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Endocrinology, doi:10.1210/en.2003-0563
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Endocrinology Vol. 144, No. 11 5058-5064
Copyright © 2003 by The Endocrine Society

Effects of Insulin-Like Growth Factor I on Steroidogenic Enzyme Expression Levels in Mouse Leydig Cells

Gui-Min Wang, Peter J. O’Shaughnessy, Curtis Chubb, Bernard Robaire and Matthew P. Hardy

Center for Biomedical Research, The Population Council, and Rockefeller University (G.M.W., M.P.H.), New York, New York 10021; Division of Veterinary Preclinical Study, University of Glasgow Veterinary School (P.J.O.), Glasgow, Scotland G61 1QH, United Kingdom; Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center (C.C.), Dallas, Texas 75390; and Department of Pharmacology and Therapeutics, McGill University (B.R.), Montréal, Québec, Canada H3G 1Y6

Address all correspondence and requests for reprints to: Matthew P. Hardy, Ph.D., The Population Council, 1230 York Avenue, New York, New York 10021. E-mail: mhardy{at}popcbr.rockefeller.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The role of IGF-I in Leydig cell maturation was studied by evaluation of: 1) steady state levels for nine mRNA species expressed specifically in Leydig cells of 35- and 50-d-old IGF-I-null mice and wild-type controls; 2) protein levels for 17{alpha}-hydroxylase/C17–20 lyase, cholesterol side-chain cleavage, and type I 5{alpha}-reductase (5{alpha}R-1) in Leydig cells by immunocytochemistry; and 3) serum testosterone (T) and testicular interstitial fluid IGF-I levels. Expression levels of all mRNA species associated with T biosynthesis were lower in the absence of IGF-I stimulation. In contrast, androgen-metabolizing enzyme mRNA species had either normal (3{alpha}-hydroxysteroid dehydrogenase) or higher expression (5{alpha}R-1) levels in IGF-I-null mice (P < 0.05) relative to wild-type controls. None of the mRNA species studied changed developmentally in the mutant, whereas there were increases or decreases between d 35 and 50 in normal controls. Parallel trends were observed for average Leydig cell 5{alpha}R-1 immunostaining intensity. T levels in mutants were initially higher during d 14–21, equivalent to normal on d 28, and then failed to increase pubertally, remaining at 30% of control levels (P < 0.01) in 90-d-old adult animals. In normal wild-type mice, interstitial fluid and plasma IGF-I levels were highest (P < 0.05) on d 24, indicating that the action of this growth factor on the testis peaks during pubertal development. These results show that in the absence of IGF-I, there is a failure of adult Leydig cells to mature, and that the reduced capacity for T production is caused by disproportionate expression of T biosynthetic and metabolizing enzymes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ANDROGEN PRODUCTION and fertility in the sexually mature male are dependent on the postnatal development of adult Leydig cells, which is achieved by morphological differentiation from spindle-shaped progenitor cells. The progenitor cells first transform into round immature Leydig cells and then, with further increases in size, mature into adult Leydig cells, acquiring the capacity for testosterone (T) production (1, 2, 3, 4). The amount of T secreted by Leydig cells is determined by a balance between T biosynthetic and metabolizing enzyme activities. In general, the mRNA levels for T biosynthetic enzymes are increased, and androgen-metabolizing enzymes decline postnatally as adult Leydig cell mature (5, 6, 7, 8, 9). Recently, the trends for gene expression in mouse Leydig cells were subdivided into four categories: 1) cholesterol side-chain cleavage (P450scc), 17{alpha}-hydroxylase/C17–20 lyase (P450c17{alpha}), steroidogenic acute regulatory (StAR) protein, and relaxin-like factor (RLF), which are expressed at relatively high levels in both fetal and adult Leydig cells; 2) type VI 3ß-hydroxysteroid dehydrogenase (3ßHSD VI) and type III 17ß-hydroxysteroid dehydrogenase (17ßHSD III), which are expressed only in adult Leydig cells; 3) genes encoding androgen-metabolizing enzymes, type I 5{alpha}-reductase (5{alpha}R-1) and 3{alpha}-hydroxysteroid dehydrogenase (3{alpha}HSD), which attain peak expression during puberty; and 4) genes such as 3ßHSD I, which show little change in expression during development (9). The trends for the steady state levels of mRNAs that encode these enzymes corresponded closely with their protein and activity levels (8, 10). Therefore, all of the above genes are useful as markers to chart the progress of Leydig cell differentiation and maturation.

