RESUMEN
Genomic imprinting is an epigenetic phenomenon known to regulate fetal growth and development. Studies from our laboratory have demonstrated that treatment of adult male rats with tamoxifen increased postimplantation loss around mid gestation. Further studies demonstrated the aberrant expression of transcripts of several imprinted genes in the resorbing embryos at days 11 and 13 of gestation including IGF2. In addition, decreased methylation at the Igf2-H19 imprint control region was observed in spermatozoa and in resorbing embryos sired by tamoxifen-treated males. In this study, methylation analysis of the imprinted genes, which were found to be differentially expressed, was done using EpiTYPER in the spermatozoa of tamoxifen-treated rats and in postimplantation embryos sired by tamoxifen-treated rats. Differentially methylated regions (DMRs) for most imprinted genes have not been identified in the rats. Hence, initial experiments were performed to identify the putative DMRs in the genes selected for the study. Increased methylation at CpG islands present in the putative DMRs of a number of imprinted genes was observed in the resorbing embryos sired by tamoxifen-treated male rats. This increase in methylation is associated with the downregulation of most of these genes at the transcript level in resorbing embryos. No change in the methylation status of these genes was observed in spermatozoa. These observations suggest that a deregulation of mechanisms protecting unmethylated alleles from a wave of de novo methylation occurs following implantation.
Asunto(s)
Metilación de ADN/efectos de los fármacos , Embrión de Mamíferos/efectos de los fármacos , Desarrollo Embrionario/efectos de los fármacos , Impresión Genómica/efectos de los fármacos , Factor II del Crecimiento Similar a la Insulina/metabolismo , Exposición Paterna/efectos adversos , Moduladores Selectivos de los Receptores de Estrógeno/efectos adversos , Animales , Proteínas de Unión al Calcio , Islas de CpG/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Pérdida del Embrión/inducido químicamente , Pérdida del Embrión/metabolismo , Embrión de Mamíferos/metabolismo , Femenino , Factor II del Crecimiento Similar a la Insulina/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Masculino , Embarazo , Ratas , Ratas Sprague-Dawley , Espermatozoides/efectos de los fármacos , Espermatozoides/metabolismo , Tamoxifeno/efectos adversos , Regulación hacia Arriba/efectos de los fármacos , ras-GRF1/genética , ras-GRF1/metabolismoRESUMEN
OBJECTIVE: To study H19 ICR methylation levels in association with sperm parameters routinely analyzed in idiopathic recurrent spontaneous miscarriage cases. DESIGN: Case-control study. SETTING: Academic research setting. PATIENT(S): Male partners of couples with a history of idiopathic recurrent spontaneous miscarriage (RSM group) and male partners of couples with proven fertility (control group). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Paternal age, sperm concentration, motility, chromatin compaction status, morphology, and H19 ICR methylation were assessed in control and idiopathic RSM group participants. RESULT(S): Paternal age and basic semen parameters analyzed did not show any significant difference between the two groups; however H19 ICR methylation levels were reduced significantly in the idiopathic RSM group compared with the control group. CONCLUSION(S): Significant reduction in the H19 ICR methylation without significant difference in the sperm parameters demonstrates aberrant imprinting to be associated with idiopathic RSM.
