Hyperactivation of Leydig Cells

REPRODUCTION-DEVELOPMENT

Male 41, XXY* Mice as a Model for Klinefelter Syndrome: Hyperactivation of Leydig Cells Joachim Wistuba,* C. Marc Luetjens,* Jan-Bernd Stukenborg, Andreas Poplinski, Steffi Werler, Matthias Dittmann, Oliver S. Damm, Tuula Ha¨ma¨la¨inen, Manuela Simoni, and Jo¨rg Gromoll Centre of Reproductive Medicine and Andrology (J.W., C.M.L., J.-B.S., A.P., S.W., M.D., O.S.D., M.S., J.G.), University Clinics, 48149 Muenster, Germany; and Institute of Biomedicine (T.H.), Department of Physiology, University of Turku, 20520 Turku, Finland

Sex chromosome imbalance in males is linked to a supernumerary X chromosome, a condition resulting in Klinefelter syndrome (KS; 47, XXY). KS patients suffer from infertility, hypergonadotropic hypogonadism, and cognitive impairments. Mechanisms of KS pathophysiology are poorly understood and require further exploration using animal models. Therefore, we phenotypically characterized 41, XXY* mice of different ages, evaluated observed germ cell loss, studied X-inactivation, and focused on the previously postulated impaired Leydig cell maturation and function as a possible cause of the underandrogenization seen in KS. Xist methylation analysis revealed normal X-chromosome inactivation similar to that seen in females. Germ cell loss was found to be complete and to occur during the peripubertal phase. Significantly elevated FSH and LH levels were persistent in 41, XXY* mice of different ages. Although Leydig cell hyperplasia was prominent, isolated XXY* Leydig cells showed a mature mRNA expression profile and a significantly higher transcriptional activity compared with controls. Stimulation of XXY* Leydig cells in vitro by human chorionic gonadotropin indicated a mature LH receptor whose maximal response exceeded that of control Leydig cells. The hyperactivity of Leydig cells seen in XXY* mice suggests that the changes in the endocrine milieu observed in KS is not due to impaired Leydig cell function. We suggest that the embedding of Leydig cells into the changed testicular environment in 41 XXY* males as such influences their endocrine function. (Endocrinology 151: 2898 –2910, 2010)

linefelter’s syndrome (KS, 47, XXY; prevalence 0.2% of the male population) results from meiotic I or II nondisjunction events during gametogenesis, which lead to a sex chromosomal imbalance, namely the presence of a supernumerary X chromosome (1–9). KS is characterized by hypogonadism, impaired androgenization, an increased morbidity/mortality rate, and cognitive impairments (3, 4, 10 –16). Symptoms of KS appear to be related to low androgen levels and disturbed Leydig cell steroidogenesis. The reduction in androgens could be caused by malfunctioning LH receptor signal transduction or a more general perturbation, which results in disorganized and/or incomplete Leydig cell maturation.

K

The corollary for spermatogenesis is that, besides a few exceptions in which sperm has been found in men with KS (17), most patients lose their germ cells before the onset of puberty (3). Beyond numerous investigations describing the phenotypical features of KS, experimental approaches addressing the pathophysiology have been limited due to the unavailability of suitable experimental models. Thus far, two mouse models resembling the KS karyotype have been published (5–7, 9, 18). Interestingly, both mouse lines were derived from the same genetic background (i.e. B6Ei.Lt-Y*) in which the Y chromosome lost its normal centromere by spontaneous mutation and acquired a new

ISSN Print 0013-7227 ISSN Online 1945-7170 Printed in U.S.A. Copyright © 2010 by The Endocrine Society doi: 10.1210/en.2009-1396 Received December 1, 2009. Accepted March 19, 2010. First Published Online April 28, 2010 * J.W. and C.M.L. contributed equally to this work.

Abbreviations: 1C, Postmeiotic haploid spermatids; 2C, diploid spermatogonia and somatic cells; 4C, spermatocytes in meiosis double-diploid cells; FISH, fluorescence in situ hybridization; HC, elongated spermatids with condensed DNA; hCG, human chorionic gonadotropin; 3␤-HSD, 3␤-hydroxysteroid dehydrogenase; ITT, intratesticular testosterone; KS, Klinefelter syndrome; LHR, LH receptor; PAS, periodic acid-Schiff; PI, propidium iodide; pp, postpartum.

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distal centromere, designated Y* (19). Breeding of these males with normal females has resulted in approximately one fourth of male pups possessing a 41, XXY* karyotype. Due to meiotic nondisjunction of the sex chromosomes, a substantial part of the Y chromosome is translocated and adjacent to one of the X chromosomes in these animals, resulting in a phenotype as if a completely separated Y chromosome were present. In our colony, the few males of the XYY*X karyotype were all found to be infertile and did not produce offspring. The benefit of both models is that they display the general symptoms associated with human KS such as the disturbed cognition, infertility, and hypergonadotropic hypogonadism (9). Using our 41, XXY* mice, we evaluated the impact of the second X chromosome through methylation analysis, followed the developmental time course of germ cell loss, and investigated the endocrine dynamics from puberty to aging. In particular, we focused on the characterization of Leydig cells at a molecular and cellular level to prove or disprove the hypothesis that Leydig cell malfunction is primary cause for the androgenization failure in KS.

