RESUMEN
Deca-brominated diphenyl ether (BDE-209) is a common flame retardant utilized in electronic products, textiles, furniture, and upholstery materials. Environmental BDE-209 exposure results in spermatogenesis disorder, because of the characteristics of bioaccumulation, persistence, and probably toxicity. Meiotic prophase I is a crucial phase during spermatogenesis which is a key influential factor of normal sperm production. However, the effects of BDE-209 on meiotic prophase I during spermatogenesis are poorly understood. The present study aimed to evaluate whether BDE-209 exposure impairs meiotic prophase I during spermatogenesis of spermatocytes. We validated the effects of BDE-209 on alternations of meiotic prophase I in Balb/c male mice. Firstly, we analyzed sperm quality in cauda epididymis with decreasing sperm count, increasing abnormal sperm, and male reproductive dysfunction after exposure to BDE-209. Then, reactive oxygen species (ROS) and malondialdehyde (MDA) levels in testis and GC-2spd cells were significant increased after treated with BDE-209. Furthermore, we found that meiotic prophase I arrest at early-pachytene stage during spermatogenesis with increasing of DSBs damage and trimethylated histone H3 at lysine-4 (H3K4me3) in spermatocytes exposed to BDE-209. Finally, we conducted homologous recombination (HR) analyses to identify the progression of meiosis. The recombination markers, including DMC1 and RAD51, and crossover marker MLH1 were decreased during spermatogenesis after exposure to BDE-209. Collectively, our data indicated that BDE-209 has detrimental impacts on meiotic prophase I of spermatocytes in mice.
Asunto(s)
Puntos de Control del Ciclo Celular/efectos de los fármacos , Éteres Difenilos Halogenados/toxicidad , Fase Paquiteno/efectos de los fármacos , Espermatocitos/efectos de los fármacos , Espermatogénesis/efectos de los fármacos , Testículo/efectos de los fármacos , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Retardadores de Llama , Histonas/genética , Histonas/metabolismo , Masculino , Metilación , Ratones Endogámicos BALB C , Homólogo 1 de la Proteína MutL/genética , Homólogo 1 de la Proteína MutL/metabolismo , Estrés Oxidativo/efectos de los fármacos , Proteínas de Unión a Fosfato/genética , Proteínas de Unión a Fosfato/metabolismo , Procesamiento Proteico-Postraduccional , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , Recuento de Espermatozoides , Espermatocitos/metabolismo , Espermatocitos/patología , Testículo/metabolismo , Testículo/patologíaRESUMEN
BACKGROUND: Infertility is linked to depletion of the primordial follicle pool consisting of individual oocytes arrested at the diplotene stage of meiotic prophase I surrounded by granulosa cells. Primordial germ cells, the oocyte precursors, begin to differentiate during embryonic development. These cells migrate to the genital ridge and begin mitotic divisions, remaining connected, through incomplete cytokinesis, in clusters of synchronously dividing oogonia known as germ cell cysts. Subsequently, they enter meiosis, become oocytes and progress through prophase I to the diplotene stage. The cysts break apart, allowing individual oocytes to be surrounded by a layer of granulosa cells, forming primordial follicles each containing a diplotene arrested oocyte. A large number of oocytes are lost coincident with cyst breakdown, and may be important for quality control of primordial follicle formation. Exposure of developing ovaries to exogenous hormones can disrupt cyst breakdown and follicle formation, but it is unclear if hormones affect progression of oocytes through prophase I of meiosis. METHODS: Fetal ovaries were treated in organ culture with estradiol, progesterone, or both hormones, labeled for MSY2 or Synaptonemal complex protein 3 (SYCP3) using whole mount immunocytochemistry and examined by confocal microscopy. Meiotic prophase I progression was also followed using the meiotic surface spread technique. RESULTS: MSY2 expression in oocytes was reduced by progesterone but not estradiol or the hormone combination. However, while MSY2 expression was upregulated during development it was not a precise marker for the diplotene stage. We also followed meiotic prophase I progression using antibodies against SYCP3 using two different methods, and found that the percent of oocytes at the pachytene stage peaked at postnatal day 1. Finally, estradiol and progesterone treatment together but not either alone in organ culture increased the percent of oocytes at the pachytene stage. CONCLUSIONS: We set out to examine the effects of hormones on prophase I progression and found that while MSY2 expression was reduced by progesterone, MSY2 was not a precise diplotene stage marker. Using antibodies against SYCP3 to identify pachytene stage oocytes we found that progesterone and estradiol together delayed progression of oocytes through prophase I.
