RESUMO
Cytochrome P450 family 26 subfamily B member 1 (CYP26B1) regulates the concentration of all-trans retinoic acid (RA) and plays a key role in germ cell differentiation by controlling local distribution of RA. The mechanisms regulating Cyp26b1 expression in postnatal Sertoli cells, the main components of the stem cell niche, are so far unknown. During gonad development, expression of Cyp26b1 is maintained by Steroidogenic Factor 1 (SF-1) and Sex-Determining Region Y Box-9 (SOX9), which ensure that RA is degraded and germ cell differentiation is blocked. Here, we show that the NOTCH target Hairy/Enhancer-of-Split Related with YRPW Motif 1 (HEY1), a transcriptional repressor, regulates germ cell differentiation via direct binding to the Cyp26b1 promoter and thus inhibits its expression in Sertoli cells. Further, using in vivo germ cell ablation, we demonstrate that undifferentiated type A spermatogonia are the cells that activate NOTCH signaling in Sertoli cells through their expression of the NOTCH ligand JAGGED-1 (JAG1) at stage VIII of the seminiferous epithelium cycle, therefore mediating germ cell differentiation by a ligand concentration-dependent process. These data therefore provide more insights into the mechanisms of germ cell differentiation after birth and potentially explain the spatiotemporal RA pulses driving the transition between undifferentiated to differentiating spermatogonia.-Parekh, P. A., Garcia, T. X., Waheeb, R., Jain, V., Gandhi, P., Meistrich, M. L., Shetty, G., Hofmann, M.-C. Undifferentiated spermatogonia regulate Cyp26b1 expression through NOTCH signaling and drive germ cell differentiation.
Assuntos
Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Receptores Notch/metabolismo , Ácido Retinoico 4 Hidroxilase/biossíntese , Transdução de Sinais , Espermatogônias/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteína Jagged-1/genética , Proteína Jagged-1/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas , Receptores Notch/genética , Ácido Retinoico 4 Hidroxilase/genética , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Espermatogônias/citologia , Fator Esteroidogênico 1/genética , Fator Esteroidogênico 1/metabolismoRESUMO
STUDY QUESTION: What effect does cancer treatment have on levels of spontaneous selfish fibroblast growth factor receptor 2 (FGFR2) point mutations in human sperm? SUMMARY ANSWER: Chemotherapy and radiotherapy do not increase levels of spontaneous FGFR2 mutations in sperm but, unexpectedly, highly-sterilizing treatments dramatically reduce the levels of the disease-associated c.755C > G (Apert syndrome) mutation in sperm. WHAT IS KNOWN ALREADY: Cancer treatments lead to short-term increases in gross DNA damage (chromosomal abnormalities and DNA fragmentation) but the long-term effects, particularly at the single nucleotide resolution level, are poorly understood. We have exploited an ultra-sensitive assay to directly quantify point mutation levels at the FGFR2 locus. STUDY DESIGN, SIZE, DURATION: 'Selfish' mutations are disease-associated mutations that occur spontaneously in the sperm of most men and their levels typically increase with age. Levels of mutations at c.752-755 of FGFR2 (including c.755C > G and c.755C > T associated with Apert and Crouzon syndromes, respectively) in semen post-cancer treatment from 18 men were compared to levels in pre-treatment samples from the same individuals (n = 4) or levels in previously screened population controls (n = 99). PARTICIPANTS/MATERIALS, SETTING, METHODS: Cancer patients were stratified into four different groups based on the treatments they received and the length of time for spermatogenesis recovery. DNA extracted from semen samples was analysed using a previously established highly sensitive assay to identify mutations at positions c.752-755 of FGFR2. Five to ten micrograms of semen genomic DNA was spiked with internal controls for quantification purposes, digested with MboI restriction enzyme and gel extracted. Following PCR amplification, further MboI digestion and a nested PCR with barcoding primers, samples were sequenced on Illumina MiSeq. Mutation levels were determined relative to the spiked internal control; in individuals heterozygous for a nearby common single nucleotide polymorphism (SNP), mutations were phased to their respective alleles. MAIN RESULTS AND THE ROLE OF CHANCE: Patients treated with moderately-sterilizing alkylating regimens and who recovered spermatogenesis within <3 years after therapy (Group 3, n = 4) or non - alkylating chemotherapy and/or low gonadal radiation doses (Group 1, n = 4) had mutation levels similar to untreated controls. However, patients who had highly-sterilizing alkylating treatments (i.e. >5 years to spermatogenesis recovery) (Group 2, n = 7) or pelvic radiotherapy (Group 4, n = 3) exhibited c.755C > G mutation levels at or below background. Two patients (A and B) treated with highly-sterilizing alkylating agents demonstrated a clear reduction from pre-treatment levels; however pre-treatment samples were not available for the other patients with low mutation levels. Therefore, although based on their age we would expect detectable levels of mutations, we cannot exclude the possibility that these patients also had low mutation levels pre-treatment. In three patients with low c.755C > G levels at the first timepoint post-treatment, we observed increasing mutation levels over time. For two such patients we could phase the mutation to a nearby polymorphism (SNP) and determine that the mutation counts likely originated from a single or a small number of mutational events. LIMITATIONS, REASONS FOR CAUTION: This study was limited to 18 patients with different treatment regimens; for nine of the 18 patients, samples from only one timepoint were available. Only 12 different de novo substitutions at the FGFR2 c.752-755 locus were assessed, two of which are known to be disease associated. WIDER IMPLICATIONS OF THE FINDINGS: Our data add to the body of evidence from epidemiological studies and experimental data in humans suggesting that male germline stem cells are resilient to the accumulation of spontaneous mutations. Collectively, these data should provide physicians and health-care professionals with reassuring experimental-based evidence for counselling of male cancer patients contemplating their reproductive options several years after treatment. STUDY FUNDING/COMPETING INTEREST(S): This work was primarily supported by grants from the Wellcome (grant 091182 to AG and AOMW; grant 102 731 to AOMW), the University of Oxford Medical Sciences Division Internal Fund (grant 0005128 to GJM and AG), the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre Programme (to AG) and the US National Institutes of Health (to MLM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. None of the authors has any conflicts of interest to declare. TRIAL REGISTRATION NUMBER: NA.
