RESUMO
The cytidine deaminases APOBEC3A (A3A) and APOBEC3B (A3B) are prominent mutators of human cancer genomes. However, tumor-specific genetic modulators of APOBEC-induced mutagenesis are poorly defined. Here, we used a screen to identify 61 gene deletions that increase A3B-induced mutations in yeast. We also determined whether each deletion was epistatic with Ung1 loss, which indicated whether the encoded factors participate in the homologous recombination (HR)-dependent bypass of A3B/Ung1-dependent abasic sites or suppress A3B-catalyzed deamination by protecting against aberrant formation of single-stranded DNA (ssDNA). We found that the mutation spectra of A3B-induced mutations revealed genotype-specific patterns of strand-specific ssDNA formation and nucleotide incorporation across APOBEC-induced lesions. Combining these three metrics, we were able to establish a multifactorial signature of APOBEC-induced mutations specific to (1) failure to remove H3K56 acetylation, (2) defective CTF18-RFC complex function, and (3) defective HR-mediated bypass of APOBEC-induced lesions. We extended these results by analyzing mutation data for human tumors and found BRCA1/2-deficient breast cancers display three- to fourfold more APOBEC-induced mutations. Mirroring our results in yeast, Rev1-mediated C-to-G substitutions are mainly responsible for increased APOBEC-signature mutations in BRCA1/2-deficient tumors, and these mutations associate with lagging strand synthesis during replication. These results identify important factors that influence DNA replication dynamics and likely the abundance of APOBEC-induced mutation during tumor progression. They also highlight a novel role for BRCA1/2 during HR-dependent lesion bypass of APOBEC-induced lesions during cancer cell replication.
Assuntos
Proteína BRCA1 , Neoplasias da Mama , Humanos , Feminino , Proteína BRCA1/genética , Saccharomyces cerevisiae/genética , Proteína BRCA2/genética , Mutagênese , Mutação , Citidina Desaminase/genética , Neoplasias da Mama/genética , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismoRESUMO
APOBEC cytidine deaminases are the second-most prominent source of mutagenesis in sequenced tumors. Previous studies have proposed that APOBEC3B (A3B) is the major source of mutagenesis in breast cancer (BRCA). We show that APOBEC3A (A3A) is the only APOBEC whose expression correlates with APOBEC-induced mutation load and that A3A expression is responsible for cytidine deamination in multiple BRCA cell lines. Comparative analysis of A3A and A3B expression by qRT-PCR, RSEM-normalized RNA-seq, and unambiguous RNA-seq validated the use of RNA-seq to measure APOBEC expression, which indicates that A3A is the primary correlate with APOBEC-mutation load in primary BRCA tumors. We also demonstrate that A3A has >100-fold more cytidine deamination activity than A3B in the presence of cellular RNA, likely explaining why higher levels of A3B expression contributes less to mutagenesis in BRCA. Our findings identify A3A as a major source of cytidine deaminase activity in breast cancer cells and possibly a prominent contributor to the APOBEC mutation signature.
Assuntos
Neoplasias da Mama/genética , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Proteínas/genética , Proteínas/metabolismo , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismo , Mutação , Análise de Sequência de RNARESUMO
Spermatogenesis in mammals is a very complex, highly organized process, regulated in part by testosterone and retinoic acid (RA). Much is known about how RA and testosterone signaling pathways independently regulate this process, but there is almost no information regarding whether these two signaling pathways directly interact and whether RA is crucial for steroidogenic cell function. This study uses a transgenic mouse line that expresses a dominant-negative form of RA receptor α (RAR-DN) and the steroidogenic cell-specific Cre mouse line, Cyp17iCre, to generate male mice with steroidogenic cells unable to perform RA signaling. Testes of mutant mice displayed increased apoptosis of pachytene spermatocytes, an increased number of macrophages in the interstitium and a loss of advanced germ cells. Additionally, blocking RA signaling in Leydig cells resulted in increased permeability of the blood-testis barrier, decreased levels of the steroidogenic enzyme cytochrome P450 17a1 and decreased testosterone levels. Surprisingly, the epididymides of the mutant mice also displayed an abnormal phenotype. This study demonstrates that RA signaling is required in steroidogenic cells for their normal function and, thus, for male fertility.