Asunto(s)
Aborto Habitual/genética , Metilación de ADN , Impresión Genómica , Factor II del Crecimiento Similar a la Insulina/genética , ARN Largo no Codificante/genética , Espermatozoides/metabolismo , Factores de Edad , Estudios de Casos y Controles , Forma de la Célula , Ensamble y Desensamble de Cromatina , Regulación hacia Abajo , Femenino , Predisposición Genética a la Enfermedad , Humanos , India , Masculino , Embarazo , Análisis de Semen/métodos , Recuento de Espermatozoides , Motilidad Espermática , Espermatozoides/patologíaRESUMEN
AIM: Imprinted genes are known regulators of embryo growth. Studies from our laboratory have demonstrated that treatment of adult male rats with tamoxifen increased post-implantation loss at around midgestation. Expression of insulin like growth factor 2 (Igf2), a paternally expressed imprinted gene was down-regulated in the resorbing embryos obtained at embryonic day 13. Hypomethylation of Igf2-H19 imprint control region was observed in the resorbing embryo sires and spermatozoa obtained from tamoxifen-treated rats thereby suggesting that errors in imprint acquisition during spermatogenesis can result in embryo loss. The present study aims at studying the expression of other imprinted genes, besides Igf2 in the embryos sired by tamoxifen-treated males. MAIN METHODS: Gene expression profiles of resorbing versus normal embryos were assessed by microarrays. Real time quantitative RT-PCR for six imprinted genes and four genes involved in cell cycle was done to validate gene expression data. The affected pathways and functions were identified in the resorbing embryos and effect on cell cycle was confirmed by flow cytometry. KEY FINDINGS: Aberrant expression of a number of imprinted genes was observed in the resorbing embryos when compared to the normal embryos at embryonic days 11 and 13. Down-regulation of Notch signaling, Wnt signaling and cell cycle pathway was observed in the resorbing embryos. SIGNIFICANCE: The study suggests that exposure of male germ cells to tamoxifen during adulthood results in aberrant expression of imprinted genes and down-regulation of development associated pathways in the F(1) progeny thereby causing embryo loss.
Asunto(s)
Pérdida del Embrión/genética , Desarrollo Embrionario/genética , Impresión Genómica , Exposición Paterna , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Tamoxifeno/farmacología , Animales , Ciclo Celular/genética , Embrión de Mamíferos/citología , Femenino , Citometría de Flujo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Edad Gestacional , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Tamoxifen is a synthetic non-steroidal Selective Estrogen Receptor Modulator used in the treatment of breast cancer and in treatment of male fertility. Earlier studies from our laboratory had demonstrated an increase in post-implantation embryo loss following tamoxifen treatment to adult male rats at a dose of 0.4mg/kg/day for 60 days. The post-implantation loss occurred at around 9-10 days of gestation suggesting that paternal factors involved in embryo development were affected by tamoxifen treatment. The present study was done to determine if any chromosomal aberrations occurred in the embryos sired by tamoxifen treated male rats. Chromosomal aberrations induced by tamoxifen treatment to adult male rats in the bone marrow (F(0) males) and in the embryos sired by these males (F(1) progeny) were determined. In addition, the reproductive performance of the F(1) progeny was assessed. A significant dose dependent reduction in mitotic activity in the bone marrow and embryonic cells was observed after tamoxifen treatment. In addition, tamoxifen also induced a significant dose dependent increase in the frequency of chromosomal aberrations, mainly gaps and breaks in bone marrow and embryonic cells. However, the embryos sired by the tamoxifen treated males had no effect on developmental milestones achieved and on their reproductive performance. The present study suggests that chromosomal aberrations observed in the embryos did not the affect their development until adulthood but could make the progeny of the tamoxifen treated males vulnerable to the development of adult onset diseases later in life.
