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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

Flow cytometric purification and immunocytochemical analysis of sorted testicular cell populations with VDG. Mice aging 10–11 dpp (a, b) and 24–25 dpp (c, d) were used. a, c. Dot plots depicting forward scatter (FSC-H) vs VDG fluorescence intensity and their corresponding histograms showing the gated cell populations. b, d. Confocal immunocytochemical analysis with anti-SYCP3 antibody (red) as a marker of the LZ (b) and PS (d) sorted fractions. Nuclei were counterstained with DAPI. Bars correspond to 10 μm
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Fig1: Flow cytometric purification and immunocytochemical analysis of sorted testicular cell populations with VDG. Mice aging 10–11 dpp (a, b) and 24–25 dpp (c, d) were used. a, c. Dot plots depicting forward scatter (FSC-H) vs VDG fluorescence intensity and their corresponding histograms showing the gated cell populations. b, d. Confocal immunocytochemical analysis with anti-SYCP3 antibody (red) as a marker of the LZ (b) and PS (d) sorted fractions. Nuclei were counterstained with DAPI. Bars correspond to 10 μm

Mentions: The optimal ages for obtaining the different cell fractions (maximum representation of each specific cell type, lowest proportion of possible contaminant cell types) were determined by monitoring the progress of the first spermatogenic wave by FCM analysis of the cell suspensions, and by microscopic observation of Epon-embedded cross sections of seminiferous tubules (Additional file 1: Figure S1A). Besides, meiotic prophase stages were scrutinized by confocal immunocytochemistry using an antibody against the main component of the lateral element of synaptonemal complexes, SYCP3 (Additional file 1: Figure S1B). Accordingly, we used 10–11 days postpartum (dpp) mice for purifying the highest amount of LZ spermatocytes without any PS contamination (Fig. 1a and b). Testes from different individuals of the same age were employed for the purification of the 2C fraction, which consists of a heterogeneous cell population containing spermatogonia and somatic cells (mostly Sertoli cells), and was used as a reference for the transcripts present in pre-meiotic and somatic testicular cell types. Besides, the choice of early prepubertal mice for the purification of the 2C fraction avoids the presence of spermatocytes II, which otherwise would co-purify with this fraction due to their DNA content.Fig. 1


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)

Flow cytometric purification and immunocytochemical analysis of sorted testicular cell populations with VDG. Mice aging 10–11 dpp (a, b) and 24–25 dpp (c, d) were used. a, c. Dot plots depicting forward scatter (FSC-H) vs VDG fluorescence intensity and their corresponding histograms showing the gated cell populations. b, d. Confocal immunocytochemical analysis with anti-SYCP3 antibody (red) as a marker of the LZ (b) and PS (d) sorted fractions. Nuclei were counterstained with DAPI. Bars correspond to 10 μm
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: Flow cytometric purification and immunocytochemical analysis of sorted testicular cell populations with VDG. Mice aging 10–11 dpp (a, b) and 24–25 dpp (c, d) were used. a, c. Dot plots depicting forward scatter (FSC-H) vs VDG fluorescence intensity and their corresponding histograms showing the gated cell populations. b, d. Confocal immunocytochemical analysis with anti-SYCP3 antibody (red) as a marker of the LZ (b) and PS (d) sorted fractions. Nuclei were counterstained with DAPI. Bars correspond to 10 μm
Mentions: The optimal ages for obtaining the different cell fractions (maximum representation of each specific cell type, lowest proportion of possible contaminant cell types) were determined by monitoring the progress of the first spermatogenic wave by FCM analysis of the cell suspensions, and by microscopic observation of Epon-embedded cross sections of seminiferous tubules (Additional file 1: Figure S1A). Besides, meiotic prophase stages were scrutinized by confocal immunocytochemistry using an antibody against the main component of the lateral element of synaptonemal complexes, SYCP3 (Additional file 1: Figure S1B). Accordingly, we used 10–11 days postpartum (dpp) mice for purifying the highest amount of LZ spermatocytes without any PS contamination (Fig. 1a and b). Testes from different individuals of the same age were employed for the purification of the 2C fraction, which consists of a heterogeneous cell population containing spermatogonia and somatic cells (mostly Sertoli cells), and was used as a reference for the transcripts present in pre-meiotic and somatic testicular cell types. Besides, the choice of early prepubertal mice for the purification of the 2C fraction avoids the presence of spermatocytes II, which otherwise would co-purify with this fraction due to their DNA content.Fig. 1

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