/kg body mass, Antisedan; Pfizer, Karlsruhe, Germany).

Collection of Blood Samples, Testis Tissues, and RNA Isolation

For the measurement of serum testosterone levels, blood samples were collected from each animal before removal of the testis. Total testes were obtained from three individual bucks for each time point. Testis and epididymidis were separated and the testis was decapsulated. Testis parenchyma was both snap frozen in liquid nitrogen for extraction of RNA and protein and fixed in Bouin solution for paraffin embedding. Total RNA was isolated with Trizol reagent (Life Technologies, Karlsruhe, Germany). The amount of mRNA isolated was determined by spectrophotometry at 260 and 280 nm [31].

Cloning of Coding Sequences for the Roe Deer Insl3 and INSL3 Receptor Lgr8

For the cloning of the full coding sequence of roe deer Insl3 and the partial coding sequences of Lgr8, mRNA was isolated from 75 µg of total testicular RNA using oligo-d(T)-coated magnetic beads according to the manufacturer's instructions (Dynal, Hamburg, Germany). Approximately 500 ng of mRNA was used for first-strand cDNA synthesis employing the Superscript reverse transcriptase kit and 500 ng/ml of oligo-d(T) primer (both Life Technologies). Reverse transcriptase-polymerase chain reactions (RT-PCR) were carried out for 40 cycles in 50-µl volumes containing 1 µl of cDNA, 5 µl of 10x Advantage cDNA polymerase mix buffer, 100 µM of dNTP, 10 pmol of each primer (Table 1) and 2.5 U Advantage cDNA mix polymerase (Clontech, Heidelberg, Germany) at annealing temperatures indicated in Table 1 for each primer. The forward and reverse Insl3 oligonucleotide primers employed flanked the putative single intron present at the N-terminus of the C-domain of Insl3 to preclude any genomic DNA amplification [32] (Table 1). For the initial nested RT-PCR cloning of the partial cDNA sequences encoding roe deer LGR8, outer oligonucleotide primers for the amplification of human Lgr sequences (Table 1) were run at 60°C annealing temperature for 40 cycles, and amplicons of the expected size were sequenced. Specific inner primers for roe deer Lgr8 located internal to the outer primers and binding to nucleic acid sequences corresponding in location to the transmembrane regions 1–5 of human Lgr8 were employed at 65°C for 40 cycles (Table 1). Single amplicons were separated on a 1% low-melting-point agarose gel, purified by Magic column extraction, and cloned into the pGEM-T vector (both Promega, Heidelberg, Germany). Employing T7- and SP6-primers and the Thermo Sequenase Dye Terminator cycle sequencing kit (Amersham, Freiburg, Germany), the sequences of the roe deer Insl3 and Lgr8 amplicons were verified bidirectionally.


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  		TABLE 1. List of oligonucleotide primers employed for the specific RT-PCR and Q-RT-PCR of the indicated roe deer target genes

Digoxygenin Labeling of cRNA and In Situ Hybridization

Synthesis of digoxygenin-labeled cRNA has been previously described [33]. Bouin-fixed paraffin-embedded testicular tissues were cut in 5-µm sections and attached to glass slides coated with 2% aminopropyltriethoxysilane (Sigma, Deisenhofen, Germany). Nonradioactive in situ hybridization was performed according to the procedure described by Lewis and Well [34] using a 1:1000 dilution of an anti-digoxigenin alkaline phosphatase conjugated Fab-antibody (Boehringer Mannheim) in 1% BSA. Specific hybridization signals were visualized using the chromogen combination 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium (both Sigma). Slides were mounted in glycerol gel and examined under bright-field microscopy.

