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Laboratory for Experimental Medicine and Endocrinology, Department of Developmental Biology, Catholic University of Leuven (K.D.G., E.D., G.V.), B-3000 Leuven, Belgium; Medical Research Council Human Reproductive Sciences Unit, Center for Reproductive Biology (N.A., K.A.L.T., C.M., R.M.S., P.T.K.S.), and Division of Reproductive and Developmental Science, University of Edinburgh (J.I.M.), Edinburgh, Scotland, EH16 4SB United Kingdom; Institute of Experimental Morphology and Anthropology, Bulgarian Academy of Science (N.A.), 1113 Sofia, Bulgaria; Laboratory of Cellular Biology, Department of Morphology, Instituto de Ciências Biológicas/Federal University of Minas Gerais (L.R.d.F., G.G.P.), Belo Horizonte-MG, Brazil; Institute for Hormone and Fertility Research, University of Hamburg (S.H.), D-20251 Hamburg, Germany; and School of Molecular and Biomedical Sciences, University of Adelaide (R.I.), Adelaide 5005, Australia
Address all correspondence and requests for reprints to: Dr. R. M. Sharpe, Medical Research Council Human Reproductive Sciences Unit, Center for Reproductive Biology, Chancellors Building, 49 Little France Crescent, Edinburgh, Scotland EH16 4SB, United Kingdom. E-mail: r.sharpe{at}hrsu.mrc.ac.uk.
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Introduction |
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Differentiation of LC precursors into immature LC is a key step in development of the adult LC population (3, 5), and androgens appear to play a positive role in this process (14). Perhaps the most dramatic demonstration of the potential importance of androgens in LC development is the observation that in mice lacking a functional AR, there is a major reduction in adult LC number and function (20). Whether this deficit arises because of the absence of effect of androgens directly on LC or their precursors or is mediated partly or wholly via the absence of effects on the SC (or even on the peritubular myoid cells) is unclear. Another complication in this model is that the testes are cryptorchid, and the consequent elevation of testicular temperature and/or the associated depletion of germ cells may also play a role. The recent generation by ourselves (21) and others (22, 23) of mice with SC-selective knockout of the AR (SCARKO mice) has opened up new possibilities for elucidating the role that androgens play in regulating the development and function of testicular cells. Moreover, by comparing these processes in SCARKO and complete AR knockout [AR0/Y; ARKO] mice, it is possible to gain insights into the relative importance of SC-mediated and non-SC-mediated effects of androgens on developing LC. We used such an approach in the present studies and show that SC-mediated effects of androgens may be important for the development of normal numbers of adult LC.
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Materials and Methods |
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Tissue and blood sampling
Urogenital systems from control, ARKO and SCARKO males (aged 12, 20, 50, or 140 d) were removed, fixed in Bouin’s fluid for 4–6 h, then transferred to 70% ethanol. Testes and seminal vesicles (if present) were dissected out and weighed. Blood was taken by cardiac puncture under ether anesthesia and allowed to clot overnight at 4 C. Serum was isolated by two centrifugal steps of 10 min at 13,000 rpm and stored at –20 C until assayed.
Antibodies for immunohistochemistry and their dilutions
Rabbit polyclonal antibody (AB1244, Chemicon International, Temecula, CA) recognizing cytochrome P450 side-chain cleavage (P450scc) was used at a 1:200 dilution. Anti-17
-hydroxylase/C17–20lyase (anti-P450c17) and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) rabbit polyclonal antibodies, used at 1:300 and 1:3000 dilutions, respectively, were generated in-house, as was the rabbit polyclonal antibody to insulin-like factor-3 (Insl3), which was used at a 1:200 dilution. Immunolocalization of AR used a rabbit polyclonal antibody (N20) raised against a peptide within the N-terminal domain of the human AR (sc-816, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and was diluted 1:500. Mouse monoclonal estrogen receptor (ER) antibody (NCL-ER-6F11/2, Novocastra Laboratories Ltd., Newcastle upon Tyne, UK) was diluted 1:40.
