# Genome-wide transcriptional silencing and mRNA stabilization allow the coordinated expression of the meiotic program in mice

Laura Bellutti, Édith Chan Sock Peng, Victoria Cluzet, Marie-Justine Guerquin, Antoine D. Rolland, Sébastien Messiaen, Elena Llano, Ihsan Dereli, Emmanuelle Martini, Attila Tóth, et al.

► To cite this version:

Laura Bellutti, Édith Chan Sock Peng, Victoria Cluzet, Marie-Justine Guerquin, Antoine D. Rolland, et al. Genome-wide transcriptional silencing and mRNA stabilization allow the coordinated expression of the meiotic program in mice. Nucleic Acids Research, 2025, 53 (5), pp.gkaf146. 10.1093/nar/gkaf146. hal-05028634

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<figure><img src="image_1.png" /></figure>
# Genome-wide transcriptional silencing and mRNA stabilization allow the coordinated expression of the meiotic program in mice

Laura Bellutti<sup>1,2,†</sup>, Edith Chan Sock Peng<sup>3,†</sup>, Victoria Cluzet<sup>1,2</sup>, Marie-Justine Guerquin<sup>1,2</sup>, Antoine Rolland<sup>3</sup>, Sébastien Messiaen<sup>1,2</sup>, Elena Llano<sup>4</sup>, Ihsan Dereli<sup>5</sup>, Emmanuelle Martini<sup>1,2</sup>, Attila Tóth<sup>5</sup>, Alberto M. Pendás<sup>4</sup>, Frederic Chalmel<sup>1,3,*</sup>, Gabriel Rivera<sup>1,2,*</sup>

## Abstract

The transcriptional dynamic of mammalian cells when these transit from the ubiquitous mitotic to a meiotic-specific program is key to understand this switch central to sexual reproduction. By quantifying active RNA polymerase II and nascent transcripts using single cell dataset and ethynyl-uridine pool-down with sorted cells from synchronized testes, we detailed the transcriptional activity of murine male germ cells. When spermatogonia differentiate, transcription slows down, reaching minimal activity at meiotic entry and resumes during pachytene stage. This event, we termed EMLT (for early meiotic low transcription), is distinct from the silencing of sex chromosomes as it is independent of Setdb1, though it is accompanied by the same chromatin mark, H3K9me3. EMLT is delayed in Stra8KO but occurs in mutants altering meiotic chromosome structure or double-strand break formation or repair. By comparing transcript abundance and nascent transcription we unveil a massive event of messenger RNA stabilization that parallels EMLT. Altogether our data indicate that meiosis is initiated with a nearly silent genome, and we propose that the stabilization of transcripts at that time facilitates the meiotic entry by synchronizing the expression of several meiotic subprograms.

## Graphical abstract

<figure><img src="image_2.png" /></figure>

## Introduction

Spermatogenesis relies on specific and complex transcriptional programs to orchestrate the production of male gametes [1]. It can be divided into a mitotic phase, a meiotic phase, and a post-meiotic phase. Spermatogonial stem cells multiply and

enter a differentiation pathway. In the mouse, this leads to a strictly regulated differentiation of type A spermatogonia into type A1–A4, intermediate, and B spermatogonia through successive cell divisions [2]. Type B spermatogonia are the precursors of preleptotene cells entering the premeiotic S phase.

<footnote n="1"><sup>1</sup>Université Paris Cité, CEA, Genetic Stability Stem Cells and Radiation, Laboratory of Development of the Gonads, F-92265 Fontenay-aux-Roses, France</footnote>

<footnote n="2"><sup>2</sup>Université Paris-Saclay, CEA, Genetic Stability Stem Cells and Radiation, LDG/IRCM/IBFJ, F-92265 Fontenay-aux-Roses, France</footnote>

<footnote n="3"><sup>3</sup>Inserm, EHESP, Univ Rennes, Irset (Institut de recherche en santé, environnement et travail)—UMR_S 1085, Rennes, France</footnote>

<footnote n="4"><sup>4</sup>Molecular Mechanism Program, Centro de Investigación del Cancer (Universidad de Salamanca-CSIC), 37007 Salamanca, Spain</footnote>

<footnote n="5"><sup>5</sup>Institute of Physiological Chemistry, Faculty of Medicine at the TU Dresden, Fiedlerstrasse 42 01307 Dresden, Germany</footnote>

<footnote>*To whom correspondence should be addressed. Email: gabriel.livera@cea.fr
Correspondence may also be addressed to Frederic Chalmel. Email: frederic.chalmel@inserm.fr
†The first two authors should be regarded as Joint First Authors.</footnote>
The spermatocytes undergo two consecutive meiotic divisions
allowing the production of haploid spermatids [3]. Finally, the
process termed spermiogenesis includes several morphological
transformations of spermatids generating the highly differen-
tiated spermatozoa [4]. Each step of spermatogenesis requires
the regulated and dynamic expression of specific transcripts,
contributing to the high diversity of testicular transcriptome
[5-7]. Additionally, several transcriptional arrests and post-
transcriptional regulations complicate the testicular transcrip-
tome [8]. Two transcriptional arrests have been exquisitely
characterized: the silencing of sex chromosomes during meio-
sis, a process termed meiotic sex chromosome inactivation
(MSCI) and a global silencing in post-meiotic elongated sper-
matids undergoing tight packaging of the genetic material [9].

A critical step of spermatogenesis consists in the conversion of mitotic spermatogonia into meiotic spermatocytes. Stimulated from retinoic acid 8 (STRA8) is a transcription factor mandatory to orchestrate the mitosis-to-meiosis transition [10]. Meiosis begins with a long prophase I that is subdivided into different stages: leptotene, zygotene, pachytene, and diplotene. During prophase I, programmed DNA double-strand breaks (DSBs) are generated at specific regions called ‘hotspots’ [11, 12]. DSBs are next repaired by meiotic homologous recombination (HR). In parallel, homologous chromosomes align, pair, and synapse thanks to the formation of a proteinaceous structure called the synaptonemal complex (SC). At the end of pachytene stage, DSBs are repaired and autosomes are fully synapsed. In contrast, X and Y chromosomes remain largely unsynapsed because of their lack of homology. They are compartmentalized in a heterochromatic structure called sex body. The sex body is transcriptionally inactive due to DNA damage response and chromatin regulation that induce the MSCI [9, 13–16].

DSBs generation, HR and SC formation are highly reg-
ulated and interdependent processes. The expression of
meiotic-specific genes allows fine regulation of these events.
Indeed, meiotic initiation is characterized by an extensive
genome reprogramming, to switch from a mitotic pro-
gram to a meiotic one. This is accompanied by elaborated
post-transcriptional mechanisms changing messenger RNA
(mRNA) stability and alternative splicing events [8, 17]. The
mechanisms governing such considerable changes in the tran-
scriptome are yet poorly understood and may be connected
to the major events occurring in the meiotic genome. Inter-
estingly, spermatocytes in the early stages of prophase I have
been reported with a very low or near-extinct transcriptional
activity [18–20]. This genome-wide low transcription level, to-
gether with the existence of specific meiotic transcripts and
transcriptional regulations, is puzzling. Currently, very little is
known about this global transcriptional reduction, contrar-
ily to MSCI, which is largely characterized. Few transcrip-
tome analyses have focused on spermatocytes initiating mei-
otic prophase I, due to the difficulty in isolating these rare
testicular cell populations. Moreover, transcriptome analyses
poorly reflect transcription, as these are in part blurred by
post-transcriptional mechanisms.

Here, we characterized the global reduction of transcrip-
tional activity in the genome of early meiotic cells. In order to
acquire a precise view of transcription dynamic around mei-
otic entry, we employed spermatogenesis synchronization, cell
sorting, and ethynyl uridine incorporation to specifically la-
bel nascent RNAs, comparing this with unspliced RNA anal-
ysis via scRNA-seq. We demonstrated that this decrease of
transcription precedes and is independent of the key events

of prophase I, DSBs, and chromosome synapsis, but depends
on the meiotic gatekeeper *Stra8*. Interestingly, this low tran-
scriptional activity is accompanied by a broad stabilization of
mRNA at the time of meiotic engagement.