Quantitative RT-PCR Analysis

For quantitation, 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 Taq polymerase (Clontech, Heidelberg, Germany), 0.2x SYBR Green (Biozym; Hess, Oldendorf, Germany), 200 µM of each dNTP, and 0.5 µM of each primer, 1/2, 5/6, and 7/8 (Table 1). A negative control without template was included. All assays were done in triplicates in a Rotor-Gene 2000 (LTF, Wasserburg, Germany) and the resulting three values for each sample were presented as an average (means ± SEM). Initial denaturation at 95°C for 300 sec 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 PCR product, was read after each elongation step at 82°C. For verification of the PCR products, melting curves were generated, amplicons were run in agarose gels for visualization of single products of the correct size, and PCR products were sequenced in both directions. Relative quantitation of gene expression was performed with the software Rotor-Gene, version 4.6, in comparative quantitation mode. This mode allowed the comparison between differently treated samples relative to a control sample. The second derivative of the raw data was taken to calculate the take-off point. The relative concentration of each sample in comparison with the control sample was calculated based on the take-off point and the reaction efficiency.

Estimation of Testosterone by Enzyme Immunoassay

Serum testosterone concentrations were measured by an enzyme immunoassay as described earlier [13]. Briefly, 0.1 ml of serum was extracted with 2 ml butyl t-methyl ether:petroleum ether 30:70 (v/v) for 30 min. The samples were frozen, and the fluid petroleum ether phase was removed and evaporated at 55°C. The steroids were re-equilibrated with 1 ml 40% methanol (v/v) and duplicates of 20 µl were analyzed. The assay used a polyclonal antibody raised in rabbits against testosterone-11-hemisuccinate-BSA and testosterone-3-carboxymethyl-oxime-horse radish peroxidase served as label. The testosterone standard curve ranged from 0.4 pg to 50 pg/20 µl. Cross-reactivity with testosterone was 100%, with 5{alpha}-dihydrotestosterone 10%, with androstenedione 2%, with estradiol <0.1% and with progesterone <0.1%. The results were presented as nanograms testosterone per milliliter of serum. The intra- and interassay coefficients of variation were 8.9% and 12.3%, respectively.

Immunohistochemistry

For the detection of immunoreactive INSL3 on Bouin-fixed, paraffin-embedded tissue of roe deer testis, a previously characterized rabbit polyclonal antiserum was used and made against a peptide epitope encompassing the putative receptor binding motif within the B-domain of human INSL3 [35, 36]. Briefly, dewaxed 2-µm sections were equilibrated in PBS containing 0.1% Tween-20 (PBS-T). Endogenous peroxidase activity was inactivated with 3% H2O2 in methanol two times for 15 min at room temperature (RT). Nonspecific binding sites were blocked for 1 h at RT with PBS-T containing 10% goat nonimmune serum. Tissue sections were incubated overnight at 4°C with the rabbit INSL3 antiserum diluted 1:800 in PBS-T with 3% BSA. After washing, sections were incubated for 1 h at RT with a peroxidase-conjugated goat anti-rabbit secondary antibody (Dianova, Hamburg, Germany) diluted 1:500 in PBS-T. Specific binding was visualized with the peroxidase substrate diaminobenzidine (DAB; Pierce, Rockford, IL). Substitution for the antiserum with rabbit nonimmune serum diluted 1:800 served as negative controls. For immunodetection of the steroidogenic enzyme 17alpha-hydroxylase, 0.1 M Tris-buffered saline (TBS) was used for washings. Antigen retrieval required boiling of the testicular sections for 15 min in citrate buffer (pH 6). Nonspecific binding was saturated with 3% BSA and 10% swine nonimmune serum applied for 30 min at RT. The primary polyclonal antibody against 17alpha-hydroxylase (kindly provided by Dr. T. Sweeney, University College Dublin, Ireland) was diluted 1:1000 in 0.5 M TBS containing 3% BSA and 3% swine nonimmune serum, and sections were incubated overnight at 4°C. Washed sections were incubated with a biotinylated swine anti-rabbit immunoglobulin at 1:500 in 0.5 M TBS for 1 h at RT and specific immunoreactivity was visualized with an avidin-biotin-complex (ABC-Kit; Vector Laboratories, Burlingame, CA) and DAB as chromogenic substrate.