Materials and Methods Animals Age-matched cohorts of juvenile [1, 3, and 10 d postpartum (pp)], pubertal (3 wk), adult (30 – 40 wk), and aged (80 –90 wk) male 41, XXY* mice, their 40, XY* littermates, and male C57BL/6 wild-type controls were obtained from our colonies. Karyotypes of each individual were determined by fluorescence in situ hybridization (FISH). Mice were housed at 24 C on a 12-h light, 12-h dark cycle and provided with food and tap-water ad libitum. As previously described (9), XXY* mice were generated by breeding male mice with a mutated Y chromosome (Y*) to wild-type females. All procedures and protocols were in accordance with the national and European (86/609/EEC) legislation for animal care and experiments (animal licenses no. A54/02, A87/05, and G24/06; RP Muenster).

Organ and tissue collection Animals were anesthetized with CO2 and killed by decapitation. Trunk blood was collected and serum was stored at ⫺20 C for later evaluation. Testes, epididymides, brains, pituitaries, and accessory sex glands were dissected and weight was recorded. Organs were either processed freshly for in vitro assays, fixed in Bouin’s fixative, or snap frozen in liquid nitrogen and subsequently stored at ⫺80 C.

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digested in 1 ml collagenase (Life Technologies, Inc., Invitrogen, Karlsruhe, Germany) to a single-cell suspension for 1.5 h and the cells dissolved in 1 ml Amniomax medium (Life Technologies). Five hundred microliters of the cells were transferred into two wells of a 12-well plate that already contained 500 ␮l pure medium and cultured at 37 C and 5% CO2 (5, 6). After 4 – 6 d, 50 ␮l Karyomax Colcemid/well were used as a blocking agent to arrest the fibroblasts in metaphase after digestion with tubes via trypsin; the cells were cracked with KCl and ethanol/glacial acetic acid and stored at ⫺20 C. The same FISH was then performed as in the interphase samples (9). Mice with a 41 XXY* karyotype were identified by the presence of a Y-chromosomal signal in close proximity to one of the X chromosomes, whereas XY* littermates and the C57BL/6 wild-type controls possessed two clearly distinct and separate signals (Fig. 1, A and B). No chromosomal mosaicisms were observed in any animals.

Hormone analysis Intratesticular testosterone (ITT) concentration was determined using a double-antibody RIA. In brief, frozen testicular tissue from XY* littermates (n ⫽ 9) and XXY* (n ⫽ 11) mice was homogenized by ultrasound in 250 ␮l PBS buffer before the addition of an iodinated tracer [testosterone-3-(O-carboxymethyl) oximino-2-[125I] iodohistamine; Amersham International, Germany] and a rabbit testosterone-3 (carboxymethyloxime)-BSA antiserum. The bound/free separation was performed by addition of a solution of solid-phase antirabbit immunoglobulins (immunobead second antibody; Bio-Rad, Munich, Germany). Recovery after ether extraction was monitored by addition of trace amounts (NET-370; NEN Life Science Products, Boston, MA). A second antibody consisting of rabbit ␥-globulin and antirabbit IgG immunoprecipitation reagent was then added. The recovery was monitored by addition of tracing amounts of [1␤, 2␤-3H] testosterone (NEN Life Science Products). The intraassay coefficient of variation was 6.0%. The total ITT content was calculated based on the weight of the testis recorded on removal. Duplicate serum samples from each animal, in all three experimental groups, were analyzed for serum testosterone levels according to the procedure of Chandolia et al. (22). The intraand interassay coefficients of variation were 4.3 and 5.6%, respectively, and the detection limit of the assay was 0.69 nmol/liter. FSH and LH levels were measured by an AutoDelfia immunoassay system [1235 AutoDelfia; LKB Wallac, Turku, Finland (23, 24)]. The intra- and interassay coefficients of variation were for FSH 4.3 and 10.4% at 4.8 ␮g/liter, respectively, and for LH 19% at 0.04 ␮g/liter and less than 5% at above 1 ␮g/liter and 12.5% at 0.24 ␮g/liter and 7.8% at 0.78 ␮g/liter, respectively. The FSH assay range was 0.04 –25 ng/ml and the LH assay range was 0.02–12.5 ng/ml.

Flow cytometric analysis of DNA content FISH The presence of sex chromosomes was determined by interphase FISH of venous blood samples (200 –300 ␮l) according to previously described protocols (9, 20, 21). Confirmation of the karyotype, and in cases in which the mice were too small to draw blood in sufficient amounts, fibroblast cultures were derived from ear or tail tip biopsies. The biopsies were minced mechanically and incubated in 1.25% trypsin for 30 min. The tissue was

Approximately 20 mg of frozen or fresh tissue were minced in a solution containing propidium iodide (PI; 1 mg/ml; Sigma Chemical Co., St. Louis, MO), ribonuclease (R 5000; type II-A 50 –100 K unit/mg; 0.1 mg/ml; Sigma), Triton X-100 (t-octophenoxypolethoxyethanol; Sigma), and PBS. Cells were released from the tissue by aspiration and then precipitated by centrifugation at 40 ⫻ g for 1 min. The supernatant was removed, the pellet resuspended in 0.5 ml staining solution, and homogenized

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(Krefeld, Germany) with a 15-mW argonion laser. Cells were recognized as elongated and round haploid spermatids [elongated spermatids with condensed DNA (HC) and postmeiotic haploid spermatids (1C)], diploid spermatogonia and somatic cells (2C), and tetraploid primary spermatocytes and G2 spermatogonia [spermatocytes in meiosis double-diploid cells (4C)]. Forward scatter and side scatter were used to remove interfering signals emanating from cell debris or aggregates. Sperm from adult mice were used to provide confirmation of the HC signal attribution. Red fluorescence signals of the PI staining were displayed on linear scale using a 620-band pass filter (605– 635 nm).

