Mouse model for men with klinefelter syndrome: a multifaceted fit for a complex disorder.

UNLABELLED: 41XXY mouse models share many characteristics of the human 47XXY Klinefelter syndrome (KS). This manuscript discusses the relative role of androgen deficiency and X chromosome genes resulting in the XXY mouse phenotype. The similarities in phenotype between 47XXY men and 41XXY mice suggest that the clinical manifestations in XXY men may be because of gene-dosage effect from genes that escape X inactivation in mouse. CONCLUSION: The 41XXY mouse is an excellent model for KS.

Genetic, hormonal, and metabolomic influences on social behavior and sex preference of XXY mice.

XXY men (Klinefelter syndrome) are testosterone deficient, socially isolated, exhibit impaired gender identity, and may experience more homosexual behaviors. Here, we characterize social behaviors in a validated XXY mouse model to understand mechanisms. Sociability and gender preference were assessed by three-chambered choice tasks before and after castration and after testosterone replacement. Metabolomic activities of brain and blood were quantified through fractional synthesis rates of palmitate and ribose (GC-MS). XXY mice exhibit greater sociability than XY littermates, particularly for male mice. The differences in sociability disappear after matching androgen exposure. Intact XXY, compared with XY, mice prefer male mice odors when the alternatives are ovariectomized female mice odors, but they prefer estrous over male mice odors, suggesting that preference for male mice may be due to social, not sexual, cues. Castration followed by testosterone treatment essentially remove these preferences. Fractional synthesis rates of palmitate are higher in the hypothalamus, amygdala, and hippocampus of XXY compared with XY mice but not with ribose in these brain regions or palmitate in blood. Androgen ablation in XY mice increases fractional synthesis rates of fatty acids in the brain to levels indistinguishable from those in XXY mice. We conclude that intact XXY mice exhibit increased sociability, differences in gender preference for mice and their odors are due to social rather than sexual cues and, these differences are mostly related to androgen deficiency rather than genetics. Specific metabolic changes in brain lipids, which are also regulated by androgens, are observed in brain regions that are involved in these behaviors.

Genetic and hormonal control of bone volume, architecture, and remodeling in XXY mice.

Klinefelter syndrome is the most common chromosomal aneuploidy in men (XXY karyotype, 1 in 600 live births) and results in testicular (infertility and androgen deficiency) and nontesticular (cognitive impairment and osteoporosis) deficits. The extent to which skeletal changes are due to testosterone deficiency or arise directly from gene overdosage cannot be determined easily in humans. To answer this, we generated XXY mice through a four-generation breeding scheme. Eight intact XXY and 9 XY littermate controls and 8 castrated XXY mice and 8 castrated XY littermate controls were euthanized at 1 year of age. Castration occurred 6 months prior to killing. A third group of 9 XXY and 11 XY littermates were castrated and simultaneously implanted with a 1-cm Silastic testosterone capsule 8 weeks prior to sacrifice. Tibias were harvested from all three groups and examined by micro-computed tomography and histomorphometry. Blood testosterone concentration was assayed by radioimmunoassay. Compared with intact XY controls, intact androgen-deficient XXY mice had lower bone volume (6.8% +/- 1.2% versus 8.8% +/- 1.7%, mean +/- SD, p = .01) and thinner trabeculae (50 +/- 4 µm versus 57 +/- 5 µm, p = .007). Trabecular separation (270 +/- 20 µm versus 270 +/- 20 µm) or osteoclast number relative to bone surface (2.4 +/- 1.0/mm2 versus 2.7 +/- 1.5/mm2) did not differ significantly. Testosterone-replaced XXY mice continued to show lower bone volume (5.5% +/- 2.4% versus 8.1% +/- 3.5%, p = .026). They also exhibited greater trabecular separation (380 +/- 69 µm versus 324 +/- 62 µm, p = .040) but equivalent blood testosterone concentrations (6.3 +/- 1.8 ng/mL versus 8.2 +/- 4.2 ng/mL, p = .28) compared with testosterone-replaced XY littermates. In contrast, castration alone drastically decreased bone volume (p < .001), trabecular thickness (p = .05), and trabecular separation (p < .01) to such a great extent that differences between XXY and XY mice were undetectable. In conclusion, XXY mice replicate many features of human Klinefelter syndrome and therefore are a useful model for studying bone. Testosterone deficiency does not explain the bone phenotype because testosterone-replaced XXY mice show reduced bone volume despite similar blood testosterone levels.

