Transgenic Mice Expressing P450 Aromatase as a Model for Male Infertility Associated with Chronic Inflammation in the Testis
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《内分泌学杂志》
Departments of Physiology (X.L., L.S., A.K., I.H., M.P.) and Anatomy (R.S., S.M.), Institute of Biomedicine and Functional Foods Forum, University of Turku, FIN-20520 Turku, Finland
Anatomical Institute (A.M.), Ludwig-Maximilians-University, D-80802 Munich, Germany
Institute of Reproductive and Developmental Biology (I.H.), Imperial College London, London W12 ONN, United Kingdom
Abstract
Our previous studies have shown that transgenic male mice expressing human P450 aromatase (AROM+) are infertile. In the present study, we followed the testis phenotype up to 15 months of age in these mice. The testes of the old AROM+ mice showed Leydig cell hypertrophy and hyperplasia, as indicated by the staining for steroidogenic enzymes and androgen and estrogen receptors. However, the Leydig cell adenomas did not show signs of malignization. In contrast, we observed a marked increase in the number of activated macrophages in the testicular interstitium of the aging AROM+ mice. The macrophages were further shown to express high levels of CD68 (a monocyte/macrophage marker) and secrete TNF, indicating strong activation, presumably by estrogen exposure. The increased activity of the macrophages was associated with Leydig cell depletion (analyzed at the age of 9 and 15 months) and an increased number of mast cells and fibrosis in the testicular interstitium. Interestingly, similar findings have been made in testes of infertile men. Hence, the aging AROM+ males present with a phenocopy of inflammation-associated infertility in men, providing a model for further studies on the putative link among estrogens, orchitis, and infertility.
Introduction
MALE REPRODUCTIVE HEALTH has deteriorated in many countries in recent decades. In addition, the incidence of testicular cancer (1, 2), hypospadias, and cryptorchidism appear to be increasing (3, 4), and testicular tumors have been reported to be the leading form of cancer in men between 20 and 39 yr of age. It has been hypothesized that exposure to estrogens or estrogenic environmental chemicals during the fetal period, and childhood could be one of the reasons (5) for the increased incidence of testicular disorders and testicular cancer. Ninety-five percent of all testicular neoplasms are testicular germinal cell tumors, whereas those in Sertoli and Leydig cells account for the remainder (6). Although Leydig cell tumors are generally small, 40% of adult men with such tumors have gynecomastia and other feminizing features (7), suggested to arise as a result of altered androgen/estrogen balance (8). Accordingly, patients with Leydig cell tumors frequently have elevated estrogen levels in urine and plasma (9).
Autoimmune orchitis is a common cause of male infertility. Studies on experimental autoimmune orchitis (EAO) have shown an increased number of macrophages in the testicular interstitium of rats (10). Recently estrogens have been proposed to act as immunomodulators with contradictory effects on autoimmune diseases. The incidence of autoimmune diseases, systemic lupus erythematosus, Sjgren’s syndrome, and rheumatoid arthritis is significantly higher in females than males (11, 12), whereas estrogens seem to offer protection in multiple sclerosis (13) and experimental autoimmune encephalomyelitis (14). Interestingly, mice lacking the aromatase enzyme gene have been shown to spontaneously develop a lymphoproliferative autoimmune disease resembling Sjgren’s syndrome (15). Although a subset of infertile men have significantly lower testosterone and higher estradiol concentrations in serum (16) and testicular macrophages have been shown to be involved in the pathogenesis or maintenance of states of infertility in the human testes (17), no connection between estrogens and idiopathic chronic orchitis has so far been demonstrated.
Leydig cells and macrophages are the two major cell types in the testicular interstitium. Testicular macrophages are divided into two different subpopulations with different functions. The CD68-expressing macrophages have been suggested to preserve the proinflammatory profile of circulating monocytes, whereas the CD163-expressing cells sensitive to the testicular microenvironment may have an antiinflammatory profile (18, 19). Testicular inflammation in vivo causes an influx of new monocytes and macrophages into the testis, drastically altering the composition of the macrophage population (20). Immunoactivated macrophages begin to secrete high amounts of proinflammatory cytokines, e.g. TNF (21, 22). The macrophages are mainly derived from the circulating monocyte pool, but the resident testicular macrophages do not reenter the circulating pool (23). The number of the macrophages and their functions are largely determined by the local environment (24, 25). During testicular development, the increase in macrophage number closely follows the increase in adult-type Leydig cell number (26, 27). There is increasing evidence that these cells are closely associated with each other and form specialized junctions, suggesting a functional relationship between the two cell types (28, 29, 30). However, the physiological significance of this association has remained obscure.
We recently developed a transgenic mouse model, AROM+, for studying the role of estrogen excess in the male (31). These mice are characterized by the expression of human P450 aromatase (P450arom) in a variety of tissues under the human ubiquitin C-promoter, resulting in increased conversion of testosterone (T) to estradiol (E2). The increased serum E2 to T ratio detected in the AROM+ mice results in a multitude of abnormalities in the structure and function of male reproductive tissues, whereas no obvious phenotype exists in female reproduction. Interestingly, AROM+ males also develop mammary epithelial structures typically seen only in female mice (32), confirming a major role of estrogens in the development of gynecomastia in males. Another typical feature of the estrogen stimulus in these mice is the induction of pituitary lactotrope adenomas. We have also shown that AROM+ males have multiple disorders in testicular function at the age of 4 months, with arrested spermatogenesis and Leydig cell hyperplasia and hypertrophy (31). The present study was carried out to further characterize the testis phenotype and follow up the long-term consequences of increased estrogen to androgen ratio in the testis of aging mice.