Histology Organs were placed in Bouins’ fixative solution for up to 24 h before being dehydrated, infiltrated, and embedded in paraffin. Multiple 5-␮m sections of each sample were obtained, stained with periodic acidSchiff (PAS)/hematoxylin, and examined using an Axiovert 200 microscope and Axiovision 3.1 software (Zeiss, Jena, Germany). The whole testis of immature animals was processed, serial sections obtained, and the section with greatest diameter selected for subsequent evaluation.

Stereology and optical dissection for analysis of Leydig cell hyperplasia Testes were removed and fixed in Bouin’s solution for up to 24 h before being washed in 70% ethanol then embedded in resin (Technovit 7100; Heraeus Kulzer, GmbH, Wehrheim, Germany). Twentymicrometer sections were cut and stained with PAS and hematoxylin. Leydig cells were evaluated in two slides each from two differing pieces to calculate the absolute Leydig cell number using the optical dissecY FIG. 1. A and B, FISH for the detection of a supernumerary X chromosome in XX * mice tor (25–28). Testicular tissue of four adult (A) and littermate control XY* (B). The red probe detects the X chromosome, the green animals per group was assessed with as probe the Y chromosome. Cell nuclei are counterstained with 4⬘,6⬘-diamino-2-phenylindole. FISH many fields as possible being evaluated per identification shows the close association of the Y* chromosome to one X chromosome volume by focusing through the section and in XXY* nuclei (A), which is absent in XY* control animals (B). C and D, Reproductive tract Y counting the Leydig cell nuclei in a defined of XX * mice (C) and their XY* littermates (D). All reproductive organs appeared normal area that appeared within planes at 4-␮m with the exception of the small and firm testes (circle) in XXY* mice. Efp, Epididymal fat intervals along the z-axis. Nuclei were pad; sv, seminal vesicle; b, bladder; e, epididymis; t, testis; p, prostate. E and F, Crosssections of the adult testis, PAS staining; histology: XY* mice (F) display normal and counted (using a Zeiss Axioskop, ⫻63 obcomplete spermatogenesis (E). Sertoli cell-only syndrome (SCO; lack of all germ cells) jective) once the nucleus and the cell margin can be found in the seminiferous tubules of XXY* mice. bv, Blood vessel; LC, Leydig cells. came simultaneously sharply into focus. For Bar, 50 ␮m. the systematic analysis of the samples, a starting point outside the tissue was chosen and then the volumes scored by random further to release cells containing higher quantities of condensed sampling using a motorized MCP4 U (Zeiss), which moved the DNA. The resultant mixture was added to the previously reslides in predetermined steps along the x- and y-axis. As a result, moved supernatant and placed in the dark for 30 min at room all areas were evaluated in proportion to their distribution temperature. Ploidy was determined based on the levels of PI throughout the testis. incorporation using a Beckman Coulter flow cytometer FC500

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Xist methylation analysis To determine the degree of X-inactivation, the methylation of the promoter region of Xist was analyzed (29 –31). Genomic DNA was extracted from blood samples using the FlexiGene kit (QIAGEN, Hilden, Germany), converted using a standard bisulfite protocol, desalted using DNA cleanup Wizard (Promega, Madison, WI), and finally desulfonated. PCR amplification of the Xist fragment was conducted using mouse forward primer 5⬘-GTT AGA TTT AAA GGT TTA ATA AGA TG and mouse reverse: 5⬘-AAA AC(AG) AAC AAA CAT AAC TAA AAC A. Cycling conditions were 2 min at 94 C (initial denaturation) followed by 40 cycles of denaturation (94 C, 25 sec), primer annealing (54 –50 C decrement 0.1 centigrade per cycle; 25 sec), elongation (72 C, 25 sec), and a final extension at 72 C for 10 min. PCR products were separated by agarose gel electrophoresis and the desired 133-bp fragment (⫺104 to ⫹29 relative to transcription start site; Eurofins MWG, Ebersberg, Germany) excised, purified using the High Pure PCR product purification kit (Roche, Basel, Switzerland), and then cloned. At least 10 clones from six male wild-type controls, six female and six male littermates, and 10 male XXY* mice were isolated and sequenced for the determination of methylation status.

Isolation, normalization of Leydig cell number, and analysis of Leydig cell status by 3␤-hydroxysteroid dehydrogenase (3␤-HSD) staining Testes were decapsulated, the tissue minced in ice-cold DMEM (Life Technologies,) and the cells separated by Percoll gradient centrifugation (21– 60% Percoll) (32, 33). Fractions containing Leydig cells were first washed and then resuspended in MEM before the Leydig cell number being counted in an improved Neubauer chamber. An aliquot of Leydig cells was removed and put on the glass slide and air dried for 15 min. Staining was performed using nitroblue tetrazolium (Sigma) as a substrate for 3␤-HSD and Hoechst for cell nuclei (34, 35). Numbers of positive 3␤-HSD Leydig cells and Hoechst staining-positive cells were counted and the percentage of Leydig cells calculated. Each experiment was performed twice with at least six animals per group.