LH, secreted by the pituitary in response to GnRH, stimulates the maturation of adult Leydig cells. This is demonstrated by the lack of androgen production postnatally in mice lacking either the GnRH peptide or the LH receptor (LHR) (11, 12, 13). With the exception of LH, the roles of other hormones and growth factors in the development of adult Leydig cells remain uncertain. Of the locally produced growth factors that are known to be expressed in the testis, IGF-I is of particular note for its action on Leydig cells (14, 15, 16). The hypothesis that IGF-I facilitates Leydig cell differentiation and maturation in conjunction with LH is based on the fact that 1) progenitor Leydig cells, fibroblastic precursors observed in early neonatal testes, possess few LHR and are relatively insensitive to LH (3, 17); 2) Leydig cells will differentiate in GnRHhpg mice, which are deficient in circulating LH (18); 3) IGF-I and its receptor mRNAs are highly expressed in progenitor and immature Leydig cells; and 4) IGF-I enhances hCG-stimulated T formation (19). This suggests that there is a requirement for IGF-I preceding LH-mediated events of Leydig cell differentiation, and that IGF-I acts in conjunction with LH to further stimulate Leydig cell maturation.

In vitro studies have also shown that IGF-I stimulates maturation of rat immature Leydig cells by increasing the expression of steroidogenic enzymes and T production (20, 21). GH-deficient Snell dwarf mice have very low circulating IGF-I levels, and treatment of these mice with IGF-I in vivo induces a marked increase in the numbers of LHRs and in the steroidogenic response (22), further indicating the importance of IGF-I for Leydig cell maturation. We have shown previously that testis weight and Leydig cell numbers are proportionally reduced to 30% of wild-type levels compared with reduced body weight. However, serum T in adult animals is only 18% of normal, suggesting that IGF-I deficiency impairs Leydig cell function (23). We hypothesize that the dramatic declines seen in circulating T levels in adult IGF-I-null mutants result from abnormal testis development, and specifically from an imbalance in T biosynthetic and metabolizing enzyme activities in Leydig cells. The underlying causes of decreased androgen production in IGF-I-null mice were analyzed by measuring 1) the levels of specific testicular mRNAs and proteins, focusing on T biosynthetic and metabolizing enzyme expression; 2) developmental trends for the T-metabolizing enzyme, 5{alpha}R-1, in Leydig cells of wild-type and IGF-I-null mice during d 14–90 postpartum; 3) developmental profiles of serum T levels during d 14–90 postpartum; and 4) IGF-I levels of serum and testicular interstitial fluid in normal mice at various ages postpartum. The data showed that in the absence of IGF-I, there was abnormal development of adult Leydig cells characterized by imbalance of T biosynthetic and metabolizing enzyme expression associated with low T levels in adult animals. In particular, Leydig cell levels of 5{alpha}R-1 were disproportionately high in the absence of IGF-I. The timing of peak IGF-I concentrations in testicular interstitial fluid was consistent with a role for IGF-I in stimulating postnatal Leydig cell development.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
IGF-I-null mice were obtained by mating heterozygous mice with a predominantly MF1 x 129/Sv background strain bearing the targeted gene IGF-I deletion (provided by Dr. Argiris Efstratiadis, Columbia University, New York, NY). Wild-type control mice were from the MF1 x 129/Sv background strain for the IGF-I knockout and GnRHhpg wild-type or unaffected heterozygotes for the serum and testicular IGF-I analysis. Animals were maintained in accordance with the regulations established by the institutional animal care and use committee of Rockefeller University (Protocol 91200-R2) and were used for study at the ages indicated in the text. Mice were observed twice daily (morning and evening) for litters. The day of birth was designated d 1 of age. Pup genotypes were determined by multiplex PCR (24).