For the detection of smooth-muscle alpha-actin, endogenous alkaline phosphatase was inactivated with 4°C 20% acidic acid for 30 sec. Nonspecific binding was saturated for 1 h at RT with 3% BSA and 10% rabbit normal serum in PBS-T. Sections were incubated overnight at 4°C with a mouse monoclonal antibody to alpha-actin (DAKO, Hamburg, Germany) diluted 1:50 in PBS-T containing 3% BSA. Sections were then incubated at RT with a rabbit anti-mouse IgG bridging antibody at 1:50 for 1 h and subsequently with the mouse alkaline phosphatase-anti-alkaline phosphatase complex (both reagents from DAKO) for 30 min at 1:100. Specific binding was visualized with the alkaline phosphatase substrate Histo Mark Red (Kirkegaard Perry Laboratories, Gaithersburg, MD). Some sections were counterstained with hematoxylin. All sections were mounted and examined under bright-field microscopy.

Estimation of the INSL3-Immunopositive Interstitial Cell Fraction

Following INSL3 immunostaining of testicular tissue from the different animals, the samples were assessed using a Leica image-analyzing system including DMLB microscope, DC camera, computer, and DHS software package (Leica Microsystems, Bensheim, Germany). Ten fields of view at 400-fold magnification were randomly chosen per section. The total amount of cells and INSL3-immunostained cells within the interstitial testicular areas were counted. The total number of cells as well as the number and the proportion of INSL3-immunopositive cells were calculated in relation to a standardized interstitial area of 1 mm2 [37].

Western Analysis

Snap-frozen roe deer testicular tissues were employed for protein extractions using a lysis buffer containing 2% SDS and 10% saccharose in 62 mM Tris-HCl. The protein lysate was boiled for 5 min at 90°C, centrifuged to pellet cell debris, and protein concentrations in the supernatants were determined using a protein assay kit (Bio-Rad, Munich, Germany). For Western analysis, 30 µg of each protein lysate were separated on a 15% SDS-polyacrylamide gel under nonreducing and reducing conditions and blotted onto a Hybond nitrocellulose membrane (Amersham). For INSL3 immunodetection, blots were incubated overnight at 4°C with the rabbit polyclonal INSL-3-antiserum diluted at 1:10 000 in PBS-T containing 5% milk powder. After washing in PBS-T, specific binding was detected using a peroxidase-conjugated goat anti-rabbit IgG antibody (Life Technologies) at 1:20 000 in PBS-T and visualized with the ECL detection reagent (Amersham). Blots incubated with a rabbit nonimmune serum (DAKO) diluted 1:10 000 in PBS-T served as negative controls. To determine equal protein loading of the gel, membranes were incubated at 70°C in stripping solution (10% sodium dodecylsulfate, 100 mM Tris [pH 6.8], and 7 µl/ml mercaptoethanol), washed three times 10 min in PBS-T, and incubated with a monoclonal antibody to human ß-actin (Sigma) diluted 1:5000 in the same buffer for 1 h at RT followed by washings and an incubation with a peroxidase-conjugated goat anti-mouse IgG secondary antibody (Dianova) at 1:20 000 in PBS-T.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
RT-PCR was employed on mRNA derived from roe deer testis collected during the rutting season to clone the cDNA coding sequence of roe deer Insl3, which consisted of 396 base pairs (bp) encoding 131 amino acids (aa; accession number: AY377785; Fig. 1). The putative signal peptide and B-, C-, and A-domains of the roe deer INSL3 were composed of 24, 31, 50, and 26 aa residues, respectively, and were found to be highly conserved on the RNA and protein level among ruminants (Figs. 1 and 2). The number and location of the cysteine residues within the B- and A-domain of roe deer INSL3, the receptor binding motif -R-A-L-V-R-, and the Trp-residue within the B-domain of INSL3 were conserved, suggesting the presence of a potentially functional INSL3 hormone within the roe deer testis [38–41] (Fig. 1). Nested RT-PCR was employed on total RNA from roe deer testis collected in June to obtain the partial cDNA sequence for the INSL3 receptor Lgr8. The partial cDNA sequence of the roe deer Lgr8 amplicon consisted of 507 bp (169 aa; accession number: AY377787) and was homologous to aa positions 429–597 in the human LGR8.