In vitro Leydig cell stimulation assay Cells (104 isolated Leydig) were dissolved in 100 ␮l medium (MEM, 1% fetal calf serum, 0.5 g NaHCO3, and 2.98g HEPES; Life Technologies) and pipetted in a glass tube (type 2203113; Hilgenberg GmbH, Malsfeld, Germany). Several dilutions of human chorionic gonadotropin (hCG; 0.04 –100 ␮IU/␮l; Choragon, Ferring, Kiel, Germany) in MEM were prepared. One hundred microliters of the dilutions were added to the cell suspensions and incubated for 3 h at 37 C in a shaking water bath. The incubation was terminated by heating the samples to 100 C for 15 min. After inactivation, 500 ␮l MEM were added per glass tube, and samples were stored at 4 C until testosterone was measured in a sample volume of 100 ␮l (see above). All measurements were performed in triplicates. Two independent experiments were performed. The intra- and interassay coefficients of variation of the testosterone measurement were 8.47 ⫾ 1.53 and 11.47 ⫾ 4.58%, respectively.

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Analysis of marker gene expression in isolated Leydig cells RNA was extracted from isolated Leydig cells (1 ⫻ 105 cells/ preparation) by Ultraspec (AMS Biotechnology, Wiesbaden, Germany). Genomic DNA was removed with the DNA-free kit (Ambion, Austin, TX) and the purified RNA (1 ␮g per reverse transcriptase reaction) was transcribed using hexamer primers and reverse transcriptase SuperScript II (Life Technologies). Relative quantification was performed using TaqMan technology and the 2(⫺␦␦ cycle threshold) method according to Livak and Schmittgen (36). Primer and probe sequences including the fluorescent dyes are given in Supplemental Table 1, published on The Endocrine Society’s Journals Online web site at http:// endo.endojournals.org. Samples were normalized using 18s RNA as housekeeping gene, and samples without reverse transcriptase were used as internal negative controls. Two microliters of cDNA were used for each PCR with the following amplification protocol: 10 min at 50 C and 5 min at 95 C followed by 40 cycles of 15 sec at 95 C and 1 min at 60 C. Each RT-PCR was performed in triplicates. Data were analyzed using the ABI relative quantification software (Applied Biosystems, Foster City, CA). Primer and genes were chosen based on a study from O’Shaughnessy et al. (37), describing gene expression patterns of Leydig cells during mouse development. Specifically the following genes were selected based on the status of Leydig cell maturation: Tsp2 (thrombospondin 2, NM_011581)-fetal/neonatal expression; Est (estrogen sulfotransferase, S78182) adult Leydig cells; Lhr (LH receptor, NM_013582.2) fetal and adult Leydig cells, and Rlf (relaxin-like factor, NM_013564), fetal and adult Leydig cells.

Statistics Data were analyzed by one-way ANOVA (all pair wise, multiple comparison by Tukey test) or Student’s t test. X-chromosome methylation analysis was evaluated by ANOVA with Bonferroni posttest. Computations were performed using the statistical software package SigmaStat 2.03 (SPSS Inc., Chicago, IL). Values were considered statistically significantly different if P ⬍ 0.05.

Results Morphology of the reproductive tract and testicular histology of adult animals All glands and organs of the reproductive tract with the exception of the testes in adult and aged (80 –90 wk) XXY* animals were similar in morphological appearance and weight to those of the controls (Fig. 1B). The testes of mice with a supernumerary X chromosome were significantly reduced in size and weight (Tables 1 and Fig. 1, C and D) compared with controls. Testicular histology of the XY* and wild-type controls revealed complete spermatogenesis in all seminiferous tubules with the cells of the interstitium appearing mature and normally distributed. In contrast, all tubules of XXY* animals had smaller diameters, were devoid of germ cells, and contained Sertoli cells only. The appearance of the Sertoli cells and peritu-bular cells was

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TABLE 2. Intratesticular testosterone Adult

XXY*

XY*

Intratesticular testosterone (pmol/testis) pmol ITT in the testis/ ng LH in a ml serum

218.8 ⫾ 9.5 (n ⫽ 11) 119.5 ⫾ 45.7

244.3 ⫾ 20.6 (n ⫽ 9) 327.1 ⫾ 94.7

Values are mean ⫾ SEM. Measurements resulting in significant differences between XXYⴱ and the control groups are given in bold (ANOVA; P ⬍ 0.05).

morphologically normal (Fig. 1, E and F), whereas the number of Leydig cell was increased.

Values are mean ⫾ SEM. Measurements resulting in significant differences between XXYⴱ and the control groups are given in bold (ANOVA; P ⬍ 0.05).

11.6 ⫾ 3.8 (n ⫽ 12) 7.8 ⫾ 2.3 (n ⫽ 12) 1.5 ⫾ 1 (n ⫽ 3) 3.4 ⫾ 0.7 (n ⫽ 3)

8.9 ⫾ 1.9 (n ⫽ 29)

12.2 ⫾ 2.4 (n ⫽ 30)

16.1 ⫾ 4.3 (n ⫽ 20)

2.8 ⫾ 0.6* (n ⫽ 15)

XY (C57/BL6)

201 ⫾ 2.4 (n ⫽ 12)

XY*

18.8 ⫾ 0.9 (n ⫽ 18)

XX * XY (C57/BL6) XX * XY (C57/BL6)

61.8 ⫾ 8.5 (n ⫽ 3)

XY*

44.1 ⫾ 23.7 (n ⫽ 3)

XX *

Combined testis weight (mg) Testosterone (nmol/liter) serum

15.6 ⫾ 5.3 (n ⫽ 3) (vs. wild type) 4.6 ⫾ 1.4 (n ⫽ 3)

Y Y

Pubertal

28.2 ⫾ 0.9 (n ⫽

29)

XY*

205.2 ⫾ 5.7 (n ⫽ 20)