Assuntos
Antineoplásicos/administração & dosagem , Sobreviventes de Câncer , Neoplasias/terapia , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Espermatozoides/efeitos da radiação , Adulto , Antineoplásicos/uso terapêutico , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Humanos , Masculino , Mutação/efeitos dos fármacos , Mutação/efeitos da radiação , Neoplasias/tratamento farmacológico , Neoplasias/radioterapia , Radioterapia , Análise do Sêmen , Contagem de Espermatozoides , Espermatogênese/efeitos dos fármacos , Espermatogênese/efeitos da radiação , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismoRESUMO
STUDY QUESTION: Can transplanted primate testicular cells form seminiferous tubules de novo, supporting complete spermatogenesis? SUMMARY ANSWER: Cryopreserved testicular cells from a prepubertal monkey can reorganize in an adult monkey recipient testis forming de novo seminiferous tubular cords supporting complete spermatogenesis. WHAT IS KNOWN ALREADY: De novo morphogenesis of testicular tissue using aggregated cells from non-primate species grafted either subcutaneously or in the testis can support spermatogenesis. STUDY DESIGN, SIZE, DURATION: Two postpubertal rhesus monkeys (Macaca mulatta) were given testicular irradiation. One monkey was given GnRH-antagonist treatment from 8 to 16 weeks after irradiation, while the other received sham injections. At 16 weeks, cryopreserved testicular cells from two different prepubertal monkeys [43 × 106 viable (Trypan-blue excluding) cells in 260 µl, and 80 × 106 viable cells in 400 µl] were transplanted via ultrasound-guided injections to one of the rete testis in each recipient, and immune suppression was given. The contralateral testis was sham transplanted. Testes were analyzed 9 months after transplantation. PARTICIPANTS/MATERIALS, SETTING, METHODS: Spermatogenic recovery was assessed by testicular volume, weight, histology and immunofluorescence. Microsatellite genotyping of regions of testicular sections obtained by LCM determined whether the cells were derived from the host or transplanted cells. MAIN RESULTS AND THE ROLE OF CHANCE: Transplanted testis of the GnRH-antagonist-treated recipient, but not the sham-treated recipient, contained numerous irregularly shaped seminiferous tubular cords, 89% of which had differentiating germ cells, including sperm in a few of them. The percentages of donor genotype in different regions of this testis were as follows: normal tubule, 0%; inflammatory, 0%; abnormal tubule region, 67%; whole interior of abnormal tubules, >99%; adluminal region of the abnormal tubules, 92%. Thus, these abnormal tubules, including the enclosed germ cells, were derived de novo from the donor testicular cells. LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: The de novo tubules were observed in only one out of the two monkeys transplanted with prepubertal donor testicular cells. WIDER IMPLICATIONS OF THE FINDINGS: These findings may represent a promising strategy for restoration of fertility in male childhood cancer survivors. The approach could be particularly useful in those exposed to therapeutic agents that are detrimental to the normal development of the tubule somatic cells affecting the ability of the endogenous tubules to support spermatogenesis, even from transplanted spermatogonial stem cells. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by research grants P01 HD075795 from Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD/NIH) to K.E.O and Cancer Center Support Grant P30 CA016672 from NCI/NIH to The University of Texas MD Anderson Cancer Center. The authors declare that they have no competing interests.
Assuntos
Túbulos Seminíferos/fisiologia , Espermatogênese/fisiologia , Testículo/citologia , Testículo/transplante , Animais , Hormônio Liberador de Gonadotropina/antagonistas & inibidores , Antagonistas de Hormônios/farmacologia , Macaca mulatta , MasculinoRESUMO
High quality fixation often inactivates epitopes and gentler fixation can fail to preserve biological structure at the required resolution. For studies of male reproduction, immunofluorescence techniques using paraformaldehyde fixation associated with paraffin as an embedding medium gives good epitope preservation, although the cell becomes morphologically compromised. On the other hand, glutaraldehyde associated with a plastic resin has been used with success to recognize and distinguish each spermatogonial cell subtype, but the antigenic sites become inaccessible to antibodies. Here we describe a new method that provides excellent morphological details of testicular cells while preserving the binding capacity of epitopes. Using a combination of glutaraldehyde and paraformaldehyde as a fixative and LR White resin for embedding, we show that it is possible to clearly recognize spermatogonial subtypes (Aund, A-A4, In and B spermatogonia) on 1-µm thick-sections and to label epitopes such as bromodeoxyuridine, a marker used for cellular cycle studies in the testis. The information gained from this procedure can be critical for understanding spermatogonial process of self-renewal and differentiation.
