Residual network improves the prediction accuracy of genomic selection.

Genetic improvement of complex traits in animal and plant breeding depends on the efficient and accurate estimation of breeding values. Deep learning methods have been shown to be not superior over traditional genomic selection (GS) methods, partially due to the degradation problem (i.e. with the increase of the model depth, the performance of the deeper model deteriorates). Since the deep learning method residual network (ResNet) is designed to solve gradient degradation, we examined its performance and factors related to its prediction accuracy in GS. Here we compared the prediction accuracy of conventional genomic best linear unbiased prediction, Bayesian methods (BayesA, BayesB, BayesC, and Bayesian Lasso), and two deep learning methods, convolutional neural network and ResNet, on three datasets (wheat, simulated and real pig data). ResNet outperformed other methods in both Pearson's correlation coefficient (PCC) and mean squared error (MSE) on the wheat and simulated data. For the pig backfat depth trait, ResNet still had the lowest MSE, whereas Bayesian Lasso had the highest PCC. We further clustered the pig data into four groups and, on one separated group, ResNet had the highest prediction accuracy (both PCC and MSE). Transfer learning was adopted and capable of enhancing the performance of both convolutional neural network and ResNet. Taken together, our findings indicate that ResNet could improve GS prediction accuracy, affected potentially by factors such as the genetic architecture of complex traits, data volume, and heterogeneity.

Selection of reference genes for RT-qPCR analysis in developing chicken embryonic ovary.

BACKGROUND: Normalization of the expression profiling of target genes, in a tissue-specific manner and under different experimental conditions, requires stably expressed gene(s) to be used as internal reference(s). However, to study the molecular regulation of oocyte meiosis initiation during ovary development in chicken embryos, stable reference gene(s) still need to be compared and confirmed. METHODS AND RESULTS: Six candidate genes previously used as internal references for the chicken embryo (Actb, Cvh, Dazl, Eef1a, Gapdh and Rpl15) were chosen, and their expression profiles in left ovaries dissected at five chicken embryonic days (E12.5, E15.5, E17.5, E18.5 and E20.5) were evaluated, respectively. Separately, GeNorm, NormFinder, BestKeeper and Comparative ΔCt methods were used to assess the stability of candidate reference genes, and all results were combined to give the final rank by RefFinder. All methods identified that Eef1a and Rpl15 were the two most stable internal reference genes, whereas Cvh is the most unstable one. Moreover, expression levels of three marker genes for chicken oocyte meiosis entry (Stra8, Scp3 and Dmc1) were normalized, based on Eef1a, Rpl15, or their combinations, respectively. CONCLUSION: Our findings provide the most suitable internal reference genes (Eef1a and Rpl15), to investigate further molecular regulation of ovary development and oocyte meiosis initiation in chicken embryos.

Single-cell RNA sequencing reveals blastomere heterogeneity of 2-cell embryos in pigs.

In mammals, single blastomeres from as early as 2-cell embryos demonstrate heterogeneous developmental capacity and fate decision into different cell lineages. However, mechanisms underlying blastomere heterogeneity of 2-cell embryos remain largely unresolved. Here, we analysed the molecular heterogeneity of full-length mRNAs and their 3'UTR regions, based on the single-cell RNA-seq data of pig 2-cell embryos generated from in vivo fertilization (in vivo), in vitro fertilization (in vitro) and parthenogenetic activation (PA), respectively. First, unsupervised clustering helped discover two different groups of blastomeres for 2-cell pig embryos. Between these two groups of blastomeres in pig 2-cell embryos, 35, 301 and 428 full-length mRNAs respectively in in vivo, in vitro and PA embryo types were identified to be differentially expressed (padj ≤ .05 and |log2 [fold change]| ≥1) (DE mRNAs), while 92, 89 and 42 mRNAs were shown to be with significantly different 3'UTR lengths (3'UTR DE) (padj ≤ .05). Gene enrichment for both DE mRNAs and 3'UTR DE mRNAs found multiple signalling pathways, including cell cycle, RNA processing. Few numbers of common DE mRNAs and 3'UTR DE mRNAs existed between in vitro and in vivo blastomeres derived from 2-cell embryos, indicating the larger differences between in vitro and in vivo fertilized embryos. Integrative genomics viewer analysis further identified that 3'UTRs of HSDL2 and SGTA (in vivo), FAM204A and phosphoserine phosphatase (in vitro), PRPF40A and RPIA (PA) had >100 nt average length changes. Moreover, numbers and locations of regulatory elements (polyadenylation site, cytoplasmic polyadenylation element and microRNA binding sites) within 3'UTRs of these DE mRNAs were predicted. These results indicate that molecular heterogeneity existed among blastomeres from different types of pig 2-cell embryos, providing useful information and novel insights into future functional investigation on its relationship with the subsequent embryo development and differentiation.