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  		FIG. 1. The cDNA of testicular tissue collected before the rut in June was employed to determine the nucleic acid sequence (414 bp) and deduced amino acid (aa) sequence (138 bp) of roe deer preproINSL3. The putative intron site (arrow) and the putative receptor binding motif—RALVR (bold)— in the B domain of INSL3 are shown



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  		FIG. 2. Comparative alignment of the putative amino acid sequences derived from cDNA sequencing confirms high homology of INSL3 in ruminant species [33, 59–61]

The expression of Insl3 mRNA showed distinct transcriptional changes during seasonal transitions between testis growth and involution. Employing quantitative RT-PCR analysis (Q-RT-PCR), an upregulation of Insl3 gene activity was observed in April for all three bucks investigated. Shortly before and during the rut (June and August), however, Insl3 transcriptional activity decreased in five out of six bucks studied, and reached basal levels in October to February in the remaining nine bucks (Fig. 3a). Lgr8 transcripts were found to be expressed in roe deer testis throughout the year. The observed variation in Lgr8 mRNA levels during the annual cycle was below significance (Fig. 3b).



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  		FIG. 3. Relative expression levels detected by quantitative RT-PCR analysis of the genes for Insl3 (a) and Lgr8 (b) in roe deer testicular tissue during the seasonal reproductive cycle. The circannual variations in the expression levels of these target genes obtained for all 18 animals investigated was standardized against the corresponding December mRNA values from animal 1 (first December column). Transcript levels are shown separately for each of the three animals investigated per month with the three bars per month representing three different animals. Error bars were determined from three independent PCR runs of three different cDNA preparations each of the testicular mRNA preparation investigated. Thus, the differences between the three Q-PCR bars within each month derive from the relative quantitation of gene expression from each individual relative to the December control sample. To confirm the identity of the amplicons, PCR products were run on an agarose gel and the single band was excised and sequenced bidirectionally

Nonradioactive in situ hybridization with a roe deer Lgr8 cRNA probe revealed specific hybridization signals for Lgr8 mRNA in Sertoli cells in the testis sections investigated (April, June, August, October; Fig. 4D) but not in the corresponding sections treated with the sense roe deer Lgr8 probe (Fig. 4E). Similar localization of Lgr8 mRNA was detected with a bovine Lgr8 cRNA probe (data not shown). Employing a roe deer Insl3 cRNA probe, Insl3 transcripts were exclusively detected within roe deer testicular Leydig cells during the months of April, June, August, and October (Fig. 4, A and B). Testicular sections derived from testes collected in December or tissue sections treated with the sense cRNA probe as a negative control were devoid of Insl3 hybridization signals (Fig. 4C). Testis tissues collected in February revealed either very faint hybridization signals or were negative.



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  		FIG. 4. In situ hybridization (ISH; A–E) and immunohistochemistry (IHC; F–M) on sections of roe deer testicular tissue. Positive hybridization signals in Leydig cells with a roe deer Insl3 antisense-cRNA probe in testis collected in April (A) and October (B). Employing a roe deer Lgr8 antisense-cRNA probe, weak hybridization signals were observed in Sertoli cells as shown for testis tissue collected in October (D). Treatment with corresponding Insl3 and Lgr8 sense cRNA probes was devoid of hybridization signals, as shown for October testis tissue (C, E). Positive immunostaining of Leydig cells in testis tissues collected in April (F) and October (G) employing an antibody to 17alpha-hydroxylase. Detection of immunoreactive INSL3 localized to interstitial Leydig cells within testis tissues of April (H), June (I), October (J), December (M), and February (L). Sections treated with rabbit nonimmune serum as a negative control were devoid of any staining, as shown for June testis tissue (K; inset). Double immunostaining for INSL3 and alpha-actin in testis collected in December showed INSL3 immunopositive spindle-shaped cells (brown staining) in the peritubular interstitial compartment adjacent to alpha-actin immunopositive peritubular myofibroblast cells (red staining) (M). Hematoxylin counterstaining was performed on A–C, F–K. Original magnification, x1000 for I and x400 for all other pictures