Assuntos
Espermatogônias/citologia , Coloração e Rotulagem/métodos , Testículo/citologia , Inclusão do Tecido/métodos , Fixação de Tecidos/métodos , Animais , Masculino , Camundongos Endogâmicos C57BLRESUMO
Ionizing radiation has been shown to arrest spermatogenesis despite the presence of surviving stem spermatogonia, by blocking their differentiation. This block is a result of damage to the somatic environment and is reversed when gonadotropins and testosterone are suppressed, but the mechanisms are still unknown. We examined spermatogonial differentiation and Sertoli cell factors that regulate spermatogonia after irradiation, during hormone suppression, and after hormone suppression combined with Leydig cell elimination with ethane dimethane sulfonate. These results showed that the numbers and cytoplasmic structure of Sertoli cells are unaffected by irradiation, only a few type A undifferentiated (Aund) spermatogonia and even fewer type A1 spermatogonia remained, and immunohistochemical analysis showed that Sertoli cells still produced KIT ligand (KITLG) and glial cell line-derived neurotrophic factor (GDNF). Some of these cells expressed KIT receptor, demonstrating that the failure of differentiation was not a result of the absence of the KIT system. Hormone suppression resulted in an increase in Aund spermatogonia within 3 days, a gradual increase in KIT-positive spermatogonia, and differentiation mainly to A3 spermatogonia after 2 weeks. KITL (KITLG) protein expression did not change after hormone suppression, indicating that it is not a factor in the stimulation. However, GDNF increased steadily after hormone suppression, which was unexpected since GDNF is supposed to promote stem spermatogonial self-renewal and not differentiation. We conclude that the primary cause of the block in spermatogonial development is not due to Sertoli cell factors such (KITL\GDNF) or the KIT receptor. As elimination of Leydig cells in addition to hormone suppression resulted in differentiation to the A3 stage within 1 week, Leydig cell factors were not necessary for spermatogonial differentiation.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Células Intersticiais do Testículo/metabolismo , Células de Sertoli/metabolismo , Espermatogênese/fisiologia , Espermatogônias/fisiologia , Fator de Células-Tronco/metabolismo , Testosterona/farmacologia , Androgênios/farmacologia , Animais , Diferenciação Celular/efeitos da radiação , Células Cultivadas , Técnicas Imunoenzimáticas , Células Intersticiais do Testículo/efeitos dos fármacos , Células Intersticiais do Testículo/efeitos da radiação , Masculino , Ratos , Células de Sertoli/efeitos dos fármacos , Células de Sertoli/efeitos da radiação , Espermatogênese/efeitos dos fármacos , Espermatogênese/efeitos da radiação , Espermatogônias/efeitos dos fármacos , Espermatogônias/efeitos da radiaçãoRESUMO
Meiosis is unique to germ cells and essential for reproduction. During the first meiotic division, homologous chromosomes pair, recombine, and form chiasmata. The homologues connect via axial elements and numerous transverse filaments to form the synaptonemal complex. The synaptonemal complex is a critical component for chromosome pairing, segregation, and recombination. We previously identified a novel germ cell-specific HORMA domain encoding gene, Hormad1, a member of the synaptonemal complex and a mammalian counterpart to the yeast meiotic HORMA domain protein Hop1. Hormad1 is essential for mammalian gametogenesis as knockout male and female mice are infertile. Hormad1 deficient (Hormad1(-/) (-)) testes exhibit meiotic arrest in the early pachytene stage, and synaptonemal complexes cannot be visualized by electron microscopy. Hormad1 deficiency does not affect localization of other synaptonemal complex proteins, SYCP2 and SYCP3, but disrupts homologous chromosome pairing. Double stranded break formation and early recombination events are disrupted in Hormad1(-/) (-) testes and ovaries as shown by the drastic decrease in the γH2AX, DMC1, RAD51, and RPA foci. HORMAD1 co-localizes with γH2AX to the sex body during pachytene. BRCA1, ATR, and γH2AX co-localize to the sex body and participate in meiotic sex chromosome inactivation and transcriptional silencing. Hormad1 deficiency abolishes γH2AX, ATR, and BRCA1 localization to the sex chromosomes and causes transcriptional de-repression on the X chromosome. Unlike testes, Hormad1(-/) (-) ovaries have seemingly normal ovarian folliculogenesis after puberty. However, embryos generated from Hormad1(-/) (-) oocytes are hyper- and hypodiploid at the 2 cell and 8 cell stage, and they arrest at the blastocyst stage. HORMAD1 is therefore a critical component of the synaptonemal complex that affects synapsis, recombination, and meiotic sex chromosome inactivation and transcriptional silencing.