Materials and Methods
Transgenic mouse model expressing human P450arom
The generation of transgenic mice expressing human P450arom cDNA under the control of the ubiquitin C promoter (AROM+) has been previously described (31). AROM+ female mice are phenotypically normal and were used as breeders. These mice had free access to soy-free food pellets (SDS; Witham, Essex, UK) and tap water. All mice were handled in accordance with the institutional animal care policies of the University of Turku (Turku, Finland). Genotyping of the AROM+ mice was carried out as described previously (31).
Hormone assays
For intratesticular T and E2 assays, whole testes were weighed and homogenized in 1.0 ml PBS. T concentrations were measured by RIA from diethyl ether-extracted testis homogenates as previously described (33). Intratesticular E2 concentrations were measured by immunofluorometric assay from diethyl ether-extracted testis homogenates, following the protocol for serum samples recommended by the manufacturer (Wallac Oy, Turku, Finland). The serum concentrations of LH and FSH were analyzed using immunofluorometric assays as described previously (34).
Quantitative RT-PCR
Total RNA was isolated from testes using the RNeasy minikit (QIAGEN, Valencia, CA) according to the manufacturer’s protocol. The mRNA analysis was carried out by quantitative real-time RT-PCR by using the Engine Opticon system (MJ Research, Inc., Waltham, MA) for continuous fluorescent detection. Reactions were performed using QuantiTect SYBR Green RT-PCR kit (QIAGEN) according to the manufacturer’s instructions, using 50 ng total RNA from the testis. All samples and standards were amplified in triplicate. The expression level of L19 or -actin was used as a reference to adjust for an equal amount of sample RNA. The sequences of primers were: TNF (GenBank accession no. NM_013693), 5'-GAACTGGCAGAAGAGGCACT-3' (forward primer) and 5'-AGGGTCTGGGCCATAGAACT-3' (reverse primer) and CD68 (GenBank accession no. NM_009853), 5'-CCAGCTGTTCACCTTGACCT-3' (forward primer) and 5'-AGAGGGGCTGGTAGGTTGAT-3' (reverse primer). The annealing temperatures were 60 and 58 C and the sizes of the products 205 and 208 bp, respectively.
Morphological and histological analyses
Wild-type (WT) and AROM+ mice were obtained at similar ages of 4, 9, and 15 months and were anesthetized by ip injection of 300–600 μl of 2.5% Avertin. Blood was collected by cardiac puncture, and tissues were dissected out for macroscopic analyses. For histological evaluation, the testes were fixed in 4% paraformaldehyde. The tissues were then dehydrated, embedded in paraffin, and sectioned. Five-micrometer-thick sections were deparaffinized in xylene and stained with hematoxylin and eosin. For detecting collagen fibers, the sections were stained with Van Gieson’s solution. The Leydig cell adenomas were classified according to the criteria described by the National Toxicology Program (35) in which the adenoma classification is warranted if the tumor diameter exceeds one seminiferous tubule cross-section. An aggregation of Leydig cells smaller than the diameter of a seminiferous tubule was classified as focal hyperplasia. To visualize mast cells, deparaffinized sections were stained with Giemsa or toluidine blue.
Immunohistochemistry
Four-micrometer-thick sections were cut from paraffin-embedded testis tissues and mounted on slides. After deparaffinization and rehydration, they were placed in a 10-mM citrate buffer (pH 6.0), followed by heating in a microwave oven for antigen retrieval. For this, three periods of 5 min each were used, after which the sections were treated with 3% H2O2 in PBS (pH 7.6) for 20 min. The sections were then incubated overnight at 4 C in PBS containing 3% BSA and one of the following antibodies: 1) antibody against F4/80 (macrophage-specific marker; rat antimouse, Serotec, Oxford, UK) used at 1:20 dilution; 2) antibody against P450 side chain cleavage (P450scc) (rabbit antimouse) used at 1:1500 dilution (provided by Professor Anita Payne, Department of Gynecology and Obstetrics, School of Medicine, Stanford University, Stanford, CA); 3) antibody against TNF (goat polyclonal IgG; Santa Cruz Biotechnology, Santa Cruz, CA) used at 1:50 dilution; 4) antibody against androgen receptor (rabbit polyclonal IgG; Santa Cruz Biotechnology) used at 1:400 dilution; 5) antibody against estrogen receptor (ER)- (rabbit polyclonal IgG; sc-542; Santa Cruz Biotechnology) used at 1:500 dilution; and 6) antibody against ER (chicken polyclonal IgG; provided by J. . Gustafsson, Department of Medical Nutrition, Karolinska Institute, Stockholm, Sweden). The bound primary antibody was detected by using biotinylated goat antirabbit, goat antirat, or horse antigoat IgG, followed by incubation with avidin-biotin-peroxidase complex (Vector Laboratories, Burlingame, CA), and visualized by using 3'-3' diaminobenzidine tetrahydrochloride. Sections were slightly counterstained with Mayer’s hematoxylin and mounted.
Statistical analyses
Statistical analyses were performed by the SigmaStat program (version 3.1 for Windows 2000 and XP; SPSS Inc., Chicago, IL). For real-time RT-PCR results, Student’s t test or the Mann-Whitney rank sum test was performed for analyzing the statistical significance (P < 0.05) between WT and AROM+ mice, whereas one-way ANOVA or Kruskal-Wallis one-way ANOVA on ranks was used for analyzing different age groups. For intratesticular E2 and T levels, the Mann-Whitney rank sum test was used for analyzing statistical significance between the 15-month-old AROM+ and WT mice.