Assuntos
Barreira Hematotesticular/metabolismo , Fertilidade/fisiologia , Receptor alfa de Ácido Retinoico/metabolismo , Transdução de Sinais/fisiologia , Espermatócitos/metabolismo , Espermatogênese/fisiologia , Animais , Barreira Hematotesticular/citologia , Masculino , Camundongos , Camundongos Transgênicos , Receptor alfa de Ácido Retinoico/genética , Espermatócitos/citologia , Esteroide 17-alfa-Hidroxilase/genética , Esteroide 17-alfa-Hidroxilase/metabolismoRESUMO
The onset of spermatogenesis occurs in response to retinoic acid (RA), the active metabolite of vitamin A. However, whether RA plays any role during establishment of the spermatogonial stem cell (SSC) pool is unknown. Because designation of the SSC population and the onset of RA signaling in the testis that induces differentiation have similar timing, this study asked whether RA influenced SSC establishment. Whole mount immunofluorescence and flow cytometric analysis using the Id4-eGfp transgenic reporter mouse line revealed an enrichment for ID4-EGFP+ cells within the testis following inhibition of RA synthesis by WIN 18,446 treatment. Transplantation analyses confirmed a significant increase in the number of SSCs in testes from RA-deficient animals. Conversely, no difference in the ID4-EGFP+ population or change in SSC number were detected following exposure to an excess of RA. Collectively, reduced RA altered the number of SSCs present in the neonatal testis but precocious RA exposure in the neonatal testis did not, suggesting that RA deficiency causes a greater proportion of progenitor undifferentiated spermatogonia to retain their SSC state past the age when the pool is thought to be determined.
Assuntos
Espermatogênese/fisiologia , Tretinoína/metabolismo , Células-Tronco Germinativas Adultas/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Masculino , Camundongos , Camundongos Transgênicos , Transdução de Sinais/efeitos dos fármacos , Espermatogênese/genética , Espermatogônias/citologia , Testículo/metabolismoRESUMO
Spermatogenesis in mammals occurs in a very highly organized manner within the seminiferous epithelium regulated by different cell types in the testis. Testosterone produced by Leydig cells regulates blood-testis barrier formation, meiosis, spermiogenesis, and spermiation. However, it is unknown whether Leydig cell function changes with the different stages of the seminiferous epithelium. This study utilized the WIN 18,446 and retinoic acid (RA) treatment regime combined with the RiboTag mouse methodology to synchronize male germ cell development and allow for the in vivo mapping of the Leydig cell translatome across the different stages of one cycle of the seminiferous epithelium. Using microarrays analysis, we identified 11 Leydig cell-enriched genes that were expressed in stage-specific manner such as the glucocorticoid synthesis and transport genes, Cyp21a1 and Serpina6. In addition, there were nine Leydig cell transcripts that change their association with polysomes in correlation with the different stages of the spermatogenic cycle including Egr1. Interestingly, the signal intensity of EGR1 and CYP21 varied among Leydig cells in the adult asynchronous testis. However, testosterone levels across the different stages of germ cell development did not cycle. These data show, for the first time, that Leydig cell gene expression changes in a stage-specific manner during the cycle of the seminiferous epithelium and indicate that a heterogeneous Leydig cell population exists in the adult mouse testis.
