XXY Mice Exhibit Gonadal and Behavioral Phenotypes Similar to Klinefelter Syndrome
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《内分泌学杂志》
Division of Endocrinology (Y.L., C.W., A.P.S.H., W.S., R.S.S.), Department of Medicine, Harbor-UCLA Medical Center and Los Angeles Biomedical Research Institute, Torrance, California 90509
Departments of Psychology (J.D.J.), Pathology and Laboratory Medicine (P.N.R.), University of California at Los Angeles, Los Angeles, California 90095
Abstract
Klinefelter syndrome (XXY males) is the most common sex chromosome aneuploidy. XXY mice were generated by using a four-generation breeding scheme that involves the use of a structurally rearranged Y chromosome, Y*, yielding approximately 50% of the live-born male offspring in the fourth generation with a XXY karyotype. Adult XXY mice have small testes, decreased plasma T levels, and elevated plasma FSH levels. The testes of adult XXY mice contained small seminiferous tubules with intraepithelial vacuolization and absence of germ cells, whereas Leydig cells appeared to be more abundant than their XY littermates. Androgen receptor immunoexpression was localized in Leydig cells and peritubular myoid cells in both XY and XXY mice. Androgen receptor immunoexpression was abundant in the Sertoli cells of XY mice but nearly absent in those of XXY mice. The testicular phenotype was marked by a 23.1% decrease in testis weights in XXY pups beginning at d 7 after birth. Gonocyte numbers were similar in XY and XXY mice at d 1 of age, followed by a 62.6% decrease in the number of gonocytes in the XXY mice on d 3 and further progressive loss in spermatogonia by d 5 and 7. On d 10, only a few spermatogonia remained in the XXY mice. To determine whether the phenotype of XXY mice extended into the neurobehavioral domain, studies were conducted demonstrating impairment of learning and memory function in XXY mice. We conclude that adult XXY mice have testicular failure and learning deficits, similar to its human counterpart, Klinefelter syndrome.
Introduction
KLINEFELTER SYNDROME (KS) is the most common sex chromosome aneuploidy, occurring in about 1.2 per 1000 live-born male births (1). Adult KS males are characterized by infertility, small testes, azoospermia, gynecomastia, elevated gonadotropin levels, and serum testosterone concentrations usually below or in the low adult male range. Many KS patients have learning disabilities and impairments of cognitive and executive functions (2, 3, 4, 5). Testicular biopsies from most adult patients demonstrate complete absence of germ cells, seminiferous tubule hyalinization, and fibrosis, whereas Leydig cells show hyperplasia (6). In contrast, testicular histology obtained from infant boys with KS appears to be nearly normal, with primary germ cells present (7, 8). Although the molecular mechanisms causing the KS phenotype are often ascribed to a gene dosage effect from the extra X chromosome, the specific genes responsible for germ cell loss in the testis and neurobehavioral deficits of KS patients remain unknown.
In prior studies, we characterized XXY mice produced by mating wild-type males with chimeric females carrying male embryonic stem cells as a model for KS (9, 10). We found that the testicular histology of adult XXY mice showed small seminiferous tubules with varying degree of intraepithelial vacuolization, complete absence of germ cells, nests of apparently degenerating Sertoli cells, and hypertrophy and hyperplasia of Leydig cells. Similar to men, spermatogonia were present in juvenile XXY mice, but progressive loss of germ cells occurred within 10 d after birth (10). In this study, we generated XXY mice by using a more efficient four-generation breeding scheme described by Hunt and colleagues (11, 12) involving the use of a structurally rearranged Y chromosome, Y*. Approximately 50% of the live-born male offspring in the fourth generation were XXY mice. In contrast to the Bronson model (9, 10), the Hunt model (11, 12) is self-replenishing and allows for a more economical creation of a colony for biological and psychological studies.
In earlier studies, the Hunt group reported that normal number of germ cells arrived at the genital ridges in the XXY embryo, but the mitotic proliferation of XXY germ cells became impaired as the testis differentiated (12). They believed that the impaired proliferation of XXY germ cells was caused by the reactivation of one of the two X chromosomes in XXY germ cells occurring when germ cells reached the genital ridge (13). Our objectives were: 1) to produce an adequate number of XXY mice by using Hunt’s breeding scheme (11, 12); 2) to characterize the testicular phenotype of adult XXY mice; 3) to examine the ontogeny of massive germ cell loss in neonatal XXY mice; and 4) to determine learning capability in adult XXY mice. Our goal was to demonstrate and establish the XXY mouse as an experimental murine model for its human counterpart, patients with Klinefelter syndrome.
Materials and Methods
Animals
Breeding pairs of XY* male and XX female were purchased from The Jackson Laboratory (Bar Harbor, ME). The animal breeding colony was established and housed in a standard animal facility under controlled temperature (22 C) and photoperiod (12-h light, 12-h darkness) with free access to water and mouse chow. XXY mice (41, XXY) and their littermate XY (40, XY) mice from the fourth generation produced from our breeding colony were used in this study. Seven adult XXY mice (8 wk old) and 7 littermate controls (XY) were used to characterize the testicular phenotype of adult XXY mice. Thirty-two juvenile XXY mice and an equivalent number of XY littermates (1 d old; n = 5; 3 d old; n = 5; 5 d old; n = 4; 7 d old; n = 5; 10 d old; n = 5; 14 d old; n = 4; and 20 d old; n = 4) were used to examine the onset of germ cell loss in XXY mice. Using a Pavlovian appetitive approach procedure (14, 15) that measures stimulus-reward learning, we determined the learning ability of seven adult XXY and seven XY littermate mice (4–12 months old at the time of testing). Animal breeding, handling and experimentation were in accordance with the recommendation of the American Veterinary Medical Association and were approved by the Harbor-University of California-Los Angeles Biomedical Research Institute Animal Care and Use Review Committee.
Sample collection
Adult XXY mice and their littermate controls were injected with heparin [130 IU per 100 g body weight (BW), ip] 15 min before being killed by lethal injections of sodium pentobarbital (100 mg/kg BW, ip) to facilitate testicular perfusion using a whole body perfusion technique (10, 16). Body and testis weights were recorded at autopsy. Blood samples were collected from the inferior vena cava of each animal immediately after death, and plasma was separated and stored at –20 C for subsequent hormone assays. The testes were then fixed by vascular perfusion with Bouin’s solution (Sigma Co., St. Louis, MO) for 30 min, preceded by a brief saline wash. The testes were removed and placed into the same fixative overnight. One slice from the middle region of the testis was processed for routine paraffin embedding for testicular histology and immunohistochemistry detecting androgen receptor expression and localization in the testis. All the male juvenile mice in the fourth generation were killed by lethal injections of sodium pentobarbital (100 mg/kg BW, ip) on d 1, 3, 5, 7, 10, 14, and 20 after birth. The testes were dissected out and immersed overnight in Bouin’s solution. The testes were then processed for routine paraffin embedding and sectioning for histologic examination and immunohistochemistry.