Asunto(s)
Estradiol/farmacología , Profase Meiótica I/efectos de los fármacos , Oocitos/efectos de los fármacos , Ovario/efectos de los fármacos , Progesterona/farmacología , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Feto , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Células de la Granulosa/metabolismo , Ratones Endogámicos C57BL , Oocitos/citología , Oocitos/metabolismo , Técnicas de Cultivo de Órganos , Ovario/embriología , Ovario/metabolismo , Fase Paquiteno/efectos de los fármacos , Embarazo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
The N-end rule pathway is a proteolytic system in which its recognition components (N-recognins) recognize destabilizing N-terminal residues of short-lived proteins as an essential element of specific degrons, called N-degrons. The RING E3 ligases UBR2 and UBR1 are major N-recognins that share size (200 kDa), conserved domains and substrate specificities to N-degrons. Despite the known function of the N-end rule pathway in degradation of cytosolic proteins, the major phenotype of UBR2-deficient male mice is infertility caused by arrest of spermatocytes at meiotic prophase I. UBR2-deficient spermatocytes are impaired in transcriptional silencing of sex chromosome-linked genes and ubiquitylation of histone H2A. In this study we show that the recruitment of UBR2 to meiotic chromosomes spatiotemporally correlates to the induction of chromatin-associated ubiquitylation, which is significantly impaired in UBR2-deficient spermatocytes. UBR2 functions as a scaffold E3 that promotes HR6B/UbcH2-dependent ubiquitylation of H2A and H2B but not H3 and H4, through a mechanism distinct from typical polyubiquitylation. The E3 activity of UBR2 in histone ubiquitylation is allosterically activated by dipeptides bearing destabilizing N-terminal residues. Insufficient monoubiquitylation and polyubiquitylation on UBR2-deficient meiotic chromosomes correlate to defects in double strand break (DSB) repair and other meiotic processes, resulting in pachytene arrest at stage IV and apoptosis. Some of these functions of UBR2 are observed in somatic cells, in which UBR2 is a chromatin-binding protein involved in chromatin-associated ubiquitylation upon DNA damage. UBR2-deficient somatic cells show an array of chromosomal abnormalities, including hyperproliferation, chromosome instability, and hypersensitivity to DNA damage-inducing reagents. UBR2-deficient mice enriched in C57 background die upon birth with defects in lung expansion and neural development. Thus, UBR2, known as the recognition component of a major cellular proteolytic system, is associated with chromatin and controls chromatin dynamics and gene expression in both germ cells and somatic cells.
Asunto(s)
Inestabilidad Cromosómica/fisiología , Fibroblastos/metabolismo , Histonas/metabolismo , Espermatocitos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/fisiología , Ubiquitinación , Animales , Animales Recién Nacidos , Cromatina/metabolismo , Reparación del ADN/fisiología , Humanos , Masculino , Meiosis , Ratones , Fase Paquiteno/efectos de los fármacos , Alveolos Pulmonares/anomalías , Ubiquitina-Proteína Ligasas/deficienciaRESUMEN
Functional analysis of cytoplasmic dynein in Caenorhabditis elegans has revealed a wide range of cellular functions for this minus-end-directed motor protein. Dynein transports a variety of cargos to diverse cellular locations, and thus cargo selection and destination are likely regulated by accessory proteins. The microtubule-associated proteins LIS-1 and dynein interact, but the nature of this interaction remains poorly understood. Here we show that both LIS-1 and the dynein heavy-chain DHC-1 are required for integrity of the actin cytoskeleton in C. elegans. Although both dhc-1(or195ts) and lis-1 loss-of-function disrupt the actin cytoskeleton and produce embryonic lethality, a double mutant suppresses these defects. A targeted RNA interference screen revealed that knockdown of other actin regulators, including actin-capping protein genes and prefoldin subunit genes, suppresses dhc-1(or195ts)-induced lethality. We propose that release or relocation of the mutant dynein complex mediates this suppression of dhc-1(or195ts)-induced phenotypes. These results reveal an unexpected direct or indirect interaction between the actin cytoskeleton and dynein activity.
