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Alternative polyadenylation affects the length and composition of 3′‐untranslated region (3′‐UTR) and regulates mRNA stability or translational activity to affect important biological processes. However, global 3′‐UTR landscape and its relationship with gamete maturation remain less studied. Here, we analysed our previously reported single‐cell RNA‐seq data of germinal vesicle and metaphase II stage oocytes in pigs to systematically catalogue the 3′‐UTR dynamics during oocyte maturation. Two softwares (DaPars and APAtrap) were employed and identified 110 and 228 mRNAs with significantly different 3′‐UTRs (adjusted p ≤.05), respectively. Gene enrichment analyses found signalling pathways related with biological processes of female gametophyte production, methyltransferase activity and mRNA surveillance (DaPars) and cell cycle process, regulation of ERK1 and ERK2 cascade, regulation of translation, spindle organization, kinetochore, condensed chromosome and progesterone‐mediated oocyte maturation (APAtrap), respectively. Moreover, 18 of 110 mRNAs (|△PDUI| ≥ 0.25 and |log2PDUI ratio| ≥ 0.59) and 15 of 228 mRNAs (Perc. diff. ≥ 0.5) were with greater difference of 3′‐UTR length or abundance, and integrative genomics viewer analysis further identified 4 (Alg10, Hadhb, Hsd17b4 and Sbds) of 18 mRNAs to be with 3′‐UTR length differed ≥150 bp and 6 (Gcc1, Hnrnpa2b1, Lsm6, Prpf18, Sfr1 and Ust) of 15 mRNAs to be with 3′‐UTR abundance extremely differed. Furthermore, the location, sequences and number of cis‐elements were predicted, which were shown to derange cytoplasmic polyadenylation element, poly(A) site and microRNA binding sites within 3′‐UTRs of Alg10, Hadhb, Hsd17b4 and Sbds mRNAs. Taken together, global 3′‐UTR landscape changes dynamically with oocyte meiotic maturation, potentially involved in regulating oocyte meiotic process in pigs. [ABSTRACT FROM AUTHOR]

The faithful execution of molecular programme underlying oocyte maturation and meiosis is vital to generate competent haploid gametes for efficient mammalian reproduction. However, the organization and principle of molecular circuits and modules for oocyte meiosis remain obscure. Here, we employed the recently developed single‐cell RNA‐seq technique to profile the transcriptomes of germinal vesicle (GV) and metaphase II (MII) oocytes, aiming to discover the dynamic changes of mRNAs and long non‐coding RNAs (lncRNAs) during oocyte in vitro meiotic maturation. During the transition from GV to MII, total number of detected RNAs (mRNAs and lncRNAs) in oocytes decreased. Moreover, 1,807 (602 up‐ and 1,205 down‐regulated) mRNAs and 313 (177 up‐ and 136 down‐regulated) lncRNAs were significantly differentially expressed (DE), i.e., more mRNAs down‐regulated, but more lncRNAs up‐regulated. During maturation of pig oocytes, mitochondrial mRNAs were actively transcribed, eight of which (ND6, ND5, CYTB, ND1, ND2, COX1, COX2 and COX3) were significantly up‐regulated. Both DE mRNAs and targets of DE lncRNAs were enriched in multiple biological and signal pathways potentially associated with oocyte meiosis. Highly abundantly expressed mRNAs (including DNMT1, UHRF2, PCNA, ARMC1, BTG4, ASNS and SEP11) and lncRNAs were also discovered. Weighted gene co‐expression network analysis (WGCNA) revealed 20 hub mRNAs in three modules to be important for oocyte meiosis and maturation. Taken together, our findings provide insights and resources for further functional investigation of mRNAs/lncRNAs in in vitro meiotic maturation of pig oocytes. [ABSTRACT FROM AUTHOR]