Fibroblast culture and karyotype analysis
Standard karyotyping was performed on cultured fibroblasts obtained from ear-clips in adult mice or pieces of skin in juvenile mice. Briefly, a 1–2 mm2 section of tissue was dissected from a piece of ear in a sterile manner. The sample was minced with a dissecting scissors and digested with 1.25% trypsin (Gibco, Invitrogen Co., New York, NY) for 30 min, followed by collagenase digestion (Gibco Invitrogen) for an hour and half at 37 C. The dispersed cells were suspended in Amino-max-II medium (Gibco, Invitrogen). Aminomax supports the growth of anchorage-dependent fibroblast cells. The cells were placed in flasks and cultured for 5–7 d at 37 C in a CO2 incubator. Once appropriate colony formation was observed, KaryoMAX Colcemid solution (Gibco, Invitrogen) was added into flask to cease the mitotic division. The cultured fibroblasts were harvested after a minor digestion with trypsin-EDTA solution (Gibco, Invitrogen). The harvested cells were suspended in 0.075 M potassium chloride solution (Gibco, Invitrogen) and incubated in water bath (37 C) for 20 min, fixed in the mixture of methanol and acetic acid (3:1 methano-acetic acid), spread on clean glass slides, and air dried for fluorescence in situ hybridization (FISH) analysis.
FISH
X and Y chromosome-specific whole chromosome painting was performed on metaphases derived from the cultured fibroblasts (10, 17). After following the standard cytogenetic techniques of harvesting and slide making, the slides were aged at room temperature for 2–3 d. FISH with STAR*FISH mouse whole chromosome-specific X- and Y-chromosome painting (Open Biosystems, Huntsville, AL) was performed following the protocol provided by the manufacturer. After dehydration by serial ethanol washing and air drying, the slides were denatured by incubation in 70% formamide/2x saline sodium citrate at 65 C for 2 min. About 15 μl of denatured X-paint probe directly labeled with fluorescein isothiocyanate and Y-paint probe labeled with cyanine 3 were applied to the slides and hybridized overnight at 37 C in a humidified chamber. After washing slides in 50% formamide/1x saline sodium citrate at 45 C for 5 min, the slides were dried, the chromosomes were stained with 4',6'-diamino-2-phenylindole and the green signal from the X-paint probe and red signal from Y-paint probe were visualized under a fluorescence microscope (Carl Zeiss, Melville, NY) equipped with appropriate filters and images captured with an FISH analysis system (Applied Imaging Corp., San Jose, CA).
Hormone assays
Testosterone concentrations in plasma were measured by RIA, as reported previously (18, 19). The minimal detection limit in the assay was 0.25 ng/ml. The intra- and interassay coefficients of variations were 8 and 11%, respectively. Plasma LH levels were measured by modified immunofluorometric assay as described previously (19). The minimal detection limit of the assay was 0.02 ng/ml. The intra- and interassay coefficients of variation were 6.9 and 12.3%, respectively. Plasma FSH levels were measured by immunofluorometric assay (19) using reagents supplied by Organon (Os, The Netherlands). The minimal detection limit in the assay was 0.04 ng/ml. The intra- and interassay coefficients of variations were 4.7 and 6.0%, respectively.
Immunohistochemical analysis
Bouin’s-fixed, paraffin-embedded testicular sections were deparaffinized, hydrated by successive series of ethanol, rinsed in distilled water, and then incubated in 2% H2O2 to quench endogenous peroxidases. Sections were blocked with 5% normal goat serum for 20 min to suppress the nonspecific binding of IgG and subsequently incubated with a 1:100 dilution of affinity-purified rabbit polyclonal androgen receptor (AR) antibody (1:100 AR; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) (20). Immunoreactivity was detected using biotinylated goat antirabbit IgG secondary antibody followed by avidin-biotinylated horseradish peroxidase complex visualized with diaminobenzidine tetrahydrochloride as per the manufacturer’s instructions (rabbit united immunohistochemistry detection system; Oncogene, Boston, MA). Slides were counterstained with hematoxylin and reviewed with the Zeiss microscope (Carl Zeiss) (19, 21).
Morphometric assessment of germ cells in juvenile mice
The method used for germ cell quantitation was similar to that described previously (10). In brief, testicular sections were examined with an American Optical Microscope (Buffalo, NY) with a x100 objective and a x10 eyepiece. A square grid fitted within the eyepiece provided a reference area of 10,000 μm2. Germ cells within the frame of grid were counted. To correct for shrinkage (if any) in the reference area, the diameters of 10 randomly selected transverse sections of the seminiferous tubules were measured from each of XY and XXY animals across the minor axes of their cross-sectioned profiles. There was no difference in the tubular diameter between XY and XXY mice within 10 d after birth.
Pavlovian appetitive approach procedure
The acquisition of a Pavlovian tone-food association was assessed in groups of seven adult (4–12 months old) XXY and XY littermates. Standard aluminum and Plexiglas operant chambers with a photocell-equipped pellet delivery magazine on one side and a curved panel with five photocell-equipped apertures on the opposite side (Med Associates, Mount Vernon, VT) were used. The boxes were housed inside of a sound-attenuating cubicle; background white noise was broadcast, and the environment was illuminated with a house light (a light diffuser that was located outside of the operant chamber but within the cubicle). Before training, the mice were food restricted to 90% of their free-feeding weights over a 3-d period. Subsequently, hungry mice were trained in four daily sessions in which 20-kHz tones (20 sec in duration) were presented on a variable time 20-sec schedule; a total of 10 tones were presented each day. During the period of each tone, food pellets (20 mg Bioserv dustless precision pellets) were dropped into the infra-red beam equipped magazine on a random time 5-sec schedule. Total duration of head pokes into the magazine during the conditioned stimulus (CS) interval, as well as latency to approach the magazine after CS onset, were measured (14, 15).
Statistical analysis
Statistical analyses were performed using the SigmaStat 2.0 Program (Jandel Corp., San Rafael, CA). Data were assessed by Student’s t test or one-way repeated-measures ANOVA followed by Student-Newman-Keuls method test or two-way ANOVA followed by Student-Newman-Keuls method test. Differences were considered significant if P < 0.05.