The animals were killed under deep anesthesia by ip injection of sodium pentobarbital (25 mg/100 g body weight; Abbott Laboratories, North Chicago, IL), and one testis from each animal was removed and frozen in liquid N2 for subsequent study of mRNA levels. The contralateral testis from each animal was fixed by whole body perfusion through the left ventricle of the heart with Bouin’s solution (25). Testes from IGF-I-null mice were small enough to permit overnight immersion fixation. After dehydration in ethanol and xylene, the testes were embedded in paraffin. Transverse sections were cut at a thickness of 5 µm and mounted on glass slides (catalog no. 12-550-15, Fisher Scientific Co., Fairlawn, NJ) for immunocytochemical analysis.

Collection of testis interstitial fluid
Testicular interstitial fluid was collected from wild-type animals on d 7 (n = 10), 11 (n = 2), 24 (n = 4), 30 (n = 8), and 160 (n = 16). Testes were 50% decapsulated (starting at the caudal pole), placed into a 1.5-ml microcentrifuge tube with the exposed parenchyma up, and centrifuged (12,000 x g, 30 min, 4 C). Supernatants were collected with a glass micropipette, snap-frozen, and stored at -70 C. Collections averaged 4.0 ± 0.3 µl/100 mg testis (n = 29). Serum was collected from the same animals, and IGF-I concentrations were measured by RIA. The T levels in testis interstitial fluid (231 ± 16 ng/ml) were not different from those reported for mouse testis surface venous blood (150 ± 59 ng/ml) (26). Further confirmation that blood did not contaminate interstitial fluid samples came from our observation that IGF-I levels did not differ significantly in interstitial fluid from testes before (141 ± 24 ng/ml; n = 5) or after (170 ± 44 ng/ml; n = 5) perfusion of the testicular artery with 0.25 M sucrose to remove red blood cells.

RT and real-time PCR
For quantification of the content of specific mRNA species in testes, a real-time PCR approach was used, with RT of the isolated RNA, followed by TaqMan PCR (27). Testis RNA was extracted using phenol/guanidium isothiocyanate (Molecular Research Center, Inc., Cincinnati, OH), and residual genomic DNA was removed by deoxyribonuclease treatment (DNA free, Ambion, Inc., supplied by AMS Biotechnology, Carterton, UK). The RNA was reverse transcribed using random hexamers and Moloney murine leukemia virus reverse transcriptase (Superscript II, Life Technologies, Paisley, Scotland, UK) as described previously (28).

Primers and probes for use in the TaqMan method have been described previously (9). Real-time PCR was carried out in a 25-µl volume using a 96-well plate format. Components for real-time PCR were purchased from PE Applied Biosystems (Warrington, UK), except for the primers and probes, which came from MWG Biotech (Milton Keynes, UK). Each PCR well contained reaction buffer, 5 mM MgCl2, 200 µM deoxy-NTPs, 300 nM of each primer, 200 nM probe, and 0.02 U/µl enzyme (AmpliTaq Gold). Reactions were carried out and fluorescence was detected on a GeneAmp 5700 system (PE Applied Biosystems). For each sample a replicate was run, omitting the RT step, and a template negative control was run for each primer-probe combination. The mRNA levels were expressed relative to an internal control Wbscr1 (Williams-Beuren syndrome chromosome region 1) gene, the mouse homolog of KIAA0038 (9).