Long noncoding RNAs (lncRNAs) regulate a variety of physiological and pathological processes. However, the biological function of lncRNAs in mammalian germ cells remains largely unexplored. Here we identified one novel lncRNA (lncRNA2193) from single‐cell RNA sequencing performed on porcine oocytes and investigated its function in oocyte meiosis. During in vitro maturation (IVM), from germinal vesicle (GV, 0 hr), GV breakdown (GVBD, 24 hr), to metaphase II stage (MII, 44 hr), the transcriptional abundance of lncRNA2193 remained stable and high. LncRNA2193 interference by small interfering RNA microinjection into porcine GV oocytes could significantly inhibit rates of GVBD and the first polar body extrusion, but enhance the rates of oocytes with a nuclear abnormality. Moreover, lncRNA2193 knockdown disturbed cytoskeletal organization (F‐actin and spindle), and decreased DNA 5‐methylcytosine (5mC) and histone trimethylation (H3K4me3, H3K9me3, H3K27me3, and H3K36me3) levels. The lncRNA2193 downregulation induced a decrease of 5mC level could be partially due to the reduction of DNA methyltransferase 3A and 3B, and the elevation of 5mC‐hydroxylase ten‐11 translocation 2 (TET2). After parthenogenetic activation of MII oocytes, parthenotes exhibited higher fragmentation but lower cleavage rates in the lncRNA2193 downregulated group. However, lncRNA2193 interference performed on mature MII oocytes and parthenotes at 1‐cell stage did not affect the cleavage and blasctocyst rates of pathenotes. Taken together, lncRNA2193 plays an important role in porcine oocyte maturation, providing more insights for relevant investigations on mammalian germ cells. [ABSTRACT FROM AUTHOR]

Heat shock protein 90 (Hsp90) functions as a molecular chaperone in its interaction with clients to influence multiple cellular and physiological processes. However, our current understanding on Hsp90's relationship with mammalian oocyte maturation is still very limited. Here, we aimed to investigate Hsp90's effect on pig oocyte meiotic maturation. Endogenous Hsp90a was constantly expressed at both mRNA and protein levels in porcine maturing oocytes. Addition of 2 µM 17-allylamino-17-demethoxygeldanamycin (17-AAG), the Hsp90 inhibitor, to in vitro mature cumulus-oocyte complexes (COC) significantly decreased Hsp90a protein level (P < 0.05), delayed germinal vesicle breakdown (GVBD) (P < 0.05), and impeded the first polar body (PB1) extrusion (P < 0.01) of porcine oocytes. 2 µM 17-AAG treatment during in vitro maturation also decreased the subsequent development competence as indicated by the lower cleavage (P < 0.001) and higher fragmentation (P < 0.001) rates of parthenotes, whereas no effects on the percentage and average cell number of blastocysts were found. Immunodepletion of Hsp90a by antibody microinjection into porcine oocytes at germinal vesicle and metaphase II stages induced similar defects of meiotic maturation and parthenote development, to that resulted from 2 µM inhibitor 17-AAG. For oocytes treated by 2 µM 17-AAG, the cytoplasm and membrane actin levels were weakened (P < 0.01), and the spindle assembly was disturbed (P < 0.05), due to decreased p-ERK1/2 level (P < 0.05). However, the mitochondrial function and early apoptosis were not affected, as demonstrated by rhodamine 123 staining and Annexin V assays. Our findings indicate that Hsp90a can couple with mitogen-activated protein kinase to regulate cytoskeletal structure and orchestrate meiotic maturation of porcine oocytes. [ABSTRACT FROM AUTHOR]

Coenzyme Q10 (CoQ10) is essential to many fundamental biological processes. However, the effect of CoQ10 on meiotic maturation of pig oocytes still remains elusive. In the present study we aimed to understand the effects of CoQ10 on porcine oocyte maturation, by supplementing different concentrations of CoQ10 (25, 50 and 100 μM) into the maturation medium. We showed that CoQ10 at 50 μM had better capacity to promote the nuclear maturation of pig oocytes derived from both small and large antral follicles. Though the cleavage and blastocyst rates of parthenotes stayed stable, 50 μM CoQ10 treatment could accelerate the development of parthenotes to blastocyst stage, and increase the average cell number of blastocyst. For cumulus-oocyte complexes from large antral follicles categorized by the brilliant cresyl blue (BCB) test, 50 μM CoQ10 treatment could specifically promote the nuclear maturation of poor-quality oocytes in the BCB-negative group. Mitochondrial function of oocytes treated by 50 μM CoQ10 could be boosted, through increasing the levels of mitochondrial membrane potential, ATP production and CoQ6, and changing the pattern of mitochondrial distribution as well. Moreover, 50 μM CoQ10 treatment suppressed the level of reactive oxygen species and reduced the percentage of oocytes with early apoptosis signal. Taken together, CoQ10 could improve the meiotic maturation of pig oocytes, especially for poor-quality oocytes, mainly through enhancing mitochondrial function and suppressing oxidative stress to reduce apoptosis. • CoQ10 (50 μM) could improve the nuclear maturation of pig oocytes, especially the poor-quality oocytes. • CoQ10 (50 μM) promotes the developmental competence of oocytes. • CoQ10 (50 μM) reduces the ROS level, suppresses apoptosis and boosts the mitochondrial function of pig oocytes. [ABSTRACT FROM AUTHOR]