Results
In the first generation, XY* males were mated to normal XX females to produce XY*X females. In the second generation, XY*X females were mated to normal XY males to produce XYY*X males. In the third generation, XYY*X males were mated to normal XX females to produce XXY male mice. In the fourth generation, over 50% of male offspring were XXY mice. Adult XXY mice were analyzed by G-banded karyotyping from metaphase cells using cultured fibroblasts and further confirmed by X and Y chromosome paints using the FISH technique (Fig. 1).
Body weight, testis weight, and plasma testosterone levels
The comparisons of body weight, testis weight, and hormone levels between adult XY and XXY mice are shown in Table 1. No significant difference was observed in the body weight. The mean weight of the testis in XXY adult mice was reduced to 27.45% of their littermate XY mice. The mean level of plasma testosterone in adult XXY mice was significantly decreased, compared with XY mice (P < 0.05), whereas mean plasma FSH level was significantly elevated in adult XXY mice (P < 0.05). Whereas there was a trend toward higher plasma LH level in the XXY mice, the difference from XY mice was not significant.
Testicular histological examination
Testicular histological examination showed that seminiferous tubules of XY mice (Fig. 2A) displayed active spermatogenesis with normal appearing Sertoli cells and various classes of germ cells. In contrast, the testicular histologic examination of adult XXY mice (Fig. 2B) showed small seminiferous tubules, intraepithelial vacuolization, degenerating Sertoli cells, and Leydig cells that appeared to be more abundant in XXY compared with XY littermates. Interestingly, in one XXY mouse, we found a few seminiferous tubules containing zygotene and pachytene spermatocytes but few if any round spermatids.
Testicular histology and morphometric observations in juvenile XXY mice
All the male pups produced in the fourth generation were studied at different age groups (d 1, 3, 5, 7, and 10) without prior knowledge of their karyotype. Karyotyping was performed later using cultured fibroblasts from a piece of skin in mice under 10 d old and a piece of ear loop in mice older than 14 d old. The XXY mice identified by karyotyping were further confirmed by FISH analysis using mouse X and Y chromosome-specific paint probes. The mean testis weight was significantly lower in the XXY pups, compared with XY pups, beginning at d 7 after birth (Fig. 3). The number of gonocytes was similar in XY and XXY mice at d 1 of age. As early as d 3, a significant decrease in the number of gonocytes in the XXY pups was observed, which was followed by progressive loss in the number of spermatogonia on d 5 and 7. By d 10, when meiosis initiation should have occurred in XY mice, only a few spermatogonia remained in the XXY mice (Fig. 4). Leydig cells appeared to be normal in appearance both in XY and XXY juvenile mice.
AR immunoexpression in XXY mice
Using immunohistochemistry in Bouin’s fixed testicular sections, AR expression was present in Leydig cells and peritubular myoid cells in both XXY and their littermate XY mice (2–3 months, n = 4). Abundant AR expression was observed in the Sertoli cells of XY mice (Fig. 2A) but was nearly absent in Sertoli cells in adult XXY mice (Fig. 2B). The loss of AR expression also was demonstrated in the Sertoli cells of the single XXY mouse that had a few seminiferous tubules containing pachytene spermatocytes and a few spermatids. Whereas AR was present in the Leydig cells of both XXY and XY mice, their intracellular localization was different. ARs were localized in cytoplasm of Leydig cells in XXY mice in contrast to their localization in the nuclei of XY mice. To determine the onset of the loss AR expression in Sertoli cells of XXY mice, we examined expression of AR in the testicular sections from groups of four XXY and their XY siblings at d 1, 3, 5, 7, 10, 14, and 20 after birth. AR was expressed in Leydig cells and peritubular myoid cells in all age groups in both XY and XXY mice. In contrast, AR was not expressed in Sertoli cells in either XY or XXY mice until d 10 after birth and became more evident in both XY and XXY mice at 14 and 20 d of age. AR remained in the Sertoli cells of adult XY mice. The ontogeny of the AR in the Sertoli cells of XXY mice was decided different from that of the XY mice. There was sporadic loss of AR expression in the Sertoli cells of the XXY mice beginning at age 20 d; further loss of AR after age 20 d resulted in their almost complete absence of AR in the Sertoli cells of adult XXY mice. AR was also expressed in gonocytes located at the center of the seminiferous cords of 1-d-old mice (Fig. 5, A and B) and in the few gonocytes in 3-d-old XY and XXY mice. Loss of AR expression in gonocytes in both XY and XXY mice was coincident with gonocytes migration to the basal lamina.
Pavlovian conditioning in XXY and XY mice
Behavioral studies were conducted to evaluate the hypothesis that XXY mice would exhibit learning deficits reminiscent of the neurobehavioral phenotypes exhibited by KS patients. Over the course of training, the latency to approach the magazine after CS onset decreased (Fig. 6). A main effect of day (F(3, 36) = 5.2, P < 0.005) was driven by a progressive reduction in magazine approach latency as a function of repeated training. A training day * genotype interaction [F(3, 36) = 4.9, P < 0.01] indicated that XXY mice exhibited longer approach latencies on d 1 and 2 of training but that the group difference disappeared by d 4. Therefore, the rate of learning was impaired in XXY mice, even though they were equally able to perform the task (once learning had occurred).
Discussion
Males with two X chromosomes in mammals are sterile in nearly all species as a result of degeneration of the germ cells before the onset of meiosis (22). The incidence of naturally occurring live 41, XXY mice is estimated at 0.02% (23). There are two approaches to generate XXY mouse in the laboratory. In the first approach (chimeric model), female chimeras were generated from the injection of male embryonic stem cells into the blastocoele of female embryos (9). About half of the embryonic stem cell-derived male offspring of these chimeric females exhibited nonmosaic aneuploidy, with XXY being the predominant karyotype. We used this approach to generate XXY mice and studied testicular histology in adult and juvenile mice. We found that the phenotype of adult XXY mice was very similar to that of KS patients (10). In the second approach, we used a four-generation breeding scheme (XY* model) established by Hunt et al. (11, 12) that involved a structurally rearranged Y chromosome to produce XXY mice. We demonstrated that over 50% of male offspring in the fourth generation were XXY mice. We confirmed that this breeding scheme of XY* model was more efficient to produce XXY mice in the laboratory. We provided additional evidence showing that adult XXY mice have small testes, decreased serum testosterone levels, and elevated serum FSH levels. Testicular histology of adult XXY mice exhibited small seminiferous tubules with varying degree of intraepithelial vacuolization, degenerating Sertoli cells, and complete absence of germ cells. Leydig cells appeared to be more abundant in the interstitium. Thus, we demonstrated a similar testicular phenotype in adult XXY mice produced by either chimeric model or XY* model.