Assuntos
Proteínas de Ciclo Celular/genética , Segregação de Cromossomos/genética , Meiose/genética , Mutação/genética , Recombinação Genética/genética , Complexo Sinaptonêmico/metabolismo , Aneuploidia , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Desenvolvimento Embrionário/genética , Feminino , Feto/citologia , Feto/metabolismo , Masculino , Camundongos , Proteínas Nucleares/metabolismo , Oócitos/citologia , Oócitos/metabolismo , Especificidade de Órgãos/genética , Ovário/crescimento & desenvolvimento , Ovário/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Cromossomos Sexuais/genética , Espermatogênese/genética , Espermatozoides/citologia , Espermatozoides/metabolismo , Espermatozoides/ultraestrutura , Complexo Sinaptonêmico/ultraestrutura , Proteínas Supressoras de Tumor/metabolismoRESUMO
Homeobox genes encode transcription factors that regulate diverse developmental events. The largest known homeobox gene cluster - the X-linked mouse reproductive homeobox (Rhox) cluster - harbors genes whose expression patterns and functions are largely unknown. Here, we report that a member of this cluster, Rhox10, is expressed in male germ cells. Rhox10 is highly transcribed in spermatogonia in vivo and is upregulated in response to the differentiation-inducing agent retinoic acid in vitro. Using a specific RHOX10 antiserum that we generated, we found that RHOX10 protein is selectively expressed in fetal gonocytes, germline stem cells, spermatogonia, and early spermatocytes. RHOX10 protein undergoes a dramatic shift in subcellular localization as germ cells progress from mitotically arrested gonocytes to mitotic spermatogonia and from mitotic spermatogonia to early meiotic spermatocytes, consistent with RHOX10 performing different functions in these stages.
Assuntos
Epididimo/metabolismo , Proteínas de Homeodomínio/metabolismo , Espermatócitos/metabolismo , Espermatogênese , Espermatogônias/metabolismo , Células-Tronco/metabolismo , Animais , Células Cultivadas , Epididimo/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Imuno-Histoquímica , Masculino , Meiose , Camundongos , Microscopia de Fluorescência , Mitose , Transporte Proteico , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espermatogênese/genética , Transcrição GênicaRESUMO
Despite the identification of H2A.Bbd as a new vertebrate-specific replacement histone variant several years ago, and despite the many in vitro structural characterizations using reconstituted chromatin complexes consisting of this variant, the existence of H2A.Bbd in the cell and its location has remained elusive. Here, we report that the native form of this variant is present in highly advanced spermiogenic fractions of mammalian testis at the time when histones are highly acetylated and being replaced by protamines. It is also present in the nucleosomal chromatin fraction of mature human sperm. The ectopically expressed non-tagged version of the protein is associated with micrococcal nuclease-refractory insoluble fractions of chromatin and in mouse (20T1/2) cell line, H2A.Bbd is enriched at the periphery of chromocenters. The exceedingly rapid evolution of this unique X-chromosome-linked histone variant is shared with other reproductive proteins including those associated with chromatin in the mature sperm (protamines) of many vertebrates. This common rate of evolution provides further support for the functional and structural involvement of this protein in male gametogenesis in mammals.
Assuntos
Genes Ligados ao Cromossomo X , Histonas/genética , Histonas/fisiologia , Espermatogênese , Animais , Linhagem Celular , Cromatina/química , Células HeLa , Histonas/metabolismo , Humanos , Masculino , Camundongos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiologia , Seleção Genética , Espermatogênese/genética , Espermatozoides/metabolismo , Testículo/metabolismoRESUMO
Concordant transcriptional regulation can generate multiple gene products that collaborate to achieve a common goal. Here we report a case of concordant transcriptional regulation that instead drives a single protein to be produced in the same cell type from divergent promoters. This gene product-the RHOX5 homeobox transcription factor-is translated from 2 different mRNAs with different 5' untranslated regions (UTRs) transcribed from alternative promoters. Despite the fact that these 2 promoters-the proximal promoter (Pp) and the distal promoter (Pd)-exhibit different patterns of tissue-specific activity, share no obvious sequence identity, and depend on distinct transcription factors for expression, they exhibit a remarkably similar expression pattern in the testes. In particular, both depend on androgen signaling for expression in the testes, where they are specifically expressed in Sertoli cells and have a similar stage-specific expression pattern during the seminiferous epithelial cycle. We report evidence for 3 mechanisms that collaborate to drive concordant Pp/Pd expression. First, both promoters have an intrinsic ability to respond to androgen receptor and androgen. Second, the Pp acts as an enhancer to promote androgen-dependent transcription from the Pd. Third, Pd transcription is positively autoregulated by the RHOX5 protein, which is first produced developmentally from the Pp. Together, our data support a model in which the Rhox5 homeobox gene evolved multiple mechanisms to activate both of its promoters in Sertoli cells to produce Rhox5 in an androgen-dependent manner during different phases of spermatogenesis.
Assuntos
Androgênios/metabolismo , Regulação da Expressão Gênica , Proteínas de Homeodomínio/genética , Regiões Promotoras Genéticas , Células de Sertoli/metabolismo , Fatores de Transcrição/genética , Regiões 5' não Traduzidas , Animais , Metilação de DNA , Genes Homeobox , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Plasmídeos/metabolismo , Isoformas de Proteínas , Receptores Androgênicos/metabolismo , Túbulos Seminíferos/metabolismo , Espermatogênese , Testículo/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Spermatogenesis, a fundamental process in the male reproductive system, requires a series of tightly controlled epigenetic and genetic events in germ cells ranging from spermatogonia to spermatozoa. Jmjd1a is a key epigenetic regulator expressed in the testis. It specifically demethylates mono- and di-methylated histone H3 lysine 9 (H3K9me1 and H3K9me2) but not tri-methylated H3K9 (H3K9me3). In this study, we generated a Jmjd1a antibody for immunohistochemistry and found Jmjd1a was specifically produced in pachytene and secondary spermatocytes. Disruption of the Jmjd1a gene in mice significantly increased H3K9me1 and H3K9me2 levels in pachytene spermatocytes and early elongating spermatids without affecting H3K9me3 levels. Concurrently, the levels of histone acetylation were decreased in Jmjd1a knock-out germ cells. This suggests Jmjd1a promotes transcriptional activation by lowering histone methylation and increasing histone acetylation. Interestingly, the altered histone modifications in Jmjd1a-deficient germ cells caused diminished cAMP-response element modulator (Crem) recruitment to chromatin and decreased expression of the Crem coactivator Act and their target genes Tnp1 (transition protein 1), Tnp2, Prm1 (protamine 1), and Prm2, all of which are essential for chromatin condensation in spermatids. In agreement with these findings, Jmjd1a deficiency caused extensive germ cell apoptosis and blocked spermatid elongation, resulting in severe oligozoospermia, small testes, and infertility in male mice. These results indicate that the Jmjd1a-controlled epigenetic histone modifications are crucial for Crem-regulated gene expression and spermatogenesis.
