3-Methyl-4-nitrophenol Exposure Deteriorates Oocyte Maturation by Inducing Spindle Instability and Mitochondrial Dysfunction.

3-methyl-4-nitrophenol (PNMC), a well-known constituent of diesel exhaust particles and degradation products of insecticide fenitrothion, is a widely distributed environmental contaminant. PNMC is toxic to the female reproductive system; however, how it affects meiosis progression in oocytes is unknown. In this study, in vitro maturation of mouse oocytes was applied to investigate the deleterious effects of PNMC. We found that exposure to PNMC significantly compromised oocyte maturation. PNMC disturbed the spindle stability; specifically, it decreased the spindle density and increased the spindle length. The weakened spindle pole location of microtubule-severing enzyme Fignl1 may result in a defective spindle apparatus in PNMC-exposed oocytes. PNMC exposure induced significant mitochondrial dysfunction, including mitochondria distribution, ATP production, mitochondrial membrane potential, and ROS accumulation. The mRNA levels of the mitochondria-related genes were also significantly impaired. Finally, the above-mentioned alterations triggered early apoptosis in the oocytes. In conclusion, PNMC exposure affected oocyte maturation and quality through the regulation of spindle stability and mitochondrial function.

MeDIP-seq and RNA-seq analysis during porcine testis development reveals functional DMR at the promoter of LDHC.

Testis development requires tight regulation of gene expression programmed by epigenetic modifiers. However, their mechanism remains to be elucidated. Here, we investigated the genome-wide DNA methylation landscape in the Duroc and Meishan boar testes using methylated DNA immunoprecipitation sequencing (MeDIP-seq). We identified over 1100 promoter differential methylation genes (DMGs) before and after puberty, most of which are associated with testis development. Furthermore, we discovered that the expression of lactate dehydrogenase C (LDHC) gene during testis development is regulated by DNA methylation. The promoter of LDHC in pre-pubertal testes is substantially methylated, whereas considerably demethylated in post-pubertal testes. Artificial demethylation with the demethylating agent 5-Aza-CdR induced LDHC expression in immature Sertoli cells (SCs). Mechanistically, we confirmed the transcription factor SP1 was recruited to bind in hypomethylated differentially methylated regions (DMRs) in LDHC promoter, which upregulated the expression of LDHC. Functionally, we demonstrated that LDHC was activated in mature SCs (mSCs) and its overexpression significantly increases lactate secretion in SCs. In conclusion, our results highlight the function and regulation of dynamic DNA methylation in testis development.

TET Family Members Are Integral to Porcine Oocyte Maturation and Parthenogenetic Pre-Implantation Embryogenesis.

The ten-eleven translocation (TET) enzyme family, which includes TET1/2/3, participates in active DNA demethylation in the eukaryotic genome; moreover, TET1/2/3 are functionally redundant in mice embryos. However, the combined effect of TET1/2/3 triple-gene knockdown or knockout on the porcine oocytes or embryos is still unclear. In this study, using Bobcat339, a specific small-molecule inhibitor of the TET family, we explored the effects of TET enzymes on oocyte maturation and early embryogenesis in pigs. Our results revealed that Bobcat339 treatment blocked porcine oocyte maturation and triggered early apoptosis. Furthermore, in the Bobcat339-treated oocytes, spindle architecture and chromosome alignment were disrupted, probably due to the huge loss of 5-hydroxymethylcytosine (5hmC)and concurrent increase in 5-methylcytosine (5mC). After Bobcat339 treatment, early parthenogenetic embryos exhibited abnormal 5mC and 5hmC levels, which resulted in compromised cleavage and blastocyst rate. The mRNA levels of EIF1A and DPPA2 (ZGA marker genes) were significantly decreased, which may explain why the embryos were arrested at the 4-cell stage after Bobcat339 treatment. In addition, the mRNA levels of pluripotency-related genes OCT4 and NANOG were declined after Bobcat339 treatment. RNA sequencing analysis revealed differentially expressed genes in Bobcat339-treated embryos at the 4-cell stage, which were significantly enriched in cell proliferation, cell component related to mitochondrion, and cell adhesion molecule binding. Our results indicated that TET proteins are essential for porcine oocyte maturation and early embryogenesis, and they act by mediating 5mC/5hmC levels and gene transcription.

Progress on Infrared Imaging Technology in Animal Production: A Review.

Infrared thermography (IRT) imaging technology, as a convenient, efficient, and contactless temperature measurement technology, has been widely applied to animal production. In this review, we systematically summarized the principles and influencing parameters of IRT imaging technology. In addition, we also summed up recent advances of IRT imaging technology in monitoring the temperature of animal surfaces and core anatomical areas, diagnosing early disease and inflammation, monitoring animal stress levels, identifying estrus and ovulation, and diagnosing pregnancy and animal welfare. Finally, we made prospective forecast for future research directions, offering more theoretical references for related research in this field.

Integrated analysis of miRNA and mRNA expression profiles in testes of Duroc and Meishan boars.