In the prior model using chimeric mice (10), plasma testosterone was not significantly lower in the XXY mice, compared with XY mice. In this study with a larger number of XXY and XY mice and more careful handling of the mice to reduce stress, mean plasma testosterone levels were significantly lower in the XXY mice. This was accompanied by elevated plasma FSH levels indicating significant seminiferous epithelium damage. Another discrepancy we observed between these two XXY mice models was the age that the germ cell loss occurred. In XXY mice produced by the chimeric model, we observed no significant differences in germ cell numbers between XXY and XY littermates until d 7 after birth. In XXY mice generated by the XY* model, we observed a significant decrease in the number of gonocytes beginning at d 3 after birth. The discrepancy may be attributed to the differences in the origin of the extra X chromosome (paternal in XY* model and maternal in chimeric model) and/or the genetic background of the animals. It is worth noting that in XXY mice generated by both schemes, massive germ cell loss occurred within 10 d after birth and before the initiation of meiosis. In addition, in contrast to the Hunt report (12) that described reduced gonocyte numbers at d 1 of age, we found no difference in the number of gonocytes in XY and XXY pups at 1 d after birth. The reason for the discrepancy is not clear. We did not study prenatal mice.
The mechanisms of germ cell loss in the XXY testis remain to be defined. It has been demonstrated that prenatal mitotic proliferation of germ cells was impaired in the XXY testis (12). The impaired proliferation of XXY germ cells resulted from reactivation of one of a pair of X chromosomes as germ cells reached the genital ridge, irrespective of the somatic events of sexual differentiation (13). Additionally by removing the germ cells from their somatic environment, XXY germ cells were able to proliferate similar to germ cells from normal XY littermate (12). The impairment of germ cell proliferation occurred in vivo but not in vitro, suggesting a defect in somatic-germ cell communication in the differentiating XXY testis from XY testis. There is available evidence to show that sex chromosome disomy and other numerical abnormalities in spermatozoa from XXY mice were significantly increased, suggesting that XY germ cells, if present, did not undergo a proper chromosome disjunction in the XXY testicular environment (24). Sertoli cell function can be further assessed by transplanting normal germ cells into testes of XXY mice of various ages and characterizing their survival; these studies are ongoing in our laboratory.
Sertoli cells support, nurture, and regulate germ cell development by forming cellular junctions with germ cells and secreting paracrine factors acting on germ cells (25). Intratesticular androgen plays an essential role for Sertoli cell function including supporting spermatogenesis (20). Binding of androgen to the intracellular AR, an X chromosome gene-encoded protein, induces a conformational change in AR leading to translocation of the receptor-steroid complex to the nucleus, binding to specific DNA regulatory elements and stimulation of gene transcription (26). In this study, we showed AR immunoexpression was absent in adult XXY Sertoli cells, suggesting dysfunction of the Sertoli cells in adult XXY mice. Dysfunctional Sertoli cells may lead to the progressive loss of germ cells in early age and may cause the rare residual germ cells to be arrested at the pachytene stage in the adult XXY mice (27, 28, 29). We further studied the ontogeny of loss of AR expression in Sertoli cells in juvenile XXY mice. AR was expressed in Sertoli cells in both XY and XXY mice only after d 10 postnatal. Sporadic loss of AR expression in some of Sertoli cells began at d 20 after birth (after most germ cells are lost) in XXY mice; AR was essentially absent in adult XXY mice. Thus, our results demonstrate that loss of AR expression in Sertoli cells may be responsive but not responsible for massive germ cell loss in juvenile XXY mice, which occurred earlier than identifiable AR loss in the Sertoli cells. AR was also expressed in gonocytes located at the central region of the seminiferous cords at 1 d and a few in 3-d-old XY and XXY mice. Depletion of AR expression in gonocytes was coincident with the gonocytes migration to the basal lamina in both XY and XXY mice. The physiological significance of this observation is not known. There were also differences in the intracellular localization of the AR in the Leydig cells in XY vs. XXY mice. AR immunostaining was limited to the cytoplasm in the Leydig cells of XXY mice, whereas AR was present in the nuclei in XY mice. This may represent dysfunction of the Leydig cells in adult XXY mice.
It is of interest that testosterone levels in neonates with human KS are higher than in postneonatal KS infants but lower than that in neonatal XY infants (30). In the rodent the surge of testosterone secretion during the neonatal period appears to play a vital role in virilizing hypothalamic function (31). Our observations led us to speculate that neonatal androgens through AR expressed in gonocytes may also promote the gonocyte migration toward the basal lamina of the seminiferous cords in the immature mouse testis. The defect in germ cell migration in neonatal XXY mice may contribute to the process of germ cell loss.
In preliminary experiments we sought to evaluate the acquisition of a Pavlovian tone-food association to determine whether XXY mice exhibited learning deficits. We demonstrated that the rate of learning of the association was significantly slower in XXY mice as compared with their XY littermates, providing evidence for impaired medial temporal lobe function in XXY mice, providing additional similarity to the human counterpart, Klinefelter syndrome. We do not know at present whether behavioral impairments in XXY mice are due to low plasma testosterone levels, defect of androgen receptor, or overdosed X-linked gene expression in specific brain regions responsible for learning. It has been established that the central nervous system is a target for sex hormones (32, 33) and that various forms of Pavlovian learning and cognitive performance depend on intact sex hormones in adult rodents (34, 35). In addition, sex steroids have a great impact on memory mechanisms, postural stability, fine motor skills, and mood (36, 37). Recently it has been proposed that sex chromosome-specific gene expression plays a role in various sexually dimorphic brain and behavioral phenotypes (38).
We demonstrate in this study that XXY mouse model can be used for studying the effect of X-linked genes that escaped X inactivation on brain structures and functions in males and uncovering the molecular mechanisms underlying XXY aneuploidy. Furthermore, comparative analysis of the human and mouse X chromosomes in sex chromosome aneuploidy (XXY) may further provide interesting clues about the genes responsible for testicular failure and learning deficits because majority of the genes on human X chromosome are preserved or have their homologues on mouse X chromosome (39, 40).
We conclude that adult XXY mice have testicular failure and learning deficits, analogous to their human counterpart, Klinefelter syndrome.
Acknowledgments
We thank Mr. Andrew Leung for performing hormone assay; Ms. Kimberley Ma for performing immunohistochemistry; and Mr. Vince Antienza, Ms. Jennifer Chiang, and Ms. Emily Wu for helping cell culture and karyotyping.
Footnotes
This work was partially supported by Los Angeles Biomedical Research Institute (11856-01, to Y.L.), and the National Institutes of Health (HD39293 to A.P.S.H.).
Presented in part at the 30th Annual Meeting of the American Society of Andrology, Seattle, Washington, April 2–5, 2005.
