Polycomb protein SCML2 mediates paternal epigenetic inheritance through sperm chromatin

Abstract Sperm chromatin retains small amounts of histones, and chromatin states of sperm mirror gene expression programs of the next generation. However, it remains largely unknown how paternal epigenetic information is transmitted through sperm chromatin. Here, we present a novel mouse model of paternal epigenetic inheritance, in which deposition of Polycomb repressive complex 2 (PRC2) mediated-repressive H3K27me3 is attenuated in the paternal germline. By applying modified methods of assisted reproductive technology using testicular sperm, we rescued infertility of mice missing Polycomb protein SCML2, which regulates germline gene expression by establishing H3K27me3 on bivalent promoters with other active marks H3K4me2/3. We profiled epigenomic states (H3K27me3 and H3K4me3) of testicular sperm and epididymal sperm, demonstrating that the epididymal pattern of the sperm epigenome is already established in testicular sperm and that SCML2 is required for this process. In F1 males of X-linked Scml2-knockout mice, which have a wild-type genotype, gene expression is dysregulated in the male germline during spermiogenesis. These dysregulated genes are targets of SCML2-mediated H3K27me3 in F0 sperm. Further, dysregulation of gene expression was observed in the mutant-derived wild-type F1 preimplantation embryos. Together, we present functional evidence that the classic epigenetic regulator Polycomb mediates paternal epigenetic inheritance through sperm chromatin.


INTRODUCTION
Emerging studies have established that the germline transmits epigenetic information to the next generation. In the male germline, paternal epigenetic states are subject to environmental and metabolic perturbation, carried through compacted sperm and impacting gene expression and disease risks in the next generation (1)(2)(3)(4)(5). In many cases, paternal epigenetic states have an intergenerational impact and ar e largely r eprogrammed in the germline, though the extent of tr ansgener ational inheritance across multiple genera tions remains deba ted ( 6 , 7 ). Se v eral mechanisms are known to be responsible for intergenerational transmission of epigenetic states, including histone modifications on retained histones, DNA methylation, and small non-coding RN A (8)(9)(10)(11). In particular, w hile the vast majority of histones are replaced with protamine in spermiogenesis, a small portion of histones ar e r etained on gene regulatory elements in sperm; the chromatin states of sperm mirror the gene expression patterns of the next generation (12)(13)(14)(15)(16)(17)(18)(19).
Howe v er, the functions of sperm chromatin remain largely undetermined.
A prominent epigenetic signature of the mammalian germline is bivalent genomic domains, characterized by concomitant enrichment in r epr essi v e Poly comb-mediated trimethylation of histone H3 at lysine 27 (H3K27me3) and acti v e di / trimethyla tion of histone H3 a t lysine 4 (H3K4me2 / 3) marks on gene promoters, which persists in mature sperm ( 12 , 13 , 15 , 20-22 ). Bivalent domains r epr esent molecular hallmarks of developmental potential in embryonic stem cells (ESC) (23)(24)(25). Thus, in the germline, bivalent domains are considered to be mediators of epigenetic inheritance to the next genera tion (26)(27)(28). In la te sperma togenesis, bivalent domains became prevalent, covering thousands of genes that are activated later in the next generation (27)(28)(29). Of note, H3K4me3 is transmitted from sperm to embryos and is associated with embryonic gene expression ( 30 ). Ov ere xpression of H3K4me2 demethylase KDM1A in the germline perturbs inter-and tr ansgener ational inheritance of paternal epigenetic states and offspring health ( 30 , 31 ), but its effect is independent of bivalent domains ( 32 ). On the other hand, paternal deletion of H3K27me3 demethylase KDM6A leads to epigenetic inheritance and increases cancer susceptibility of the next generation ( 33 ). Despite this progress, the function of H3K27me3 remains to be tested in paternal epigenetic inheritance due to the lack of mouse models, in which H3K27me3 is a ttenua ted in the male germline.
Polycomb group proteins suppress non-lineage specific genes and define cellular identity during de v elopment ( 34 , 35 ). Mammalian Polycomb proteins comprise two functionally related major comple xes --Poly comb repressi v e complex 1 (PRC1) and PRC2 --tha t ca talyze monoubiquitination of histone H2A at lysine 119 (H2AK119ub) and H3K27me3, respecti v ely ( 36 ). We identified a germlinespecific Polycomb protein, SCML2, as a critical regulator of germline transcriptomes in the later stages of spermatogenesis (37)(38)(39)(40). SCML2 interacts with both PRC1 and PRC2 and coordinates their activities to establish PRC2-mediated H3K27me3 and the bivalent domains in late spermatogenesis ( 29 , 37 ). SCML2 binding sites in spermatogonia predict the sites of H3K27me3 deposition in late spermatogenesis; loss of SCML2 resulted in H3K27me3 depletion and defecti v e bi valent domains ( 29 ). Furthermore, Scml2 -knock out ( Scml2 -KO) mice were infertile, and Scml2 -KO epididymal sperm have abnormal shape ( 37 ), with midpieces tightly associated with sperm nuclei ( 29 ). The function of H3K27me3 in paternal epigenetic inheritance could not be determined in our previous studies ( 29 , 37 ). In this study, by a ppl ying modified methods of assisted reproducti v e tech-nology (ART) using testicular sperm, w e w ere able to rescue the male infertility of Scml2 -KO mice. We performed nati v e ChIP-seq of H3K27me3 and H3K4me3 in testicular sperm and epididymal sperm, demonstrating that the epidid ymal pa ttern of sperm epigenome is alread y established in testicular sperm and that SCML2 is r equir ed for this process. Here, we determine the paternal epigenetic inheritance mediated by SCML2 through sperm chromatin.

Animals
Mice were maintained and used according to the guidelines of the Institutional Animal Care and Use Committee (protocol no. IACUC2018-0040) at Cincinnati Children's Hospital Medical Center and (r efer ence number: A29-24) Yamanashi Uni v ersity. The founder Scml2 + /female was generated in the C57BL / 6 background using zinc finger nuclease technology ( 37 ). Scml2 + /females were maintained by mating with C57BL / 6 male mice. Ther efor e, Scml2 -KO males ( Scml2 -/ y ) have a pure C57BL / 6 background. Through mating between wild-type C57BL / 6 males and Scml2 + / − females, Scml2 -KO (-/ y) male mice were born at expected ratios according to Mendel's Law. For the genotyping of Scml2 -KO mice, PCR was carried out using specific primer sets ( 37 ). Since Scml2 -KO male mice showed infertility with abnormal sperm morphology, F1 generations from littermate wild-type and Scml2 -KO males were generated using ART (intracytoplasmic sperm injection (ICSI) and round spermatids injection (ROSI)). To generate F2 mice, single F1 male progenies were continuously housed with 1to-3 C57BL / 6 females, and litters were weaned at 3 weeks of age.