Immunoreactive INSL3 was exclusively detected in Leydig cells of the roe deer testes examined. Tissue samples obtained in April (Fig. 4H) and June (Fig. 4I) displayed the strongest INSL3 immunostaining, whereas Leydig cell-derived INSL3 protein was weakly detected in October (Fig. 4J). Testis sections incubated with a rabbit nonimmune serum were devoid of specific INSL3 staining (Fig. 4K, insert). In all cases examined, immunostaining showed a cytoplasmic and granular staining for INSL3 within the Leydig cells (Fig. 4I). An identical staining pattern was also observed in bovine testicular Leydig cells (data not shown). Although testis tissues collected in December and February were devoid of INSL3-immunopositive interstitial Leydig cells, a peritubular INSL3 immunostaining was observed in spindle-shaped interstitial cells surrounding the seminiferous tubules (Fig. 4L). These INSL3 immunoreactive peritubular cells appeared to be present in testis sections throughout the season but were the only INSL3 immunopositive cell population in the roe deer testis during the winter months. These cells were further characterized by immunohistochemical double staining employing the INSL3 antiserum and an antibody to alpha-actin (Fig. 4M). The INSL3-positive but alpha-actin-negative spindle-shaped cells clearly differed from peritubular INSL3-negative, alpha-actin-positive myofibroblast cells (Fig. 4M). Thus, during the winter months of relative reproductive quiescence, a unique INSL3-producing peritubular cell population is present within the roe deer testis (Fig. 4, L and M).

We determined the seasonal expression of 17alpha-hydroxylase, the steroidogenic key enzyme of the androgenic pathway, as another parameter independent of INSL3 to assess testicular Leydig cell activity during the circannual testicular cycle. Leydig cells immunopositive for the 17alpha-hydroxylase were detected in April (Fig. 4F) and June, August, and October (Fig. 4G). In contrast, immmunoreactive 17alpha-hydroxylase was not detected in roe deer testis collected in December and February. Similar results were obtained for the specific localization of hybridization signals with an antisense cRNA probe encoding roe deer 17alpha-hydroxylase. Testis sections collected in December and February and all sections treated with the sense cRNA were devoid of hybridization signals (data not shown).

Western analysis on protein extracts of roe deer testicular tissue samples revealed an immunoreactive INSL3 protein at 14.5 kDa, indicating the presence of pro-INSL3 in the testis samples throughout the year (Fig. 5). As may be predicted from the Q-RT-PCR data (Fig. 3a), testicular tissues collected in April and June contained the highest amounts of INSL3 protein per total testicular protein content, as assessed by the beta-actin immunosignal (Fig. 5). Therefore, initiation of INSL3 production by the Leydig cells and highest intratesticular INSL3 concentrations were observed in spring before the rutting season.



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  		FIG. 5. Representative example of four Western blots of roe deer testicular protein extracts (30 µg per lane) separated on a 15% SDS-polyacrylamide gel under nonreducing conditions. Employing a previously characterized polyclonal rabbit antiserum against INSL3 [35, 36], a specific immunoreactive band at a molecular size of 14.5 kDa was detected, indicating the presence of a proINSL3 peptide. When rabbit nonimmune serum was employed as primary antiserum, the blot was devoid of specific immunoreactive bands (data not shown). Equal protein loading per lane was estimated by the detection of immunoreactive ß-actin (47 kDa) on the stripped membrane, as shown in the lane below

Serum testosterone levels in the 18 roe deer bucks investigated showed a steep rise in April, 1 mo after the reported sharp peak in the serum level of LH [14], then reached a maximum in August and rapidly declined after the rut (Fig. 6a). This increase in serum testosterone concentrations in April coincided with the presence of detectable 17alpha-hydroxylase, mRNA, and protein in roe deer testicular Leydig cells (Fig. 4F). When serum testosterone levels reached basal levels during the winter months, 17alpha-hydroxylase was not detected in roe deer testis tissues. Densitometric analysis of four independently performed INSL3 Western blots with testis extracts from the three bucks per months studied revealed increased production of immunoreactive pro-INSL3 in April and June. This coincided with an increased expression of Insl3 mRNA (Fig. 3a), INSL3 immunoprotein (Fig. 4H), and the highest percentage of INSL3-immunoreactive Leydig cells per total interstitial cell number for April (43%) and June (35%), as determined by morphometric analysis on INSL3-immunostained testicular tissue sections of the 18 buck testes (Fig. 6b). In August, October, and February, testis sections only revealed a fraction of 14%, 5%, and 2% INSL3-positive interstitial cells, respectively (Fig. 6b). INSL3 immunostaining in December testes was exclusively seen in peritubular, spindle-shaped cells and was not accounted for in the morphometric calculations.