**Materials and methods**

Mice and animals

All animal studies were conducted in accordance with the
guidelines for the care and use of laboratory animals of
the French Ministry of Agriculture (France, APAFIS#32392-
2021071211498973). Mice were housed in controlled pho-
toperiod conditions (lights on from 08:00 to 20:00) and
supplied with commercial food and tap water ad libitum.
Synchronization of spermatogenesis in NMRI mice was per-
formed as previously described [21]. Two milligram of five-
ethynyl uridine (EU, Click-iT™ Nascent RNA Capture Kit,
Invitrogen) per mouse were injected at 24 days post-partum
(dpp) to label nascent RNA. Three hours later, mice were sacri-
ficed and cells were FACS-sorted based on their ploidy to iso-
late spermatogonia, leptotene, and pachytene spermatocytes
as previously described [22, 23]. Cells from two to four an-
imals were pooled together for RNA extraction and nascent
RNA capture, representing over 700 000 cells per point. For
each cell population, experiments were run in triplicate (i.e.
three independent RNA pools for spermatogonia, leptotene,
and pachytene cells). Cells obtained through the spermatoge-
nesis synchronization protocol were used exclusively for EU-
RNA-seq. All the other experiments were performed on ani-
mals without synchronization.

To study ovary development, timed matings were set up
by housing males with females overnight. The presence of a
vaginal plug was checked the following morning. The follow-
ing midday was designated as 0.5 days post-conception (dpc).
Pregnant females were sacrificed by cervical dislocation, fe-
tuses were removed from uterine horns, and gonads were dis-
sected under a binocular microscope.

All transgenic mice used in this study have been previously
described [13, 17, 23–29]. Gonads from dogs, cats, and goats
were obtained following castrations performed in veterinary
clinics. All procedures involving non-murine species adhered
to ethical standards set by veterinary practices.

Preparation of spermatocyte chromosome spreads

Testes used for spermatocyte spread preparations were col-
lected from adult mice (2–6 months). For wild type (WT),
*Spo11*−/−, *Meiob*−/−, *Setdb1*−/−, and *Atrfl*−/−; *Atm*−/− mice,
spermatocyte spreads were prepared as follows. Spermato-
cytes were manually liberated from tubules (flat razor blades)
and resuspended in 0.1M sucrose. Ovaries used for chromo-
some spreads were collected from 14.5 dpc embryos, 18.5
dpc embryos, and 0 dpp pups. Ovaries were dilacerated in
0.1M sucrose on slides using needles. Oocytes or spermato-
cytes were then distributed on slides in humid chambers and
fixed with 1% PFA (in H2O, pH 9.2) and 0.1% Triton for
1 h 30 min. After fixation, slides were rinsed with H2O 0.4%
Photo-Flo 200 (Kodak).

Immunofluorescence on meiocyte chromosome spreads

After washing chromosome spreads from spermatocytes or
oocytes, slides were air-dried before blocking. Slides were in-
cubated for 1 h at room temperature with blocking solu-
tion (0.2% BSA, 0.2% Gelatin, 0.05% Tween in PBS) and
overnight with primary antibodies in blocking solution at
room temperature (see Table S1 for antibodies). Secondary
antibodies were incubated for 1 h 30 min at 37°C. Slides
were stained with 4',6-diamidino-2-phenylindole (DAPI) and
mounted with Prolongold medium. Imaging was performed
using a Leica DM5500 B epifluorescence microscope (Leica
Microsystems) equipped with a CoolSNAP HQ2camera (Pho-
tometrics) and Leica MMAF software (Metamorph, Molecu-
lar Devices). Images were processed with ImageJ software.

Immunohistostaining

Gonads were fixed in 4% PFA, dehydrated, embedded in
paraffin, and cut into 5-µm-thick sections. Sections were
mounted on slides, dewaxed, rehydrated, and boiled for
20 min in citrate buffer (pH 6) prior to immunostaining. For
immunofluorescence stainings, sections were blocked for 1h in
Gelatin blocking solution (0.2% BSA, 0.2% Gelatin, 0.05%
Tween in PBS) and incubated 1 h 30 min at 37°C with the pri-
mary antibody diluted in blocking solution (see Table S1 for
antibodies). After three washes in PBS 0.05% Tween, slides
were incubated with secondary antibodies for 1 h at 37°C.
Slides were treated with DAPI and mounted in ProLong Gold.
Imaging was performed using a spinning-disk confocal micro-
scope (Nikon Ti2, CSU-W1) with Metamorph software. Im-
ages were processed with ImageJ software.

RNA polymerase II quantification

For all experimental comparisons, exposure time and laser
intensity settings were kept constant, and saturation was
avoided during image acquisition. Quantitative analyses were
performed on unsaturated raw images, and representative im-
ages were adjusted to highlight faint signals while maintaining
uniform settings within each panel. Quantification of RNA
polymerase II, total (POL2) or phosphorylated (pPOL2), on
chromosome spreads was performed with ImageJ software.
DAPI staining was used to delimit nuclei of cells, and the mean
intensity of POL2 was quantified within these regions. Back-
ground intensity was subtracted from the nuclear signal to en-
sure accurate measurements.

Fluorescence intensity of POL2 and pPOL2 on testes sec-
tions was quantified with ImageJ software using line scans on
a single focal plane (plot profile). DAPI staining was employed
to identify nuclear boundaries and used to draw a line cross-
ing the nucleus of the cell. We termed this line ‘cell diameter’.
Background noise for POL2 and pPOL2 was measured using
a line scan of similar length outside the cell nucleus. Values of
the background line scan were averaged, and for each section
across the line scan, the intensity level of POL2 and pPOL2
was normalized to the mean background intensity. For POL2
and pPOL2 comparisons, quantifications were performed on
the same cells.

RNA-sequencing data analysis

For EU-RNA-seq, capture of nascent RNA was performed ac-
cording to the manufacturer instructions (Click-iT™ Nascent
RNA Capture kit, Invitrogen). RNA-seq libraries were gen-
erated using Next Ultra II Directional RNA Library Prep kit
(NEB) and ribosomal RNA depletion according to the manu-
facturer's protocols. EU-RNA-seq libraries were made like the
RNA-seq but without any depletion. Libraries were sequenced
on an Illumina HiSeq2500 aiming at an average of 60 million

100-bp reads per sample. Raw reads from the EU-RNA-seq
and RNA-seq datasets were mapped onto the mouse genome
(mm10) by using STAR (v2.5.2a). The *StringTie* tool was used
for transcript assembly while Ballgown was used for quantifi-
cation to get a FPKM expression matrix. After quantification,
the resulting FPKM expression matrix from the EU-RNA-seq
dataset was corrected according to the expression of 5S rib-
bosomal genes, which are considered transcriptionally stable
during spermatogenesis. The expression matrix was divided
by the weighted sum of all 5S ribosomal genes.

Single-cell RNA-sequencing data analysis

Pre-processing and quality control

Mouse scRNA-seq data were downloaded from the EBI (E-MTAB-6946) [20]. These included 10 samples from mice aged 5–42 dpp. Raw sequencing reads were processed and mapped to the mouse (mm10) genomes using *CellRanger* (v4) with default parameters, and all samples were aggregated with cell-ranger aggr. Outlier cells were removed from the aggregated matrices with *Scater* [30]. In parallel, *DoubletFinder* [31] was used for doublet detection and removal on each individual sample. Finally, *Scater* and *Scran* [32] were used to exclude genes detected in <10 cells.

The *Seurat* (v3) analysis pipeline [33] was then applied. Data normalization was performed with the LogNormalize function with default parameters, while the 2000 most variable genes were identified with FindVariableFeatures. A linear dimensionality reduction was then performed with RunPCA and a JackStraw procedure was used to identify the number of dimensions to be considered. The FindNeighbors and FindClusters functions were applied to determine cell clusters. Correction of batch effects was performed using the Seurat's CCA method. Data visualization was finally achieved using the uniform manifold approximation projection (UMAP) dimensional reduction technique applied to the batch effect corrected data. Following identification of main cell populations based on known marker genes, germ cell clusters were subsetted and submitted using the same pipeline as described above, i.e. applying FindVariableFeatures, RunPCA, JackStraw, FindNeighbors, FindClusters, and CellCycleScoring. Again, the clusters were annotated based on known marker genes and the resulting annotation was validated by comparison to that of Ernst and collaborators using the hypergeometric statistical test as well as by monitoring germ cell evolution according to sample ages and cell cycle phase.

Quantification of nascent transcription and correction of
ploidy effect

Next the velocity tool was used [34] to generate unspliced and
spliced count data starting from bam files from Cell Ranger, to
quantify nascent transcription and transcription, respectively.
Unspliced scRNA-seq data were used as a proxy for nascent
transcription measurement in order to study transcriptional
activity during spermatogenesis. The number of unspliced
counts for each cell was calculated independently for auto-
somes and sex chromosomes. Since the amount of DNA by
cell can directly influence the number of RNA molecules that
are produced in each cell, the DNA content of each germ cell
subpopulation was taken into account: for undifferentiated
and differentiating spermatogonia (2n/2c) and for secondary
spermatocytes (1n/2c), the number of unspliced counts was
unchanged; for primary spermatocytes (leptotene, zygotene,
pachytene, diplotene, and metaphase I; 2n/4c), the number of
unspliced counts were divided by two; while for spermatids
(1n/1c), the number of unspliced counts was multiplied by
two.