Immunocytochemistry
Five testes from each group, one per animal, were used in the immunohistochemical study by the avidin/biotin method (Vectastain, Elite, ABC Kit, PK-6101, Vector Laboratories, Inc., Burlingame, CA) according to the manufacturer’s instructions. The primary antibodies were as follows: rabbit antiamino acid sequence 132–150 antiserum raised against 5{alpha}R-1 (29), porcine P450c17{alpha} (provided by Dr. D. B. Hales, University of Illinois, Chicago, IL), and rat P450scc (catalogue no. RDI-p450sccabr, RDI Research Diagnostics, Inc., Flanders, NJ) polyclonal antibodies. Endogenous peroxidase was blocked with 0.5% H2O2 in methanol for 30 min. The sections were then incubated with 5{alpha}R-1 (diluted 1:100), P450c17{alpha} (diluted 1:1000), and P450scc polyclonal antibodies (diluted 1:1000) for 1 h at room temperature. The antibody-antigen complexes were visualized with diaminobenzidine (Peroxidase Substrate Kit, SK-4100, Vector Laboratories, Inc.) resulting in brown cytoplasmic staining in positively labeled Leydig cells, which was distinguishable from the nonstained background. The sections were counterstained with Mayer’s hematoxylin, dehydrated in graded concentrations of alcohol, and coverslipped with resin (Permount, SP15–100, Fisher Scientific Co.). In control experiments sections were incubated with nonimmune rabbit IgG using the same working dilution as the primary antibody.

Twenty randomly selected fields for each of three nonadjacent sections per testis were captured using a Nikon Eclipse E800 microscope (Nikon, Inc., Melville, NY) equipped with a x40 objective lens and a SPOT RT digital camera (model 2.3.0., Diagnostic Instruments, Inc., Michigan City, IN) interfaced to a computer. The images, displayed on a monitor, represented areas of 93,600 µm2. Average immunostaining pixel intensity was estimated using image analysis software (Image-Pro Plus, Media Cybernetics, Silver Spring, MD) as previously described (30). The intensity of the background signal was determined for each section by tracing an unlabeled area adjacent to the labeled cells. The background was then subtracted from the values obtained for the labeled cells. The resulting adjusted values, referred to as the relative signal intensities, provide a measure of the protein concentration in individual Leydig cells.

T and IGF-I concentrations
Blood was collected intraorbitally, and sera were stored at -80 C until RIA. Serum T concentrations were measured with a tritium-based RIA as previously described (31). Interassay variation was between 7–8%. The overall experimental design was performed twice to ensure that the data were repeatable.

The plasma and testicular fluid IGF-I RIA was developed based on protocols published by Incstar (Stillwater, MN), Nichols Institute Diagnostics (San Juan Capistrano, CA), and Chatelain (32). Samples were incubated in glass tubes overnight with 5000 cpm [125I]IGF-I (natural sequence; Amersham Pharmacia Biotech, Arlington Heights, IL) and then acidified (4 vol 0.5 N HCl), incubated (6 h at room temperature), and applied to an octadecasilyl-silica column (Incstar), which was prewashed with 5 ml 2-propanol, 5 ml methanol, and 10 ml 4% acetic acid. After 3 min, the column was washed with 10 ml 4% acetic acid (twice), and IGF-I was eluted with 4 ml methanol. The eluate was evaporated (N2 at 42 C), and the residue was dissolved in 1 ml buffer: 0.3 M sodium phosphate monobasic, 0.01 M EDTA, 0.02% sodium azide, 0.05% Tween 20, and 0.02% protamine sulfate (grade 2) (pH 7.5). The redissolved elute was vortexed for 4 sec and incubated at 4 C overnight. A 300-µl aliquot was used to determine recovery; the average recovery was 73 ± 1% (n = 40).

Twenty-, 40-, and 80-µl aliquots of elute were assayed in duplicate in polystyrene tubes. Standard curves (0–100 pg recombinant IGF-I; Roche, Indianapolis, IN) were run in triplicate. The primary antibody (UBK487, a gift from the National Hormone and Pituitary Program, NIDDK, NIH, Bethesda, MD) was added to the sample (1:18,000 final titer) and incubated for 2 h at 4 C. After this incubation, [125I]IGF-I (10,000 cpm) was dispensed into the tube, and the incubation was continued for 20 h at 4 C. The secondary antibody (1:30; antirabbit {gamma}-globulin; Pel-Freez, Rogers, AR) was preprecipitated by mixing with an equal volume of normal rabbit serum (1:100) at least 20 min before use. The secondary antibody was added, and the incubation was continued for 2 h at 4 C. The tubes were centrifuged (1,600 x g, 45 min, 4 C), supernatants were decanted, and pellets were counted. The within- and between-assay coefficients of variation were 1% and 4.3%, respectively. Plasma IGF-I concentrations in mice with a congenital absence of GH (little mutant mice) were low (8 ± 2 ng/ml) compared with values in control mice (206 ± 63 ng/ml).