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  		FIG. 6. a) Seasonal variation of the testosterone and LH levels in blood serum of roe deer (means ± SEM). The values of LH were taken from an earlier study with monthly collected blood samples from five roe bucks per time point [14]. b) Concentration of INSL3 testicular protein was determined by densitometry from four independent INSL3 Western blots of roe deer testis tissues from three animals per month studied (means ± SEM). This was compared with the percentage of INSL3 immunopositive interstitial cells per square millimeter of interstitial area. Testosterone and INSL3, mRNA and immunoprotein, were upregulated a month after the reported LH peak and well before the onset of rut


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The roe deer is a seasonal breeder and a unique ruminant model system, providing a rare opportunity to study the dynamic seasonal changes of the INSL3 ligand-receptor system in physiologically involuting and recrudescent testicular postpubertal Leydig cells. We observed changes in INSL3 production and number of INSL3-producing testicular Leydig cells during the reproductive season. INSL3, mRNA and protein, were maximal in April before the rut. This coincided with the first testosterone serum peak in roe deer [14–17] and the appearance of differentiated testosterone-secreting testicular Leydig cells [19], as was shown in other seasonal mammals [42, 43]. The transcriptional activity and production of 17alpha-hydroxylase protein, a key steroidogenic enzyme facilitating the transition of progesterone to androstenedione, in testicular Leydig cells reflected the observed annual profile of testosterone serum concentrations during a seasonal cycle and was strongest in April, June, and August, months with high testosterone serum levels. These hormonal changes in roe bucks were preceded by peak serum levels of LH and FSH between February and April [14–17]. In the rodent postnatal testis, transcriptional upregulation of the Insl3 gene also depends on the appearance of differentiated mature Leydig cells [26, 44] and the presence of a functional hpg axis. Differentiation of testosterone-producing [45] and Insl3-positive Leydig cells [26] in hgp mice with a defective hpg-axis was shown to be LH dependent [26, 27].

Although roe deer testicular INSL3 production and testosterone secretion were upregulated concordantly in spring, maximal expression of INSL3, mRNA and protein, occurred before the rutting period in April to reach low concentrations during the late rut in August. In contrast, the same testis tissues showed maximal testicular testosterone secretion during the rut in early August [13]. This would suggest different regulatory mechanisms for the continued testicular synthesis of both hormones. Also, testosterone or androgens other than testosterone likely produced by involuting roe deer Leydig cells in October, as shown in the rodent testis [43, 46, 47], appeared not to stimulate continued high INSL3 production nor did androgens increase or sustain the population of INSL3-immunopositive interstitial cells in the roe deer testis. Both the testicular INSL3 protein content and the number of INSL3-producing interstitial cells were low during peak testosterone levels in August. Similarly, it appears unlikely that INSL3 can stimulate testosterone production in mature Leydig cells. As previously shown in the horse [48], the presence of an INSL3 ligand-receptor system in the testis of the roe deer suggests as yet unknown intratesticular functions of INSL3. The detection of INSL3 receptor transcripts in Sertoli cells has identified this cell population as a potential INSL3 target and provided first evidence in support of an intratubular role of INSL3 as an endocrine mediator of Leydig-Sertoli cell interaction in the roe deer testis. Although this endocrine function still remains obscure, our observation of highest INSL3 expression during the peak of testicular germ cell mitosis would implicate INSL3 as a secretory hormone supporting Sertoli cell function and, thus, indirectly affecting spermatogonial division. In contrast, during the short rutting period from mid-July to mid-August, maximal total testicular volume and highest rates of spermiogenesis coincided with peak concentrations of intratesticular and serum testosterone in the roe deer buck [11, 13, 16]. Future studies are needed to further clarify the physiological role of INSL3 for Sertoli cell function.

