Cell Apoptosis In Mice Research Articles

Involvement of oxidative stress and caspase 2-mediated intrinsic pathway signaling in age-related increase in muscle cell apoptosis in mice

Apoptosis has been implicated as a mechanism of loss of muscle cells in normal aging and plays an important role in age-related sarcopenia. To test the hypothesis that caspase 2 and c-Jun NH(2)-terminal kinase (JNK)-mediated intrinsic pathway signaling contribute to skeletal muscle cell apoptosis in aging, we compared activation of caspase 2 and JNK and the in vivo expression of 4-hydroxynonenal protein adducts (4-HNE), inducible nitric oxide synthase (iNOS), glucose-6-phosphate dehydrogenase (G6PDH), B-cell lymphoma-2 (BCL-2), BAX, and phospho-BCL-2 in gastrocnemius muscles of young (5 months old) and old (25 months old) mice. A distinct age-related increase in 4-HNE and iNOS expression was readily detected in mice. Increased oxidative stress and iNOS induction were further accompanied by a decrease in G6PDH expression, activation of caspase 2 and JNK, and inactivation of BCL-2 through phosphorylation at serine 70, and caspase 9 activation. Regression analysis further revealed that increased muscle cell death in aging was significantly correlated with changes in the levels of these molecules. Taken together, our data indicate that caspase 2 and JNK-mediated intrinsic pathway signaling is one of the mechanisms involved in age-related increase in muscle cell apoptosis.

Elevated End‐of‐Treatment Serum INSL3 Is Associated With Failure to Completely Suppress Spermatogenesis in Men Receiving Male Hormonal Contraception

The administration of testosterone plus a progestogen functions as a male contraceptive by inhibiting the release of pituitary gonadotropins. After 3 to 4 months of treatment, most men are azoospermic or severely oligospermic (< or =1 million sperm/mL). However, 10% to 20% of men have persistent sperm production despite profound gonadotropin suppression. Since insulin-like factor 3 (INSL3) has been shown to prevent germ cell apoptosis in mice, we hypothesized that INSL3 might be higher in men with persistent spermatogenesis during treatment with male hormonal contraceptives. In a retrospective analysis, we measured serum INSL3 in 107 men from 3 recent male hormonal contraceptive studies and determined the relationship between suppression of spermatogenesis and serum INSL3. At the end of treatment 63 men (59%) were azoospermic and 44 men (41%) had detectable sperm in their ejaculates. Baseline INSL3 did not predict azoospermia; however, end of treatment serum INSL3 was significantly higher in nonazoospermic men compared with those with azoospermia (median [interquartile range]: 95 [73-127] pg/mL vs 80 [67-101] pg/mL; P = .03). Furthermore, serum INSL3 was positively correlated with sperm concentration (r = .25; P = .009) at the end of treatment and was significantly associated with nonazoospermia by multivariate logistic regression (P = .03). After 6 months of treatment with a hormonal male contraceptive regimen, higher serum INSL3 concentrations were associated with persistent sperm production. INSL3 may play a role in preventing complete suppression of spermatogenesis in some men on hormonal contraceptive regimens. This finding suggests that INSL3 may be a potential target for male contraceptive development.

Protective effects of ginsenoside Rg3 against cyclophosphamide-induced DNA damage and cell apoptosis in mice

Despite the significant anti-tumor activities, cyclophosphamide (CP) also shows cytotoxicity to normal cells. In order to explore the protective effects of drugs against CP-induced adverse effects, 20(S)-ginsenoside Rg(3) was tested for its possibly protective activities on CP-induced DNA damage and cell apoptosis in mouse bone marrow cells or peripheral lymphocyte cells. In the current study, the alkaline single cell gel electrophoresis (comet assay), flow cytometry assay with annexin V-FITC/PI and AO/EB staining assay were employed to measure DNA strand breakage and cell apoptosis, respectively. The activities of SOD and GPx and the contents of MDA were also tested by the various colormetric methods. The results showed that CP at a dose of 100 mg/kg, i.p. significantly caused DNA damages in both mouse bone marrow cells and peripheral lymphocyte cells, and markedly inhibited the activities of GPx and SOD and increased MDA contents in mouse blood. Moreover, CP at a dose of 200 mg/kg, i.p. triggered apoptosis in mouse bone marrow cells. On the other hand, 20(S)-ginsenoside Rg(3) orally administered at a dose of 20 mg/kg to the animals once a day for 2 days significantly inhibited CP-induced DNA damages in mouse bone marrow cells and peripheral lymphocyte cells, decrease the apoptotic numbers of bone marrow cells, antagonized the reduction of the activities of SOD and GPx, and the increase in MDA contents. In conclusion, 20(S)-ginsenoside Rg(3) showed the significant protective effects on CP-induced cell DNA damage and apoptosis. These effects might be partially attributed to its protective actions against CP-induced oxidative stress.

Molecular Cloning of &amp;lt;italic&amp;gt;MSRG-11&amp;lt;/italic&amp;gt; Gene Related to Apoptosis of Mouse Spermatogenic Cells

Abstract Beginning with a new contig of the expressed sequence tags (Mm.63892) obtained by comparing testis libraries with other tissue and cell line libraries using the digital differential display program, we cloned a new gene which is related to the apoptosis of mouse spermatogenic cells using the Genscan program and polymerase chain reaction (PCR) technology. The sequence data have been submitted to the GenBank database under accession number AY747687. The full cDNA length is 1074 bp, and the gene with 7 exons and 6 introns is located in mouse chromosome 1 H5. The protein is recognized as a new member of calmodulin (CaM) binding protein family because the sequence contains three short calmodulin-binding motifs containing conserved Ile and Gln residues (IQ motif) and is considered to play a critical role in interactions of IQ motif-containing proteins with CaM proteins. The putative protein encoded by this gene has 192 amino acid residues with a theoretical molecular mass of 23.7 kDa and a calculated isoelectric point of 9.71. The sequence shares no significant homology with any known protein in databases. RT-PCR and Northern blot analyses revealed that 1.3 kb MSRG-11 transcript was strongly expressed in adult mouse testis but weakly expressed in the spleen and thymus. The MSRG-11 gene was expressed at various levels, faintly at two weeks postpartum and strongly from three weeks postpartum in adult testes. The green fluorescence produced by pEGFP-C2/MSRG-11 was detected in the cytoplasm of COS7 cells 24 h post-transfection. The pcDNA3.1(–)/MSRG-11 plasmid was constructed and introduced into COS7 cells using Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, USA). MSRG-11 can accelerate COS7 cell apoptosis, which suggests that this gene may play an important role in the development of mouse testes and is a candidate gene of testis-specific apoptosis. Based on these observations, it was considered that we cloned a new gene which probably accelerates spermatogenetic cell apoptosis in mouse.

Molecular Cloning of MSRG-11 Gene Related to Apoptosis of Mouse Spermatogenic Cells

Beginning with a new contig of the expressed sequence tags (Mm.63892) obtained by comparing testis libraries with other tissue and cell line libraries using the digital differential display program, we cloned a new gene which is related to the apoptosis of mouse spermatogenic cells using the Genscan program and polymerase chain reaction (PCR) technology. The sequence data have been submitted to the GenBank database under accession number AY747687. The full cDNA length is 1074 bp, and the gene with 7 exons and 6 introns is located in mouse chromosome 1 H5. The protein is recognized as a new member of calmodulin (CaM) binding protein family because the sequence contains three short calmodulin-binding motifs containing conserved Ile and Gln residues (IQ motif) and is considered to play a critical role in interactions of IQ motif-containing proteins with CaM proteins. The putative protein encoded by this gene has 192 amino acid residues with a theoretical molecular mass of 23.7 kDa and a calculated isoelectric point of 9.71. The sequence shares no significant homology with any known protein in databases. RT-PCR and Northern blot analyses revealed that 1.3 kb MSRG-11 transcript was strongly expressed in adult mouse testis but weakly expressed in the spleen and thymus. The MSRG-11 gene was expressed at various levels, faintly at two weeks postpartum and strongly from three weeks postpartum in adult testes. The green fluorescence produced by pEGFP-C2/MSRG-11 was detected in the cytoplasm of COS7 cells 24 h post-transfection. The pcDNA3.1(?-)/MSRG-11 plasmid was constructed and introduced into COS7 cells using Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, USA). MSRG-11 can accelerate COS7 cell apoptosis, which suggests that this gene may play an important role in the development of mouse testes and is a candidate gene of testis-specific apoptosis. Based on these observations, it was considered that we cloned a new gene which probably accelerates spermatogenetic cell apoptosis in mouse.

