Familial primary ovarian insufficiency associated with an SYCE1 point mutation: defective meiosis elucidated in humanized mice.

More than 50% of cases of primary ovarian insufficiency (POI) and nonobstructive azoospermia in humans are classified as idiopathic infertility. Meiotic defects may relate to at least some of these cases. Mutations in genes coding for synaptonemal complex (SC) components have been identified in humans, and hypothesized to be causative for the observed infertile phenotype. Mutation SYCE1 c.721C>T (former c.613C>T)-a familial mutation reported in two sisters with primary amenorrhea-was the first such mutation found in an SC central element component-coding gene. Most fundamental mammalian oogenesis events occur during the embryonic phase, and eventual defects are identified many years later, thus leaving few possibilities to study the condition's etiology and pathogenesis. Aiming to validate an approach to circumvent this difficulty, we have used the CRISPR/Cas9 technology to generate a mouse model with an SYCE1 c.721C>T equivalent genome alteration. We hereby present the characterization of the homozygous mutant mice phenotype, compared to their wild type and heterozygous littermates. Our results strongly support a causative role of this mutation for the POI phenotype in human patients, and the mechanisms involved would relate to defects in homologous chromosome synapsis. No SYCE1 protein was detected in homozygous mutants and Syce1 transcript level was highly diminished, suggesting transcript degradation as the basis of the infertility mechanism. This is the first report on the generation of a humanized mouse model line for the study of an infertility-related human mutation in an SC component-coding gene, thus representing a proof of principle.

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 co-localization of an AS lncRNA and its host sense mRNA in the chromatoid body, a round spermatids-specific 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 chromosome.

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.

MTCH2 is differentially expressed in rat testis and mainly related to apoptosis of spermatocytes.

MTCH2 has been described in liver as a protein involved in the intrinsic apoptotic pathway, although new evidence also associates this protein with cellular metabolism. In this work, the expression of MTCH2 in testis (an organ in which high levels of apoptosis normally take place as part of the spermatogenic process) is analyzed in rat, both at the mRNA and at the protein levels. Our results showed that MTCH2 was highly expressed in testis compared with other tissues and was differentially expressed according to developmental stage and testicular cell type. Protein expression was initially detected during the first spermatogenic wave at the time of meiosis onset and its levels increased in adulthood, with the highest expression levels being detected in meiotic prophase I. Specific differences in MTCH2 expression levels at the various stages of the adult seminiferous epithelium were also observed. Co-staining with TUNEL revealed a differential MTCH2 staining pattern in TUNEL-positive cells, mainly in dying primary spermatocytes, i.e., meiotic prophase I cells. Furthermore, upon mild hyperthermia (treatment shown to increase apoptosis in testis), MTCH2 levels rose concomitantly with a massive appearance of TUNEL-positive cells within the seminiferous tubules; these cells exhibited a differential MTCH2 distribution. Thus, MTCH2 is related to testicular apoptosis, especially during meiotic prophase.

High-purity flow sorting of early meiocytes based on DNA analysis of guinea pig spermatogenic cells.

Mammalian spermatogenesis is still nowadays poorly understood at the molecular level. Testis cellular heterogeneity is a major drawback for spermatogenic gene expression studies, especially when research is focused on stages that are usually very short and poorly represented at the cellular level such as initial meiotic prophase I (i.e., leptotene [L] and zygotene [Z]). Presumably, genes whose products are involved in critical meiotic events such as alignment, pairing and recombination of homologous chromosomes are expressed during the short stages of early meiotic prophase. Aiming to characterize mammalian early meiotic gene expression, we have found the guinea pig (Cavia porcellus) as an especially attractive model. A detailed analysis of its first spermatogenic wave by flow cytometry (FCM) and optical microscopy showed that guinea pig testes exhibit a higher representation of early meiotic stages compared to other studied rodents, partly because of their longer span, and also as a result of the increased number of cells entering meiosis. Moreover, we have found that adult guinea pig testes exhibit a peculiar 4C DNA content profile, with a bimodal peak for L/Z and P spermatocytes that is absent in other rodents. Besides, we show that this unusual 4C peak allows the separation by FCM of highly pure L/Z spermatocyte populations aside from pachytene ones, even from adult individuals. To our knowledge, this is the first report on an accurate and suitable method for highly pure early meiotic prophase cell isolation from adult mammals, and thus sets an interesting approach for gene expression studies aiming at a deeper understanding of the molecular groundwork underlying male gamete production.

Intact DNA purified from flow-sorted nuclei unlocks the potential of next-generation genome mapping and assembly in Solanum species.

Next-generation genome mapping through nanochannels (Bionano optical mapping) of plant genomes brings genome assemblies to the 'nearly-finished' level for reliable and detailed gene annotations and assessment of structural variations. Despite the recent progress in its development, researchers face the technical challenges of obtaining sufficient high molecular weight (HMW) nuclear DNA due to cell walls which are difficult to disrupt and to the presence of cytoplasmic polyphenols and polysaccharides that co-precipitate or are covalently bound to DNA and might cause oxidation and/or affect the access of nicking enzymes to DNA, preventing downstream applications. Here we describe important improvements for obtaining HMW DNA that we tested on Solanum crops and wild relatives. The methods that we further elaborated and refined focus on •Improving flexibility of using different tissues as source materials, like fast-growing root tips and young leaves from seedlings or in vitro plantlets.•Obtaining nuclei suspensions through either lab homogenizers or by chopping.•Increasing flow sorting efficiency using DAPI (4',6-diamidino-2-phenylindole) and PI (propidium iodide) DNA stains, with different lasers (UV or 488 nm) and sorting platforms such as the FACSAria and FACSVantage flow sorters, thus making it appropriate for more laboratories working on plant genomics. The obtained nuclei are embedded into agarose plugs for processing and isolating uncontaminated HMW DNA, which is a prerequisite for nanochannel-based next-generation optical mapping strategies.