Immunohistochemistry
Immunohistochemistry was performed on dewaxed sections without antigen retrieval, except for sections used for ER immunostaining, which were subjected to heat-induced antigen retrieval for 5 min in 0.01 M citrate buffer, pH 6.0 (Sigma-Aldrich Corp., St. Louis, MO) using a pressure cooker. This was followed by endogenous peroxidase blocking [3% (vol/vol) H2O2 (BDH, Poole, UK)] in methanol (BDH) for 30 min at room temperature. All washes between antibody or reagent incubations comprised two washes, 5 min each time, at room temperature in Tris-(Tris-hydroxymethyl methylamine)-buffered saline [TBS; 0.05 M Tris, pH 7.4, 0.85% (vol/vol) saline] unless otherwise stated. Tissue sections were first blocked in TBS containing normal swine serum or normal rabbit serum for ER- (1:4 dilution; Diagnostics Scotland, Carluke, UK) and 5% (wt/vol) BSA [Sigma-Aldrich Corp.; normal swine serum (NSS) or normal rabbit serum (NRS)] before incubation with primary antibodies diluted in NSS or NRS. A swine antirabbit biotinylated secondary antibody (E0353, DakoCytomation, Carpinteria, CA) or rabbit antimouse biotinylated secondary antibody (E0464, DakoCytomation), both applied at 1:500, were diluted in NSS or NRS, respectively, and incubated at room temperature for 30 min. Bound antibodies were visualized by incubating the sections with avidin-biotin-horseradish peroxidase agent (K0355, DakoCytomation) for 30 min, followed by color development with 3,3'-diaminobenzidine tetrahydrochloride chromogenic substrate (K3468, Liquid DAB+ kit, DakoCytomation), monitored microscopically. Sections were counterstained with hematoxylin, dehydrated, and mounted with Pertex (Histolab, Göteborg, Sweden). Images were captured using a Provis microscope (Olympus Optical Co., London, UK) equipped with a Kodak DCS330 camera (Eastman Kodak Co., Rochester, NY). Captured images were stored on a Macintosh G4 computer (Apple Computer, Cupertino, CA) and compiled using Photoshop 7.0 (Adobe Systems, Mountain View, CA).
For double immunostaining for AR and 3ß-HSD, dewaxed sections were subjected to heat-induced antigen retrieval, as described above, before endogenous peroxidase blocking. Testis sections were blocked in normal goat serum [goat serum (Diagnostics Scotland) diluted 1:4 in TBS with 5% (wt/vol) BSA (Sigma-Aldrich Corp.); NGS] and incubated with anti-AR (Santa Cruz Biotechnology, Inc.) before the addition of a goat antirabbit peroxidase secondary antibody (P0448, DakoCytomation) diluted 1:200 and subsequent color development with DAB+ (DakoCytomation). Slides were then boiled in glycine/EDTA [0.05 M glycine and 0.01% (wt/vol) EDTA, pH 3.5] for 2 min before blocking again in NGS and incubation with 3ß-HSD antibody. A biotinylated goat antirabbit antibody (E0432, DakoCytomation), diluted 1:500, was used in combination with Strept ABComplex/alkaline phosphatase (K0391, DakoCytomation) and Fast Blue [1 mg/ml Fast Blue BB salt (Sigma-Aldrich Corp.) in 0.1 M Tris-(hydroxymethyl)methylamine (pH 8.2), 200 µg/ml Naphthol AS-MX phosphate (Sigma-Aldrich Corp.), and 2% (vol/vol) dimethylformamide buffer] to enable visualization of 3ß-HSD protein expression. Sections were counterstained with hematoxylin and aqueous mounted in Hydromount (National Diagnostics, Highland Park, NJ). All washes for double immunostaining comprise of one wash in TBS and 0.05% (vol/vol) Tween 20, followed by a second wash in TBS, and all antibodies were diluted in NGS.
To enable comparative evaluation of the immunostaining, sections of tissues from control and knockout animals were processed in parallel on at least three occasions to ensure reproducibility of results; on each occasion, tissue sections from four to six animals in each group were run. To ensure direct comparability of staining intensities, one section each from control, ARKO, and SCARKO mice was mounted on the same slide.