Assuntos
Modulador de Elemento de Resposta do AMP Cíclico/genética , Histonas/metabolismo , Infertilidade Masculina/fisiopatologia , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Espermatogênese/fisiologia , Animais , Anticorpos , Apoptose/fisiologia , Copulação/fisiologia , Modulador de Elemento de Resposta do AMP Cíclico/metabolismo , Células-Tronco Embrionárias/fisiologia , Epigênese Genética/fisiologia , Feminino , Regulação da Expressão Gênica/fisiologia , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Histona Desmetilases com o Domínio Jumonji/imunologia , Masculino , Metilação , Camundongos , Camundongos Knockout , Oligospermia/metabolismo , Oligospermia/patologia , Oligospermia/fisiopatologia , Coelhos , Espermátides/citologia , Espermátides/fisiologia , Espermatócitos/citologia , Espermatócitos/fisiologiaRESUMO
Epigenetic modifications, and methylation of histones in particular, dynamically change during spermatogenesis. Among various methylations of histone H3, methylation of histone H3 lysine 9 (H3K9) and its regulation are essential for spermatogenesis. Trimethytransferases as well as dimethyltransferase are required for meiotic progression. In addition, didemethylase of H3K9 is also critical for spermatogenesis through transcriptional regulation of spermatid-specific genes. However, the requirement for demethylation of trimethylated H3K9 (H3K9me3) during spermatogenesis remains to be elucidated. Here, we report the targeted disruption of KDM4D, a testis-enriched tridemethylase of H3K9. Kdm4d-null mice are viable and fertile and do not show any obvious phenotype. However, H3K9me3 accumulates significantly in Kdm4d-null round spermatids, and the distribution of methylated H3K9 in germ cells is dramatically changed. Nevertheless, the progression of spermatogenesis and the number of spermatozoa are normal, likely secondary to the earlier nuclear localization of another H3K9 tridemethylase, KDM4B, in Kdm4d-null elongating spermatids. These results suggest that demethylation of H3K9me3 in round spermatids is dispensable for spermatogenesis but that possible defects in Kdm4d-null elongating spermatids could be rescued by functional redundancy of the KDM4B demethylase.
Assuntos
Fertilidade/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Histona Desmetilases/metabolismo , Histonas/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Espermatogênese/fisiologia , Testículo/metabolismo , Animais , Histona Desmetilases/genética , Histona Desmetilases com o Domínio Jumonji/genética , Masculino , Metilação , Camundongos , Camundongos Knockout , Espermatozoides/fisiologiaRESUMO
Spermatogenesis is dependent primarily on testosterone action on the Sertoli cells, but the molecular mechanisms have not been identified. Attempts to identify testosterone-regulated target genes in Sertoli cells have used microarray analysis of gene expression in mice lacking the androgen receptor (AR) in Sertoli cells (SCARKO) and wild-type mice, but the analyses have been complicated both by alteration of germ cell composition of the testis when pubertal or adult mice were used and by differences in Sertoli-cell gene expression from the expression in adults when prepubertal mice were used. To overcome these limitations and identify AR-regulated genes in adult Sertoli cells, we compared gene expression in adult jsd (Utp14b jsd/jsd, juvenile spermatogonial depletion) mouse testes and with that in SCARKO-jsd mouse testes, since their cellular compositions are essentially identical, consisting of only type A spermatogonia and somatic cells. Microarray analysis identified 157 genes as downregulated and 197 genes as upregulated in the SCARKO-jsd mice compared to jsd mice. Some of the AR-regulated genes identified in the previous studies, including Rhox5, Drd4, and Fhod3, were also AR regulated in the jsd testes, but others, such as proteases and components of junctional complexes, were not AR regulated in our model. Surprisingly, a set of germ cellspecific genes preferentially expressed in differentiated spermatogonia and meiotic cells, including Meig1, Sycp3, and Ddx4, were all upregulated about 2-fold in SCARKO-jsd testes. AR-regulated genes in Sertoli cells must therefore be involved in the regulation of spermatogonial differentiation, although there was no significant differentiation to spermatocytes in SCARKO-jsd mice. Further gene ontogeny analysis revealed sets of genes whose changes in expression may be involved in the dislocation of Sertoli cell nuclei in SCARKO-jsd testes.