Identification of escaping and silenced genes during EMLT

Since ploidy-corrected unspliced count matrix was employed,
intron size may bias the number of quantified reads per gene.
To avoid this effect, a cumulative intron size correction was
applied. For each gene: (i) intron size was obtained by sub-
tracting the cumulative sum of exons using the Bedtools sub-
tractBed function, (ii) this value was then used to divide gene
count and the ratio was multiplied by an arbitrary constant
(1 000 000). This normalization allows setting of a threshold
for expressed gene detection. Genes showing a null variance
were discarded. Next, a two-step process was used to identify
potential ‘silenced’ and ‘escaping’ genes. Genes were consid-
ered ‘silenced’ if their expression values were: (i) higher than
an expression threshold (median value of the overall count
matrix = 0.07) in at least one of the relevant stages (Diff
Spg 1, Diff Spg 2, ePa 1, ePa 2, lPa, and/or Di) and, (ii) sig-
nificantly correlated (one-tailed Pearson correlation test, P -
value <.05) to the expression profiles of archetypal genes (Nd-
ufa11 and Park7) showing a silenced expression pattern in
Le and Zy. Genes were considered ‘escaping’ if their expres-
sion values were: (i) >0.07 in Le and Zy and (ii) significantly
correlated (P-value <.05) to archetypal genes (Pet2, Ccnb3,
and Prdm9) showing an escaping expression pattern in Le or
Zy. The median value of the overall count matrix was chosen
as the expression threshold because it provides a robust and
data-driven criterion for filtering extreme values while ensur-
ing reproducibility across datasets.

RNA stability

RNA stability during meiosis was investigated using both
single-cell and bulk RNA-seq approaches. For the scRNA-seq
dataset the ‘spliced’ and ‘unspliced’ expression profiles reflect
transcription and nascent transcription, respectively, while the
‘stability’ is the ratio of ‘spliced’ to ‘unspliced’ expression lev-
els. In the RNA-seq dataset ‘RNA-seq’ and ‘EU-RNA-seq’ ex-
pression profiles reflect transcription and nascent transcrip-
tion, respectively, while ‘stability’ is the ratio of ‘RNA-seq’ to
‘EU-RNA-seq’ expression levels.

Data representation: heatmaps and boxplots

Heatmaps and boxplots were generated using *Ggplot2* (R-package). For heatmaps expression values of each gene were scaled between [0;1] using a 0-max scale (0 = 0; maximum value = 1). Thanks to this scaling, zero can be regarded as an actual zero. Stability matrix was scaled between [0;1] with the Rescale () function from the *Scales* package.

Integration of gene expression with recombination hotspots
and epigenetic marks

Data on DSB hotspots as defined by anti-DMC1 single-
stranded DNA (ssDNA) sequencing data in C57BL/6J mice
were downloaded from GEO (GSE75419) [35]. The distance
of genes to the nearest hotspot out of 19 528 recombination
sites was computed with the closest function from *BedTools*
[36]. Two-tailed Wilcoxon tests were used to compare the
distance to the nearest hotspot for ‘silenced’ genes or 1000
random intergenic regions. A DSB map defined by SPO11-

oligonucleotides was also used to compare recombination
sites to transcriptional changes [12].

ChIP-seq data for seven histone marks (H3K4me3,
H3K4me1, H3K9me2, H3K9me3, H3K27me3, H3K36me3,
and H3K27ac) obtained in spermatogenic cells were down-
load from GEO (GSE132446) [37]. In this study histone mod-
ifications in homogenous synchronous spermatogenic cells
of 9 or 10 spermatogenic stages (undifferentiated spermato-
gonia, type A1 spermatogonia, type B spermatogonia, mid-
preleptotene spermatocytes, leptotene spermatocytes, mid-
zygotene spermatocytes, early-pachytene spermatocytes, mid-
pachytene spermatocytes, and diplotene spermatocytes) were
studied. *DeepTools* was used to perform histone modification
distribution analysis around ‘silenced’, ‘escaping’ genes and
random intergenic regions. To focus on the events around the
transcription start site (TSS), different upstream and down-
stream distances were used: -2000/+2000 pb for H3K9me3,
H3K9me2, H3K27me3, and H3K4me1; -1000/+1000 for
H3K27ac and H3K4me3; and 0/+2000 for H3K36me3. The
histone modification scores associated with genomic regions
of interest (‘escaped’, ‘silenced’ genes and random intergenic
regions) were generated by *ComputeMatrix* modules. Bins for
each cell type per genomic region were summed in the output
matrix. Then profiles of histone modification scores for ‘si-
lenced’, ‘escaping’ genes and random intergenic regions were
represented using mean ± sem.

Results

Transcriptional quiescence initiates prior meiotic
entry

To finely characterize transcriptional levels across spermato-
genesis, we first quantified the active form of RNA polymerase
II, phosphorylated on Serine 2 of its CTD (pPOL2), by im-
munofluorescence staining (Fig. 1A–G). Using spermatocyte
chromosome spreads (Fig. 1A and B), pPOL2 was hardly
detected on chromatin at leptotene and zygotene stages. By
mid-pachytene stage, pPOL2 intensity rose sharply, peaking
at late pachytene and diplotene stages, except on X and
Y chromosomes, where it remained undetectable (Fig. 1A
and Supplementary Fig. S1). Quantification in leptotene cells
and in the sex body from mid-pachytene cells indicated an
equivalent lack of transcriptional activity. This confirms and
extends previous observations [9, 19]. We termed this pro-
cess of genome-wide pPOL2 reduction during the beginning
of prophase I, early meiotic low transcription (EMLT), to
distinguish it from the MSCI process, which silences exclu-
sively the sex chromosomes in later stages (i.e. pachynema and
diplonema).

Next, we exploited the well-characterized seminiferous ep-
ithelium cycle to determine when EMLT initiates and ends
during spermatogenesis. Measure of pPOL2 on testis sections
show that type A spermatogonia displayed pPOL2 staining
(Fig. 1C and D, and Supplementary Fig. S2), which decreased
in intermediate and type B spermatogonia, and further dimin-
ished in preleptotene and leptotene spermatocytes. During zy-
gonema, the staining increased slightly and progressively to
reach at the early pachytene stage an intensity level similar
to type A spermatogonia. Intensity of pPOL2 staining peaked
at late prophase I and in first round spermatid stages. As ex-
pected, elongated spermatids were completely silenced and
devoid of pPOL2 staining due to their high level of DNA
<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

Sg A Sg In/B pLe Le Zy
ePa mPa IPa/Di rSd eSp

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /><figcaption>**Figure 1.** Dynamic of transcription during spermatogenesis. Characterization of RNA polymerase II phosphorylated on Serine 2 (pPOL2) profile during prophase I by co-immunolabelling of pPOL2 and SYCP3 on spermatocytes chromosome spreads (A). DNA is stained with DAPI. pPOL2 staining is very</figcaption></figure>
compaction. Total RNA polymerase II (POL2), including non-
phosphorylated and phosphorylated forms, had an expres-
sion profile similar to that of pPOL2 (Fig. 1C and D, and
**Supplementary Figs S1** and S2). Using chromosome spreads,
we evidenced that POL2 was also hardly detected on the chro-
matin during EMLT (**Supplementary Fig. S1**). This indicates
that the variations of pPOL2 intensity are due to a reduction
in RNA polymerase II availability and/or loading on chro-
matin. Interestingly, early pachytene spermatocytes showed
maximal POL2 levels, whereas pPOL2 reached its peak later,
during mid-pachynema (Fig. 1D, and **Supplementary Figs S2B**
and S3), suggesting a two-step process: first, the loading of
POL2 onto chromatin, and second, the activation of POL2
(pPOL2) to produce the hyper-transcriptional state observed
in late prophase I. Of note, after MSCI initiated, POL2 but
not pPOL2 reappeared specifically on the axes of sex chro-
mosomes (**Supplementary Fig. S1A**), while during EMLT both
were absent from the whole chromatin.

To determine accurately when EMLT was set up during spermatogonia differentiation, we monitored two spermatogonia markers: STRA8 and ZBTB16 (Fig. 1E and F). Undifferentiated spermatogonia, that expressed ZBTB16 but not STRA8, and differentiating type A spermatogonia, that expressed ZBTB16 and STRA8 at low level, were stained for POL2. Intermediate and type B spermatogonia, in which ZBTB16 was weakly detected ([38] and personal observations), were faintly stained for POL2. Thus, EMLT initiates during spermatogonia mitotic divisions, more precisely at the transition from type A spermatogonia to intermediate spermatogonia (Fig. 1G).