Asunto(s)
Actinas/metabolismo , Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Dineínas Citoplasmáticas/genética , Citoesqueleto/metabolismo , Mutación/genética , Alelos , Animales , Caenorhabditis elegans/citología , Caenorhabditis elegans/embriología , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Dineínas Citoplasmáticas/metabolismo , Citoesqueleto/efectos de los fármacos , Depsipéptidos/farmacología , Dineínas/metabolismo , Embrión no Mamífero/citología , Embrión no Mamífero/efectos de los fármacos , Embrión no Mamífero/metabolismo , Técnicas de Silenciamiento del Gen , Genes de Helminto , Genes Supresores , Gónadas/citología , Gónadas/efectos de los fármacos , Gónadas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Fase Paquiteno/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Interferencia de ARN/efectos de los fármacos , Supresión Genética/efectos de los fármacosRESUMEN
Aim of the present study was to investigate whether estrogens were able to directly activate rapid signaling pathways controlling spermatogenesis in rat pachytene spermatocytes (PS). Classically, estrogens act by binding to estrogen receptors (ERs) alpha and beta. Recently, it has been demonstrated that rapid estrogen action can also be activated through the G-protein-coupled receptor (GPR)-30. Herein, we demonstrated that rat PS express ER alpha, ER beta and GPR30. Treatment of PS with estradiol (E2), the selective GPR30 agonist G1 and the selective ER alpha agonist PPT determined activation of ERK1/2 which are part of GPR30 signaling cascade. ERK1/2 activation in response to E2 and G1 was correlated to an increased phosphorylation of c-Jun. All treatments failed to induce these responses in the presence of EGFR inhibitor AG1478, ERK inhibitor PD98059 and ER inhibitor ICI182780. mRNA expression of cell cycle regulators cyclin A1 and B1 was downregulated by E2 and G1 while an up-regulation of proapoptotic factor Bax was observed in the same conditions. These data demonstrate that E2, working through both ER alpha and/or GPR30, activates in PS the rapid EGFR/ERK/c-Jun pathway, modulating the expression of genes involved in the balance between cellular proliferation and apoptosis.
Asunto(s)
Apoptosis/efectos de los fármacos , Estradiol/farmacología , Receptor alfa de Estrógeno/metabolismo , Fase Paquiteno/efectos de los fármacos , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/efectos de los fármacos , Espermatocitos/citología , Animales , Células Cultivadas , Activación Enzimática/efectos de los fármacos , Receptor alfa de Estrógeno/genética , Receptor beta de Estrógeno/genética , Receptor beta de Estrógeno/metabolismo , Masculino , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fenoles/farmacología , Proteínas Proto-Oncogénicas c-jun/metabolismo , Pirazoles/farmacología , Ratas , Ratas Sprague-Dawley , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/genética , Espermatocitos/efectos de los fármacos , Espermatocitos/enzimología , Espermatocitos/metabolismoRESUMEN
Specific features of spermatogenesis were studied in senesce-accelerated mice of the line SAMP-1 after one-time injection of the chemical mutagen dipin. Quantitative and histomorphological changes in the spermatogenic epithelium proved to develop gradually. Cell loss and disorganization of spermatogenesis reached the peak as late as on days 28 and 35 after the injection. Differentiating spermatogonia manifested increased sensitivity to dipin. In prophase I of meiosis, developing spermatocytes proved to be less sensitive to the cytotoxic action of dipin at the pachytene than at the preleptotene-leptotene stages. Spermatogenesis in most seminiferous tubules was restored by day 56 after dipin treatment. At the end of the experiment (day 100), both quantitative parameters and morphological pattern of spermatogenesis did not differ significantly from those in the control. Thus, the cytotoxic action of dipin does not lead to irreversible structural disorganization of the spermatogenic epithelium in SAMP1 mice. Radioautography revealed a large proportion of highly differentiated Sertoli cells with 3H-thymidine-labeled nuclei in experimental animals. In some cases, structures resembling embryonic seminiferous tubules were revealed in the vicinity of rete testis in testis sections of experimental mice. These structures contained the cells morphologically similar to gonocytes and young Sertoli cells.