Measurement of Leydig (3ß-HSD-positive) cytoplasmic volume and number per testis
Testicular sections were immunostained for 3ß-HSD as described previously (19, 24) and counterstained with hematoxylin. The volume of 3ß-HSD-positive cells per testis was then determined using point-counting methods detailed previously (19, 25). In brief, testicular cross-sections from each of three to 12 animals per group and age were examined, and points falling over 3ß-HSD-positive cytoplasm or over the nuclei of cells with 3ß-HSD-positive cytoplasm were scored separately. Both were then independently expressed as relative volumes per testis. This data were converted to absolute volumes per testis by multiplying by testis weight (=volume), because shrinkage was minimal. Separately, the diameter of 100 LC nuclei in three to five animals per group was measured, and the mean value obtained was used to convert data for LC nuclear volume per testis to LC numbers per testis using standard procedures, as described previously (25).
LC morphology and ultrastructure in adult control and SCARKO mice
The testes of three control and three 50-d-old SCARKO mice were perfusion-fixed with 4% (vol/vol) glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3) preceded by a brief saline wash. The testes were then diced into small pieces, placed into the same fixative for 1 h, washed in cacodylate buffer overnight, postfixed with 1% (wt/vol) osmium/1.25% (wt/vol) potassium ferrocyanide mixture, dehydrated in ethanol, and embedded in Araldite (CY 212). Thin sections were then prepared, mounted on 200-mesh grids, stained with uranyl acetate and lead citrate, and examined on an EM-10 electron microscope (Zeiss, Oberkochen, Germany). Photomicrographs were taken at x6,500 and, with a final magnification of approximately x15,000, the volume density of mitochondria, endoplasmic reticulum, and lipid droplets was determined by point counting using a multipurpose grid (line length, 1 cm). A minimum of 10 LC were analyzed in each animal. The volume density of organelles per cell was determined as detailed previously (26).
Measurement of plasma LH and testosterone levels
Serum LH was measured via a double-antibody RIA using reagents supplied by Dr. A. F. Parlow (Harbor-University of California-Los Angeles, Torrance, CA) and the National Institute of Diabetes and Digestive and Kidney Diseases, National Hormone and Peptide Program. The standard preparation used was mLH-RP (lot AFP5306A), the tracer was prepared from rLH-I-10, and the antiserum was anti rLH-S-11. Serum testosterone levels were measured using the Testo-RIA-CT kit (BioSource International, Camarillo, CA; detection limit, 0.05 ng/ml), according to the instructions of the manufacturer. The within-assay coefficients of variation for testosterone and LH were 4.7% and 7.8%; the interassay coefficients of variation were 6.2% and 8.1% for testosterone and LH, respectively.
RNA analysis
Tissue samples were removed and snap-frozen in liquid nitrogen. RNA from control (AMH-Cre) and SCARKO testes was prepared using the RNeasy midi kit (Qiagen, Chatsworth, CA). Due to their small testicular size, RNA from ARKO testes and their appropriate control, PGK-Cre testes, were extracted using the RNeasy mini kit (Qiagen). Synthesis of cDNA from deoxyribonuclease I-treated total RNA (RNase-Free DNase I Set, Qiagen) used SuperScript II ribonuclease H– reverse transcriptase and random hexamer primers (Invitrogen Life Technologies, Carlsbad, CA). To allow specific mRNA levels to be expressed per testis and to control for the efficiency of RNA extraction, RNA degradation, and the RT step, 10 ng luciferase mRNA (Promega Corp., Madison, WI) were added to each testis at the start of the RNA extraction procedure and used as an internal standard. Gene expression was quantified using the ABI PRISM 7700 PCR detection system (Applied Biosystems, Foster City, CA) with a quantitative two-step RT-PCR protocol. Components for real-time PCR were obtained from Applied Biosystems, except for primers and probes (Eurogentec, Sar-Tilman, Belgium) and SYBR Green (Sigma-Aldrich Corp.). Each 25-µl real-time PCR contained 1x buffer A, 5 mM MgCl2, 400 µM deoxy-NTPs, 200 nM of each primer, 0.4x SYBR Green, and 0.025 U/µl AmpliTaq Gold enzyme. Amplified samples were electrophoresed on polyacrylamide gels to ensure that only a single band was amplified in each PCR. Primer sequences for platelet-derived growth factor (PDGF) receptor- were: forward, 5'-CCTTACGACTCCAGATGGGAAT-3'; reverse, 5'-ATGCACCGGATCCCAAAA-3'; those used for PDGF-A, Insl3, P450scc, 3ß-HSDI, P450c17, and estrogen sulfotransferase (EST) were described previously (20). The quantity of measured mRNA was expressed relative to the luciferase standard in the same sample. All samples and standard curves were run in triplicate.