Integrated single cell transcriptome sequencing analysis reveals species-specific genes and molecular pathways for pig spermiogenesis.

Mammalian spermatogenesis is a highly complicated and intricately organized process involving spermatogonia propagation (mitosis) and meiotic differentiation into mature sperm cells (spermiogenesis). In pigs, spermatogonia development and the role of somatic cells in spermatogenesis were previously investigated in detail. However, the characterization of key molecules fundamental to pig spermiogenesis remains less explored. Here we compared spermatogenesis between humans and pigs, focusing on spermiogenesis, by integrative testicular single-cell RNA sequencing (scRNA-seq) analysis. Human and pig testicular cells were clustered into 26 different groups, with cell-type-specific markers and signalling pathways. For spermiogenesis, pseudo-time analysis classified the lineage differentiation routes for round, elongated spermatids and spermatozoa. Moreover, markers and molecular pathways specific to each type of spermatids were examined for humans and pigs, respectively. Furthermore, high-dimensional weighted gene co-expression network analysis (hdWGCNA) identified gene modules specific for each type of human and pig spermatids. Hub genes (pig: SNRPD2.1 related to alternative splicing; human: CATSPERZ, Ca[2+] ion channel) potentially involved in spermiogenesis were also revealed. Taken together, our integrative analysis found that human and pig spermiogeneses involve specific genes and molecular pathways and provided resources and insights for further functional investigation on spermatid maturation and male reproductive ability.

Dynamic changes of 3'UTR length during oocyte-to-zygote transition of in vitro pig embryos.

Alternative polyadenylation (APA) generates different 3'-untranslated regions (3'UTRs) to regulate gene expression and localization, and affects a variety of biological processes. Here, we characterized the 3'UTR dynamics during the oocyte-to-zygote transition by analysing our previously reported porcine single-cell RNA-seq (scRNA-seq) datasets (in vitro matured metaphase II (MII) oocytes, in vitro fertilized zygotes (IVF1) and parthenogenetically activated 1-cell embryos (PA1)). After IVF1 versus MII comparison, dynamic analyses of APA from RNA-seq (DaPars) method identified 139 mRNAs with significantly different 3'UTRs (padj . ≤ .05), mainly enriched in cell cycle, regulation of cyclin-dependent protein kinase activity, histone modification, mRNA surveillance, and regulation of actin cytoskeleton. For PA1 versus MII comparison, 105 mRNAs with significantly different 3'UTRs (padj . ≤ .05) were identified to be mainly enriched in intracellular transport, mitotic spindle organization, cell cycle, pyruvate metabolism and glycolysis/gluconeogenesis. Furthermore, there were 7 mRNAs with more significant 3'UTR differences (|△PDUI| ≥ 0.45 and |log2 [PDUI ratio]| ≥ 0.59) respectively in IVF1 versus MII (Lrp2bp, Mtfr2, Nhlrc2, Psip1, Smu1, Ssr1 and Wtap) and PA1 versus MII (Asf1b, Dimt1, Nap1l1, Ncoa4, Nudt21, Pnn and Rpl15) comparisons. Integrative genomics viewer analysis further identified that 3'UTRs of Psip1, Smu1, Ssr1 and Wtap had more than 140 nt average length changes, whereas those of Dimt1, Nap1l1 and Rpl15 were shortened with more than 460 nt. Regulatory elements (PAS, CPE, microRNA binding sites and m6 A sites) in 3'UTRs of different lengths were predicted. Our findings provide useful information to further investigate the molecular mechanism of 3'UTR in regulating the oocyte-to-zygote transition of pig embryos.

Comparative transcriptome analysis identifies important maternal molecules and associated biological pathways for pig and human mature oocytes.