Abbreviations: AR, Androgen receptor; BW, body weight; CS, conditioned stimulus; FISH, fluorescence in situ hybridization; KS, Klinefelter syndrome.
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Departments of Psychology (J.D.J.), Pathology and Laboratory Medicine (P.N.R.), University of California at Los Angeles, Los Angeles, California 90095
Abstract
Klinefelter syndrome (XXY males) is the most common sex chromosome aneuploidy. XXY mice were generated by using a four-generation breeding scheme that involves the use of a structurally rearranged Y chromosome, Y*, yielding approximately 50% of the live-born male offspring in the fourth generation with a XXY karyotype. Adult XXY mice have small testes, decreased plasma T levels, and elevated plasma FSH levels. The testes of adult XXY mice contained small seminiferous tubules with intraepithelial vacuolization and absence of germ cells, whereas Leydig cells appeared to be more abundant than their XY littermates. Androgen receptor immunoexpression was localized in Leydig cells and peritubular myoid cells in both XY and XXY mice. Androgen receptor immunoexpression was abundant in the Sertoli cells of XY mice but nearly absent in those of XXY mice. The testicular phenotype was marked by a 23.1% decrease in testis weights in XXY pups beginning at d 7 after birth. Gonocyte numbers were similar in XY and XXY mice at d 1 of age, followed by a 62.6% decrease in the number of gonocytes in the XXY mice on d 3 and further progressive loss in spermatogonia by d 5 and 7. On d 10, only a few spermatogonia remained in the XXY mice. To determine whether the phenotype of XXY mice extended into the neurobehavioral domain, studies were conducted demonstrating impairment of learning and memory function in XXY mice. We conclude that adult XXY mice have testicular failure and learning deficits, similar to its human counterpart, Klinefelter syndrome.
Introduction
KLINEFELTER SYNDROME (KS) is the most common sex chromosome aneuploidy, occurring in about 1.2 per 1000 live-born male births (1). Adult KS males are characterized by infertility, small testes, azoospermia, gynecomastia, elevated gonadotropin levels, and serum testosterone concentrations usually below or in the low adult male range. Many KS patients have learning disabilities and impairments of cognitive and executive functions (2, 3, 4, 5). Testicular biopsies from most adult patients demonstrate complete absence of germ cells, seminiferous tubule hyalinization, and fibrosis, whereas Leydig cells show hyperplasia (6). In contrast, testicular histology obtained from infant boys with KS appears to be nearly normal, with primary germ cells present (7, 8). Although the molecular mechanisms causing the KS phenotype are often ascribed to a gene dosage effect from the extra X chromosome, the specific genes responsible for germ cell loss in the testis and neurobehavioral deficits of KS patients remain unknown.
In prior studies, we characterized XXY mice produced by mating wild-type males with chimeric females carrying male embryonic stem cells as a model for KS (9, 10). We found that the testicular histology of adult XXY mice showed small seminiferous tubules with varying degree of intraepithelial vacuolization, complete absence of germ cells, nests of apparently degenerating Sertoli cells, and hypertrophy and hyperplasia of Leydig cells. Similar to men, spermatogonia were present in juvenile XXY mice, but progressive loss of germ cells occurred within 10 d after birth (10). In this study, we generated XXY mice by using a more efficient four-generation breeding scheme described by Hunt and colleagues (11, 12) involving the use of a structurally rearranged Y chromosome, Y*. Approximately 50% of the live-born male offspring in the fourth generation were XXY mice. In contrast to the Bronson model (9, 10), the Hunt model (11, 12) is self-replenishing and allows for a more economical creation of a colony for biological and psychological studies.
In earlier studies, the Hunt group reported that normal number of germ cells arrived at the genital ridges in the XXY embryo, but the mitotic proliferation of XXY germ cells became impaired as the testis differentiated (12). They believed that the impaired proliferation of XXY germ cells was caused by the reactivation of one of the two X chromosomes in XXY germ cells occurring when germ cells reached the genital ridge (13). Our objectives were: 1) to produce an adequate number of XXY mice by using Hunt’s breeding scheme (11, 12); 2) to characterize the testicular phenotype of adult XXY mice; 3) to examine the ontogeny of massive germ cell loss in neonatal XXY mice; and 4) to determine learning capability in adult XXY mice. Our goal was to demonstrate and establish the XXY mouse as an experimental murine model for its human counterpart, patients with Klinefelter syndrome.
Materials and Methods
Animals
Breeding pairs of XY* male and XX female were purchased from The Jackson Laboratory (Bar Harbor, ME). The animal breeding colony was established and housed in a standard animal facility under controlled temperature (22 C) and photoperiod (12-h light, 12-h darkness) with free access to water and mouse chow. XXY mice (41, XXY) and their littermate XY (40, XY) mice from the fourth generation produced from our breeding colony were used in this study. Seven adult XXY mice (8 wk old) and 7 littermate controls (XY) were used to characterize the testicular phenotype of adult XXY mice. Thirty-two juvenile XXY mice and an equivalent number of XY littermates (1 d old; n = 5; 3 d old; n = 5; 5 d old; n = 4; 7 d old; n = 5; 10 d old; n = 5; 14 d old; n = 4; and 20 d old; n = 4) were used to examine the onset of germ cell loss in XXY mice. Using a Pavlovian appetitive approach procedure (14, 15) that measures stimulus-reward learning, we determined the learning ability of seven adult XXY and seven XY littermate mice (4–12 months old at the time of testing). Animal breeding, handling and experimentation were in accordance with the recommendation of the American Veterinary Medical Association and were approved by the Harbor-University of California-Los Angeles Biomedical Research Institute Animal Care and Use Review Committee.
Sample collection
Adult XXY mice and their littermate controls were injected with heparin [130 IU per 100 g body weight (BW), ip] 15 min before being killed by lethal injections of sodium pentobarbital (100 mg/kg BW, ip) to facilitate testicular perfusion using a whole body perfusion technique (10, 16). Body and testis weights were recorded at autopsy. Blood samples were collected from the inferior vena cava of each animal immediately after death, and plasma was separated and stored at –20 C for subsequent hormone assays. The testes were then fixed by vascular perfusion with Bouin’s solution (Sigma Co., St. Louis, MO) for 30 min, preceded by a brief saline wash. The testes were removed and placed into the same fixative overnight. One slice from the middle region of the testis was processed for routine paraffin embedding for testicular histology and immunohistochemistry detecting androgen receptor expression and localization in the testis. All the male juvenile mice in the fourth generation were killed by lethal injections of sodium pentobarbital (100 mg/kg BW, ip) on d 1, 3, 5, 7, 10, 14, and 20 after birth. The testes were dissected out and immersed overnight in Bouin’s solution. The testes were then processed for routine paraffin embedding and sectioning for histologic examination and immunohistochemistry.
