228 results on '"Dirk G. de Rooij"'
Search Results
2. The Neonatal and Adult Human Testis Defined at the Single-Cell Level
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Abhishek Sohni, Kun Tan, Hye-Won Song, Dana Burow, Dirk G. de Rooij, Louise Laurent, Tung-Chin Hsieh, Raja Rabah, Saher Sue Hammoud, Elena Vicini, and Miles F. Wilkinson
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Biology (General) ,QH301-705.5 - Abstract
Summary: Spermatogenesis has been intensely studied in rodents but remains poorly understood in humans. Here, we used single-cell RNA sequencing to analyze human testes. Clustering analysis of neonatal testes reveals several cell subsets, including cell populations with characteristics of primordial germ cells (PGCs) and spermatogonial stem cells (SSCs). In adult testes, we identify four undifferentiated spermatogonia (SPG) clusters, each of which expresses specific marker genes. We identify protein markers for the most primitive SPG state, allowing us to purify this likely SSC-enriched cell subset. We map the timeline of male germ cell development from PGCs through fetal germ cells to differentiating adult SPG stages. We also define somatic cell subsets in both neonatal and adult testes and trace their developmental trajectories. Our data provide a blueprint of the developing human male germline and supporting somatic cells. The PGC-like and SSC markers are candidates to be used for SSC therapy to treat infertility. : Sohni et al. use scRNA-seq analysis to define cell subsets in the human testis. Highlights include the identification of primordial germ cell- and spermatogonial stem cell-like cell subsets in neonatal testes, numerous undifferentiated spermatogonial cell states in adult testes, and somatic cell subsets in both neonatal and adult testes. Keywords: single-cell RNA sequencing, spermatogenesis, testes, germ cells, spermatogonial stem cells, spermatogonia, primordial germ cells, Leydig cells, Sertoli cells, peritubular myoid cells
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- 2019
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3. C14ORF39/SIX6OS1 is a constituent of the synaptonemal complex and is essential for mouse fertility
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Laura Gómez-H, Natalia Felipe-Medina, Manuel Sánchez-Martín, Owen R. Davies, Isabel Ramos, Ignacio García-Tuñón, Dirk G. de Rooij, Ihsan Dereli, Attila Tóth, José Luis Barbero, Ricardo Benavente, Elena Llano, and Alberto M. Pendas
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Science - Abstract
The synaptonemal complex is a meiosis-specific proteinaceous structure that supports homologous chromosome pairs during meiosis. Here, the authors show that SIX6OS1 (of previously unknown function) is part of the synaptonemal complex central element and upon deletion in mice, causes defective chromosome synapsis and infertility.
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- 2016
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4. The Homeobox Transcription Factor RHOX10 Drives Mouse Spermatogonial Stem Cell Establishment
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Hye-Won Song, Anilkumar Bettegowda, Blue B. Lake, Adrienne H. Zhao, David Skarbrevik, Eric Babajanian, Meena Sukhwani, Eleen Y. Shum, Mimi H. Phan, Terra-Dawn M. Plank, Marcy E. Richardson, Madhuvanthi Ramaiah, Vaishnavi Sridhar, Dirk G. de Rooij, Kyle E. Orwig, Kun Zhang, and Miles F. Wilkinson
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spermatogonial stem cells ,spermatogonia ,spermatogenesis ,germ cell ,germ line stem cell ,homeobox ,Rhox ,transcription factor ,gonocytes ,prospermatogonia ,Biology (General) ,QH301-705.5 - Abstract
The developmental origins of most adult stem cells are poorly understood. Here, we report the identification of a transcription factor—RHOX10—critical for the initial establishment of spermatogonial stem cells (SSCs). Conditional loss of the entire 33-gene X-linked homeobox gene cluster that includes Rhox10 causes progressive spermatogenic decline, a phenotype indistinguishable from that caused by loss of only Rhox10. We demonstrate that this phenotype results from dramatically reduced SSC generation. By using a battery of approaches, including single-cell-RNA sequencing (scRNA-seq) analysis, we show that Rhox10 drives SSC generation by promoting pro-spermatogonia differentiation. Rhox10 also regulates batteries of migration genes and promotes the migration of pro-spermatogonia into the SSC niche. The identification of an X-linked homeobox gene that drives the initial generation of SSCs has implications for the evolution of X-linked gene clusters and sheds light on regulatory mechanisms influencing adult stem cell generation in general.
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- 2016
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5. DNA Double Strand Break Response and Limited Repair Capacity in Mouse Elongated Spermatids
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Emad A. Ahmed, Harry Scherthan, and Dirk G. de Rooij
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DNA repair ,Rad54/Rad54B deficient mice ,SCID mice ,PARP1-inhibited mice ,elongated spermatids ,NHEJ ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
Spermatids are extremely sensitive to genotoxic exposures since during spermiogenesis only error-prone non homologous end joining (NHEJ) repair pathways are available. Hence, genomic damage may accumulate in sperm and be transmitted to the zygote. Indirect, delayed DNA fragmentation and lesions associated with apoptotic-like processes have been observed during spermatid elongation, 27 days after irradiation. The proliferating spermatogonia and early meiotic prophase cells have been suggested to retain a memory of a radiation insult leading later to this delayed fragmentation. Here, we used meiotic spread preparations to localize phosphorylate histone H2 variant (γ-H2AX) foci marking DNA double strand breaks (DSBs) in elongated spermatids. This technique enabled us to determine the background level of DSB foci in elongated spermatids of RAD54/RAD54B double knockout (dko) mice, severe combined immunodeficiency SCID mice, and poly adenosine diphosphate (ADP)-ribose polymerase 1 (PARP1) inhibitor (DPQ)-treated mice to compare them with the appropriate wild type controls. The repair kinetics data and the protein expression patterns observed indicate that the conventional NHEJ repair pathway is not available for elongated spermatids to repair the programmed and the IR-induced DSBs, reflecting the limited repair capacity of these cells. However, although elongated spermatids express the proteins of the alternative NHEJ, PARP1-inhibition had no effect on the repair kinetics after IR, suggesting that DNA damage may be passed onto sperm. Finally, our genetic mutant analysis suggests that an incomplete or defective meiotic recombinational repair of Spo11-induced DSBs may lead to a carry-over of the DSB damage or induce a delayed nuclear fragmentation during the sensitive programmed chromatin remodeling occurring in elongated spermatids.
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- 2015
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6. The Dnmt3L ADD Domain Controls Cytosine Methylation Establishment during Spermatogenesis
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Georgios Vlachogiannis, Chad E. Niederhuth, Salih Tuna, Athanasia Stathopoulou, Keijo Viiri, Dirk G. de Rooij, Richard G. Jenner, Robert J. Schmitz, and Steen K.T. Ooi
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Biology (General) ,QH301-705.5 - Abstract
A critical aspect of mammalian gametogenesis is the reprogramming of genomic DNA methylation. The catalytically inactive adaptor Dnmt3L is essential to ensuring this occurs correctly, but the mechanism by which it functions is unclear. Using gene targeting to engineer a single-amino-acid mutation, we show that the Dnmt3L histone H3 binding domain (ADD) is necessary for spermatogenesis. Genome-wide single-base-resolution DNA methylome analysis of mutant germ cells revealed overall reductions in CG methylation at repetitive sequences and non-promoter CpG islands. Strikingly, we also observe an even more severe loss of non-CG methylation, suggesting an unexpected role for the ADD in this process. These epigenetic deficiencies were coupled with defects in spermatogonia, with mutant cells displaying marked changes in gene expression and reactivation of retrotransposons. Our results demonstrate that the Dnmt3L ADD is necessary for Dnmt3L function and full reproductive fitness.
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- 2015
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7. piRNA-associated proteins and retrotransposons are differentially expressed in murine testis and ovary of aryl hydrocarbon receptor deficient mice
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Eva M. Rico-Leo, Nuria Moreno-Marín, Francisco J. González-Rico, Eva Barrasa, Cristina Ortega-Ferrusola, Patricia Martín-Muñoz, Luis O. Sánchez-Guardado, Elena Llano, Alberto Alvarez-Barrientos, Ascensión Infante-Campos, Inmaculada Catalina-Fernández, Matías Hidalgo-Sánchez, Dirk G. de Rooij, Alberto M. Pendás, Fernando J. Peña, Jaime M. Merino, and Pedro M. Fernández-Salguero
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aryl hydrocarbon receptor ,nuage proteins ,spermatogenesis ,ovary ,fertility ,repetitive elements ,Biology (General) ,QH301-705.5 - Abstract
Previous studies suggested that the aryl hydrocarbon receptor (AhR) contributes to mice reproduction and fertility. However, the mechanisms involved remain mostly unknown. Retrotransposon silencing by Piwi-interacting RNAs (piRNAs) is essential for germ cell maturation and, remarkably, AhR has been identified as a regulator of murine B1-SINE retrotransposons. Here, using littermate AhR+/+ and AhR−/− mice, we report that AhR regulates the general course of spermatogenesis and oogenesis by a mechanism likely to be associated with piRNA-associated proteins, piRNAs and retrotransposons. piRNA-associated proteins MVH and Miwi are upregulated in leptotene to pachytene spermatocytes with a more precocious timing in AhR−/− than in AhR+/+ testes. piRNAs and transcripts from B1-SINE, LINE-1 and IAP retrotransposons increased at these meiotic stages in AhR-null testes. Moreover, B1-SINE transcripts colocalize with MVH and Miwi in leptonema and pachynema spermatocytes. Unexpectedly, AhR−/− males have increased sperm counts, higher sperm functionality and enhanced fertility than AhR+/+ mice. In contrast, piRNA-associated proteins and B1-SINE and IAP-derived transcripts are reduced in adult AhR−/− ovaries. Accordingly, AhR-null female mice have lower numbers of follicles when compared with AhR+/+ mice. Thus, AhR deficiency differentially affects testis and ovary development possibly by a process involving piRNA-associated proteins, piRNAs and transposable elements.
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- 2016
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8. Closing the preparedness-response gap in time: Operational readiness at points of entry in Europe
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Dirk G. de Rooij, Aura Timen, Jörg Raab, and J. Janse
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Flexibility (engineering) ,medicine.medical_specialty ,Process management ,Strategic thinking ,Public health ,media_common.quotation_subject ,Closing (real estate) ,Public Health, Environmental and Occupational Health ,Workload ,Conceptual framework ,Preparedness ,Added value ,medicine ,Business ,media_common - Abstract
Background As points of entry (POE) - ports, airports and ground-crossings - are important structures for international travel and trade, their timely activated capacity to respond to infectious disease threats is of utmost importance. How can POE timely adjust their capacity from generic preparedness to specific response? Methods We developed a conceptual framework for public health response at POE, based on a military used model, leading to a step-wise approach from preparedness to response. Subsequently, our framework has been used to analyze interview data of professionals working at European ports (n = 12), airports (n = 15) and ground-crossings (n = 4) in 11 European countries in June - August 2020 covering the activation of the response to COVID-19. Results By integrating the military model, we learned that in preparedness frameworks the pre-defined time expected to activate required capacity is missing. While combining time with capacity creates the possibility of defining a readiness range for different capacity requirements. E.g. information provision to passengers is arranged within hours, while entry-screening may take up to days to be operational. We hypothesized that this readiness range provides the opportunity to split strategic thinking from operational readiness, and may support proactive thinking. The interviews confirmed a very implicit and non-formalized reference to the time needed to activate the response. POE differed highly as regards proactive or reactive response strategies. A much stated problem was fast and often changing strategies on measures, leading to extreme required flexibility and workload in operations. Conclusions Explicitly acknowledging the time needed to activate appropriate capacity during the transition from preparedness to response can contribute to a better definition of the operational consequences of readiness. These findings may also be of added value in other public health area than POE.
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- 2021
9. The testis-specific transcription factor TCFL5 responds to A-MYB to elaborate the male meiotic program in placental mammals
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Haiwei Mou, Dirk G. de Rooij, Katharine Cecchini, Zhiping Weng, Cansu Colpan, Ildar Gaitendinov, Tianxiong Yu, Adriano Biasini, Phillip D. Zamore, Amena Arif, and Deniz M. Ozata
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Meiosis ,Transcription (biology) ,Gene expression ,Piwi-interacting RNA ,MYB ,Biology ,Spermatogenesis ,Transcription factor ,Gene ,Cell biology - Abstract
In male mice, the transcription factor (TF) A-MYB initiates reprogramming of gene expression after spermatogonia enter meiosis. We report that A-MYB activates Tcfl5, a testis-specific TF first produced in pachytene spermatocytes. Subsequently, A-MYB and TCFL5 reciprocally reinforce their own transcription to establish an extensive circuit that regulates meiosis. TCFL5 promotes transcription of genes required for mRNA turnover, pachytene piRNA production, meiotic exit, and spermiogenesis. This transcriptional architecture is conserved in rhesus macaque, suggesting TCFL5 plays a central role in meiosis and spermiogenesis in placental mammals. Tcfl5em1/em1 mutants are sterile, and spermatogenesis arrests at the mid- or late-pachytene stage of meiosis.
