key: cord-0271545-yjpuugju
authors: Hasuwa, Hidetoshi; Iwasaki, Yuka W.; Wan Kin, Au Yeung; Ishino, Kyoko; Masuda, Harumi; Sasaki, Hiroyuki; Siomi, Haruhiko
title: Production of functional oocytes requires maternally expressed PIWI genes and piRNAs in golden hamsters
date: 2021-01-27
journal: bioRxiv
DOI: 10.1101/2021.01.27.428354
sha: 170051ac8e00219ceb0f02d0381a44f36c173872
doc_id: 271545
cord_uid: yjpuugju

Many animals have a conserved adaptive genome defense system known as the Piwi-interacting RNA (piRNA) pathway which is essential for germ cell development and function. Disruption of individual mouse Piwi genes results in male but not female sterility, leading to the assumption that PIWI genes play little or no role in mammalian oocytes. Here, we report generation of PIWI-defective golden hamsters, which reveals defects in the production of functional oocytes. The mechanisms involved vary among the hamster PIWI genes; lack of PIWIL1 has a major impact on gene expression, including hamster-specific young transposon de-silencing, whereas PIWIL3 deficiency has little impact on gene expression in oocytes, although DNA methylation was found to be reduced to some extent in PIWIL3-defecient oocytes. Our findings serve as the foundation for developing useful models to study the piRNA pathway in mammalian oocytes, including humans, which is not possible with mice.

piRNAs are derived from specific genomic loci termed piRNA clusters and form effector 15 complexes with PIWI proteins, a germline-specific class of Argonaute proteins, to guide recognition and silencing of their targets, mostly transposable elements (TEs) (1) (2) (3) . Mammalian PIWI-piRNA pathways have mostly been studied in mice, which express three PIWI proteins (MIWI/PIWIL1, MILI/PIWIL2, and MIWI2/PIWIL4) abundantly in the testis, but only weakly in the ovary (1) (2) (3) . These PIWI proteins bind distinct classes of piRNAs which direct chromatin 20 modifications of target TE sequences during embryogenesis and guide silencing of target TEs at the posttranscriptional level later in spermatogenesis, to ensure completion of meiosis and successful sperm production. Deficiency of Piwi genes in mice is characterized by 3 spermatogenesis arrest and infertility in males, but females with deficiencies in these genes remain fertile (4) (5) (6) (7) (8) (9) with limited impact on TE silencing (10, 11) . These findings clearly demonstrate that mouse Piwi genes are essential for male, but not female, germ cell development.

However, most mammalian species including humans possess an additional PIWI gene, termed 5 PIWIL3, in addition to the three PIWI genes described above (12, 13) . Thus, piRNA-mediated silencing may differ between mice and other mammals with four PIWI genes. However, little is known about the piRNA pathway in mammalian species with the four PIWI genes, particularly their potential roles in functional oocyte production. Golden Syrian hamsters (golden hamster, Mesocricetus auratus) have been used as an experimental rodent model for studying human 10 diseases, particularly, cancer and infectious diseases, including the recent Covid-19, because they display physiological and pharmacological responses resembling those of humans (14, 15 ). In addition, genome editing using the CRISPR/Cas9 system was recently enabled in golden hamsters to engineer the genes of interest (16, 17) . Unlike laboratory mice and rats, which belong to the Muridae family of rodents lacking PIWIL3, the golden hamster belongs to the Cricetidae family 15 of rodents and has four distinct PIWI genes. We recently found that hamster PIWIL1 and PIWIL2 are expressed in both the testis and the ovary, whereas PIWIL3 and PIWIL4 are exclusively expressed in the ovary and testis, respectively (18) .