Results
As we have previously reported (31), at 4 months of age, the AROM+ mice present with Leydig cell hyperplasia and hypertrophy (Fig. 1B). In the present study, we further analyzed the testicular phenotype in mice at the age of 9 and 15 months. Histologically defined Leydig cell adenomas were present in only a limited number of AROM+ testes at the age of 9 (one of five mice) and 15 (three of nine mice) months (Fig. 1E), whereas no histologically malignant or metastatic tumors were observed.
The most profound phenotype was the severe progression of the spermatogenic disruption during the aging. At 9 months of age, most of the seminiferous tubules in AROM+ mice had only a small number of germ cells (Fig. 1C), and at the age of 15 months, most of the tubular cords totally lacked germ cells (Fig. 1D). Variable amounts of yellowish giant multinucleated cells were present in the testicular interstitium of AROM+ testes at different ages (Fig. 1, B–D). At the age of 9 and 15 months, the size and number of the multinucleated cells was markedly greater than at 4 months. The cells were irregular in shape and had multiple nuclei arranged in the periphery of the cytoplasm (Fig. 1, C and D).
We have previously reported that intratesticular E2 levels are markedly elevated, and T levels reduced in AROM+ males at 5 months of age (36). Similarly, there was a significant increase in intratesticular E2 and reduction in T level in 15-month-old AROM+ mice (Table 1). The serum gonadotrophin levels were also in line with the results reported at 4 months of age (31), showing no significant differences in the average LH concentrations between 15-month-old AROM+ and WT mice, whereas the level of FSH was significantly reduced in the AROM+ mice, compared with WT (Table 1). Both ER (Fig. 2, A–C) and ER (Fig. 2, D–H) were present in AROM+ testes, confirming that the estrogen-dependent signal transduction pathway was functional. Staining for androgen receptor (AR) revealed positive staining of the nuclei of Leydig, Sertoli, and myoid cells at the ages of 4, 9, and 15 months (Fig. 2, I–M). This, together with the measurable levels of T present, indicated that the Leydig and Sertoli cells were not devoid of androgen action.
The hyperplastic Leydig cells present at 9 months of age were confirmed by strong immunoreactivity toward antibodies against P450scc (Fig. 3B) and 3-hydroxysteroid dehydrogenase (data not shown). The stainings also confirmed the steroidogenic capacity of the hyperplastic interstitial tissue of AROM+ testes.
Immunohistochemical analysis with antibodies against F4/80 (a macrophage marker) indicated that the giant multinucleated cells were macrophages (Fig. 3, C–H). These F4/80-positive macrophages formed a strong network, with an increased number of cells present in 9- and 15-month-old AROM+ mice (Fig. 3, F–H). In the WT testes, only a slight increase in the number of F4/80-positive cells was detected during aging (Fig. 3, C–E). Taken together, the data suggest that the elevated levels of E2 in AROM+ males resulted in a marked increase in the number of macrophages in the testicular interstitium. To further analyze the activation of macrophages, we examined the expression of TNF in the testes. Immunohistochemical analyses showed a low level of TNF expression in the Leydig cells of WT mice at 15 months of age (Fig. 3K). In contrast, in AROM+ mice, marked staining was detected and there was an increase in the TNF-positive cells during the aging of AROM+ mice (Fig. 3, L–N). Using quantitative RT-PCR, an increased amount at TNF mRNA was detected in AROM+ males (Fig. 4A). No change in IL-1 expression was detected, in either real-time RT-PCR or immunohistochemistry, and it was similarly identified in the spermatogonia both at WT and AROM+ mice (data not shown). To analyze the type of the macrophages present, the expression of monocyte/macrophage marker CD68 was measured by quantitative RT-PCR. A significantly higher expression of CD68 in AROM+ mice, compared with WT mice, indicated that the macrophages were recently recruited from the circulating monocyte pool and represent the immunologic active population of macrophages (Fig. 4B). Van Gieson staining demonstrated markedly increased fibrosis (Fig. 5, B–E) in the interstitium of AROM+ mice, which strongly indicates a chronic inflammation in the AROM+ testes. Additionally, mast cells were regularly found by Giemsa staining in the interstitial spaces of the older, 9- and 15-month AROM+ mice (Fig. 5, F and G). Most of these cells had an elongated, rather than round, shape, indicating activation.
Microscopic evaluation revealed evidence for direct engulfment of Leydig cells into macrophages (Fig. 5, H and I). Clearly different stages of this process could be observed. Some engulfed cells were still intact and without signs of membrane disruption, whereas some were swollen, had lost their membrane integrity, and were ruptured within the macrophages.
Discussion
We have previously shown that AROM+ mice expressing human P450arom under the human ubiquitin C promoter display multiple abnormalities in the male urogenital region, such as decreased size of testes, cryptorchidism, and Leydig cell hyperplasia (31).
In the present study, the AROM+ males had an elevated amount of intratesticular E2, combined with reduced amount of T at the age of 15 months, similar to males we described previously at 5 months of age (36). Whereas there were no significant differences in LH levels between AROM+ and WT groups, the reduced intratesticular androgen concentrations indicate testicular failure independent of feedback inhibition through the hypothalamus-pituitary axis. In fact, estrogen has been shown to directly affect steroidogenesis in the rat testis via accumulation of estrogen-regulated protein (molecular weight 27,000) and consequential desensitization of 17-hydroxylase and 17–20 desmolase (37, 38). Likewise, recent studies with mouse Leydig cells isolated from estrogen sulfotransferase knockout mice showed that accumulation of estrogen leads to decreased expression of P450 17-hydroxylase (39). Whereas ER and AR were expressed in the AROM+ Leydig cells in all age groups, we hypothesize that E2 is the driving force behind the testicular phenotype and that mouse Leydig cells are a target of estrogen action. This hypothesis is supported by previous findings showing that estrogen excess stimulates Leydig cell hyperplasia in rodents and has been associated with cryptorchidism, testicular cancer, and impaired spermatogenesis (40).