Statistical analysis
With the exception of the real-time PCR data, for which a two-sample t test was employed, statistical analysis was performed using one-way ANOVA supplemented with Fisher’s multiple comparison test using NCSS 2000 software (NCSS Statistical Analysis and Data Analysis Software, Kaysville, UT).
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). In ARKO mice, testis weight was already reduced to 43% of the control value on d 12, and the magnitude of this decrease increased progressively thereafter, such that by d 140, testis weight was less than 6% of that in controls (Table 1).
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). In SCARKO animals on d 12, LC number (mean ± SEM, 0.36 ± 0.05 x 106; n = 5) was comparable to the control value (0.40 ± 0.06 x 106; n = 10), but on d 20, 50, and 140, LC number was reduced by 42–54% compared with controls (Fig. 1A), although the temporal pattern of change in LC number was broadly similar in SCARKO mice and controls. In ARKO mice, LC number was already reduced (by 37%, although nonsignificantly) on d 12 (0.25 ± 0.03 x 106; n = 5) compared with controls, and a major decrease in LC number was evident at all later ages, such that the LC number achieved on d 140 was only 22% of that in controls (Fig. 1A). On d 50 and 140, LC number was significantly higher in SCARKO than in ARKO animals (Fig. 1A).
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). Between d 50 and 140, LC cytoplasmic volume in controls remained constant, but there was additionally a 32% increase (P < 0.05) in cytoplasmic volume of LC in SCARKO animals between d 50 and 140, such that at the latter age their LC cytoplasmic volume was 23% greater than that in controls (P < 0.05; Fig. 1B). In contrast, LC cytoplasmic volume in ARKO animals remained virtually unchanged from d 12 to 140, and at the latter age it was only 60% of that in controls and 48% of that in SCARKO mice (Fig. 1B).
LC function in adulthood in SCARKO and ARKO mice
To evaluate the function of LC in adulthood (d 50–140), the mRNA expression and immunoexpression of four proteins connected to LC hormone secretory function (P450ssc, 3ß-HSD, P450c17, and Insl3) were studied (Fig. 2) as well as the expression of the mRNA for EST; data for mRNA expression were expressed relative to LC number (Fig. 3).
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). This has to be taken into account when evaluating whether the immunoexpression of steroidogenic enzymes in LC is altered in such testes. The intensity of immunoexpression of P450ssc and P450c17 in SCARKOs displayed little change from controls, whereas immunoexpression of P450ssc was increased and that of P450c17 was reduced in ARKO mice (Fig. 2). Immunoexpression of 3ß-HSD revealed no major difference in the intensity of expression among the three groups (Fig. 2); it should also be noted that the antibody used was not 3ß-HSD type specific. Immunoexpression of Insl3 was comparable to that in controls in SCARKO mice, but was reduced markedly in intensity in ARKO testes (Fig. 2).
Expression per LC of the five mRNAs studied revealed major differences between SCARKOs and ARKOs. Thus, mRNA expression for each of the three steroidogenic enzymes investigated (P450ssc, 3ß-HSD type 1, and P450c17) was approximately doubled in SCARKO mice compared with controls, whereas ARKO mice showed a different pattern of change for each enzyme; namely, expression of 3ß-HSD type 1 mRNA was increased 5-fold, and that of P450ssc was increased by 2-fold, whereas mRNA for P450c17 was reduced by approximately 70% (Fig. 3). In SCARKO mice, the expression per LC of Insl3 was normal, and that of EST increased more than 3-fold compared with controls, whereas in ARKO mice, the expression of Insl3 and EST was reduced by more than 90% compared with controls (Fig. 3).