Fibroblast culture and karyotype analysis
Standard karyotyping was performed on cultured fibroblasts obtained from ear-clips in adult mice or pieces of skin in juvenile mice. Briefly, a 1–2 mm2 section of tissue was dissected from a piece of ear in a sterile manner. The sample was minced with a dissecting scissors and digested with 1.25% trypsin (Gibco, Invitrogen Co., New York, NY) for 30 min, followed by collagenase digestion (Gibco Invitrogen) for an hour and half at 37 C. The dispersed cells were suspended in Amino-max-II medium (Gibco, Invitrogen). Aminomax supports the growth of anchorage-dependent fibroblast cells. The cells were placed in flasks and cultured for 5–7 d at 37 C in a CO2 incubator. Once appropriate colony formation was observed, KaryoMAX Colcemid solution (Gibco, Invitrogen) was added into flask to cease the mitotic division. The cultured fibroblasts were harvested after a minor digestion with trypsin-EDTA solution (Gibco, Invitrogen). The harvested cells were suspended in 0.075 M potassium chloride solution (Gibco, Invitrogen) and incubated in water bath (37 C) for 20 min, fixed in the mixture of methanol and acetic acid (3:1 methano-acetic acid), spread on clean glass slides, and air dried for fluorescence in situ hybridization (FISH) analysis.
FISH
X and Y chromosome-specific whole chromosome painting was performed on metaphases derived from the cultured fibroblasts (10, 17). After following the standard cytogenetic techniques of harvesting and slide making, the slides were aged at room temperature for 2–3 d. FISH with STAR*FISH mouse whole chromosome-specific X- and Y-chromosome painting (Open Biosystems, Huntsville, AL) was performed following the protocol provided by the manufacturer. After dehydration by serial ethanol washing and air drying, the slides were denatured by incubation in 70% formamide/2x saline sodium citrate at 65 C for 2 min. About 15 μl of denatured X-paint probe directly labeled with fluorescein isothiocyanate and Y-paint probe labeled with cyanine 3 were applied to the slides and hybridized overnight at 37 C in a humidified chamber. After washing slides in 50% formamide/1x saline sodium citrate at 45 C for 5 min, the slides were dried, the chromosomes were stained with 4',6'-diamino-2-phenylindole and the green signal from the X-paint probe and red signal from Y-paint probe were visualized under a fluorescence microscope (Carl Zeiss, Melville, NY) equipped with appropriate filters and images captured with an FISH analysis system (Applied Imaging Corp., San Jose, CA).
Hormone assays
Testosterone concentrations in plasma were measured by RIA, as reported previously (18, 19). The minimal detection limit in the assay was 0.25 ng/ml. The intra- and interassay coefficients of variations were 8 and 11%, respectively. Plasma LH levels were measured by modified immunofluorometric assay as described previously (19). The minimal detection limit of the assay was 0.02 ng/ml. The intra- and interassay coefficients of variation were 6.9 and 12.3%, respectively. Plasma FSH levels were measured by immunofluorometric assay (19) using reagents supplied by Organon (Os, The Netherlands). The minimal detection limit in the assay was 0.04 ng/ml. The intra- and interassay coefficients of variations were 4.7 and 6.0%, respectively.
Immunohistochemical analysis
Bouin’s-fixed, paraffin-embedded testicular sections were deparaffinized, hydrated by successive series of ethanol, rinsed in distilled water, and then incubated in 2% H2O2 to quench endogenous peroxidases. Sections were blocked with 5% normal goat serum for 20 min to suppress the nonspecific binding of IgG and subsequently incubated with a 1:100 dilution of affinity-purified rabbit polyclonal androgen receptor (AR) antibody (1:100 AR; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) (20). Immunoreactivity was detected using biotinylated goat antirabbit IgG secondary antibody followed by avidin-biotinylated horseradish peroxidase complex visualized with diaminobenzidine tetrahydrochloride as per the manufacturer’s instructions (rabbit united immunohistochemistry detection system; Oncogene, Boston, MA). Slides were counterstained with hematoxylin and reviewed with the Zeiss microscope (Carl Zeiss) (19, 21).
Morphometric assessment of germ cells in juvenile mice
The method used for germ cell quantitation was similar to that described previously (10). In brief, testicular sections were examined with an American Optical Microscope (Buffalo, NY) with a x100 objective and a x10 eyepiece. A square grid fitted within the eyepiece provided a reference area of 10,000 μm2. Germ cells within the frame of grid were counted. To correct for shrinkage (if any) in the reference area, the diameters of 10 randomly selected transverse sections of the seminiferous tubules were measured from each of XY and XXY animals across the minor axes of their cross-sectioned profiles. There was no difference in the tubular diameter between XY and XXY mice within 10 d after birth.
Pavlovian appetitive approach procedure
The acquisition of a Pavlovian tone-food association was assessed in groups of seven adult (4–12 months old) XXY and XY littermates. Standard aluminum and Plexiglas operant chambers with a photocell-equipped pellet delivery magazine on one side and a curved panel with five photocell-equipped apertures on the opposite side (Med Associates, Mount Vernon, VT) were used. The boxes were housed inside of a sound-attenuating cubicle; background white noise was broadcast, and the environment was illuminated with a house light (a light diffuser that was located outside of the operant chamber but within the cubicle). Before training, the mice were food restricted to 90% of their free-feeding weights over a 3-d period. Subsequently, hungry mice were trained in four daily sessions in which 20-kHz tones (20 sec in duration) were presented on a variable time 20-sec schedule; a total of 10 tones were presented each day. During the period of each tone, food pellets (20 mg Bioserv dustless precision pellets) were dropped into the infra-red beam equipped magazine on a random time 5-sec schedule. Total duration of head pokes into the magazine during the conditioned stimulus (CS) interval, as well as latency to approach the magazine after CS onset, were measured (14, 15).
Statistical analysis
Statistical analyses were performed using the SigmaStat 2.0 Program (Jandel Corp., San Rafael, CA). Data were assessed by Student’s t test or one-way repeated-measures ANOVA followed by Student-Newman-Keuls method test or two-way ANOVA followed by Student-Newman-Keuls method test. Differences were considered significant if P < 0.05.
Results
In the first generation, XY* males were mated to normal XX females to produce XY*X females. In the second generation, XY*X females were mated to normal XY males to produce XYY*X males. In the third generation, XYY*X males were mated to normal XX females to produce XXY male mice. In the fourth generation, over 50% of male offspring were XXY mice. Adult XXY mice were analyzed by G-banded karyotyping from metaphase cells using cultured fibroblasts and further confirmed by X and Y chromosome paints using the FISH technique (Fig. 1).
