21 research outputs found
Rapid preparation of rodent testicular cell suspensions and spermatogenic stages purification by flow cytometry using a novel blue-laser-excitable vital dye
Availability of purified or highly enriched fractions representing the various spermatogenic stages is a usual requirement to study mammalian spermatogenesis at the molecular level. Fast preparation of high quality testicular cell suspensions is crucial when flow cytometry (FCM) is chosen to accomplish the stage/s purification. Formerly, we reported a method to rapidly obtain good quality rodent testicular cell suspensions for FCM analysis and sorting. Using that method we could distinguish and purify early meiocytes (leptotene/zygotene stages, L/Z) from more advanced ones (pachytene, P) in guinea pig, which presents an unusually high content of early stages. Here we present an upgrade of that method with improvements that have enabled the obtainment of high-purity meiotic substages also from mouse testis, namely:
• Shortening of the mechanical disaggregation time and elimination of a 25µm-filtration step, to optimize the integrity of the suspension and ensure the presence of large P cells.
• Inclusion of a non-cytotoxic, DNA-specific, 488_nm-excitable vital fluorochrome (Vybrant-DyeCycle-Green [VDG], Invitrogen) instead of Hoechst 33342 (requires UV laser, which can damage nucleic acids) or propidium iodide (usually related to dead/damaged cells). As far as we know, this is the first report on the use of this fluorochrome on testicular cells.Agencia Nacional de Investigación e Innovació
Transcriptome analysis of highly purified mouse spermatogenic cell populations: gene expression signatures switch from meiotic-to postmeiotic-related processes at pachytene stage
Top five canonical pathways and molecular and cellular functions from pairwise comparisons of the four cell populations in chronological order (LZ relative to 2C; PS relative to LZ; RS relative to PS). (XLSX 10Â kb
Poly(ADP-ribosylation) is present in murine sciatic nerve fibers and is altered in a Charcot-Marie-Tooth-1E neurodegenerative model
Background. Poly-ADP-ribose (PAR) is a polymer synthesized by poly-ADP-ribose
polymerases (PARPs) as a postranslational protein modification and catabolized
mainly by poly-ADP-ribose glycohydrolase (PARG). In spite of the existence of
cytoplasmic PARPs and PARG, research has been focused on nuclear PARPs and PAR,
demonstrating roles in the maintenance of chromatin architecture and the participation
in DNA damage responses and transcriptional regulation. We have recently detected
non-nuclear PAR structurally and functionally associated to the E-cadherin rich zonula
adherens and the actin cytoskeleton of VERO epithelial cells. Myelinating Schwann cells
(SC) are stabilized by E-cadherin rich autotypic adherens junctions (AJ). We wondered
whether PAR would map to these regions. Besides, we have demonstrated an altered
microfilament pattern in peripheral nerves of Trembler-J (Tr-J) model of CMT1-E. We
hypothesized that cytoplasmic PAR would accompany such modified F-actin pattern.
Methods. Wild-type (WT) and Tr-J mice sciatic nerves cryosections were subjected to
immunohistofluorescence with anti-PAR antibodies (including antibody validation),
F-actin detection with a phalloidin probe and DAPI/DNA counterstaining. Confocal
image stacks were subjected to a colocalization highlighter and to semi-quantitative
image analysis.
Results. We have shown for the first time the presence of PAR in sciatic nerves.
Cytoplasmic PAR colocalized with F-actin at non-compact myelin regions in WT
nerves. Moreover, in Tr-J, cytoplasmic PAR was augmented in close correlation with
actin. In addition, nuclear PAR was detected in WT SC and was moderately increased
in Tr-J SC.
Discussion. The presence of PAR associated to non-compact myelin regions (which
constitute E-cadherin rich autotypic AJ /actin anchorage regions) and the co-alterations
experienced by PAR and the actin cytoskeleton in epithelium and nerves, suggest that PAR may be a constitutive component of AJ /actin anchorage regions. Is PAR
stabilizing the AJ -actin complexes? This question has strong implications in structural
cell biology and cell signaling networks. Moreover, if PAR played a stabilizing role,
such stabilization could participate in the physiological control of axonal branching.
