The rapidly evolving X-linked MIR-506 family fine-tunes spermatogenesis to enhance sperm competition.

Despite rapid evolution across eutherian mammals, the X-linked MIR-506 family miRNAs are located in a region flanked by two highly conserved protein-coding genes (SLITRK2 and FMR1) on the X chromosome. Intriguingly, these miRNAs are predominantly expressed in the testis, suggesting a potential role in spermatogenesis and male fertility. Here, we report that the X-linked MIR-506 family miRNAs were derived from the MER91C DNA transposons. Selective inactivation of individual miRNAs or clusters caused no discernible defects, but simultaneous ablation of five clusters containing 19 members of the MIR-506 family led to reduced male fertility in mice. Despite normal sperm counts, motility, and morphology, the KO sperm were less competitive than wild-type sperm when subjected to a polyandrous mating scheme. Transcriptomic and bioinformatic analyses revealed that these X-linked MIR-506 family miRNAs, in addition to targeting a set of conserved genes, have more targets that are critical for spermatogenesis and embryonic development during evolution. Our data suggest that the MIR-506 family miRNAs function to enhance sperm competitiveness and reproductive fitness of the male by finetuning gene expression during spermatogenesis.

The Rapidly Evolving X-linked miR-506 Family Finetunes Spermatogenesis to Enhance Sperm Competition.

Despite rapid evolution across eutherian mammals, the X-linked miR-506 family miRNAs are located in a region flanked by two highly conserved protein-coding genes (Slitrk2 and Fmr1) on the X chromosome. Intriguingly, these miRNAs are predominantly expressed in the testis, suggesting a potential role in spermatogenesis and male fertility. Here, we report that the X-linked miR-506 family miRNAs were derived from the MER91C DNA transposons. Selective inactivation of individual miRNAs or clusters caused no discernable defects, but simultaneous ablation of five clusters containing nineteen members of the miR-506 family led to reduced male fertility in mice. Despite normal sperm counts, motility and morphology, the KO sperm were less competitive than wild-type sperm when subjected to a polyandrous mating scheme. Transcriptomic and bioinformatic analyses revealed that these X-linked miR-506 family miRNAs, in addition to targeting a set of conserved genes, have more targets that are critical for spermatogenesis and embryonic development during evolution. Our data suggest that the miR-506 family miRNAs function to enhance sperm competitiveness and reproductive fitness of the male by finetuning gene expression during spermatogenesis.

The Influence of Race/Ethnicity on the Transcriptomic Landscape of Uterine Fibroids.

The objective of this study was to determine if the aberrant expression of select genes could form the basis for the racial disparity in fibroid characteristics. The next-generation RNA sequencing results were analyzed as fold change [leiomyomas/paired myometrium, also known as differential expression (DF)], comparing specimens from White (n = 7) and Black (n = 12) patients. The analysis indicated that 95 genes were minimally changed in tumors from White (DF ≈ 1) but were significantly altered by more than 1.5-fold (up or down) in Black patients. Twenty-one novel genes were selected for confirmation in 69 paired fibroids by qRT-PCR. Among these 21, coding of transcripts for the differential expression of FRAT2, SOX4, TNFRSF19, ACP7, GRIP1, IRS4, PLEKHG4B, PGR, COL24A1, KRT17, MMP17, SLN, CCDC177, FUT2, MYO5B, MYOG, ZNF703, CDC25A, and CDCA7 was significantly higher, while the expression of DAB2 and CAV2 was significantly lower in tumors from Black or Hispanic patients compared with tumors from White patients. Western blot analysis revealed a greater differential expression of PGR-A and total progesterone (PGR-A and PGR-B) in tumors from Black compared with tumors from White patients. Collectively, we identified a set of genes uniquely expressed in a race/ethnicity-dependent manner, which could form the underlying mechanisms for the racial disparity in fibroids and their associated symptoms.

Differential Expression of MED12-Associated Coding RNA Transcripts in Uterine Leiomyomas.

Recent studies have demonstrated that somatic MED12 mutations in exon 2 occur at a frequency of up to 80% and have a functional role in leiomyoma pathogenesis. The objective of this study was to elucidate the expression profile of coding RNA transcripts in leiomyomas, with and without these mutations, and their paired myometrium. Next-generation RNA sequencing (NGS) was used to systematically profile the differentially expressed RNA transcripts from paired leiomyomas (n = 19). The differential analysis indicated there are 394 genes differentially and aberrantly expressed only in the mutated tumors. These genes were predominantly involved in the regulation of extracellular constituents. Of the differentially expressed genes that overlapped in the two comparison groups, the magnitude of change in gene expression was greater for many genes in tumors bearing MED12 mutations. Although the myometrium did not express MED12 mutations, there were marked differences in the transcriptome landscape of the myometrium from mutated and non-mutated specimens, with genes regulating the response to oxygen-containing compounds being most altered. In conclusion, MED12 mutations have profound effects on the expression of genes pivotal to leiomyoma pathogenesis in the tumor and the myometrium which could alter tumor characteristics and growth potential.

Triptonide is a reversible non-hormonal male contraceptive agent in mice and non-human primates.

There are no non-hormonal male contraceptives currently on the market despite decades of efforts toward the development of "male pills". Here, we report that triptonide, a natural compound purified from the Chinese herb Tripterygium Wilfordii Hook F displays reversible male contraceptive effects in both mice and monkeys. Single daily oral doses of triptonide induces deformed sperm with minimal or no forward motility (close to 100% penetrance) and consequently male infertility in 3-4 and 5-6 weeks in mice and cynomolgus monkeys, respectively. Male fertility is regained in ~4-6 weeks after cessation of triptonide intake in both species. Either short- or long-term triptonide treatment causes no discernable systematic toxic side effects based on histological examination of vital organs in mice and hematological and serum biochemical analyses in monkeys. Triptonide appears to target junction plakoglobin and disrupts its interactions with SPEM1 during spermiogenesis. Our data further prove that targeting late spermiogenesis represents an effective strategy for developing non-hormonal male contraceptives.

Efficient genome editing by CRISPR-Mb3Cas12a in mice.

As an alternative and complementary approach to Cas9-based genome editing, Cas12a has not been widely used in mammalian cells largely due to its strict requirement for the TTTV protospacer adjacent motif (PAM) sequence. Here, we report that Mb3Cas12a (Moraxella bovoculi AAX11_00205) can efficiently edit the mouse genome based on the TTV PAM sequence with minimal numbers of large on-target deletions or insertions. When TTTV PAM sequence-targeting CRISPR (cr)RNAs of 23 nt spacers are used, >70% of the founders obtained are edited. Moreover, the use of Mb3Cas12a tagged to monomeric streptavidin (mSA) in conjunction with biotinylated DNA donor template leads to high knock-in efficiency in two-cell mouse embryos, with 40% of founders obtained containing the desired knock-in sequences.

Insertion of a chimeric retrotransposon sequence in mouse Axin1 locus causes metastable kinky tail phenotype.

BACKGROUND: Transposable elements (TEs) make up > 50% of the human genome, and the majority of retrotransposon insertions are truncated and many are located in introns. However, the effects of retrotransposition on the host genes remain incompletely known. RESULTS: We report here that insertion of a chimeric L1 (cL1), but not IAP solo LTR, into intron 6 of Axin1 using CRIPSR/Cas9 induced the kinky tail phenotype with ~ 80% penetrance in heterozygous Axin cL1 mice. Both penetrant (with kinky tails) and silent (without kinky tails) Axin cL1 mice, regardless of sex, could transmit the phenotype to subsequent generations with similar penetrance (~ 80%). Further analyses revealed that a longer Axin1 transcript isoform containing partial cL1-targeted intron was present in penetrant, but absent in silent and wild type mice, and the production of this unique Axin1 transcript appeared to correlate with altered levels of an activating histone modification, H3K9ac. CONCLUSIONS: The mechanism for Axin cL1 mice is different from those previously identified in mice with spontaneous retrotransposition of IAP, e.g., Axin Fu and A vy , both of which have been associated with DNA methylation changes. Our data suggest that Axin1 locus is sensitive to genetic and epigenetic alteration by retrotransposons and thus, ideally suited for studying the effects of new retrotransposition events on target gene function in mice.

Pervasive Genotypic Mosaicism in Founder Mice Derived from Genome Editing through Pronuclear Injection.

Genome editing technologies, especially the Cas9/CRISPR system, have revolutionized biomedical research over the past several years. Generation of novel alleles has been simplified to unprecedented levels, allowing for rapid expansion of available genetic tool kits for researchers. However, the issue of genotypic mosaicism has become evident, making stringent analyses of the penetrance of genome-edited alleles essential. Here, we report that founder mice, derived from pronuclear injection of ZFNs or a mix of guidance RNAs and Cas9 mRNAs, display consistent genotypic mosaicism for both deletion and insertion alleles. To identify founders with greater possibility of transmitting the mutant allele through the germline, we developed an effective germline genotyping method. The awareness of the inherent genotypic mosaicism issue with genome editing will allow for a more efficient implementation of the technologies, and the germline genotyping method will save valuable time and resources.