To obtain a measure of transcription, we first analysed pub-
lished dataset from single cells (scRNA-seq). The study of
Ernst *et al*. did not discard droplets containing very few RNAs
and was thus well suited to analyse cells with a low transcrip-
tion [20]. Data were downloaded from the EBI database and
visualized with UMAP to identify germ cell stages. Thirty-
four thousand seventy-eight germ cells were selected and par-
titioned into 30 clusters (Supplementary Figs S4, S5, and S6).

We then analysed unspliced RNAs as a proxy to monitor nascent transcription. Ploidy effect was corrected as a function of the spermatogenic stage as transcription is strongly associated to ploidy. Gene with null variance were removed, and scaled expression of the 17 233 remaining genes was visualized as a heatmap according to their chromosomal location (autosomes and sex chromosomes). This approach validated the two well-known transcriptional arrests during spermatogenesis, the MSCI during the pachytene stage and the global inactivation during spermiogenesis (Supplementary Fig. S6C). Global silencing was observed at the leptotene and zygotene stages (Fig. 1H and Supplementary Fig. S6C). Total nascent transcription decreased 12-fold between spermatogonia (Diff Sg1) and leptonema.

Second, we directly captured nascent transcripts using
ethynyl-uridine incorporation (EU-RNA-seq).
EU incorporation was performed in post-natal testes with syn-
chronized spermatogenesis and spermatogonia, leptotene and
pachytene cells were sorted simultaneously (Supplementary
Fig. S7A). This approach allowed minimizing contaminations
and enrichment of rare stages. Given the fact that EU-RNA-
seq relies on artificially synchronized spermatogenesis we en-
sure that EMLT takes place similarly (Supplementary Fig.
S7B) and carefully compare data obtained from this ap-
proach with scRNA-seq performed in non-synchronized an-
imals. EU-RNA-seq identified nascent RNA and also evi-
denced EMLT and MSCI at the leptotene and pachytene
stages, respectively (Fig. 1H). EU-RNA-seq allowed the anal-
ysis of nearly twice as much genes (31 258) in comparison to
scRNA-seq. Comparison of gene transcription with both ap-
proaches revealed similar profiles as exemplified by a set of ro-
bustly expressed genes (reference genes or germ cell markers,
Fig. 1I).

Altogether, staining for pPOL2 and POL2, pre-mRNA anal-
ysis (unspliced scRNA-seq), and nascent RNA analysis (EU-
RNA-seq) confirmed the existence of EMLT. This silencing is
initiated prior to meiotic entry and is most pronounced at the
leptotene stage in mouse testicular cells.

low at leptotene (Le), zygotene (Zy), and early-pachytene stages (ePa) and is obvious at mid-pachytene (mPa), late-pachytene (IPa), and diplotene (Di) stages. Sex body (orange dashed circle) is not stained for pPOL2. Quantification of pPOL2 staining (B). pPOL2 intensity is normalized to SYCP3-negative cells (SYCP3-, 100%, black dashed line). Each dot represents one spermatocyte. Spermatocytes were harvested from seven mice; black bars represent means ± sem. For each column, different letters indicate significantly different values [ordinary one-way analysis of variance (ANOVA), multiple comparisons]. Immunofluorescence analysis of pPOL2 and total RNA polymerase II (POL2) in testis sections (C). Roman numbers indicate the stage of seminiferous epithelium cycle. Prophase I cells are identified thanks to SYCP3 and DNA is stained with DAPI. For panels (A), (E), and (F), dotted white lines delimit seminiferous tubules. Scale bar = 5 µm. Quantification of pPOL2 (upper) or POL2 (lower) intensity at each stage of spermatogenesis by line plot analysis in the nuclei (D). Each line represents the average of several cells at a specific spermatogenic stage. Sg A = Type A spermatogonia, n = 19; Sg Int/B = intermediate and type B spermatogonia, n = 26; pLe = preleptotene, n = 27; Le = leptotene, n = 27; Zy = zygotene, n = 22; ePa = early pachytene, n = 27; mPa = mid pachytene, n = 50; IPa/Di = late pachytene/diplotene, n = 32; rSd = round spermatids, n = 50; and eSd = elongated spermatids, n = 4. Black dashed line represents pPOL2 intensity in Sertoli cells (S, n = 28). AU = arbitrary units. Immunofluorescence analysis of POL2 and STRA8 in testis sections (E). DNA is stained with DAPI. Differentiated A spermatogonia (Sg Adiff), pLe and early-leptotene (eLe) cells are stained for STRA8 and show different levels of POL2 intensity. Immunofluorescence analysis of POL2 and ZBTB16 in testis sections (F). DNA is stained with DAPI. Sg A and Sp Int/B are stained for ZBTB16 and show different levels of POL2 intensity. Schematic representation of EMLT during spermatogenesis, with a focus on meiotic prophase I (G). Transcription level decreases during spermatogonia differentiation to reach minimum at preleptotene and leptotene stages. Transcription is hyperactivated at mid-pachytene and diplotene stages when sex chromosomes remain silent (MSCI). Heatmaps and boxplots of the expression of 17 233 and 31 258 genes distributed according to their chromosomal location (autosomes and sex chromosomes) from the unspliced scRNA-seq dataset (average count matrix) and the EU-RNA-seq dataset (FPKM matrix), respectively (H). These datasets reflect nascent transcription, and are used to study transcriptional activity during spermatogenesis. In the heatmaps, each row represents a gene, and each column represents a specific spermatogenic cell population (scRNA-seq) or a sample (EU-RNA-seq). For heatmaps, expression values (mean counts or FPKM) of each gene were scaled between [0;1] using a 0-max scale (0 = 0; maximum value = 1). Boxplots display the mean counts per gene and the FPKM values for unspliced scRNA-seq and for EU-RNA-seq, respectively. One-tailed Wilcoxon tests were used to compare the average counts or FPKM of each spermatogenic stage to the first spermatogenic cells analysed; a second axis representing the -log10 (P-value) indicates the test significance for scRNA-seq; **** indicates P < .0001 for EU-RNA-seq. Diff Sg = differentiated spermatogonia. For EU-RNA-seq, 1, 2, and 3 correspond to three independent experiments. Heatmaps representing the gene expression profiles of a subset of emblematic genes robustly expressed in the unspliced scRNA-seq and EU-RNA-seq datasets (I).
## EMLT is conserved in mammalian testes and is observed in female mice

Since this meiotic genome-wide transcription arrest has, apart from *Mus musculus*, only been reported in males of a few species [39, 40], we sought to investigate its conservation in mammals. Interestingly, pPOL2 was almost undetectable at the beginning of prophase I in three different mammals: goat, rabbit, and dog (Supplementary Fig. S8A). As in mice, EMLT was also followed by a hyperactive transcriptional state in pachynema in these mammals. Altogether, this reflects a conserved dynamism of germ cells transcriptional state during spermatogenesis.

Meiosis occurs both during male and female gametogenesis. In females, meiosis begins during fetal life and arrests at the diplotene stage around birth. Transcriptional levels during prophase I have been poorly described in females. Thus, we analysed pPOL2 intensity in oocytes during prophase I (Supplementary Fig. S8B and C). We collected embryos at different fetal ages 14.5 and 18.5 days post-conception and at birth and performed chromosome spreads. pPOL2 staining was least abundant in leptotene and zygotene oocytes, lower than in somatic cells, and increased during late prophase I stages. However, there was a large heterogeneity in pPOL2 intensity in female meiocytes, whatever the stage, and many oocytes seem to escape a complete transcriptional repression. Thus, a process analogous to EMLT appears to also occur at meiotic entry in the female germline, albeit this phenomenon is less pronounced in the mouse developing ovary.

## EMLT mechanism differs from MSCI

In order to search for epigenetic marks correlated to EMLT, we first partitioned genes in two opposite categories based on unspliced RNAs from scRNA-seq. 'Silenced' genes exhibited higher transcription levels both before and after EMLT, whereas 'escaping' genes were predominantly transcribed specifically during EMLT. Genes were identified by using a correlation-based analysis with archetypal genes, i.e. *Ndufa11* and *Park7* for the 'silenced' genes (n = 11 660), and *Pet2*, *Ccnb3*, and *Prdm9* for the 'escaping' genes (n = 79) (Fig. 2A and Supplementary Fig. S4B). We compared 'silenced' genes identified by scRNA-seq and EU-RNA-seq and all presented a low transcription in leptotene cells (Supplementary Fig. S9). As scRNA-seq presented a fine resolution of stages we next focus on silenced and escaping genes defined by this approach. More than 99% of 'silenced' genes were located on autosomes (11 590/11 660), whereas over 40% of 'escaping' genes (33/79) were found on sex chromosomes (32 on the X), and their profiles were roughly similar when assessed with EU-RNA-seq (Supplementary Fig. S10). Escaping genes are found scattered throughout the genome and are mostly protein coding genes (Table S2, Supplementary Fig. S11A). Most have a low expression in pachynema (including for escaping genes on autosomes) and are expressed again later in spermatids cells (Supplementary Fig. S11B).

We next analysed the profile of several histone marks associated with activation or repression of transcription using published ChIP-seq data [37] (Fig. 2B and Supplementary Fig. S12). None distinguished the 11 660 'silenced' from the 79 'escaping' genes. The histone H3K9me2 and me3 modifications around the TSS correlated with the timing of transcriptional quiescence. These marks are known to silence transcription. As H3K9me3 has been proposed to be involved

in MSCI, we focused on this mark, it presented a gradual increase from undifferentiated spermatogonia to zygotene stages. This was confirmed by immunostaining in testicular sections (Fig. 2C). H3K9me3 staining was observed in patchy areas that likely correspond to pericentromeric heterochromatin. In the rest of the nucleus, staining was faint in A spermatogonia and pachytene spermatocytes and most abundant in leptotene and zygotene stages. As H3K9me3 has been proposed to repress transcription and is involved in MSCI, we sought to determine whether the same mechanisms govern MSCI and EMLT. During MSCI, sex chromosomes inactivation is primarily triggered by DNA damage response pathway and SETDB1 trimethylates H3K9 leading to the transient accumulation of H3K9me3 on sex chromosomes during early pachytene stages [13, 16, 19, 41]. We thus analysed pPOL2 in chromosome spreads from conditional KO obtained with the *Ngn3-cre* for *Setdb1* (*Setb1*<sup>-/-</sup>) and for both *Atm* and *Atr* (*Atm*<sup>fl/fl</sup>; *Atr*<sup>-/-</sup>). In these animals, MSCI has been reported greatly hampered at pachytene stage. At the leptotene stage, no pPOL2 was detected in the chromatin of both mutant mice indicating that impairment of key factors that govern MSCI do not alter EMLT (Fig. 2D). At the early pachytene stage, pPOL2 staining was retrieved in mutants as in WT animals indicating no defect in resuming transcription. To further compare MSCI and EMLT, expression profiles of 612 and 1293 genes located on the sex chromosomes were investigated in the unspliced scRNA-seq and the EU-RNA-seq datasets, respectively (Fig. 2E). scRNA-seq evidenced that nearly all genes investigated were extinct at the beginning of pachytene stage, many of which had transcriptional depression initiated before meiotic entry. In the EU-RNA-seq, three expression patterns were discernible. The first concerned genes silenced during EMLT but escaping MSCI, the second and predominant cluster was composed of genes silenced during both EMLT and MSCI, and the last cluster consisted of genes escaping EMLT but not MSCI (Fig. 2E, and c1, c2, and c3). Next, we focused on specific genes and gene families on sex chromosomes (Fig. 2F). The *androgen receptor* (*Ar*) gene was silenced both during EMLT and MSCI, a profile representative of most genes on sex chromosomes. Nascent RNAs of *Tex16*, *Dusp9*, and most genes of the *Mage* family were detected during EMLT, albeit possibly at lower levels than in spermatogonia, but not during MSCI. Genes from the Spin2 family on the contrary were specifically expressed during MSCI. Of note, Spin2 genes are mono-exonic and were thus discarded from the unspliced scRNA-seq data analysis. Altogether, these data indicate that MSCI is not just a mere extension of EMLT, as some genes on sex chromosomes display different expression dynamics and since key regulators of MSCI do not affect EMLT. Nonetheless repression of transcription may be associated in both cases with H3K9 methylation, possibly using different methylases.

## EMLT is unaffected by cohesin or HORMAD1 depletion

At the beginning of prophase I, spermatocytes undergo specific chromatin rearrangements. Chromosomes form an axe/loop-structure. Sister chromatids are held together by cohesins allowing establishment of axial elements (AEs) at early leptotene [42]. To investigate the link between EMLT and chromosome structure, we analysed *Rad21L*<sup>-/-</sup> and *Stag3*<sup>-/-</sup> mice, which lack key meiotic-specific cohesins [27, 28, 42]. Mutation of these cohesins caused prophase I arrest at or
<figure><img src="image_12.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_13.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_15.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_18.png" /></figure>

**Figure 2.** Gene expression profiles of 11 660 'silenced' and 79 'escaping' genes in the unspliced scRNA-seq dataset represented as heatmaps (scaled mean counts) and profile (mean counts per gene ± sem) (A). 'Silenced' and 'escaping' genes were identified by using a correlation-based analysis with archetypal genes. Quantification of the histone H3K9me3 modification around the TSS of the 11 660 'silenced' and 79 'escaping' genes according to ChIP-seq data from Chen *et al.*, (2020; GSE132446) (B). Heatmaps show the scaled tag density around the TSS from spermatogonia to diplotene spermatocytes: Sg 0 = undifferentiated spermatogonia; Sg A1 = type A1 spermatogonia; Sg B = type B spermatogonia; mpLe = mid-preleptotene spermatocytes; Le = leptotene spermatocytes; mZy = mid-zygotene spermatocytes; IZy = late zygotene spermatocytes; mPa = mid-pachytene spermatocytes; and Di = diplotene spermatocytes. The enrichment profiles of 'silenced' genes (blue) and 'escaping' genes (red) are represented using mean ± sem. Immunostaining for H3K9me3 and SYCP3 in testis sections from adult mice (C). DNA is stained with DAPI. Quantification of H3K9me3 intensity at each stage of spermatogenesis was performed (lower panel). Each line represents the average of several cells at a specific spermatogenic stage and the dashed line represents intensity in Sertoli cells (Ser). Legend is the same as in Fig. 1C. Co-labelling with SYCP3 and pPOL2 of spermatocyte chromosome spreads of WT, conditional KO mice for Setdb1 (Setdb1-/-), and conditional double KO for Atr and Atm (Atr fl/- Atm -/-) (D). Conditional inactivation of one Setb1 and one Atr allele was mediated by Ngn3-cre (Ngn3-cre, Setdb1 fl/- and Ngn3-cre, Atr fl/-, Atm -/-). All mice show weak pPOL2 staining at leptotene (Le) stage and an increase at early pachytene (ePa) stages. Heatmaps show the scaled expression profiles of genes located on the sex chromosomes in the unspliced scRNA-seq (scaled mean counts) and the EU-RNA-seq (scaled FPKM) datasets according to the scale bars (E). Clusters c1, c2, and c3 represent respectively genes escaping only MSCI, genes silenced during both EMLT and MSCI, and genes escaping EMLT but not MSCI. Heatmap representing the expression profiles of selected genes of interest for EMLT and for MSCI in the EU-RNA-seq (scaled FPKM) dataset (F). This dataset contains mono-exonic genes unlike the unspliced scRNA-seq dataset. The legends of heatmaps in panels (A), (E), and (F) are the same as for heatmaps in Fig. 1H.
before early pachynema. In both *Rad21L*⁻/⁻ and *Stag3*⁻/⁻, leptotene-like and zygotene-like spermatocytes displayed a low level of pPOL2, similar to that of WT mice (Fig. 3A and B). In *Rad21L* mutants, some spermatocytes resembling early pachytene stages presented a more intense staining for pPOL2 (Fig. 3B and C), albeit Rad21L mutation renders the staging of these cells uncertain.

Proper formation of AEs is mandatory to allow the synapsis of homologous chromosomes through completion of the SC. The HORMA domains protein HORMAD1 localises to unsynapsed chromosome axes and is removed soon after synapsis [26]. The disruption of *Hormad1* caused pairing defects and pachytene arrest. Leptotene and zygotene-like *Hormad1*⁻/⁻ spermatocytes were faintly stained for pPOL2, with a quantified intensity similar to WT (Fig. 3A and B). Zygotene-like and early pachytene-like mutant spermatocytes appeared more robustly stained for pPOL2 that WT cells (Fig. 3C). However, the precise identification of these stages is uncertain in *Hormad1* mutants in which synapsis signaling is defective. Of note, in WT early pachynema pPOL2 initially reappeared in highly localized areas emanating from the axes of the fully synapsed chromosomes (Supplementary Fig. S13). In *Hormad1*⁻/⁻ spermatocytes, the reappearance of pPOL2 commenced on both synapsed and unsynapsed axes. This indicates that local synapsis is not a prerequisite for the reinitiation of transcription.

These results indicate that establishment of the meiotic-specific chromatin structure does not drive EMLT. In addition, completion of chromosome synapsis is not essential for concluding EMLT and transcriptional reactivation.

