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An orchestrated intron retention program in meiosis controls timely usage of transcripts during germ cell differentiation (RNA-Seq)

ABSTRACT: Alternative splicing (AS) represents a powerful resource to amplify the coding potential of eukaryotic genomes and to finely tune gene expression during cell differentiation and development. Notably, high-throughput transcriptome analyses identified testis among the tissues displaying the highest proportion of AS events and profiling of whole testis transcriptome during the first wave of mouse spermatogenesis highlighted some AS events that are timely regulated during this process. Thus, AS modulation likely contributes to the fine-tuned regulation of gene expression that occurs during the male germ cell differentiation. In particular, meiotic spermatocytes and post-meiotic spermatids are known to express a highly specific repertoire of protein variants. However, no study has directly investigated whether global splicing-changes occur during the trans-meiotic phases of spermatogenesis to date. Spermatocytes undergo two consecutive meiotic divisions to yield haploid post-meiotic spermatids, which then differentiate into highly specialized and motile spermatozoa. Herein, to achieve a comprehensive characterization of the splicing changes occurring during trans-meiotic differentiation of male germ cells, we performed high-throughput RNA sequencing (RNA-seq) analyses of polyA+ RNA isolated from highly purified populations of meiotic spermatocytes and post-meiotic spermatids. Our analysis uncovered a robust intron retention (IR) program in meiotic spermatocytes. Intron-retaining genes were strongly enriched in functional categories related to differentiation and properties of the male gamete. We found that meiotic intron-retaining transcripts are long-lived mRNAs, which are preserved in the nucleus until they need to be translated. Thus, our study reveals an unexpected physiological role for IR in ensuring proper and timely control of gene expression during male germ cell differentiation.

ORGANISM(S): Mus musculus

PROVIDER: GSE95138 | GEO | 2017/06/13

SECONDARY ACCESSION(S): PRJNA376175

REPOSITORIES: GEO

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Project description:Meiosis is a specialized form of cell division essential for sexual reproduction. The unique cellular events of meiosis have been extensively studied in model eukaryotes, including mammals. In mammals, the molecular and cellular changes that germ cells must undergo to transition from mitotic spermatogonia to meiotic spermatocytes remain largely undefined. Retinoic acid (RA) has been proposed as the meiosis inducing substance (MIS) required for female and male meiotic initiation. However, evidence for this presumption is based on studies of fetal gonads – meiosis is initiated in fetal ovaries but suppressed in fetal testes. Critically, the requirement for RA in meiosis has never been formally tested because germ cell differentiation and meiotic initiation in the fetal ovary are temporally inseparable. Using the postnatal mouse testis, where differentiation and meiotic initiation are temporally distinct (8.6 days apart), we test the hypothesis that RA is dispensable for initiation and progression through meiosis. Complementary in vivo experimental models were designed to address the hypothesis. In the first, “RA-sufficient” (RA-suf) model, spermatogenesis was synchronized by administration of potent and selective RA synthesis inhibitor; as a result, testes contained only undifferentiated spermatogonia. Then, the inhibitor was discontinued, and mice were given a single dose of exogenous RA to initiate spermatogonial differentiation. Germ cells completed differentiation, entered and completed meiosis, and formed haploid gametes at predicted timepoints. In the second, “RA-deficient” (RA-def) model, mice were treated as above, except the inhibitor was administered continuously. The lack of testicular RA in this model was confirmed in multiple ways, including assessment of RA-responsive gene expression, in vitro serum-free cultures, and dose-response experiments. To our surprise, germ cells in RA-def testes completed differentiation, underwent meiotic DNA replication, completed the landmark cytological processes of meiosis, and formed postmeiotic haploid spermatids, all on the predicted timeline of the RA-suf model. To define the effect of RA-deficiency on gene expression during meiotic initiation, large, highly enriched populations of premeiotic spermatogonia from RA-suf mice, and meiotic preleptotene spermatocytes from both models were FACS-isolated and subjected to bulk RNA-seq. Interestingly, analyses revealed few significant differences in expression of characteristic meiotic genes at the mRNA level. However, genes that were significantly different between RA-suf and RA-def preleptotene spermatocytes were associated with spermiogenesis functions. Taken together, our data reveal that, after initiating spermatogonial differentiation, RA is dispensable for initiation, progression through, and completion of meiosis in the male germline.

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An orchestrated intron retention program in meiosis controls timely usage of transcripts during germ cell differentiation (RNA-Seq)

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