Assuntos
Células Intersticiais do Testículo/metabolismo , Polirribossomos/metabolismo , Espermatogênese/fisiologia , Testículo/metabolismo , Animais , Barreira Hematotesticular , Expressão Gênica , Células Intersticiais do Testículo/citologia , Masculino , Camundongos , Epitélio Seminífero/citologia , Epitélio Seminífero/metabolismo , Esteroide 21-Hidroxilase/genética , Esteroide 21-Hidroxilase/metabolismo , Testículo/citologia , Transcortina/genética , Transcortina/metabolismoRESUMO
Retinoic acid (RA), the active metabolite of vitamin A, is known to be required for the differentiation of spermatogonia. The first round of spermatogenesis initiates in response to RA and occurs in patches along the length of the seminiferous tubule. However, very little is known about the individual differentiating spermatogonial populations and their progression through the cell cycle due to the heterogeneous nature of the onset of spermatogenesis. In this study, we utilized WIN 18,446 and RA as tools to generate testes enriched with different populations of spermatogonia to further investigate 1) the undifferentiated to differentiating spermatogonial transition, 2) the progression of the differentiating spermatogonia through the cell cycle, and 3) Sertoli cell number in response to altered RA levels. WIN 18,446/RA-treated neonatal mice were used to determine when synchronous S phases occurred in the differentiating spermatogonial population following treatment. Five differentiating spermatogonial S phase windows were identified between spermatogonial differentiation and formation of preleptotene spermatocytes. In addition, a slight increase in Sertoli cell number was observed following RA treatment, possibly implicating a role for RA in Sertoli cell cycle progression. This study has enhanced our understanding of the spermatogonial populations present in the neonatal testis during the onset of spermatogenesis by mapping the cell cycle kinetics of both the undifferentiated and the differentiating spermatogonial populations and identifying the precise timing of when specific individual differentiating spermatogonial populations are enriched within the testis following synchrony, thus providing an essential tool for further study of the differentiating spermatogonia.
Assuntos
Espermatogênese/efeitos dos fármacos , Espermatogônias/efeitos dos fármacos , Tretinoína/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Animais Recém-Nascidos , Biomarcadores/metabolismo , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/fisiologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Diaminas/farmacologia , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Túbulos Seminíferos/metabolismo , Células de Sertoli/citologia , Células de Sertoli/efeitos dos fármacos , Transdução de Sinais , Espermatogênese/fisiologia , Espermatogônias/citologia , Espermatogônias/fisiologia , Testículo/citologia , Testículo/efeitos dos fármacos , Testículo/fisiologia , Tretinoína/fisiologiaRESUMO
Perturbations in the vitamin A metabolism pathway could be a significant cause of male infertility, as well as a target toward the development of a male contraceptive, necessitating the need for a better understanding of how testicular retinoic acid (RA) concentrations are regulated. Quantitative analyses have recently demonstrated that RA is present in a pulsatile manner along testis tubules. However, it is unclear if the aldehyde dehydrogenase (ALDH) enzymes, which are responsible for RA synthesis, contribute to the regulation of these RA concentration gradients. Previous studies have alluded to fluctuations in ALDH enzymes across the spermatogenic cycle, but these inferences have been based primarily on qualitative transcript localization experiments. Here, we show via various quantitative methods that the three well-known ALDH enzymes (ALDH1A1, ALDH1A2, and ALDH1A3), and an ALDH enzyme previously unreported in the murine testis (ALDH8A1), are not expressed in a stage-specific manner in the adult testis, but do fluctuate throughout juvenile development in perfect agreement with the first appearance of each advancing germ cell type. We also show, via treatments with a known ALDH inhibitor, that lowered testicular RA levels result in an increase in blood-testis barrier permeability, meiotic recombination, and meiotic defects. Taken together, these data further our understanding of the complex regulatory actions of RA on various spermatogenic events and, in contrast with previous studies, also suggest that the ALDH enzymes are not responsible for regulating the recently measured RA pulse.