Assuntos
Expressão Gênica , Mutação , Receptores Androgênicos/deficiência , Ribonucleoproteínas Nucleolares Pequenas/genética , Células de Sertoli/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular/fisiologia , RNA Helicases DEAD-box/metabolismo , Proteínas de Ligação a DNA , Feminino , Masculino , Meiose , Camundongos , Camundongos Knockout , Análise em Microsséries , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Células de Sertoli/citologia , Espermatócitos/citologia , Espermatogônias/citologia , Testículo , Regulação para CimaRESUMO
To achieve the specialized nuclear structure in sperm necessary for fertilization, dramatic chromatin reorganization steps in developing spermatids are required where histones are largely replaced first by transition proteins and then by protamines. This entails the transient formation of DNA strand breaks to allow for, first, DNA relaxation and then chromatin compaction. However, the nature and origin of these breaks are not well understood. We previously reported that these DNA strand breaks trigger the activation of poly(ADP-ribose) (PAR) polymerases PARP1 and PARP2 and that interference with PARP activation causes poor chromatin integrity with abnormal retention of histones in mature sperm and impaired embryonic survival. Here we show that the activity of topoisomerase II beta (TOP2B), an enzyme involved in DNA strand break formation in elongating spermatids, is strongly inhibited by the activity of PARP1 and PARP2 in vitro, and this is in turn counteracted by the PAR-degrading activity of PAR glycohydrolase. Moreover, genetic and pharmacological PARP inhibition both lead to increased TOP2B activity in murine spermatids in vivo as measured by covalent binding of TOP2B to the DNA. In summary, the available data suggest a functional relationship between the DNA strand break-generating activity of TOP2B and the DNA strand break-dependent activation of PARP enzymes that in turn inhibit TOP2B. Because PARP activity also facilitates histone H1 linker removal and local chromatin decondensation, cycles of PAR formation and degradation may be necessary to coordinate TOP2B-dependent DNA relaxation with histone-to-protamine exchange necessary for spermatid chromatin remodeling.
Assuntos
Montagem e Desmontagem da Cromatina , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Espermátides/metabolismo , Espermatogênese , Animais , Antígenos de Neoplasias/metabolismo , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Etoposídeo/farmacologia , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Concentração Osmolar , Fenantrenos/farmacologia , Poli(ADP-Ribose) Polimerase-1 , Poli Adenosina Difosfato Ribose/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases , Poli(ADP-Ribose) Polimerases/genética , Proteínas de Ligação a Poli-ADP-Ribose , Espermátides/citologia , Espermátides/efeitos dos fármacosRESUMO
Despite numerous observations of the effects of estrogens on spermatogenesis, identification of estrogen-regulated genes in the testis is limited. Using rats in which irradiation had completely blocked spermatogonial differentiation, we previously showed that testosterone suppression with gonadotropin-releasing hormone-antagonist acyline and the antiandrogen flutamide stimulated spermatogenic recovery and that addition of estradiol (E2) to this regimen accelerated this recovery. We report here the global changes in testicular cell gene expression induced by the E2 treatment. By minimizing the changes in other hormones and using concurrent data on regulation of the genes by these hormones, we were able to dissect the effects of estrogen on gene expression, independent of gonadotropin or testosterone changes. Expression of 20 genes, largely in somatic cells, was up- or downregulated between 2- and 5-fold by E2. The unexpected and striking enrichment of transcripts not corresponding to known genes among the E2-downregulated probes suggested that these might represent noncoding mRNAs; indeed, we have identified several as miRNAs and their potential target genes in this system. We propose that genes for which expression levels are altered in one direction by irradiation and in the opposite direction by both testosterone suppression and E2 treatment are candidates for controlling the block in differentiation. Several genes, including insulin-like 3 (Insl3), satisfied those criteria. If they are indeed involved in the inhibition of spermatogonial differentiation, they may be candidate targets for treatments to enhance recovery of spermatogenesis following gonadotoxic exposures, such as those resulting from cancer therapy.
Assuntos
Estradiol/uso terapêutico , Estrogênios/uso terapêutico , Regulação da Expressão Gênica/efeitos dos fármacos , Espermatogênese/efeitos dos fármacos , Espermatogênese/efeitos da radiação , Testículo/efeitos dos fármacos , Testículo/metabolismo , Antagonistas de Androgênios/uso terapêutico , Animais , Cruzamentos Genéticos , Quimioterapia Combinada , Flutamida/uso terapêutico , Raios gama , Regulação da Expressão Gênica/efeitos da radiação , Hormônio Liberador de Gonadotropina/antagonistas & inibidores , Antagonistas de Hormônios/uso terapêutico , Insulina/genética , Insulina/metabolismo , Masculino , MicroRNAs/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Oligopeptídeos/uso terapêutico , Proteínas/genética , Proteínas/metabolismo , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos Lew , Testículo/patologia , Testículo/efeitos da radiação , Testosterona/antagonistas & inibidoresRESUMO
Sperm chromatin is organized in a protamine-based, highly condensed form, which protects the paternal chromosome complement in transit, facilitates fertilization, and supports correct gene expression in the early embryo. Very few histones remain selectively associated with genes and defined regulatory sequences essential to embryonic development, while most of the genome becomes bound to protamine during spermiogenesis. Chromatin remodeling processes resulting in the dramatically different nuclear structure of sperm are poorly understood. This study shows that perturbation of poly(ADP-ribose) (PAR) metabolism, which is mediated by PAR polymerases and PAR glycohydrolase in response to naturally occurring endogenous DNA strand breaks during spermatogenesis, results in the abnormal retention of core histones and histone linker HIST1H1T (H1t) and H1-like linker protein HILS1 in mature sperm. Moreover, genetic or pharmacological alteration of PAR metabolism caused poor sperm chromatin quality and an abnormal nuclear structure in mice, thus reducing male fertility.