Lysosome, an important organelle in eukaryotes, can sequester macromolecules submitted by the endocytosis and autophagy pathways for degradation and recycling. Massive macromolecular turnover is also vital to the growth and development of mammalian oocytes. However, the functional role of lysosomes in the meiotic maturation of mammalian oocytes remains largely unexplored. Here, by treating in vitro matured porcine cumulus-oocyte complexes (COCs) with chloroquine (CQ), a lysosome inhibitor, we showed that regardless of CQ concentration, lysosomal inhibition affected neither the extrusion of the first polar body (PB1), nor the ROS levels. However, CQ treatment dramatically decreased the rates of oocytes with normal chromosome alignment and cytoskeleton organization (P < 0.05), but boosted the rates of oocytes with apoptosis (P < 0.05). Subsequently, after pathenogenetic activation or in vitro fertilization, the death or fragmentation rates of oocytes treated by CQ (both 35 μM and 45 μM) were significantly higher (P < 0.05), whereas the rates of embryo cleavage, embryos developed to blastocysts, and average blastomere number per blastocyst, were all significantly lower (P < 0.05), respectively. Furthermore, CQ (35 μM) treatment activated the autophagy pathway by elevating the LC3 II/I ratio. Taken together, lysosomes could affect porcine oocyte maturation and subsequent developmental capacity partially through the chromosome organization/cytoskeleton assembly and autophagy/apoptosis pathways. • Lysosomal inhibition during IVM of pig oocytes neither affected the extrusion of PB1, nor changed the ROS level. • Lysosomal inhibition induced by chloroquine suppressed the developmental competence of pig oocytes. • Lysosomal dysfunction disturbed chromosomal alignment/cytoskeleton organization, and induced oocyte apoptosis in pig. • Chloroquine treatment could activate the autophagy pathway by elevating the LC3 II/I ratio in pig oocytes. [ABSTRACT FROM AUTHOR]

Abstract Oocyte meiosis is a complex process coordinated by multiple endocrinal and molecular circuits. Recently, N6-methyladenosine (m6A) epigenetic modification on RNA is revealed to be important for meiotic maturation. However, the molecular mechanism of how m6A modification exerts its effect on oocyte maturation is largely unknown. Here, we showed that endogenous m6A writers (Mettl3 and Wtap) and eraser (Fto) elevated their transcript levels during meiotic maturation of pig oocytes. From germinal vesicle (GV) to metaphase II (MII) stages, global m6A level significantly increased, and existed mostly in ooplasm. Methyl donor (betaine, 16 mM) treatment of porcine cumulus-oocyte complexes (COCs) during in vitro maturation (IVM) significantly boosted nucleic acid m6A level within oocytes, but unchanged meiotic process and oocyte subsequent development. By contrast, methylation inhibitor (cycloleucine, 20 mM) reduced nucleic acid m6A level, and significantly decreased the germinal vesicle breakdown (GVBD) rate, the extrusion rate of the first polar body, and the cleavage and blastocyst rates of parthenotes. In addition, in cycloleucine-treated oocytes Wtap increased but Lin28 decreased their abundances significantly, along with the higher incidence of spindle defects and chromosome misalignment. Furthermore, pT161-CDK1 protein level in pig oocytes was confirmed to be decreased after cycloleucine treatment for 24 h. Taken together, chemical induced reduction of nucleic acid m6A methylation during pig oocyte meiosis could impair meiotic maturation and subsequent development potency, possibly through down-regulating pluripotency marker Lin28 mRNA abundance and disturbing MPF-regulated chromosome/spindle organization. Highlights • Endogenous m6A writers Mettl3 and Wtap, eraser Fto elevated their transcript levels in maturing pig oocytes. • Global m6A modification level significantly increased in maturing pig oocytes. • Betaine treatment significantly boosted m6A level, but unaffected oocyte meiosis and subsequent development ability. • Cycloleucine treatment significantly reduced m6A level, that impaired meiotic maturation and subsequent development potency. • Cycloleucine could down-regulate Lin28 and disturb MPF-regulated chromosome/spindle organization to affect porcine oocytes. [ABSTRACT FROM AUTHOR]