Asunto(s)
Aberraciones Cromosómicas/inducido químicamente , Embrión de Mamíferos/efectos de los fármacos , Antagonistas de Estrógenos/toxicidad , Preñez , Tamoxifeno/toxicidad , Animales , Células de la Médula Ósea/efectos de los fármacos , Implantación del Embrión , Femenino , Masculino , Exposición Paterna , Embarazo , Efectos Tardíos de la Exposición Prenatal/inducido químicamente , Ratas , Ratas Sprague-Dawley , Reproducción/efectos de los fármacosRESUMEN
BACKGROUND: The identification of estrogen receptors alpha and beta and aromatase in the testis has highlighted the important role of estrogens in regulating spermatogenesis. There is a wealth of information on the deleterious effects of fetal and neonatal exposure of estrogens and xenoestrogens in the testis, including spermiation failure and germ cell apoptosis. However, very little is known about gene transcripts affected by exogenous estradiol exposure in the testis. The objective of the present study was to unveil global gene expression profiles and testicular cell number changes in rats after estradiol treatment. METHODS: 17beta-estradiol was administered to adult male rats at a dose of 100 micrograms/kg body weight in saline daily for 10 days; male rats receiving only saline were used as controls. Microarray analysis was performed to examine global gene expression profiles with or without estradiol treatment. Real time RT-PCR was conducted to verify the microarray data. In silico promoter and estrogen responsive elements (EREs) analysis was carried out for the differentially expressed genes in response to estradiol. Quantitation of testicular cell number based on ploidy was also performed using flow cytometry in rats with or without estradiol treatment. RESULTS: We found that 221 genes and expressed sequence tags (ESTs) were differentially expressed in rat testes treated with estradiol compared to the control; the microarray data were confirmed by real time RT-PCR. Gene Ontology analysis revealed that a number of the differentially expressed genes are involved in androgen and xenobiotic metabolism, maintenance of cell cytoskeleton, endocytosis, and germ cell apoptosis. A total of 33 up-regulated genes and 67 down-regulated genes showed the presence of EREs. Flow cytometry showed that estradiol induced a significant decrease in 2n cells (somatic and germ cells) and 4n cells (pachytene spermatocytes) and a marked increase in the number of elongated and elongating spermatids. CONCLUSIONS: This study provides a novel insight into the molecular basis for spermiation failure and apoptosis caused by 17beta-estradiol and it also offers new mechanisms by which adult exposure to environmental estrogens can affect spermatogenesis and fertility.
Asunto(s)
Estrógenos/metabolismo , Estrógenos/farmacología , Expresión Génica/efectos de los fármacos , Testículo/citología , Testículo/metabolismo , Animales , Recuento de Células , Análisis por Conglomerados , Estrógenos/análisis , Perfilación de la Expresión Génica , Células Germinativas/citología , Células Germinativas/efectos de los fármacos , Células Germinativas/metabolismo , Células Intersticiales del Testículo/citología , Células Intersticiales del Testículo/efectos de los fármacos , Células Intersticiales del Testículo/metabolismo , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Concentración Osmolar , Ratas , Células de Sertoli/citología , Células de Sertoli/efectos de los fármacos , Células de Sertoli/metabolismo , Regulación hacia Arriba , Estudios de Validación como AsuntoRESUMEN
OBJECTIVE: To determine the effect of tamoxifen treatment on global and insulin-like growth factor 2-H19 imprinting control region (Igf2-H19 ICR)-specific DNA methylation in rat spermatozoa and analyze its association with postimplantation loss. DESIGN: Experimental prospective study. SETTING: Animal research and academic research facility. SUBJECT(S): Male and female 75-day-old Holtzman rats. INTERVENTION(S): Global and Igf2-H19 ICR-specific DNA methylation was analyzed in an epididymal sperm sample in control and tamoxifen-treated rats at a dose of 0.4 mg tamoxifen/kg/day. DNA methylation status was correlated to postimplantation loss in females mated with tamoxifen-treated males. MAIN OUTCOME MEASURE(S): Global sperm DNA methylation level, methylation status of Igf2-H19 ICR in sperm, postimplantation loss. RESULT(S): Tamoxifen treatment significantly reduced methylation at Igf2-H19 ICR in epididymal sperm. However, the global methylation level was not altered. A mating experiment confirmed a significant increase in postimplantation loss upon tamoxifen treatment and showed significant correlation with methylation at Igf2-H19 ICR. CONCLUSION(S): Reduced DNA methylation at Igf2-H19 ICR in rat spermatozoa upon tamoxifen treatment indicated a role of estrogen-associated signaling in the acquisition of paternal-specific imprints during spermatogenesis. In addition, association between DNA methylation and postimplantation loss suggests that errors in paternal imprints at Igf2-H19 ICR could affect embryo development.