Uncovering a multitude of stage-specific splice variants and putative protein isoforms generated along mouse spermatogenesis.

Background: Mammalian testis is a highly complex and heterogeneous tissue. This complexity, which mostly derives from spermatogenic cells, is reflected at the transcriptional level, with the largest number of tissue-specific genes and long noncoding RNAs (lncRNAs) compared to other tissues, and one of the highest rates of alternative splicing. Although it is known that adequate alternative-splicing patterns and stage-specific isoforms are critical for successful spermatogenesis, so far only a very limited number of reports have addressed a detailed study of alternative splicing and isoforms along the different spermatogenic stages.

SPATS1 (spermatogenesis-associated, serine-rich 1) is not essential for spermatogenesis and fertility in mouse.

SPATS1 (spermatogenesis-associated, serine-rich 1) is an evolutionarily conserved, testis-specific protein that is differentially expressed during rat male meiotic prophase. Some reports have suggested a link between SPATS1 underexpression/mutation and human pathologies such as male infertility and testicular cancer. Given the absence of functional studies, we generated a Spats1 loss-of-function mouse model using CRISPR/Cas9 technology.

Transcriptomics of Meiosis in the Male Mouse.

Molecular studies of meiosis in mammals have been long relegated due to some intrinsic obstacles, namely the impossibility to reproduce the process , 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.

Contributions of Flow Cytometry to the Molecular Study of Spermatogenesis in Mammals.

Mammalian testes are very heterogeneous organs, with a high number of different cell types. Testicular heterogeneity, together with the lack of reliable in vitro culture systems of spermatogenic cells, have been an obstacle for the characterization of the molecular bases of the unique events that take place along the different spermatogenic stages. In this context, flow cytometry has become an invaluable tool for the analysis of testicular heterogeneity, and for the purification of stage-specific spermatogenic cell populations, both for basic research and for clinical applications.

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.

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.

Flow Cytometry for the Isolation and Characterization of Rodent Meiocytes.

Molecular analyses in mammalian meiotic cells have been hindered by the difficulty in isolating stage-specific cell populations, and this is especially true for early meiotic prophase stages (leptotene and zygotene). Here, we describe a method for obtaining cells in different spermatogenic stages from rodents including lepto-zygotene meiocytes at very high purity levels. The procedure includes an approach for the mechanical disaggregation of the testicular tissue, staining with a vital, noncytotoxic dye that is excitable with a blue laser, isolation of the cell populations by flow sorting, and different alternative protocols for the collection of the sorted cells.

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.

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.

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.

Simple and efficient technique for the preparation of testicular cell suspensions.

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline cells. This heterogeneity is an important drawback concerning the study of the bases of mammalian spermatogenesis, as pure or enriched cell populations in certain stages of sperm development are needed for most molecular analyses. Various strategies such as Staput, centrifugal elutriation, and flow cytometry (FC) have been employed to obtain enriched or purified testicular cell populations in order to enable differential gene expression studies.

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.

Ultra-fast and optimized method for the preparation of rodent testicular cells for flow cytometric analysis.

Unlabelled: 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.