Collection of testicular spermatozoa and round spermatids for ICSI and ROSI
To obtain testicular spermatozoa and round spermatids, a single cell suspension from whole adult testis (wild-type and Scml2 -KO) was pr epar ed as described ( 29 ). Briefly, individual testes from wild-type and Scml2 -KO mice at 8-12 weeks of age were dissected with fine forceps using sterile procedures in a 35-mm Petri dish containing 1 × Enriched Krebs-Ringer bicarbonate (EKRB, containing 2.12 g / l sodium bicarbonate, Sigma) buffer, supplemented with 1 × GlutaMax (Thermo Fisher Scientific), 1 × nonessential amino acids (Thermo Fisher Scientific), 1 × essential amino acid (Thermo Fisher Scientific), 1.3 mM calcium chloride, 1.2 mM magnesium sulfate and 1 × penicillinstreptomycin (Thermo Fisher Scientific). After removing tunica albuginea membrane and unraveling seminiferous tub ules, tub ules were digested with collagenase (5 mg / ml, Sigma) at 35 • C for 20 min with gentle pipetting e v ery 5 min and then centrifuged at 188 xg for 5 min. The pellets were washed with the 1 × EKRB buffer and then digested with trypsin (2.5 mg / ml, Sigma) at 35 • C for 15 min. After incubation, Soybean trypsin inhibitor (50 g / ml, Sigma) was added to neutralize the trypsin. Cells wer e filter ed with a 40m strainer and resuspended with 1 ml of CELLBANKER 1 (Zenogen Pharma testicular spermatozoa and round spermatids were selected under the microscope based on their morphological features. These sperm nuclei were detached from midpieces after cryopreservation, prior to ICSI. This was not possible in our pre vious ICSI e xperiments using Scml2 -KO fresh epidid ymal sperm, since epidid ymal sperm were surrounded by their midpieces that were tightly associated with nuclei ( 29 ). We were not able to dissociate midpieces from sperm nuclei, which might hav e pre v ented ICSI from wor king in our previous study ( 29 ).

ICSI and ROSI
Oocytes and cumulus cells complexes were obtained from BDF1 female mice (SLC, Shizuoka, Japan) for Figure 1 and Supplementary Table S1 and from C57BL / 6 female (SLC, Shizuoka, Japan) for Figure 7 and Supplementary  Table S2, which had been injected with 7.5 IU of equine chorionic gonadotropin (ASKA Pharmaceutical Co., Ltd, Tok yo , Japan) and human chorionic gonadotropin (ASKA Pharmaceutical). Obtained oocytes and cumulus cells complex es wer e tr eated with hyaluronidase for 10 min; then the oocytes wer e r etrie v ed. The cryopreserv ed testicular pellet w as w ashed two times with D-MEM supplemented with 10% fetal bovine serum. After washing, the pellet was transferred to 10% PVP containing HEPES-buffered CZB-medium (CZB-HEPES) ( 41 ). Prior to cytosolic injection, zona pellucida and cytosolic membrane were disrupted with a piezo dri v e micromanipulator (Prime Tech Ltd, Ibaraki, Japan). Round spermatids or testicular spermatozoa were injected into the oocytes in CZB-HEPES. Ten min after injection, oocytes were washed and cultured in CZB-HEPES for 10 min. To obtain higher activation rate, 10 min after culturing in CZB-HEPES, round spermatidinjected oocytes as well as spermatozoa-injected oocytes were subjected to activ ation b y culturing in Ca 2+ -free CZB-HEPES containing 5 mM SrCl 2 for 1-2 h.

Embryo culture and transfer
ICSI and ROSI-deri v ed embryos wer e cultur ed in droplets of CZB-HEPES under paraffin oil in a 5% CO 2 atmosphere at 37.5 • C, until the desired de v elopmental stage. To obtain full-term li v e offsprings, on the f ollowing da y of ICSI and ROSI, the embryos that reached the two-cell stage wer e transferr ed into the oviducts of pseudopregnant female ICR mice at 0.5 days post-coitum (dpc), which had mated with a vasectomized ICR male overnight on the day bef ore embry o transfer. At 18.5 dpc, the pups were obtained by the caesarean section (though some ones were deli v ered by natural parturition) and nursed by foster mothers. For the RNA-seq experiments in Figure 7 , ICSI-deri v ed embryos were kept in culturing for 4 days until they became blastocysts.

RNA-seq
Total RNA from the li v er tissue was extracted with TRIzol reagent (Thermo Fisher Scientific) and purified using an RNeasy Plus Mini Kit (QIAGEN) with genomic DNA elimination according to the manufacturer's instructions. RNA-seq library preparation was carried out using a TruSeq Stranded mRNA Library Prep Kit (Illumina), following the manufacturer's instructions. For the preparation of the sperm RNA-seq library, we extracted total RNA from the swim-up separated motile sperm from the cauda epididymis using an RNeasy Plus Mini Kit (QIA-GEN) with genomic DNA elimination according to the manufacturer's instructions. cDNA synthesis and preamplification were performed with total RNA using a SMART-Seq v4 Ultra Low Input RNA Kit and an Advantage 2 PCR Kit (Clontech), respecti v el y. Preamplified cDN As were supplied for RNA-seq library preparation with Nextera XT Library Prep Kit (Illumina). Indexed libraries were pooled and sequenced using an Illumina HiSeq-4000 or NextSeq-500 sequencer (single-end, 75 bp). Two independent biological replicates were generated for each sample. The protocol for the construction of the embryo total RNA-seq libraries was adapted from a previous report with minor modifications ( 42 ). Briefly, the ICSI-deri v ed two-cell and blastocyst embryos sired by wild-type control (Ctrl) and Scml2 -KO sperm wer e tr eated with Acidic Tyrode's solution (Sigma) containing 0.5% polyvinylpyrrolidone and 50 mM NaCl to remove the zona pellucida (ZP) from embryos before sample collection, and then washed with PBS containing 0.2% polyvinyl alcohol. Pooled 30 each of ZP-free two-cells per replicate, and e v ery single blastocyst in Ctrl-F1 and Scml2 -KO-F1 embryos were lysed in 1 × Lysis Buffer containing RNase inhibitor (0.2 IU / l, from SMART-Seq Stranded Kit, Clontech), directly and stored at -80 • C until subsequent libr ary prepar ation. For PCR-based sexing, the lysates of e v ery single b lastocyst were boiled at 95 • C for 10 min and subjected to PCR with KOD One polymerase (TOYOBO) and a specific primer set: Zfy1 Fw (5 -GA CTAGA CATGTCTTAA CAT CTGT CC-3 ) and Zfy1 Rv (5 -CCTA TTGCA TGGACA GCA GCTTATG-3 ). PCR was carried out using the following conditions: 95 • C f or 2 min, f ollowed by 40 cycles each of 98 • C for 10 s and 68 • C for 30 s, on a ProFlex PCR System (Thermo Fisher Scientific). PCR products were visualized with electrophoresis on a 2% TAE agarose gel, and the genetic sex of each blastocyst was determined by the presence of PCR products. According to the manufacturer's instructions, the total RNA-seq libraries of pooled two-cell and single XY blastocyst embryos wer e pr epar ed using SMART-Seq Stranded Kit (Clontech). RNAs were randomly sheared by hea ting a t 85 • C for 6 min and subjected to re v erse tr anscription with r andom hexamers and PCR amplification. Ribosomal fragments were depleted from each cDN A sample with scZa pR and scR-Probes. Indexed total RNA-seq libraries were enriched through a second PCR amplification and sequenced using an Illumina HiSeqX sequencer (paired-end, 150 bp).