Asunto(s)
Envejecimiento/efectos de los fármacos , Aziridinas/toxicidad , Mutágenos/toxicidad , Espermatogénesis/efectos de los fármacos , Envejecimiento/patología , Animales , Masculino , Ratones , Fase Paquiteno/efectos de los fármacos , Epitelio Seminífero/metabolismo , Epitelio Seminífero/patología , Células de Sertoli/metabolismo , Células de Sertoli/patología , Espermatocitos/metabolismo , Espermatocitos/patología , Espermatogonias/metabolismo , Espermatogonias/patología , Factores de TiempoRESUMEN
Estrogen plays an essential role in the growth and maturation of the mammalian oocyte, and recent studies suggest that it also influences follicle formation in the neonatal ovary. In the course of studies designed to assess the effect of the estrogenic chemical bisphenol A (BPA) on mammalian oogenesis, we uncovered an estrogenic effect at an even earlier stage of oocyte development--at the onset of meiosis in the fetal ovary. Pregnant mice were treated with low, environmentally relevant doses of BPA during mid-gestation to assess the effect of BPA on the developing ovary. Oocytes from exposed female fetuses displayed gross aberrations in meiotic prophase, including synaptic defects and increased levels of recombination. In the mature female, these aberrations were translated into an increase in aneuploid eggs and embryos. Surprisingly, we observed the same constellation of meiotic defects in fetal ovaries of mice homozygous for a targeted disruption of ERbeta, one of the two known estrogen receptors. This, coupled with the finding that BPA exposure elicited no additional effects in ERbeta null females, suggests that BPA exerts its effect on the early oocyte by interfering with the actions of ERbeta. Together, our results show that BPA can influence early meiotic events and, importantly, indicate that the oocyte itself may be directly responsive to estrogen during early oogenesis. This raises concern that brief exposures during fetal development to substances that mimic or antagonize the effects of estrogen may adversely influence oocyte development in the exposed female fetus.
Asunto(s)
Feto/efectos de los fármacos , Meiosis/efectos de los fármacos , Oogénesis/efectos de los fármacos , Fenoles/farmacología , Proteínas Adaptadoras Transductoras de Señales/genética , Aneuploidia , Animales , Compuestos de Bencidrilo , Segregación Cromosómica/efectos de los fármacos , Cromosomas de los Mamíferos/efectos de los fármacos , Cromosomas de los Mamíferos/genética , Intercambio Genético/efectos de los fármacos , Receptor beta de Estrógeno/deficiencia , Femenino , Feto/metabolismo , Feto/patología , Metafase/efectos de los fármacos , Ratones , Ratones Noqueados , Homólogo 1 de la Proteína MutL , Proteínas Nucleares/genética , Oocitos/citología , Oocitos/efectos de los fármacos , Fase Paquiteno/efectos de los fármacos , Embarazo , Recombinación Genética/efectos de los fármacos , Recombinación Genética/genéticaRESUMEN
The aim of this work was to assess the effect of metronidazole (MTZ) on the stages of the seminiferous epithelial cycle and spermatozoa morphology when the drug is administered in human therapeutic doses to 60-day-old CFW male mice. The frequency of the stages was established by counting spermatocytes in pachytene and spermatids. Abnormalities in the flagellum or the head, lack of maturity and multiple malformations, were considered in the morphological analysis. Murine control strain was compared with MTZ treated group (v.ip 130 mg/kg/bw) both kept in standard captivity conditions. Cellular composition or number of stages in the seminiferous tubules were not altered in MTZ exposed animals, though the number of cells in stages I, V and XII was increased. The sperm cell morphology was severely affected by the treatment with potentially serious consequences on the normal fertilization process. Thus, the MTZ has to be considered as a conceivable thread regarding male fertility.