It is well established that estradiol plays an important role in regulating gonadotrophin secretion in both male rodents and men. The studies have also demonstrated that, compared with LH, the circulating FSH concentration is more effectively reduced by estradiol (41, 42). Our study with significantly decreased FSH levels and unaltered LH levels in AROM+ males is in line with these previous observations. The data, furthermore, suggest that a constitutive exposure to increased estrogens effectively inhibits FSH secretion in male mice, also in the presence of severely suppressed spermatogenesis.
One of the most striking features of the AROM+ testes was the presence of a high number of yellow multinucleated giant cells in the interstitium. These cells were identified as macrophages by immunohistochemical analysis of a macrophage-specific marker (F4/80). Similar histological findings (i.e. the presence of multinucleated giant cells in the interstitium) have been reported in old mice with chronic estrogen stimulation caused by targeted disruption of the estrogen sulfotransferase gene (43). This together with the present data suggests a role for estrogen stimulus in the macrophage activation of the testis. Accordingly, the increased size and number of macrophages observed at 9 and 15 months of age were accompanied by an increase in E2 concentrations. Because of the high background staining with the antibody used, we could not conclude whether the macrophages in AROM+ testes expressed ER. However, previous studies have demonstrated the expression of ER (44, 45, 46), but not ER (46), in murine macrophages, giving a possibility for direct effect of estrogens on macrophages. Furthermore, Guo et al. (47) reported that E2 is able to induce the activation of RAW-fos13 macrophages (RAW 264.7 macrophage cell line stable transfected with c-fos promoter) via the nongenomic Ca2+ signaling pathway. However, the mechanisms behind the E2-associated increase in testicular macrophages still remain open. One such mechanism could be that E2-induced proliferation of Leydig cells stimulates macrophage proliferation and activation, and the activated macrophages control the number of Leydig cells. There is a connection between the numbers of Leydig cells and testicular macrophages. Previous studies have namely shown that Leydig cells regenerate more slowly if the testis is depleted of macrophages, and T production is decreased after macrophage depletion (48, 49). The fact that in rats the total number of testicular macrophages declines if Leydig cells are ablated by treatment with the specific Leydig cell cytotoxin ethane dimethane sulfonate (18) further supports our finding of a strong association between the numbers of Leydig cells and testicular macrophages.
Our results indicate signs of severe inflammation in the testis of aging AROM+ mice. The high expression of CD68 indicates the presence of recently arrived monocyte-macrophages (18). This, together with increased expression of the proinflammatory cytokine, TNF, demonstrates that the macrophages in AROM+ testis are immunoactivated. Testicular macrophages produce undetectable levels of TNF under nonimmune conditions (50), whereas the studies with an experimental model for EAO have proposed a role for TNF in the pathogenesis of the disease (22, 21). The main target of the immunological attack in EAO are the germ cells that undergo apoptosis and sloughing, probably through an activated Fas-Fas ligand system (51). However, we could not detect an increased rate of apoptosis by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling staining or caspase-3 immunohistochemistry in the seminiferous tubules of AROM+ testis at any ages studied (data not shown). Nevertheless, severe fibrosis and an increased number of mast cells, in particular in the older groups of transgenic mice, was observed, providing further evidence for chronic inflammation reactions. Based on the elongated form of most of the mast cells, it can be concluded that they are activated, i.e. releasing their secretory products into the surrounding interstitial space. Although the precise nature of the putative mast cell products is not known, interaction between mast cells and macrophages have been shown to lead directly or indirectly to proliferation of fibroblast and collagen formation (52, 53), a likely response in the AROM+ mice as well. Interestingly, very similar inflammation-related changes have been reported in the testes of infertile men. Interstitial fibrosis, together with increased number of activated, degranulated mast cells, secreting proteases and histamine (54), and CD68-positive macrophages expressing TNF and IL-1 (17), has been observed in the human testes in connection with infertility.
The connection among estrogens, orchitis, and infertility still remains unknown, although the interaction of estrogens and the immune system has recently been discussed extensively (55). The AROM+ transgenic mouse model is a novel tool for analysis of the role of estrogens in testicular function, and it provides a useful model to better understand the interaction between macrophages and Leydig cells in vivo. Taken together, our data suggest that the elevated testicular E2 in AROM+ males results in disrupted spermatogenesis, a marked increase in the number of immunoactive macrophages and Leydig cell hyperplasia. Furthermore, an interaction between macrophages and Leydig cells in the interstitium was shown. Further studies are needed to clarify the exact mechanisms causing the disruption of spermatogenesis associated with E2-induced chronic inflammation in the testes of AROM+ mice.
Acknowledgments
We thank Ms. Tarja Laiho; Ms. Hannele Rekola; Ms. Saija Savolainen, M.Sc.; Ms. Katja Suomi; and Ms. Johanna Lahtinen for technical assistance.
Footnotes
This work was supported by Center of Excellence Grants 211480 and 207028 from The Academy of Finland and Grant FOOD-CT-2004-506319 from the European Commission [CASCADE (Chemicals as Contaminants in the Food Chain, a Network of Excellence for Research, Risk Assessment, and Education)].
The authors have no conflict of interest.
First Published Online November 23, 2005
1 X.L. and L.S. contributed equally to this work.
Abbreviations: AR, Androgen receptor; AROM+, human P450 aromatase; E2, estradiol; EAO, experimental autoimmune orchitis; ER, estrogen receptor; P450arom, P450 aromatase; P450scc, P450 side chain cleavage; T, testosterone; WT, wild type.