Y

Adult

168.6 ⫾ 3.8 (n ⫽ 30)

Aged

159.6 ⫾ 6.5 (n ⫽ 14)

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TABLE 1. Testis weight and serum testosterone

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Developmental time course of germ cell loss Chromosomal complement did not affect testicular weight during early development because no difference was found in testicular weights irrespective of age (i.e. d 1, 3, or 10 pp) or experimental group (data not shown). Similarly in the three time points examined, no difference was seen in testicular contents with all animals having germ cells up to the spermatogonial stage (Fig. 2). Flow cytometric evaluation of spermatogenesis in pubertal and adult XXY* animals (n ⫽ 3/group) revealed that by d 21 pp, the testes were devoid of 4C, 1C, and HC (Fig. 3). The absence of germ cells was confirmed by the histological examination of 3-wk-old animals and the significantly decreased testicular weight compared with wildtype controls (Table 1). Because the loss of germ cells occurred between 10 and 21 pp, it is unlikely that even the first spermatogenic wave at the onset of puberty was successfully concluded. X-chromosome inactivation Female mice were found to have a Xist mean methylation status of 55.14 ⫾ 2% (mean ⫾ SEM), which is consistent with the presence of one active and one inactivated X chromosome. In contrast, normal male mice and their littermate controls were found to have a high degree of Xist-methylation (XY*: 82.66 ⫾ 3.6%; XY: 82.93 ⫾ 5.3%), which is indicative of an inactivated Xist gene. XXY* males were found to possess a degree of Xist methylation (57.85 ⫾ 2.95%), statistically different from the male controls (P ⬍ 0.01; Fig. 4) but not different from the females and which is consistent with the correct inactivation of the supernumerary X-chromosome. Endocrine state of the XXY* mice LH and FSH serum levels were significantly (P ⬍ 0.05) elevated in both serum and the pituitary (LH) homogenates of adult XXY* animals when compared with controls (Table 1). Serum testosterone was reduced but not significantly different in adult XXY* mice (Table 1). In the

XY*

47.9 ⫾ 5.8 (n ⫽ 3) 43.4 ⫾ 8 (n ⫽ 4)

and the control groups are given in bold (ANOVA; P ⬍ 0.05). Values are mean ⫾ SEM. Measurements resulting in significant differences between XX

Y*

3 ⫾ 1.6 (n ⫽ 4)

0.47 ⫾ 0.1 (n ⫽ 19) 490.4 ⫾ 43.8 (n ⫽ 19) 49.63 ⫾ 1.9 (n ⫽ 19) 1.8 ⫾ 0.4 (n ⫽ 19) 897.9 ⫾ 67.1 (n ⫽ 19) 65.57 ⫾ 2 (n ⫽ 21)

0.75 ⫾ 0.2 (n ⫽ 18) 459.5 ⫾ 39.7 (n ⫽ 14) 51.44 ⫾ 6.5 (n ⫽ 14)

XY* XXY*

LH (ng/ml) serum LH (ng/pituitary) FSH (ng/ml) serum

TABLE 3. Gonadotropins

Adult

XY (C57/BL6)

XXY*

Aged

1.7 ⫾ 1.1 (n ⫽ 3)

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aged animal group, serum testosterone concentrations were significantly reduced in 41, XXY* mice when compared with their littermates, whereas LH and FSH levels were found to be no longer significantly changed between the XXY*males and their littermates (Table 3). No significant differences of serum testosterone were found between the groups at the onset of puberty when androgens of all karyotypes were still in a similar range (Table 1). Interestingly, ITT measurements of adult XXY* mice and their littermates revealed similar androgen levels, indicating that the much smaller testes of XXY* animals contained the same amount of testosterone as the littermate controls (Table 2). Nevertheless, a significant difference was found between XXY* males and their littermate controls when a ratio of ITT and serum LH levels was calculated. Testes of XY* controls produced 2.7-fold more ITT per nanogram serum LH compared with the testes of XXY* males under the same LH stimulation in vivo (Table 2). Determination of Leydig cell number No significant difference in the number of Leydig cells per microliter of testicular tissue was found between XY* (mean ⫾ SEM; 42,835 ⫾ 9,508) and XY (17,193 ⫾ 3,432) males. In contrast, in XXY* males, a large (947,792 ⫾ 59,938) and statistically significant (t test; P ⬍ 0.05) increase (Fig. 5A) equating to 32 times the number of Leydig cells found in the littermates and 57 times those in the wild-type controls was observed. A calculation of Leydig cells per organ revealed a significant 5.3-fold higher number of Leydig cells in XXY* mice when compared with their XY* littermates and an 8.2-fold higher number compared with XY C57BL/6 controls (Fig. 5A). Additionally, when the absolute Leydig cell numbers were calculated per picomole ITT produced per testis, the number of Leydig cells needed in XXY* per picomole testosterone was 4.3fold higher compared with littermate controls. In the aged 41, XXY* mice group, Leydig cell hyperplasia persisted, although the differences between the 41, XXY* males and the controls were smaller than during adulthood. Compared with the controls, the number of Leydig cells present in the XXY* testes increased by approximately 20-fold per volume unit and 2-fold per testis (per microliter: XXY* vs. XY* 24-fold; vs. XY 21-fold; per organ: XXY* vs. XY* 2.30-fold; vs. XY 2.04-fold, Fig. 5B). Leydig cell-specific marker expression Leydig cell preparations were adjusted among the groups to allow normalization of expression levels. Percentages of morphologically identified and the proportion of 3␤-HSD-positive Leydig cells were similar between the experimental groups (XY 77.09 ⫾ 10.8% of all cells in the

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Leydig cells from XXY* mice with a prominent increase of adult Leydig cell marker genes (Fig. 6). In vitro stimulation of Leydig cells All Leydig cells tested displayed a sigmoidal dose-response curve on stimulation with hCG with no difference in the EC50 values (Fig. 7A) among the groups (5.812 ⫾ 1.75 in XXY*, 5.014 ⫾ 0.247 in XY*, and 4.56 ⫾ 0.498 in XY males), consistent with the presence of mature LH receptor (LHR) responding normally. However, XXY* Leydig cells produced significantly more testosterone per dose of hCG compared with Leydig cells from either control group (Fig. 7B).