Statistical analysis
Data are expressed as the mean ± SEM. Differences between IGF-I-null animals and the appropriate control group at each age were assessed by t test, and differences among groups were identified by ANOVA using a software package (InStat, GraphPad, Inc., San Diego, CA). Differences were regarded as significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Leydig cell-specific mRNAs
Of the nine Leydig cell-specific mRNA species measured, six, including P450scc, P450c17{alpha}, 17ßHSD III, 3ßHSD VI, StAR, and RLF, had lower steady state levels on d 35 and 50 in IGF-I-null mice relative to their age-matched controls (P < 0.05; Fig. 1Go, A and B). Three of these, including StAR, 3ßHSD VI, and RLF, were increased by 40–100% from d 35–50 in control animals (P < 0.05), whereas these same three mRNAs were unchanged in IGF-I-null testes (Fig. 1BGo). Two of the nine Leydig cell mRNA species, 3ßHSD I and 3{alpha}HSD mRNA, had equivalent levels in IGF-I-null compared with wild-type control mice (Fig. 1CGo). The androgen-metabolizing enzyme, 5{alpha}R-1, was unusual in that it was higher in IGF-I-null mice than in normal mice on both d 35 and 50 (P < 0.05). In control animals, 5{alpha}R-1 mRNA levels decreased from d 35–50, whereas a decrease did not occur in the mutant (Fig. 1CGo).



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  		FIG. 1. Quantification of Leydig cell-specific mRNAs in testes of wild-type control and IGF-I-null mice. RNA was extracted from whole testes of 35- and 50-d-old animals and reverse transcribed to generate cDNA. Levels of specific mRNA species were measured by real-time PCR and expressed relative to an internal (Wbscr1) control. Results show the means ± SEM of five animals in each group. Groups with different letter superscripts were significantly different (P < 0.05). A, Genes with reduced levels of expression on d 35 and 50 in IGF-I-null mice (P450scc, P450c17{alpha}, and 17ßHSD III); B, genes with increased expression from d 35–50 in control animals, but reduced levels of expression on d 35 and 50 in IGF-I-null mice (StAR, 3ßHSD VI, and RLF); C, genes expressed at normal (3ßHSD I and 3{alpha}HSD) or increased levels (5{alpha}R-1) in IGF-I-null mice.

 
P450scc and P450c17{alpha} protein levels
Using immunohistochemistry, the steroidogenic enzymes P450scc and P450c17{alpha} were examined in situ on d 35 and 50 and were detected only in Leydig cells. Consistent with the mRNA levels, Leydig cells were intensely stained for both P450scc and P450c17{alpha} protein in wild-type testes, but were weakly stained in the testes of IGF-I-null mice (Fig. 2Go, A–D).



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  		FIG. 2. Localization of P450scc (A and B), P450c17{alpha} (C and D), and 5{alpha}R-1 (E and F) in normal (A, C, and E) and IGF-I-null (B, D, and F) mouse testes on d 35 (A, B, C, and D) and 50 (E and F). Leydig cells had weak immunostaining intensities for P450c17{alpha} and P450scc in IGF-I-null testes (B and D) compared with their age-matched controls (A and C). In contrast, Leydig cells in IGF-I-null testes showed strong immunostaining intensity for 5{alpha}R-1 compared with their age-matched controls. The arrows above represent interstitial areas containing Leydig cells. Scale bar, 50 µm (bottom right, the same for all panels).

