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.


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.
Department of Genomics, Instituto de Investigaciones Biológicas Clemente Estable (IIBCE), Av. Italia 3318, 11,600, Montevideo, Uruguay.|Department of Molecular Biology, Instituto de Investigaciones Biológicas Clemente Estable (IIBCE), Av. Italia 3318, 11,600, Montevideo, Uruguay.
Irene
I
da Cruz
Department of Molecular Biology, Instituto de Investigaciones Biológicas Clemente Estable (IIBCE), Av. Italia 3318, 11,600, Montevideo, Uruguay.
Rosana
R
Rodríguez-Casuriaga
Flow Cytometry and Cell Sorting Core, IIBCE, Montevideo, Uruguay.
Federico F
FF
Santiñaque
Department of Proteins and Nucleic Acids, IIBCE, Montevideo, Uruguay.
Joaquina
J
Farías
Department of Molecular Biology, Instituto de Investigaciones Biológicas Clemente Estable (IIBCE), Av. Italia 3318, 11,600, Montevideo, Uruguay.
Gianni
G
Curti
Department of Molecular Biology, Instituto de Investigaciones Biológicas Clemente Estable (IIBCE), Av. Italia 3318, 11,600, Montevideo, Uruguay.
Carlos A
CA
Capoano
Flow Cytometry and Cell Sorting Core, IIBCE, Montevideo, Uruguay.|Department of Genetics, IIBCE, Montevideo, Uruguay.
Gustavo A
GA
Folle
Department of Cell and Developmental Biology, Biocenter, University of Würzburg, D-97074, Würzburg, Germany.
Ricardo
R
Benavente
Department of Genomics, Instituto de Investigaciones Biológicas Clemente Estable (IIBCE), Av. Italia 3318, 11,600, Montevideo, Uruguay. sotelojos@gmail.com.|Department of Cell and Molecular Biology, Facultad de Ciencias, Universidad de la República (UDELAR), 11,400, Montevideo, Uruguay. sotelojos@gmail.com.
José Roberto
JR
Sotelo-Silveira
Department of Molecular Biology, Instituto de Investigaciones Biológicas Clemente Estable (IIBCE), Av. Italia 3318, 11,600, Montevideo, Uruguay. adriana.geisinger@gmail.com.|Biochemistry-Molecular Biology, Facultad de Ciencias, UDELAR, Montevideo, Uruguay. adriana.geisinger@gmail.com.
Adriana
A
Geisinger
D000818:Animals; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D059014:High-Throughput Nucleotide Sequencing; D008297:Male; D049469:Meiotic Prophase I / Q000235:genetics; D051379:Mice; D049471:Pachytene Stage / Q000235:genetics*; D015203:Reproducibility of Results; D017423:Sequence Analysis, RNA; D013091:Spermatogenesis / Q000235:genetics*; D013093:Spermatogonia / Q000166:cytology; D059467:Transcriptome; D014960:X Chromosome / Q000235:genetics
Flow cytometry; RNAseq; Spermatogenesis; Transcriptome