Assuntos
Aldeído Desidrogenase/biossíntese , Espermatogênese/genética , Aldeído Desidrogenase/antagonistas & inibidores , Aldeído Desidrogenase/genética , Animais , Biotina/metabolismo , Barreira Hematotesticular/efeitos dos fármacos , Pareamento Cromossômico/efeitos dos fármacos , Diaminas/farmacologia , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica/genética , Isoenzimas/metabolismo , Masculino , Meiose/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Espermatogênese/efeitos dos fármacos , Testículo/efeitos dos fármacos , Testículo/crescimento & desenvolvimento , Testículo/metabolismo , Tretinoína/metabolismoRESUMO
The active metabolite of vitamin A, retinoic acid (RA), is known to be essential for spermatogenesis. Changes to RA levels within the seminiferous epithelium can alter the development of male germ cells, including blocking their differentiation completely. Excess RA has been shown to cause germ cell death in both neonatal and adult animals, yet the cells capable of degrading RA within the testis have yet to be investigated. One previous study alluded to a requirement for one of the RA degrading enzymes, CYP26B1, in Sertoli cells but no data exist to determine whether germ cells possess the ability to degrade RA. To bridge this gap, the roles of CYP26A1 and CYP26B1 within the seminiferous epithelium were investigated by creating single and dual conditional knockouts of these enzymes in either Sertoli or germ cells. Analysis of these knockout models revealed that deletion of both Cyp26a1 and Cyp26b1 in either cell type resulted in increased vacuolization within the seminiferous tubules, delayed spermatid release, and an increase in the number of STRA8-positive spermatogonia, but spermatozoa were still produced and the animals were found to be fertile. However, elimination of CYP26B1 activity within both germ and Sertoli cells resulted in severe male subfertility, with a loss of advanced germ cells from the seminiferous epithelium. These data indicate that CYP26 activity within either Sertoli or germ cells is essential for the normal progression of spermatogenesis and that its loss can result in reduced male fertility.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Epitélio Seminífero/enzimologia , Espermatogênese/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Sistema Enzimático do Citocromo P-450/genética , Células Germinativas/metabolismo , Masculino , Camundongos , Camundongos Knockout , Ácido Retinoico 4 Hidroxilase , Células de Sertoli/metabolismo , Espermatogônias/metabolismo , Espermatozoides/metabolismoRESUMO
The asynchronous cyclic nature of spermatogenesis is essential for continual sperm production and is one of the hallmarks of mammalian male fertility. While various mRNA and protein localization studies have indirectly implicated changing retinoid levels along testis tubules, no quantitative evidence for these changes across the cycle of the seminiferous epithelium currently exists. This study utilized a unique mouse model of induced synchronous spermatogenesis, localization of the retinoid-signaling marker STRA8, and sensitive quantification of retinoic acid concentrations to determine whether there are fluctuations in retinoid levels at each of the individual stages of germ cell differentiation and maturation to sperm. These data show that processive pulses of retinoic acid are generated during spermatogonial differentiation and are the likely trigger for cyclic spermatogenesis and allow us, for the first time, to understand how the cycle of the seminiferous epithelium is generated and maintained. In addition, this study represents the first direct quantification of a retinoid gradient controlling cellular differentiation in a postnatal tissue.
Assuntos
Espermatogênese/efeitos dos fármacos , Testículo/efeitos dos fármacos , Tretinoína/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Masculino , Camundongos , Receptores do Ácido Retinoico/metabolismo , Testículo/metabolismo , Tretinoína/metabolismoRESUMO
Continual sperm production relies on germ cells undergoing spermatogenesis asynchronously. As a result, the testis always contains a mixed population of germ cells at different stages of their differentiation process. The heterogeneous nature of the testis makes profiling gene expression within Sertoli cells or specific populations of germ cells impossible when a wild-type testis is assessed. We recently reported a unique method for synchronizing spermatogenesis without affecting fertility by manipulating RA levels within the neonatal testis. Using this protocol, combined with the RiboTag transgenic mouse line, we have mapped the Sertoli and germ cell translatome during the initial synchronized wave of