Fate of bone marrow stem cells transplanted into the testis: potential implication for men with testicular failure.

To assess adult stem cell differentiation in the testis, we injected bone marrow cells from adult green fluorescent protein (GFP) transgenic mice into the seminiferous tubules and the testicular interstitium of busulfan-treated wild-type or c-kit mutant (W/W(v)) mice. Ten to 12 weeks after transplantation, we examined the fate of the transplanted bone marrow cells and found that they survived in recipient testes. In both the busulfan-treated and W/W(v) mice, some of the GFP-positive donor cells had a Sertoli cell appearance and expressed follicle-stimulating hormone receptor within the seminiferous tubules. In addition, GFP-positive donor cells were found in the interstitium of recipient testes, and they expressed the cytochrome P450 side chain cleavage enzyme (P450scc). In the seminiferous tubules of busulfan-treated mice, GFP-positive donor cells had the appearance of spermatogonia or spermatocytes and expressed VASA. However, this was not found in the seminiferous tubules of W/W(v) mice. We conclude that adult bone marrow cells, in a favorable testicular environment, differentiate into somatic and germ cell lineages. The resident neighboring cells in the recipient testis may control site-appropriate stem cell differentiation. This clinically relevant finding raises the possibility for treatment of male infertility and testosterone deficiency through the therapeutic use of stem cells.

Involvement of p38 mitogen-activated protein kinase and inducible nitric oxide synthase in apoptotic signaling of murine and human male germ cells after hormone deprivation.

This study investigates the role of p38 MAPK, inducible nitric oxide synthase (iNOS), and the intrinsic pathway signaling in male germ cell death in rats after hormonal deprivation by a potent GnRH antagonist treatment. Germ cell apoptosis, involving exclusively middle (VII-VIII) stages, was activated by d 5 after GnRH antagonist treatment. Initiation of germ cell apoptosis was preceded by p38 MAPK activation and induction of iNOS. p38 MAPK activation and iNOS induction were further accompanied by a marked perturbation of the BAX/BCL-2 rheostat, cytochrome c, and DIABLO release from mitochondria, caspase activation, and poly(ADP-ribose) polymerase cleavage. Concomitant administration of aminoguanidine, a selective iNOS inhibitor, significantly prevented hormone deprivation-induced germ cell apoptosis. Inhibitors of iNOS or p38 MAPK were also effective in preventing human male germ cell apoptosis induced by hormone-free culture conditions. Together, these results establish a new signal transduction pathway involving p38 MAPK and iNOS that, through activation of the intrinsic pathway signaling, promotes male germ cell death in response to a lack of hormonal stimulation across species.

Regulation of human male germ cell death by modulators of ATP production.

The understanding of testicular physiology, pathology, and male fertility issues requires knowledge of male germ cell death and energy production. Here, we induced human male germ cell apoptosis (detected by Southern blot analysis of DNA fragmentation, TUNEL, activation of caspases-3 and -9, and electron microscopy) by incubating seminiferous tubule segments under hormone- and serum-free conditions. Inhibitors of complexes I to IV of mitochondrial respiration, exposure to anoxia, and inhibition of F0F1-ATPase (with oligomycin) decreased the ATP levels (analyzed by HPLC) and suppressed apoptosis at 4 h. Uncoupler 2,4-dinitrophenol (DNP) and oligomycin combination also suppressed death at 4 h, as did the DNP alone. Inhibition of glycolysis by 2-deoxyglucose neither suppressed nor further induced apoptosis nor altered the antiapoptotic effects of the mitochondrial inhibitors. Furthermore, Fas system activation did not modify the effects of mitochondrial modulators. After 24 h, delayed male germ cell apoptosis was observed despite the presence of the mitochondrial inhibitors. We conclude that the mitochondrial ATP production machinery plays an important role in regulating in vitro-induced primary pathways of human male germ apoptosis. The ATP synthesized by the F0F1-ATPase seems to be the crucial death regulator, rather than any of the complexes (I-IV) alone, the functional electron transport chain, or the membrane potential. We also conclude that there seem to be secondary pathways of human testicular cell apoptosis that do not require mitochondrial ATP production. The present study emphasizes the role of the main catabolic pathways in the complex network of regulating events of male germ cell life and death.