 
Developmental analysis of 5{alpha}R-1 immunostaining intensity
Developmental profiles in 5{alpha}R-1 protein expression were assessed by immunohistochemistry in combination with image analysis (Fig. 2Go, E and F, and Fig. 3Go). In wild-type animals, the intensities increased steadily from d 14 onward and reached a peak on d 28. This was followed by a progressive decrease in signal intensity between d 28 and 90. Most of the intensely immunoreactive interstitial cells on d 21 and 28 resembled progenitor and immature Leydig cells based on their cytomorphology (respectively, spindle-shaped and round) (33). In IGF-I-null males, 5{alpha}R-1 immunostaining intensity followed a similar developmental pattern, but with a delayed start after 14 d; the values were lower on d 21–28 (P < 0.05 compared with age-matched controls). Signal intensity reached the control level from d 35 onward and peaked later, on d 50. A low level nonspecific signal was detected in the seminiferous tubules, consistent with previous observations (29).



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  		FIG. 3. Levels of 5{alpha}R-1 protein in testes from wild-type control (black bar) and IGF-I-null (open bar) mice at different ages. Results represented are means ± SEM of five animals in each group. Groups with shared superscript letters did not have a difference in staining intensity (P < 0.05).

 
Serum and testicular hormone profiles during postnatal development
T levels were measured in control and IGF-I-null mice at different ages (Fig. 4Go). Compared with those in age-matched wild-type animals, serum T levels in IGF-I-null mice were higher during d 14–21 (P < 0.05). Levels in the mutant were then reduced to normal on d 28 and failed to increase subsequently, being only 30% of control values in adult animals (P < 0.05). The levels of IGF-I in plasma and interstitial fluid had similar trends, varying significantly with age. There was a steady increase starting on d 7, with the highest concentrations occurring on d 24 (P < 0.05; Fig. 5Go), which is concurrent with the timing of the developmental process for adult Leydig cells: differentiation from progenitor Leydig cells starting on d 7 and 10, and immature Leydig cell maturation after d 28 (33, 34, 35, 36). IGF-I is undetectable in IGF-I mutant mice (24) and therefore is not shown in Fig. 5Go.



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  		FIG. 4. Serum testosterone (T) profiles in control and IGF-I-null mice at various ages. Serum T concentrations in IGF-I-null mice were higher on d 14 and 21, and lower on d 56 and 90 (P < 0.05). Data are presented as the means ± SEM (n = 3–5, each pooled from two to three animals for IGF-I-null mice under age d 35, and wild-type under age d 21. n = 5–10 animals for all other groups. *, Significant difference in T levels (P < 0.05) within each age group.

 


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  		FIG. 5. IGF-I concentrations in testicular interstitial fluid and plasma collected from normal mice at various ages. Testicular interstitial fluid IGF-I levels on d 24 (two pools of two testes) were higher (P < 0.05) than those on d 7 (pool of 10 testes), 11 (pool of two testes), 30 (four pools of two testes) and 160 (eight pools of two testes). Plasma IGF-I concentrations on d 24 (n = 2) were higher (P < 0.05) than those of d 7 (n = 3), 11 (n = 3), 30 (n = 4), and 160 (n = 7). Data are represented as the means ± SEM. *, Significant difference in IGF-I levels (P < 0.05) compared with the other age groups.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Targeted deletion of IGF-I results in a dwarf phenotype, with the adult mutant being about 30% the body weight of age-matched, wild-type controls. A more severe effect occurs in the male reproductive system, resulting in 80% decreases in the weights of androgen-dependent accessory sex glands. Leydig cell numbers are reduced, and their steroidogenic function is low; the male mutant is typically infertile (23). Results from the present study show that adult Leydig cells develop abnormally in IGF-I-null mice. Most notably, the androgen-metabolizing enzyme, 5{alpha}R-1, was distinct from other Leydig cell mRNAs and proteins included in our analysis in that it was maintained at disproportionately high levels, whereas the others were extremely low in the absence of IGF-I stimulation. These defects in the mutant are consistent with the hypothesis that IGF-I stimulates Leydig cell differentiation (progenitor into immature) and maturation (immature into adult). Moreover, the concentrations of IGF-I in testicular interstitial fluid progressively increased after d 7 postpartum and peaked on d 24, coincident with the timing of adult Leydig cell differentiation and maturation, suggesting a pubertal requirement for IGF-I action in the testis.

