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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

Dynamic expression patterns of 13 selected genes representative of the different expression profiles. The analyses were carried out by RNAseq (CLC bio and edgeR) and qPCR. a. Expression profiles obtained by RNAseq analysis (CLC bio: orange; edgeR: blue) and qPCR analysis (yellow). Col1a1: collagen, type I, alpha 1; Sycp3: synaptonemal complex protein 3; Dazl: deleted in azoospermia-like; Tex15: testis expressed 15; Top2a: DNA topoisomerase II, alpha isozyme; Dnahc8: dynein, axonemal, heavy chain 8; Tcte3: T-complex-associated-testis-expressed 3; Atp8b3: ATPase, aminophospholipid transporter, class I, type 8B, member 3; Clgn; calmegin; Spa17: sperm autoantigenic protein 17; Ldhc: lactate dehydrogenase c; Tnp1: transition protein 1; Prm1: protamine 1. b. Correlation between the expression levels in RNAseq analysis (RPKM values of CLC bio analysis) and those obtained by qPCR analysis for the 13 selected genes in the four testicular cell populations
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Fig6: Dynamic expression patterns of 13 selected genes representative of the different expression profiles. The analyses were carried out by RNAseq (CLC bio and edgeR) and qPCR. a. Expression profiles obtained by RNAseq analysis (CLC bio: orange; edgeR: blue) and qPCR analysis (yellow). Col1a1: collagen, type I, alpha 1; Sycp3: synaptonemal complex protein 3; Dazl: deleted in azoospermia-like; Tex15: testis expressed 15; Top2a: DNA topoisomerase II, alpha isozyme; Dnahc8: dynein, axonemal, heavy chain 8; Tcte3: T-complex-associated-testis-expressed 3; Atp8b3: ATPase, aminophospholipid transporter, class I, type 8B, member 3; Clgn; calmegin; Spa17: sperm autoantigenic protein 17; Ldhc: lactate dehydrogenase c; Tnp1: transition protein 1; Prm1: protamine 1. b. Correlation between the expression levels in RNAseq analysis (RPKM values of CLC bio analysis) and those obtained by qPCR analysis for the 13 selected genes in the four testicular cell populations

Mentions: The dynamic expression patterns of all genes in the four cell populations were consistent with both RNAseq analyses (CLC bio and edgeR) (Fig. 6a). Additionally, there was a high correlation between RNAseq and qRT-PCR data (Pearson r2 between 0.81-0.93 for CLC bio -Fig. 6b, and 0.73-0.93 for edgeR-derived results), thus supporting the reliability of our RNAseq data.Fig. 6


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)

Dynamic expression patterns of 13 selected genes representative of the different expression profiles. The analyses were carried out by RNAseq (CLC bio and edgeR) and qPCR. a. Expression profiles obtained by RNAseq analysis (CLC bio: orange; edgeR: blue) and qPCR analysis (yellow). Col1a1: collagen, type I, alpha 1; Sycp3: synaptonemal complex protein 3; Dazl: deleted in azoospermia-like; Tex15: testis expressed 15; Top2a: DNA topoisomerase II, alpha isozyme; Dnahc8: dynein, axonemal, heavy chain 8; Tcte3: T-complex-associated-testis-expressed 3; Atp8b3: ATPase, aminophospholipid transporter, class I, type 8B, member 3; Clgn; calmegin; Spa17: sperm autoantigenic protein 17; Ldhc: lactate dehydrogenase c; Tnp1: transition protein 1; Prm1: protamine 1. b. Correlation between the expression levels in RNAseq analysis (RPKM values of CLC bio analysis) and those obtained by qPCR analysis for the 13 selected genes in the four testicular cell populations
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig6: Dynamic expression patterns of 13 selected genes representative of the different expression profiles. The analyses were carried out by RNAseq (CLC bio and edgeR) and qPCR. a. Expression profiles obtained by RNAseq analysis (CLC bio: orange; edgeR: blue) and qPCR analysis (yellow). Col1a1: collagen, type I, alpha 1; Sycp3: synaptonemal complex protein 3; Dazl: deleted in azoospermia-like; Tex15: testis expressed 15; Top2a: DNA topoisomerase II, alpha isozyme; Dnahc8: dynein, axonemal, heavy chain 8; Tcte3: T-complex-associated-testis-expressed 3; Atp8b3: ATPase, aminophospholipid transporter, class I, type 8B, member 3; Clgn; calmegin; Spa17: sperm autoantigenic protein 17; Ldhc: lactate dehydrogenase c; Tnp1: transition protein 1; Prm1: protamine 1. b. Correlation between the expression levels in RNAseq analysis (RPKM values of CLC bio analysis) and those obtained by qPCR analysis for the 13 selected genes in the four testicular cell populations
Mentions: The dynamic expression patterns of all genes in the four cell populations were consistent with both RNAseq analyses (CLC bio and edgeR) (Fig. 6a). Additionally, there was a high correlation between RNAseq and qRT-PCR data (Pearson r2 between 0.81-0.93 for CLC bio -Fig. 6b, and 0.73-0.93 for edgeR-derived results), thus supporting the reliability of our RNAseq data.Fig. 6

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