Serum testosterone levels were only determined on d 50 and 140 and showed very wide variation between animals within genotype groups. Although mean values in both SCARKO and ARKO animals were lower than those in controls, there was no significant difference from controls (Fig. 4). In contrast, serum LH levels were elevated more than 8-fold in ARKO mice compared with controls, presumably due to the lack of androgen negative feedback via ARs, but LH levels were normal in SCARKO mice, consistent with there being a normal level of androgen feedback (Fig. 4). Additional evidence of normal serum testosterone levels overall in SCARKO mice was that seminal vesicle weight on d 50 and 140 was comparable to that in controls (Table 2); the seminal vesicles were absent in ARKOs due to failure of Wolffian duct development (21).
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), and the volume of cytoplasmic organelles per LC was evaluated. This demonstrated that the volumes of mitochondria and lipid droplets were approximately doubled in SCARKO animals compared with controls (Fig. 5D), although only the increase in lipid droplets was statistically significant (P < 0.05). In contrast, the volume of endoplasmic reticulum per LC was nonsignificantly decreased in SCARKO mice compared with controls (Fig. 5D).
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); other interstitial cells, which were immunonegative for 3ß-HSD, were AR immunopositive. In contrast, by d 20 and through to d 50, most 3ß-HSD-immunopositive cells were AR immunopositive in both control and SCARKO mice (Fig. 6); there was no AR immunoexpression at any age in ARKO testes (Fig. 6). Second, immunostaining for ER revealed that the intensity of ER immunoexpression was reduced in SCARKO LC and increased in ARKO LC compared with controls (supplementary figure), perhaps indicating parallel alterations in estrogen action within LC.
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, when expressed per LC, was unaltered in SCARKO mice, except for a small increase on d 50 (Fig. 7). It was notable that the expression of mRNA for PDGF receptor- declined more than 10-fold from d 12 to 50 in both control and SCARKO mice (Fig. 7).
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The major observed differences in LC development between ARKO and SCARKO mice were 1) LC number was reduced only by about 50% in SCARKO mice compared with about 75% in ARKO mice; and 2) LC size was normal or increased in SCARKO mice compared with controls, whereas LC size was consistently reduced in ARKO mice. The reduced LC numbers in ARKO animals are not explained by the concurrent cryptorchidism (20). At face value, the comparison of LC number in the two knockout models could indicate that the development of approximately 50% of adult LC number is dependent on androgen action via the AR in SC, whereas the development of normal adult LC size is not dependent on such action. In contrast, androgen action on testicular cell types other than SC is essential for the development of normal adult LC size and also accounts for approximately 25% of the final adult LC number. The latter finding would be consistent with evidence that androgen action on LC precursors plays a role in their differentiation into immature LC (14), and the present observation that the AR was mainly expressed in 3ß-HSD-immunonegative cells (i.e. not in immature and adult LC, which are both 3ß-HSD immunopositive) in control and SCARKO animals at 12 d of age is consistent with this. Although these straightforward interpretations may be partly true, there are numerous publications that attest to potentially important roles for locally produced hormones and growth factors in regulating LC development and function (3, 5, 7, 10), and the expression of at least two of these were altered dramatically in SCARKO and ARKO mice, namely, EST and PDGF-A, as discussed below.
EST, which is expressed in LC, plays a key role in the inactivation of biologically active estrogens. In EST knockout mice, there is reduced testicular expression of P450c17 (28), reduced testosterone production (28), and progressive LC hyperplasia/hypertrophy with aging (29). These changes can be recapitulated by administering estrogens to mice and these exert their effect via ER (30, 31). Conversely, knockout of aromatase, leading to ablation of estrogen action in the testis, leads to LC hyperplasia and/or hypertrophy and elevated testosterone levels (32). The dramatic down-regulation of EST expression in ARKO mice in the present studies and also reported in tfm mice in other studies (33) may therefore account for their reduced expression of P450c17, although the categorical absence of LC hyperplasia and hypertrophy in ARKO mice means that altered EST expression does not provide a complete explanation of their LC phenotype. In any case, the reduced EST expression in ARKO mice is probably explained by the abdominal location of the testes (20). In contrast to ARKO animals, the expression of EST in LC of adult SCARKO mice was increased 3-fold, a change that would be expected to increase the rate of estrogen catabolism within LC and thus reduce local estrogen action within the LC. ER knockout mice, in which estrogen action on LC via ER is prevented, exhibit increased testosterone production per LC in fetal (31) as well as in adult (30) life together with increased expression of P450scc and P450c17, changes also observed in adult LC of SCARKO mice in the present studies. Estrogen action in mice can also lead to LC hyperplasia and hypertrophy (33, 34), but whether underactivity of estrogens can lead to reduced LC number in mice, as found presently in SCARKO animals, is unknown. In view of the evidence just discussed, it seems reasonable to propose that some of the LC changes observed in both SCARKO and ARKO mice are attributable to altered EST expression and consequent alteration in local estrogen action within LC. Our observations of increased intensity of ER immunoexpression in LC nuclei of ARKO mice and reduced ER immunoexpression in LC nuclei of SCARKO mice are consistent with this interpretation, because estrogens are recognized to positively regulate ER immunoexpression (35). Although EST expression in the epididymis (36) and LC (33) is reported to be androgen dependent, the mechanism by which androgen action on SC is able to alter EST expression in LC in SCARKO mice remains to be defined, but one potential candidate for mediating SC-LC communication is PDGF-A, which is expressed predominantly in SC in the testis (27, 37).