Minocycline up-regulates BCL-2 levels in mitochondria and attenuates male germ cell apoptosis.

In this study, we determined the efficacy of minocycline, a second generation tetracycline, in preventing male germ cell apoptosis after withdrawal of gonadotropins and intratesticular testosterone (T). Groups of 5 male rats received one of the following treatments daily for 5 days: (i) daily sc injection of GnRH-A (1.6 mg/kg BW), (ii) oral administration of 30% gum acacia as a vehicle control, and (iii) GnRH-A + oral administration of 50 or 100 mg/kg BW of minocycline. Minocycline at both 50 and 100 mg dose levels significantly (P < 0.05) prevented GnRH-A -induced germ cell apoptosis by 59.4% and 62.2%, respectively, and fully prevented PARP cleavage. Minocycline-mediated protection occurred at the mitochondria, involving the restoration of the BCL-2 levels and, in turn, suppression of cytochrome c and DIABLO release. Minocycline was also effective in preventing human male germ cell apoptosis induced by hormone free culture condition.

Radiation sensitizing effect of estramustine is not dependent on apoptosis.

BACKGROUND: Estramustine is an anti-mitotic cytostatic drug that also enhances the effect of radiotherapy. The mechanism of radiosensitization is not thoroughly known. Since both radiotherapy and estramustine induce apoptosis in prostate cancer cells, we conducted an experiment to show whether radiosensitization is mediated by apoptosis. MATERIALS AND METHODS: DU-145 human prostate cancer cells were xenografted to nude mice and treated with estramustine for 2 weeks and external radiation for 3 to 6 days (18 to 36 Gy). Tumor regression was measured mechanically and the rate of apoptosis defined by the amount of low molecular weight DNA fragmentation. Follow-up time was 1 to 18 days. RESULTS: The tumor size regressed in the group of mice receiving both radiotherapy and estramustine. Four weeks after the treatment, apoptosis was accentuated in the tumors treated with estramustine or radiation but not with their combination. CONCLUSION: Estramustine potentiates radiotherapy, but not by enhancing radiation-induced apoptosis.

Effects of acid sphingomyelinase deficiency on male germ cell development and programmed cell death.

Deficiency of acid sphingomyelinase (ASM), an enzyme responsible for producing a pro-apoptotic second messenger ceramide, has previously been shown to promote the survival of fetal mouse oocytes in vivo and to protect oocytes from chemotherapy-induced apoptosis in vitro. Here we investigated the effects of ASM deficiency on testicular germ cell development and on the ability of germ cells to undergo apoptosis. At the age of 20 weeks, ASM knock-out (ASMKO) sperm concentrations were comparable with wild-type (WT) sperm concentrations, whereas sperm motility was seriously affected. ASMKO testes contained significantly elevated levels of sphingomyelin at the age of 8 weeks as detected by high-performance, thin-layer chromatography. Electron microscopy revealed that the testes started to accumulate pathological vesicles in Sertoli cells and in the interstitium at the age of 21 days. Irradiation of WT and ASMKO mice did not elevate intratesticular ceramide levels at 16 h after irradiation. In situ end labeling of apoptotic cells also showed a similar degree of cell death in both groups. After a 21-day recovery period, the numbers of primary spermatocytes and spermatogonia at G2 as well as spermatids were essentially the same in the WT and ASMKO testes, as detected by flow cytometry. In serum-free cultures both ASMKO and WT germ cells showed a significant increase in the level of ceramide, as well as massive apoptosis. In conclusion, ASM is required for maintenance of normal sphingomyelin levels in the testis and for normal sperm motility, but not for testicular ceramide production or for the ability of the germ cells to undergo apoptosis.

Activator protein-1 in human male germ cell apoptosis.

Apoptosis limits germ cell number in the testis, and its dysregulation is associated with male infertility. Here, we evaluated the role of the transcription factor activator protein 1 (AP-1) in male germ cell apoptosis in a culture of human seminiferous tubules. AP-1 DNA-binding activity increased in cultured tubules within 2.5 h, which was earlier than the onset of apoptosis as detected by caspase 3 activation and apoptotic DNA fragmentation. The c-Jun, c-Fos and JunD proteins were detected in the Sertoli cell nuclei, whereas apoptosis occurred in the germ cells. Follicle-stimulating hormone (FSH), whose receptors are expressed in the Sertoli cells, inhibited germ cell apoptosis and concomitantly suppressed AP-1 DNA-binding activity, but had no effect on nuclear factor kappaB (NF-kappaB) activation. These results suggest that AP-1 transcription factors are involved in the Sertoli cell-mediated control of germ cell apoptosis, and that inhibition of germ cell apoptosis by FSH appears to involve suppression of AP-1 activation.