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- 2021
10. A missense in HSF2BP causing primary ovarian insufficiency affects meiotic recombination by its novel interactor C19ORF57/BRME1
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Rodrigo Garcia-Valiente, Stavit A. Shalev, Reiner A. Veitia, Fernando Sánchez-Sáez, Dirk G. de Rooij, Alberto M. Pendás, Natalia Felipe-Medina, Anne-Laure Todeschini, Laura Gómez-H, Manuel Sánchez-Martín, Paloma Duque, Yazmine B Condezo, Sandrine Caburet, Elena Llano, Université Paris Diderot, Fondation pour la Recherche Médicale, Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), Junta de Castilla y León, European Commission, Ministerio de Economía y Competitividad (España), Centro de Investigación del Cáncer (España), Instituto de Salud Carlos III, Sub Developmental Biology, and Developmental Biology
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0301 basic medicine ,Mouse ,RAD51 ,Primary Ovarian Insufficiency ,Mice ,0302 clinical medicine ,Human genetics ,Recombinase ,Missense mutation ,meiosis ,Biology (General) ,Heat-Shock Proteins ,Genetics ,Mice, Knockout ,Recombination, Genetic ,fertility ,meiotic recombination ,Reproduction ,General Neuroscience ,General Medicine ,DNA-Binding Proteins ,Meiosis ,Medicine ,Female ,Research Article ,Cell biology ,QH301-705.5 ,PALB2 ,Science ,Mutation, Missense ,human genetics ,Biology ,General Biochemistry, Genetics and Molecular Biology ,reproduction ,03 medical and health sciences ,Exome Sequencing ,Animals ,Gene ,Meiotic recombination ,General Immunology and Microbiology ,Cell Biology ,Fertility ,030104 developmental biology ,DMC1 ,Rad51 Recombinase ,Homologous recombination ,Carrier Proteins ,030217 neurology & neurosurgery - Abstract
© Felipe-Medina et al., Primary Ovarian Insufficiency (POI) is a major cause of infertility, but its etiology remains poorly understood. Using whole-exome sequencing in a family with three cases of POI, we identified the candidate missense variant S167L in HSF2BP, an essential meiotic gene. Functional analysis of the HSF2BP-S167L variant in mouse showed that it behaves as a hypomorphic allele compared to a new loss-of-function (knock-out) mouse model. Hsf2bpS167L/S167L females show reduced fertility with smaller litter sizes. To obtain mechanistic insights, we identified C19ORF57/BRME1 as a strong interactor and stabilizer of HSF2BP and showed that the BRME1/HSF2BP protein complex co-immunoprecipitates with BRCA2, RAD51, RPA and PALB2. Meiocytes bearing the HSF2BP-S167L variant showed a strongly decreased staining of both HSF2BP and BRME1 at the recombination nodules and a reduced number of the foci formed by the recombinases RAD51/DMC1, thus leading to a lower frequency of crossovers. Our results provide insights into the molecular mechanism of HSF2BP-S167L in human ovarian insufficiency and sub(in)fertility., This study was supported by Université Paris Diderot and the Fondation pour la Recherche Médicale (Labelisation Equipes DEQ20150331757, SC, A-LT and RAV). This work was supported by MINECO (BFU2017-89408-R) and by Junta de Castilla y León (CSI239P18). NFM, FSS and LGH are supported by European Social Fund/JCyLe grants (EDU/310/2015, EDU/556/2019 and EDU/1083/2013). YBC is funded by a grant from MINECO (BS-2015–073993). The proteomic analysis was performed in the Proteomics Facility of Centro de Investigación del Cáncer, Salamanca, Grant PRB3(IPT17/0019 - ISCIII-SGEFI/ERDF). CIC-IBMCC is supported by the Programa de Apoyo a Planes Estratégicos de Investigación de Estructuras de Investigación de Excelencia cofunded by the Castilla–León autonomous government and the European Regional Development Fund (CLC–2017–01).
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- 2020
11. Author response: A missense in HSF2BP causing primary ovarian insufficiency affects meiotic recombination by its novel interactor C19ORF57/BRME1
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Natalia Felipe-Medina, Sandrine Caburet, Fernando Sánchez-Sáez, Yazmine B Condezo, Dirk G de Rooij, Laura Gómez-H, Rodrigo Garcia-Valiente, Anne Laure Todeschini, Paloma Duque, Manuel Adolfo Sánchez-Martin, Stavit A Shalev, Elena Llano, Reiner A Veitia, and Alberto M Pendás
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- 2020
12. Author response: DAZL mediates a broad translational program regulating expansion and differentiation of spermatogonial progenitors
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Yuting Fan, Dirk G. de Rooij, David C. Page, Peter K. Nicholls, Emily K. Jackson, Sarah A Cobb, Tsutomu Endo, and Maria M. Mikedis
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DAZL ,Progenitor cell ,Biology ,Cell biology - Published
- 2020
13. DAZL mediates a broad translational program regulating expansion and differentiation of spermatogonial progenitors
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Peter K. Nicholls, Maria M. Mikedis, Yuting Fan, Tsutomu Endo, Dirk G. de Rooij, David C. Page, Emily K. Jackson, and Sarah A Cobb
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0301 basic medicine ,Untranslated region ,Male ,Mouse ,QH301-705.5 ,Science ,Population ,translation ,Biology ,Ribosome ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,DAZL ,Mice ,0302 clinical medicine ,Transcription (biology) ,Animals ,Progenitor cell ,Biology (General) ,education ,Gene ,3' Untranslated Regions ,Progenitor ,education.field_of_study ,General Immunology and Microbiology ,General Neuroscience ,RNA-Binding Proteins ,Cell Differentiation ,General Medicine ,Dazl ,Spermatogonia ,spermatogenesis ,Cell biology ,030104 developmental biology ,Medicine ,Developmental biology ,030217 neurology & neurosurgery ,Research Article ,Developmental Biology - Abstract
Fertility across metazoa requires the germline-specific DAZ family of RNA-binding proteins. Here we examine whether DAZL directly regulates progenitor spermatogonia using a conditional genetic mouse model and in vivo biochemical approaches combined with chemical synchronization of spermatogenesis. We find that the absence of Dazl impairs both expansion and differentiation of the spermatogonial progenitor population. In undifferentiated spermatogonia, DAZL binds the 3’ UTRs of ∼2,500 protein-coding genes. Some targets are known regulators of spermatogonial proliferation and differentiation while others are broadly expressed, dosage-sensitive factors that control transcription and RNA metabolism. DAZL binds 3’ UTR sites conserved across vertebrates at a UGUU(U/A) motif. By assessing ribosome occupancy in undifferentiated spermatogonia, we find that DAZL increases translation of its targets. In total, DAZL orchestrates a broad translational program that amplifies protein levels of key spermatogonial and gene regulatory factors to promote the expansion and differentiation of progenitor spermatogonia.
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- 2020
14. Author response: Sox8 and Sox9 act redundantly for ovarian-to-testicular fate reprogramming in the absence of R-spondin1 in mouse sex reversals
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Alain de Bruin, Sameh A. Youssef, Isabelle Gillot, Elodie P. Gregoire, Marie-Cécile De Cian, Dirk G. de Rooij, Marie-Christine Chaboissier, and Nainoa Richardson
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R spondin1 ,SOX9 ,Biology ,Reprogramming ,Cell biology - Published
- 2020
15. A missense in HSF2BP causing Primary Ovarian Insufficiency affects meiotic recombination by its novel interactor C19ORF57/MIDAP
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Natalia Felipe-Medina, Fernando Sánchez-Sáez, Dirk G. de Rooij, Reiner A. Veitia, Yazmine B Codezo, Paloma Duque, Stavit A. Shalev, Alberto M. Pendás, Laura Gómez-H, Elena Llano, Rodrigo Garcia-Valiente, Manuel Sánchez-Martín, Anne-Laure Todeschini, and Sandrine Caburet
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Genetics ,Mutation ,PALB2 ,Recombinase ,RAD51 ,medicine ,Missense mutation ,DMC1 ,Interactor ,Biology ,Homologous recombination ,medicine.disease_cause - Abstract
Primary Ovarian Insufficiency (POI) is a major cause of infertility, but its etiology remains poorly understood. Using whole-exome sequencing in a family with 3 cases of POI, we identified the candidate missense variant S167L in HSF2BP, an essential meiotic gene. Functional analysis of the HSF2BP-S167L variant in mouse, compared to a new HSF2BP knock-out mouse showed that it behaves as a hypomorphic allele. HSF2BP-S167L females show reduced fertility with small litter sizes. To obtain mechanistic insights, we identified C19ORF57/MIDAP as a strong interactor and stabilizer of HSF2BP by forming a higher-order macromolecular structure involving BRCA2, RAD51, RPA and PALB2. Meiocytes bearing the HSF2BP-S167L mutation showed a strongly decreased expression of both MIDAP and HSF2BP at the recombination nodules. Although HSF2BP-S167L does not affect heterodimerization between HSF2BP and MIDAP, it promotes a lower expression of both proteins and a less proficient activity in replacing RPA by the recombinases RAD51/DMC1, thus leading to a lower frequency of cross-overs. Our results provide insights into the molecular mechanism of two novel actors of meiosis underlying non-syndromic ovarian insufficiency.SummaryFelipe-Medina et al. describe a missense variant in the meiotic gene HSF2BP in a consanguineous family with Premature Ovarian Insufficiency, and characterize it as an hypormorphic allele, that in vivo impairs its dimerization with a novel meiotic actor, MIDAP/ C19ORF57, and affect recombination at double-strand DNA breaks.
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- 2020
16. Dynamic and regulated TAF gene expression during mouse embryonic germ cell development
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Megan A. Gura, David C. Page, Kimberly A. Seymour, Richard N. Freiman, Maria M. Mikedis, and Dirk G. de Rooij
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Male ,Cancer Research ,Gene Expression ,QH426-470 ,Biochemistry ,Gametogenesis ,DAZL ,Mice ,0302 clinical medicine ,Animal Cells ,Spermatocytes ,Transcriptional regulation ,Medicine and Health Sciences ,Cell Cycle and Cell Division ,Genetics (clinical) ,Regulation of gene expression ,0303 health sciences ,Chromosome Biology ,Messenger RNA ,Gene Expression Regulation, Developmental ,Animal Models ,3. Good health ,Cell biology ,Ovaries ,Nucleic acids ,Meiosis ,medicine.anatomical_structure ,Experimental Organism Systems ,Cell Processes ,Cellular Types ,Anatomy ,Reprogramming ,Genital Anatomy ,Germ cell ,Research Article ,Mouse Models ,Biology ,Research and Analysis Methods ,03 medical and health sciences ,Model Organisms ,medicine ,Genetics ,Animals ,RNA, Messenger ,Gonads ,Molecular Biology ,Gene ,Ecology, Evolution, Behavior and Systematics ,030304 developmental biology ,Adaptor Proteins, Signal Transducing ,TATA-Binding Protein Associated Factors ,Cell growth ,Reproductive System ,Biology and Life Sciences ,Deleted in Azoospermia 1 Protein ,Cell Biology ,Embryonic stem cell ,Sperm ,Mice, Inbred C57BL ,Germ Cells ,Animal Studies ,RNA ,Transcription Factor TFIID ,030217 neurology & neurosurgery - Abstract
Germ cells undergo many developmental transitions before ultimately becoming either eggs or sperm, and during embryonic development these transitions include epigenetic reprogramming, quiescence, and meiosis. To begin understanding the transcriptional regulation underlying these complex processes, we examined the spatial and temporal expression of TAF4b, a variant TFIID subunit required for fertility, during embryonic germ cell development. By analyzing published datasets and using our own experimental system to validate these expression studies, we determined that both Taf4b mRNA and protein are highly germ cell-enriched and that Taf4b mRNA levels dramatically increase from embryonic day 12.5–18.5. Surprisingly, additional mRNAs encoding other TFIID subunits are coordinately upregulated through this time course, including Taf7l and Taf9b. The expression of several of these germ cell-enriched TFIID genes is dependent upon Dazl and/or Stra8, known regulators of germ cell development and meiosis. Together, these data suggest that germ cells employ a highly specialized and dynamic form of TFIID to drive the transcriptional programs that underlie mammalian germ cell development., Author summary Assisted reproductive therapy and fertility preservation are increasingly used to improve human reproduction across the world, yet there are still many unanswered questions regarding what factors govern the development of eggs and sperm and how these factors work together. We previously identified a subunit of the general transcription factor TFIID, TAF4b, that is essential for fertility. However, many basic characteristics of how Taf4b and its associated TFIID family members contribute to the formation of healthy sperm and eggs in mice and humans remain unknown. In this study, we find that mouse Taf4b and several closely related TFIID subunits become highly abundant during mouse embryonic gonad development, specifically in the cells that ultimately become eggs and sperm. Here, we analyzed data from public repositories and isolated these developing cells to examine their gene expression patterns throughout embryonic development. Together these data suggest that the dynamic expression of Taf4b and other TFIID family members are dependent on the well-established reproductive cell regulators Dazl and Stra8. This understanding of Taf4b gene expression and regulation in mouse reproductive cell development is likely conserved during development of human cells and offers novel insights into the interconnectedness of the factors that govern the formation of healthy eggs and sperm.
