Background Cryopreservation induces transcriptomic and epigenetic modifications that strongly impairs sperm quality and function, and thus decrease reproductive performance. N6-methyladenosine (m6A) RNA methylation varies in response to stress and has been implicated in multiple important biological processes, including post-transcriptional fate of mRNA, metabolism, and apoptosis. This study aimed to explore whether cryopreservation induces m6A modification of mRNAs associated with sperm energy metabolism, cryoinjuries, and freezability. Results The mRNA and protein expression of m6A modification enzymes were significantly dysregulated in sperm after cryopreservation. Furthermore, m6A peaks were mainly enriched in coding regions and near stop codons with classical RRACH motifs. The mRNAs containing highly methylated m6A peaks (fts vs. fs) were significantly associated with metabolism and gene expression, while the genes with less methylated m6A peaks were primarily involved in processes regulating RNA metabolism and transcription. Furthermore, the joint analysis of DMMGs and differentially expressed genes indicated that both of these play a vital role in sperm energy metabolism and apoptosis. Conclusions Our study is the first to reveal the dynamic m6A modification of mRNAs in boar sperm during cryopreservation. These epigenetic modifications may affect mRNA expression and are closely related to sperm motility, apoptosis, and metabolism, which will provide novel insights into understanding of the cryoinjuries or freezability of boar sperm during cryopreservation.

Overproduction of pro-inflammatory cytokines in the aged, which is called inflammaging, leads to the deterioration of periodontitis. Toll-like receptor 4 (TLR4) plays a role in the regulation of cellular senescence, and its expression increases with age. However, there has been limited research into the molecular mechanisms underlying the onset of periodontal inflammaging, and the interplay between TLR4 and inflammaging. In the present study, wild-type and TLR4 gene knockout mice were used to investigate the activation of the TLR4 pathway in mouse periodontitis and the expression of the nucleotide-binding and oligomerization domain-like receptor 3 (NLRP3) inflammasome, an upstream immune checkpoint during the development of inflammaging. Activation of TLR4 in a mouse model of periodontitis enhanced the expression of a senescence-associated secretory phenotype (SASP), which boosted the inflammaging process. Conversely, TLR4 activation downregulated the expression of B cell-specific Moloney murine leukemia virus integration site 1 (Bmi-1) and promoted the priming of NLRP3 inflammasome, both of which are regulators of SASP. Treating gingival fibroblasts with Bmi-1 inhibitor PTC209, it was demonstrated that TLR4 activated the NLRP3 pathway and the inflammaging process by suppressing Bmi-1. In addition, there was a significant reduction in the expression of Bmi-1 expression in the gingiva of patients with periodontitis compared with healthy controls. In conclusion, the present study demonstrated that TLR4 acted by inhibiting Bmi-1 to enhance the NLRP3 pathway and SASP factors. This cascade of reactions may contribute to the senescence of the periodontium.

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Sperm chemotaxis, which guide sperm toward oocyte, is tightly associated with sperm capacitation, motility, and fertility. However, the molecular mechanism of sperm chemotaxis is not known. Reproductive odorant and taste receptors, belong to G-protein-coupled receptors (GPCR) super-family, cause an increase in intracellular Ca2+ concentration which is pre-requisite for sperm capacitation and acrosomal reaction, and result in sperm hyperpolarization and increase motility through activation of Ca2+-dependent Cl¯ channels. Recently, odorant receptors (ORs) in olfactory transduction pathway were thought to be associated with post-thaw sperm motility, freeze tolerance or freezability and cryo-capacitation-like change during cryopreservation. Investigation of the roles of odorant and taste receptors (TRs) is important for our understanding of the freeze tolerance or freezability mechanism and improve the motility and fertility of post-thaw sperm. Here, we reviewed the roles, mode of action, impact of odorant and taste receptors on sperm chemotaxis and post-thaw sperm quality.