Asunto(s)
Antiprotozoarios/toxicidad , Metronidazol/toxicidad , Espermatozoides/efectos de los fármacos , Animales , Recuento de Células , Fertilidad/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos , Fase Paquiteno/efectos de los fármacos , Túbulos Seminíferos/efectos de los fármacos , Túbulos Seminíferos/patología , Espermátides/efectos de los fármacos , Espermátides/patología , Espermatozoides/patologíaRESUMEN
Previous studies of the toxicity of candidate fuel additives identified severe testicular toxicity in animals exposed to 1,6-dimethoxyhexane (DMH). A series of studies were conducted to characterize the effects of DMH on spermatogenesis and to compare the effects of DMH with responses to structural similar aliphatic ethers. In the first study, sexually mature male rats were administered a single dose (600 mg/kg) of DMH, and subsets of animals were sampled at intervals post exposure (PE). Both testis and thymus weight declined steadily after DMH exposure, both being significantly lower than control by 7 days PE. Treatment with DMH led, at 24 to 48 h PE, to an increase in dying primary spermatocytes in seminiferous tubule stages I-IV and stages XII-XIV, but not intervening stages. The affected cohort of germ cells was seen progressing through the developmental sequence of spermatogenesis as numbers of dying cells returned to control levels by 7 days PE, coincident with a significant decline in the proportion of round spermatids among germ cells as determined by flow cytometry. Resolution of round spermatids to control levels by day 21 PE coincided with a reduction in condensed spermatids (homogenization-resistant spermatid nuclei) and was followed at 28 days PE by a significant reduction in cauda epididymal sperm counts. Further studies of repeated exposures to DMH (200 mg.kg(-1).day(-1), 5 days per week for 4 weeks) confirmed the significant testis toxicity of this material. In contrast, similar treatment with any of a variety of structurally similar aliphatic ethers had little or no impact on testis function. Methoxyacetic acid, previously shown to cause rapid death of some meiotic germ cell stages, was found at high concentrations in the urine of DMH-treated rats but not in rats treated with other ethers, suggesting that DMH exerts its testis toxicity via metabolism to this substance. These results demonstrate that DMH selectively deletes germ cells from the testis At the very early or very late pachytene, diplotene, or M-phase spermatocyte stages, possibly through conversion to MAA.
Asunto(s)
Éteres de Etila/toxicidad , Gasolina , Hexanos/toxicidad , Espermatogénesis/efectos de los fármacos , Testículo/efectos de los fármacos , Acetatos/orina , Animales , Apoptosis/efectos de los fármacos , Éteres de Etila/química , Éteres de Etila/metabolismo , Citometría de Flujo , Hexanos/química , Hexanos/metabolismo , Masculino , Tamaño de los Órganos/efectos de los fármacos , Fase Paquiteno/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Túbulos Seminíferos/efectos de los fármacos , Túbulos Seminíferos/patología , Espermatogénesis/fisiología , Relación Estructura-Actividad , Testículo/patología , Timo/efectos de los fármacos , Timo/patologíaRESUMEN
OBJECTIVE: The present study evaluated the role of experimental oxidative stress (induced by feeding diets with different concentrations of selenium [Se], a trace nutrient and potent antioxidant) on male reproductive activity in mice. METHODS: To create different levels of oxidative stress in male mice, three diets with different levels of Se were fed to different groups for 8 wk. Mice in group 1 were fed a yeast-based diet, which is considered a Se-deficient diet (0.02 ppm). Mice in groups 2 and 3 were fed with an Se-deficient diet supplemented with 0.2 and 1 ppm Se as sodium selenite, respectively. RESULTS: After completion of the feeding schedule, a significant decrease in Se levels were observed in Se-deficient mice (group 1), whereas Se levels greatly increased in the Se-excess mice (group 3). Glutathione peroxidase activity was greatly decreased in the liver and testis in group 1, whereas glutathione-S-transferase activity was significantly increased in the testis. No significant change was found in activities of glutathione peroxidase and glutathione-S-transferase in group 3 compared with group 2. Cell kinetics showed a significant decrease in the number of pachytene spermatocytes and young and mature spermatids in group 1 compared with group 2. No appreciable change was observed in the germinal cell population in group 3. A significant decrease in sperm number was observed in group 1 compared with group 2. No change in these parameters was observed in group 3. The fertility status of mice in terms of percent fertility and litter size also exhibited a significant decrease in the reproductive ability of group 1. No change in these parameters was observed in group 3 compared with group 2. CONCLUSION: The present results clearly demonstrate the effect of oxidative stress generated by feeding different concentrations of Se on cell kinetics in the testis and, hence, its effect on the reproductive ability of male mice.
Asunto(s)
Antioxidantes/administración & dosificación , División Celular/efectos de los fármacos , Fertilidad/efectos de los fármacos , Estrés Oxidativo/fisiología , Selenio/administración & dosificación , Testículo/efectos de los fármacos , Animales , Relación Dosis-Respuesta a Droga , Glutatión Peroxidasa/efectos de los fármacos , Glutatión Transferasa/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/enzimología , Masculino , Ratones , Oxidación-Reducción/efectos de los fármacos , Fase Paquiteno/efectos de los fármacos , Selenio/deficiencia , Motilidad Espermática/efectos de los fármacos , Espermátides/efectos de los fármacos , Espermatocitos/efectos de los fármacos , Espermatozoides/efectos de los fármacos , Testículo/citología , Testículo/enzimologíaRESUMEN
In experiments involving different germ-cell stages, we had previously found meiotic prophase of the male mouse to be vulnerable to the induction of several types of genetic damage by the topoisomerase-II inhibitor etoposide. The present study of etoposide effects involved two end points of meiotic events known to occur in primary spermatocytes--chromosomal crossing-over and segregation. By following assortment of 13 microsatellite markers in two chromosomes (Ch 7 and Ch 15) it was shown that etoposide significantly affected crossing-over, but did not do so in a uniform fashion. Treatment generally changed the pattern for each chromosome, leading to local decreases in recombination, a distal shift in locations of crossing-over, and an overall decrease in double crossovers; at least some of these results might be interpreted as evidence for increased interference. Two methods were used to explore etoposide effects on chromosome segregation: a genetic experiment capable of detecting sex-chromosome nondisjunction in living progeny; and the use of FISH (fluorescence in situ hybridization) technology to score numbers of Chromosomes X, Y, and 8 in spermatozoa. Taken together these two approaches indicated that etoposide exposure of pachytene spermatocytes induces malsegregation, and that the findings of the genetic experiment probably yielded a marked underestimate of nondisjunction. As indicated by certain segregants, at least part of the etoposide effect could be due to disrupted pairing of achiasmatic homologs, followed by precocious sister-centromere separation. It has been shown for several organisms that absent or reduced levels of recombination, as well as suboptimally positioned recombination events, may be associated with abnormal segregation. Etoposide is the only chemical tested to date for which living progeny indicates an effect on both male meiotic crossing-over and chromosome segregation. Whether, however, etoposide-induced changes in recombination patterns are direct causes of the observed malsegregation requires additional investigation.