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- 2020
17. GCNA Interacts with Spartan and Topoisomerase II to Regulate Genome Stability
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Dirk G. de Rooij, Hannah R. Tatnell, Gregoriy A Dokshin, Craig C. Mello, Matthew D. Eldridge, Ahmet R. Ozturk, David C. Page, Gregory J. Hannon, Katherine A. Romer, Gregory M. Davis, Michelle A. Carmell, Peter K. Nicholls, Luke W. Molesworth, Taylin E. Gourley, Ashley Sawle, Whitehead Institute for Biomedical Research, Academic Medical Center, Developmental Biology, and Sub Developmental Biology
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germ cells ,DNA damage ,Mutant ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Germline ,03 medical and health sciences ,SprT ,0302 clinical medicine ,Homologous chromosome ,Germ cells ,Top1 ,Molecular Biology ,Mitosis ,030304 developmental biology ,Top2 ,topoisomerase ,0303 health sciences ,Topoisomerase ,DNA replication ,Spartan ,Embryo ,Cell Biology ,GCNA ,Cell biology ,DNA-protein crosslink (DPC) repair ,biology.protein ,DVC-1 ,030217 neurology & neurosurgery ,Developmental Biology - Abstract
GCNA proteins are expressed across eukarya in pluripotent cells and have conserved functions in fertility. GCNA homologs Spartan (DVC-1) and Wss1 resolve DNA-protein crosslinks (DPCs), including Topoisomerase-DNA adducts, during DNA replication. Here, we show that GCNA mutants in mouse and C. elegans display defects in genome maintenance including DNA damage, aberrant chromosome condensation, and crossover defects in mouse spermatocytes and spontaneous genomic rearrangements in C. elegans. We show that GCNA and topoisomerase II (TOP2) physically interact in both mice and worms and colocalize on condensed chromosomes during mitosis in C. elegans embryos. Moreover, C. elegans gcna-1 mutants are hypersensitive to TOP2 poison. Together, our findings support a model in which GCNA provides genome maintenance functions in the germline and may do so, in part, by promoting the resolution of TOP2 DPCs. DNA topoisomerases help unwind DNA but occasionally get trapped, resulting in DNA-protein crosslinks (DPCs). DPCs damage DNA and threaten genomic integrity. Dokshin et al. find that GCNA protein family complements standard DPC processing machinery in resolving topoisomerase II DPCs to ensure heritable genome stability and germline immortality., National Institutes of Health (U.S.) (Grant P40 OD010440)
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- 2020
18. Effective training in cross-border infection prevention and response
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E Belfroid, Jörg Raab, Dirk G. de Rooij, Aura Timen, and Corien Swaan
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Nursing ,business.industry ,Needs assessment ,Public Health, Environmental and Occupational Health ,MEDLINE ,Infection control ,Medicine ,business ,Organizational performance ,Training (civil) - Abstract
Introduction Competent personnel at points of entry is important to prevent international spreading of disease. Education, training and exercises (ETE) are commonly used to secure this competency. We studied what effective training looks like, by conducting a literature review on effective ETE, conducting a training needs assessment. Methodology A systematic search in Embase, Medline, Web of Science, ERIC, Cinahl, and PsycInfo, to identify effective ETE on infectious disease control was performed. We integratively analysed effectiveness of ETE methods on different outcome levels: satisfaction, learning, behavior and organizational performance. Also, we assessed training needs and -preferences among professionals involved in infectious disease control at points of entry in Europe. They completed a digital questionnaire about previously received ETE, importance of topics, highest training needs, and their preferred ETE methodologies. Results We identified a range of effective ETEs in 62 studies. However, details on specific ETE methodologies are scarce, thwarting to link effectiveness to methodology. Also, long term and high level outcomes like behavioral change or public health system performance are reported less frequently than mere satisfaction or knowledge change, while these former often have higher relevance in real practice. Respondents (n = 59) had highest training needs concerning handling ill or exposed persons, and design and use of the contingency plan. Training needs correlated with importance of topics. Highest preferred training methods were presentations and e-modules. Conclusions We call for more extensive evaluations of education, training and exercises in infectious disease control, with measurement of high level outcomes and long term effects. Results from the current review and training needs from the field could be combined to design most effective ETE at points of entry.
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- 2019
19. Retinoic Acid and Germ Cell Development in the Ovary and Testis
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Maria M. Mikedis, David C. Page, Tsutomu Endo, Peter K. Nicholls, Dirk G. de Rooij, Developmental Biology, and Sub Developmental Biology
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0301 basic medicine ,Male ,endocrine system ,germ cells ,Gonad ,Retinoic acid ,Ovary ,Tretinoin ,Review ,Biology ,testis ,Biochemistry ,Gametogenesis ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Meiosis ,Testis ,medicine ,Germ cells ,retinoic acid ,meiosis ,Animals ,Humans ,Spermatogenesis ,Molecular Biology ,Sexual differentiation ,urogenital system ,Sperm ,Spermatozoa ,spermatogenesis ,Cell biology ,030104 developmental biology ,medicine.anatomical_structure ,chemistry ,Oocytes ,Female ,030217 neurology & neurosurgery ,Germ cell - Abstract
Retinoic acid (RA), a derivative of vitamin A, is critical for the production of oocytes and sperm in mammals. These gametes derive from primordial germ cells, which colonize the nascent gonad, and later undertake sexual differentiation to produce oocytes or sperm. During fetal development, germ cells in the ovary initiate meiosis in response to RA, whereas those in the testis do not yet initiate meiosis, as they are insulated from RA, and undergo cell cycle arrest. After birth, male germ cells resume proliferation and undergo a transition to spermatogonia, which are destined to develop into haploid spermatozoa via spermatogenesis. Recent findings indicate that RA levels change periodically in adult testes to direct not only meiotic initiation, but also other key developmental transitions to ensure that spermatogenesis is precisely organized for the prodigious output of sperm. This review focuses on how female and male germ cells develop in the ovary and testis, respectively, and the role of RA in this process.
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- 2019
20. Spermatogonial kinetics in humans
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Mario Stefanini, Carla Boitani, Bartolomeo P. Berloco, Rossana Saracino, Barbara Muciaccia, Francesco Nudo, Elena Vicini, Stefania Fera, Dirk G. de Rooij, Sara Di Persio, G. Spadetta, and Valentina Esposito
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Adult ,Male ,0301 basic medicine ,endocrine system ,Glial Cell Line-Derived Neurotrophic Factor Receptors ,Lineage (genetic) ,Cell Count ,stem cell renewal ,Biology ,GFRA1 ,Models, Biological ,Protein expression ,spermatogonial differentiation ,models ,Young Adult ,03 medical and health sciences ,0302 clinical medicine ,UCL-H1 ,Humans ,Compartment (development) ,human ,Cell Self Renewal ,Molecular Biology ,reproductive and urinary physiology ,Aged ,Cell Proliferation ,Genetics ,030219 obstetrics & reproductive medicine ,Daily production ,urogenital system ,Nuclear Proteins ,KIT ,Cell Differentiation ,Epithelial Cells ,UTF1 ,Middle Aged ,Cell cycle ,Sperm ,Spermatogonia ,Cell biology ,Kinetics ,030104 developmental biology ,Trans-Activators ,Human testis ,spermatogonia ,adult ,aged ,cell count ,cell differentiation ,cell proliferation ,cell self renewal ,epithelial cells ,glial cell line-derived neurotrophic factor receptors ,humans ,kinetics ,male ,middle aged ,models, biological ,nuclear proteins ,trans-activators ,young adult ,molecular biology ,developmental biology ,Spermatogenesis ,biological ,Developmental Biology - Abstract
The human spermatogonial compartment is essential for daily production of millions of sperm. Despite this crucial role, the molecular signature, kinetic behavior and regulation of human spermatogonia are poorly understood. Using human testis biopsies with normal spermatogenesis and by studying marker protein expression, we have identified for the first time different subpopulations of spermatogonia. MAGE-A4 marks all spermatogonia, KIT marks all B spermatogonia and UCLH1 all Apale-dark (Ap-d) spermatogonia. We suggest that at the start of the spermatogenic lineage there are Ap-d spermatogonia that are GFRA1High, likely including the spermatogonial stem cells. Next, UTF1 becomes expressed, cells become quiescent and GFRA1 expression decreases. Finally, GFRA1 expression is lost and subsequently cells differentiate into B spermatogonia, losing UTF1 and acquiring KIT expression. Strikingly, most human Ap-d spermatogonia are out of the cell cycle and even differentiating type B spermatogonial proliferation is restricted. A novel scheme for human spermatogonial development is proposed that will facilitate further research in this field, the understanding of cases of infertility and the development of methods to increase sperm output.
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- 2017
21. Unraveling transcriptome dynamics in human spermatogenesis
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Dirk G. de Rooij, Tinke L. Vormer, Sabrina Z. Jan, Aldo Jongejan, Sjoerd Repping, Sherman J. Silber, Geert Hamer, Ans M.M. van Pelt, Michael D. Röling, Graduate School, Center for Reproductive Medicine, APH - Methodology, Epidemiology and Data Science, Other Research, APH - Personalized Medicine, and ARD - Amsterdam Reproduction and Development
- Subjects
0301 basic medicine ,Adult ,Male ,Cellular differentiation ,Human Development ,Biopsy ,Cell ,RNA-sequencing ,RNA-binding protein ,RNA-binding proteins ,Laser Capture Microdissection ,Biology ,Transcriptome ,03 medical and health sciences ,Mice ,Prophase ,Testis ,medicine ,Animals ,Humans ,RNA, Messenger ,Spermatogenesis ,Molecular Biology ,Gene ,Gamete development ,Genetics ,Gene Expression Regulation, Developmental ,Cell Differentiation ,Middle Aged ,Spermatozoa ,Chromatin ,Spermatogonia ,Cell biology ,Meiosis ,030104 developmental biology ,medicine.anatomical_structure ,Multigene Family ,Stem cell ,Germ cell ,Developmental Biology ,Human - Abstract
Spermatogenesis is a dynamic developmental process that includes stem cell proliferation and differentiation, meiotic cell divisions and extreme chromatin condensation. Although studied in mice, the molecular control of human spermatogenesis is largely unknown. Here, we developed a protocol that enables next-generation sequencing of RNA obtained from pools of 500 individually laser-capture microdissected cells of specific germ cell subtypes from fixed human testis samples. Transcriptomic analyses of these successive germ cell subtypes reveals dynamic transcription of over 4000 genes during human spermatogenesis. At the same time, many of the genes encoding for well-established meiotic and post-meiotic proteins are already present in the pre-meiotic phase. Furthermore, we found significant cell type-specific expression of post-transcriptional regulators, including expression of 110 RNA-binding proteins and 137 long non-coding RNAs, most of them previously not linked to spermatogenesis. Together, these data suggest that the transcriptome of precursor cells already contains the genes necessary for cellular differentiation and that timely translation controlled by post-transcriptional regulators is crucial for normal development. These established transcriptomes provide a reference catalog for further detailed studies on human spermatogenesis and spermatogenic failure., Highlighted Article: Using laser capture microscopy, a comprehensive transcriptomic dataset of well-defined and distinct germ cell subtypes based on morphology and localization in the human testis is generated.
- Published
- 2017
22. PD09-11 THE NEONATAL AND ADULT HUMAN TESTIS DEFINED AT THE SINGLE-CELL LEVEL
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Elena Vicini, Raja Rabah, Miles F. Wilkinson, Louise C. Laurent, Tung-Chin Hsieh, Sue Hammoud, Abhishek Sohni, Kun Tan, Dirk G. de Rooij, Dana A. Burow, and Hye-Won Song
- Subjects
Infertility ,business.industry ,Urology ,medicine ,Human testis ,Cellular level ,Bioinformatics ,medicine.disease ,business - Abstract
INTRODUCTION AND OBJECTIVES:Over 100 million men worldwide suffer from infertility. In most cases, the underlying cause is unknown. Treating human infertility requires detailed knowledge of human s...
