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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

Enriched GO categories and differential expression of genes in early meiotic prophase (LZ). a. Enrichment analysis of biological process GO terms of up-regulated genes in 2C/LZ comparison. The fold enrichment shows the ratio of observed vs expected genes for each category, with an adjusted p-value <0.01. b. Venn diagram showing separate and overlapping expression between comparisons of the lists “2C/LZ up” (i.e. genes that are up-regulated in LZ compared to 2C) and “LZ/PS down” (i.e. genes that are down-regulated in PS compared to LZ). The intersection contains a subset of DEG whose expression peaks in early meiotic prophase. c. Graphical representation of the expression levels (RPKM) of the genes within the LZ peak in the four cell populations. The fifteen top genes are listed to the right in decreasing order according to their expression levels in LZ. d. Heat maps showing relative expression levels of the genes contained within GO categories “reciprocal meiotic recombination” (GO# 0007131) and “meiotic chromosome segregation” (GO# 0045132). High expression levels are indicated in red and low expression levels in green
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Fig4: Enriched GO categories and differential expression of genes in early meiotic prophase (LZ). a. Enrichment analysis of biological process GO terms of up-regulated genes in 2C/LZ comparison. The fold enrichment shows the ratio of observed vs expected genes for each category, with an adjusted p-value <0.01. b. Venn diagram showing separate and overlapping expression between comparisons of the lists “2C/LZ up” (i.e. genes that are up-regulated in LZ compared to 2C) and “LZ/PS down” (i.e. genes that are down-regulated in PS compared to LZ). The intersection contains a subset of DEG whose expression peaks in early meiotic prophase. c. Graphical representation of the expression levels (RPKM) of the genes within the LZ peak in the four cell populations. The fifteen top genes are listed to the right in decreasing order according to their expression levels in LZ. d. Heat maps showing relative expression levels of the genes contained within GO categories “reciprocal meiotic recombination” (GO# 0007131) and “meiotic chromosome segregation” (GO# 0045132). High expression levels are indicated in red and low expression levels in green

Mentions: To understand the underlying biological processes in the LZ cell population, we carried out gene ontology (GO) analysis of the DEG. For up-regulated genes, terms related to cellular metabolism, RNA and DNA metabolic processes, reproduction, chromatin organization, cell cycle, spermatogenesis, microtubule cytoskeleton organization, DNA repair, mitosis, meiosis, meiotic recombination, chromosome condensation and segregation, and synaptonemal complex, were among the most significant (p < 0.01) (Fig. 4a). On the other hand, down-regulated genes from 2C to LZ mostly fell into organ and tissue development-related, and extracellular matrix and cell adhesion-associated categories (p < 10−7). Besides, genes with shared expression between 2C and LZ (i.e. those that remained fairly constant in both lists, −2 < FC < 2), were mostly related to translation, intracellular transport, mRNA processing, cellular macromolecule localization, generation of metabolites and energy, chromatin organization, cellular respiration, and other general metabolic processes (p < 10−5). Thus, meiosis-related GO terms show significant enrichment during early meiotic prophase and not before.Fig. 4


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)

Enriched GO categories and differential expression of genes in early meiotic prophase (LZ). a. Enrichment analysis of biological process GO terms of up-regulated genes in 2C/LZ comparison. The fold enrichment shows the ratio of observed vs expected genes for each category, with an adjusted p-value <0.01. b. Venn diagram showing separate and overlapping expression between comparisons of the lists “2C/LZ up” (i.e. genes that are up-regulated in LZ compared to 2C) and “LZ/PS down” (i.e. genes that are down-regulated in PS compared to LZ). The intersection contains a subset of DEG whose expression peaks in early meiotic prophase. c. Graphical representation of the expression levels (RPKM) of the genes within the LZ peak in the four cell populations. The fifteen top genes are listed to the right in decreasing order according to their expression levels in LZ. d. Heat maps showing relative expression levels of the genes contained within GO categories “reciprocal meiotic recombination” (GO# 0007131) and “meiotic chromosome segregation” (GO# 0045132). High expression levels are indicated in red and low expression levels in green
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4837615&req=5

Fig4: Enriched GO categories and differential expression of genes in early meiotic prophase (LZ). a. Enrichment analysis of biological process GO terms of up-regulated genes in 2C/LZ comparison. The fold enrichment shows the ratio of observed vs expected genes for each category, with an adjusted p-value <0.01. b. Venn diagram showing separate and overlapping expression between comparisons of the lists “2C/LZ up” (i.e. genes that are up-regulated in LZ compared to 2C) and “LZ/PS down” (i.e. genes that are down-regulated in PS compared to LZ). The intersection contains a subset of DEG whose expression peaks in early meiotic prophase. c. Graphical representation of the expression levels (RPKM) of the genes within the LZ peak in the four cell populations. The fifteen top genes are listed to the right in decreasing order according to their expression levels in LZ. d. Heat maps showing relative expression levels of the genes contained within GO categories “reciprocal meiotic recombination” (GO# 0007131) and “meiotic chromosome segregation” (GO# 0045132). High expression levels are indicated in red and low expression levels in green
Mentions: To understand the underlying biological processes in the LZ cell population, we carried out gene ontology (GO) analysis of the DEG. For up-regulated genes, terms related to cellular metabolism, RNA and DNA metabolic processes, reproduction, chromatin organization, cell cycle, spermatogenesis, microtubule cytoskeleton organization, DNA repair, mitosis, meiosis, meiotic recombination, chromosome condensation and segregation, and synaptonemal complex, were among the most significant (p < 0.01) (Fig. 4a). On the other hand, down-regulated genes from 2C to LZ mostly fell into organ and tissue development-related, and extracellular matrix and cell adhesion-associated categories (p < 10−7). Besides, genes with shared expression between 2C and LZ (i.e. those that remained fairly constant in both lists, −2 < FC < 2), were mostly related to translation, intracellular transport, mRNA processing, cellular macromolecule localization, generation of metabolites and energy, chromatin organization, cellular respiration, and other general metabolic processes (p < 10−5). Thus, meiosis-related GO terms show significant enrichment during early meiotic prophase and not before.Fig. 4

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