Accepted for publication November 17, 2005.
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Anatomical Institute (A.M.), Ludwig-Maximilians-University, D-80802 Munich, Germany
Institute of Reproductive and Developmental Biology (I.H.), Imperial College London, London W12 ONN, United Kingdom
Abstract
Our previous studies have shown that transgenic male mice expressing human P450 aromatase (AROM+) are infertile. In the present study, we followed the testis phenotype up to 15 months of age in these mice. The testes of the old AROM+ mice showed Leydig cell hypertrophy and hyperplasia, as indicated by the staining for steroidogenic enzymes and androgen and estrogen receptors. However, the Leydig cell adenomas did not show signs of malignization. In contrast, we observed a marked increase in the number of activated macrophages in the testicular interstitium of the aging AROM+ mice. The macrophages were further shown to express high levels of CD68 (a monocyte/macrophage marker) and secrete TNF, indicating strong activation, presumably by estrogen exposure. The increased activity of the macrophages was associated with Leydig cell depletion (analyzed at the age of 9 and 15 months) and an increased number of mast cells and fibrosis in the testicular interstitium. Interestingly, similar findings have been made in testes of infertile men. Hence, the aging AROM+ males present with a phenocopy of inflammation-associated infertility in men, providing a model for further studies on the putative link among estrogens, orchitis, and infertility.
Introduction
MALE REPRODUCTIVE HEALTH has deteriorated in many countries in recent decades. In addition, the incidence of testicular cancer (1, 2), hypospadias, and cryptorchidism appear to be increasing (3, 4), and testicular tumors have been reported to be the leading form of cancer in men between 20 and 39 yr of age. It has been hypothesized that exposure to estrogens or estrogenic environmental chemicals during the fetal period, and childhood could be one of the reasons (5) for the increased incidence of testicular disorders and testicular cancer. Ninety-five percent of all testicular neoplasms are testicular germinal cell tumors, whereas those in Sertoli and Leydig cells account for the remainder (6). Although Leydig cell tumors are generally small, 40% of adult men with such tumors have gynecomastia and other feminizing features (7), suggested to arise as a result of altered androgen/estrogen balance (8). Accordingly, patients with Leydig cell tumors frequently have elevated estrogen levels in urine and plasma (9).
Autoimmune orchitis is a common cause of male infertility. Studies on experimental autoimmune orchitis (EAO) have shown an increased number of macrophages in the testicular interstitium of rats (10). Recently estrogens have been proposed to act as immunomodulators with contradictory effects on autoimmune diseases. The incidence of autoimmune diseases, systemic lupus erythematosus, Sjgren’s syndrome, and rheumatoid arthritis is significantly higher in females than males (11, 12), whereas estrogens seem to offer protection in multiple sclerosis (13) and experimental autoimmune encephalomyelitis (14). Interestingly, mice lacking the aromatase enzyme gene have been shown to spontaneously develop a lymphoproliferative autoimmune disease resembling Sjgren’s syndrome (15). Although a subset of infertile men have significantly lower testosterone and higher estradiol concentrations in serum (16) and testicular macrophages have been shown to be involved in the pathogenesis or maintenance of states of infertility in the human testes (17), no connection between estrogens and idiopathic chronic orchitis has so far been demonstrated.
Leydig cells and macrophages are the two major cell types in the testicular interstitium. Testicular macrophages are divided into two different subpopulations with different functions. The CD68-expressing macrophages have been suggested to preserve the proinflammatory profile of circulating monocytes, whereas the CD163-expressing cells sensitive to the testicular microenvironment may have an antiinflammatory profile (18, 19). Testicular inflammation in vivo causes an influx of new monocytes and macrophages into the testis, drastically altering the composition of the macrophage population (20). Immunoactivated macrophages begin to secrete high amounts of proinflammatory cytokines, e.g. TNF (21, 22). The macrophages are mainly derived from the circulating monocyte pool, but the resident testicular macrophages do not reenter the circulating pool (23). The number of the macrophages and their functions are largely determined by the local environment (24, 25). During testicular development, the increase in macrophage number closely follows the increase in adult-type Leydig cell number (26, 27). There is increasing evidence that these cells are closely associated with each other and form specialized junctions, suggesting a functional relationship between the two cell types (28, 29, 30). However, the physiological significance of this association has remained obscure.
We recently developed a transgenic mouse model, AROM+, for studying the role of estrogen excess in the male (31). These mice are characterized by the expression of human P450 aromatase (P450arom) in a variety of tissues under the human ubiquitin C-promoter, resulting in increased conversion of testosterone (T) to estradiol (E2). The increased serum E2 to T ratio detected in the AROM+ mice results in a multitude of abnormalities in the structure and function of male reproductive tissues, whereas no obvious phenotype exists in female reproduction. Interestingly, AROM+ males also develop mammary epithelial structures typically seen only in female mice (32), confirming a major role of estrogens in the development of gynecomastia in males. Another typical feature of the estrogen stimulus in these mice is the induction of pituitary lactotrope adenomas. We have also shown that AROM+ males have multiple disorders in testicular function at the age of 4 months, with arrested spermatogenesis and Leydig cell hyperplasia and hypertrophy (31). The present study was carried out to further characterize the testis phenotype and follow up the long-term consequences of increased estrogen to androgen ratio in the testis of aging mice.