- Published
- 2019
23. GCNA interacts with Spartan and Topoisomerase II to regulate genome stability
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Luke W. Molesworth, Katherine A. Romer, Gregory M. Davis, Ashley Sawle, Hannah R. Tatnell, Craig C. Mello, Gregory J. Hannon, Ahmet R. Ozturk, David C. Page, Michelle A. Carmell, Matthew D. Eldridge, Taylin E. Gourley, Gregoriy A Dokshin, and Dirk G. de Rooij
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Prophase ,DNA damage ,Mutant ,Homologous chromosome ,DNA replication ,Biology ,Genome ,Mitosis ,Germline ,Cell biology - Abstract
SummaryGCNA proteins are expressed across eukarya in pluripotent cells and have conserved functions in fertility. GCNA homologs Spartan/DVC-1 and Wss1 resolve DNA-protein crosslinks (DPCs), including Topoisomerase-DNA adducts, during DNA replication. We show that GCNA and Topoisomerase 2 (Top2) physically interact and colocalize on condensed chromosomes during mitosis, when Spartan is not present. We show thatC. elegans gcna-1mutants are sensitive to Top2 poison and accumulate mutations consistent with low fidelity repair of DNA damage, leading to loss of fitness and fertility over generations. We also demonstrate that mouse GCNA interacts with TOP2, andGcna-mutant mice exhibit abnormalities consistent with the inability to process DPCs, including chromatin condensation and crossover defects. Together, our findings provide evidence that GCNA maintains genomic integrity by processing Top2 DPCs in the germline and early embryo, where the genome is challenged with an increased DPC burden.
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- 2019
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24. Amplification of a broad transcriptional program by a common factor triggers the meiotic cell cycle in mice
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Dirk G. de Rooij, David C. Page, and Mina L Kojima
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0301 basic medicine ,germ cells ,Mouse ,QH301-705.5 ,Science ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Germline ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Prophase ,Meiosis ,Transcription (biology) ,Gene expression ,Animals ,Gene Regulatory Networks ,Biology (General) ,Promoter Regions, Genetic ,Gene ,Adaptor Proteins, Signal Transducing ,General Immunology and Microbiology ,General Neuroscience ,Cell Cycle ,DNA replication ,Stra8 ,General Medicine ,DNA ,Cell cycle ,Chromosomes and Gene Expression ,Cell biology ,030104 developmental biology ,Gene Expression Regulation ,Medicine ,transcription ,030217 neurology & neurosurgery ,Research Article ,Developmental Biology ,Protein Binding - Abstract
The germ line provides the cellular link between generations of multicellular organisms, its cells entering the meiotic cell cycle only once each generation. However, the mechanisms governing this initiation of meiosis remain poorly understood. Here, we examined cells undergoing meiotic initiation in mice, and we found that initiation involves the dramatic upregulation of a transcriptional network of thousands of genes whose expression is not limited to meiosis. This broad gene expression program is directly upregulated by STRA8, encoded by a germ cell-specific gene required for meiotic initiation. STRA8 binds its own promoter and those of thousands of other genes, including meiotic prophase genes, factors mediating DNA replication and the G1-S cell-cycle transition, and genes that promote the lengthy prophase unique to meiosis I. We conclude that, in mice, the robust amplification of this extraordinarily broad transcription program by a common factor triggers initiation of meiosis.
- Published
- 2019
25. Author response: Amplification of a broad transcriptional program by a common factor triggers the meiotic cell cycle in mice
- Author
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Mina L Kojima, Dirk G. de Rooij, and David C. Page
- Subjects
Meiotic cell cycle ,Biology ,Cell biology - Published
- 2019
26. Author response: An ancient germ cell-specific RNA-binding protein protects the germline from cryptic splice site poisoning
- Author
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Dirk G. de Rooij, Ingrid Ehrmann, Tomaž Curk, Yoseph Barash, Ian R. Adams, David J. Elliott, Matthew R. Gazzara, Simon Cockell, Jannetta S. Steyn, James H. Crichton, Yilei Liu, Sushma Nagaraja Grellscheid, and Katherine James
- Subjects
medicine.anatomical_structure ,Cryptic splice site ,medicine ,RNA-binding protein ,Biology ,Germline ,Germ cell ,Cell biology - Published
- 2018
27. Corrigendum to 'Isolating mitotic and meiotic germ cells from male mice by developmental synchronization, staging, and sorting' [Dev. Biol. 443 (2018) 19-34]
- Author
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David C. Page, Mina L Kojima, Katherine A. Romer, and Dirk G. de Rooij
- Subjects
Meiosis ,Synchronization (computer science) ,Sorting ,Male mice ,Germ ,Cell Biology ,Biology ,Molecular Biology ,Mitosis ,Developmental Biology ,Cell biology - Published
- 2018
28. ATR is a multifunctional regulator of male mouse meiosis
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Obah A. Ojarikre, Scott Keeney, Valdone Maciulyte, Julian Lange, Elias ElInati, Sarai Pacheco, Shantha K. Mahadevaiah, Dirk G. de Rooij, Jasmin Zohren, James M. A. Turner, Attila Tóth, Alexander Widger, Takayuki Hirota, Andros Maldonado-Linares, Ignasi Roig, Marcello Stanzione, Academic Medical Center, and ARD - Amsterdam Reproduction and Development
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Male ,0301 basic medicine ,Science ,RAD51 ,General Physics and Astronomy ,Cell Cycle Proteins ,Mice, Transgenic ,Ataxia Telangiectasia Mutated Proteins ,Biology ,Article ,General Biochemistry, Genetics and Molecular Biology ,Germline ,03 medical and health sciences ,Meiosis ,Spermatocytes ,Homologous chromosome ,medicine ,Recombinase ,Animals ,DNA Breaks, Double-Stranded ,Meiotic Prophase I ,lcsh:Science ,In Situ Hybridization, Fluorescence ,Mice, Knockout ,Multidisciplinary ,Synapsis ,Nuclear Proteins ,General Chemistry ,Phosphate-Binding Proteins ,Chromosomes, Mammalian ,Cell biology ,Mice, Inbred C57BL ,Chromosome Pairing ,030104 developmental biology ,medicine.anatomical_structure ,lcsh:Q ,DMC1 ,Rad51 Recombinase ,biological phenomena, cell phenomena, and immunity ,Germ cell - Abstract
Meiotic cells undergo genetic exchange between homologs through programmed DNA double-strand break (DSB) formation, recombination and synapsis. In mice, the DNA damage-regulated phosphatidylinositol-3-kinase-like kinase (PIKK) ATM regulates all of these processes. However, the meiotic functions of the PIKK ATR have remained elusive, because germline-specific depletion of this kinase is challenging. Here we uncover roles for ATR in male mouse prophase I progression. ATR deletion causes chromosome axis fragmentation and germ cell elimination at mid pachynema. This elimination cannot be rescued by deletion of ATM and the third DNA damage-regulated PIKK, PRKDC, consistent with the existence of a PIKK-independent surveillance mechanism in the mammalian germline. ATR is required for synapsis, in a manner genetically dissociable from DSB formation. ATR also regulates loading of recombinases RAD51 and DMC1 to DSBs and recombination focus dynamics on synapsed and asynapsed chromosomes. Our studies reveal ATR as a critical regulator of mouse meiosis., ATR kinase is required for meiosis in non-mammalian model organisms. Here the authors demonstrate, using a tissue-specific knockout approach, that ATR is also essential for male meiosis in mouse, regulating meiotic recombination and synapsis.
- Published
- 2018
29. Isolating mitotic and meiotic germ cells from male mice by developmental synchronization, staging, and sorting
- Author
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David C. Page, Katherine A. Romer, Dirk G. de Rooij, Mina L Kojima, Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology. Computational and Systems Biology Program, Massachusetts Institute of Technology. Department of Biology, Academic Medical Center, and Amsterdam Reproduction & Development (AR&D)
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0301 basic medicine ,Male ,endocrine system ,Somatic cell ,Tretinoin ,Biology ,Germline ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Prophase ,Meiosis ,Testis ,medicine ,Animals ,Epigenetics ,Spermatogenesis ,Molecular Biology ,Mitosis ,Cell Biology ,Cell sorting ,Flow Cytometry ,Spermatogonia ,Cell biology ,Mice, Inbred C57BL ,030104 developmental biology ,medicine.anatomical_structure ,Germ Cells ,Stem cell ,Developmental biology ,030217 neurology & neurosurgery ,Germ cell ,Developmental Biology - Abstract
Isolating discrete populations of germ cells from the mouse testis is challenging, because the adult testis contains germ cells at every step of spermatogenesis, in addition to somatic cells. We present a novel method for isolating precise, high-purity populations of male germ cells. We first synchronize germ cell development in vivo by manipulating retinoic acid metabolism, and perform histological staging to verify synchronization. We use fluorescence-activated cell sorting to separate the synchronized differentiating germ cells from contaminating somatic cells and undifferentiated spermatogonia. We achieve ~90% purity at each step of development from undifferentiated spermatogonia through late meiotic prophase. Utilizing this “3 S” method (synchronize, stage, and sort), we can separate germ cell types that were previously challenging or impossible to distinguish, with sufficient yield for epigenetic and biochemical studies. 3 S expands the toolkit of germ cell sorting methods, and should facilitate detailed characterization of molecular and biochemical changes that occur during the mitotic and meiotic phases of spermatogenesis., National Institutes of Health (U.S.). Pre-Doctoral Training Grant (T32GM007287)
- Published
- 2018
30. SETDB1 Links the Meiotic DNA Damage Response to Sex Chromosome Silencing in Mice
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James M. A. Turner, Vesela Encheva, Obah A. Ojarikre, Paul Blakeley, Ambrosius P. Snijders, Elias ElInati, Mahesh N. Sangrithi, Kathy K. Niakan, Dirk G. de Rooij, Takayuki Hirota, Shantha K. Mahadevaiah, ARD - Amsterdam Reproduction and Development, and Center for Reproductive Medicine
- Subjects
Male ,0301 basic medicine ,DNA Repair ,meiotic silencing ,Apoptosis ,Tripartite Motif-Containing Protein 28 ,Biology ,DNA damage response ,Article ,General Biochemistry, Genetics and Molecular Biology ,H3K9me3 ,Histones ,Mice ,03 medical and health sciences ,Meiosis ,Homologous chromosome ,Animals ,Gene silencing ,Gene Silencing ,Molecular Biology ,X chromosome ,mouse ,MSCI ,sex chromosomes ,Synapsis ,Chromosome ,Histone-Lysine N-Methyltransferase ,Cell Biology ,Cell biology ,Chromatin ,Histone Code ,Mice, Inbred C57BL ,body regions ,Chromosome Pairing ,030104 developmental biology ,Histone ,biology.protein ,DNA Damage ,Developmental Biology - Abstract
Summary Meiotic synapsis and recombination ensure correct homologous segregation and genetic diversity. Asynapsed homologs are transcriptionally inactivated by meiotic silencing, which serves a surveillance function and in males drives meiotic sex chromosome inactivation. Silencing depends on the DNA damage response (DDR) network, but how DDR proteins engage repressive chromatin marks is unknown. We identify the histone H3-lysine-9 methyltransferase SETDB1 as the bridge linking the DDR to silencing in male mice. At the onset of silencing, X chromosome H3K9 trimethylation (H3K9me3) enrichment is downstream of DDR factors. Without Setdb1, the X chromosome accrues DDR proteins but not H3K9me3. Consequently, sex chromosome remodeling and silencing fail, causing germ cell apoptosis. Our data implicate TRIM28 in linking the DDR to SETDB1 and uncover additional factors with putative meiotic XY-silencing functions. Furthermore, we show that SETDB1 imposes timely expression of meiotic and post-meiotic genes. Setdb1 thus unites the DDR network, asynapsis, and meiotic chromosome silencing., Graphical Abstract, Highlights • The histone methyltransferase SETDB1 is essential for male mouse meiosis • The meiotic DDR network recruits SETDB1 to the XY pair, where it induces H3K9me3 • SETDB1 deletion perturbs meiotic sex chromosome remodeling and silencing • SETDB1 ensures timely expression of meiotic and post-meiotic genes, During male meiosis in mammals, the asynapsed regions of the X and Y chromosomes retain DNA double-strand breaks (DSBs), which triggers silencing of the sex chromosomes, a process essential for fertility. Hirota et al. show that meiotic DSB factors recruit the H3K9-methyltransferase SETDB1, inducing XY-chromatin remodeling and silencing.