RNA-seq analysis
RN A-seq anal ysis was performed as described previously ( 43 ). Raw single-end RNA-seq reads from F1-li v er, F1sperm and F2-sperm samples were aligned to the mouse genome (GRCm38 / mm10) using Hisat2 version 2.1.0 ( 44 ) with default settings. Alternati v ely, raw paired-end RNA-seq reads from two-cell and blastocyst embryos sired by Ctrl and Scml2 -KO sperm, were aligned to mouse genome (GRCm38 / mm10) using STAR aligner version 2.5.3a ( 45 ) with following options, --twopassMode Basic; --outSAMtype BAM SortedByCoordinate; --outFilterType BySJout; --outFilterMultimapNmax 1; --winAnchorMultimapNmax 1; --alignSJoverhangMin 8; --alignSJDBoverhangMin 1; --outFilterMismatchNmax 1; --outFilterMismatchNoverReadLmax 0.04; --alignIntr onMin 20; --alignIntr onMax 1000000; --alignMatesGapMax 1000000 for unique alignments. To visualize read enrichments over r epr esentati v e genomic loci, coverage files in TDF and bigWig formats were created from sorted BAM files using the IGVTools count function (Broad Institute) and bamCoverage program as implemented in deepTools (version 3.1.3) ( 46 ). Figures of continuous tag counts over selected genomic intervals were created in the IGV br owser (Br oad Institute). For in silico genotyping of each single blastocyst, we manually checked the cover age tr acks of RNA-seq reads on Y chromosome, and then excluded dataset (XX blastocysts) in which we observed almost no aligned reads to Y chromosome. To quantify uniquely aligned reads on respecti v e annotated gene transcript and repetiti v e loci, the featureCounts function, part of the Subread package ( 33 ), was used. The RPKM expression levels for each transcript were calculated using StringTie version 1.3.4 ( 47 ). Pearson correla tion coef ficients between biological replica tes of RNA-seq profiles were calculated using SeqMonk (Babraham Bioinformatics). The gene expression matrix of RNA-seq datasets was applied to bidimensional PCA with R programming. Each sample score from the covariance matrix was plotted in the two eigenvectors PC1 and PC2 with ggplot2 ( https://github.com/tidyverse/ggplot2 ). To detect differ entially expr essed genes between two biological samples, a read count output file was input to the DESeq2 package version 1.16.1 ( 48 ); program functions DESeqDa taSetFromMa trix and DESeq were then used to compare each gene's expression le v el between two biolo gical samples. Differentiall y expr essed genes wer e identified through two criteria: (i) ≥2-fold change and (ii) binominal tests ( P adj < 0.01; P values were adjusted for multiple testing using the Benjamini-Hochberg method, or P < 0.01). To perform gene ontolo gy anal ysis of gene sets, differ entially expr essed between two biological samples, the functional annotation clustering tool in DAVID version 6.8 ( 49 ) and Enrichr ( 50 ) were used, and a background of all mouse genes was a pplied. Biolo gical process term groups with a P < 0.05 (modified Fisher's exact test) were considered significant. Further analysis was performed with R (version 3.4.0) and visualized as heat maps using Morpheus ( https://software .broadinstitute .org/morpheus , Broad Institute).

Collection of sperm from the testis and cauda epididymis for native ChIP-seq
For testicular sperm collection, tunica albuginea were removed, and seminiferous tubules were physically dissociated with tweezers. Tubules were further dissociated by 10 ml of EKRB Buffer, supplemented with 400 g / ml Collagenase (Worthington Biochemical Corp., NJ), 80 g / ml Hyaluronidase (Sigma), and 1.6 g / ml DNase (Sigma), at 35 • C for 10 min. Tubules were then centrifuged at 100 g at room temperature for 5 min. Then, the supernatant fraction was further centrifuged at 200 g at room temperature for 5 min. This supernatant fraction was filtered with a 25m mesh and centrifuged at 2500 rpm at room temperature for 5min. The pellet was used as testicular sperm for nati v e ChIP-seq.
For epididymal sperm collection, cauda epididymides wer e transferr ed in a 5% CO 2 -equillibrated M2 medium (Sigma). Each epididymis was thoroughly pierced with a 27G needle and incubated at 37 • C in a 5% CO 2 condition for 30 min. M2 medium containing the swim-up sperm was collected carefully and filtered with a 25-m mesh. Filtered sperms were centrifuged at 200 g at room temperature for 5 min, and the supernatant was further centrifuged at 2500 rpm at room temperature for 5min. The pellet was used as epididymal sperm for nati v e ChIP-seq.