Effect of electromagnetic field exposure on spermatogenesis and sexual activity

We read with interest the paper by Lee et al. [1]. They reported that continuous exposure to an electromagnetic field with extremely low frequency may induce testicular germ cell apoptosis in mice. Our group performed a series of experiments on the effect of exposure to an electrostatic field on spermatogenesis and sexual activity [2–7]. Polyester (polyethylene terphthalate) exhibited electrostatic potentials (ESPs) in the range of 306–376 V/cm2 (mean 338.9 ± 25.0) [2, 3]. The effects of polyester underpants on testicular temperature and sperm count was tested on a dog and a human volunteer. Both subjects wore a pair polyester underpants, which fitted loosely around the scrotum [2– 5]. The testicular temperature showed no significant changes during the period when the pants were worn. At the end of 24 months, the sperm counts and the motile sperms were significantly decreased and the abnormal forms increased (P < 0.001); the testicular biopsy revealed degenerative changes. However, the semen patterns normalized in both the dog and the human once they ceased wearing the polyester pants. Regarding the effect of the ESPs generated by the polyester underwear on the sexual activity, sexual behavior in rats was assessed before and after 6 and 12 months of wearing the pants and 6 months after their removal [7]. With the pants on, the rate of intromission to mounting (I/M) was significantly reduced compared to the pre-test levels and controls (P < 0.0001). Six months after removal of the pants, the I/M ratio returned to the pre-test levels. The polyester containing pants generated ESPs, which seem to create ‘electrostatic fields’ in the scrotum and the penile structures, that presumably induce diminished spermatogenesis and sexual activity.

Inhibition of T–Cell Responses by Hepatic Stellate Cells Via B7–H1-Mediated T–Cell Apoptosis in Mice

In the injured liver, hepatic stellate cells (HSCs) secrete many different cytokines, recruit lymphocytes, and thus participate actively in the pathogenesis of liver disease. Little is known of the role of HSCs in immune responses. In this study, HSCs isolated from C57BL/10 (H2b) mice were found to express scant key surface molecules in the quiescent stage. Activated HSCs express major histocompatibility complex class I, costimulatory molecules, and produce a variety of cytokines. Stimulation by interferon gamma (IFN-gamma) or activated T cells enhanced expression of these molecules. Interestingly, addition of the activated (but not quiescent) HSCs suppressed thymidine uptake by T cells that were stimulated by alloantigens or by anti-CD3-mediated T-cell receptor ligation in a dose-dependent manner. High cytokine production by the T cells suggests that the inhibition was probably not a result of suppression of their activation. T-cell division was also found to be normal in a CFSE dilution assay. The HSC-induced T-cell hyporesponsiveness was associated with enhanced T-cell apoptosis. Activation of HSCs was associated with markedly enhanced expression of B7-H1. Blockade of B7-H1/PD-1 ligation significantly reduced HSC immunomodulatory activity, suggesting an important role of B7-H1. In conclusion, the bidirectional interactions between HSCs and immune cells may contribute to hepatic immune tolerance.

Male infertility due to germ cell apoptosis in mice lacking the thiamin carrier, Tht1. A new insight into the critical role of thiamin in spermatogenesis

A mouse model of thiamin-responsive megaloblastic anemia (diabetes mellitus, deafness, megaloblastic anemia) lacking functional Slc19a2 has been generated and unexpectedly found to have a male-specific sterility phenotype. We describe here the characterization of the testis-specific effects of absence of the high-affinity thiamin transporter, Tht1. Null males were found to have hypoplastic testes secondary to germ cell depletion. Morphologic and expression analysis revealed that under conditions of standard thiamin intake, tissues affected in the syndrome (pancreatic β-cell, hematopoietic cells, auditory nerve) maintained normal function but pachytene stage spermatocytes underwent apoptosis. Under conditions of thiamin challenge, the apoptotic cell loss extended to earlier stages of germ cells but spared Sertoli cells and Leydig cells. Injection of high-dose thiamin was effective in reversing the spermatogenic failure, suggesting that the absence of the thiamin carrier could be overcome by diffusion-mediated transport at supranormal thiamin concentrations. These observations demonstrated that male germ cells, particularly those with high thiamin transporter expression beyond the blood–testis barrier, were more susceptible to apoptosis triggered by intracellular thiamin deficiency than any other tissue type. The findings described here highlight an unexpected and critical role for thiamin transport and metabolism in spermatogenesis.