## EMLT is independent of meiotic DSBs

As DSBs can be associated to transcriptional modifications in somatic cells, we speculated that in meiotic cells the formation of hundreds of DSBs might affect transcriptional activity. At the beginning of prophase I, DSBs are generated by the topoisomerase-like transesterase SPO11 at specific sites defined by the methyltransferase PRDM9 (PR/SET domain 9). DSBs are rapidly signaled by phosphorylation of H2AX (γH2AX) by the kinase ATM at leptotene stage. Interestingly, cells stained for γH2AX harbored low pPOL2 (Supplementary Fig. S14); this was obvious in early spermatocytes I and in intermediate and B spermatogonia that presented a weak but distinct γH2AX signal as previously reported [43]. To investigate if DSBs formation induces or maintains EMLT, we quantified pPOL2 in *Prdm9* and *Spo11* null spermatocytes (Fig. 4). In *Prdm9*⁻/⁻ mice, DSBs are catalysed but do not localize correctly [25, 44, 45]. In *Spo11*⁻/⁻ animals, programmed DSBs are not generated [46, 47]. In both cases, mutant spermatocytes arrest at zygotene/pachytene-like stage. Quantification of pPOL2 in section of *Prdm9*⁻/⁻ testes revealed that EMLT was unchanged as hardly any staining was detected in leptotene and zygotene spermatocytes as in WT cells (Fig. 4A and B). This result is in accordance with EMLT being present in dog, which lacks *Prdm9* (Supplementary Fig. S8A). EMLT was also retrieved similarly in chromosome spreads from *Spo11*⁻/⁻ spermatocytes (Fig. 4C and D). These data are consistent with a previous report [48]. Of note, in leptotene-like spermatocytes from *Spo11*⁻/⁻ mice, pPOL2 intensity is slightly reduced compared to WT (Supplementary Fig. S15). This might indicate that in absence

of DSB, transcription is even more repressed. Overall, we concluded that EMLT is not induced by programmed DSB.

Though *Prdm9*⁻/⁻ and *Spo11*⁻/⁻ mice are defective for DSBs localization and formation, respectively, in both mutants, γH2AX is still present, albeit at low level in *Spo11*⁻/⁻ spermatocytes. We thus decided to analyse the transcriptional activity in *Atm* null mutants. We used *Atm*⁻/⁻; *Spo11*⁺/⁻ mice as these progress up to the diplotene stage allowing the study of transcriptional arrest (EMLT) and transcriptional reactivation. In these mutants, γH2AX signaling of DSBs occurring at leptotene stage is absent [49]. At leptotene and zygotene stages pPOL2 intensity was equivalent in WT and *Atm*⁻/⁻; *Spo11*⁺/⁻ spermatocytes (Fig. 4C and D). Reactivation of transcription at pachytene stage also occurs timely (Fig. 4E and F). This result indicates that DSB signaling does not influence EMLT.

In meiocytes, DSBs are rapidly repaired by HR and when global transcription is hyperactivated, few DSBs remain on autosomes from pachynemas. Meiosis specific with OB domains (MEIOB) is essential for completing meiotic recombination [23]. To assess the dependence of EMLT on HR, we analysed *Meiob* null mutants, which exhibit persistent DSBs. *Meiob*⁻/⁻ spermatocytes showed a clear induction of EMLT, with minimal pPOL2 staining, similarly to WT leptonema and zygonema (Fig. 4C and D). In the absence of *Meiob*, prophase I arrested at early pachytene-like stage. The few mutant spermatocytes that reached this stage showed a mild elevation of pPOL2 signal, indicating that in the absence of DSB repair, transcription could be reactivated. Interestingly, pPOL2 reappeared as small patches in *Meiob*⁻/⁻ spermatocytes and these localized both in synapsed and unsynapsed chromosomal domains (Supplementary Fig. S13B). This confirms the observation from *Hormad1* mutant revealing that synapsis is not required to terminate EMLT and to resume transcription.

Since DSBs do not appear to affect EMLT, we conjectured the reverse: that transcription might shape meiotic DSBs. Consequently, we thus tested the hypothesis that an association between recombination sites and transcriptional silencing may exist. To evaluate this hypothesis, the distance of the ‘silenced’ genes and 1000 random intergenic regions to the nearest recombination site was computed based on DSB hotspot data defined by anti-DMC1 ssDNA sequencing [35]. The distance distribution of the genes or regions to the nearest hotspots was plotted in a boxplot according to their chromosomal location (Fig. 4G). The ‘silenced’ genes were closer to the recombination sites than the random intergenic regions both on autosomes and sex chromosomes. These results suggest a possible link between distance to recombination sites and EMLT with DSBs occurring preferentially in transcriptionally silent domains. Nevertheless, given that both DSBs and silencing events occur throughout the whole genome, it was challenging to further test this relationship. Indeed, a detailed examination of a high-resolution DSB map provided by *Spo11*-oligonucleotides [12], revealed that distribution of DSBs and that of silenced genes largely overlapped. We exemplified this using chromosome 17 that contains several ‘escaping’ genes including the *Prdm9* (Fig. 4H). Albeit *Prdm9* is indeed located between two DSB hotspots, the presence of actively transcribed genes in leptotene is rare and is not systematic between distant hotspots.

Altogether these results indicate that EMLT is independent of the induction, signaling and repair of DSBs though it may be related to the localization of programmed DSBs.
<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /><figcaption>**Figure 3.** Co-labelling with SYCP3 and pPOL2 on spermatocyte chromosome spreads of WT and KO mice *Rad21L-/-*, *Stag3-/-*, and *Hormad1-/-* (**A**). Quantification of pPOL2 intensity evidenced a weak signal at leptotene (Le) or leptotene-like (Le-L) stage (**B**). pPOL2 intensity is normalized to mitotic cells (100%, dashed line). Black bars represent mean ± sem. WT: *n* = 5; *Hormad1-/-*: *n* = 3; *Stag3-/-*: *n* = 3; and *Rad21L-/-*: *n* = 3. \* *P* < .05, **** *P* < .0001 (unpaired *t*-test). *In Hormad1-/-*, some early pachytene (ePa) spermatocytes are strongly stained for pPOL2 in comparison to WT and *Rad21L-/-* (**C**).</figcaption></figure>

## *Stra8* regulates EMLT induction

Given that EMLT occurs around meiotic entry, we analysed pPOL2 in germ cells mutant for the main regulators of meiotic initiation. *Stra8* is a key gene involved in mitotic to meiotic transition [50,51]. It is initially expressed in A spermatogonia following their differentiation and it is strongly expressed during pre-leptotene stage. STRA8 regulates the transcription of large sets of genes. Analysis of *Stra8* null mutant mice revealed the absence of the primary induction of EMLT. In fact, quantification of pPOL2 in testis sections of *Stra8*⁻/⁻ mice showed a high level of pPOL2 in intermediate and B spermatogonia and in pre-leptotene spermatocytes when compared to corresponding stages in WT (Fig. 5 and Supplementary Fig. S16 and S17A). Interestingly, measure of pPOL2 intensity in *Stra8*⁻/⁻ ovaries also showed a higher level of pPOL2 in leptotene-like oocytes (Supplementary Fig. S17B). MEIOC is another key factor that regulates meiosis initiation through modulation of post-transcriptional events. Contrarily to *Stra8*, ge-

netic depletion of *Meioc* did not modify EMLT (Fig. 5 and Supplementary Fig. S17A), indicating that simply impairing meiotic entry was insufficient to delay the transcriptional arrest. Thus, establishment of meiotic program through STRA8 is important for the correct temporal regulation of EMLT.