Sperm native ChIP-seq
Sperm nati v e ChIP-seq was perf ormed by f ollowing a previously published protocol ( 51 ) with minor modifications. Sperm (2.6-4.9 × 10 5 cells per sample) were suspended in PBS and treated with 50 mM DTT a t room tempera ture for 2 h. DTT was quenched by 100 mM N -ethylmaleimide. After washing with PBS, sperm were treated with a buffer, containing 15 mM Tris-HCl (pH 7.5), 60 mM KCl, 5 mM MgCl 2 , 0.1 mM EGTA, 1% NP-40, and 1% sodium deoxycholate, on ice for 10 min. MNase treatment was performed using detergent-treated sperms with 6000 gel units of MNase (New England Biolabs, MA) per sample at 37 • C for 5 min and was immediately inhibited by 5 mM EDTA. After MNase treatment, 10% of total chromatin was separated and used as an input sample. Sperm chromatin was first pr e-clear ed with protein A / G (1:1) Dynabeads (Life Technologies, CA), then reacted with 1 l of anti-H3K27me3 antibody (C15410195, Diagenode, Belgium) or anti-H3K4me3 antibody (39155, Acti v e Motif, CA) at 4 • C overnight with gentle rotation. For antibody-beads conjugation, protein A / G Dynabeads were added, and samples were incubated at 4 • C for 4 h with gentle rotation. After incubation, to remove non-specific bound chromatin, samples were washed once with a low-salt washing buffer, containing 50 mM Tris-HCl (pH 7.5), 10 mM EDTA and 75 mM NaCl, and twice with a high-salt washing buffer, containing 50 mM Tris-HCl (pH 7.5), 10 mM EDTA and 125 mM NaCl. ChIP sample was then eluted with 1% SDS / PBS, treated with 200 g / ml of RNase A (Thermo Fisher Scientific) at 37 • C for 30 min and further treated with 200 g / ml of Pr oteinase K (Invitr o gen, CA) at 55 • C overnight. Finall y, DNA was purified with phenol / chloroform / isoamyl alcohol and precipitated with ethanol.
ChIP-seq libr ary prepar ation was carried out using a NEBNext Ultra II DNA Library Prep Kit (New England Biolabs) according to the manufacturers' instructions. Index ed libraries wer e pooled and sequenced using an Illumina Hiseq X sequencer (paired-end, 150 bp). Two independent biological replicates were generated for each sample. For the analysis of Figures 2 , 4 , 5 and Supplementary Figure S3, we analyzed representati v e data sets (all data for rep1 except for wild-type epididymal sperm H3K4me3 and H3K27me3: rep2).

ChIP-seq analyses
Raw paired-end ChIP-seq reads were aligned to the mouse genome (GRCm38 / mm10) using bowtie2 version 2.3.3.1 with default settings ( 52 ); the reads were filtered to Nucleic Acids Research, 2023, Vol. 51, No. 13 6673 remove alignments mapped to multiple locations by calling grep with the -v option. Peak calling for ChIP-seq data was performed by MACS2 version 2.1.4 with default arguments ( 53 ); a cut-off P -value of 10 −2 was used. Relati v e ChIP-seq enrichments in respecti v e loci were calculated by dividing input controls. The program ngs.plot ( 54 ) was used to draw tag density plots and heatmaps for H3K4me3, H3K27me3, H3.3, and H3.1 / H3.2 ChIP-seq read enrichment within ±10 kb of TSS. For k-means clustering of all H3K4me3 and H3K27me3 peaks in testicular and epididymal sperm from wild-type and Scml2 -KO males, we firstly generated a set of regions that were called peaks with MACS2 in at least one of the datasets, by merging peak regions of all biological replicates using the mergedBed function from BEDTools (version 2.30.0) ( 55 ). Using respecti v e merged peak files from H3K4me3 and H3K27me3 ChIPseq data, we ran k-means clustering analysis using com-puteMa trix and plotHea tma p pro gram as implemented in deepTools (version 3.1.3) ( 46 ) and determined that k = 4 was suitable for our data. To evaluate the functional annotation of each cluster, we utilized Genomic Region Enrichment of Annotation Tool (GREAT, version 4.0.4) ( 56 ) and HOMER (version 4.9) ( 57 ), both of which associates ChIPseq peak regions in each cluster with their genomic feature and ontology of their putati v e target genes adjacent to peaks. To visualize read enrichment over representati v e genomic loci, coverage files in TDF and bigWig formats were created from sorted BAM files using the IGVTools count function (Broad Institute) and bamCoverage program as implemented in deepTools (version 3.1.3) ( 46 ). Figures for continuous tag counts over selected genomic intervals were created in the IGV browser (Broad Institute). Pearson correla tion coef ficients between biological replicates of ChIPseq profiles (bin = 10 kb) were calculated using SeqMonk (Babraham Bioinformatics).

ICSI or ROSI rescued infertility of Scml2 -KO mice
Because Scml2 is an X-linked gene, all-male pups sired from Scml2 -KO males are wild-type, while all-female pups are heterozygous Scml2 mutants (Figure 1 A). Therefore, Scml2 -KO mice are an ideal model to study epigenetic inheritance from the paternal germline to the descendant male germline without transmission of the mutant allele. To test the functions of SCML2 in paternal epigenetic inheritance, we sought to recover the infertility of Scml2 -KO mice by ART. Because our previous attempts of in vitro fertilization (IVF) or ICSI using Scml2 -KO epididymal sperm with wild-type oocytes were not successful ( 29 ), we tried a modified method of ICSI using testicular sperm that had not yet undergone later maturation processes. Notably, ICSI using testicular sperm rescued infertility of Scml2 -KO males, and these pups underwent full-term de v elopment with a pparentl y normal placentas at a comparable frequency with that of controls (Figure 1 B, Supplementary Table S1). As an alternative approach, we further performed ROSI using Scml2 -KO round spermatids and recovered pups with full-term development at the comparable frequency with that of controls (Figure 1 C, Supplementary Table S1). Body weight and placental weight were sim-ilar between wild-type-and Scml2 -KO-deri v ed pups from ICSI or ROSI ( Figure 1 D and E). Consistently, Scml2 -KOderi v ed embryos normally reached the two-cell stage prior to the transfer to pseudopregnant surrogate females (Supplementary Table S1). In this study, we focused on Scml2 -KO-deri v ed males sired by ICSI as a model of epigenetic inheritance.