Materials and Methods
Transgenic mouse model expressing human P450arom
The generation of transgenic mice expressing human P450arom cDNA under the control of the ubiquitin C promoter (AROM+) has been previously described (31). AROM+ female mice are phenotypically normal and were used as breeders. These mice had free access to soy-free food pellets (SDS; Witham, Essex, UK) and tap water. All mice were handled in accordance with the institutional animal care policies of the University of Turku (Turku, Finland). Genotyping of the AROM+ mice was carried out as described previously (31).
Hormone assays
For intratesticular T and E2 assays, whole testes were weighed and homogenized in 1.0 ml PBS. T concentrations were measured by RIA from diethyl ether-extracted testis homogenates as previously described (33). Intratesticular E2 concentrations were measured by immunofluorometric assay from diethyl ether-extracted testis homogenates, following the protocol for serum samples recommended by the manufacturer (Wallac Oy, Turku, Finland). The serum concentrations of LH and FSH were analyzed using immunofluorometric assays as described previously (34).
Quantitative RT-PCR
Total RNA was isolated from testes using the RNeasy minikit (QIAGEN, Valencia, CA) according to the manufacturer’s protocol. The mRNA analysis was carried out by quantitative real-time RT-PCR by using the Engine Opticon system (MJ Research, Inc., Waltham, MA) for continuous fluorescent detection. Reactions were performed using QuantiTect SYBR Green RT-PCR kit (QIAGEN) according to the manufacturer’s instructions, using 50 ng total RNA from the testis. All samples and standards were amplified in triplicate. The expression level of L19 or -actin was used as a reference to adjust for an equal amount of sample RNA. The sequences of primers were: TNF (GenBank accession no. NM_013693), 5'-GAACTGGCAGAAGAGGCACT-3' (forward primer) and 5'-AGGGTCTGGGCCATAGAACT-3' (reverse primer) and CD68 (GenBank accession no. NM_009853), 5'-CCAGCTGTTCACCTTGACCT-3' (forward primer) and 5'-AGAGGGGCTGGTAGGTTGAT-3' (reverse primer). The annealing temperatures were 60 and 58 C and the sizes of the products 205 and 208 bp, respectively.
Morphological and histological analyses
Wild-type (WT) and AROM+ mice were obtained at similar ages of 4, 9, and 15 months and were anesthetized by ip injection of 300–600 μl of 2.5% Avertin. Blood was collected by cardiac puncture, and tissues were dissected out for macroscopic analyses. For histological evaluation, the testes were fixed in 4% paraformaldehyde. The tissues were then dehydrated, embedded in paraffin, and sectioned. Five-micrometer-thick sections were deparaffinized in xylene and stained with hematoxylin and eosin. For detecting collagen fibers, the sections were stained with Van Gieson’s solution. The Leydig cell adenomas were classified according to the criteria described by the National Toxicology Program (35) in which the adenoma classification is warranted if the tumor diameter exceeds one seminiferous tubule cross-section. An aggregation of Leydig cells smaller than the diameter of a seminiferous tubule was classified as focal hyperplasia. To visualize mast cells, deparaffinized sections were stained with Giemsa or toluidine blue.
Immunohistochemistry
Four-micrometer-thick sections were cut from paraffin-embedded testis tissues and mounted on slides. After deparaffinization and rehydration, they were placed in a 10-mM citrate buffer (pH 6.0), followed by heating in a microwave oven for antigen retrieval. For this, three periods of 5 min each were used, after which the sections were treated with 3% H2O2 in PBS (pH 7.6) for 20 min. The sections were then incubated overnight at 4 C in PBS containing 3% BSA and one of the following antibodies: 1) antibody against F4/80 (macrophage-specific marker; rat antimouse, Serotec, Oxford, UK) used at 1:20 dilution; 2) antibody against P450 side chain cleavage (P450scc) (rabbit antimouse) used at 1:1500 dilution (provided by Professor Anita Payne, Department of Gynecology and Obstetrics, School of Medicine, Stanford University, Stanford, CA); 3) antibody against TNF (goat polyclonal IgG; Santa Cruz Biotechnology, Santa Cruz, CA) used at 1:50 dilution; 4) antibody against androgen receptor (rabbit polyclonal IgG; Santa Cruz Biotechnology) used at 1:400 dilution; 5) antibody against estrogen receptor (ER)- (rabbit polyclonal IgG; sc-542; Santa Cruz Biotechnology) used at 1:500 dilution; and 6) antibody against ER (chicken polyclonal IgG; provided by J. . Gustafsson, Department of Medical Nutrition, Karolinska Institute, Stockholm, Sweden). The bound primary antibody was detected by using biotinylated goat antirabbit, goat antirat, or horse antigoat IgG, followed by incubation with avidin-biotin-peroxidase complex (Vector Laboratories, Burlingame, CA), and visualized by using 3'-3' diaminobenzidine tetrahydrochloride. Sections were slightly counterstained with Mayer’s hematoxylin and mounted.
Statistical analyses
Statistical analyses were performed by the SigmaStat program (version 3.1 for Windows 2000 and XP; SPSS Inc., Chicago, IL). For real-time RT-PCR results, Student’s t test or the Mann-Whitney rank sum test was performed for analyzing the statistical significance (P < 0.05) between WT and AROM+ mice, whereas one-way ANOVA or Kruskal-Wallis one-way ANOVA on ranks was used for analyzing different age groups. For intratesticular E2 and T levels, the Mann-Whitney rank sum test was used for analyzing statistical significance between the 15-month-old AROM+ and WT mice.
Results
As we have previously reported (31), at 4 months of age, the AROM+ mice present with Leydig cell hyperplasia and hypertrophy (Fig. 1B). In the present study, we further analyzed the testicular phenotype in mice at the age of 9 and 15 months. Histologically defined Leydig cell adenomas were present in only a limited number of AROM+ testes at the age of 9 (one of five mice) and 15 (three of nine mice) months (Fig. 1E), whereas no histologically malignant or metastatic tumors were observed.