Discussion Although recognized for decades, KS is still a very poorly understood condition. Beyond the presence of a supernumerary X chromosome, little in-depth knowledge exists about the molecular and cellular mechanisms of the phenotypic, physiological, and biochemical characteristics of KS. Questions such as why the FIG. 2. Germ cell development from d 1–10 pp in XXY* mice determined by endocrine feedback is disturbed resulting in hyhistological analysis (cell counts): percentages of seminiferous tubules according pergonadotropic hypogonadism and why to the germ cell stage observed. Between d 1 pp and d 10 pp, no significant sperm can be recovered from some KS patients differences were found concerning the testicular germ cell content (XY, d 1, 3, and 10 pp, n ⫽ 2; XY*, d 1, 3, and 10 pp, n ⫽ 2; XXY*, d 1 pp (n ⫽ 4), d 3 and but not from all cannot be simply answered by 10 pp (n ⫽ 2). the presence of an additional X chromosome. Although the complexity of symptoms is suspected to be related to a general disturbance in preparation were morphologically identified as Leydig androgenization, we aimed at the examination of Leydig cells and 45.16 ⫾ 12.72% of those were steroidogenically Y Y active, XY* 57.42 ⫾ 9.63 and 76.73 ⫾ 8.56%, XX * cell maturation and function in XX * mice, the prime 38.46 ⫾ 23.15 and 58.98 ⫾ 22.79%, respectively; mean ⫾ source of androgens. In our colony we have not been able to produce XXY SD). Two independent experiments were performed and in each experiment Leydig cells from six animals per group offspring, although in other colonies, male mice with a were pooled. Data on relative expression were calibrated completely separated Y chromosome have been reported by setting the expression of Leydig cells from adult (5–7). In a previous publication (9), we could demonstrate Y C57BL/6 males as 1. The expression of all genes assessed that we achieved results in the 41, XX * model, which was found to be between 3- and 20-fold higher in Leydig were consistent with those obtained from the XXY mouse, cells from XXY* males than in the controls (Fig. 6). We accepted as a good model for KS (6, 7). Comparison with detected a slight but significant (P ⬍ 0.05) increase in Tsp2 the previously published X/X sxr mouse model in which mRNA, which is indicative of fetal/neonatal Leydig cells, only the sex-determining part of the Y chromosome is and a significant (P ⬍ 0.05) elevation in the expression of translocated, revealed some similarities such as low serum Lhr and Rlf mRNAs, both markers of fetal and adult Ley- testosterone and ITT and germ cell depleted testes (38, 39) dig cells. The largest increase in mRNA levels (20-fold) but also important differences such as elevated LH levels was detected for the expression of Est, a marker for adult and Leydig cell hyperplasia in the XXY* mice, both typical and mature Leydig cells only. Taken together these results features of KS. We concluded that the part of Y chromoindicated an overall increased gene expression pattern by some present in 41, XXY* mice, although closely attached

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FIG. 3. Flow cytometric analysis of germ cells in XXY* mice. Comparison of relative proportions of testicular cell fractions (means ⫾ SD) [HC represents counts of testicular elongated spermatozoa with condensed DNA, 1C the haploid (postmeiotic) cells, 2 C the diploid germ cells and somatic testicular cells, 4C the counts of double diploid cells (spermatocytes in meiosis)] revealed no ongoing spermatogenesis in pubertal and adult XXY* animals, indicating that germ cell loss occurred between d 10 pp (see Fig. 2) and the age of 3 wk in male XXY* mice.

to an X chromosome, is functionally comparable with a fully separated Y chromosome, making the 41, XXY* mice a suitable model for KS. As a prerequisite for the understanding of the genetic mechanism possibly underlying the pathophysiology, we analyzed X-inactivation, as indicated by aberrant methylation. Male XXY* mice have a Xist methylation pattern very similar to that of normal females with approximately 50% methylation compared with approximately 45% methylation respectively. A pattern consistent with a normal inactivation of the second X chromosome and in strong contrast to the highly methylated Xist (approximately 85%) seen in XY* and XY males. It is therefore safe to conclude that X chromosomal inactivation is correctly performed in XXY* mice and cannot therefore account for the observed KS phenotype. Interestingly, recent findings in the human mirror our results. We and others have found the epigenetic pattern of the X chromosome of Klinefelter patients to be extremely similar to that of women but different from 46, XX men (40, 41). However, in contrast to the female, in the male

environment, an elevated expression of certain X-linked genes escaping from the normal X-inactivation might contribute to the observed KS phenotype (42– 44). In KS patients it has been suggested that the expression of these escapees may contribute to the features of the syndrome (45); therefore, future studies should address the expression of theses genes in the available animal models. Our model closely resembles the dynamics of germ cell loss seen in humans with the diminition beginning at the onset of puberty (3). In the XXY* mice, germ cell loss was found to occur between d 10 and 21 pp (i.e. the peri- and pubertal phase). Previously, it has been suggested that the endocrine and testicular alterations occurring in the postpubertal Klinefelter patients might reflect precocious reproductive aging (46). Hypergonadotropic hypogonadism as well as increasing Leydig cell numbers characterizes Klinefelter patients, also being general symptoms of the aging male (47). Consequently, differences between chronologically aged men and older Klinefelter become smaller than during adulthood, and KS patients do not show age-related