In the present study, we aimed to investigate the roles of PIWI genes in female reproduction by studying PIWIL1 and PIWIL3 genes, both of which were highly expressed in oocytes (18; see also Fig . S3A and B). We employed direct injection of Cas9 mRNA or Cas9 protein together with 4 sgRNAs into the pronucleus (PN) and/or cytoplasm of an embryo to generate PIWIL1-and PIWIL3-deficient hamsters. We injected the cells under a microscope with red filters (600 nm) in a dark room, because even brief exposure of hamster zygotes to light results in total developmental arrest (16) . Injected embryos with a normal morphology were transferred to each oviduct (10-15 embryos per oviduct) of pseudo-pregnant recipient females. In total, 46 pups were obtained from 5 the PIWIL1-sgRNA injected embryos, of which 23 carried a mutant allele, as demonstrated by genomic PCR and sequencing using the tail tissue (Fig. S1 ). However, nine female founder hamsters were infertile and could only establish three frameshift mutant hamster lines. For the production of PIWIL3-deficient hamsters, 18 pups were obtained from the injected embryos.

Genomic sequencing revealed some mosaicism and identified a total of 13 types of PIWIL3 mutant 10 alleles (Fig. S2) . We crossed F1 heterozygous mutant hamsters to generate F2 homozygous mutant hamsters, which yielded offspring with genotypes that segregated in a Mendelian distribution.

Western blots confirmed the lack of PIWIL1 or PIWIL3 in the mutant ovaries. Immunostaining also demonstrated the loss of PIWIL1 or PIWIL3 protein in the cytoplasm of mutant growing oocytes ( Fig. S3A and B) . 15 Both PIWIL1-and PIWIL3-deficient hamsters appeared normal without outwardly discernible morphological and behavioral abnormalities. PIWIL1-deficient male hamsters displayed small testes and lack of mature sperm in the cauda epididymis ( Fig. S4A -E). DDX4 (mouse Vasa) and acrosome staining with lectin peanut agglutinin (PNA) revealed spermatogenesis arrest in the 20 pachytene stage and a complete lack of S2 spermatids in the PIWIL1 mutant testis ( Fig. S4F and G). These developmental abnormalities resembled those observed in Miwi/Piwil1-deficient male mice (4). PIWIL3 was not expressed in the testis and PIWIL3-deficient male hamsters displayed 5 no overt phenotype. Histological examination of ovaries from PIWIL1-and PIWIL3-deficient adult females revealed no gross abnormalities (Fig. S3C) . PIWIL1-and PIWIL3-deficient female hamsters displayed complete sterility and reduced fertility, respectively (Fig. 1) . When PIWIL1deficient females were crossed with heterozygous males, they never became pregnant despite successful coitus demonstrated by the presence of sperm in the vagina ( Fig. 1A and B) . PIWIL3-5 deficent females, when mated with heterozygous males, displayed reduced fertility with both reduced pregnancy rate (43.5% versus 75% in homozygous and heterozygous females, respectively) and smaller litter size (4.6 versus 6.8 in homozygous and heterozygous females, respectively) ( Fig. 1A and B). The homozygous PIWIL3 mutant pups proceeded into adulthood without expressing any overt developmental abnormalities. 10 To examine how embryogenesis proceeds after fertilization with PIWIL1-or PIWIL3-deficient oocytes, we isolated two-cell (2C) embryos from PIWIL1-or PIWIL3-deficient females crossed with wild-type males and cultured them in vitro. All maternal PIWIL1-deficient 2C embryos remained at the 2C stage and apparently died after one day of in vitro culture, while 2C embryos 15 isolated from heterozygous females developed into four-cell to eight-cell embryos under the same culture conditions (Fig. 1C) . A high proportion (55%) of maternal PIWIL3-deficient 2C embryos showed a 2C arrest phenotype even after two days of in vitro culture and a significant portion of the others were arrested at the three-or five-cell stages, indicating that one blastomere of 2C was arrested and the other divided (Fig. 1D) . These results indicate that both PIWIL1-and PIWIL3-20 deficent oocytes can be fertilized and can proceed through the first cell division, but they fail to develop, either completely or partially, at subsequent developmental stages. These results also highlight the non-redundant essential role of PIWI genes in hamster oogenesis. 6 To gain insight into the molecular defects leading to the observed abnormalities, we performed small RNA sequencing (small RNA-seq) of PIWIL1-and PIWIL3-deficient oocytes; results revealed a decrease in specific populations of small RNAs ( Fig. 2A) . In both heterozygous control oocytes, three peaks at 19 nucleotide (nt), 23 nt, and 29 nt were observed. We compared the small 5 RNA length distribution between heterozygous and homozygous oocytes by normalizing the whole small RNA population using the detected miRNA reads. This revealed a significant decrease in the population of 23-and 29-nt small RNAs in PIWIL1-deficient oocytes. In contrast, only the 19-nt small RNA population was decreased in PIWIL3-deficient oocytes. This is consistent with our recent findings that PIWIL1 binds to two populations of small RNAs, 23 and 29 nt (PIWIL1- 10 bound piRNAs), and PIWIL3 binds to 19-nt small RNAs (PIWIL3-bound piRNAs) in metaphase II (MII) oocytes (18) . It has been recently shown that human PIWIL3 binds a class of ~20-nt small RNAs in oocytes (19) , suggesting that hamster PIWIL3 resembles the human ortholog.