H3K27me3 and H3K4me3 distribution in testicular and epididymal sperm
Since we were able to rescue the infertility of Scml2 -KO male with ICSI using testicular sperm, we sought to determine how the sperm epigenome is regulated in testicular sperm prior to its ma tura tion to epidid ymal sperm. Although many previous studies have reported the epigenomic status of epididymal sperm in mice (12)(13)(14)(15)(16)(17)(18)(19), the epigenomic status of testicular sperm has ne v er been reported. To test whether epigenomic states alter from testicular sperm to epididymal sperm and whether SCML2 is r equir ed for this process, we examined distributions of H3K4me3 and H3K27me3 in testicular sperm and cauda epidid ymal sperm isola ted from wild-type and Scml2 -KO males by nati v e ChIP-seq e xperiments. We confirmed nearly 100% purity of our isolated sperm fractions (Supplementary Figure S1) and reproducibility of our nati v e ChIPseq experiments of H3K4me3 and H3K27me3 in testicular sperm and cauda epididymal sperm between biological replicates (Supplementary Figure S2). We detected 78037 peaks among all samples, categorized into 4 clusters through k-means clustering (Figure 2 A). Cluster 1 and 3 peaks show enrichment of H3K4me3, while Cluster 2 peaks are enriched with H3K27me3 but modestly marked with H3K4me3. Cluster 4 peaks are marked with both H3K4me3 and H3K27me3. Overall, the distribution patterns of H3K4me3 and H3K27me3 were largely unchanged from testicular sperm to epididymal sperm in wild-type males, demonstrating that the epididymal pattern of sperm epigenome is already established in testicular sperm.
In Scml2 -KO males, distribution patterns of these marks ov erall resemb le that of wild-type sperm. Howe v er, the intensity of these marks changed in Scml2 -KO males. On Cluster 2 peaks, H3K4me3 was increased, but H3K27me3 was a ttenua ted in Scml2 -KO testicular sperm; H3K27me3 was further a ttenua ted in Scml2 -KO epidid ymal sperm (Figure 2 B). Cluster 2 peaks are associated with de v elopmental regulator genes such as neuron, gland, and heart de v elopment (Figure 2 C). Consistent with these results, our promoter-specific analysis further confirmed that epigenetic states of de v elopmental regulator genes were altered in Scml2 -KO testicular and epididymal sperm (Supplementary Figure S3). We further found an e xtensi v e reduction of H3K27me3 in Scml2 -KO epididymal sperm on Cluster 4 peaks (Figure 2 D), which is associated with other somatic cellular processes (Figure 2 E). Taken together, we conclude that SCML2 is r equir ed for the establishment of the proper epigenome in testicular sperm and epididymal sperm and tha t Scml2 -KO epidid ymal sperm has more pronounced defects compared to Scml2 -KO testicular sperm. Paternal Scml2 deficiency leads to altered sperm RNA content in the next generation To determine whether SCML2 mediates epigenetic inheritance between generations (i.e. intergenerational inheritance), we sought to determine changes in gene expression in Scml2 -KO-deri v ed F1 (KO-F1) males obtained from ICSI with testicular sperm. To this end, we performed RN A-seq anal yses of li v ers and spermatozoa in KO-F1 males in comparison with the control F1 (Ctrl-F1) males that deri v ed from wild type ( Scml2 + / y ) males using ICSI (Figure 3 A, Supplementary Figure S4A); these comparisons were made between wild-type mice generated from ICSI to reflect true epigenetic differences. We selected the li v er because the li v er is a r epr esentati v e organ that shows the effect of paternal epigenetic inheritance in the case of low-protein diet-fed fathers ( 3 ). Howe v er, we detected only a few differentially expressed d ysregula ted genes between KO-F1 and Ctrl-F1 li v er (Figure 3 B, Supplementary Dataset S1), suggesting that paternal Scml2 deficiency has a minor impact on gene expression in the F1 liver. We next analyzed F1 spermatozoa isolated from the cauda epididymis. Because SCML2 is a regulator of gene expression in germ cells ( 37 ), we suspect that SCML2-dependent paternal epigenetic states may persist on sperm chromatin to regulate gene expression in the germline of the next generation. We first confirmed that our sperm RNA-seq did not exhibit expression of somatic markers in the epididymis ( 58 ) (Supplementary Figure S4B). We found that 253 genes were defined as differentially expressed genes (DEGs) between KO-F1 and Ctrl-F1 spermatozoa (Figure 3 C, Supplementary Dataset S1). Because acti v e transcription is ceased in spermatozoa, these results suggest that SCML2-dependent paternal epigenetic states alter gene expression in spermiogenesis in KO-F1 males, leading to altered RNA content in KO-F1 spermatozoa. Despite the altered RNA content, KO-F1 males were fertile and produced offspring in natural mating (F2 analysis is described in a later section).
Among 253 DEGs in KO-F1 spermatozoa, 84 genes were upregulated in KO-F1 spermatozoa, while 169 genes were downregulated. Gene ontology (GO) enrichment analysis re v ealed that these upregulated genes are enriched with gene functions in various biological processes such as glutathione metabolic process and oxidation-reduction process (Figure 3 D). By contrast, down-regulated genes were highly enriched with gene functions in spermatogenesis and sperm motility (Figure 3 D). Further, we e xamined e xpression of these DEGs in many other tissues and different stages of isolated germ cells and confirmed la te-sperma togenesis specific expression of 169 downregulated genes (down-DEGs)     ( 15 )), WT and Scml2 -KO testicular sperm (H3K4me2 and H3K27me3, generated in this study), and RNA-seq reads from WT (adapted from ( 70 )), Ctrl-and KO-ICSI deri v ed F1 sperm (generated in this study). Tnp2 , Prm1 , Prm2 and Prm3 genes were extracted as down-DEGs in KO-F1 spermatozoa. Data ranges are shown in brackets. The Prm1 and Prm2 gene loci were highlighted with blue. (B-E) Average tag density plots and heatmaps around TSS ±10 kb, as well as whisker plots of enrichment around TSSs ±1 kb of H3.3, H3.1 / H3.2, H3K4me3 and H3K27me3 in wild-type sperm ChIP-seq reads on gene groups shown in upper color insets. * P < 0.05; ** P < 0.01; *** P < 0.001; NS, not significant; Wilco x on r ank-sum test. Whisker plots: Centr al bars r epr esent medians, the box es encompass 50% of the data points, and the whiskers indicate 90% of the data points. ( F ) The proportion of genes with H3.3 retention in spermatozoa on gene groups shown in upper color insets. Genes with > 1.5-fold enrichment compared to inputs were defined as genes with H3.3 retention. * P < 0.05; *** P < 0.001. Chi-square test with Yates correction.
in KO-F1 sperm; these downregulated genes are highly expressed in meiotic pachytene spermatocytes (PS), round spermatids (RS), and spermatozoa (Figure 3 E). On the other hand, many of the 84 upregulated genes (up-DEGs) in KO-F1 spermatozoa are highly expressed in various somatic tissues and some stages of dif ferentia ted germ cells (such as KIT + spermatogonia and spermatozoa), though these 84 up-DEGs are not highly expressed in undifferentiated cells (such as ESC and THY1 + undifferentiated spermatogonia) and in later spermatogenesis (PS and RS). Consistent with the late spermatogenesis functions of 169 down-DEGs in KO-F1 sperm, these genes tend not to be highly conserved in mammals. Among them, 20 rodent-specific genes are observed, which are in line with the rapid evolution of late spermatogenesis genes (Supplementary Figure  S5). Together, we conclude that paternal Scml2 deficiency leads to altered sperm RNA content in the next generation in two major groups: downregulation of late spermatogenesis genes and upregulation of differ entiation-r elated genes. These results indica te tha t SCML2 mediates intergenerational inheritance of epigenetic information.