Recent researches reveal that during oocyte maturation, species-specific molecular profile exists and has important functional roles. However, molecular differences between pig (a larger animal model for human reproduction) and human mature oocytes remain unknown. Here, by comparative transcriptome analyses of single-cell RNA-seq data, we aimed to identify the common and unique maternal factors and associated biological processes between in vivo and in vitro matured pig oocytes, and between in vitro matured human and pig oocytes. Annotated protein-coding mRNAs were identified in pig in vivo (11,147) and in vitro (11,997), and human in vitro (14,491) MII oocytes, respectively. For in vivo and in vitro derived pig MII oocytes, 10,551 annotated maternal mRNAs were common, mainly enriched in signalling pathways such as cell cycle, oocyte meiosis, microtubule cytoskeleton, MAPK, RNA processing/binding. Besides, in vivo (596) and in vitro (1446) pig MII-specific mRNAs and their involved signalling pathways (in vivo: Bmp, calcium-mediated signalling, PI3K-Akt; in vitro: growth factor activity, JAK-STAT, cytokine-cytokine receptor interaction, calcium signalling pathway) were also found. As for in vitro derived human and pig MII oocytes, 10,285 annotated mRNAs were common, enriched in a variety of signalling pathways (cell cycle, oocyte meiosis, microtubule, AMPK, RNA splicing, protein serine/threonine kinase activity, etc). In vitro MII-specific mRNAs were found for humans (4206) and pigs (1712), which were also enriched in species-specific signalling pathways (humans: golgi-related terms, transcription repressor and hormone activity; pigs: ATP biosynthetic process, G protein-coupled peptide receptor activity, animoacyl-tRNA biosynthesis), respectively. These findings improve our understanding on oocyte maturation, and also the limitations of pig model for human oocyte maturation and fertilization.

Identification of lncRNAs involved in maternal-to-zygotic transition of in vitro-produced porcine embryos by single-cell RNA-seq.

Long non-coding RNAs (lncRNAs) function through multiple tiers of molecular circuits and are vital to gamete maturation and early embryo development. However, in pig early embryos, identification and expression dynamics of lncRNAs remain less studied. Here, we systematically analysed the expression dynamics of lncRNAs based on our previously published single-cell RNA-seq data from pig mature oocytes (GSE160334), and single blastomeres biopsied from pig in vitro fertilized (IVF) and early parthenogenetically activated (PA) embryos (1- to 8-cell stages; GSE164812). With the progression of embryo development, the total number of expressed lncRNAs gradually decreased and showed great variation at each developmental stage for both IVF and PA groups. Consecutive stage pairwise comparison of MII oocytes, 1-cell zygotes, 2-cell, 4-cell and 8-cell IVF embryos identified 151, 245, 1119 and 188 differentially expressed (DE) lncRNAs, including 119, 80, 867, 77 up-regulated and 32, 165, 252, 111 down-regulated, while 289, 437, 895 and 495 DE lncRNAs (141, 89, 768, 97 up-regulated and 148, 348, 127, 398 down-regulated) were identified in PA embryos at the same stages. The DE lncRNAs identified within IVF embryos were much different from that identified within PA embryos, showing embryo type-specific manner. Further cross-comparison between PA and IVF embryos identified 184, 656, 2502 and 266 DE lncRNAs for the 1- to 8-cell embryo stages, respectively. Further GO and KEGG enrichment analysis of DE mRNAs targeted by DELs indicated that different signalling pathways were involved in maternal-only and bi-parental embryo development. Collectively, comparative profiling of lncRNA expression dynamics between pig IVF and PA embryos provides a valuable resource, to investigate further regulatory mechanisms of lncRNAs associated with ZGA and maternal RNA decay during early embryo development.

Global 3'-untranslated region landscape mediated by alternative polyadenylation during meiotic maturation of pig oocytes.

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 |log2 PDUI 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.

Identification of internal reference genes for porcine immature Sertoli cells under heat stress.