The most profound phenotype was the severe progression of the spermatogenic disruption during the aging. At 9 months of age, most of the seminiferous tubules in AROM+ mice had only a small number of germ cells (Fig. 1C), and at the age of 15 months, most of the tubular cords totally lacked germ cells (Fig. 1D). Variable amounts of yellowish giant multinucleated cells were present in the testicular interstitium of AROM+ testes at different ages (Fig. 1, B–D). At the age of 9 and 15 months, the size and number of the multinucleated cells was markedly greater than at 4 months. The cells were irregular in shape and had multiple nuclei arranged in the periphery of the cytoplasm (Fig. 1, C and D).
We have previously reported that intratesticular E2 levels are markedly elevated, and T levels reduced in AROM+ males at 5 months of age (36). Similarly, there was a significant increase in intratesticular E2 and reduction in T level in 15-month-old AROM+ mice (Table 1). The serum gonadotrophin levels were also in line with the results reported at 4 months of age (31), showing no significant differences in the average LH concentrations between 15-month-old AROM+ and WT mice, whereas the level of FSH was significantly reduced in the AROM+ mice, compared with WT (Table 1). Both ER (Fig. 2, A–C) and ER (Fig. 2, D–H) were present in AROM+ testes, confirming that the estrogen-dependent signal transduction pathway was functional. Staining for androgen receptor (AR) revealed positive staining of the nuclei of Leydig, Sertoli, and myoid cells at the ages of 4, 9, and 15 months (Fig. 2, I–M). This, together with the measurable levels of T present, indicated that the Leydig and Sertoli cells were not devoid of androgen action.
The hyperplastic Leydig cells present at 9 months of age were confirmed by strong immunoreactivity toward antibodies against P450scc (Fig. 3B) and 3-hydroxysteroid dehydrogenase (data not shown). The stainings also confirmed the steroidogenic capacity of the hyperplastic interstitial tissue of AROM+ testes.
Immunohistochemical analysis with antibodies against F4/80 (a macrophage marker) indicated that the giant multinucleated cells were macrophages (Fig. 3, C–H). These F4/80-positive macrophages formed a strong network, with an increased number of cells present in 9- and 15-month-old AROM+ mice (Fig. 3, F–H). In the WT testes, only a slight increase in the number of F4/80-positive cells was detected during aging (Fig. 3, C–E). Taken together, the data suggest that the elevated levels of E2 in AROM+ males resulted in a marked increase in the number of macrophages in the testicular interstitium. To further analyze the activation of macrophages, we examined the expression of TNF in the testes. Immunohistochemical analyses showed a low level of TNF expression in the Leydig cells of WT mice at 15 months of age (Fig. 3K). In contrast, in AROM+ mice, marked staining was detected and there was an increase in the TNF-positive cells during the aging of AROM+ mice (Fig. 3, L–N). Using quantitative RT-PCR, an increased amount at TNF mRNA was detected in AROM+ males (Fig. 4A). No change in IL-1 expression was detected, in either real-time RT-PCR or immunohistochemistry, and it was similarly identified in the spermatogonia both at WT and AROM+ mice (data not shown). To analyze the type of the macrophages present, the expression of monocyte/macrophage marker CD68 was measured by quantitative RT-PCR. A significantly higher expression of CD68 in AROM+ mice, compared with WT mice, indicated that the macrophages were recently recruited from the circulating monocyte pool and represent the immunologic active population of macrophages (Fig. 4B). Van Gieson staining demonstrated markedly increased fibrosis (Fig. 5, B–E) in the interstitium of AROM+ mice, which strongly indicates a chronic inflammation in the AROM+ testes. Additionally, mast cells were regularly found by Giemsa staining in the interstitial spaces of the older, 9- and 15-month AROM+ mice (Fig. 5, F and G). Most of these cells had an elongated, rather than round, shape, indicating activation.
Microscopic evaluation revealed evidence for direct engulfment of Leydig cells into macrophages (Fig. 5, H and I). Clearly different stages of this process could be observed. Some engulfed cells were still intact and without signs of membrane disruption, whereas some were swollen, had lost their membrane integrity, and were ruptured within the macrophages.
Discussion
We have previously shown that AROM+ mice expressing human P450arom under the human ubiquitin C promoter display multiple abnormalities in the male urogenital region, such as decreased size of testes, cryptorchidism, and Leydig cell hyperplasia (31).
In the present study, the AROM+ males had an elevated amount of intratesticular E2, combined with reduced amount of T at the age of 15 months, similar to males we described previously at 5 months of age (36). Whereas there were no significant differences in LH levels between AROM+ and WT groups, the reduced intratesticular androgen concentrations indicate testicular failure independent of feedback inhibition through the hypothalamus-pituitary axis. In fact, estrogen has been shown to directly affect steroidogenesis in the rat testis via accumulation of estrogen-regulated protein (molecular weight 27,000) and consequential desensitization of 17-hydroxylase and 17–20 desmolase (37, 38). Likewise, recent studies with mouse Leydig cells isolated from estrogen sulfotransferase knockout mice showed that accumulation of estrogen leads to decreased expression of P450 17-hydroxylase (39). Whereas ER and AR were expressed in the AROM+ Leydig cells in all age groups, we hypothesize that E2 is the driving force behind the testicular phenotype and that mouse Leydig cells are a target of estrogen action. This hypothesis is supported by previous findings showing that estrogen excess stimulates Leydig cell hyperplasia in rodents and has been associated with cryptorchidism, testicular cancer, and impaired spermatogenesis (40).
