DNA methylation dynamics during spermatogenesis in ruminants

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DNA methylation dynamics during spermatogenesis in ruminants

Aurélie Allais-Bonnet, Sébastien Messiaen, Marjolaine André, Béatrice Mandon-Pepin, Gabriel Livera, Laurent Schibler, Hélène Jammes, Eric

Pailhoux, Hélène Kiefer, Maëlle Pannetier

To cite this version:

Aurélie Allais-Bonnet, Sébastien Messiaen, Marjolaine André, Béatrice Mandon-Pepin, Gabriel Livera, et al.. DNA methylation dynamics during spermatogenesis in ruminants. Mammalian Reproductive Gordon Research, Jul 2018, Lucca, Italy. 2018. �hal-02790316�

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DNA methylation dynamics during spermatogenesis in ruminants

Aurélie Allais-Bonnet

^1,2

, Sebastien Messiaen

³

, Marjolaine André

¹

, Béatrice Mandon-Pépin

¹

, Gabriel Livera

³

, Laurent Schibler

²

, Hélène Jammes

¹

, Eric Pailhoux

¹

, Hélène Kiefer

¹

and Maëlle Pannetier

¹

1 UMR BDR, INRA, ENVA, Université Paris-Saclay, 78350, Jouy-en-josas, France.

2 ALLICE, Département R&D, 75012 Paris, France.

3 Laboratoire de Développement des Gonades, UMR967, INSERM-CEA-Universités Paris Diderot et Paris-Saclay, 92265 Fontenay-aux-Roses, France.

2017-2019

INTRODUCTION

1. DNA methylation in male germ line

RESULTS

OBJECTIVES

 5mC detection during spermatogenesis

PERSPECTIVES

 Spermatogenic cells purification

 Optimisation of the flow cytometry-based method (Hoechst-FACS)

 Caprine and bovine testis (X4)

 Enrichment analysis

 DNA methylation analyses of purified spermatogenic cells

 RRBS analysis

 RNA sequencing

[1] Perrier et al., BMC Genomic. 2018

Determination and comparison of DNA methylation dynamics in bovine and caprine germ cells during spermatogenesis.

1. DNA methylation marks observation  IF 2. Germ cells purification  flow cytometry 3. DNA methylation analyses  RRBS

 DNA methylation of cytosines is a critical epigenetic modification in mammals that plays crucial roles in transcriptional regulation, chromatin remodelling and genomic imprinting. Dynamic erasure and reestablishment of DNA methylation marks are required for spermatogenesis and the normal function of mature sperm.

 DNA methylation is catalysed by DNA methyltransferase enzymes (DNMT) providing either maintenance (DNMT1) or de novo (DNMT3A/B/L) DNA methylation processes.



3. DNA Methylation analysis on

purified spermatogenic cells

 DNMT3s expression by real time PCR (RT-qPCR)

 Reduced-Representation Bisulfite Sequencing

(RRBS)

Adult stage

2. Spermatogenic cells purification from seminiferous tubules:

 flow cytometry-based method (Hoechst-FACS)

65-70% 40-50%



1. DNA methylation mark detection in testis

 5-methylcytosine (5-mC) Immunofluorescence

Spermatocytes I (pachytène)

Round Spermatids

 In caprine testis , DNA methylation is highly detected in germ cells of all stages of spermatogenesis (VASA+).

 Differences in DNA methylation dynamics in spermatogenic cells between this two species of ruminants.

5mC / VASA (DDX4) double immunodetection

 What are the differentially methylated regions in testis?

2. Male germ cells purification

3. DNA methylation analysis in spermatogenic cells

 Flow cytometry isolation

- Collection of fresh testicular tissue - Cellular dissociation

- Cell staining (+Ho +PI)

- Fluorescence – Activated Cell Sorting

SP = Spgonia; SC I= Spcyte I SC II= Spcyte II; ST=Sptid

0,00 0,10 0,20 0,30 0,40 0,50 0,60 0,70 0,80 0,90 1,00

Testis Spgonia Spcyte I Spcyte II Sptid

rELATIVE EXPRESSION

PRM1

0,00 0,10 0,20 0,30 0,40 0,50 0,60 0,70 0,80 0,90 1,00

Relative expression

SYCP3

0,00 0,50 1,00 1,50 2,00 2,50

Relative expression

DDX4

0,00 0,10 0,20 0,30 0,40 0,50 0,60 0,70 0,80 0,90 1,00

Relative expression

SOX9

SG SC I

SC II ST

Specific markers of spermatogenic cells analyzed in RT-qPCR

50µm 50µm

 DNA methylation dynamics during spermatogenesis has been previously described in mice and humans. Nothing is known in productive livestock.

 Recent study from our laboratory pointed DNA undermethylation of bull spermatozoa compared to other mammals such as humans, mice, sheep or goats [1]

 DNA methylation description in ruminants would determine methylation state of male germ line during spermatogenesis and could be helpful to determine its impact on male fertily in productive livestock.

 DNMT3s expression quantification – De novo methylation

0,00 0,50 1,00 1,50 2,00 2,50

Relative expression

DNMT3A

0,00 0,20 0,40 0,60 0,80 1,00 1,20 1,40

Relative expression

DNMT3B

0,00 20,00 40,00 60,00 80,00 100,00 120,00 140,00

Relative expression

DNMT3L

 DNMT3A/B = de novo DNA methyltransferase

 DNMT3L = catalytically inactive regulatory factor of DNMT3A and DNMT3B. Specifically expressed in the germline of both sexes.

 Expression level of DNMT3s fluctuates within stages of spermatogenic cells indicating methylation variations during spermatogenesis progression.

Quantification of DNMT3s by RT-qPCR on FACS- purified spermatogenic cells.



Sequencing

 RRBS analysis – Protocol (Gu et al., 2011)



 SYCP3 and PRM1 expression seems to be higher in Spermatocyte I and Spermatid cells respectively indicating a specific enrichment of these spermatogenic cells in each fraction.

 DDX4 is expressed at all stages of spermatogenesis and the quantification of SOX9 expression shows that purified cells are mostly germ cells.

Spermatocytes I (pachytène)

Round Spermatids

 In bovine testis, DNA methylation is clearly detected in Spermatogonia and Spermatocyte I nuclei, while low to negative signal is observed at later stages.

 Caprine testis

IF = high 5mC staining at all stages of spermatogenesis

 Stability in DNA methylation ?

RT-qPCR = DNMT3s expression variation

 New events of DNA methylation

 Specific regions ?

 Bovine testis

IF = 5mC staining only in Spgonia and Spcyte I

 Strong variation in DNA methylation ?

 Studies on purified germ cell fractions are highly required,

CONCLUSION

Repeated Sequences MspI cutoff

frequency

CpG rich regions

Fragment size CpG deserts