Leydig cell-specific mRNAs can be assigned to four categories according to their developmental trends for expression in mouse testis (9). The mRNA species in category 1 (e.g. encoding P450c17{alpha}, P450scc, and StAR protein) and category 2 (17ßHSD III and 3ßHSD VI) are known to be required for androgen synthesis and are thus fundamental to Leydig cell function. Steady state mRNA levels for these genes begin to increase shortly after initial differentiation of adult Leydig cells about d 15 and are highest at sexual maturity from d 50 onward. The mRNA species in category 3 (3{alpha}HSD and 5{alpha}R-1) encode enzymes required for androgen metabolism, and expression levels peak between d 20–30. The mRNA levels of these enzymes corresponded closely with their protein and activity levels (8, 10, 29). Our results show that steroidogenic mRNA and protein levels (including category 2) were low as a group, whereas androgen-metabolizing enzyme mRNA and protein levels were normal (3{alpha}HSD) or higher (5{alpha}R-1) in 50-d-old IGF-I-null mice. We counted Leydig cell numbers in IGF-I-null testes and found that they were proportionally reduced to 25% of wild-type control levels (i.e. by the same extent that testis size and body weight were reduced in the mutant). Therefore, the representation of Leydig cell mRNAs in samples of whole testis total RNA from wild-type and mutant animals should be similar. Preliminary data on isolated Leydig cells in IGF-I-null mice also showed reduced mRNA levels for P45017{alpha} and 17ßHSD (data not shown), consistent with results obtained using whole testis samples. Moreover, 5{alpha}-R1 mRNA was elevated in whole testis despite the reductions in Leydig cell numbers. Therefore, the differences in mRNA levels between control and IGF-I-null mice measured in whole testis are probably due not only to reduced Leydig cell numbers, but also to impaired steroidogenic function of Leydig cells in mutant testes. Decreased steroidogenic enzyme protein levels and the disproportionately increased 5{alpha}-R1 levels further confirm the hypothesis that adult Leydig cells underwent initial differentiation, but then failed to mature in the absence of IGF-I stimulation.

In wild-type males, steroidogenic mRNAs typically increase while metabolism by 5{alpha}R-1 is simultaneously decreased between 35–50 d, providing overt evidence of the Leydig cell maturational process. However, no apparent developmental trends in mRNA and protein levels in IGF-I-null testes were observed in this study, in marked contrast to wild-type controls. mRNA levels for RLF, a marker for fully differentiated adult Leydig cells (37, 38, 39), were low, whereas those for 5{alpha}R-1, a marker of immature Leydig cells, were simultaneously high in mutant Leydig cells on d 50, corroborating the hypothesis that IGF-I deficiency causes a development block of Leydig cells at the immature stage. This is consistent with morphological studies showing that Leydig cells in these animals lack the characteristic growth in cytoplasmic lipid content and smooth endoplasmic reticulum profile area (23). Moreover, cytological analysis indicates that Leydig cells in adult IGF-I-null mice attain only the immature stage of differentiation (23, 40). As peak intratesticular IGF-I concentrations occur before these events on d 24, it is possible that IGF-I acts as an inductive signal to stimulate the precursor stage of the Leydig cell. The mechanism of IGF-I action on the expression of steroidogenic and metabolizing enzymes is not clear. The fact that IGF-I modulates several different steroidogenic enzymes makes it unlikely that the action is direct. In vitro studies have shown that IGF-I potentiates LH stimulation by increasing the number of LHRs. IGF-I may therefore affect Leydig cell steroidogenesis by increasing LHR numbers. Taken together, the evidence leads to the conclusion that IGF-I and LH, acting in concert, facilitate Leydig cell proliferation and differentiation. Each acting alone is unable to establish normal Leydig cell numbers and steroidogenic function, as demonstrated by studies of IGF-I-null and LHR-null mice (12, 23).