Assuntos
Nucleoproteínas/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Espermatogênese/fisiologia , Animais , Animais Geneticamente Modificados , Núcleo Celular/metabolismo , Montagem e Desmontagem da Cromatina/fisiologia , Quebras de DNA , Proteínas de Ligação a DNA/metabolismo , Glicosídeo Hidrolases/metabolismo , Histonas/metabolismo , Masculino , Camundongos , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/metabolismo , Espermátides/fisiologia , Espermatozoides/metabolismoRESUMO
Despite the knowledge of spermatogonial biology in adult mice, spermatogonial development in immature animals has not been fully characterized. Thus, the aim of this study was to evaluate the ontogeny of the morphological development of the spermatogonial lineage in C57BL/6 mouse testis, using high-resolution light microscopy. Spermatogonial morphology, chronology, and absolute number were determined for different ages postpartum (pp). The morphology of spermatogonia in immature mice was similar to that of adult spermatogonia, although their nuclear diameter was slightly smaller. The A(1) spermatogonia were first observed on day 2 pp, and only 24 h later, differentiating type A(3) and A(4) spermatogonia were observed in the seminiferous cords. This result indicated a shortening of the spermatogonial phase for immature mice of about â¼2.5 days when compared with adult mice and suggests that gonocytes and/or A(1) spermatogonia could directly become A(4) spermatogonia, skipping the developmental sequence of type A spermatogonia. These A(4) spermatogonia are functional as they develop into type B spermatogonia by day 5 pp. At day 8 pp, while differentiation to spermatocytes begins, the A(und) spermatogonia reach their maximal numbers, which are maintained through adulthood. The various details of the spermatogonial behavior in immature normal mice described in this study can be used as a baseline for further studies under experimental or pathological conditions.
Assuntos
Espermatogênese , Espermatogônias/crescimento & desenvolvimento , Espermatogônias/ultraestrutura , Testículo/crescimento & desenvolvimento , Testículo/ultraestrutura , Animais , Animais Recém-Nascidos , Apoptose , Peso Corporal , Contagem de Células , Tamanho do Núcleo Celular , Forma Celular , Cinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia/métodos , Índice Mitótico , Tamanho do Órgão , Espermatozoides/crescimento & desenvolvimento , Espermatozoides/ultraestruturaRESUMO
BACKGROUND: Cancer treatment of prepubertal patients impacts future fertility due to the abolition of spermatogonial stem cells (SSCs). In macaques, spermatogenesis could be regenerated by intratesticular transplantation of SSCs, but no studies have involved cytotoxic treatment before puberty and transplantation after puberty, which would be the most likely clinical scenario. OBJECTIVES: To evaluate donor-derived functional sperm production after SSC transplantation to adult monkeys that had received testicular irradiation during the prepubertal period. MATERIALS AND METHODS: We obtained prepubertal testis tissue by unilaterally castrating six prepubertal monkeys and 2 weeks later irradiated the remaining testes with 6.9 Gy. However, because spermatogenic recovery was observed, we irradiated them again 14 months later with 7 Gy. Three of the monkeys were treated with GnRH-antagonist (GnRH-ant) for 8 weeks. The cryopreserved testis cells from the castrated testes were then allogeneically transplanted into the intact testes of all monkeys. Tissues were harvested 10 months later for analyses. RESULTS: In three of the six monkeys, 61%, 38%, and 11% of the epididymal sperm DNA were of the donor genotype. The ability to recover donor-derived sperm production was not enhanced by the GnRH-ant pretreatment. However, the extent of filling seminiferous tubules during the transplantation procedure was correlated with the eventual production of donor spermatozoa. The donor epididymal spermatozoa from the recipient with 61% donor contribution were capable of fertilizing rhesus eggs and forming embryos. Although the transplantation was done into the rete testis, two GnRH-ant-treated monkeys, which did not produce donor-derived epididymal spermatozoa, displayed irregular tubular cords in the interstitium containing testicular spermatozoa derived from the transplanted donor cells. DISCUSSION AND CONCLUSION: The results further support that sperm production can be restored in non-human primates from tissues cryopreserved prior to prepubertal and post-pubertal gonadotoxic treatment by transplantation of these testicular cells after puberty into seminiferous tubules.
Assuntos
Células-Tronco Germinativas Adultas/transplante , Puberdade/efeitos da radiação , Lesões Experimentais por Radiação/terapia , Espermatogênese/efeitos da radiação , Transplante de Células-Tronco , Animais , Criopreservação , Hormônio Liberador de Gonadotropina/antagonistas & inibidores , Antagonistas de Hormônios/administração & dosagem , Macaca mulatta , Masculino , Lesões Experimentais por Radiação/fisiopatologia , Túbulos Seminíferos , Espermatozoides/efeitos da radiação , Testículo/fisiopatologia , Testículo/efeitos da radiaçãoRESUMO
Spermatogenesis is dependent primarily on testosterone action on the Sertoli cells, but the molecular mechanisms have not been identified. Attempts to identify testosterone-regulated target genes in Sertoli cells have used microarray analysis of gene expression in mice lacking the androgen receptor (AR) in Sertoli cells (SCARKO) and wild-type mice, but the analyses have been complicated both by alteration of germ cell composition of the testis when pubertal or adult mice were used and by differences in Sertoli-cell gene expression from the expression in adults when prepubertal mice were used. To overcome these limitations and identify AR-regulated genes in adult Sertoli cells, we compared gene expression in adult jsd (Utp14b(jsd/jsd), juvenile spermatogonial depletion) mouse testes and with that in SCARKO-jsd mouse testes, since their cellular compositions are essentially identical, consisting of only type A spermatogonia and somatic cells. Microarray analysis identified 157 genes as downregulated and 197 genes as upregulated in the SCARKO-jsd mice compared to jsd mice. Some of the AR-regulated genes identified in the previous studies, including Rhox5, Drd4, and Fhod3, were also AR regulated in the jsd testes, but others, such as proteases and components of junctional complexes, were not AR regulated in our model. Surprisingly, a set of germ cell-specific genes preferentially expressed in differentiated spermatogonia and meiotic cells, including Meig1, Sycp3, and Ddx4, were all upregulated about 2-fold in SCARKO-jsd testes. AR-regulated genes in Sertoli cells must therefore be involved in the regulation of spermatogonial differentiation, although there was no significant differentiation from spermatocytes in SCARKO-jsd mice. Further gene ontogeny analysis revealed sets of genes whose changes in expression may be involved in the dislocation of Sertoli cell nuclei in SCARKO-jsd testes.