In a recent study of SC development in SCARKO and ARKO mice, we reported that both models exhibited a major decrease in the expression of PDGF-A (24), a change confirmed in the present study for SCARKO animals. Because the reduction in PDGF-A expression occurred in both models, it is unlikely that the cryptorchidism in ARKOs (20, 21) is a major factor in this decrease. The reduction in PDGF-A could account for the reduced LC number in SCARKO and ARKO mice described in this study, because interference with PDGF-A action via knockout of its receptor results in impairment of development of fetal LC (38), and knockout of PDGF-A itself leads to gross impairment of adult LC development (27). In this regard, the relative degree of reduction in PDGF-A gene expression in SCARKO mice (present study and Ref. 24) and ARKO mice (24) parallels the magnitude of reduction in LC number.
Another SC-derived factor that has been shown to affect LC development in rodents is AMH. LC development is slightly compromised in AMH-deficient mice (39), whereas aberrantly high/continued expression of AMH beyond early puberty inhibits the development of adult LC generation and steroidogenesis (40, 41). However, our previous study indicated that the expression of AMH by SC showed a normal age-related decline in both SCARKO and ARKO mice (24), so AMH is unlikely to be involved in the LC changes in these animals.
In view of the key role that SC appear to play in regulating LC development and function, it has been suggested that an alteration of SC number, such as occurs in animals in which thyroid hormones are manipulated perinatally could lead to a parallel change in LC numbers (11, 18). In ARKO animals, the approximately 75% reduction in LC number shown here and previously (20) parallels a similar reduction in SC number (24). However, SC number remains largely unchanged from control values in SCARKO mice (24), yet these same animals exhibit an approximately 50% reduction in adult LC number. The present data together with other findings (19) therefore do not support the concept of a simple relationship between SC and LC numbers in the adult testis.
One of the most puzzling observations of the present studies was the apparent disparity between the major reduction in LC number in SCARKO and ARKO mice and the lack of change in blood levels of testosterone. Although the latter measurements showed huge between-animal variation, which may have obscured a reduction in testosterone levels in ARKO mice, three independent lines of evidence support the normality of testosterone levels in SCARKO animals; thus, serum testosterone levels were normal, serum LH levels were normal, and seminal vesicle weight was normal. The ability of LC in SCARKO mice to maintain normal testosterone levels despite an approximately 50% reduction in LC number and no increase in LH levels can only indicate that testosterone production per LC must be increased, perhaps doubled, in young adult SCARKO animals. Our observations of increased LC cytoplasmic volume in adulthood, increased volume of lipid droplets and mitochondria, and increased expression per LC of several of the key steroidogenic enzymes (P450ssc, 3ß-HSD, and P450c17) in adult SCARKO animals are consistent with this interpretation. The finding that testosterone levels are normal in SCARKO mice means that, physiologically, androgen-dependent processes outside the SC may be entirely normal in these animals. However, the fact that major adaptive changes have to occur in LC function/gene expression in SCARKO mice to achieve normal testosterone levels despite the halving of LC number, emphasizes the importance of a thorough analysis of testicular cell development in transgenic models, because there may be secondary consequences of these adaptive changes (e.g. altered estrogen action within the testis).