## RNAs are massively stabilized during EMLT

The combined duration of leptotene and zygotene stages is expected to be ~3 days. How do cells survive and progress through these stages with hardly any transcription is intriguing. Comparing the ‘spliced’ and ‘unspliced’ expression profiles, which reflect the presence of transcripts and nascent transcription, respectively, we observed that both decreased notably at meiotic entry (Fig. 6A). However, the decrease of ‘spliced’ RNAs was less pronounced indicating that some transcripts persisted. As an example, the *Rplp0* housekeeping gene displayed a 20-fold decrease in transcription while
<figure><img src="image_22.png" /><figcaption>**Figure 4.** WT and Prdm9-/- testis sections stained for SYCP3 (red) and pPOL2 (green) (A). Prophase I cells are identified thanks to SYCP3 and DNA is stained with DAPI. SgA = spermatogonia A; eZy = early zygotene; and Di = diplotene. Dotted white curbs delimit semiferous tubules. Scale bar = 10 µm. Line plot quantification of pPOL2 intensity in Prdm9 null mutants (Prdm9-/-, *n* = 2 mice) (B). For quantification, a line was traced randomly through nucleus and for each point along the line, intensity was quantified using ImageJ software (plot profile). The horizontal axis represents the diameter of the cells. Each line represents the average of pPOL2 intensity of several cells. Leptotene-like (Le-L, *n* = 16), zygotene-like (Zy-L, *n* = 38), and early-pachytene like (ePaL, *n* = 14) cells are less stained for pPOL2 than A spermatogonia (Sg A, *n* = 9). AU = arbitrary units. Immunofluorescence staining for SYCP3 and pPOL2 of spermatocyte chromosome spreads of WT and KO mice *Meiob-/-*, *Spo11-/-*, and double transgenic KO mice *Atm-/- Spo11+/-* (C). Quantification of pPOL2 intensity indicates that all mice show weak pPOL2 signal at leptotene (Le) stage (D). pPOL2 intensity is normalized to mitotic cells (100%, black dashed line). Black bars represent mean ± sem. WT: *n* = 5; *Meiob-/-*: *n* = 3; *Spo11-/-*: *n* = 3; and *Atm-/- Spo11+/-*: *n* = 3.
***P < .001, ****P < .0001 (unpaired t-test). Both WT and *Atm-/- Spo11+/-* mice show high pPOL2 staining at diplotene stage (Di), except for sex body (orange dotted line) (E). Quantification of pPOL2 intensity on spermatocyte chromosome spreads (F). Boxplot of the distance (log10) to the closest hotspot (in bp) for the 11 660 'silenced' genes and 1000 random intergenic regions according to their chromosomal location (autosomes in green and sex chromosomes in purple) (G). DSBs hotspot data as defined by anti-DMC1 ssDNA sequencing data in C57BL/6J mice was downloaded from GEO (GSE75419). Two-tailed Wilcoxon tests were used to compare the distance to the nearest hotspot in the different sets of genes. ****P < 0.0001.
Illustration of the distribution of DNA DSBs defined by mapping SPO11 oligonucleotides (Spo11 oligo, purple) and transcriptional changes between leptotene stage and spermatogonia (Ratio Le/Sg, black) along mouse chromosome 17 (H). Bottom image is a two Mb zoom centered on the 'escaping' gene *Prdm9*. Coordinate of SPO11-oligo were obtained from Lange *et al*. Transcriptional changes were calculated from scRNA-seq data (ratio Le/Diff Sg1).</figcaption></figure>
<figure><img src="image_23.png" /><figcaption>**Figure 5.** Co-immunofluorescence staining of pPOL2 with SYCP3 on testis sections from WT and *Stra8*-/- and *Meioc*-/- mice (A). Preleptotene (pLe) cells are indicated and DNA is stained with DAPI (panels (A) and (D)). Scale bar = 10 µm. Line plot quantification of pPOL2 intensity in WT (upper), *Stra8*-/- (middle), and *Meioc*-/- (lower) (B). The horizontal axis represents the diameter of the cells. (AU = arbitrary units, normalization was based on background level). Legend is as in Fig. 1B. The number of analysed cells is indicated in brackets for each cell type. Mean pPOL2 intensity within nuclei (C). Columns represent mean ± sem. In *Stra8*-/-, Sg Int/B, and pLe cells are more stained for pPOL2 than in WT and *Meioc*-/-, AU = arbitrary units. ****P < 0.0001 (ordinary one-way ANOVA, multiple comparisons). Co-labelling of pPOL2, γH2AX, and SYCP3 in *Stra8*-/- testes sections (D). pLe cells in *Stra8*-/- testes, stained for γH2AX and SYCP3, are not subjected to EMLT and show pPOL2 staining. Scale bar = 5 µm.</figcaption></figure>

its mRNA merely decreased two-fold during EMLT (i.e. in leptotene, unspliced mRNA equal to 4.3% versus spliced mRNA equal to 39% of the value in undifferentiated spermatogonia). We thus considered the ratio of ‘spliced’ to ‘unspliced’ expression levels to obtain a proxy of RNA stability. An increase of RNA stability was observed during EMLT for both ‘silenced’ and ‘escaping’ genes in scRNA-seq data (Fig. 6A). Similar results were observed using the EU-RNA-seq dataset (Supplementary Fig. S18). We hypothesized that

this stabilization may be involved in launching the meiotic program and thus focused on a list of genes highly expressed during meiotic entry (Fig. 6B and Supplementary Table S3); these were previously published genes expressed specifically from pre-meiotic cells and onwards [52] completed by five additional genes we systematically observed at meiotic entry in our transcriptomic analyses (*Fmr1*, *Ly6k*, *Sirt7*, *Taf4b*, and *Terb2*). These 91 genes were grouped based on their nascent transcription profile (set 1 = early transcribed genes;
<figure><img src="image_24.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_25.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_26.png" /><figcaption>**Figure 6.** Heatmaps and boxplots of the spliced (left) and unspliced (middle) expression profiles and the stability (right) of 'Silenced' (top) and 'Escaping' (bottom) genes (A), and of four sets of early meiotic genes (B). Set 1 = early transcribed genes; set 2 = genes transcribed during Le/Zy; set 3 = late transcribed genes; and set 4 = genes silenced during Le/Zy. The 'spliced' and 'unspliced' expression profiles reflect transcription and nascent transcription, respectively, while the 'stability' is the ratio of 'spliced' to 'unspliced' expression levels. In the heatmaps, each row represents a gene, and each column represents a specific spermatogenic cell population. For heatmaps, 'spliced' and 'unspliced' expression values of each gene were scaled between [0;1] using a zero-max scale (0 = 0; maximum value = 1), stability of each gene was scaled between [0;1] using a conventional scaling (minimum value = 0; maximum value = 1). Boxplots display the average counts per gene for the 'spliced' and 'unspliced' dataset, and stability per gene. Undiff Sg 0 = undifferentiated spermatogonia; Diff Sg = differentiated spermatogonia; Le = leptotene; Zy = zygotene; ePa = early pachytene spermatocytes; IPA = late pachytene; Di = diplotene spermatocytes; and MI = metaphase I spermatids. Gene expression and stability profiles of four representative genes from undifferentiated spermatogonia to metaphase I spermatocytes (C). Plots represent the scaled average counts per gene from spliced (left) and unspliced (middle) data, and the resulting stability (right). Each of the selected genes (*Dmc1*, *Prdm9*, *Meioc*, and *Hormad2*) belong to one of the four sets of meiotic genes presented in panel (B).</figcaption></figure>
set 2 = genes transcribed during Le/Zy; set 3 = late tran-
scribed genes; and set 4 = genes silenced during Le/Zy). RNA
stability during EMLT was also observed for the four sets of
genes. These observations were also confirmed in the bulk ap-
proach with sorted cells (Supplementary Fig. S19). Four well-
known meiotic genes (*Dmc1 Prdm9*, *Meioc*, and *Hormad2*),
representing each set, were used to exemplify this (Fig. 6C).
Although these displayed distinct nascent transcription pro-
files (unspliced RNA), they were robustly and simultaneously
expressed in early meiosis (spliced RNA), with their stabil-
ity sharply increasing in leptotene and zygotene cells. These
results suggest that the global stabilization of mRNA during
EMLT and meiotic entry may facilitate the synchronized rise
of meiotic gene expression.

Discussion

During the reproductive cycle, several transcriptional silenc-
ing events occur in germ cells and in the zygote [53–55]. In
this study, we characterized EMLT, a genome-wide silencing
phenomenon during early meiotic prophase I. EMLT was de-
pendent of the meiotic gatekeeper STRA8 but did not rely on
other key events of meiotic prophase I for its establishment.

Examples of genome-wide transcriptional arrest are un-
common and notably associated with gametogenesis. Such oc-
currences have been well-documented post-fertilization dur-
ing the early embryonic development and in primordial germ
cells. The tight packaging of chromatin in the spermatozoa
also induces a transcriptional silencing, though the cause is
evident in this context [56]. Here, we provide a detailed char-
acterization of the silencing event occurring at the time of
meiotic entry, adding new insights to its understanding. The
reason why germ cells are that frequently subjected to tran-
scriptional arrests remains unclear. One may consider that
among all cells, germ cells must transition between highly dif-
ferent states, justifying an extreme transcriptional dynamic.
Indeed, germ cells need to undergo various specifications: first
PGCs are committed to the germ lineage; then they shift from
a mitotic to a meiotic program; and later, post-meiotic male
germ cells undergo a massive cellular reorganization. Finally,
after the fusion of gamete genomes, the zygote must regain
totipotency. Interestingly, all these changes are accompanied
by major chromatin remodelling events, such as DNA methy-
lation erasure [57], meiotic recombination [58], histone to
protamine transition [59], or diploidization. Of note, methyla-
tion erasure occurs in PGCs, during the first embryonic cleav-
ages and at the time of meiotic entry [60]. A simple view may
accept that a quiet chromatin is a conductive environment
for such events, and that an active transcriptional machinery
could potentially interfere with these processes.