To further characterize the decreased population of small RNAs, changes in the expression level 15 of each small RNA were analyzed in PIWIL1 and PIWIL3 mutants. A decrease in expression level was observed in populations of small RNAs (Fig. S5) , consistent with our observation of the length distribution ( Fig. 2A) . Comparisons with the previously identified PIWIL1-bound/PIWIL3-bound piRNAs (18) indicated that the small RNAs, the populations of which were found to decrease, were identical to these PIWIL1/PIWIL3-bound piRNAs (Fig. 2B) , showing that the lack of PIWI 20 proteins depleted piRNAs that bind to them. In contrast, the expression levels of a minor population of the PIWIL1/PIWIL3-piRNAs were not decreased in homozygous mutants. This may be because these piRNAs can be bound by other PIWI proteins and/or can be stably present in a 7 PIWI-unassociated manner. Small RNAs identical to the PIWIL1-bound piRNAs/PIWIL3-bound piRNAs (18) and those that decreased by over 4-fold in homozygous mutants were defined as PIWIL1-piRNA or PIWIL3-piRNA. As expected, PIWIL1-piRNAs were mostly 23-and 29-nt small RNAs, and PIWIL3-piRNAs were mostly 19-nt small RNAs (Fig. 2C) . They both possessed uracil (U) at their 5' end, which is a conserved characteristic of piRNAs (1, 2) (Fig. 2D ). Genome 5 mapping and annotation of PIWIL1-and PIWIL3-piRNAs revealed that they were mainly mapped to unannotated regions of the genome, as in the case of mouse pachytene piRNAs (3). A total of 12.4% PIWIL1-piRNAs and 15.5% of PIWIL3-piRNAs were mapped to the antisense orientation of TEs, suggesting that they could target TEs (Fig. 2E ). 10 We also performed RNA sequencing (RNA-seq) using samples isolated from PIWIL1-and PIWIL3-deficient MII oocytes. This revealed that the lack of PIWIL1 had a significant impact on the oocyte transcriptome (Fig. 3A) . We detected 1612 differentially expressed genes (DEGs) in PIWIL1-deficient oocytes, 66.13% of which were up-regulated, indicating possible silencing of these genes by the PIWI-piRNA pathway. However, only 0.02% of the PIWIL1-piRNAs were 15 mapped to the antisense direction of protein-coding genes (Fig. 2E) , suggesting that they cannot directly target the mRNA of these DEGs. In contrast, the level of most genes remained unchanged in PIWIL3-deficient oocytes, with only 21 DEGs detected (Fig. 3A) . These results indicate that a number of genes are regulated by PIWIL1, but not PIWIL3, in hamster oocytes. 20 The impact of the loss of PIWIL1 or PIWIL3 on the expression level of TEs was further analyzed using RNA-seq. We recently re-sequenced the golden hamster genome and detected speciesspecific TEs (18) . Notably, ~80% of the expressed TEs (TPM >1) were newly detected members 8 of the TEs. Analysis of the TE expression levels revealed that the loss of PIWIL1 resulted in the increased expression of 29 families of TEs (> 2-fold). In sharp contrast, the loss of PIWIL3 had little impact on the expression levels of TEs ( Fig. 3B and Fig. S6 ). In PIWIL1-deficient oocytes, TE family members, including ERV2-9, ERV2-14b, and ERV2-14a, were up-regulated the most ( Fig. 3C and D) . Of the 29 up-regulated TE families, 18 were LTR and 11 were LINE TEs. In 5 addition, 27 out of the 29 up-regulated TE families were golden hamster specific TEs (18), suggesting that PIWIL1 regulates active TEs that were recently added to the golden hamster genome. We then compared the abundance of the PIWIL1-piRNA population corresponding to each TE family with the change in expression of that TE in PIWIL1-deficient oocytes (Fig. 3C ).