- Published
- 2018
31. Periodic production of retinoic acid by meiotic and somatic cells coordinates four transitions in mouse spermatogenesis
- Author
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Elizaveta Freinkman, David C. Page, Dirk G. de Rooij, and Tsutomu Endo
- Subjects
Male ,0301 basic medicine ,Somatic cell ,Retinoic acid ,Mice ,chemistry.chemical_compound ,0302 clinical medicine ,Spermatocytes ,retinoic acid ,Mice, Knockout ,Genetics ,Multidisciplinary ,digestive, oral, and skin physiology ,Gene Expression Regulation, Developmental ,Cell Differentiation ,Biological Sciences ,Spermatids ,Spermatozoa ,Cell biology ,Meiosis ,medicine.anatomical_structure ,PNAS Plus ,Stem cell ,Ploidy ,Signal Transduction ,endocrine system ,Tretinoin ,testis ,Biology ,Aldehyde Dehydrogenase 1 Family ,ALDH1A2 ,03 medical and health sciences ,medicine ,Animals ,Spermatogenesis ,mouse ,Adaptor Proteins, Signal Transducing ,Spermatid ,urogenital system ,Retinal Dehydrogenase ,Aldehyde Dehydrogenase ,Spermatogonia ,Mice, Inbred C57BL ,030104 developmental biology ,chemistry ,Pachytene Stage ,030217 neurology & neurosurgery ,Developmental Biology - Abstract
Significance Male mouse sex cells mature into sperm through a 35-d process punctuated by four transitions, two occurring before meiosis (spermatogonial differentiation and meiotic initiation) and two after meiosis (spermatid elongation and sperm release). The four transitions occur in proximity spatially and temporally, with an 8.6-d periodicity. We describe how this coordination is achieved. The premeiotic transitions were known to be regulated by retinoic acid (RA). We show that RA also regulates the two postmeiotic transitions. RA levels change periodically, and meiotic cells contribute to its production. The two postmeiotic transitions require RA from meiotic cells while the premeiotic transitions require RA from somatic cells. These elements underpin the spatiotemporal coordination of spermatogenesis to ensure constant sperm production throughout adult life., Mammalian spermatogenesis is an elaborately organized differentiation process, starting with diploid spermatogonia, which include germ-line stem cells, and ending with haploid spermatozoa. The process involves four pivotal transitions occurring in physical proximity: spermatogonial differentiation, meiotic initiation, initiation of spermatid elongation, and release of spermatozoa. We report how the four transitions are coordinated in mice. Two premeiotic transitions, spermatogonial differentiation and meiotic initiation, were known to be coregulated by an extrinsic signal, retinoic acid (RA). Our chemical manipulations of RA levels in mouse testes now reveal that RA also regulates the two postmeiotic transitions: initiation of spermatid elongation and spermatozoa release. We measured RA concentrations and found that they changed periodically, as also reflected in the expression patterns of an RA-responsive gene, STRA8; RA levels were low before the four transitions, increased when the transitions occurred, and remained elevated thereafter. We found that pachytene spermatocytes, which express an RA-synthesizing enzyme, Aldh1a2, contribute directly and significantly to RA production in testes. Indeed, chemical and genetic depletion of pachytene spermatocytes revealed that RA from pachytene spermatocytes was required for the two postmeiotic transitions, but not for the two premeiotic transitions. We conclude that the premeiotic transitions are coordinated by RA from Sertoli (somatic) cells. Once germ cells enter meiosis, pachytene spermatocytes produce RA to coordinate the two postmeiotic transitions. In combination, these elements underpin the spatiotemporal coordination of spermatogenesis and ensure its prodigious output in adult males.
- Published
- 2017
32. TAF4b is Required for Mouse Spermatogonial Stem Cell Development
- Author
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Richard N. Freiman, Eric A. Gustafson, Kimberly A. Seymour, Dirk G. de Rooij, Lindsay A. Lovasco, Other Research, and Center for Reproductive Medicine
- Subjects
Male ,endocrine system ,Cellular differentiation ,Biology ,Article ,Mice ,Gonocyte ,medicine ,Animals ,Progenitor cell ,Spermatogenesis ,Mice, Knockout ,TATA-Binding Protein Associated Factors ,Cell Differentiation ,Cell Biology ,Embryonic stem cell ,Spermatogonia ,Cell biology ,Mice, Inbred C57BL ,Adult Stem Cells ,medicine.anatomical_structure ,Animals, Newborn ,Immunology ,Molecular Medicine ,Transcription Factor TFIID ,Germ line development ,Stem cell ,Germ cell ,Developmental Biology ,Adult stem cell - Abstract
Long-term mammalian spermatogenesis requires proper development of spermatogonial stem cells (SSCs) that replenish the testis with germ cell progenitors during adult life. TAF4b is a gonadal-enriched component of the general transcription factor complex, TFIID, which is required for the maintenance of spermatogenesis in the mouse. Successful germ cell transplantation assays into adult TAF4b-deficient host testes suggested that TAF4b performs an essential germ cell autonomous function in SSC establishment and/or maintenance. To elucidate the SSC function of TAF4b, we characterized the initial gonocyte pool and rounds of spermatogenic differentiation in the context of the Taf4b-deficient mouse testis. Here, we demonstrate a significant reduction in the late embryonic gonocyte pool and a deficient expansion of this pool soon after birth. Resulting from this reduction of germ cell progenitors is a developmental delay in meiosis initiation, as compared to age-matched controls. While GFRα1+ spermatogonia are appropriately present as Asingle and Apaired in wild-type testes, TAF4b-deficient testes display an increased proportion of long and clustered chains of GFRα1+ cells. In the absence of TAF4b, seminiferous tubules in the adult testis either lack germ cells altogether or are found to have missing generations of spermatogenic progenitor cells. Together these data indicate that TAF4b-deficient spermatogenic progenitor cells display a tendency for differentiation at the expense of self-renewal and a renewing pool of SSCs fail to establish during the critical window of SSC development. Stem Cells 2015;33:1267–1276
- Published
- 2015
33. ATR is a multifunctional regulator of male mouse meiosis
- Author
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Obah A. Ojarikre, Elias ElInati, Julian Lange, Takayuki Hirota, Shantha K. Mahadevaiah, Dirk G. de Rooij, Scott Keeney, Valdone Maciulyte, Jasmin Zohren, Attila Tóth, Alexander Widger, James M. A. Turner, and Marcello Stanzione
- Subjects
0303 health sciences ,Synapsis ,RAD51 ,Spermatocyte ,Biology ,Molecular biology ,03 medical and health sciences ,0302 clinical medicine ,medicine.anatomical_structure ,Meiosis ,Homologous chromosome ,medicine ,Recombinase ,biological phenomena, cell phenomena, and immunity ,Ataxia telangiectasia and Rad3 related ,030217 neurology & neurosurgery ,Germ cell ,030304 developmental biology - Abstract
Meiotic cells undergo genetic exchange between homologous chromosomes through programmed DNA double-strand break (DSB) formation, recombination and synapsis1, 2. In mice, the DNA damage-regulated phosphatidylinositol-3-kinase-like kinase (PIKK) ATM regulates all of these processes3-6. However, the meiotic functions of another major PIKK, ATR, have remained elusive, because germ line-specific depletion of this kinase is challenging. Using an efficient conditional strategy, we uncover roles for ATR in male mouse prophase I progression. Deletion of ATR causes chromosome axis fragmentation and germ cell elimination at mid pachynema. ATR is required for homologous synapsis, in a manner genetically dissociable from DSB formation. In addition, ATR regulates loading of recombinases RAD51 and DMC1 to DSBs and maintenance of recombination foci on synapsed and asynapsed chromosomes. Mid pachytene spermatocyte elimination in ATR deficient mice cannot be rescued by deletion of ATM and the third DNA damage-regulated PIKK, PRKDC, consistent with the existence of a PIKK-independent surveillance mechanism in the mammalian germ line. Our studies identify ATR as a multifunctional regulator of mouse meiosis.
- Published
- 2017
34. Author response: The conserved RNA helicase YTHDC2 regulates the transition from proliferation to differentiation in the germline
- Author
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Pedro J. Batista, Dirk G. de Rooij, Howard Y. Chang, Y. Grace Chen, Alexis S Bailey, Rebecca S Gold, and Margaret T. Fuller
- Subjects
Transition (genetics) ,Biology ,RNA Helicase A ,Germline ,Cell biology - Published
- 2017
35. Meioc maintains an extended meiotic prophase I in mice
- Author
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Mina L Kojima, David C. Page, Alexander K. Godfrey, Dirk G. de Rooij, Maria M. Mikedis, Y. Q. Shirleen Soh, Massachusetts Institute of Technology. Department of Biology, Whitehead Institute for Biomedical Research, Soh, Ying Qi Shirleen, Mikedis, Maria M., Kojima, Mina, Godfrey, Alexander Kamitsuka, Page, David C, and de Rooij, Dirk G.
- Subjects
Male ,0301 basic medicine ,Cancer Research ,Cell Cycle Proteins ,Prophase ,Mice ,Animal Cells ,Spermatocytes ,Medicine and Health Sciences ,Cell Cycle and Cell Division ,Testes ,Genetics (clinical) ,Chromosome Biology ,Zygotene Stage ,Synapsis ,Gene Expression Regulation, Developmental ,RNA-Binding Proteins ,Cell cycle ,Precipitation Techniques ,Cell biology ,Ovaries ,Meiosis ,Cell Processes ,Cellular Types ,Anatomy ,Genital Anatomy ,Research Article ,lcsh:QH426-470 ,Mitosis ,Biology ,Research and Analysis Methods ,03 medical and health sciences ,Meiotic Prophase I ,Genetics ,Immunoprecipitation ,Animals ,Spermatogenesis ,Molecular Biology ,Metaphase ,Ecology, Evolution, Behavior and Systematics ,urogenital system ,Reproductive System ,Biology and Life Sciences ,Cell Biology ,Spermatogonia ,Chromosome Pairing ,lcsh:Genetics ,Germ Cells ,030104 developmental biology ,Cyclin A2 ,Meiotic Prophase - Abstract
The meiosis-specific chromosomal events of homolog pairing, synapsis, and recombination occur over an extended meiotic prophase I that is many times longer than prophase of mitosis. Here we show that, in mice, maintenance of an extended meiotic prophase I requires the gene Meioc, a germ-cell specific factor conserved in most metazoans. In mice, Meioc is expressed in male and female germ cells upon initiation of and throughout meiotic prophase I. Mouse germ cells lacking Meioc initiate meiosis: they undergo pre-meiotic DNA replication, they express proteins involved in synapsis and recombination, and a subset of cells progress as far as the zygotene stage of prophase I. However, cells in early meiotic prophase—as early as the preleptotene stage—proceed to condense their chromosomes and assemble a spindle, as if having progressed to metaphase. Meioc-deficient spermatocytes that have initiated synapsis mis-express CYCLIN A2, which is normally expressed in mitotic spermatogonia, suggesting a failure to properly transition to a meiotic cell cycle program. MEIOC interacts with YTHDC2, and the two proteins pull-down an overlapping set of mitosis-associated transcripts. We conclude that when the meiotic chromosomal program is initiated, Meioc is simultaneously induced so as to extend meiotic prophase. Specifically, MEIOC, together with YTHDC2, promotes a meiotic (as opposed to mitotic) cell cycle program via post-transcriptional control of their target transcripts., Author summary Meiosis is the specialized cell division that halves the genetic content of germ cells to produce haploid gametes. This reductive division is preceded by a preparative phase of the cell cycle, meiotic prophase I, during which several meiosis-specific chromosomal events occur. Across sexually reproducing organisms, prophase of meiosis I is dramatically longer than mitotic prophase. However, it was not known in mammals how and why meiotic prophase I is extended. We have identified a mouse mutant in which this extended prophase I is disrupted: germ cells lacking Meioc initiate meiosis, but prematurely proceed to metaphase. Mutant male meiotic germ cells mis-express a cell cycle regulator that is normally expressed in mitotic male germ cells, suggesting that Meioc is required for germ cells to properly transition to a meiotic cell cycle program. Biochemical analyses of proteins and transcripts that associate with MEIOC protein suggest that MEIOC may promote the transition from a mitotic to meiotic cell cycle program by post-transcriptionally regulating target transcripts. Our studies indicate that in mammals, as in other sexually reproducing organisms, meiotic prophase I must be extended to allow time for meiotic chromosomal events to reach completion.
- Published
- 2017
36. A Neofunctionalized X-Linked Ampliconic Gene Family Is Essential for Male Fertility and Equal Sex Ratio in Mice
- Author
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Yueh-Chiang Hu, Alyssa N. Kruger, Jamie L. Huizinga, Dirk G. de Rooij, Jeffrey M. Kidd, Michele Brogley, Jacob L. Mueller, Developmental Biology, Sub Developmental Biology, and Academic Medical Center
- Subjects
Male ,0301 basic medicine ,Protein family ,Gene Expression ,gene dosage ,Biology ,Biochemistry ,Gene dosage ,Article ,General Biochemistry, Genetics and Molecular Biology ,male infertility ,X chromosome ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Genes, X-Linked ,Gene duplication ,Animals ,Gene family ,Sex Ratio ,Gene ,Genetics ,Sex Chromosomes ,Agricultural and Biological Sciences(all) ,Biochemistry, Genetics and Molecular Biology(all) ,meiotic drive ,Mice, Inbred C57BL ,Synaptonemal complex ,Fertility ,030104 developmental biology ,Meiotic drive ,Mice, Inbred DBA ,Multigene Family ,Female ,General Agricultural and Biological Sciences ,030217 neurology & neurosurgery ,Genetics and Molecular Biology(all) - Abstract
Summary The mammalian sex chromosomes harbor an abundance of newly acquired ampliconic genes, although their functions require elucidation [ 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 ]. Here, we demonstrate that the X-linked Slx and Slxl1 ampliconic gene families represent mouse-specific neofunctionalized copies of a meiotic synaptonemal complex protein, Sycp3. In contrast to the meiotic role of Sycp3, CRISPR-loxP-mediated multi-megabase deletions of the Slx (5 Mb) and Slxl1 (2.3Mb) ampliconic regions result in post-meiotic defects, abnormal sperm, and male infertility. Males carrying Slxl1 deletions sire more male offspring, whereas males carrying Slx and Slxl1 duplications sire more female offspring, which directly correlates with Slxl1 gene dosage and gene expression levels. SLX and SLXL1 proteins interact with spindlin protein family members (SPIN1 and SSTY1/2) and males carrying Slxl1 deletions downregulate a sex chromatin modifier, Scml2, leading us to speculate that Slx and Slxl1 function in chromatin regulation. Our study demonstrates how newly acquired X-linked genes can rapidly evolve new and essential functions and how gene amplification can increase sex chromosome transmission.
