Dynamics of Meiotic Sex Chromosome Inactivation and Pachytene Activation in Mice Spermatogenesis

Background & Summary:

Germ cell formation requires a regulated transition from mitosis to meiosis to generate haploid cells: oocytes and sperm. In males, there are specific features of meiosis given that the X and Y chromosomes only share the homologous region, and the non-homologous region stays unpaired. X and Y chromosomes undergo a process of silencing called meiotic sex chromosome inactivation (MSCI) and a subsequent reactivation of transcription of genes involved in spermiogenesis (final stage of spermatogenesis) (Turner, 2015).

In this work, Vertesy and colleagues use single-cell transcriptomics and single-molecule fluorescent in situ hybridisation (smFISH) to resolve the temporal dynamics that orchestrate this transition. The authors took advantage of a Dazl-GFP reporter mice which expresses the reporter gene in male germ cells spans from the spermatogonia up to the initiation of meiosis in spermatocytes (Chen et al., 2014). After validating a reconstructed temporal trajectory from their transcriptomic data, in-depth computational analysis allowed the authors to detect a functional order in the timing of silencing and reactivation of sex chromosome genes, rather than a positional order.

 

Key findings of the preprint:

• Sharply timed gene activation during spermatogenesis.

Changes in gene expression programs driving the different steps of spermatogenesis take place in a sequential but sharp way temporally, rather than genes being progressively activated. Three different waves of gene activation can be clearly distinguished which correlate with three specific stages of differentiation: spermatogonia, (pre)leptotene and pachytene.

 

• A transcriptional activation peak precedes meiotic sex chromosome inactivation.

MSCI is a silencing event and takes places rapidly. Many sex chromosomal genes are specifically activated right before they get silenced, dismissing the idea that a lack of transcription drives MSCI. Importantly, many of these genes are involved in gene regulation.

 

• Genes escaping MSCI are different from genes that escape X-chromosome inactivation in females.

A group of X chromosome genes are detected upon MSCI, and with an increased number of transcripts than before the general silencing. Interestingly, out of the 27 genes, only 1 is shared with known genes that escape the process of X-chromosome inactivation in female cells to balance dosage compensation. This suggests that the ability of certain genes to escape inactivation is not only based on their position in the chromosome.

• The reactivation pattern of genes after MSCI follows a functional order.

Three behaviours of gene activation are detected: a cluster of genes that are gradually activated, another cluster of genes that are sharply activated (referred to as immediate genes) and a third one with an intermediate behaviour. The immediate genes were enriched in transcriptional activation and energy production functions but, interestingly, there was also a correlation with the order they will be used in the spermatozoa. Genes playing a role in flagella formation and cell movement were mostly found in the immediate group, while late activated genes are involved in fertilization.

 

Why I chose this work:

Once the snapshots of different processes are known, the dynamics underlying these events are key to understand how they are regulated. The different steps that male germ cells undergo until the formation of haploid cells, including wide transcriptional silencing and reactivation, are a very good model to study dynamic transitions.

I chose this preprint because the authors nicely use state-of-the-art techniques, such as single-cell RNAseq and single-molecule FISH, to address those questions. They combine three reconstruction methods to generate a more reliable pseudotemporal trajectory which then, they validated by analysing expression of key genes by smFISH. Also, they have built an online tool that will help other interested researchers to explore the dynamic expression of specific genes: http://bit.ly/male_meiosis. After their first validation and general analyses, the authors go in depth to find detailed cues of how this process is temporally regulated, thus, harnessing the value of the data they have generated.

Questions to the authors:

• The authors use single-molecule FISH to beautifully validate different patterns of gene expression according mainly to spatial changes. Does this technique allow to go deeper and quantify transcriptional differences within a spatially-separated population to try to detect a temporal scale? If this technique could detect finer differences, it could be used to see how synchronised are neighbouring cells in terms of the sequential peaks of gene activation or the transition to MSCI for instance.
• Given that escapee genes after MSCI are different from escapee genes from other known X-chromosome silencing events (such as XCI in female cells), do the authors think those genes have specific functions in each case? Or could it be due to the lack of Xist expression in males that make the mechanism of silencing completely different? Since many escapee genes from the Xist locus are non-coding genes they could be involved in gene silencing directly, but if this is their function it could be the same than in other inactivation processes.
• It is very curious that although genes involved in flagella formation and fertilization are transcribed in a specific order according to their function, they will not be used until a week later. Do the authors have an idea of why they follow this order to then be stored? Could this storage also be ordered to stay in a “prepared” mode and act more efficiently?

 

References:

Chen HH, Welling M, Bloch DB, Muñoz J, Mientjes E, Chen X, Tramp C, Wu J, Yabuuchi A, Chou YF, Buecker C, Krainer A, Willemsen R, Heck AJ, Geijsen N. 2014. DAZL limits pluripotency, differentiation, and apoptosis in developing primordial germ cells. Stem Cell Reports 3(5):892-904.

Turner JM. 2015. Meiotic silencing in mammals. Annu Rev Genet 49:395-412.

 

doi: https://doi.org/10.1242/prelights.11559

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