Histone retention at Scml2 KO-F1 dysregulated gene loci in wild-type spermatozoa
To delineate mechanisms associated with SCML2-mediated paternal epigenetic inheritance, we examined the chromatin status of the DEGs in wild-type spermatozoa. A r epr esenta-ti v e locus of down-DEGs of KO-F1 spermatozoa comprises the Protamine gene cluster, which contains three protamine genes ( Prm1 , Prm2 and Prm3 ) and a transition nuclear protein gene ( Tnp2 ) on chromosome 16, all of which are coregulated as a cluster and highly expressed in spermiogenesis ( 59 ). In KO-F1 sperm, these genes were significantly downr egulated (Figur e 4 A, Supplementary Dataset S1), raising the possibility that SCML2 regulates the Protamine gene cluster. We reanalyzed the published ChIP-seq data set ( 15 ) and found that Prm1 , Prm2 , Prm3 and Tnp2 loci are enriched with histone variants H3.3, which is a major component of retained histones in spermatozoa ( 15 ). These gene loci were associated with H3.3 in spermatozoa ( 15 ). Further, the Protamine gene cluster is broadly enriched with H3K27me3, a PRC2 mediated r epr essi v e mar k regulated by SCML2 in the germline ( 29 ). Indeed, H3K27me3 was attenua ted a t the Prm1 and Prm2 gene loci in Scml2 -KO testicular sperm (highlighted with blue bars in Figure 4 A). Thus, histone retention at the Protamine gene cluster and the presence of H3K27me3 in spermatozoa is a possible mechanism by which SCML2 media tes pa ternal epigenetic inheritance.
We further investigated the genome-wide features of these DEGs in wild-type spermatozoa. We compared average tag density of H3.3, canonical histone H3 (H3.1 / H3.2), H3K4me3 and H3K27me3 in wild-type spermatozoa over the four groups of genes: up-and down-DEGs in KO-F1 spermatozoa, canonical Polycomb target genes (3599 genes) identified in ESC ( 60 ), and other genes in the genome.
Compared to other genes, up-and down-DEGs in KO-F1 spermatozoa are enriched with H3.3 (Figure 4 B, while canonical H3.1 / H3.2 did not show notable enrichment on these genes (Figure 4 C). Consistent with the example of the Protamine gene cluster, up-and down-DEGs in KO-F1 spermatozoa are enriched with H3K4me3 to a higher degree than Polycomb target genes (Figure 4 D). H3K27me3 was also significantly enriched on up-and down-DEGs in KO-F1, but to a lesser extent compared to canonical Polycomb target genes (Figure 4 E). Further, proportions of genes with histone r etention wer e estimated based on H3.3 enrichment: 46.3% and 50.3% of up-and do wn-DEGs sho w > 1.5-fold enrichment of H3.3 (normalized to input read) in spermatozoa (Figure 4 F). These analyses demonstra te tha t, in wild-type sperma tozoa, histone retention takes place on d ysregula ted gene loci detected in KO-F1, raising the possibility that SCML2 media tes intergenera tional epigenetic inheritance through sperm chromatin.

SCML2 establishes H3K27me3 on Scml2 -KO-F1 dysregulated gene loci in late spermatogenesis
We next examined how the Scml2 -KO-F1 d ysregula ted gene loci are modified in the male germline. Of note, H3K4me3 and H3K27me3 are present on up-and down-DEGs loci in E13.5 primordial germ cells, which undergo extensive epigenetic reprogramming (reanalysis from ( 61 ): Figure 5 A and Supplementary Figure S6). At these gene loci, H3K4me3 and H3K27me3 are present in THY1 + undif ferentia ted spermatogonia and KIT + differentiating spermatogonia (reanalysis from ( 29 )). Subsequently, H3K4me3 increases in PS in meiosis, followed by an increase of H3K27me3 in RS in the postmeiotic stage (reanalysis from ( 29 ) : Figure 5 A). After RS, the intensity of H3K27me3 decreases in testicular sperm but remains present in epididymal sperm, while H3K4me3 remains high after RS (original data in this study: Figure 5 A). The late establishment of H3K27me3 after H3K4me3 appears to be explained by the function of SCML2 as the regulator of bivalent chromatin; SCML2 binds to h ypometh ylated promoter marked with H3K4me3 and induces H3K27me3 to establish bivalent chromatin status ( 29 ). This notion is further supported by the analysis of SCML2-binding sites. We reanalyzed the SCML2 binding sites detected in cultured germline stem cells ( 37 ) and found that SCML2 directly binds to promoters of both up-and down-DEGs, and the intensity of SCML2 signals is high on these DEG loci compared to other gene loci (Figure  5 B). We next examined the function of SCML2 using the ChIP-seq data from Scml2 -KO PS and RS ( 29 ) and nati v e ChIP-seq da ta genera ted in this stud y. Although sta tistically significant changes were not observed on Scml2 -KO PS for H3K4me3 and H3K27me3 enrichment on down-DEGs, H3K27me3 enrichment on up-DEGs in PS , RS , testicular sperm, and epididymal sperm was decreased in Scml2 -KO (Figure 5 C). H3K27me3 enrichment on down-DEGs in RS, testicular sperm and epididymal sperm was also decr eased in Scml2 -KO (Figur e 5 C). These r esults suggest that the SCML2-dependent establishment of H3K27me3 in the male germline is correlated with SCML2 mediated paternal epigenetic inheritance.