Recently, low transcription during leptonema and zy-
gonema has also been reported in a study proposing it may
reflect a promoter proximal paused state of POL2 [61]. An-
other recent study described a POL2 pausing in spermatogo-
nia [62]. It is therefore conceivable that the absence of POL2
detection on chromatin in our work merely reflects a paused
state.

EMLT occurring around the mitotic–meiotic switch is in-
triguing. Here we reported, through the analysis of several
meiotic mutants, that this transcriptional arrest occurred sim-
ilarly when the specific meiotic chromosomal architecture
or DSB program was impaired. Similarly, it has previously
been reported that unresolved DSBs or severe synaptic de-

fects do not alter the pattern of pPOL2 in leptotene- and
zygotene-like spermatocytes in various mutant mice [48]. Co-
hesins shape the chromosomal architecture and have been
proposed as key regulators of transcription [63–65]. The in-
volvement of specific cohesins during meiosis would be a ten-
tative hypothesis to explain a change in transcriptional activ-
ity. However, neither *Stag3* nor *Rad21L* depletion impacted
EMLT, despite the structure of meiotic chromosomes being
deeply altered in such mutants. On the other hand, transcrip-
tion has been proven to move cohesins [66, 67]. One may thus
speculate that a reverse relationship exists between EMLT and
cohesins. During early meiosis, the lack of transcription may
provide some stability to cohesins to initiate the axial-loop
structure and the formation of the axial element formation.
Another significant event occurring during early meiosis is the
formation and repair of hundreds of programmed DSBs. In
somatic cells, DNA damage repair has been reported to inter-
fere with transcription in many ways, likely because both phe-
nomena require an accessible chromatin [68]. Initially, a rather
straight hypothesis would have been that DSB patterning, for-
mation, signaling or repair might trigger a transcriptional ar-
rest. The analysis of *Prdm9*, *Spo11*, *Atm/Atr*, and *Meiob* mu-
tants contradicts such hypothesis. A reverse relationship, i.e.
that active transcription might interfere with the proper gener-
ation or repair of programmed DSBs, may then be considered.
In this line, it is worth quoting that DSB sites are marked by
H3K4me3, this histone modification is also the one retrieved
in the promoters of transcriptionally active genes. A specula-
tive assumption could be that sharing such a mark is risky and
may confuse the recruitment of the DSB forming machinery.
Therefore, a state of near-extinct transcription might favour
the proper localization of DSB in hotspots outside gene pro-
moters. This hypothesis gains support from our observation
that recombination occurred preferentially in transcription-
ally silent regions and is in agreements with a publication indi-
cating that DSBs occur in untranscribed genomic regions [61].
In the same line, it was also recently proposed that paused
POL2 sites in spermatogonia correlate with those of DSBs in-
duced by SPO11 in spermatocytes [62].

To test such hypotheses, it would be necessary to manip-
ulate EMLT and this implies understanding its mechanisms
or trigger. Here we report that EMLT is significantly delayed
in absence of *Stra8*. STRA8 controls the expression of the
near complete meiotic gene network. Specifically, it has re-
cently been shown to amplify a pre-existing meiotic program
[10]. This aligns with the concept that STRA8 might regulate
a meiotic factor that causes EMLT. In the absence of *Stra8*,
such a factor would be expressed at low levels, thus delay-
ing EMLT. Unfortunately, identifying this factor within the
thousands of genes that compose the STRA8-dependent mei-
otic program remains a challenge. The mechanism causing
EMLT also remains evasive. We report here that this mech-
anism differs from MSCI that is regulated by DSB signal-
ing, asynapsis and the SETDB1 histone methylase. However,
the absence of recruitment of POL2 and pPOL2 is a com-
mon feature shared by MSCI and EMLT. This reinforces the
view that MSCI might be a gonosome-specific prolongation
of a wider phenomenon, EMLT [19]. In this line, it could
be conceived that the histone mark repressing transcription
during MSCI would also be shared with EMLT. H3K9me3 is
the mark deposited by SETDB1 involved in instructing MSCI
[13], though its exact role has recently been debated [16]. In-
terestingly, H3K9me3 is also present on silent chromosomes
during early prophase I, making it a likely candidate to in-
duce EMLT. In this scenario, EMLT would rely on a different
methylase which may explain that few genes on sex chromo-
somes are differentially regulated during EMLT and MSCI. Of
note, H3K9me3 may not be the sole signal involved and ad-
ditional chromatin modifications may be involved. Notably,
mono-methylation of H3K4 may also be correlated to EMLT
[19, 48]. Furthermore, other differences, especially regarding
POL2 dynamics, exist between EMLT and MSCI.

Gene expression can be considered as the outcome of tran-
scription and mRNA decay rates. Our work also revealed that
EMLT is accompanied by a wide event of RNA stabilization.
This stabilization event likely buffered the decrease in tran-
scription at meiotic onset. This aligns with a report indicating
that in human cell lines, global transcriptional shutdown leads
to stabilization of nearly all of transcripts through inhibition
of the mRNA degradation machinery [69]. Whether a simi-
lar relationship between low transcription and RNA stability
exist in germ cells is a point that warrants future studies. Strik-
ingly, when assessing the transcription of genes specifically ex-
pressed at the onset of meiosis, we observed that many (84/91)
were predominantly transcribed at other stages. For most of
them (sets 1, 3, and 4), their transcription decreased at the
time of meiotic entry. Thus, the increased expression of these
meiotic genes is mainly due to changes in RNA stability. Mei-
otic licensing depends upon *Dazl* that is expressed early dur-
ing fetal life, yet male germ cells do not initiate meiosis before
post-natal life [70]. This allows speculation that part of the
meiotic program might be set up early, and that the decision
to actually initiate meiosis requires the stabilization of RNA to
allow meiotic genes transcribed at low levels to reach a suffi-
cient level. The second effect of this massive RNA stabilization
we observed is likely to synchronize various 'meiotic subpro-
grams' to provide a timely expression of all genes needed in
early meiotic prophase I. Such a hypothesis positions mRNA
stabilization as a key player orchestrating meiotic entry. In
*Schizosaccharomyces pombe*, the proper timing of meiosis is
controlled by the sequestration of the RNA-binding protein
Mmi1, that triggers the degradation of some meiotic RNA
during mitotic growth [71]. In *Caenorhabditis elegans*, mei-
otic entry is partly controlled by a poly (A)-polymerase, GLD-
2, that increases mRNA stability [72, 73]. Thus, a change of
RNA stability might be an ancestral mechanism controlling
the mitotic-meiotic switch that has yet poorly been investi-
gated in mammals.

In conclusion, our study provides an in-depth description
of the genome-wide silencing event reported to occur during
leptotene and zygotene stages. We quantified this event and
proved it initiates prior to meiotic entry and is regulated by
the meiotic gatekeeper, STRA8. This silencing and the accom-
panying RNA stabilization are key to ensure a robust switch
from the mitotic to the meiotic program. Future research will
have to identify the specific mechanisms responsible for EMLT
and through their manipulation it is expected that the reasons
for such a major genome silencing will be unveiled.

## Acknowledgements

The authors sincerely thank Dr J.M. Turner for helpful dis-
cussion, advices, and providing the samples from Setb1 and
Atr/Atm mutant mice. We are indebted to Dr F. Baudat and Dr
B. de Massy and Dr C. Grey for the generous provision of tes-
ticular samples from Spo11, Prdm9, and mice. Flow cytometry

and cell sorting were carried out at the iRCM Flow Cytometry
Shared Resource. We also thank the team within the animal
housing facility at the iRCM, particularly V. Neuville and the
Centre National de Recherche en Génomique Humaine for
sequencing, D. Moison and S. Tourpin for her technical assis-
tance and A. Gouret for her secretarial assistance.

Author contributions: L.B. designed and performed most of the molecular, cell, and animal experiments with V.C., M-J.G., S.M., E.L., and I.D. E.C.S.P. and A.R. performed the single-cell transcriptome analysis and nascent RNA analysis. L.B., A.T., and A.M.P. helped design the animal study. A.R., E.M., A.T., and A.M.P. monitored the study. F.C. and G.L. conceived the study, interpreted the experiments, and wrote the manuscript with inputs from the other authors.

## Supplementary data

Supplementary data is available at NAR online.

## Conflict of interest

All authors declare that they have no conflicts of interest.

## Funding

This research was supported by the Agence Nationale
de la Recherche (ANR-18-CE14-0038 and ANR-20-CE14-
0022) and by the Ministerio de Ciencia e Innovación
(PID2020-120326RB-I00) and Junta de Castilla y León
(Unidad de Investigación Consolidada 066, CSI148P20 and
CSI017P23). Funding to pay the Open Access publication
charges for this article was provided by University dotation
or research contract.

## Data availability

The data underlying this article will be shared upon request to the corresponding authors. The sequencing datasets have been deposited in the GEO with accession number GSE261062.

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