The Leydig cell is a metabolizing site for androgens via the 5{alpha}-reduction pathway, which converts T into the more potent androgen, dihydrotestosterone. Dihydrotestosterone is then further catalyzed by 3{alpha}HSD into a weak androgen, androstane-3{alpha},17-diol. In rats and wild-type mice, levels of 5{alpha}-reductase activity are highest during the midpubertal period (9, 41). Under normal conditions, a midpubertal rise in 5{alpha}R-1 activity could enhance androgen action before the development of full steroidogenic potential in Leydig cells. A changing balance in the expression of T biosynthetic and metabolizing enzyme activities undoubtedly serves to regulate testicular T levels maintained by the Leydig cell (8, 42, 43). In the IGF-I-null mutant, decreased P450scc and P450c17{alpha} mRNA levels were associated with disproportionately higher 5{alpha}R-1 expression. This accounts for the extremely low T production in adult IGF-I-null testes and further supports the hypothesis that the absence of IGF-I stimulation results in disproportionate androgen-metabolizing enzyme expression in these animals. Dihydrotestosterone is more potent than T as an androgen, but may be immediately converted by 3{alpha}HSD to androstane-3{alpha},17ß-diol, a weak androgen (44). Thus, depending on its level of expression relative to enzymes of T biosynthesis and 3{alpha}-hydroxysteroid dehydrogenase, 5{alpha}R-1 may be an intensifier or an inhibitor of androgen action. As androgen plays a stimulatory role in Leydig cell maturation (45), it will be of interest to evaluate testicular concentrations of dihydrotestosterone and androstane-3{alpha},17ß-diol in IGF-I-null males during pubertal development. Our prediction is that androstane-3{alpha},17ß-diol concentrations will be relatively higher in mutant males, in keeping with the failure of their Leydig cells to fully differentiate.

GH receptor gene-disrupted male mice have undetectable circulating IGF-I levels and delayed puberty (46, 47). This agrees with the developmental profile of 5{alpha}R-1 protein levels in the present study. Signal intensities for 5{alpha}R-1 in mutant Leydig cells appeared later, peaked later, and remained higher relative to those in wild-type controls. Therefore, we infer that the absence of IGF-I stimulation delays the initial differentiation of progenitor Leydig cells and blocks further maturation of immature Leydig cells. This conclusion is also consistent with our observations on circulating T levels at the different ages in the mutant. It has been established that serum T levels in mice begin to decline after birth, when fetal Leydig cells atrophy (34, 35, 36). In IGF-I-null mice, higher serum T concentrations are found on postnatal d 14 and 21, potentially resulting from lower metabolism by 5{alpha}R-1 at these ages. The subsequent failure of the T levels to increase, presumably arising from higher metabolism by 5{alpha}R-1, results in low androgen status in adult mutant males. IGF-I levels in testicular interstitial fluid progressively increased from d 7 and were highest on d 24 postpartum. This apparent association between local IGF-I levels and Leydig cell development suggests a critical role for increased testicular IGF-I levels in adult Leydig cell differentiation and maturation. Taken together, these data suggest that IGF-I 1) facilitates precursor cell differentiation preceding LH-mediated events during puberty, and 2) stimulates maturation of adult Leydig cells in conjunction with LH by increasing steroidogenic enzyme expression and decreasing androgen metabolism by 5{alpha}R-1.


   Acknowledgments
 
The technical assistance of Chantal Manon Sottas and Enmei Niu is gratefully acknowledged. We are indebted to Argiris Efstratiadis (Columbia University) for providing the IGF-I knockout mouse line. We also thank Buck Hales (University of Illinois-Chicago) for the gift of the anti-P450c17{alpha} antibody.


   Footnotes
 
Preliminary results from this study were presented at the 85th Annual Meeting of The Endocrine Society, Philadelphia, PA, 2003; the 28th Annual Meeting of American Society of Andrology, Phoenix, AZ, 2003; and the 25th Annual Meeting of Society for the Study of Reproduction, Raleigh, NC, 1992.

Abbreviations: 3ßHSD IV, Type VI 3ß-hydroxysteroid dehydrogenase; LHR, LH receptor; P450c17{alpha}, 17{alpha}-hydroxylase/C17–20 lyase; P450scc, cholesterol side-chain cleavage; 5{alpha}R-1, type I 5{alpha}-reductase; RLF, relaxin-like factor; StAR, steroidogenic acute regulatory.

Received May 6, 2003.

Accepted for publication August 1, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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