Assuntos
Regulação da Expressão Gênica , Receptores Androgênicos/fisiologia , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Células de Sertoli/metabolismo , Espermatogênese , Animais , Perfilação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Camundongos Transgênicos , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Especificidade de Órgãos , RNA Mensageiro/metabolismo , Receptores Androgênicos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonucleoproteínas Nucleolares Pequenas/genética , Testículo/citologia , Testículo/metabolismo , Testosterona/metabolismoRESUMO
Although gonadotropins and androgen are required for normal spermatogenesis and both testosterone and follicle-stimulating hormone (FSH) are responsible for the inhibition of spermatogonial differentiation that occurs in irradiated rats, it has been difficult to identify the specific genes involved. To study specific hormonally regulated changes in somatic cell gene expression in the testis that may be involved in these processes, without the complication of changing populations of germ cells, we used irradiated LBNF(1) rats, the testes of which contain almost exclusively somatic cells except for a few type A spermatogonia. Three different groups of these rats were treated with various combinations of gonadotropin-releasing hormone antagonist, an androgen receptor antagonist (flutamide), testosterone, and FSH, and we compared the gene expression levels 2 wk later to those of irradiated-only rats by microarray analysis. By dividing the gene expression patterns into three major patterns and 11 subpatterns, we successfully distinguished, in a single study, the genes that were specifically regulated by testosterone, by luteinizing hormone (LH), and by FSH from the large number of genes that were not hormonally regulated in the testis. We found that hormones produced more dramatic upregulation than downregulation of gene expression: Testosterone had the strongest upregulatory effect, LH had a modest but appreciable upregulatory effect, and FSH had a minor upregulatory effect. We also separately identified the somatic cell genes that were chronically upregulated by irradiation. Thus, the present study identified gene expression changes that may be responsible for hormonal action on somatic cells to support normal spermatogenesis and the hormone-mediated block in spermatogonial development after irradiation.
Assuntos
Hormônio Foliculoestimulante/metabolismo , Regulação da Expressão Gênica , Hormônio Luteinizante/metabolismo , Testículo/metabolismo , Testosterona/farmacologia , Animais , Flutamida/farmacologia , Raios gama , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos da radiação , Células Germinativas/efeitos dos fármacos , Células Germinativas/metabolismo , Células Germinativas/efeitos da radiação , Hormônio Liberador de Gonadotropina/antagonistas & inibidores , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Oligopeptídeos/farmacologia , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Testículo/efeitos dos fármacos , Testículo/efeitos da radiação , Testosterona/sangueRESUMO
BACKGROUND: Men who have just started cytotoxic therapy for cancer are uncertain and concerned about whether spermatozoa collected or pregnancies occurring during therapy might be transmitting genetic damage to offspring. There are no comprehensive guidelines on the risks of different doses of the various cytotoxic, and usually genotoxic, antineoplastic agents. OBJECTIVES: To develop a schema showing the risks of mutagenic damage when spermatozoa, exposed to various genotoxic agents during spermatogenesis, are collected or used to produce a pregnancy. MATERIALS AND METHODS: A comprehensive literature review was performed updating the data on genetic and epigenetic effects of genotoxic agents on animal and human spermatozoa exposed during spermatogenic development. RESULTS: Relevant data on human spermatozoa and offspring are extremely limited, but there are extensive genetic studies in experimental animals that define sensitivities for specific drugs and times. The animal data were extrapolated to humans based on the stage when the cells were exposed and the relative kinetics of spermatogenesis and were consistent with the limited human data. In humans, alkylating agents and radiation should already induce a high risk of mutations in spermatozoa produced within 1 or 2 weeks after initiation of therapy. Topoisomerase II inhibitors and possibly microtubule inhibitors produce the greatest risk at weeks 5-7 of therapy. Nucleoside analogs, antimetabolites, and bleomycin exert their mutagenic effects on spermatozoa collected at 7-10 weeks of therapy. DISCUSSION AND CONCLUSIONS: A schema showing the time from initiation of therapy at which specific antineoplastic agents can cause significant levels of genetic damage in conceptuses and live offspring was developed. The estimates and methods for computing the level of such risk from an individual patient's treatment regimen will enable patients and counselors to make informed decisions on the use of spermatozoa or continuation of a pregnancy.