spermatogenesis. Using microarray analysis, we identified 392 and 194 germ cell and Sertoli cells transcripts, respectively, that dynamically change during spermatogonial differentiation, division, and the onset of meiosis. Functional annotation clustering revealed that transcripts enriched in germ cells were mostly associated with meiosis (21 transcripts), chromatin organization (12 transcripts), and cell cycle (3 transcripts). In addition, glycoproteins (65 transcripts), cell adhesion (15 transcripts), and cell junction (13 transcripts) transcripts were overrepresented in the Sertoli cell-enriched list. These datasets represent the first transcriptional analysis of spermatogonial differentiation, division, and meiotic onset. These data suggest that several of the genes encoding meiotic proteins are expressed and are actively being translated well before germ cells enter meiosis. In addition, this study provides novel candidate genes, Asf1b and Esyt3, that may be involved in the regulation of spermatogonial chromatin reorganization, germ-Sertoli cell interactions, and/or blood-testis barrier formation.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Células de Sertoli/fisiologia , Espermatogênese/fisiologia , Espermatogônias/fisiologia , Testículo/fisiologia , Animais , Animais Recém-Nascidos , Biologia Computacional , Diaminas/farmacologia , Feminino , Perfilação da Expressão Gênica/métodos , Imuno-Histoquímica , Masculino , Camundongos Knockout , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos , RNA/química , RNA/genética , Células de Sertoli/citologia , Espermatogônias/citologia , Testículo/citologia , Transcriptoma/fisiologia , Tretinoína/farmacologiaRESUMO
The BDADs (bis-[dichloroacetyl]-diamines) are compounds that can inhibit spermatogenesis via blocking the metabolism of vitamin A. We utilized one specific BDAD, WIN 18,446, to manipulate the endogenous production of retinoic acid (RA) in the testis to further investigate the action of this compound on mammalian sperm production. Transient treatment of adult male mice with WIN 18,446 blocked spermatogonial differentiation and induced significant changes in the cycle of the seminiferous epithelium. WIN 18,446 treatment of neonatal mice also blocked spermatogonial differentiation and, followed by injection of RA, induced synchronous spermatogenesis in adulthood. The net result was pulsatile, rather than normal continuous, release of sperm from the seminiferous epithelium. This study describes a novel technique that can enrich for specific germ cell populations within the testis, representing a valuable new tool for studying spermatogenesis.
Assuntos
Diaminas/farmacologia , Espermatogênese/efeitos dos fármacos , Espermatogênese/fisiologia , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Masculino , Camundongos , Modelos Animais , Testículo/citologia , Testículo/embriologia , Testículo/metabolismo , Tretinoína/metabolismoRESUMO
Ultraviolet (UV) light primarily causes C > T substitutions in lesion-forming dipyrimidine sequences. However, many of the key driver mutations in melanoma do not fit this canonical UV signature, but are instead caused by T > A, T > C, or C > A substitutions. To what extent exposure to the UVB or UVA spectrum of sunlight can induce these noncanonical mutation classes, and the molecular mechanism involved is unclear. Here, we repeatedly exposed wild-type or repair-deficient yeast (Saccharomyces cerevisiae) to UVB or UVA light and characterized the resulting mutations by whole genome sequencing. Our data indicate that UVB induces C > T and T > C substitutions in dipyrimidines, and T > A substitutions that are often associated with thymine-adenine (TA) sequences. All of these mutation classes are induced in nucleotide excision repair-deficient cells and show transcriptional strand asymmetry, suggesting they are caused by helix-distorting UV photoproducts. In contrast, UVA exposure induces orders of magnitude fewer mutations with a distinct mutation spectrum. UVA-induced mutations are elevated in Ogg1-deficient cells, and the resulting spectrum consists almost entirely of C > A/G > T mutations, indicating they are likely derived from oxidative guanine lesions. These mutations show replication asymmetry, with elevated G > T mutations on the leading strand, suggesting there is a strand bias in the removal or bypass of guanine lesions during replication. Finally, we develop a mutation reporter to show that UVA induces a G > T reversion mutation in yeast that mimics the oncogenic NRAS Q61K mutation in melanoma. Taken together, these findings indicate that UVA and UVB exposure can induce many of the noncanonical mutation classes that cause driver mutations in melanoma.