Protection from radiation-induced male germ cell loss by sphingosine-1-phosphate.

Male germ cells are susceptible to radiation-induced injury, and infertility is a common problem after total-body irradiation. Here we investigated, first, the effects of irradiation on germ cells in mouse testis and, second, the role of sphingosine-1-phosphate (S1P) treatment in radiation-induced male germ cell loss. Irradiation of mouse testes mainly damaged the early developmental stages of spermatogonia. The damage was seen by means of DNA flow cytometry 21 days after irradiation as decreasing numbers of spermatocytes and spermatids with increasing amounts of ionizing radiation (0.1-2.0 Gy). Intratesticular injections of S1P given 1-2 h before irradiation (0.5 Gy) did not protect against short-term germ cell loss as measured by in situ end labeling of DNA fragmentation 16 h after irradiation. However, after 21 days, in the S1P-treated testes, the numbers of primary spermatocytes and spermatogonia at G2 (4C peak as measured by flow cytometry) were higher at all stages of spermatogenesis compared with vehicle-treated testes, indicating protection of early spermatogonia by S1P, whereas the spermatid (1C) populations were similar. In conclusion, S1P appears to protect partially (16%-47%) testicular germ cells against radiation-induced cell death. This warrants further studies aimed at development of therapeutic agents capable of blocking sphingomyelin-induced pathways of germ cell loss.

Sphingosine-1-phosphate in inhibition of male germ cell apoptosis in the human testis.

It has been suggested that apoptosis is controlled by two intracellular sphingolipids, ceramide and sphingosine-1-phosphate (S1P), which are widely distributed in mammalian tissues. In the ovary, S1P was found to effectively block apoptosis caused by cancer therapies. Its role in male germ cell death, however, was unknown. In this study, we investigated the effects of ceramide and S1P on human male germ cell apoptosis. Germ cell death was induced by incubation of segments of seminiferous tubules in vitro. During apoptosis, ceramide levels increased rapidly before appearance of caspase 3 activation and DNA laddering, suggesting a role for ceramide in the induction of germ cell death. Ceramide appeared to regulate an early step of apoptosis because n-acetyl-L-cysteine and blockade of mitochondrial respiration inhibited apoptosis but had no effect on ceramide levels. Moreover, fumonisin B1 (ceramide synthetase inhibitor) did not significantly affect testicular apoptosis. Therefore, elevated ceramide levels are likely to result from breakdown of sphingomyelin rather than from de novo synthesis. Finally, we found that S1P at 1 and 10 micromol/liter suppressed germ cell apoptosis by 30% (P < 0.001). Taken together, sphingolipids appear to play a role in male germ cell apoptosis and can partly be inhibited by S1P.

Nuclear factor-kappa B activation in human testicular apoptosis.

Apoptotic cell death plays an important role in limiting testicular germ cell population during spermatogenesis and its dysregulation has been shown to be associated with male infertility. The growing evidence on the role of the transcription factor nuclear factor (NF)-kappa B in controlling apoptosis prompted us to investigate NF-kappa B activity in the normal human testis and its role in testis tissue undergoing excessive apoptosis in vitro. In electrophoretic mobility shift assays, low-level constitutive NF-kappa B DNA-binding activity was found and, by immunostaining of the RelA and p50 NF-kappa B subunits, was localized to Sertoli cell nuclei. During in vitro-induced testicular apoptosis, the Sertoli cell nuclear NF-kappa B levels and whole seminiferous tubule NF-kappa B DNA-binding activity increased previous detection of germ cells undergoing apoptosis. The anti-inflammatory drug sulfasalazine effectively suppressed stress-induced NF-kappa B DNA binding and NF-kappa B-mediated I kappa B alpha gene expression. Importantly, concomitantly with inhibiting NF-kappa B, sulfasalazine blocked germ cell apoptosis. These results suggest that during testicular stress Sertoli cell NF-kappa B proteins exert proapoptotic effects on germ cells, which raises the possibility that pharmacological inhibition of NF-kappa B could be a therapeutic target in transient stress situations involving excessive germ cell death.