- Published
- 2019
37. The RHOX homeobox gene cluster is selectively expressed in human oocytes and male germ cells
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R A L Bayne, Hye-Won Song, Louise C. Laurent, Richard A. Anderson, R.J. Chang, Miles F. Wilkinson, Mana M. Parast, Dirk G. de Rooij, Tung-Chin Hsieh, Shunichi Shimasaki, Jörg Gromoll, Other Research, and Center for Reproductive Medicine
- Subjects
Adult ,Male ,Cell type ,Placenta ,Blotting, Western ,Molecular Sequence Data ,Ovary ,Biology ,Gonocyte ,Pregnancy ,Gene expression ,Gene cluster ,Testis ,medicine ,Humans ,Amino Acid Sequence ,Homeodomain Proteins ,Rehabilitation ,Obstetrics and Gynecology ,Gene Expression Regulation, Developmental ,Original Articles ,Middle Aged ,Molecular biology ,Immunohistochemistry ,Spermatozoa ,Cell biology ,Pregnancy Trimester, First ,medicine.anatomical_structure ,Reproductive Medicine ,Multigene Family ,Pregnancy Trimester, Second ,Oocytes ,Homeobox ,Female ,Spermatogenesis ,Germ cell - Abstract
study question: What human tissues and cell types express the X-linked reproductive homeobox (RHOX) gene cluster? summary answer: The RHOX homeobox genes and proteins are selectively expressed in germ cells in both the ovary and testis. what is known already: The RHOX homeobox transcription factors are encoded by an X-linked gene cluster whose members are selectively expressed in the male and female reproductive tract of mice and rats. The Rhox genes have undergone strong selection pressure to rapidly evolve, making it uncertain whether they maintain their reproductive tissue-centric expression pattern in humans, an issue we address in this report. study design, size, duration: We examined the expression of all members of the human RHOX gene cluster in 11 fetal and 8 adult tissues. The focus of our analysis was on fetal testes, where we evaluated 16 different samples from 8 to 20 weeks gestation. We also analyzed fixed sections from fetal testes, adult testes and adult ovaries to determine the cell type-specific expression pattern of the proteins encoded by RHOX genes. participants/materials, setting, methods: We used quantitative reverse transcription –polymerase chain reaction analysis to assay human RHOX gene expression. We generated antisera against RHOX proteins and used them for western blotting, immunohistochemical and immunofluorescence analyses of RHOXF1 and RHOXF2/2B protein expression. main results and the role of chance: We found that the RHOXF1 and RHOXF2/2B genes are highly expressed in the testis and exhibit low or undetectable expression in most other organs. Using RHOXF1- and RHOXF2/2B-specific antiserum, we found that both RHOXF1 and RHOXF2/2B are primarily expressed in germ cells in the adult testis. Early stage germ cells (spermatogonia and early spermatocytes) express RHOXF2/2B, while later stage germ cells (pachytene spermatocytes and round spermatids) express RHOXF1. Both RHOXF1 and RHOXF2/2B are expressed in prespermatogonia in human fetal testes. Consistent with this, RHOXF1 and RHOXF2/2B mRNA expression increases in the second trimester during fetal testes development when gonocytes differentiate into prespermatogonia. In the human adult ovary, we found that RHOXF1 and RHOXF2/2B are primarily expressed in oocytes. limitations, reasons for caution: While the average level of expression of RHOX genes was low or undetectable in all 19 human tissues other than testes, it is still possible that RHOX genes are highly expressed in a small subset of cells in some of these nontesticular tissues. As a case in point, we found that RHOX proteins are highly expressed in oocytes within the human ovary, despite low levels of RHOX mRNA in the whole ovary. wider implications of the findings: The cell type-specific and developmentally regulated expression pattern of the RHOX transcription factors suggests that they perform regulatory functions during human fetal germ cell development, spermatogenesis and
- Published
- 2013
38. ATR acts stage specifically to regulate multiple aspects of mammalian meiotic silencing
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James M. A. Turner, Haydn M. Prosser, Christer Höög, Yaroslava Ruzankina, Attila Tóth, Eric J. Brown, Dirk G. de Rooij, Shantha K. Mahadevaiah, Tomoyuki Fukuda, Jeffrey M. Cloutier, Hélène Royo, Allan Bradley, Marek Baumann, Other Research, and Center for Reproductive Medicine
- Subjects
Male ,Mad2 ,Cell Cycle Proteins ,Ataxia Telangiectasia Mutated Proteins ,Protein Serine-Threonine Kinases ,Chromosomes ,Histones ,Mice ,Genetics ,Animals ,Gene silencing ,Gene Silencing ,Epigenetics ,Phosphorylation ,Adaptor Proteins, Signal Transducing ,biology ,Intracellular Signaling Peptides and Proteins ,G2-M DNA damage checkpoint ,Chromatin ,MDC1 ,Repressor Proteins ,Meiosis ,Protein Transport ,Histone ,Gene Expression Regulation ,H2AFX ,biology.protein ,biological phenomena, cell phenomena, and immunity ,Research Paper ,Developmental Biology - Abstract
In mammals, homologs that fail to synapse during meiosis are transcriptionally inactivated. This process, meiotic silencing, drives inactivation of the heterologous XY bivalent in male germ cells (meiotic sex chromosome inactivation [MSCI]) and is thought to act as a meiotic surveillance mechanism. The checkpoint protein ATM and Rad3-related (ATR) localizes to unsynapsed chromosomes, but its role in the initiation and maintenance of meiotic silencing is unknown. Here we show that ATR has multiple roles in silencing. ATR first regulates HORMA (Hop1, Rev7, and Mad2) domain protein HORMAD1/2 phosphorylation and localization of breast cancer I (BRCA1) and ATR cofactors ATR-interacting peptide (ATRIP)/topoisomerase 2-binding protein 1 (TOPBP1) at unsynapsed axes. Later, it acts as an adaptor, transducing signaling at unsynapsed axes into surrounding chromatin in a manner that requires interdependence with mediator of DNA damage checkpoint 1 (MDC1) and H2AFX. Finally, ATR catalyzes histone H2AFX phosphorylation, the epigenetic event leading to gene inactivation. Using a novel genetic strategy in which MSCI is used to silence a chosen gene in pachytene, we show that ATR depletion does not disrupt the maintenance of silencing and that silencing comprises two phases: The first is dynamic and reversible, and the second is stable and irreversible. Our work identifies a role for ATR in the epigenetic regulation of gene expression and presents a new technique for ablating gene function in the germline.
- Published
- 2013
39. C14ORF39/SIX6OS1 is a constituent of the synaptonemal complex and is essential for mouse fertility
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Manuel Sánchez-Martín, Elena Llano, Ignacio García-Tuñón, Attila Tóth, Dirk G. de Rooij, Laura Gómez-H, Alberto M. Pendás, Owen R. Davies, Isabel Ramos, Ihsan Dereli, José Luis Barbero, Ricardo Benavente, Natalia Felipe-Medina, Ministerio de Economía y Competitividad (España), German Research Foundation, European Commission, Wellcome Trust, and Royal Society (UK)
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Male ,0301 basic medicine ,Transcription, Genetic ,Chromosomal Proteins, Non-Histone ,General Physics and Astronomy ,Cell Cycle Proteins ,Haploidy ,Chromosomal crossover ,Mice ,Testis ,Crossing Over, Genetic ,Recombination, Genetic ,Genetics ,Genome ,Multidisciplinary ,Synapsis ,Nuclear Proteins ,3. Good health ,DNA-Binding Proteins ,Meiosis ,Synaptonemal complex ,Electroporation ,Female ,Mouse fertility ,Science ,2415 Biología Molecular ,Biology ,Article ,Chromosomes ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Meiotic Prophase I ,Two-Hybrid System Techniques ,ddc:570 ,Homologous chromosome ,Animals ,Humans ,Spermatogenesis ,Central element ,Genetic Variation ,General Chemistry ,Chromosome Pairing ,Fertility ,HEK293 Cells ,cromosomas ,030104 developmental biology ,espermatogénesis ,Homologous recombination - Abstract
Meiotic recombination generates crossovers between homologous chromosomes that are essential for genome haploidization. The synaptonemal complex is a ‘zipper’-like protein assembly that synapses homologue pairs together and provides the structural framework for processing recombination sites into crossovers. Humans show individual differences in the number of crossovers generated across the genome. Recently, an anonymous gene variant in C14ORF39/SIX6OS1 was identified that influences the recombination rate in humans. Here we show that C14ORF39/SIX6OS1 encodes a component of the central element of the synaptonemal complex. Yeast two-hybrid analysis reveals that SIX6OS1 interacts with the well-established protein synaptonemal complex central element 1 (SYCE1). Mice lacking SIX6OS1 are defective in chromosome synapsis at meiotic prophase I, which provokes an arrest at the pachytene-like stage and results in infertility. In accordance with its role as a modifier of the human recombination rate, SIX6OS1 is essential for the appropriate processing of intermediate recombination nodules before crossover formation., This work was supported by BFU_2014-59307-R, MEIONet and JCyLe (CSI052U16). LGH and NFM are supported by European Social Fund/JCyLe grants (EDU/1083/2013 and EDU/310/2015). ORD is a Sir Henry Dale Fellow jointly funded by the Wellcome Trust and Royal Society (Grant Number 104158/Z/14/Z). RB is funded by DFG (grant Be1168/8-1). AT and ID were supported by DFG grants TO421/8-2 and TO421/6-1, respectively.
- Published
- 2016
40. Zfy genes are required for efficient meiotic sex chromosome inactivation (MSCI) in spermatocytes
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Dirk G. de Rooij, Paul S. Burgoyne, Nadège Vernet, Shantha K. Mahadevaiah, and Peter J.I. Ellis
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0301 basic medicine ,Male ,Cell cycle checkpoint ,X Chromosome ,Biology ,03 medical and health sciences ,Mice ,Meiosis ,Spermatocytes ,X Chromosome Inactivation ,Genetics ,medicine ,Animals ,QP506 ,Spermatogenesis ,Molecular Biology ,Gene ,QH426 ,Genetics (clinical) ,QH581.2 ,General Medicine ,Articles ,Chromosome inactivation ,DNA-Binding Proteins ,030104 developmental biology ,medicine.anatomical_structure ,Germ cell ,Transcription Factors - Abstract
During spermatogenesis, germ cells that fail to synapse their chromosomes or fail to undergo meiotic sex chromosome inactivation (MSCI) are eliminated via apoptosis during mid-pachytene. Previous work showed that Y-linked genes Zfy1 and Zfy2 act as ‘executioners’ for this checkpoint, and that wrongful expression of either gene during pachytene triggers germ cell death. Here, we show that in mice, Zfy genes are also necessary for efficient MSCI and the sex chromosomes are not correctly silenced in Zfy-deficient spermatocytes. This unexpectedly reveals a triple role for Zfy at the mid-pachytene checkpoint in which Zfy genes first promote MSCI, then monitor its progress (since if MSCI is achieved, Zfy genes will be silenced), and finally execute cells with MSCI failure. This potentially constitutes a negative feedback loop governing this critical checkpoint mechanism.