This study aimed to determine whether Bmi-1 deficiency leads to intestinal epithelial barrier destruction and microbiota dysfunction, which members of the microbial community alter barrier function with age, and whether p16INK4a deletion could reverse the damage of intestinal epithelial barrier and microbial dysbiosis. Intestines from Bmi-1–deficient (Bmi-1–/–), Bmi-1 and p16INK4a double-knockout (Bmi-1–/–p16INK4a–/–), and wild-type mice were observed for aging and inflammation. Duolink Proximity Ligation Assay, immunoprecipitation, and construction of p16INK4a overexpressed adenovirus and the overexpressed plasmids of full-length, mutant, or truncated fragments for occludin were used for analyzing the interaction between p16INK4a and occludin. High-throughput sequencing of V4 region amplicon of 16S ribosomal RNA was conducted using intestinal microbiota. We found Bmi-1 deficiency destructed barrier structure, barrier function, and tight junction (TJ) in intestinal epithelium; decreased the TJ proteins; increased tumor necrosis factor α (TNF-α)–dependent barrier permeability; and up-regulated proinflammatory level of macrophages induced by intestinal microbial dysbiosis. The transplantation of fecal microbiota from wild-type mice ameliorated TJ in intestinal epithelium of Bmi-1–/– and Bmi-1–/–p16INK4a–/– mice. Harmful bacteria including Desulfovibrio, Helicobacter, and Oscillibacter were at a higher level in Bmi-1–/– mice. More harmful bacteria Desulfovibrio entered the epithelium and promoted macrophages-secreted TNF-α and caused TNF-α–dependent barrier permeability and aging. Accumulated p16INK4a combined with occludin at the 1st–160th residue in cytoplasm of intestinal epithelium cells from Bmi-1–/– mice, which blocked formation of TJ and the repair of intestinal epithelium barrier. P16INK4a deletion could maintain barrier function and microbiota balance in Bmi-1–/– mice through strengthening formation of TJ and decreasing macrophages-secreted TNF-α induced by Desulfovibrio entering the intestinal epithelium. Thus, Bmi-1 maintained intestinal TJ, epithelial barrier function, and microbiota balance through preventing senescence characterized by p16INK4a accumulation. The clearance of p16INK4a-positive cells in aging intestinal epithelium would be a new method for maintaining barrier function and microbiota balance. The residues 1–160 of occludin could be a novel therapeutic target for identifying small molecular antagonistic peptides to prevent the combination of p16INK4a with occludin for protecting TJ.

Cryopreservation induces sperm cryoinjuries, including physiological and functional changes. However, the molecular mechanisms of sperm cryoinjury and cryoresistance are still unknown. Cryoresistance or the freeze tolerance of sperm varies across species, and boar sperm is more susceptible to cold stress. Contrary to boar sperm, giant panda sperm appears to be strongly freeze-tolerant and is capable of surviving repeated cycles of freeze-thawing. In this study, differentially expressed (DE) PIWI-interacting RNAs (piRNAs) of fresh and frozen-thawed sperm with different freeze tolerance capacity from giant panda and boar were evaluated. The results showed that 1,160 (22 downregulated and 1,138 upregulated) and 384 (110 upregulated and 274 downregulated) DE piRNAs were identified in giant panda and boar sperm, respectively. Gene ontology (GO) enrichment analysis revealed that the target DE messenger RNAs (mRNAs) of DE piRNAs were mainly enriched in biological regulation, cellular, and metabolic processes in giant panda and boar sperm. Moreover, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that the target DE mRNAs of DE piRNAs were only distributed in DNA replication and the cyclic adenosine monophosphate (cAMP) signaling pathway in giant panda, but the cAMP, cyclic guanosine monophosphate (cGMP), and mitogen-activated protein kinase (MAPK) signaling pathways in boar sperm were considered as part of the olfactory transduction pathway. In conclusion, we speculated that the difference in the piRNA profiles and the DE piRNAs involved in the cAMP signaling pathway in boar and giant panda may have contributed to the different freeze tolerance capacities between giant panda and boar sperm, which helps to elucidate the molecular mechanism behind sperm cryoinjury and cryoresistance.

Diabetes mellitus (DM)-induced glucolipotoxicity is a factor strongly contributing to alveolar bone deficiency. Parathyroid hormone (PTH) has been identified as a main systemic mediator to balance physiological calcium in bone. This study aimed to uncover PTH's potential role in ameliorating the osteogenic capacity of human bone marrow mesenchymal stem cells (HBMSCs) against glucolipotoxicity. Optimal PTH concentrations and high glucose and palmitic acid (GP) were administered to cells, followed by alkaline phosphatase (ALP) staining and ALP activity assay. Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) and Immunoblot were carried out for assessing mRNA and protein amounts, respectively. Cell counting kit-8 (CCK-8) and flow cytometry were performed for quantitating cell proliferation. Osteogenesis and oxidative stress were determined, and the involvement of mitogen-activated protein kinase (MAPK) signaling was further verified. About 1-50 mmol/ml GP significantly inhibited the osteogenic differentiation of HBMSCs. 10 −9 mol/L PTH was found to be the optimal concentration for HBMSC induction.

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