Ablation of the miR-465 Cluster Causes a Skewed Sex Ratio in Mice

Abstract

The X-linked miR-465 cluster is highly expressed in the testis, sperm, newborn ovary, and blastocysts as well as in 8-16 cell embryos. However, the physiological role of the miR-465 cluster is still largely unknown. This study aims to dissect the role of the miR-465 cluster in murine development. Despite abundant expression in the testis, ablation of the miR-465 miRNA cluster using CRISPR-Cas9 did not cause infertility. Instead, a skewed sex ratio biased toward males (60% males) was observed among miR-465 KO mice. Further analyses revealed that the female conceptuses selectively degenerated as early as embryonic day 8.5 (E8.5). Small RNA deep sequencing, qPCR, and in situ hybridization analyses revealed that the miRNAs encoded by the miR-465 cluster were mainly localized to the extraembryonic tissue/developing placenta. RNA-seq analyses identified altered mRNA transcriptome characterized by the dysregulation of numerous critical placental genes, e.g., Alkbh1, in the KO conceptuses at E7.5. Taken together, this study showed that the miR-465 cluster is required for normal female placental development, and ablation of the miR-465 cluster leads to a skewed sex ratio with more males (~60%) due to selective degeneration and resorption of the female conceptuses.

The Rapidly Evolving X-linkedmiR-506Family Finetunes Spermatogenesis to Enhance Sperm Competition

“…For example, the mature miR-465a , miR-465b , and miR-465c only have a few mismatches outside of the seed region, but only miR-465c exerts functional activation of Egr1 . A similar effect has also been reported in the miR-465 cluster on the Alkbh1 3’UTR activity (18). Similarly, despite the same seed sequences in the miR-465 or miR-743 cluster, miR-465a and miR-465c have differential activating effects on the 3’UTR of Crisp1 , and miR-743a and miR-743b exert opposite effects on the Crisp1 3’UTR, further confirming their functional divergence.…”

“…Similarly, despite the same seed sequences in the miR-465 or miR-743 cluster, miR-465a and miR-465c have differential activating effects on the 3’UTR of Crisp1 , and miR-743a and miR-743b exert opposite effects on the Crisp1 3’UTR, further confirming their functional divergence. Therefore, the sequences outside of the miRNA seed region may play an important role in their functions, which have also been observed in C. elegans and human HEK293 cells (18, 78, 79). To unequivocally demonstrate the physiological role of miRNAs, it would be ideal to delete not only the miRNAs but also their binding sites in their target transcripts in vivo .…”

Epididymal acquired sperm microRNAs modify post-fertilization embryonic gene expression

“…Future studies to determine whether gene expression changes are a result of the collective down regulation of these miRNAs or driven primarily by only a few individual miRNAs warrants investigation. Notably, the ablation of one Fx-miR family, the miR-465 family in mice does not impair male fertility but leads to skewed sex ratios in resulting offspring due to dysfunction in the placentas of females (Wang et al, 2022).…”

“…Fascinatingly, the introduction of sperm RNA to parthenotes results in changes in expression of hundreds of genes to more closely resemble the expression of a sperm fertilized embryo (Conine et al, 2020). In narrowing the role of individual small RNA classes in regulating post-fertilization gene expression, several additional studies have specifically identified post-fertilization functions for sperm microRNAs (miRNAs) in the early embryo (Wang et al, 2022; Yuan et al, 2016). Embryos produced via intracytoplasmic sperm injection (ICSI) of sperm conditionally depleted of miRNAs during spermatogenesis display reduced preimplantation development which was partially rescued by microinjection of sperm small RNA (Yuan et al, 2016).…”

A cluster of X-linked miRNAs are de-repressed with age in mouse liver and target growth hormone signaling

“…Interestingly, they may also regulate stem cell differentiation, as members of the X-chromosomal cluster are upregulated during embryonic stem cell differentiation ( Tay et al, 2008 ), and knocking out the cluster in rat fibroblasts inhibits reprogramming to induced pluripotent stem cells ( Sherstyuk et al, 2019 ). The mir-465 family may also be involved in normal female placental development, as knocking out these miRNAs results in the sex ratio skewing towards males in mouse litters due to degeneration and resorption of female embryos ( Wang et al, 2022 ). These studies indicate that the miRNAs in the X-chromosomal cluster can regulate developmental pathways.…”