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FIG. 4. Hypomethylation of Xist in XXY* mice. The CpG region of the Xist gene was analyzed in XXY* (n ⫽ 10) mice and compared with female (XX, n ⫽ 6), normal males (XY, n ⫽ 6), and the XY* littermates (XY* n ⫽ 6). The columns represent the mean degree of methylation calculated from six CpG sites. XXY* mice show a methylation pattern highly similar to female mice that therefore resemble closely the proper inactivation of the second X chromosome. Stars mark significant differences. ANOVA followed by a Bonferroni posttest (***, ⬍ 0.001).

changes but a very stable phenotype over the entire postpubertal life span. In our mouse model, we also analyzed aged animals to examine whether the phenotype is also

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stable over lifetime and whether the differences between the aged 41, XXY* males and old controls become smaller over time. Although serum testosterone was more reduced in aged XXY* males, the gonadotropin serum levels were not significantly different. Testicular histology confirmed that the Leydig cell hyperplasia, although still present in old males with a supernumerary X-chromosome, is less notable when compared with the aged matched controls. In summary, these observations indicate that the 41, XXY* mice also exhibit a stable phenotype after puberty that is maintained over the entire life span and therefore closely resemble KS in this regard. When measured at the onset of puberty, testosterone levels of 41, XXY* males were not different from the controls, although testicular weight was already smaller and germ cells were lacking. The fact that during puberty the endocrine response is still similar suggests that the hypogonadism occurs secondary or independently from the germ line failure in the 41, XXY* males. It is at the onset of puberty that spermatogonia enter meiosis in normal mice; however, due to an unknown mechanism, all germ cells in XXY* mice become apoptotic and the stem cell pool decreases and is finally lost during this time. This process is brought to completion because in

FIG. 5. A, Leydig cell hyperplasia in adult XXY* animals. Left, Leydig cell number per volume unit: the number of Leydig cells per volume unit was almost 32-fold increased in male mice of the karyotype XXY* compared with littermates and 57-fold increased compared with wild-type controls. Right, Leydig cell number per organ: although the testis volumes of XXY* mice were reduced compared with controls, the hyperplasia was still about 8-fold. Leydig cell numbers found in the XY* and the XY controls are in agreement with published reports (52, 53). B, Leydig cell hyperplasia in aged XXY* animals. The hyperplasia persisted in the aged males, although the differences became smaller when compared with those occurring between the adult animals. Left, Leydig cell number per volume unit: an approximately 20-fold higher number of Leydig cells is present in the XXY* testes. Right, Leydig cell number per organ: there is double the amount of Leydig cells compared with the control animals.

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pected Leydig cell impairment was absent. The analysis of ITT values revealed androgen biosynthesis comparable with that of fertile littermates. Although smaller, testes of XXY* males are able to produce sufficient amounts of testosterone in vivo to maintain normal androgenization in androgen-dependent organs such as the accessory sex glands and the epididymis. However, to obtain these androgen levels, elevated LH levels and a higher number of Leydig cells per unit volume than those in the normal testis are needed (Fig. 3). The concept of disturbed regulation is further strengthened by FIG. 6. mRNA expression profile of marker genes from isolated Leydig cells. the finding that the number of Leydig cells Expression of genes specific for Leydig cell development was analyzed by relative ⫺⌬⌬Ct needed in XXY* per picomole testosterone was quantification (RQ) using the 2 method. Data were calibrated by setting the expression of adult XY Leydig cells (controls) as 1. Tsp2, Thromospondin-2, Est, 4.3-fold higher compared with XY* littermate estrogen sulfotransferase, Rlf, Relaxin like factor, Lhr, LH receptor. Expression is controls. significantly higher (ANOVA, P ⬍ 0.05, error bars, SEM) in Leydig cells from 41, Y However, our studies on isolated Leydig XX * males compared with Leydig cells from their XY* littermates. cells show that the situation is not as clear-cut as the previous results may indicate. In contrast all XXY* testes studied, not a single germ cell beyond to what is expected, expression analysis of Leydig cells meiotic phase 1 or 2 could be detected. This developmental isolated from XXY* mice show Est to be present at high failure of the germ line is most probably caused by the inability to handle the second X-chromosome during mei- levels, indicating that the cells are mature adult type. Adosis. Similarly, in the female chromosomal aberration dis- ditionally, Est and LHR mRNA were higher than in Leyorder of 45, X0 (Turner syndrome), germ cells are also lost dig Ycells obtained from the XY* littermates, showing at the same stage (48), implying that meiosis is generally XX * Leydig cells to be functionally hyperactive. Furalthough EC50 values were similar among prone to failure when the number of sex chromosomes is thermore, Y XX * animals and their XY* littermates with regard to changed. In a very recent study, Lue et al. (7) demonstrated that the dose response to hCG, the testosterone production of Y transplantation of euploid germ cells into germ cell-deficient XX * isolated cells significantly exceeded that of control Y XXY mice could restore spermatogenesis, indicating that the Leydig cells. Thus, Leydig cells of XX * mice are also somatic milieu of the testis is capable of supporting germ cell hypersensitive to stimulation. Taken together, Leydig cells Y development. This is consistent with another study in which in XX * mice appear to be normally matured, which are Klinefelter patients who possess seminiferous tubules con- able to express a functional LHR, and are hypersensitive taining focal spermatogenic activity had euploid germ cells to external stimuli such as hCG, which is probably medithat were capable of undergoing meiosis. Patients could ated by the elevated Lhr mRNA expression (Fig. 6). As a therefore produce sperm that could in turn be used for as- consequence the contention that there is a maturation or Y sisted reproduction techniques. The origin of these euploid functional defect in XX * Leydig cells seems unlikely. Y cells is most likely due to the random expulsion of the su- Given that in our XX * mouse model the ability of the pernumerary X chromosome during the mitotic activity of Leydig cells to function is not intrinsically impaired, it is more feasible that other external factors present in their the early spermatogonia (49). Our study focused on the characterization of Leydig testicular environment are responsible for the disturbed cell function because of their essential role in normal an- LH action (i.e. the feed-forward regulation). Currently, we favor a model in which the transportadrogenization and the completion of spermatogenesis [for review see Wistuba et al. (50)]. We postulated that an tion of LH via the blood supply to its endocrine target cells association with abnormal function or maturation of the is hampered. In such a scenario, the blood vessel architecLeydig cells was the most plausible cause for the hyper- ture could be changed by a reduction of diameter, the lack gonadotropic hypogonadism seen in our XXY* mice, an of vessels, or the hindrance of LH transportation into assumption that was driven by the reduced Leydig cell and/or androgen transport out of the testis. Interestingly, function reported in KS patients and the observed Leydig a close relationship between Leydig cells and the microcell hyperplasia. We were therefore surprised to see that in vasculature has previously been demonstrated, providing XXY* mice when cell numbers were normalized, the ex- some support for this hypothesis (48, 49). A possible al-