Identification of stably expressed gene(s) as internal reference(s) for different experimental conditions is key to the accurate normalization and quantification of target transcripts. Previously, our RNA-seq study showed that Hprt1, Actb, and 18S rRNA abundances were all significantly altered in porcine immature Sertoli cells (iSCs) during acute heat stress (HS). In the current study, we aimed to identify stable reference gene(s) to study the gene expression dynamics of quick and delayed responses after acute HS treatment of porcine iSCs. A total of six genes previously used in pig testis or Sertoli cells (Hprt1, Top2b, Actb, Rpl32, Gapdh, and 18S rRNA) were chosen to perform RT-qPCR for the control (before acute HS), HS0.5 (acute HS at 43°C for 0.5 h), and HS0.5-R36 (36 h recovery following acute HS) groups. The stability of candidate reference genes was examined by the GeNorm, NormFinder, BestKeeper and Comparative ΔCt methods, and RefFinder to obtain the final rank. Rpl32 and Actb were the two most stable internal reference genes as found by all methods, whereas Hprt1 and 18S rRNA were the two most unstable as ranked by RefFinder. Moreover, expression of six target mRNAs (Ccn1, Ccnb1, Eif4g1, Hdac6, Plk2, and Ptma) was normalized using Rpl32, Actb, or the combination of Rpl32 and Actb, respectively. Therefore, our findings that the most suitable internal references are Rpl32 and Actb provide useful information for further functional investigation on genes regulating the acute HS of porcine iSCs.

Network analysis reveals different hub genes and molecular pathways for pig in vitro fertilized early embryos and parthenogenotes.

Maternal-to-zygotic transition (MZT) occurs when maternal transcripts decay and zygotic genome is activated gradually at the early stage of embryo development. Previously, single-cell RNA-seq (scRNA-seq) has helped us to uncover the MZT-associated mRNA dynamics of in vitro-produced pig early embryos. Here, to further investigate functional modules and hub genes associated with MZT process, the weighted gene co-expression network analysis (WGCNA) was performed on our previously generated 45 scRNA-seq datasets. For the in vitro fertilized embryo (IVF) group, 5 significant modules were identified (midnight blue/black/red and blue/brown modules, positively correlated with 1-cell (IVF1) and 8-cell (IVF8), respectively), containing genes mainly enriched in signalling pathways such as Wnt, regulation of RNA transcription, fatty acid metabolic process, poly(A) RNA binding and lysosome. For the parthenogenetically activated embryo (PA) group, 9 significant modules were identified (black/purple/red, brown/turquoise/yellow, and magenta/blue/green modules, positively correlated with MII oocytes, 1-cell (PA1) and 8-cell (PA8), respectively), mainly enriched in extracellular exosome, poly(A) RNA binding, mitochondrion and transcription factor activity. Moreover, some of identified hub genes within 3 IVF and 9 PA significant modules, including ADCY2, DHX34, KDM4A, GDF10, ABCC10, PAFAH2, HEXIM2, COQ9, DCAF11, SGK1 and ESRRB, have been reported to play vital roles in different biological processes. Our findings provide information and resources for subsequent in-depth study on the regulation and function of MZT in pig embryos.

Benzophenone-3 breaches mouse Sertoli cell barrier and alters F-actin organization without evoking apoptosis.

Benzophenone-3 (BP-3), one of the most commonly utilized ultraviolet filters in personal care products, has aroused public concern in recent years for its high chances of human exposure. Previous studies have found that BP-3 can impair testes development and spermatogenesis, but the targets of BP-3 are still unknown. In this study, primary Sertoli cells from 20-day-old mice were treated in vitro with 0-100 µM BP-3 for 24 h to identify its toxicity on Sertoli cells and Sertoli cell barrier. Results demonstrated that BP-3 could induce a notable change in cell morphology and impair Sertoli cell viability. The analysis of transepithelial electrical resistance showed that the integrity of the Sertoli cell barrier was destroyed by BP-3 (100 µM). Some structural proteins of the barrier including ZO-1, Occludin, and Connexin43 were lower expressed and the localization of basal ectoplasmic specializations protein ß-catenin was altered because of BP-3 treatment. Further exploration suggested that BP-3 led to Sertoli cell F-actin disorganization by affecting the expression of Rictor, a key component of the mTORC2 complex. Moreover, although increased DNA damage marker γH2A.X was observed in the treatment group, the cell apoptosis rate was changeless which was further confirmed by increased BAX and stable Bcl-2 (two primary apoptosis regulating proteins). In conclusion, this study revealed that BP-3 had the potential to perturb the Sertoli cell barrier through altered junction proteins and disorganized F-actin, but it could hardly evoke Sertoli cell apoptosis.

Bisphenol F exposure affects mouse oocyte in vitro maturation through inducing oxidative stress and DNA damage.