PARP and PAR alterations exist in several neurodegenerative pathologies including
Alzheimer's, Parkinson's and Hungtington's diseases. Conversely, PARP inhibition
decreases PAR and promotes neurite outgrowth in cortical neurons in vitro. Coherently,
the PARP inhibitor XAV939 improves myelination in vitro, ex vivo and in vivo. Until
now such results have been interpreted in terms of nuclear PARP activity. Our results
indicate for the first time the presence of PARylation in peripheral nerve fibers, in
a healthy environment. Besides, we have evidenced a PARylation increase in Tr-J,
suggesting that the involvement of cytoplasmic PARPs and PARylation in normal and
neurodegenerative conditions should be re-evaluated
Revealing stage-specific expression patterns of long noncoding RNAs along mouse spermatogenesis
The discovery of a large number of long noncoding RNAs (lncRNAs), and the finding that they may play
key roles in different biological processes, have started to provide a new perspective in the understanding of gene regulation. It has been shown that the testes express the highest amount of lncRNAs among different vertebrate tissues. However, although some studies have addressed the characterization of lncRNAs along spermatogenesis, an exhaustive analysis of the differential expression of lncRNAs at its different stages is still lacking. Here, we present the results for lncRNA transcriptome profiling along mouse spermatogenesis, employing highly pure flow sorted spermatogenic stage-specific cell populations, strand-specific RNAseq, and a combination of up-to-date bioinformatic pipelines for analysis. We found that the vast majority of testicular lncRNA genes are expressed at post-meiotic stages (i.e. spermiogenesis), which are characterized by extensive post-transcriptional regulation. LncRNAs at different spermatogenic stages shared common traits in terms of transcript length, exon number, and biotypes. Most lncRNAs were lincRNAs, followed by a high representation of antisense (AS) lncRNAs. Co-expression analyses showed a high correlation along the different spermatogenic stage transitions between the expression patterns of AS lncRNAs and their overlapping protein-coding genes, raising possible clues about lncRNA-related regulatory mechanisms. Interestingly, we observed the colocalization of an AS lncRNA and its host sense mRNA in the chromatoid body, a round spermatidsspecific organelle that has been proposed as a reservoir of RNA-related regulatory machinery. An additional, intriguing observation is the almost complete lack of detectable expression for Y-linked testicular lncRNAs, despite that a high number of lncRNA genes are annotated for this chromosom
Impact of EMS outreach: successful developments in Latin America
This collection of articles was inspired by the long-standing relationship between the Environmental Mutagen Society and Latin American scientists, and by the program for the 39th Environmental Mutagen Society meeting in Puerto Rico in 2008, which included a symposium featuring “South of the border” scientists. This collection, compiled by Graciela Spivak and Ofelia Olivero, both originally from Argentina, highlights scientists who work in or were trained in Latin American countries and in Puerto Rico in a variety of scientific specialties related to DNA repair and cancer susceptibility, genomic organization and stability, genetic diversity, and environmental contaminants.Facultad de Ciencias Naturales y Muse
Transcriptome analysis of highly purified mouse spermatogenic cell populations: gene expression signatures switch from meiotic-to postmeiotic-related processes at pachytene stage
Background: Spermatogenesis is a complex differentiation process that involves the successive and simultaneous execution of three different gene expression programs: mitotic proliferation of spermatogonia, meiosis, and spermiogenesis. Testicular cell heterogeneity has hindered its molecular analyses. Moreover, the characterization of short, poorly represented cell stages such as initial meiotic prophase ones (leptotene and zygotene) has remained elusive, despite their crucial importance for understanding the fundamentals of meiosis. Results: We have developed a flow cytometry-based approach for obtaining highly pure stage-specific spermatogenic cell populations, including early meiotic prophase. Here we combined this methodology with next generation sequencing, which enabled the analysis of meiotic and postmeiotic gene expression signatures in mouse with unprecedented reliability. Interestingly, we found that a considerable number of genes involved in early as well as late meiotic processes are already on at early meiotic prophase, with a high proportion of them being expressed only for the short time lapse of lepto-zygotene stages. Besides, we observed a massive change in gene expression patterns during medium meiotic prophase (pachytene) when mostly genes related to spermiogenesis and sperm function are already turned on. This indicates that the transcriptional switch from meiosis to post-meiosis takes place very early, during meiotic prophase, thus disclosing a higher incidence of post-transcriptional regulation in spermatogenesis than previously reported. Moreover, we found that a good proportion of the differential gene expression in spermiogenesis corresponds to up-regulation of genes whose expression starts earlier, at pachytene stage this includes transition protein-and protamine-coding genes, which have long been claimed to switch on during spermiogenesis. In addition, our results afford new insights concerning X chromosome meiotic inactivation and reactivation. Conclusions: This work provides for the first time an overview of the time course for the massive onset and turning off of the meiotic and spermiogenic genetic programs. Importantly, our data represent a highly reliable information set about gene expression in pure testicular cell populations including early meiotic prophase, for further data mining towards the elucidation of the molecular bases of male reproduction in mammals
Ultra-Fast and Optimized Method for the Preparation of Rodent Testicular Cells for Flow Cytometric Analysis
Homogeneity of cell populations is a prerequisite for the analysis of biochemical and molecular events during male gamete differentiation. Given the complex organization of the mammalian testicular tissue, various methods have been used to obtain enriched or purified cell populations, including flow cell sorting. Current protocols are usually time-consuming and may imply loss of short-lived RNAs, which is undesirable for expression profiling. We describe an optimized method to speed up the preparation of suitable testicular cell suspensions for cytometric analysis of different spermatogenic stages from rodents. The procedure takes only 15 min including testis dissection, tissue cutting, and processing through the Medimachine System (Becton Dickinson). This method could be a substitute for the more tedious and time-consuming cell preparation techniques currently in use