This revealed a correlation between the abundance of PIWIL1-piRNAs antisense to particular TEs 10 and up-regulation of these TEs with a lack of PIWIL1. Together, these results show that PIWIL1

can regulate recently identified active TEs via their targeting by piRNAs in oocytes.

We performed gene ontology (GO) enrichment analysis of the DEGs identified with PIWIL1 deficiency and found enrichment of terms related to nucleosome assembly and transcriptional and 15 epigenetic regulation ( Fig. S7A and B) . This suggests that a loss of PIWIL1 may cause defects in chromatin and/or the genome integrity network. Consistent with this notion, PIWIL1-deficient 2C nuclei displayed a single enlarged nucleolus with altered nuclear DNA enrichment, while 2C nuclei of PIWIL1 heterozygous and PIWIL3 homozygous oocytes displayed multiple nucleoli ( Fig.   S7C and D) . This suggests that a loss of PIWIL1 may induce the nucleolar stress response, which 20 is often associated with cell cycle arrest and cell death (20, 21).

In mice, nuclear Miwi2/Piwil4 acts as an effector for de novo DNA methylation of target TEs in 9 embryonic male germ cells (7, 22, 23) . Recent studies indicated that other PIWI proteins, which are predominantly cytoplasmic, may also function in DNA methylation in a Miwi2-independent manner in male germ cells (24, 25) . These findings prompted us to examine DNA methylation in PIWIL1-and PIWIL3-deficient oocytes, although both PIWIL1 and PIWIL3 are predominantly cytoplasmic in oocytes (18) (also see Fig. S3B ). Because it is known that de novo DNA 5 methylation is complete by the germinal vesicle (GV) stage in mouse oocytes (26), we stained hamster PIWI mutant and control GV oocytes with an anti-5-methylcytosine (5mC) antibody. The DNA methylation level was approximately the same in PIWIL1-deficient and control GV oocytes ( Fig. 4A and Fig. S8 ). However, PIWIL3-defficient oocytes had a significantly reduced DNA methylation level compared to control oocytes ( Fig. 4B and Fig. S8 ). This suggests the 10 involvement of PIWIL3 in DNA methylation during oocyte development.

To explore the DNA methylation status in greater detail, we performed whole genome bisulfite sequencing. This analysis revealed that the level of global CG methylation (DNA methylation at CG sites, which are the major methylation target) was significantly decreased in PIWIL3-defficient 15 GV oocytes compared to that in heterozygous control oocytes (36.4% versus 39.1%, respectively) ( Fig. 4C ). Our recent study showed that piRNA loading onto PIWIL3 is visible only in ovulated oocytes (18) , suggesting a piRNA-independent role of PIWIL3 in DNA methylation. The level of non-CG methylation was unchanged. Measurement of CG methylation at piRNA clusters, TEs, piRNA target TEs and satellite repeats revealed that their total CG methylation level was not 20 significantly altered in PIWIL3 mutants (Fig. S9A-C) .