Body weight, testis weight, and plasma testosterone levels
The comparisons of body weight, testis weight, and hormone levels between adult XY and XXY mice are shown in Table 1. No significant difference was observed in the body weight. The mean weight of the testis in XXY adult mice was reduced to 27.45% of their littermate XY mice. The mean level of plasma testosterone in adult XXY mice was significantly decreased, compared with XY mice (P < 0.05), whereas mean plasma FSH level was significantly elevated in adult XXY mice (P < 0.05). Whereas there was a trend toward higher plasma LH level in the XXY mice, the difference from XY mice was not significant.
Testicular histological examination
Testicular histological examination showed that seminiferous tubules of XY mice (Fig. 2A) displayed active spermatogenesis with normal appearing Sertoli cells and various classes of germ cells. In contrast, the testicular histologic examination of adult XXY mice (Fig. 2B) showed small seminiferous tubules, intraepithelial vacuolization, degenerating Sertoli cells, and Leydig cells that appeared to be more abundant in XXY compared with XY littermates. Interestingly, in one XXY mouse, we found a few seminiferous tubules containing zygotene and pachytene spermatocytes but few if any round spermatids.
Testicular histology and morphometric observations in juvenile XXY mice
All the male pups produced in the fourth generation were studied at different age groups (d 1, 3, 5, 7, and 10) without prior knowledge of their karyotype. Karyotyping was performed later using cultured fibroblasts from a piece of skin in mice under 10 d old and a piece of ear loop in mice older than 14 d old. The XXY mice identified by karyotyping were further confirmed by FISH analysis using mouse X and Y chromosome-specific paint probes. The mean testis weight was significantly lower in the XXY pups, compared with XY pups, beginning at d 7 after birth (Fig. 3). The number of gonocytes was similar in XY and XXY mice at d 1 of age. As early as d 3, a significant decrease in the number of gonocytes in the XXY pups was observed, which was followed by progressive loss in the number of spermatogonia on d 5 and 7. By d 10, when meiosis initiation should have occurred in XY mice, only a few spermatogonia remained in the XXY mice (Fig. 4). Leydig cells appeared to be normal in appearance both in XY and XXY juvenile mice.
AR immunoexpression in XXY mice
Using immunohistochemistry in Bouin’s fixed testicular sections, AR expression was present in Leydig cells and peritubular myoid cells in both XXY and their littermate XY mice (2–3 months, n = 4). Abundant AR expression was observed in the Sertoli cells of XY mice (Fig. 2A) but was nearly absent in Sertoli cells in adult XXY mice (Fig. 2B). The loss of AR expression also was demonstrated in the Sertoli cells of the single XXY mouse that had a few seminiferous tubules containing pachytene spermatocytes and a few spermatids. Whereas AR was present in the Leydig cells of both XXY and XY mice, their intracellular localization was different. ARs were localized in cytoplasm of Leydig cells in XXY mice in contrast to their localization in the nuclei of XY mice. To determine the onset of the loss AR expression in Sertoli cells of XXY mice, we examined expression of AR in the testicular sections from groups of four XXY and their XY siblings at d 1, 3, 5, 7, 10, 14, and 20 after birth. AR was expressed in Leydig cells and peritubular myoid cells in all age groups in both XY and XXY mice. In contrast, AR was not expressed in Sertoli cells in either XY or XXY mice until d 10 after birth and became more evident in both XY and XXY mice at 14 and 20 d of age. AR remained in the Sertoli cells of adult XY mice. The ontogeny of the AR in the Sertoli cells of XXY mice was decided different from that of the XY mice. There was sporadic loss of AR expression in the Sertoli cells of the XXY mice beginning at age 20 d; further loss of AR after age 20 d resulted in their almost complete absence of AR in the Sertoli cells of adult XXY mice. AR was also expressed in gonocytes located at the center of the seminiferous cords of 1-d-old mice (Fig. 5, A and B) and in the few gonocytes in 3-d-old XY and XXY mice. Loss of AR expression in gonocytes in both XY and XXY mice was coincident with gonocytes migration to the basal lamina.
Pavlovian conditioning in XXY and XY mice
Behavioral studies were conducted to evaluate the hypothesis that XXY mice would exhibit learning deficits reminiscent of the neurobehavioral phenotypes exhibited by KS patients. Over the course of training, the latency to approach the magazine after CS onset decreased (Fig. 6). A main effect of day (F(3, 36) = 5.2, P < 0.005) was driven by a progressive reduction in magazine approach latency as a function of repeated training. A training day * genotype interaction [F(3, 36) = 4.9, P < 0.01] indicated that XXY mice exhibited longer approach latencies on d 1 and 2 of training but that the group difference disappeared by d 4. Therefore, the rate of learning was impaired in XXY mice, even though they were equally able to perform the task (once learning had occurred).
Discussion
Males with two X chromosomes in mammals are sterile in nearly all species as a result of degeneration of the germ cells before the onset of meiosis (22). The incidence of naturally occurring live 41, XXY mice is estimated at 0.02% (23). There are two approaches to generate XXY mouse in the laboratory. In the first approach (chimeric model), female chimeras were generated from the injection of male embryonic stem cells into the blastocoele of female embryos (9). About half of the embryonic stem cell-derived male offspring of these chimeric females exhibited nonmosaic aneuploidy, with XXY being the predominant karyotype. We used this approach to generate XXY mice and studied testicular histology in adult and juvenile mice. We found that the phenotype of adult XXY mice was very similar to that of KS patients (10). In the second approach, we used a four-generation breeding scheme (XY* model) established by Hunt et al. (11, 12) that involved a structurally rearranged Y chromosome to produce XXY mice. We demonstrated that over 50% of male offspring in the fourth generation were XXY mice. We confirmed that this breeding scheme of XY* model was more efficient to produce XXY mice in the laboratory. We provided additional evidence showing that adult XXY mice have small testes, decreased serum testosterone levels, and elevated serum FSH levels. Testicular histology of adult XXY mice exhibited small seminiferous tubules with varying degree of intraepithelial vacuolization, degenerating Sertoli cells, and complete absence of germ cells. Leydig cells appeared to be more abundant in the interstitium. Thus, we demonstrated a similar testicular phenotype in adult XXY mice produced by either chimeric model or XY* model.