During the winter months, December and February, when serum levels of pituitary gonadotropins and testosterone are low, a small population of spindle-shaped interstitial cells adjacent to the seminiferous tubules within the involuted roe deer testis continued to exhibit immunoreactive INSL3. In rodents, peritubular mesenchymal cells have been shown to act as Leydig cell precursors [46, 47, 49, 50], become Leydig progenitor cells in an LH-independent manner, and enter, in an LH-dependent manner, the final differentiation processes during puberty to become mature, adult, INSL3-producing Leydig cells [49]. In the mature rat, this spindle-shaped peritubular cell population appears to have the potential to regenerate the Leydig cell compartment. Upon selective destruction of rat testicular Leydig cells by a single treatment with ethylene dimethane sulfonate (EDS), mature rat Leydig cells were shown to regenerate from spindle-shaped peritubular LH-receptor-positive cells derived from mesenchymal precursor cells [49, 51–53]. Renewed detection of INSL3 within the EDS-treated rat testis coincided with the appearance of interstitial cells phenotypically resembling mature Leydig cells [27]. The roe deer testis appeared to retain an interstitial peritubular cell population distinct from alpha-actin-immunopositive myoepithelial cells and these cells could represent INSL3-positive, partially dedifferentiated testicular Leydig cells. Thus, in seasonal breeders, INSL3 may be regarded a suitable marker not only for mature Leydig cells of the spermatogenically active testis but also for a specific subpopulation of Leydig progenitor cells in the involuted testis.

The limited available data on the number of differentiated Leydig cells during a reproductive season is inconsistent [3]. Leydig cell numbers were found unaltered during the seasonal changes in the testis of Syrian hamster [43], but other studies on regressed testis reported a reduction of Leydig cells per testis to 50% or 71% in this species [9, 54], to 42% in woodchuck [55], and to 58.6% in camel [56]. Potential pitfalls in the calculation of cell numbers from parameters estimated in tissue sections emphasized by Mendis-Handagama and Ewing [57] had been accounted for in this study. In a parallel study, we compared the observed changes in cell numbers per interstitial area unit with the theoretically expected values, assuming a constant interstitial cell number in roe deer, and determined a high concordance of counted and calculated interstitial cell numbers [37]. This is in agreement with the hypothesis of a constant interstitial cell number and suggests that the considerable seasonal variation in INSL3-positive cell number was caused by de- and redifferentiation rather than by a change in total Leydig cell number. Additional evidence to conclude that the Leydig cell population remains constant during the season in the roe deer testis was provided by our observation that the testis interstitium did not show an upregulation of the proliferation markers Ki-67 or PCNA antigen during testis recrudescence nor did we observe an increase of apoptosis markers during the involution phase (unpublished results). Similarly, no increase in cell death or proliferation of interstitial or Sertoli cells was detected during the seasonal changes in the European brown hare [58].

In conclusion, the seasonal changes of INSL3 expression in recrudescent and involuting Leydig cells and the discovery of Sertoli cells as a potential INSL3 target within the roe deer testis have provided further evidence implicating the INSL3 ligand-receptor system as a new molecular player in testis physiology.


   ACKNOWLEDGMENTS
 
The authors would like to thank Dr. Frank Göritz for the castration of roe bucks. We are grateful to Ms. E. Schlueter and Ms. C. Fröhlich for excellent technical assistance. We also thank Dr. Torres Sweeney, University College Dublin, Ireland, for generously providing the primary polyclonal antibody against 17alpha-hydroxylase and Dr. Paola Pocar, Department of Anatomy and Cell Biology, Martin Luther University, for providing digoxigenin-labeled cRNA probes for bovine Lgr8.


   FOOTNOTES
 
1 Correspondence and current address: Department of Human Anatomy and Cell Science, Faculty of Medicine, University of Manitoba, Winnipeg, MB, R3E 0W3, Canada. FAX: 204 789 3920; hombach{at}cc.manitoba.ca and klonisch{at}manitoba.ca Back

Received: 27 October 2003.

First decision: 17 November 2003.

Accepted: 17 May 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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