Although direct measurement of serum testosterone levels did not show any significant reduction in ARKO mice, our suspicion is that a reduction was masked by the high variability in control values, because earlier studies of tfm and ARKO male mice have reported up to 80% reductions in testosterone levels (42, 43). Because there were no seminal vesicles in ARKO mice, and serum LH levels were grossly elevated due to lack of viable androgen negative feedback, there was no independent measure of the normality of testosterone levels in ARKO animals. In fact, the marked reduction in LC cytoplasmic volume and in the expression of P450c17 per LC in ARKO mice would fit more readily with reduced testosterone production; in this regard, it is possible that the reduction in P450c17 expression per LC may result from the almost complete suppression of EST expression in ARKO animals, because a similar reduction in P450c17 expression was observed in EST null mice (28); the reduced EST expression in ARKO mice is probably a consequence of the cryptorchid position of the testes (20).
The present data also show that Insl3 expression in adult LC is almost obliterated in ARKO mice, but is normal in SCARKO animals; the massive reduction in ARKO mice is not explained by cryptorchid position of the testes (20), but is consistent with the view that fully adult LC do not differentiate in ARKO/tfm testes (20), although a role for androgens in regulating Insl3 expression is also possible. In this regard, the normal LC expression of Insl3 in SCARKO mice shows that androgen action on SC is not required for the expression of this protein in LC.
In conclusion, the present findings demonstrate that the absence of AR-mediated androgen action selectively on SC has important consequences for LC development, in particular for the development of normal LC numbers. In terms of LC function and LC-specific gene expression, there are major differences between SCARKO and ARKO animals, which may indicate that androgen action via cell types other than SC, probably including LC and their precursors, is important for the development of normal LC hormone (testosterone and Insl3) secretory function; however, this conclusion is tempered by the occurrence of cryptorchidism in ARKO mice, which itself is undoubtedly an important cause of changes in LC gene expression (20). Nevertheless, it is likely that estrogens (via altered EST expression) and PDGF-A may play some part in the LC changes, in SCARKO animals at least, although it is likely that factors other than these are also involved. The altered LC function in SCARKO mice, in particular the likelihood of reduced estrogen levels intratesticularly due to elevation of EST expression, must be taken into account when using this model for investigation of the regulation of spermatogenesis.
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Acknowledgments |
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Footnotes |
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First Published Online May 26, 2005
1 K.D.G. and N.A. contributed equally to this study. 
Abbreviations: AMH, Anti-Mullerian hormone; AR, androgen receptor; ARKO, complete androgen receptor knockout; Cre recombinase, cyclization recombination recombinase; EST, estrogen sulfotransferase; 3ß-HSD, 3ß-hydroxysteroid dehydrogenase; Insl3, insulin-like factor-3; LC, Leydig cell; NGS, normal goat serum; NRS, normal rabbit serum; NSS, normal swine serum; PDGF-A, platelet-derived growth factor-A; P450scc, P450 cholesterol side chain cleavage enzyme; P450c17 17-hydroxylase/C17–20lyase; PGK, phosphoglycerate kinase-1; SC, Sertoli cell; SCARKO, Sertoli cell-selective androgen receptor knockout; TBS, Tris-buffered saline; tfm, testicular feminized mice; WT, wild type.
Received March 15, 2005.
Accepted for publication May 16, 2005.
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gene deficiency enhances androgen biosynthesis in the mouse Leydig cell. Endocrinology 144:84–93. Endocrinology 146:2454–2461 gene promoter region. Mol Endocrinol 15:2057–2063 mediates testis cord organization and fetal Leydig cell development in the XY gonad. Genes Dev 17:800–810This article has been cited by other articles:
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S. Huyghe, H. Schmalbruch, K. De Gendt, G. Verhoeven, F. Guillou, P. P. Van Veldhoven, and M. Baes Peroxisomal Multifunctional Protein 2 Is Essential for Lipid Homeostasis in Sertoli Cells and Male Fertility in Mice Endocrinology, May 1, 2006; 147(5): 2228 - 2236. [Abstract] [Full Text] [PDF]
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