It is well established that estradiol plays an important role in regulating gonadotrophin secretion in both male rodents and men. The studies have also demonstrated that, compared with LH, the circulating FSH concentration is more effectively reduced by estradiol (41, 42). Our study with significantly decreased FSH levels and unaltered LH levels in AROM+ males is in line with these previous observations. The data, furthermore, suggest that a constitutive exposure to increased estrogens effectively inhibits FSH secretion in male mice, also in the presence of severely suppressed spermatogenesis.
One of the most striking features of the AROM+ testes was the presence of a high number of yellow multinucleated giant cells in the interstitium. These cells were identified as macrophages by immunohistochemical analysis of a macrophage-specific marker (F4/80). Similar histological findings (i.e. the presence of multinucleated giant cells in the interstitium) have been reported in old mice with chronic estrogen stimulation caused by targeted disruption of the estrogen sulfotransferase gene (43). This together with the present data suggests a role for estrogen stimulus in the macrophage activation of the testis. Accordingly, the increased size and number of macrophages observed at 9 and 15 months of age were accompanied by an increase in E2 concentrations. Because of the high background staining with the antibody used, we could not conclude whether the macrophages in AROM+ testes expressed ER. However, previous studies have demonstrated the expression of ER (44, 45, 46), but not ER (46), in murine macrophages, giving a possibility for direct effect of estrogens on macrophages. Furthermore, Guo et al. (47) reported that E2 is able to induce the activation of RAW-fos13 macrophages (RAW 264.7 macrophage cell line stable transfected with c-fos promoter) via the nongenomic Ca2+ signaling pathway. However, the mechanisms behind the E2-associated increase in testicular macrophages still remain open. One such mechanism could be that E2-induced proliferation of Leydig cells stimulates macrophage proliferation and activation, and the activated macrophages control the number of Leydig cells. There is a connection between the numbers of Leydig cells and testicular macrophages. Previous studies have namely shown that Leydig cells regenerate more slowly if the testis is depleted of macrophages, and T production is decreased after macrophage depletion (48, 49). The fact that in rats the total number of testicular macrophages declines if Leydig cells are ablated by treatment with the specific Leydig cell cytotoxin ethane dimethane sulfonate (18) further supports our finding of a strong association between the numbers of Leydig cells and testicular macrophages.
Our results indicate signs of severe inflammation in the testis of aging AROM+ mice. The high expression of CD68 indicates the presence of recently arrived monocyte-macrophages (18). This, together with increased expression of the proinflammatory cytokine, TNF, demonstrates that the macrophages in AROM+ testis are immunoactivated. Testicular macrophages produce undetectable levels of TNF under nonimmune conditions (50), whereas the studies with an experimental model for EAO have proposed a role for TNF in the pathogenesis of the disease (22, 21). The main target of the immunological attack in EAO are the germ cells that undergo apoptosis and sloughing, probably through an activated Fas-Fas ligand system (51). However, we could not detect an increased rate of apoptosis by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling staining or caspase-3 immunohistochemistry in the seminiferous tubules of AROM+ testis at any ages studied (data not shown). Nevertheless, severe fibrosis and an increased number of mast cells, in particular in the older groups of transgenic mice, was observed, providing further evidence for chronic inflammation reactions. Based on the elongated form of most of the mast cells, it can be concluded that they are activated, i.e. releasing their secretory products into the surrounding interstitial space. Although the precise nature of the putative mast cell products is not known, interaction between mast cells and macrophages have been shown to lead directly or indirectly to proliferation of fibroblast and collagen formation (52, 53), a likely response in the AROM+ mice as well. Interestingly, very similar inflammation-related changes have been reported in the testes of infertile men. Interstitial fibrosis, together with increased number of activated, degranulated mast cells, secreting proteases and histamine (54), and CD68-positive macrophages expressing TNF and IL-1 (17), has been observed in the human testes in connection with infertility.
The connection among estrogens, orchitis, and infertility still remains unknown, although the interaction of estrogens and the immune system has recently been discussed extensively (55). The AROM+ transgenic mouse model is a novel tool for analysis of the role of estrogens in testicular function, and it provides a useful model to better understand the interaction between macrophages and Leydig cells in vivo. Taken together, our data suggest that the elevated testicular E2 in AROM+ males results in disrupted spermatogenesis, a marked increase in the number of immunoactive macrophages and Leydig cell hyperplasia. Furthermore, an interaction between macrophages and Leydig cells in the interstitium was shown. Further studies are needed to clarify the exact mechanisms causing the disruption of spermatogenesis associated with E2-induced chronic inflammation in the testes of AROM+ mice.
Acknowledgments
We thank Ms. Tarja Laiho; Ms. Hannele Rekola; Ms. Saija Savolainen, M.Sc.; Ms. Katja Suomi; and Ms. Johanna Lahtinen for technical assistance.
Footnotes
This work was supported by Center of Excellence Grants 211480 and 207028 from The Academy of Finland and Grant FOOD-CT-2004-506319 from the European Commission [CASCADE (Chemicals as Contaminants in the Food Chain, a Network of Excellence for Research, Risk Assessment, and Education)].
The authors have no conflict of interest.
First Published Online November 23, 2005
1 X.L. and L.S. contributed equally to this work.
Abbreviations: AR, Androgen receptor; AROM+, human P450 aromatase; E2, estradiol; EAO, experimental autoimmune orchitis; ER, estrogen receptor; P450arom, P450 aromatase; P450scc, P450 side chain cleavage; T, testosterone; WT, wild type.
Accepted for publication November 17, 2005.
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