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cell depleted tissue that can be overcome by an additional increase of gonadotropin concentrations or hormone differences. Because Sertoli cell function has not been studied in detail in males with a supernumerary X chromosome, one could speculate that Sertoli cell function might also be changed with respect to the production of androgen binding protein or other Sertoli cell-specific products. Independent of the cause for the hypogonadism observed in our XXY* mice, the fact that Leydig cells in these animals are apparently normal has implications for our understanding of the KS pathophysiology and might influence the development of future treatment designs for KS patients. Our mouse model offers new avenues to understand the molecular and cellular mechanisms underlying KS. We have shown that the inactivation of the supernumerary X chromosome is normal, thereby excluding major epigenetic alterations as a possible cause and that the observed hypergonadotropic hypogonadism is not due to impaired Leydig cell function. On the contrary, a hyperactive adult Leydig cell type is present in XXY* mice, which indicates that the interaction between the hormones and their endocrine target cells is disturbed, not the cell functions as such. FIG. 7. In vitro testosterone production in response to increasing doses of hCG of isolated Leydig cells. A, Cultured Leydig cells displayed a sigmoidal dose-response curve on stimulation and EC50 values did not differ, indicating a mature LHR. B, The XXY* Leydig cells produced significantly more testosterone when compared with Leydig cells from their littermates and C57BL/6 wild-type controls. Values are means from duplicate experiments. Error bars, SEM. Stars mark significant higher testosterone amounts produced by XXY* Leydig cells compared with the controls under identical stimulation (t test, P ⬍ 0.05).

ternative cause could be that the blood supply in the testes of XXY* males is altered due to the lack of germ cells, thereby leading to packed and condensed organ architecture, meaning that the endocrine phenotype might have occurred secondary to or independently of the germ cell loss. Interestingly, clinical studies in which hCG was administered to KS patients (51) resulted in a testosterone increase, which has been explained by an steroidogenic reserve of the Leydig cells in the Klinefelter testis but might be reinterpreted as being due to insufficient LH concentrations because of the structural conditions in the germ

Acknowledgments

We thank Martin Heuermann and Gu¨nter Stelke for animal care taking. The authors are indebted to Reinhild Sandhowe-Klaverkamp, Nicole Terwort, Petra Ko¨ckemann, and Jutta Salzig for excellent technical assistance and Dr. Con Mallidis for language editing. We thank Professor Eberhard Nieschlag (Centre of Reproductive Medicine and Andrology) for the support of this work and fruitful discussions. Address all correspondence and requests for reprints to: Dr. Joachim Wistuba, Centre of Reproductive Medicine and Andrology, University Clinics, Domagkstrasse 11, 48149 Muenster, Germany. E-mail: [email protected]. This work was supported by Deutsche Forschungsgemeinschaft, DFG Grant WI 27-23/1-1 and Grant IMF LU 1 2 03 05 from the Medical Faculty Muenster.

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Present address for C.M.L.: Covance Laboratories, Kesselfeld 29, 48155 Muenster, Germany. Present address for J.-B.S.: Pediatric Endocrinology Unit, Department of Women’s and Children’s Health, Astrid Lindgren Children’s Hospital, Karolinska Institutet, Stockholm, Sweden. Present address for M.S.: Chair of Endocrinology, University of Modena and Reggio Emilia, Department of Medicine, Endocrinology, Metabolism, and Geriatrics, Via Giardini 1355, I-41126 Modena, Italy. Disclosure Summary: The authors have nothing to disclose.

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