Bisphenol F (BPF), a substitute for bisphenol A (BPA), is progressively used to manufacture various consumer products. Despite the established reproductive toxicity of BPF, the underlying mechanisms remain to elucidate. This in-vitro study deep in sighted the BPF toxicity on mouse oocyte meiotic maturation and quality. After treating oocytes with BPF (300 µM), the oocyte meiotic progression was blocked, accentuated by a reduced rate in the first polar body extrusion (PBE). Next, we illustrated that BPF induced α-tubulin hyper-acetylation disrupted the spindle assembly and chromosome alignment. Concurrently, BPF resulted in severe oxidative stress and DNA damage, which triggered the early apoptosis in mouse oocytes. Further, altered epigenetic modifications following BPF exposure were proved by increased H3K27me3 levels. Concerning the toxic effects on spindle structure, oxidative stress, and DNA damage in mouse oocytes, BPF toxicity was less severe to oocyte maturation and spindle structure than BPA and induced low oxidative stress. However, compared with BPA, oocytes treated with BPF were more prone to DNA damage, indicating not less intense or even more severe toxic effects of BPF than BPA on some aspects of oocytes maturation. In brief, the present study established that like wise to BPA, BPF could inhibit meiotic maturation and reduce oocyte quality, suggesting it is not a safe substitute for BPA.

Single-cell RNA-seq reveals mRNAs and lncRNAs important for oocytes in vitro matured in pigs.

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.

Long non-coding RNA MALAT1 targeting STING transcription promotes bronchopulmonary dysplasia through regulation of CREB.

Bronchopulmonary dysplasia (BPD) is a severe complication of preterm infants characterized by increased alveolarization and inflammation. Premature exposure to hyperoxia is believed to be a key contributor to the pathogenesis of BPD. No effective preventive or therapeutic agents have been created. Stimulator of interferon gene (STING) is associated with inflammation and apoptosis in various lung diseases. Long non-coding RNA MALAT1 has been reported to be involved in BPD. However, how MALAT1 regulates STING expression remains unknown. In this study, we assessed that STING and MALAT1 were up-regulated in the lung tissue from BPD neonates, hyperoxia-based rat models and lung epithelial cell lines. Then, using the flow cytometry and cell proliferation assay, we found that down-regulating of STING or MALAT1 inhibited the apoptosis and promoted the proliferation of hyperoxia-treated cells. Subsequently, qRT-PCR, Western blotting and dual-luciferase reporter assays showed that suppressing MALAT1 decreased the expression and promoter activity of STING. Moreover, transcription factor CREB showed its regulatory role in the transcription of STING via a chromatin immunoprecipitation. In conclusion, MALAT1 interacts with CREB to regulate STING transcription in BPD neonates. STING, CREB and MALAT1 may be promising therapeutic targets in the prevention and treatment of BPD.

Long noncoding RNA 2193 regulates meiosis through global epigenetic modification and cytoskeleton organization in pig oocytes.

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.

Kupffer Cells Regulate Natural Killer Cells Via the NK group 2, Member D (NKG2D)/Retinoic Acid Early Inducible-1 (RAE-1) Interaction and Cytokines in a Primary Biliary Cholangitis Mouse Model.

BACKGROUND Kupffer cells and natural killer (NK) cells has been identified as contributing factors in the pathogenesis of hepatitis, but the detailed mechanism of these cell types in the pathogenesis of primary biliary cholangitis (PBC) is poorly understood. MATERIAL AND METHODS In this study, polyinosinic: polycytidylic acid (poly I: C), 2-octynoic acid-bovine serum albumin (2OA-BSA) and Freund's adjuvant (FA) were injected to establish a murine PBC model, from which NK cells and Kupffer cells were extracted and isolated. The cells were then co-cultivated in a designed culture system, and then NK group 2, member D (NKG2D), retinoic acid early inducible-1 (RAE-1), F4/80, and cytokine expression levels were detected. RESULTS The results showed close crosstalk between Kupffer cells and NK cells. PBC mice showed increased surface RAE-1 protein expression and Kupffer cell cytokine secretion, which subsequently activated NK cell-mediated target cell killing via NKG2D/RAE-1 recognition, and increased inflammation. NK cell-derived interferon-γ (IFN-γ) and Kupffer cell-derived tumor necrosis factor alpha (TNF-alpha) were found to synergistically regulate inflammation. Moreover, interleukin (IL)-12 and IL-10 improved the crosstalk between NK cells and Kupffer cells. CONCLUSIONS Our findings in mice are the first to suggest the involvement of the NKG2D/RAE-1 interaction and cytokines in the synergistic effects of NK and Kupffer cells in PBC.