BACKGROUND: MicroRNAs (miRNAs) are small non-coding RNAs playing vital roles in regulating posttranscriptional gene expression. Elucidating the expression regulation of miRNAs underlying pig testis development will contribute to a better understanding of boar fertility and spermatogenesis. RESULTS: In this study, miRNA expression profile was investigated in testes of Duroc and Meishan boars at 20, 75, and 270 days of age by high-throughput sequencing. Forty-five differentially expressed miRNAs were identified from testes of Duroc and Meishan boars before and after puberty. Integrated analysis of miRNA and mRNA profiles predicted many miRNA-mRNA pairs. Gene ontology and biological pathway analyses revealed that predicted target genes of ssc-mir-423-5p, ssc-mir-34c, ssc-mir-107, ssc-mir-196b-5p, ssc-mir-92a, ssc-mir-320, ssc-mir-10a-5p, and ssc-mir-181b were involved in sexual reproduction, male gamete generation, and spermatogenesis, and GnRH, Wnt, and MAPK signaling pathway. Four significantly differentially expressed miRNAs and their predicted target genes were validated by quantitative real-time polymerase chain reaction, and phospholipase C beta 1 (PLCß1) gene was verified to be a target of ssc-mir-423-5p. CONCLUSIONS: This study provides an insight into the functional roles of miRNAs in testis development and spermatogenesis and offers useful resources for understanding differences in sexual function development caused by the change in miRNAs expression between Duroc and Meishan boars.

The Interaction of lncRNA XLOC-2222497, AKR1C1, and Progesterone in Porcine Endometrium and Pregnancy.

The endometrium is an important tissue for pregnancy and plays an important role in reproduction. In this study, high-throughput transcriptome sequencing was performed in endometrium samples of Meishan and Yorkshire pigs on days 18 and 32 of pregnancy. Aldo-keto reductase family 1 member C1 (AKR1C1) was found to be a differentially expressed gene, and was identified by quantitative real-time PCR (qRT-PCR) and Western blot. Immunohistochemistry results revealed the cellular localization of the AKR1C1 protein in the endometrium. Luciferase activity assay demonstrated that the AKR1C1 core promoter region was located in the region from -706 to -564, containing two nuclear factor erythroid 2-related factor 2 (NRF2) binding sites (antioxidant response elements, AREs). XLOC-2222497 was identified as a nuclear long non-coding RNA (lncRNA) highly expressed in the endometrium. XLOC-2222497 overexpression and knockdown have an effect on the expression of AKR1C1. Endocrinologic measurement showed the difference in progesterone levels between Meishan and Yorkshire pigs. Progesterone treatment upregulated AKR1C1 and XLOC-2222497 expression in porcine endometrial epithelial cells. In conclusion, transcriptome analysis revealed differentially expressed transcripts during the early pregnancy process. Further experiments demonstrated the interaction of XLOC-2222497/AKR1C1/progesterone in the endometrium and provided new potential targets for pregnancy maintenance and its control.

Circ0001470 Acts as a miR-140-3p Sponge to Facilitate the Progression of Embryonic Development through Regulating PTGFR Expression.

Embryonic implantation and development are vital in early pregnancy and assisted reproduction. Circular RNAs (circRNAs) are involved in the two physiological processes and thus regulate animal reproduction. However, their specific regulatory functions and mechanisms remain unclear. Here, a novel circ0001470, originating from the porcine GRN gene, differentially expressed on day 18 versus day 32 of gestation in Meishan and Yorkshire pigs was screened. The circularization characteristic of circ0001470 was identified based on divergent primer amplification, Sanger sequencing, RNase digestion, and RNA nuclear-cytoplasmic fractionation. Functionally, circ0001470 can promote cell proliferation and cycle progression of endometrial epithelial cells (EECs) and also inhibit apoptosis of EECs using CCK-8 assays and flow cytometry analyses. Mechanistically, bioinformatics database prediction, luciferase screening, RNA immunoprecipitation (RIP), RNA-pull down, and FISH co-localization experiments revealed that the circ0001470 acted as a competing endogenous RNA (ceRNA) through sponging miR-140-3p to regulate downstream PTGFR expression. Moreover, in vivo assays revealed that mmu_circGRN promoted embryonic development by affecting the expression of PTGFR, which can activate the MAPK reproduction pathway and facilitate pregnancy maintenance. This study enriched our understanding of circRNAs in embryo implantation and development by deciding the fate of EECs.

Expression and regulation of GnRHR2 gene and testosterone secretion mediated by GnRH2 and GnRHR2 within porcine testes.

Gonadotropin-releasing hormone 2 receptor (GnRHR2) together with its cognate ligand involves in regulating reproductive behavior. However, little is known concerning the effect of transcription factor steroidogenic factor1 (SF-1) regulation on porcine GnRHR2 gene expression and GnRH2 regulation mechanism in testosterone secretion through GnRHR2. Our study demonstrated that GnRHR2 transcription levels were high in porcine testis. Immunohistochemistry analyses showed that GnRHR2 immunoreactivity was strong in the Leydig cells in boar testes. Two SF-1 binding sites were predicted in GnRHR2 promoter and the second site (-159/-149) was considered to be important for GnRHR2 promoter activity through site-directed mutagenesis. The binding of SF-1 to GnRHR2 promoter was confirmed by electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP). Overexpression and knockdown experiments revealed that SF-1 could up-regulate porcine GnRHR2 expression. DNA methylation of GnRHR2 promoter CpG island also specifically regulated GnRHR2 expression. Meanwhile, our study also demonstrated GnRH2 treatment promoted the expression of SF-1 and steroidogenic acute regulatory protein (StAR), and that this treatment stimulated cAMP responsive element binding protein (CREB) phosphorylation, regulated the expression of GnRHR2, especially that of GnRHR2-X1, and promoted testosterone secretion in porcine Leydig cells. We speculated that testosterone secretion mediated by GnRH2 and GnRHR2 (mainly GnRHR2-X1) was regulated by phosphorylated CREB interacting with SF-1 to control StAR expression. Taken together, the present study indicates that SF-1 and GnRH2 are the essential regulatory factors for GnRHR2 expression. This study also explores the regulation mechanism of testosterone secretion mediated by GnRH2 and GnRHR2 in porcine Leydig cells.