Assuntos
Melanoma , Saccharomyces cerevisiae , Humanos , Saccharomyces cerevisiae/genética , Dano ao DNA , Mutação , Mutagênese , Reparo do DNA/genética , Raios Ultravioleta/efeitos adversos , Melanoma/genética , GuaninaRESUMO
The cytidine deaminases APOBEC3A and APOBEC3B (A3B) are prominent mutators of human cancer genomes. However, tumor-specific genetic modulators of APOBEC-induced mutagenesis are poorly defined. Here, we utilized a screen to identify 61 gene deletions that increase A3B-induced mutations in yeast. Also, we determined whether each deletion was epistatic with UNG1 loss, which indicated whether the encoded factors participate in the error-free bypass of A3B/Ung1-dependent abasic sites or suppress A3B-catalyzed deamination by protecting against aberrant formation of single stranded DNA (ssDNA). Additionally, we determined that the mutation spectra of A3B-induced mutations revealed genotype-specific patterns of strand-specific ssDNA formation and nucleotide incorporation across APOBEC-induced lesions. Combining these three metrics we were able to establish a multifactorial signature of APOBEC-induced mutations specific to (1) failure to remove H3K56 acetylation, which results in extremely high A3B-induced mutagenesis, (2) defective CTF18-RFC complex function, which results in high levels of A3B induced mutations specifically on the leading strand template that synergistically increase with loss of UNG1, and (3) defective HR-mediated bypass of APOBEC-induced lesions, which were epistatic with Ung1 loss and result from increased Rev1-mediated C-to-G substitutions. We extended these results by analyzing mutation data for human tumors and found BRCA1/2-deficient breast cancer tumors display 3- to 4-fold more APOBEC-induced mutations. Mirroring our results in yeast, for BRCA1/2 deficient tumors Rev1-mediated C-to-G substitutions are solely responsible for increased APOBEC-signature mutations and these mutations occur on the lagging strand during DNA replication. Together these results identify important factors that influence the dynamics of DNA replication and likely the abundance of APOBEC-induced mutation during tumor progression as well as a novel mechanistic role for BRCA1/2 during HR-dependent lesion bypass of APOBEC-induced lesions during cancer cell replication.
RESUMO
UV exposure induces a mutation signature of C > T substitutions at dipyrimidines in skin cancers. We recently identified additional UV-induced AC > TT and A > T substitutions that could respectively cause BRAF V600K and V600E oncogenic mutations. The mutagenic bypass mechanism past these atypical lesions, however, is unknown. Here, we whole genome sequenced UV-irradiated yeast and used reversion reporters to delineate the roles of replicative and translesion DNA polymerases in mutagenic bypass of UV-lesions. Our data indicates that yeast DNA polymerase eta (pol η) has varied impact on UV-induced mutations: protecting against C > T substitutions, promoting T > C and AC > TT substitutions, and not impacting A > T substitutions. Surprisingly, deletion rad30Δ increased novel UV-induced C > A substitutions at CA dinucleotides. In contrast, DNA polymerases zeta (pol ζ) and epsilon (pol ε) participated in AC > TT and A > T mutations. These results uncover lesion-specific accurate and mutagenic bypass of UV lesions, which likely contribute to key driver mutations in melanoma.
Assuntos
Dano ao DNA , Mutagênicos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Raios Ultravioleta/efeitos adversos , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Replicação do DNA/genéticaRESUMO
Meiosis is essential for generation of healthy gametes in both sexes and involves recombination and segregation of homologous chromosomes to produce haploid gametes. The initiation of meiosis in both sexes relies upon retinoic acid (RA) (Griswold MD, Hogarth CA, Bowles J, Koopman P. Initiating Meiosis: The Case for Retinoic Acid. Biol Reprod 2012; 86(35):1-7). Previous studies have demonstrated that the stimulated by retinoic acid gene 8 (Stra8) was required for meiotic progression in both the mouse ovary and postnatal testis. To identify additional candidates that may play a role during meiosis, we used microarray databases to generate lists of transcripts with expression profiles similar to that of Stra8 in the embryonic ovary and postnatal testis. One such gene, establishment of cohesion 1 homolog 2 (Saccharomyces cerevisiae) (Esco2), has been described as a regulator of sister chromatid cohesion during mitosis. This study describes the first in-depth analysis of ESCO2 localization and regulation during meiosis in both males and females. ESCO2 colocalized with the gamma H2A histone family member X (H2AFX) in pachytene spermatocytes, indicating that ESCO2 is a component of the XY body. In pachytene cells of the embryonic ovary, ESCO2 colocalized with H2AFX, which is consistent with the presence of ESCO2 in areas of double-stranded breaks. In addition, the expression of Esco2 was found to be regulated by RA in the postnatal testis. These data indicate that ESCO2 may play a vital role in meiosis in both males and females.
