BackgroundSpermatogenesis 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.ResultsWe 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.ConclusionsThis 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2618-1) contains supplementary material, which is available to authorized users.
Human infertility is often classified as idiopathic in both males and females. Meiotic errors may account for at least part of these cases. As the synaptonemal complex (SC, a meiosis-specific protein scaffold) is essential for successful meiosis progression, in this paper, we analyzed the mutations in genes coding for SC components described in infertile patients to assess to what extent alterations in the SC can be related to human infertility. So far, mutations in SYCP3 and SYCE1 genes have been reported. While most SYCP3 mutations are heterozygous mutations with dominant-negative effect on the region encoding the C-terminal coiled coil of the protein, SYCE1 mutations are homozygous, which is consistent with a recessive inheritance. Similarities and differences between males and females as well as between mice and humans have been found and are discussed herein. The results suggest that a low percentage of human infertility cases may be explained by mutations in genes coding for SC components. The characterization of these mutations, together with available information from the study of knockout mice, will enable a deeper understanding of the underlying molecular bases for some of the cases of idiopathic infertility.
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 chromosome.
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. ' International Society for Advancement of CytometryKey terms meiosis; spermatogenesis; flow cytometry; cell sorting; guinea pig; meiotic prophase; gene expression SPERMATOGENESIS can be viewed as the simultaneous and coordinated execution of three individual programs of gene expression: somatic proliferation of spermatogonia, meiosis, and spermiogenesis (1). In the seminiferous tubules of adult mammals, germ cells in different maturation steps coexist, with C (round spermatids, elongating and elongated spermatids, spermatozoa), 2C (several types of G1 spermatogonia, secondary spermatocytes) and 4C (different stages of primary spermatocytes, G2 spermatogonia) DNA content. Moreover, somatic Sertoli cells (2C) coexist with germ cells inside the tubules, which are surrounded by peritubular myoid cells and immersed in a stroma containing fibroblasts, lymphocytes, mastocytes, macrophages, and Leydig cells (all of them 2C).Beside the obvious importance of spermatogenesis as a source of male reproductive cells, along the lengthy first meiotic prophase significant events such as the recognition, alignment, pairing (synapsis), and recombination (crossing over) of homologous chromosomes take place (2-5). Particularly, meiotic homologous recombination has vital importance for genetic variability (6). Furthermore, mutations of
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.
Mammalian spermatogenesis is nowadays still poorly understood at the molecular level, mainly due to the heterogeneous nature of testes, which contain a high number of different cell types, and to the lack of spermatogenic cell culture systems for in vitro studies. As a consequence, the development and/or application of methodological approaches aiming at the enrichment or purification of specific testicular cell types are of great interest and have been addressed by scientists for at least 4 decades. Among the many applications that flow cytometry (FC) has gained since its invention, analysis and sorting of spermatogenic cell populations represent a promising strategy to efficiently overcome testis heterogeneity drawback. Surprisingly, FC has been only rarely used as a preparative method for downstream gene expression studies in specific spermatogenic stages. This work aims to provide an overview of FC for spermatogenic studies including preparation of testicular single cell suspensions, dyes for DNA staining, and our own experience with rodent testis material.
Molecular studies of meiosis in mammals have been long relegated due to some intrinsic obstacles, namely the impossibility to reproduce the process in vitro, and the difficulty to obtain highly pure isolated cells of the different meiotic stages. In the recent years, some technical advances, from the improvement of flow cytometry sorting protocols to single-cell RNAseq, are enabling to profile the transcriptome and its fluctuations along the meiotic process. In this mini-review we will outline the diverse methodological approaches that have been employed, and some of the main findings that have started to arise from these studies. As for practical reasons most studies have been carried out in males, and mostly using mouse as a model, our focus will be on murine male meiosis, although also including specific comments about humans. Particularly, we will center on the controversy about gene expression during early meiotic prophase; the widespread existing gap between transcription and translation in meiotic cells; the expression patterns and potential roles of meiotic long non-coding RNAs; and the visualization of meiotic sex chromosome inactivation from the RNAseq perspective.
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