Intergener ational effects w er e largely corr ected in F2 mice sired from Scml2-KO males
Next, we sought to address whether SCML2-mediated epigenetic states persist across generations (i.e. tr ansgener ational inheritance). To this end, we obtained F2 males from the KO-F1 males sired from Scml2 -KO males (KO-F2; Figure 6 A); the KO-F1 males were fully fertile, despite the d ysregula tion of gene expression in spermatozoa ( Figure  3 ). To test the germline gene expression d ysregula ted in KO-F2 spermatozoa, we performed RNA-seq of KO-F2 spermatozoa and compared it with F1 control spermatozoa (Supplementary Figure S7A, B). We detected 161 dysregulated genes Scml2 -KO F2 spermatozoa; 75 genes were up-DEGs and 86 genes were down-DEGs in KO-F2 sperma tozoa (Figure 6 B , Supplementary Da taset S2). Of note, only two genes were overlapped between down-DEGs in KO-F1 spermatozoa and KO-F2 spermatozoa, while there was no overlap between up-DEGs in KO-F1 spermatozoa and KO-F2 spermatozoa (Figure 6 C, D). This suggests that, in KO-F2, there are no consistent set of DEGs tha t correla te with the effects observed in the first generation (KO-F1) and that epigenetic abnormally detected in KO-F1 spermatozoa were largely corrected in the F2 generation. Consistent with this idea, GO enrichment analysis re v ealed distinct classes of genes ar e dysr egulated in between KO-F1 spermatozoa and KO-F2 spermatozoa, and we did not observe an enrichment of spermatogenesisrelated genes in down-DEGs of KO-F2 spermatozoa (Supplementary Figure S7C). Howe v er, SCML2 peaks were detected at the promoters of up-and down-DEGs of KO-F2 spermatozoa in cultured germline stem cells ( Figure  6 E). In our experiments, we used a consistent genetic background, and we confirmed that biological replicates for our data sets are similar to each other ( Supplementary Figure S7B), excluding the possibility that the KO-F2 results were caused by differences in genetic background or individual differences. An alternati v e possibility could be instability of epigenetic information in the F2 mice sired from Scml2-KO males, while similar patterns of gene dysregulation were not observed between KO-F1 spermatozoa and KO-F2 spermatozoa. Ther efor e, intergenerational effects wer e largely corr ected in F2 mice sir ed from Scml2-KO males. It is still possible that SCML2 may impact transgenerational inheritance of epigenetic information. Howe v er, the instability is unlikely to be explained by stable transmission of epigenetic sta tes a t specific target loci across generations.

Dysregulation of gene expression in preimplantation embryos deriv ed fr om Scml2 -KO testicular sperm
Finally, we sought to determine how paternal chromatin defects in Scml2 -KO testicular sperm impact the next generation after fertilization. To this end, we performed ICSI experiments using Scml2 -KO and wild-type control testicular sperm and examined gene expression in preimplantation embryos deri v ed from them. To precisely evaluate the gene expression, we used oocytes from C57BL / 6 female mice, which have the same genetic background as the Scml2 -KO male mice (Figure 7 A). After ICSI, KO-deri v ed (KO-F1) and wild-type control-deri v ed (Ctrl-F1) embryos were cultured for a day to obtain two-cell embryos and for 4 days to obtain blastocysts (Figure 7 B). KO-F1 embryos de v eloped into blastocysts, which showed abnormal morphology (Figure 7 B)  . We previously showed that SCML2 regulates H3K27me3 on two distinct classes of bivalent genes; a class includes de v elopmental regulator genes that are not acti v e in the germline (Class I) and somatic / mitotic genes that ar e suppr essed in la te sperma togenesis (Class II) ( 29 ). Based on these findings, we ne xt e xamined whether these target genes are d ysregula ted in the embryos. Notably, the Class I genes were specifically upregulated in KO-F1 blastocysts (Figure 7 F). A GO term analysis re v ealed that the upregulated genes in KO-F1 blastocysts are enriched with genes related to de v elopmental processes, including the GO terms 'regulation of neuron differentiation', 'cardiocyte dif ferentia tion', and 'car diac cell de v elopment' (Supplementary Figure S8D). Further, these DEGs in KO-F1 male blastocysts were the SCML2 target genes in the male germline; SCML2 accumulated on DEGs in germline stem cells, and H3K27me3 accumulation on these genes was SCML2-dependent in r epr esentati v e stages of the male germline (Supplementary Figure S9). Therefore, these results suggest tha t pa ternal epigenetic defects in Scml2 -KO sperm were associated with the misregulation of de v elopmental regulator genes in KO-F1 blastocysts.