The methylating agent streptozotocin induces persistent telomere dysfunction in mammalian cells.

We analyzed chromosomal aberrations involving telomeres in the progeny of mammalian cells exposed to the methylating agent and antineoplastic/diabetogenic drug streptozotocin (STZ), to test whether it induces long-term telomere instability (by chromosome end loss and/or telomere dysfunction). Rat cells (ADIPO-P2 cell line, derived from Sprague-Dawley rat adipose cells) were treated with a single concentration of STZ (2mM). Chromosomal aberrations were analyzed 18h, 10 days, and 15 days after treatment, using PNA-FISH with a pan-telomeric probe [Cy3-(CCCTAA)3] to detect (TTAGGG)n repeats. Cytogenetic analysis revealed a higher frequency of chromosomal aberrations in STZ-exposed cultures vs. untreated cultures at each time point analyzed. The yield of induced aberrations was very similar at each time point. Induction of aberrations not involving telomere dysfunction was only observed 18h and 15 days after treatment, whereas induction of telomere dysfunction-related aberrations by STZ (mainly in the form of telomere FISH signal loss and duplications, most of them chromatid-type aberrations) was observed at each time point. Our results show that STZ induces persistent telomere instability in mammalian cells, cytogenetically manifested as telomere dysfunction-related chromosomal aberrations. Neither telomere length nor telomerase activity is related to the telomere dysfunction.

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 enabled the obtainment of high-purity meiotic substages also from mouse testis, namely:•Shortening of the mechanical disaggregation time to optimize the integrity of the suspension.•Elimination of the 25 µm-filtration step to 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 for the discrimination and purification of meiotic prophase I substages.

Non-random distribution of high density chromatin detected at opposite ends of T-banded human metaphase chromosomes

Previous research using microdensitometric scanning and computer graphic image analysis showed that T-banded segments of human metaphase chromosomes usually exhibit an asymmetrical distribution of high density (HD) chromatin between sister chromatids. Here, we employed the same methods to analyze HD chromatin distribution at opposite ends of T-banded human lymphocyte chromosomes. This study revealed that in most chromosomes with an asymmetrical distribution of HD chromatin at both ends, the highest densities of each arm were located in opposite chromatids. The frequency of this configuration was 0.792 per chromosome, indicating that the highest chromatin densities of the terminal segments of T-banded human chromosomes were non-randomly distributed at opposite chromosome arms. The possible relationship of this observation to the mode of replication of the terminal chromosome region is briefly discussed.

Retardo mental, malformaciones congénitas y aberraciones cromosómicas subteloméricas crípticas

Los reciente progresos tecnológicos ocurridos en citogenética molecular han permitido la detección de numerosas minúsculas aberraciones en la región cromosómica contigua al telómero o segmento subtelomérico, que han sido relacionadas con diversos cuadros de retardo mental, malformaciones congénitas y otros síndromes de interés médico. Investigaciones previas, llevadas a cabo mediante exploración microfotométrica y análisis gráfico computacional del subtelómero, revelaron diferentes patrones de distribución de las densidades de la cromatina y la existencia de intercambios sumamente pequeños entre cromátidas hermanas en dicho segmento cromosómico. La detección de aberraciones crípticas sindromáticas, el elevado número de intercambios cromosómicos y las observaciones microscópicas sobre la estructura subtelomérica sugieren que reflejan la gran actividad genómica y la complejidad estructural prevalente en la región. En la presente revisión se describen brevemente varios síndromes cromosómicos subteloméricos, así como la estructura molecular y citológica de la región subtelomérica y las principales funciones del segmento cromosómico terminal a fin de brindar un panorama general sobre ésta área de investigaciones en rápido crecimiento de considerable significación biomédica

A proposal of a standardised nomenclature for terminal minute sister chromatid exchanges

We described spontaneous minute sister chromatid exchanges (SCE) in telomeric regions of human and Chinese hamster ovary (CHO) chromosomes more than 10 years ago. These structures, which we called t-SCE, were detected by means of highly precise quantitative microphotometrical scanning and computer graphic image analysis. Recently, several authors using the CO-FISH method also found small SCEs in telomeric regions and called them T-SCE. The use of different terms for designating the same phenomenon should be avoided. We propose ter SCE as a uniform nomenclature for minute telomeric SCEs.

Retardo mental, malformaciones congénitas y aberraciones cromosómicas subteloméricas crípticas / Mental retardation, congenital malformations and subtelomeric chromosome aberrations

Los recientes progresos tecnológicos ocurridos en citogenética molecular han permitido la detección de numerosas minúsculas aberraciones en la región cromosómica contigua al telómero o segmento subtelomérico, que han sido relacionadas con diversos cuadros de retardo mental, malformaciones congénitas y otros síndromes de interés médico. Investigaciones previas, llevadas a cabo mediante exploración microfotométrica y análisis gráfico computacional del subtelómero, revelaron diferentes patrones de distribución de las densidades de la cromatina y la existencia de intercambios sumamente pequeños entre cromátidas hermanas en dicho segmento cromosómico. La detección de aberrraciones crípticas sindromáticas, el elevado número de intercambios cromosómicos y las observaciones microscópicas sobre la estructura subtelomérica sugieren que reflejan la gran actividad genómica y la complejidad estructural prevalente en la región. En la presente revisión se describen brevemente varios síndromes cromosómicos subteloméricos, así como la estructura molecular y citológica de la región subtelomérica y las principales funciones del segmento cromosómico terminal a fin de brindar un panorama general sobre esta área de investigaciones en rápido crecimiento de considerable significación biomédica.