- Published
- 2016
41. piRNA-associated proteins and retrotransposons are differentially expressed in murine testis and ovary of aryl hydrocarbon receptor deficient mice
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Alberto M. Pendás, Cristina Ortega-Ferrusola, Pedro M. Fernández-Salguero, Inmaculada Catalina-Fernández, Francisco J. González-Rico, Jaime M. Merino, Matías Hidalgo-Sánchez, Dirk G. de Rooij, Nuria Moreno-Marín, Elena Llano, Ascensión Infante-Campos, Patricia Martín-Muñoz, Alberto Álvarez-Barrientos, Fernando J. Peña, Eva M. Rico-Leo, Luis Óscar Sánchez-Guardado, Eva Barrasa, Instituto de Salud Carlos III, Junta de Castilla y León, Ministerio de Educación (España), Ministerio de Educación, Cultura y Deporte (España), European Commission, Red Temática de Investigación Cooperativa en Cáncer (España), Ministerio de Economía y Competitividad (España), Ministerio de Ciencia e Innovación (España), and Junta de Extremadura
- Subjects
0301 basic medicine ,Male ,Retrotransposon ,Oogenesis ,DEAD-box RNA Helicases ,Gene Knockout Techniques ,Mice ,0302 clinical medicine ,Testis ,Basic Helix-Loop-Helix Transcription Factors ,RNA, Small Interfering ,lcsh:QH301-705.5 ,Aryl hydrocarbon receptor ,fertility ,biology ,aryl hydrocarbon receptor ,General Neuroscience ,Gene Expression Regulation, Developmental ,Nuage proteins ,respiratory system ,Up-Regulation ,repetitive elements ,Meiosis ,medicine.anatomical_structure ,Argonaute Proteins ,Female ,Germ cell ,Research Article ,nuage proteins ,endocrine system ,Retroelements ,Immunology ,Piwi-interacting RNA ,Ovary ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,MiWi ,medicine ,Animals ,Repetitive elements ,Spermatogenesis ,urogenital system ,Research ,Molecular biology ,spermatogenesis ,respiratory tract diseases ,030104 developmental biology ,Fertility ,lcsh:Biology (General) ,Receptors, Aryl Hydrocarbon ,biology.protein ,030217 neurology & neurosurgery - Abstract
Previous studies suggested that the aryl hydrocarbon receptor (AhR) contributes to mice reproduction and fertility. However, the mechanisms involved remain mostly unknown. Retrotransposon silencing by Piwi-interacting RNAs (piRNAs) is essential for germ cell maturation and, remarkably, AhR has been identified as a regulator of murine B1-SINE retrotransposons. Here, using littermate AhR and AhR mice, we report that AhR regulates the general course of spermatogenesis and oogenesis by a mechanism likely to be associated with piRNA-associated proteins, piRNAs and retrotransposons. piRNA-associated proteins MVH and Miwi are upregulated in leptotene to pachytene spermatocytes with a more precocious timing in AhR than in AhR testes. piRNAs and transcripts from B1-SINE, LINE-1 and IAP retrotransposons increased at these meiotic stages in AhR-null testes. Moreover, B1-SINE transcripts colocalize with MVH and Miwi in leptonema and pachynema spermatocytes. Une pectedly, AhR males have increased sperm counts, higher sperm functionality and enhanced fertility than AhR mice. In contrast, piRNA-associated proteins and B1-SINE and IAP-derived transcripts are reduced in adult AhR2/2 ovaries. Accordingly, AhR-null female mice have lower numbers of follicles when compared with AhR\+/\+ mice. Thus, AhR deficiency differentially affects testis and ovary development possibly by a process involving piRNA-associated proteins, piRNAs and transposable elements., This work was supported by grants to P.M.F-S. from the Spanish Ministry of Science and Innovation (BFU2011-22678) and Economy and Competitiveness (SAF2014-51813-R) and from the Junta de Extremadura (GR15008). Research at the F.J.P. laboratory was supported by grant no. AGL2013-43211-R from the Spanish Ministry of Economy and Competitiveness. Research at the P.M.F-S laboratory was also supported by the Red Tematica de Investigacion Cooperativa en Cancer (RTICC), Carlos III Institute, Spanish Ministry of Economy and Competitiveness (RD12/0036/0032). E.R.L., F.J.G-R and E.B. were supported by the RTICC, MICINN and Junta de Extremadura, respectively. N.M.M. was an FPI fellow of the Spanish Ministry of Education. P.M.M. was supported by a pre-doctoral grant from the Spanish Ministry of Education, Culture and Sport (FPU13/03991) C.O.F. was supported by a postdoctoral Juan de la Cierva (IJCI-2014-21671) grant from the Spanish Ministry of Economy and Competitiveness. A.M.P. and E.L. were supported by grants from the Spanish Ministry of Economy and Competitiveness (BFU2014-59307-R), MEIONet and the Junta de Castilla y Leon. All Spanish funding is co-sponsored by the European Union FEDER programme.
- Published
- 2016
42. The Antagonistic Gene Paralogs Upf3a and Upf3b Govern Nonsense-Mediated RNA Decay
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Kevin J. Peterson, Samantha H Jones, Eleen Shum, Ada Shao, Hye-Won Song, Josh L. Espinoza, Dirk G. de Rooij, Lulu Huang, Wai Kin Chan, Heidi Cook-Andersen, Matthew D. Krause, Jennifer N Dumdie, Mimi H. Phan, Miles F. Wilkinson, John R. McCarrey, Madhuvanthi Ramaiah, and Chih-Hong Lou
- Subjects
0301 basic medicine ,Evolution ,1.1 Normal biological development and functioning ,Nonsense-mediated decay ,Duplicate ,Repressor ,Embryonic Development ,RNA-binding protein ,Biology ,Medical and Health Sciences ,General Biochemistry, Genetics and Molecular Biology ,Article ,Gametogenesis ,Cell Line ,Evolution, Molecular ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Genes, Duplicate ,Underpinning research ,Cell Line, Tumor ,Gene duplication ,Genetics ,Animals ,Humans ,RNA, Messenger ,Gene ,Tumor ,Mechanism (biology) ,RNA ,RNA-Binding Proteins ,Molecular ,Biological Sciences ,Stem Cell Research ,Nonsense Mediated mRNA Decay ,030104 developmental biology ,Fertility ,Genes ,Hela Cells ,Adaptation ,030217 neurology & neurosurgery ,HeLa Cells ,Developmental Biology - Abstract
Gene duplication is a major evolutionary force driving adaptation and speciation, as it allows for the acquisition of new functions and can augment or diversify existing functions. Here, we report a gene duplication event that yielded another outcome--the generation of antagonistic functions. One product of this duplication event--UPF3B--is critical for the nonsense-mediated RNA decay (NMD) pathway, while its autosomal counterpart--UPF3A--encodes an enigmatic protein previously shown to have trace NMD activity. Using loss-of-function approaches in vitro and in vivo, we discovered that UPF3A acts primarily as a potent NMD inhibitor that stabilizes hundreds of transcripts. Evidence suggests that UPF3A acquired repressor activity through simple impairment of a critical domain, a rapid mechanism that may have been widely used in evolution. Mice conditionally lacking UPF3A exhibit "hyper" NMD and display defects in embryogenesis and gametogenesis. Our results support a model in which UPF3A serves as a molecular rheostat that directs developmental events.
- Published
- 2016
43. DNA Double Strand Break Response and Limited Repair Capacity in Mouse Elongated Spermatids
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Dirk G. de Rooij, Harry Scherthan, and Emad A. Ahmed
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Male ,Chromosomal Proteins, Non-Histone ,Spermiogenesis ,Mice, SCID ,PARP1-inhibited mice ,Histones ,lcsh:Chemistry ,Spermatocytes ,Radiation, Ionizing ,elongated spermatids ,DNA Breaks, Double-Stranded ,Phosphorylation ,lcsh:QH301-705.5 ,Spectroscopy ,Mice, Knockout ,Recombination, Genetic ,Rad54/Rad54B deficient mice ,Antigens, Nuclear ,General Medicine ,Spermatids ,Computer Science Applications ,DNA-Binding Proteins ,Non-homologous end joining ,Meiosis ,Histone ,DNA fragmentation ,Tumor Suppressor p53-Binding Protein 1 ,endocrine system ,DNA damage ,DNA repair ,Biology ,Article ,Catalysis ,Inorganic Chemistry ,SCID mice ,Prophase ,Animals ,NHEJ ,Physical and Theoretical Chemistry ,Fragmentation (cell biology) ,Ku Autoantigen ,Molecular Biology ,urogenital system ,Organic Chemistry ,fungi ,Molecular biology ,Kinetics ,lcsh:Biology (General) ,lcsh:QD1-999 ,biology.protein - Abstract
Spermatids are extremely sensitive to genotoxic exposures since during spermiogenesis only error-prone non homologous end joining (NHEJ) repair pathways are available. Hence, genomic damage may accumulate in sperm and be transmitted to the zygote. Indirect, delayed DNA fragmentation and lesions associated with apoptotic-like processes have been observed during spermatid elongation, 27 days after irradiation. The proliferating spermatogonia and early meiotic prophase cells have been suggested to retain a memory of a radiation insult leading later to this delayed fragmentation. Here, we used meiotic spread preparations to localize phosphorylate histone H2 variant (γ-H2AX) foci marking DNA double strand breaks (DSBs) in elongated spermatids. This technique enabled us to determine the background level of DSB foci in elongated spermatids of RAD54/RAD54B double knockout (dko) mice, severe combined immunodeficiency SCID mice, and poly adenosine diphosphate (ADP)-ribose polymerase 1 (PARP1) inhibitor (DPQ)-treated mice to compare them with the appropriate wild type controls. The repair kinetics data and the protein expression patterns observed indicate that the conventional NHEJ repair pathway is not available for elongated spermatids to repair the programmed and the IR-induced DSBs, reflecting the limited repair capacity of these cells. However, although elongated spermatids express the proteins of the alternative NHEJ, PARP1-inhibition had no effect on the repair kinetics after IR, suggesting that DNA damage may be passed onto sperm. Finally, our genetic mutant analysis suggests that an incomplete or defective meiotic recombinational repair of Spo11-induced DSBs may lead to a carry-over of the DSB damage or induce a delayed nuclear fragmentation during the sensitive programmed chromatin remodeling occurring in elongated spermatids.
- Published
- 2015
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44. The Homeobox Transcription Factor RHOX10 Drives Mouse Spermatogonial Stem Cell Establishment
- Author
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Adrienne H. Zhao, Eric Babajanian, Eleen Shum, Vaishnavi Sridhar, Marcy E. Richardson, Terra-Dawn M. Plank, Hye-Won Song, Meena Sukhwani, Dirk G. de Rooij, Anilkumar Bettegowda, David Skarbrevik, Blue B. Lake, Mimi H. Phan, Kyle E. Orwig, Kun Zhang, Madhuvanthi Ramaiah, and Miles F. Wilkinson
- Subjects
0301 basic medicine ,Male ,Medical Physiology ,Regenerative Medicine ,Mice ,0302 clinical medicine ,Single-cell analysis ,Genes, X-Linked ,2.1 Biological and endogenous factors ,Protein Isoforms ,Developmental ,Aetiology ,gonocytes ,skin and connective tissue diseases ,lcsh:QH301-705.5 ,transcription factor ,Genetics ,Mice, Knockout ,030219 obstetrics & reproductive medicine ,integumentary system ,Adult Germline Stem Cells ,homeobox ,Gene Expression Regulation, Developmental ,Phenotype ,Rhox ,medicine.anatomical_structure ,Multigene Family ,Stem Cell Research - Nonembryonic - Non-Human ,prospermatogonia ,Stem cell ,Single-Cell Analysis ,Sequence Analysis ,Germ cell ,Adult stem cell ,endocrine system ,Knockout ,1.1 Normal biological development and functioning ,Biology ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Rare Diseases ,Underpinning research ,medicine ,Animals ,Genes, Developmental ,Spermatogenesis ,Transcription factor ,spermatogonial stem cells ,Homeodomain Proteins ,Sequence Analysis, RNA ,germ line stem cell ,germ cell ,X-Linked ,Stem Cell Research ,Spermatogonia ,030104 developmental biology ,Gene Expression Regulation ,Genes ,lcsh:Biology (General) ,RNA ,Homeobox ,Generic health relevance ,Biochemistry and Cell Biology - Abstract
SummaryThe developmental origins of most adult stem cells are poorly understood. Here, we report the identification of a transcription factor—RHOX10—critical for the initial establishment of spermatogonial stem cells (SSCs). Conditional loss of the entire 33-gene X-linked homeobox gene cluster that includes Rhox10 causes progressive spermatogenic decline, a phenotype indistinguishable from that caused by loss of only Rhox10. We demonstrate that this phenotype results from dramatically reduced SSC generation. By using a battery of approaches, including single-cell-RNA sequencing (scRNA-seq) analysis, we show that Rhox10 drives SSC generation by promoting pro-spermatogonia differentiation. Rhox10 also regulates batteries of migration genes and promotes the migration of pro-spermatogonia into the SSC niche. The identification of an X-linked homeobox gene that drives the initial generation of SSCs has implications for the evolution of X-linked gene clusters and sheds light on regulatory mechanisms influencing adult stem cell generation in general.