Single-cell transcriptome sequencing reveals that cell division cycle 5-like protein is essential for porcine oocyte maturation.

The brilliant cresyl blue (BCB) test is used in both basic biological research and assisted reproduction to identify oocytes likely to be developmentally competent. However, the underlying molecular mechanism targeted by the BCB test is still unclear. To explore this question, we first confirmed that BCB-positive porcine oocytes had higher rates of meiotic maturation, better rates of cleavage and development into blastocysts, and lower death rates. Subsequent single-cell transcriptome sequencing on porcine germinal vesicle (GV)-stage oocytes identified 155 genes that were significantly differentially expressed between BCB-negative and BCB-positive oocytes. These included genes such as cdc5l, ldha, spata22, rgs2, paip1, wee1b, and hsp27, which are enriched in functionally important signaling pathways including cell cycle regulation, oocyte meiosis, spliceosome formation, and nucleotide excision repair. In BCB-positive GV oocytes that additionally had a lower frequency of DNA double-strand breaks, the CDC5L protein was significantly more abundant. cdc5l/CDC5L inhibition by short interference (si)RNA or antibody microinjection significantly impaired porcine oocyte meiotic maturation and subsequent parthenote development. Taken together, our single-oocyte sequencing data point to a potential new role for CDC5L in porcine oocyte meiosis and early embryo development, and supports further analysis of this protein in the context of the BCB test.

Primary familial brain calcifications linked with a novel SLC20A2 gene mutation in a Chinese family.

It has been recently reported that mutations in SLC20A2 gene are a major cause of primary familial brain calcifications, a rare neurodegenerative disorder characterized by symmetrical and bilateral intracranial calcification. We conducted a pedigree study by performing next Generation Sequencing in a Chinese family with three generations. Three members in this family developed Parkinsonism in their sixth decade, also, the proband presented with schizophrenia for 40 years. Next Generation Sequencing identified a novel nonsense heterozygous substitution c.1158C > A (p.Thr 386*) of SLC20A2 gene, introducing a stop codon in exon 10. The mutation was present in symptomatic and asymptomatic individuals with intracranial calcification, but absent in the individual without calcification, suggesting the mutation segregates with brain calcification. mRNA expression was decreased by 35% in the proband. We are the first to demonstrate a novel c.1158C > A mutation of SLC20A2 gene in a Chinese family with primary familial brain calcifications.

MicroRNA-21 and PDCD4 expression during in vitro oocyte maturation in pigs.

BACKGROUND: MicroRNA (miRNA) are small non-coding RNA molecules critical for regulating cellular function, and are abundant in the maturing oocyte and developing embryo. MiRNA-21 (MIR21) has been shown to elicit posttranscriptional gene regulation in several tissues associated with rapid cell proliferation in addition to demonstrating anti-apoptotic features through interactions with PDCD4 mRNA and other targets. In many tissues, MIR21 interacts and suppresses PDCD4 due to the strong complementation between MIR21 and the PDCD4 3'UTR. METHODS: The objective of this project was to examine the relationship between MIR21 and PDCD4 expression in porcine oocytes during in vitro maturation and assess the impact of MIR21 inhibition during oocyte maturation on early embryo development. Additionally, we evaluated the effect of gonadotropins in maturation media and the presence of cumulus cells to determine their ability to contribute to MIR21 abundance in the oocyte during maturation. RESULTS: During in vitro maturation, expression of MIR21 increased approximately 6-fold in the oocyte and 25-fold in the cumulus cell. Temporally associated with this was the reduction of PDCD4 protein abundance in MII arrested oocytes compared with GV stage oocytes, although PDCD4 mRNA was not significantly different during this transition. Neither the presence of cumulus cells nor gonadotropins during in vitro maturation affected MIR21 abundance in those oocytes achieving MII arrest. However, inhibition of MIR21 activity during in vitro maturation using antisense MIR21 suppressed embryo development to the 4-8 cell stage following parthenogenetic activation. CONCLUSIONS: MIR21 is differentially expressed in the oocyte during meiotic maturation in the pig and inhibition of MIR21 during this process alters PDCD4 protein abundance suggesting posttranscriptional regulatory events involving MIR21 during oocyte maturation may impact subsequent embryonic development in the pig.