Assuntos
Acetiltransferases/genética , Acetiltransferases/metabolismo , Células Germinativas/metabolismo , Meiose/genética , Animais , Animais Recém-Nascidos , Células Cultivadas , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Células Germinativas/enzimologia , Células Germinativas/fisiologia , Gônadas/embriologia , Gônadas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Distribuição TecidualRESUMO
Increasing evidence indicates that microRNAs (miRNAs) may be critical players in spermatogenesis. The miRNA expression profiles of THY1(+)-enriched undifferentiated spermatogonia were characterized, and members of Mir-17-92 (Mirc1) and its paralog Mir-106b-25 (Mirc3) clusters are significantly downregulated during retinoic acid-induced spermatogonial differentiation, both in vitro and in vivo. The repression of microRNA clusters Mir-17-92 (Mirc1) and Mir-106b-25 (Mirc3) by retinoic acid in turn potentially upregulates the expression of Bim, Kit, Socs3, and Stat3. The male germ cell-specific Mir-17-92 (Mirc1) knockout mice exhibit small testes, a lower number of epididymal sperm, and mild defect in spermatogenesis. Absence of Mir-17-92 (Mirc1) in male germ cells dramatically increases expression of Mir-106b-25 (Mirc3) cluster miRNAs in the germ cells. These results suggest that Mir-17-92 (Mirc1) cluster and Mir-106b-25 (Mirc3) cluster miRNAs possibly functionally cooperate in regulating spermatogonial development.
Assuntos
Diferenciação Celular/genética , MicroRNAs/fisiologia , Espermatogênese/fisiologia , Animais , Regulação para Baixo/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Espermatogênese/genética , Testículo/citologia , Testículo/fisiologia , Tretinoína/farmacologiaRESUMO
Meiosis is unique to germ cells and occurs in a sex-specific manner. The genes regulating meiotic initiation in either sex are yet to be fully elucidated. Recent studies have revealed the importance of retinoic acid and one of its target genes, Stra8, in meiotic initiation in both sexes. Microarray analysis of whole murine embryonic ovary and postnatal testis time course data revealed a single peak of Stra8 expression in each organ at the onset of meiosis; at Embryonic Day 14.5 in the ovary and 10 days postpartum in the testis. In order to identify other genes involved in the initiation of meiosis in mammals, murine testis and ovary microarray data were examined more closely for transcripts with expression profiles similar to Stra8. Three such candidates include establishment of cohesion 1 homolog 2 (Esco2), encoding a protein essential for sister chromatid cohesion; SET domain, bifurcated 2 (Setdb2), the mouse ortholog of Eggless, which is essential for oogenesis in Drosophila; and ubiquitin-activating enzyme 6 (Uba6), a gene with fivefold higher expression in human and mouse testes than any other organ. In situ hybridization and immunohistochemistry or immunofluorescence were performed to localize Esco2, Setbd2, and Uba6 expression in the developing testis. The cellular expression pattern localized all three of these transcripts and their respective proteins to germ cells transitioning from mitosis to meiosis, hence supporting the hypothesis of their involvement in the initiation of meiosis. Future research will be directed at determining a specific role for these three proteins in germ cell differentiation.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Meiose/fisiologia , Mitose/fisiologia , Animais , Bases de Dados Factuais , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos , Ovário/embriologia , Ovário/metabolismo , Análise Serial de Proteínas , Transporte Proteico/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Testículo/embriologia , Testículo/metabolismoRESUMO
Spermatogonial differentiation is orchestrated by the precise control of gene expression involving retinoic acid signaling. MicroRNAs have emerged as important regulators of spermatogenesis, and here we show that the Mirlet7 family miRNAs are expressed in mouse spermatogonia and spermatocytes. Retinoic acid significantly leads to the induction of Mirlet7 miRNAs through suppression of Lin28. We further confirmed both in vitro and in vivo that expressions of Mycn, Ccnd1, and Col1a2, which are targets of Mirlet7, were downregulated during spermatogonial differentiation. These results suggest that Mirlet7 family miRNAs play a role in retinoic acid-induced spermatogonial differentiation.