- Published
- 2015
45. The cohesin subunit RAD21L functions in meiotic synapsis and exhibits sexual dimorphism in fertility
- Author
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Teresa Hernández, Manuel Sánchez-Martín, Alberto Viera, Cristina Gutiérrez-Caballero, Yurema Herrán, Alberto M. Pendás, José A. Suja, Dirk G. de Rooij, Enrique de Álava, Elena Llano, José Luis Barbero, Other Research, and Center for Reproductive Medicine
- Subjects
Male ,Cohesin complex ,Chromosomal Proteins, Non-Histone ,Cell Cycle Proteins ,Biology ,Chromosomes ,Article ,General Biochemistry, Genetics and Molecular Biology ,Mice ,Sex Factors ,Meiosis ,Testis ,Homologous chromosome ,Animals ,Molecular Biology ,Anaphase ,Mice, Knockout ,Genetics ,General Immunology and Microbiology ,Cohesin ,Histocytochemistry ,General Neuroscience ,Ovary ,Age Factors ,Synapsis ,Protein Subunits ,Synaptonemal complex ,Infertility ,Cohesion ,Female ,biological phenomena, cell phenomena, and immunity ,Separase - Abstract
The cohesin complex is a ring-shaped proteinaceous structure that entraps the two sister chromatids after replication until the onset of anaphase when the ring is opened by proteolytic cleavage of its α-kleisin subunit (RAD21 at mitosis and REC8 at meiosis) by separase. RAD21L is a recently identified α-kleisin that is present from fish to mammals and biochemically interacts with the cohesin subunits SMC1, SMC3 and STAG3. RAD21L localizes along the axial elements of the synaptonemal complex of mouse meiocytes. However, its existence as a bona fide cohesin and its functional role awaits in vivo validation. Here, we show that male mice lacking RAD21L are defective in full synapsis of homologous chromosomes at meiotic prophase I which provokes an arrest at zygotene and leads to total azoospermia and consequently infertility. In contrast, RAD21L-deficient females are fertile but develop an age-dependent sterility. Thus, our results provide in vivo evidence that RAD21L is essential for male fertility and in females for the maintenance of fertility during natural aging., This work was supported by SAF (2008-0317), J CyLe (SA), BFU (2008- 00300/BCM) and BFU (2009-08975/BMC). CGC and YS are supported by FIS and FPI fellowships, respectively. EL is recipient of a Ramo´n y Cajal Research contract.
- Published
- 2011
46. BMP4-Induced Differentiation of a Rat Spermatogonial Stem Cell Line Causes Changes in Its Cell Adhesion Properties1
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Dirk G. de Rooij, Sjoerd Repping, Cindy M. Korver, Ans M.M. van Pelt, Gianfranco Carlomagno, and Maaike P.A. van Bragt
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endocrine system ,education.field_of_study ,Cell adhesion molecule ,Population ,Cell Biology ,General Medicine ,Biology ,Actin cytoskeleton ,Cell biology ,Adherens junction ,Reproductive Medicine ,Cell culture ,Immunology ,Stem cell ,Signal transduction ,education ,Cell adhesion - Abstract
Spermatogonial stem cells (SSCs) are at the basis of the spermatogenic process and are essential for the continuous lifelong production of spermatozoa. Although several factors that govern SSC self-renewal and differentiation have been investigated, the direct effect of such factors on SSCs has not yet been studied, mainly because of the absence of markers to identify SSCs and the lack of effective methods to obtain and culture a pure population of SSCs. We now have used a previously established rat SSC cell line (GC-6spg) to elucidate the role of BMP4 in SSC differentiation. We found that GC-6spg cells cultured in the presence of BMP4 upregulate KIT expression, which is an early marker for differentiating spermatogonia. GC-6spg cells were found to express three BMP4 receptors and the downstream SMAD1/5/8 proteins were phosphorylated during BMP4-induced differentiation. A time-course DNA micro-array analysis revealed a total of 529 differentially regulated transcripts (≥2-fold), including several known downstream targets of BMP4 such as Id2 and Gata2. Pathway analysis revealed that the most affected pathways were those involved in adherens junctions, focal junctions, gap junctions, cell adhesion molecules, and regulation of actin cytoskeleton. Interestingly, among the genes belonging to the most strongly affected adhesion pathways was Cdh1 (known as E-cadherin), an adhesion molecule known to be expressed by a subpopulation of spermatogonia including SSCs. Overall, our results suggest that BMP4 induces early differentiation of SSCs in a direct manner by affecting cell adhesion pathways.
- Published
- 2010
47. Parp1-XRCC1 and the repair of DNA double strand breaks in mouse round spermatids
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Emad A. Ahmed, Peter Boer, Dirk G. de Rooij, Marielle E.P. Philippens, Henk B. Kal, Other departments, Other Research, and Center for Reproductive Medicine
- Subjects
Male ,endocrine system ,DNA Repair ,Somatic cell ,DNA repair ,Health, Toxicology and Mutagenesis ,Blotting, Western ,Poly (ADP-Ribose) Polymerase-1 ,Fluorescent Antibody Technique ,DNA-Activated Protein Kinase ,Mice, SCID ,Biology ,Histones ,Immunoenzyme Techniques ,XRCC1 ,Mice ,Genetics ,medicine ,Animals ,DNA Breaks, Double-Stranded ,Spermatogenesis ,Molecular Biology ,Cell Nucleus ,Spermatid ,Nuclear Proteins ,Cell cycle ,Molecular biology ,Spermatids ,Non-homologous end joining ,DNA-Binding Proteins ,Human Reproduction [NCEBP 12] ,enzymes and coenzymes (carbohydrates) ,medicine.anatomical_structure ,Germ Cells ,X-ray Repair Cross Complementing Protein 1 ,Gamma Rays ,Poly(ADP-ribose) Polymerases ,Homologous recombination - Abstract
Contains fulltext : 89842.pdf (Publisher’s version ) (Closed access) The repair of DNA double strand breaks (DSBs) in male germ cells is slower and differently regulated compared to that in somatic cells. Round spermatids show DSB repair and are radioresistant to apoptosis induction. Mutation induction studies using ionizing irradiation, indicated a high frequency of chromosome aberrations (CA) in the next generation. Since they are in a G1 comparable stage of the cell cycle, haploid spermatids are expected to repair DSBs by the non-homologous end-joining pathway (NHEJ). However, immunohistochemical evidence indicates that not all components of the classical NHEJ pathway are available since the presence of DNA-PKcs cannot be shown. Here, we demonstrate that round spermatids, as well as most other types of male germ cells express both Parp1 and XRCC1. Therefore, we have determined whether the alternative Parp1/XRCC1 dependent NHEJ pathway is active in these nuclei and also have tested for classical NHEJ activity by a genetic method. To evaluate DSB repair in SCID mice, deficient for DNA-PKcs, and to study the involvement of the Parp1/XRCC1 dependent NHEJ pathway in round spermatids, the loss of gamma-H2AX foci after irradiation has been determined in nucleus spreads of round spermatids of SCID mice and in nucleus spreads and histological sections of Parp1-inhibited mice and their respective controls. Results show that around half of the breaks in randomly selected round spermatids are repaired between 1 and 8h after irradiation. The repair of 16% of the induced DSBs requires DNA-PKcs and 21% Parp1. Foci numbers in the Parp1-inhibited testes tend to be higher in spermatids of all epithelial stages reaching significance in stages I-III which indicates an active Parp1/XRCC1 pathway in round spermatids and a decreased repair capacity in later round spermatid stages. In Parp1-inhibited SCID mice only 14.5% of the breaks were repaired 8h after irradiation indicating additivity of the two NHEJ pathways in round spermatids.
- Published
- 2010
48. DNA polymerase β is critical for mouse meiotic synapsis
- Author
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Scott Keeney, Dirk G. de Rooij, Jing Pan, Joann B. Sweasy, Agnes Keh, Yanfeng Liu, Terry Ashley, Ivailo S. Mihaylov, Urmi Banerjee, Dawit Kidane, Timothy S Gorton, Daniel Zelterman, Alan S. Jonason, Other Research, and Center for Reproductive Medicine
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Male ,Spo11 ,DNA Repair ,DNA polymerase ,Spermatocyte ,Chromosomes ,Article ,General Biochemistry, Genetics and Molecular Biology ,Mice ,Meiosis ,Spermatocytes ,medicine ,Animals ,DNA Breaks, Double-Stranded ,Molecular Biology ,Replication protein A ,DNA Polymerase beta ,Endodeoxyribonucleases ,General Immunology and Microbiology ,biology ,General Neuroscience ,fungi ,Esterases ,Synapsis ,Seminiferous Tubules ,Molecular biology ,Chromosome Pairing ,Synaptonemal complex ,medicine.anatomical_structure ,biology.protein ,Female ,DMC1 ,Gene Deletion - Abstract
We have shown earlier that DNA polymerase beta (Pol beta) localizes to the synaptonemal complex (SC) during Prophase I of meiosis in mice. Pol beta localizes to synapsed axes during zygonema and pachynema, and it associates with the ends of bivalents during late pachynema and diplonema. To test whether these localization patterns reflect a function for Pol beta in recombination and/or synapsis, we used conditional gene targeting to delete the PolB gene from germ cells. We find that Pol beta-deficient spermatocytes are defective in meiotic chromosome synapsis and undergo apoptosis during Prophase I. We also find that SPO11-dependent gammaH2AX persists on meiotic chromatin, indicating that Pol beta is critical for the repair of SPO11-induced double-strand breaks (DSBs). Pol beta-deficient spermatocytes yielded reduced steady-state levels of the SPO11-oligonucleotide complexes that are formed when SPO11 is removed from the ends of DSBs, and cytological experiments revealed that chromosome-associated foci of replication protein A (RPA), RAD51 and DMC1 are less abundant in Pol beta-deficient spermatocyte nuclei. Localization of Pol beta to meiotic chromosomes requires the formation of SPO11-dependent DSBs. Taken together, these findings strongly indicate that Pol beta is required at a very early step in the processing of meiotic DSBs, at or before the removal of SPO11 from DSB ends and the generation of the 3' single-stranded tails necessary for subsequent strand exchange. The chromosome synapsis defects and Prophase I apoptosis of Pol beta-deficient spermatocytes are likely a direct consequence of these recombination defects.
- Published
- 2009
49. Proliferative Activity In Vitro and DNA Repair Indicate that Adult Mouse and Human Sertoli Cells Are Not Terminally Differentiated, Quiescent Cells1
- Author
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Emad A. Ahmed, S. Canan Mizrak, Henk B. Kal, Hooman Sadri-Ardekani, Angelique D. Barten-Van Rijbroek, Ans M. M. van Pelt, and Dirk G. de Rooij
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endocrine system ,urogenital system ,Cell growth ,Somatic cell ,Cellular differentiation ,Cell Biology ,General Medicine ,Testicle ,Cell cycle ,Biology ,Sertoli cell ,Cell biology ,Comet assay ,chemistry.chemical_compound ,medicine.anatomical_structure ,Reproductive Medicine ,chemistry ,Immunology ,medicine ,Bromodeoxyuridine - Abstract
Sertoli cells isolated from the adult mouse and human testis resume proliferation in culture. After 20 days of culture in Dulbecco modified Eagle medium/Ham F12 (DMEM/F12) medium containing 5% fetal calf serum, about 36% of the mouse Sertoli cells, identified by their immunohistochemical staining for the Sertoli cell marker vimentin, incorporated bromodeoxyuridine (BrdU). The renewed proliferation was associated with a 70% decrease in expression of the cell cycle inhibitor CDKN1B (P27 kip1 ) and a 2-fold increase in the levels of the proliferation inducer ID2. In vivo, the balance between cell cycle inhibitors and inducers probably is such that the cells remain quiescent, whereas in culture the balance is disturbed such that Sertoli cells start to proliferate again. The renewed proliferative activity of Sertoli cells in culture was further confirmed by double staining for BrdU and the Sertoli cell marker clusterin (CLU), showing about 25% of the CLU-positive Sertoli cells to be also positive for BrdU after 13 days of culture. Radiobiologically, Sertoli cells are also different from other quiescent somatic cells in the testis because they express several DNA repair proteins (XRCC1, PARP1, and others). Indeed, a comet assay on irradiated Sertoli cells revealed a 70% reduction in tail length and tail moment at 20 h after irradiation. Hence, Sertoli cells repair DNA damage, whereas other quiescent somatic testicular cells do not. This repair may be accomplished by nonhomologous end joining via XRCC1 and PARP1. In conclusion, cell kinetic and radiobiological data indicate that Sertoli cells more resemble arrested proliferating cells than the classic postmitotic and terminally differentiated somatic cells that they have always been assumed to be. cell cycle genes, culture, DNA repair, Sertoli cells, testis
- Published
- 2009
50. Deriving multipotent stem cells from mouse spermatogonial stem cells: a new tool for developmental and clinical research
- Author
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Dirk G. de Rooij and S. Canan Mizrak
- Subjects
Male ,endocrine system ,Biomedical Research ,Multipotent Stem Cells ,Cellular differentiation ,Cell Culture Techniques ,Cell Differentiation ,Developmental research ,Cell Separation ,Germ layer ,Biology ,Embryonic stem cell ,Regenerative medicine ,Spermatogonia ,Cell biology ,Mice ,Multipotent Stem Cell ,Immunology ,Animals ,Humans ,Spermatogonial stem cells ,Stem cell ,Molecular Biology ,Developmental Biology - Abstract
In recent years, embryonic stem (ES) cell-like cells have been obtained from cultured mouse spermatogonial stem cells (SSCs). These advances have shown that SSCs can transition from being the stem cell-producing cells of spermatogenesis to being multipotent cells that can differentiate into derivatives of all three germ layers. As such, they offer new possibilities for studying the mechanisms that regulate stem cell differentiation. The extension of these findings to human SSCs offers a route to obtaining personalized ES-like or differentiated cells for use in regenerative medicine. Here, we compare the different approaches used to derive ES-like cells from SSCs and discuss their importance to clinical and developmental research.
- Published
- 2008
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