DISCUSSION
In this study, we present functional evidence that the classic epigenetic regulator Polycomb mediates paternal epigenetic inheritance through sperm chromatin. We rescued the infertility of Scml2 -KO males and demonstrated that the Scml2 -KO mouse line serves as a novel model of epigenetic inheritance, particularly that of paternal epigenetic states. We further provide novel epigenomic r esour ces for testicular sperm. We show that epididymal pattern of the sperm epigenome is already established in testicular sperm. Although w e w ere not able to determine an exact reason why testicular sperm of Scml2 -KO males w ork ed with ICSI, possible reasons include deterioration of chromatin states or genome integrity from testicular sperm to epididymal sperm of Scml2 -KO males. In line with this possibility, H3K27me3 was further a ttenua ted from testicular sperm to epididymal sperm of Scml2 -KO males at specific loci (Figure 2 B). Another alternati v e possibility could be structural defects of Scml2 -KO epididymal sperm. Scml2 -KO epididymal sperm have midpieces that are tightly associated with nuclei; w e w ere not able to dissociate their midpieces from sperm nuclei, which might have prevented ICSI from working in our previous study ( 29 ). In this study, we used nuclei of cryopreserved Scml2 -KO testicular sperm for ICSI.   ( 29 ); Class I, de v elopmental regula tor genes; Class II, soma tic genes) in ICSI-deri v ed Ctrl-and Scml2 -KO-F1 embryos at two-cell and blastocyst stages. Central bars r epr esent medians, box es encompass 50% of the data points, and the whiskers indicate 90% of the data points. NS, not significant; *** P < 0.001; Wilco x on rank-sum test.
These sperm nuclei were detached from midpieces after cryopreservation and prior to ICSI.
In la te sperma togenesis, SCML2 binds to h ypometh ylated promoters of target genes where H3K4me3 is enriched and interacts with PRC2 to establish H3K27me3 on its target gene loci, leading to the establishment of bivalent genomic domains ( 29 ) (Figure 8 A). SCML2 interacts with both PRC1 and PRC2 ( 29 , 37 ). Howe v er, PRC1-mediated H2AK119ub was not observed in normal late spermatogenesis ( 37 ). On the other hand, PRC2-mediated H3K27me3 is e xtensi v ely estab lished during la te sperma togenesis in an SCML2-dependent manner ( 29 ). Ther efor e, it is r easonable to postulate that the Scml2 -KO phenotypes emanate from SCML2-dependent regulation of PRC2 in late sperma togenesis. We found tha t histone H3.3 was retained, and H3K4me3 and H3K27me3 were present in spermatozoa, whereas H3.1 / 2 le v els were v ery low on the promoters of d ysregula ted gene loci of our Scml2 -KO-F1 model. These findings support the possibility that H3.3 in sperm may be modified with trimethylation at K4 and K27 sites and is responsible for the inheritance of paternal epigenetic states. In spermatogenesis, testis-specific histone variant H3T is a major isoform of histone H3 and is considered to be replaced by H3.3 in late spermatogenesis ( 62 ). Thus, SCML2 media ted trimethyla tion a t K27 may occur on H3.3 after replacing H3T at the promoter regions of target loci, and the epigenetic states may persist into the next genera tion to regula te gene expression in la te sperma togenesis of F1.
A recent study showed that multiple histone modifications undergo changes during sperm ma tura tion in the epididymis, some of which occurred on H3.3 ( 63 ). In accord with this observation, we show that H3K27me3 is further a ttenua ted in epidid ymal sperm of Scml2 -KO compared to testicular sperm (Figure 2 B), raising the possibility that alterations of H3.3 modifications during sperm maturation are affected in Scml2 -K O sperm. Further , SCML2 is a regulator of germline gene expression ( 37 ); thereby, it is possible that SCML2 establishes chromatin states that modulate germline gene expression in the next generation. This possibility is in line with the abnormal gene expression in Scml2 -KO-F1 blastocysts. We speculate that SCML2 deletion in the paternal germline causes epigenetic instability on target loci, which are again regulated in the germline of the offsprings. To test this possibility in future studies, it would be important to determine the epigenetic states of Scml2 -KO-F1. In addition, there remains an important question as to the impact of paternal SCML2 loss on the soma of the offsprings, although we found it has a minor impact on the li v er.
Because acti v e H3K4me2 / 3 and silent H3K27me3 counteract each other at bivalent chromatin in the germline ( 29 ), it is possible that the balanced establishment of bivalent domains may be r equir ed for the proper inheritance of epigenetic states for gene regulation in the germline. In Scml2 -KO-F1 b lastocysts, de v elopmental regulator genes that carry bivalent chromatin were dysregulated, although such e xtensi v e d ysregula tion could lead to embryonic dea th and the survival of relati v ely normal embryos after implantation. Accor dingly, a possib le e xplanation for the F1 germline phenotype is that the attenuation of H3K27me3 may result in abnormal gene expression of F1 spermiogenesis (Figure 8 A). Although bivalent chromatin is a persistent feature in the germline, a major unsolved mystery is how epigenetic states are inherited and maintained throughout the germline after fertiliza tion. W hile pa ternal H3.3 on promoters disappears after fertilization ( 64 ) and H3K27me3 is r eprogrammed in pr eimplantation de v elopment ( 60 ), a recent study showed that paternal H3K4me3 persists through preimplantation de v elopment ( 30 ). Thus, it is possib le that the paternal epigenetic states, reflecting the memory of paternal H3K27me3, escape epigenetic reprogramming and persist at the promoter regions of the target genes throughout germ cell de v elopment.
In support of this idea, we found that H3K4me3 and H3K27me3 persist on the target genes, albeit at low le v els, in E13.5 PGC ( Figure 5 A and Supplementary Figure S6), where e xtensi v e epigenetic reprogramming takes place. Because SCML2 is r equir ed for H3K27me3 establishment in the male germline, SCML2 deletion in the paternal germline may perturb epigenetic states in offspring. This notion is in part supported by the analyses of Scml2 -KO-F1 blastocysts. Our recent study compared embryos deri v ed from ROSI and ICSI using epididymal sperm, and we found that pa-ternal H3K27me3 is linked to the gene expression change in early embryos ( 65 ). This study further supports our conclusion tha t pa ternal H3K27me3 is involved in intergenerational epigenetic inheritance. On the other hand, the KO-F1 and KO-F2 showed a pparentl y distinct patterns of gene expression, suggesting that epigenetic states may be largely reprogrammed in each generation, though a sort of epigenetic instability may be transmitted across generations.
Of note, our new model of epigenetic inheritance may hav e some rele vance with the pre viously estab lished model of paternal epigenetic inheritance through sperm chromatin: the Kdm1a ov ere xpression model, which reduces H3K4me2 in the germline ( 30 , 31 ) (Figure 8 B), and the Kdm6a conditional deletion model, which potentially increases H3K27me3 in the germline ( 33 ) (Figure 8 C). The precise mechanisms of action underlying these mouse models remain elusi v e; yet, together with our study, it is tempting to specula te tha t the balance between Polycomb-media ted H3K27me3 and H3K4me2 / 3 a t regula tory elements may be responsible for the establishment of proper gene expression in the next generation.
At the end of this study, we note that there remains a major unsolved question as to how chromatin states can be bookmarked, escape epigenetic reprogramming, and persist at the promoter regions of the target throughout the germline. A possibility is that the abnormal H3K27me3 / H3K4me3 could affect DNA methylation or transcription factor binding to gene regulatory elements in the germline ( 66 ). Another possibility could be that these sites may escape epigenetic reprogramming, as some TE-deri v ed locus esca pe epigenetic repro gramming in PGC ( 67 ). The notion of chromatin-based persistent epigenetic abnormality is in line with the other emerging cases of epigenetic inheritance ( 68 , 69 ). Further, the sev ere b lastocyst phenotype of Scml2 -KO-F1 blastocysts suggests that there could be a broad impact on F1 soma via Nucleic Acids Research, 2023, Vol. 51, No. 13 6681 inter generational inheritance. Ho we v er, the se v ere b lastocyst phenotype could be enhanced due to in vitro culture because we did not observe a significant difference in reproduction outcomes in two-cell transferred embryos (Supplementary Table S1). Although much remains unknown in paternal epigenetic inheritance, our de v elopment of a new tractable mouse model will open up new avenues for future investigations.

DA T A A V AILABILITY
NGS datasets used in this study are publicly available and r efer enced within the article. All 59 datasets from other resour ces ar e listed and r efer enced in Supplementary Dataset S4. All the RNA-seq and nati v e ChIP-seq data generated in this study are deposited to the Gene Expression Omnibus (GEO) under accession code GSE183994.