Unlike any other differentiated mammalian cells, oocytes have DNA methylation predominantly 10 within gene bodies with almost no methylation occurring in intergenic regions (24, 25) . Therefore, we analyzed the DNA methylation status of gene bodies and gene promoters in PIWIL3-deficient oocytes and found that a number of genes showed altered levels of CG methylation at their gene bodies or promoters, with a larger population having reduced methylation, in PIWIL3-deficent GV oocytes ( Fig. 4D and E) . In addition, although there was no significant difference in oocyte 5 transcripts between PIWIL3 mutant and controls (Fig. 3A) , genes having increased DNA methylation at their gene bodies tended to be expressed at higher levels (Fig. S9D) . These results show that PIWIL3 plays a role in DNA methylation in oocytes and suggest that the 3D chromatin structure could be altered in PIWIL3-deficient oocytes. The significant decrease in 5mC staining intensity in PIWIL3 mutant GV oocytes ( Fig. 4B and Fig. S8 ) may also be explained by possible 10 changes in antibody accessibility due to altered chromatin structure. We found that meiosis occurs earlier in PIWIL3-deficient oocytes (Fig. S10A) , suggesting either premature entry into meiosis or accelerated post-ovulatory aging. The morphology of the MII spindle was also altered in PIWIL3deficient oocytes, with wider metaphase plates and longer acute-angled poles observed (Fig. S10B) .

Together, these findings suggest that the loss of PIWIL3 may affect chromosome segregation in 15 oocytes and early embryos, accounting for the appearance of three-cell or five-cell embryos that lack PIWIL3 (Fig. 1D ).

The establishment of DNA methylation in mouse oocytes and sperm is primarily due to the activity 20 of DNMT3A and its cofactor DNMT3L (28-31). DNMT3A-and DNMT3L-deficient mouse oocytes can be fertilized normally but embryos derived from these oocytes die by E10.5, largely due to the absence of DNA methylation at maternally imprinted regions, leading to the notion that 11 the only definitive roles for oocyte DNA methylation are in post-fertilization contexts (28-30).

Nearly half of the embryos derived from PIWIL3-deficient hamster oocytes have a 2C block phenotype, but the observed DNA hypomethylation alone cannot account for the observed phenotype. Since transcripts from both TEs and protein-coding regions were not significantly affected in PIWIL3-deficent MII oocytes, PIWIL3 may be involved in the formation of 5 developmentally competent chromatin, which could be inherited by the zygote.

We analyzed PIWIL1and PIWIL3-deficient hamsters and showed that maternally expressed PIWIs are important for the development of preimplantation embryos. PIWIL1 regulated the expression of genes, including transposons, while PIWIL3 had completely different functions, such 10 as DNA methylation, meiosis and cell division. The origins of the piRNAs that bind to each PIWI are almost the same (18) , suggesting that the length of the piRNA, the PIWI-piRNA complex, or PIWI protein itself was responsible for its respective biological characteristics.

Although mice have contributed immensely to our understanding of the physiology of humans, it 15 is also increasingly clear that the genetic and physiological differences between humans and mice hamper the extrapolation of the results obtained in mouse models to direct applications in humans.

We infer that the emergence of an oocyte specific Dicer isoform (32) could reduce the impact of the Piwi genes on regulation of gene expression, leading to the lack of the dependence on the piRNA pathway in the female germline of mice. The lack of discernible abnormalities in Piwi-20 deficient female mice may represent a special case of the piRNA pathway in mammals. Indeed, our study demonstrates that PIWI-deficient hamsters show abnormalities in oocyte function. This also suggests that PIWI genes should be studied as human infertility genes in women. (16) and those that decreased by >4-fold in homozygous mutants 16 were defined as PIWIL1-piRNA or PIWIL3-piRNA (indicated by blue lines). The red dots indicate piRNA bound to PIWIs described previously (18) . as genes with >20% CG methylation differences between heterozygous and homozygous mutants, and p values between all heterozygous and homozygous replicates were <0.05. Among the 10 differentially methylated genes, significantly less methylated genes were plotted in blue, whereas more methylated genes were plotted in red. p values were calculated by t test.

Acknowledgments: We thank all members of the Siomi laboratory, especially H. Ishizu and K

Nanjing Medical University) for sharing unpublished data. We also thank J. Oishi of the Sasaki 15 laboratory and T. Akinaga (Laboratory for Research Support

wrote the paper.; Competing interests: The authors declare no competing interests.; Data and materials availability: Raw sequence data generated during this study are available in the Gene Expression Omnibus (GEO) repository under accession no. GSE164356. Requests for materials