In the prior model using chimeric mice (10), plasma testosterone was not significantly lower in the XXY mice, compared with XY mice. In this study with a larger number of XXY and XY mice and more careful handling of the mice to reduce stress, mean plasma testosterone levels were significantly lower in the XXY mice. This was accompanied by elevated plasma FSH levels indicating significant seminiferous epithelium damage. Another discrepancy we observed between these two XXY mice models was the age that the germ cell loss occurred. In XXY mice produced by the chimeric model, we observed no significant differences in germ cell numbers between XXY and XY littermates until d 7 after birth. In XXY mice generated by the XY* model, we observed a significant decrease in the number of gonocytes beginning at d 3 after birth. The discrepancy may be attributed to the differences in the origin of the extra X chromosome (paternal in XY* model and maternal in chimeric model) and/or the genetic background of the animals. It is worth noting that in XXY mice generated by both schemes, massive germ cell loss occurred within 10 d after birth and before the initiation of meiosis. In addition, in contrast to the Hunt report (12) that described reduced gonocyte numbers at d 1 of age, we found no difference in the number of gonocytes in XY and XXY pups at 1 d after birth. The reason for the discrepancy is not clear. We did not study prenatal mice.
The mechanisms of germ cell loss in the XXY testis remain to be defined. It has been demonstrated that prenatal mitotic proliferation of germ cells was impaired in the XXY testis (12). The impaired proliferation of XXY germ cells resulted from reactivation of one of a pair of X chromosomes as germ cells reached the genital ridge, irrespective of the somatic events of sexual differentiation (13). Additionally by removing the germ cells from their somatic environment, XXY germ cells were able to proliferate similar to germ cells from normal XY littermate (12). The impairment of germ cell proliferation occurred in vivo but not in vitro, suggesting a defect in somatic-germ cell communication in the differentiating XXY testis from XY testis. There is available evidence to show that sex chromosome disomy and other numerical abnormalities in spermatozoa from XXY mice were significantly increased, suggesting that XY germ cells, if present, did not undergo a proper chromosome disjunction in the XXY testicular environment (24). Sertoli cell function can be further assessed by transplanting normal germ cells into testes of XXY mice of various ages and characterizing their survival; these studies are ongoing in our laboratory.
Sertoli cells support, nurture, and regulate germ cell development by forming cellular junctions with germ cells and secreting paracrine factors acting on germ cells (25). Intratesticular androgen plays an essential role for Sertoli cell function including supporting spermatogenesis (20). Binding of androgen to the intracellular AR, an X chromosome gene-encoded protein, induces a conformational change in AR leading to translocation of the receptor-steroid complex to the nucleus, binding to specific DNA regulatory elements and stimulation of gene transcription (26). In this study, we showed AR immunoexpression was absent in adult XXY Sertoli cells, suggesting dysfunction of the Sertoli cells in adult XXY mice. Dysfunctional Sertoli cells may lead to the progressive loss of germ cells in early age and may cause the rare residual germ cells to be arrested at the pachytene stage in the adult XXY mice (27, 28, 29). We further studied the ontogeny of loss of AR expression in Sertoli cells in juvenile XXY mice. AR was expressed in Sertoli cells in both XY and XXY mice only after d 10 postnatal. Sporadic loss of AR expression in some of Sertoli cells began at d 20 after birth (after most germ cells are lost) in XXY mice; AR was essentially absent in adult XXY mice. Thus, our results demonstrate that loss of AR expression in Sertoli cells may be responsive but not responsible for massive germ cell loss in juvenile XXY mice, which occurred earlier than identifiable AR loss in the Sertoli cells. AR was also expressed in gonocytes located at the central region of the seminiferous cords at 1 d and a few in 3-d-old XY and XXY mice. Depletion of AR expression in gonocytes was coincident with the gonocytes migration to the basal lamina in both XY and XXY mice. The physiological significance of this observation is not known. There were also differences in the intracellular localization of the AR in the Leydig cells in XY vs. XXY mice. AR immunostaining was limited to the cytoplasm in the Leydig cells of XXY mice, whereas AR was present in the nuclei in XY mice. This may represent dysfunction of the Leydig cells in adult XXY mice.
It is of interest that testosterone levels in neonates with human KS are higher than in postneonatal KS infants but lower than that in neonatal XY infants (30). In the rodent the surge of testosterone secretion during the neonatal period appears to play a vital role in virilizing hypothalamic function (31). Our observations led us to speculate that neonatal androgens through AR expressed in gonocytes may also promote the gonocyte migration toward the basal lamina of the seminiferous cords in the immature mouse testis. The defect in germ cell migration in neonatal XXY mice may contribute to the process of germ cell loss.
In preliminary experiments we sought to evaluate the acquisition of a Pavlovian tone-food association to determine whether XXY mice exhibited learning deficits. We demonstrated that the rate of learning of the association was significantly slower in XXY mice as compared with their XY littermates, providing evidence for impaired medial temporal lobe function in XXY mice, providing additional similarity to the human counterpart, Klinefelter syndrome. We do not know at present whether behavioral impairments in XXY mice are due to low plasma testosterone levels, defect of androgen receptor, or overdosed X-linked gene expression in specific brain regions responsible for learning. It has been established that the central nervous system is a target for sex hormones (32, 33) and that various forms of Pavlovian learning and cognitive performance depend on intact sex hormones in adult rodents (34, 35). In addition, sex steroids have a great impact on memory mechanisms, postural stability, fine motor skills, and mood (36, 37). Recently it has been proposed that sex chromosome-specific gene expression plays a role in various sexually dimorphic brain and behavioral phenotypes (38).
We demonstrate in this study that XXY mouse model can be used for studying the effect of X-linked genes that escaped X inactivation on brain structures and functions in males and uncovering the molecular mechanisms underlying XXY aneuploidy. Furthermore, comparative analysis of the human and mouse X chromosomes in sex chromosome aneuploidy (XXY) may further provide interesting clues about the genes responsible for testicular failure and learning deficits because majority of the genes on human X chromosome are preserved or have their homologues on mouse X chromosome (39, 40).
We conclude that adult XXY mice have testicular failure and learning deficits, analogous to their human counterpart, Klinefelter syndrome.
Acknowledgments
We thank Mr. Andrew Leung for performing hormone assay; Ms. Kimberley Ma for performing immunohistochemistry; and Mr. Vince Antienza, Ms. Jennifer Chiang, and Ms. Emily Wu for helping cell culture and karyotyping.
Footnotes
This work was partially supported by Los Angeles Biomedical Research Institute (11856-01, to Y.L.), and the National Institutes of Health (HD39293 to A.P.S.H.).
Presented in part at the 30th Annual Meeting of the American Society of Andrology, Seattle, Washington, April 2–5, 2005.
Abbreviations: AR, Androgen receptor; BW, body weight; CS, conditioned stimulus; FISH, fluorescence in situ hybridization; KS, Klinefelter syndrome.
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