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Transcriptome analysis of highly purified mouse spermatogenic cell populations: gene expression signatures switch from meiotic-to postmeiotic-related processes at pachytene stage.

da Cruz I, Rodríguez-Casuriaga R, Santiñaque FF, Farías J, Curti G, Capoano CA, Folle GA, Benavente R, Sotelo-Silveira JR, Geisinger A - BMC Genomics (2016)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Genomics, Instituto de Investigaciones Biológicas Clemente Estable (IIBCE), Av. Italia 3318, 11,600, Montevideo, Uruguay.

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus

Heat maps showing relative expression levels of X-linked protein-coding genes in the four cell populations. a. The genes were ordered according to their position on the chromosome from p to q. Chromosome bands are indicated to the left of the figure. b. Hierarchical clustering. High expression levels are indicated in red and low expression levels in green for both heat maps
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Fig7: Heat maps showing relative expression levels of X-linked protein-coding genes in the four cell populations. a. The genes were ordered according to their position on the chromosome from p to q. Chromosome bands are indicated to the left of the figure. b. Hierarchical clustering. High expression levels are indicated in red and low expression levels in green for both heat maps

Mentions: Regarding MSCI, we analyzed the expression of all X chromosome-linked protein coding genes present in our RNAseq data (812 genes). We generated two heat maps representing the expression levels in the four populations: a first one ordered according to the position of the genes on the chromosome from p to q (Fig. 7a), and a second one showing their hierarchical clustering (Fig. 7b). This allowed to observe the inactivation of the X chromosome, which further validates our data. As previously reported in a microarray study [35], we detected a massive switch off of X-linked genes between LZ and PS cell populations (see Fig. 7a). Interestingly, we identified a cluster of ~70 genes that showed an opposite behavior to that of most X-linked genes, as they escaped MSCI being up-regulated in PS (see Fig. 7b). Nearly half of these genes showed a 5-to 1000-fold increase from LZ to PS, and were clearly identified as differential of PS in our comparative analysis (Additional file 4: Dataset S3). Among those genes with a known function, many code for sperm-related proteins such as AKAP4 that is involved in sperm motility [67], CYPT1 that is a sperm-specific component of the post-acrosomal perinuclear theca [68], CYLC1, which forms part of the sperm head cytoskeleton, and SPACA5, a sperm acrosome-associated protein, among others. Besides, five predicted proteins contain a H2A conserved domain, and are putative histone cluster 2 family members.Fig. 7


Transcriptome analysis of highly purified mouse spermatogenic cell populations: gene expression signatures switch from meiotic-to postmeiotic-related processes at pachytene stage.

da Cruz I, Rodríguez-Casuriaga R, Santiñaque FF, Farías J, Curti G, Capoano CA, Folle GA, Benavente R, Sotelo-Silveira JR, Geisinger A - BMC Genomics (2016)

Heat maps showing relative expression levels of X-linked protein-coding genes in the four cell populations. a. The genes were ordered according to their position on the chromosome from p to q. Chromosome bands are indicated to the left of the figure. b. Hierarchical clustering. High expression levels are indicated in red and low expression levels in green for both heat maps
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4837615&req=5

Fig7: Heat maps showing relative expression levels of X-linked protein-coding genes in the four cell populations. a. The genes were ordered according to their position on the chromosome from p to q. Chromosome bands are indicated to the left of the figure. b. Hierarchical clustering. High expression levels are indicated in red and low expression levels in green for both heat maps
Mentions: Regarding MSCI, we analyzed the expression of all X chromosome-linked protein coding genes present in our RNAseq data (812 genes). We generated two heat maps representing the expression levels in the four populations: a first one ordered according to the position of the genes on the chromosome from p to q (Fig. 7a), and a second one showing their hierarchical clustering (Fig. 7b). This allowed to observe the inactivation of the X chromosome, which further validates our data. As previously reported in a microarray study [35], we detected a massive switch off of X-linked genes between LZ and PS cell populations (see Fig. 7a). Interestingly, we identified a cluster of ~70 genes that showed an opposite behavior to that of most X-linked genes, as they escaped MSCI being up-regulated in PS (see Fig. 7b). Nearly half of these genes showed a 5-to 1000-fold increase from LZ to PS, and were clearly identified as differential of PS in our comparative analysis (Additional file 4: Dataset S3). Among those genes with a known function, many code for sperm-related proteins such as AKAP4 that is involved in sperm motility [67], CYPT1 that is a sperm-specific component of the post-acrosomal perinuclear theca [68], CYLC1, which forms part of the sperm head cytoskeleton, and SPACA5, a sperm acrosome-associated protein, among others. Besides, five predicted proteins contain a H2A conserved domain, and are putative histone cluster 2 family members.Fig. 7

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Genomics, Instituto de Investigaciones Biológicas Clemente Estable (IIBCE), Av. Italia 3318, 11,600, Montevideo, Uruguay.

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus