Arginase 1 does not affect RNA m6A methylation in mouse fetal lung.

BACKGROUND: Arginase 1 (Arg1) encodes a key enzyme that catalyzes the metabolism of arginine to ornithine and urea. In our recent study, we found that knockdown of Arg1 in the lungs of fetal mice induces apoptosis of epithelial cells and dramatically delays initiation of labor. As the most abundant internal mRNA modification, N6 -methyladenosine (m6 A) has been found to play important roles in lung development and cellular differentiation. However, if the knockdown of Arg1 affects the RNA m6A modification in fetal lungs remains unknown. METHODS: In the current study, the RNA m6A levels and the expression of RNA m6A related enzymes were validated in 13.0 dpc fetal lungs that Arg1 was knocked down by adeno-associated virus carrying Arg1-shRNA, using western blot, immunofluorescence, and RT-qPCR. RESULTS: No statistical differences were found in the expression of methyltransferase, demethylases, and binding proteins in the fetal lungs between AAV-shArg1-injected mice and AAV-2/9-injected mice. Besides, there is no significant change of overall RNA m6A level in fetal lungs from AAV-shArg1-injected mice, compared with that from AAV-2/9-injected mice. CONCLUSIONS: These results indicate that arginase 1 does not affect RNA m6A methylation in mouse fetal lung, and the mechanisms other than RNA m6A modification underlying the effects of Arg1 knockdown on the fetal lung development and their interaction with labor initiation need to be further explored.

Arginase 1 and L-arginine coordinate fetal lung development and the initiation of labor in mice.

Fetal development and parturition are precisely regulated processes that involve continuous crosstalk between the mother and the fetus. Our previous discovery that wild-type mice carrying steroid receptor coactivator (Src)-1 and Src-2 double-deficient fetuses exhibit impaired lung development and delayed labor, which indicates that the signals for parturition emanate from the fetus. In this study, we perform RNA sequencing and targeted metabolomics analyses of the lungs from fetal Src-1/-2 double-knockout mice and find that expression of arginase 1 (Arg1) is significantly decreased, accompanied by increased levels of the Arg1 substrate L-arginine. Knockdown of Arg1 in the lungs of fetal mice induces apoptosis of epithelial cells and dramatically delays initiation of labor. Moreover, treatment of human myometrial smooth muscle cells with L-arginine significantly inhibits spontaneous contractions by attenuating activation of NF-κB and downregulating expression of contraction-associated protein genes. Transcription factors GR and C/EBPß increase transcription of Arg1 in an Src-1/Src-2-dependent manner. These findings provide new evidence that fetus-derived factors may play dual roles in coordinating fetal lung development and the initiation of labor.

RNA m6A modifications in mammalian gametogenesis and pregnancy.

In brief: RNA modifications play key roles in regulating various biological processes. This article discusses and summarizes the recent advances of RNA m6A modifications related to mammalian gametogenesis, early embryonic development, and miscarriage. Abstract: The epitranscriptome is defined as the collection of post-transcriptional chemical modifications of RNA in a cell. RNA methylation refers to the chemical post-transcriptional modification of RNA by selectively adding methyl groups under the catalysis of a methyltransferase. The N6 methyladenosine (m6A) is one of the most common of the more than 100 known RNA modifications. Recent research has revealed that RNA m6A modifications are reversible. Additionally, m6A containing RNA can be selectively identified by immunoprecipitation followed by high-throughput sequencing (MeRIP-SEQ). These two developments have inspired a tremendous effort to unravel the biological role of m6A. The role of RNA m6A modifications in immune regulation, cell division, stem cell renewal, gametogenesis, embryonic development, and placental function has gradually emerged, which is of great significance for the study of post-transcriptional regulation of gene expression in reproductive biology. This review summarizes the current knowledge about RNA m6A modification in a variety of mammalian reproductive events.

Oocyte competence is maintained by m6A methyltransferase KIAA1429-mediated RNA metabolism during mouse follicular development.

KIAA1429 (also known as vir-like m6A methyltransferase-associated protein (VIRMA)), a newly identified component of the RNA m6A methyltransferase complex, plays critical roles in guiding region-selective m6A deposition. However, in mammals, whether KIAA1429 mediates RNA m6A regulatory pathway functions in vivo remains unknown. Here, we show that the Kiaa1429-specific deficiency in oocytes resulted in female infertility with defective follicular development and fully grown germinal vesicle (GV) oocytes failing to undergo germinal vesicle breakdown (GVBD) and consequently losing the ability to resume meiosis. The oocyte growth is accompanied by the accumulation of abundant RNAs and posttranscriptional regulation. We found that the loss of Kiaa1429 could also lead to abnormal RNA metabolism in GV oocytes. RNA-seq profiling revealed that Kiaa1429 deletion altered the expression pattern of the oocyte-derived factors essential for follicular development. In addition, our data show that the conditional depletion of Kiaa1429 decreased the m6A levels in oocytes and mainly affected the alternative splicing of genes associated with oogenesis. In summary, the m6A methyltransferase KIAA1429-mediated RNA metabolism plays critical roles in folliculogenesis and the maintenance of oocyte competence.

METTL3-mediated m6A is required for murine oocyte maturation and maternal-to-zygotic transition.

N6-methyladenosine (m6A) is the most prevalent epigenetic modification of messenger RNA (mRNA) in higher eukaryotes; this modification is mainly catalyzed by a methyltransferase complex including methyltransferase-like 3 (METTL3) as a key factor. Although m6A modification has been proven to play an essential role in diverse biological processes, our knowledge of Mettl3 is still limited because Mettl3 mutations are lethal to embryos in both mammals and plants. In this study, we knocked down Mettl3 by microinjection of its specific short interfering RNAs (siRNAs) or morpholino into fully grown germinal vesicle (GV) oocytes. As a result, we demonstrated that knocking down Mettl3 in female germ cells severely inhibited oocyte maturation by decreasing mRNA translation efficiency and led to defects in the maternal-to-zygotic transition, probably due to its interference in disrupting mRNA degradation. The discovery from this study suggests that the reversible m6A modification has vital functions in mammalian oocyte maturation and pre-implantation embryonic development processes.

Deletion of maternal UHRF1 severely reduces mouse oocyte quality and causes developmental defects in preimplantation embryos.

The ubiquitin-like, containing PHD and RING finger domains, 1 (UHRF1) protein recognizes DNA methylation and histone modification and plays a critical role in epigenetic regulation. Recently, UHRF1 was shown to have a role in DNA methylation in oocytes and early embryos. Here, we reveal that maternal UHRF1 determines the quality of mouse oocytes. We generated oocyte-specific Uhrf1-knockout mice and found that females were sterile, and few maternal UHRF1-null embryos developed into blastocysts. The UHRF1-null oocytes had an increased incidence of aneuploidy and DNA damage. In addition to defective DNA methylation, histone modification was affected during oogenesis, with UHRF1-null germinal vesicle and metaphase II-stage oocytes exhibiting reduced global histone H3 lysine 9 dimethylation levels and elevated acetylation of histone H4 lysine 12. Taken together, our results suggest that UHRF1 plays an important role in determining oocyte quality and affects epigenetic regulation of oocyte maturation as a maternal protein, which is crucial for embryo developmental potential. Further exploration of the biologic function and underlying mechanisms of maternal UHRF1 will enhance our understanding of the maternal control of the oocyte and early embryonic development.-Cao, Y., Li, M., Liu, F., Ni, X., Wang, S., Zhang, H., Sui, X., Huo, R. Deletion of maternal UHRF1 severely reduces mouse oocyte quality and causes developmental defects in preimplantation embryos.

CAV1 regulates primordial follicle formation via the Notch2 signalling pathway and is associated with premature ovarian insufficiency in humans.

STUDY QUESTION: What is the function of CAV1 in folliculogenesis and female reproduction? SUMMARY ANSWER: CAV1 regulates germline cyst breakdown and primordial follicle (PF) formation in mice, and CAV1 mutation may be related to premature ovarian insufficiency (POI). WHAT IS KNOWN ALREADY: Pre-granulosa cells are essential for the establishment of the PF pool, which determines female fertility and reproductive lifespan. Cav1 participates in vascularization in fetal mouse ovaries. However, the role of CAV1 in early folliculogenesis and POI pathogenesis remains unclear. STUDY DESIGN, SIZE, DURATION: Cav1 function was investigated in mice and Human Embryonic Kidney 293 cells. Ovaries (six per group) were randomly assigned to Cav1-vivo-morpholino, control and control-morpholino groups, and all experiments were repeated at least three times. To investigate CAV1 mutations in women, 200 Chinese women with POI and 200 control individuals with regular menstrual cycles and normal endocrine profiles were recruited from the Center for Reproductive Medicine of Shandong University between September 2012 and December 2013. PARTICIPANTS/MATERIALS, SETTING, METHODS: Wild-type CD1 mice, Lgr5-EGFP-ires-CreERT2 (Lgr5-KI) reporter mice and Human Embryonic Kidney 293 cells were used for these experiments. Protein expression was detected by Western blot, and quantitative RT-PCR was used to detect gene expression. The expression pattern of CAV1 in mouse ovaries and the phenotype of Cav1 deficiency in mice were detected by immunofluorescence. Pre-granulosa cell proliferation in ovaries was detected by bromodeoxyuridine (BrdU) assay and immunofluorescence. The coding region of the CAV1 gene was sequenced in 200 women with POI and 200 controls. The functional effect of the novel mutation c.142 G > C (p.Glu48Gln) was investigated by Cell Counting Kit-8 (CCK8) assays and Western blot. MAIN RESULTS AND THE ROLE OF CHANCE: We confirmed that Cav1 deficiency in mouse ovary induced by CAV1-vivo-morpholino resulted in more multi-oocyte follicles than in the control and control-morpholino groups (P < 0.01). Suppression of Cav1 decreased Leucine rich repeat containing G protein coupled receptor 5 (Lgr5)-positive cell proliferation (P < 0.01) and reduced the number of Lgr5 and Forkhead box L2 (Foxl2) double-positive cells (P < 0.01). Furthermore, suppression of Cav1 inhibited ovarian epithelial Lgr5-positive cell proliferation and differentiation through the Notch2 signalling pathway. Two of the POI women carried novel CAV1 mutations (c.45 C > G synonymous and c.142 G > C [Glu48Gln]). The deleterious effect of p.Glu48Gln was corroborated by showing that it adversely affected the function of CAV1 in cell proliferation and NOTCH2 expression in HEK293FT cells. LARGE-SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The novel Glu48Gln mutation was only detected in one of 200 POI patients and we were unable to investigate its effects in the ovary. WIDER IMPLICATIONS OF THE FINDINGS: The identification of CAV1 as a potentially causative gene for POI provides a theoretical basis to devise treatments for POI in women. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Basic Research Program of China (973 Programs: 2012CB944700; 2013CB945501; 2013CB911400; 2014CB943202), the National Key Research and Development Program of China (2016YFC1000604, 2017YFC1001301), the State Key Program of National Natural Science Foundation of China (81430029), and the National Natural Science Foundation of China (31571540, 81522018, 81471509, 81601245, 81701406, 81571406). The authors declare no competing financial interests.

Fatty acid degradation plays an essential role in proliferation of mouse female primordial germ cells via the p53-dependent cell cycle regulation.

Primordial germ cells (PGCs) are embryonic founders of germ cells that ultimately differentiate into oocytes and spermatogonia. Embryonic proliferation of PGCs starting from E11.5 ensures the presence of germ cells in adulthood, especially in female mammals whose total number of oocytes declines after this initial proliferation period. To better understand mechanisms underlying PGC proliferation in female mice, we constructed a proteome profile of female mouse gonads at E11.5. Subsequent KEGG pathway analysis of the 3,662 proteins profiled showed significant enrichment of pathways involved in fatty acid degradation. Further, the number of PGCs found in in vitro cultured fetal gonads significantly decreased with application of etomoxir, an inhibitor of the key rate-limiting enzyme of fatty acid degradation carnitine acyltransferase I (CPT1). Decrease in PGCs was further determined to be the result of reduced proliferation rather than apoptosis. The inhibition of fatty acid degradation by etomoxir has the potential to activate the Ca(2+)/CamKII/5'-adenosine monophosphate-activated protein kinase (AMPK) pathway; while as an upstream activator, activated AMPK can function as activator of p53 to induce cell cycle arrest. Thus, we detected the expressional level of AMPK, phosphorylated AMPK (P-AMPK), phosphorylated p53 (P-p53) and cyclin-dependent kinase inhibitor 1 (p21) by Western blots, the results showed increased expression of them after treatment with etomoxir, suggested the activation of p53 pathway was the reason for reduced proliferation of PGCs. Finally, the involvement of p53-dependent G1 cell cycle arrest in defective proliferation of PGCs was verified by rescue experiments. Our results demonstrate that fatty acid degradation plays an important role in proliferation of female PGCs via the p53-dependent cell cycle regulation.