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Paternally inherited H3K27me3 impacts zygotic genome activation in round spermatid injection [RNA-seq]

ABSTRACT: Round spermatid injection (ROSI) gives rise to a lower birth rate than intracytoplasmic sperm injection (ICSI), which uses mature spermatozoa and has limited clinical application. Inefficient development of ROSI-embryos has been attributed to epigenetic abnormalities. However, the chromatin-based mechanism that underpins the low birth rate of ROSI remains to be determined. Here we show that a repressive histone mark H3K27me3 persists from doner round spermatids into zygotes in ROSI and that paternally derived H3K27me3 is associated with less accessible chromatin and impaired zygotic genome activation (ZGA) in ROSI-derived zygotes. These H3K27me3 marked loci in the paternal germline undergo histone turnover in spermiogenesis, resulting in the reduced paternal H3K27me3 in normal spermatozoa. Thus, the lack of histone turnover during spermiogenesis led to the dysregulation of chromatin accessibility and ZGA in ROSI-embryos. Our results unveil a molecular logic by which chromatin states in round spermatids impinge on chromatin states and ZGA in ROSI-embryos, highlighting the importance of chromatin remodeling in spermiogenesis in successful reproduction.

ORGANISM(S): Mus musculus

PROVIDER: GSE196858 | GEO | 2022/08/23

REPOSITORIES: GEO

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Project description:Pachytene piRNAs are PIWI-interacting small RNAs abundantly expressed in pachytene spermatocytes and spermatids in adult mouse testes. Both MIWI and MILI-bound pachytene piRNAs have been found enriched in round spermatids. Miwi-null male mice are sterile due to spermiogenic arrest. In C. elegans, sperm-borne piRNAs appear to have an epigenetic role during fertilization and development because progeny of offspring derived from piRNA-deficient sperm display a progressive fertility loss after several generations. In mice, it remains unknown whether MIWI-bound pachytene piRNA-deficient round spermatids can produce offspring, and whether the progeny of offspring derived from MIWI-bound pachytene piRNA-deficient round spermatids also exhibit transgenerational loss of fertility. Here, we report that Miwi KO round spermatids could fertilize both wild type (WT) and Miwi KO oocytes through round spermatid microinjection (ROSI), and produce healthy and fertile offspring despite the aberrant pachytene piRNA profiles in those Miwi KO spermatids. Progeny of ROSI-derived heterozygotes, both male and female, displayed normal fertility for at least three generations when bred with either WT or Miwi KO females. Our data indicate that aberrant MIWI-bound pachytene piRNAs profiles in spermatids do not affect fertilization, early embryonic development, or fertility of the offspring, suggesting a normal pachytene piRNAs profile is not required for paternal transgenerational epigenetic inheritance in mice. Method: Round spermatids were purified from WT and Miwi KO adult testes using a mini-STA-PUT method[Methods Enzymol 1993; 225:84-113.]，the purity of round spermatids was >90% based on our previous report[ J Biol Chem 2012; 287:25173-25190.]. Small RNA was isolated from round spermatids using the mirVana RNA isolation kit (Ambion) according to the manufacturer’s instructions. RNA quality and quantity were assessed using the Agilent 2100 Bioanalyzer. Small RNA-Seq was performed on an Ion Proton sequencer (Life Technologies). Libraries were prepared using the Ion Total RNA-Seq Kit v2 (Invitrogen) with biological triplicates for WT and Miwi KO samples. Resutls:Our data indicate that aberrant MIWI-bound pachytene piRNAs profiles in spermatids do not affect fertilization, early embryonic development, or fertility of the offspring, suggesting a normal pachytene piRNAs profile is not required for paternal transgenerational epigenetic inheritance in mice.

Project description:Acf1 was isolated almost 15 years ago from Drosophila embryo extracts as a subunit of several complexes that possess chromatin assembly activity. Acf1 binds to the ATPase ISWI (SNF2H in mammals) to form the ACF/CHRAC chromatin remodeling complexes. Studies in Drosophila and in human and mouse cell culture implicate Acf1 in many cellular processes including transcriptional repression, heterochromatin formation and replication, and DNA damage checkpoints and repair. However, the in vivo function of Acf1 in mammals is unknown. We generated mice deficient for Baz1a, the mammalian homolog of Drosophila Acf1. In contrast to partially penetrant lethality of the mutation in flies, Baz1a-deficient mice are viable. The mutant mice show no obvious defects in B or T cell lineages, class switch recombination in cultured B cells, or meiotic recombination. Thus, BAZ1A is dispensable in vivo for the survival and differentiation of cells that require the repair of developmentally programmed DNA double-strand breaks (DSBs). However, Baz1a-/- male mice are sterile because of a severe defect in spermiogenesis that results in fewer and non-motile sperm with morphological defects. Baz1a-deficient round spermatids display widespread perturbation of gene expression. The mutant cells faithfully execute the widespread reprogramming of gene expression that normally accompanies the developmental transition from meiosis to postmeiotic differentiation, but overlaid on this normal developmental program is mis-regulation of a large number of additional genes. The inappropriate expression of this eclectic group of genes is likely the cause of the pleiotropic spermiogenesis defects in the mutant. We propose that the dramatic changes in chromatin composition that occur in late meiotic prophase and early spermiogenesis create a window of vulnerability during which BAZ1A-targeted chromatin assembly functions are needed to prevent inappropriate transcription changes. This appears to be the sole essential function of the ACF and CHRAC complexes in mouse. total RNA isolated from FACS-enriched pachytene/diplotene spermatocytes and round spermatids from three littermate pairs of Baz1a-/- and Baz1a-/+ animals. Data from all samples are provided, but expression data for one Baz1a-/- spermatocyte sample (683_PD) was censored because of inferred high degree of contamination with round spermatids

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Paternally inherited H3K27me3 impacts zygotic genome activation in round spermatid injection [RNA-seq]

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