Assuntos
Diferenciação Celular , MicroRNAs/metabolismo , Espermatogônias/metabolismo , Animais , Células Cultivadas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/metabolismo , Receptores do Ácido Retinoico/metabolismo , Espermatogônias/citologia , TretinoínaRESUMO
Bis-(dichloroacetyl)-diamines (BDADs) are compounds that inhibit spermatogenesis and function as male contraceptives in many species; however, their mechanism of action has yet to be fully investigated. It has been proposed that BDADs may function via inhibition of testicular retinoic acid (RA) biosynthesis. We employed an organ culture technique and the expression of a marker for RA activity, Stra8 (stimulated by retinoic acid gene 8), to investigate if the BDAD WIN 18,446 inhibited the biosynthesis of RA from retinol (ROL) in neonatal and adult murine testis and in the embryonic murine gonad. After culturing either whole testes or germ cells isolated from mice at 2 days postpartum (dpp) with WIN 18,446 or with WIN 18,446 plus ROL, Stra8 expression was suppressed, demonstrating that WIN 18,446 inhibited the conversion of ROL to RA in both systems. We also utilized a transgenic mouse containing an RA-responsive LacZ reporter gene to demonstrate limited RA induction of LacZ expression in 2-dpp testes cultured with WIN 18,446 plus ROL. The expression of Stra8 was downregulated in adult mouse testis tubules cultured with WIN 18,446 when compared to tubules cultured with the vehicle control. WIN 18,446 also inhibited the conversion of ROL to RA in embryonic ovaries and testes cultured for 48 h. These murine results provide critical insights regarding how the BDADs can inhibit spermatogenesis by blocking the ability of vitamin A to drive germ cell development. In addition, these techniques will be useful for screening novel inhibitors of RA biosynthesis as potential male contraceptives.
Assuntos
Anticoncepcionais Masculinos/farmacologia , Diaminas/farmacologia , Regulação para Baixo/efeitos dos fármacos , Proteínas/metabolismo , Testículo/efeitos dos fármacos , Testículo/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Animais Recém-Nascidos , Células Cultivadas , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/metabolismo , Feminino , Genes Reporter , Masculino , Camundongos , Camundongos Transgênicos , Técnicas de Cultura de Órgãos , Ovário/efeitos dos fármacos , Ovário/metabolismo , Receptores do Ácido Retinoico/genética , Elementos de Resposta , Espermatogênese/efeitos dos fármacos , Espermatozoides/citologia , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismo , Testículo/citologia , Testículo/crescimento & desenvolvimento , Tretinoína/metabolismo , Vitamina A/metabolismoRESUMO
Spermatogenesis is an intricately regulated process of cellular differentiation transforming spermatogonial stem cells to spermatozoa. Elimination of the transcription factor EGR4 generates subfertile male mice yet the expression and function of EGR4 in the mammalian testis has yet to be fully investigated. We performed in situ hybridization and immunofluorescence to identify Egr4 transcript and protein localization in the developing murine testis. EGR4 was detected in both germ and somatic cells in the neonatal testis but was specific to germ cells inside the seminiferous epithelium from juvenile development onward. EGR4 also displayed distinct intracellular localization patterns within specific cell populations of the testis. In addition, Egr4-deficient testis tubules regress from relatively normal to Sertoli cell and undifferentiated spermatogonia only over time. Taken together, these data suggest that Egr4 may regulate spermatogenesis at multiple steps, with roles in the dividing Sertoli cells, peritubular myoid cells, and the meiotic and elongating haploid germ cell populations.