Late Arrest of Spermiogenesis and Germ Cell Apoptosis in Mice Lacking the TBP-like TLF/TRF2 Gene

Metazoan genomes encode two related proteins, TBP and the TBP-like factor (TLF/TRF2), sharing a highly conserved saddle-like domain. TLF is highly expressed in a finely regulated pattern in the mouse testis during spermatogenesis. The murine TLF gene has been inactivated using homologous recombination. TLF −/− mice are viable, but mutant male mice are sterile due to a late, complete arrest of spermiogenesis. In mutant animals, spermatogonia and spermatocytes develop normally, but round spermatids undergo apoptosis at step 7. Although the expression of the transcriptional activator CREM and many other postmeiotic genes was unaltered in TLF null mice, several spermiogenesis genes transcribed in late round spermatids appeared to be under TLF control. Hence, TLF is not required for embryonic development in the mouse but is essential for spermiogenesis.

A Chromosome 15 Quantitative Trait Locus Controls Levels of Radiation-Induced Jejunal Crypt Cell Apoptosis in Mice

Jejunal crypt cells undergo apoptosis in response to ionizing radiation exposure. In mice the number of cells deleted by apoptosis is determined by several factors including the dose of radiation, the time of day the apoptosis level is quantified, and the strain of mouse irradiated. We previously found that the difference in radiation-induced apoptosis levels between C57BL/6J (B6) and C3Hf/Kam (C3H) mice is controlled by multiple genes, and this set of genes is distinct from that controlling thymocyte apoptosis levels in the same strain combination. Here, we report that a new quantitative trait locus on chromosome 15, Rapop5, partly accounts for the murine strain difference in susceptibility to radiation-induced jejunal crypt cell apoptosis. In addition, we show sexual dimorphism in the extent of radiation-induced jejunal crypt cell apoptosis, with female mice having higher levels.

Defective liver formation and liver cell apoptosis in mice lacking the stress signaling kinase SEK1/MKK4

The stress signaling kinase SEK1/MKK4 is a direct activator of stress-activated protein kinases (SAPKs; also called Jun-N-terminal kinases, JNKs) in response to a variety of cellular stresses, such as changes in osmolarity, metabolic poisons, DNA damage, heat shock or inflammatory cytokines. We have disrupted the sek1 gene in mice using homologous recombination. Sek1(-/- )embryos display severe anemia and die between embryonic day 10.5 (E10.5) and E12.5. Haematopoiesis from yolk sac precursors and vasculogenesis are normal in sek1(-/- )embryos. However, hepatogenesis and liver formation were severely impaired in the mutant embryos and E11.5 and E12.5 sek1(-/- )embryos had greatly reduced numbers of parenchymal hepatocytes. Whereas formation of the primordial liver from the visceral endoderm appeared normal, sek1(-/-) liver cells underwent massive apoptosis. These results provide the first genetic link between stress-responsive kinases and organogenesis in mammals and indicate that SEK1 provides a crucial and specific survival signal for hepatocytes.

Agonistic anti-Fas antibodies induce glomerular cell apoptosis in mice in vivo

Recent studies suggest that apoptotic cell death regulates the cell complement in glomerular diseases. However, little is-known about the factors that promote glomerular cell apoptosis. Activation of the Fas receptor by the Fas ligand or agonistic antibodies triggers apoptosis in some cell types that express Fas. Cultured human mesangial cell are among the cells that undergo apoptosis upon Fas activation, but it is unclear whether mesangial cells are sensitive to death induced by Fas in vivo. We have now explored the role of Fas in experimental glomerular injury. Murine mesangial cells in culture express fas and undergo apoptosis when stimulated with the Jo2 agonistic anti-Fas mAb. A fas mRNA transcript is present in normal murine kidney and freshly isolated glomeruli. Balb-c mice developed hematuria and proteinuria within 24 hours of the intraperitoneal injection of 10 micrograms Jo2 anti-Fas mAb. In addition to liver cell apoptosis, glomerular cell apoptosis and mesangial cell depletion were evident in the kidney at three hours and more pronounced at 24 hours. Glomerular and liver injury were not prevented by decomplementation. These data suggest that Fas activation in vivo by specific antibodies induces glomerular and mesangial cell apoptosis in mice.