Odorant Receptor OR2C1 Is an Essential Modulator of Boar Sperm Capacitation by Binding with Heparin.

Heparin, a class of glycosaminoglycans (GAGs), is widely used to induce sperm capacitation and fertilization. How heparin induces sperm capacitation remains unclear. Olfactory receptors (ORs) which are G protein-coupled receptors, have been proposed to be involved in sperm capacitation. However, the interaction between ORs and odor molecules and the molecular mechanism of ORs mediating sperm capacitation are still unclear. The present study aimed to explore the underlying interaction and mechanism between heparin and ORs in carrying out the boar sperm capacitation. The results showed that olfactory receptor 2C1 (OR2C1) is a compulsory unit which regulates the sperm capacitation by recognizing and binding with heparin, as determined by Dual-Glo Luciferase Assay and molecular docking. In addition, molecular dynamics (MD) simulation indicated that OR2C1 binds with heparin via a hydrophobic cavity comprises of Arg3, Ala6, Thr7, Asn171, Arg172, Arg173, and Pro287. Furthermore, we demonstrated that knocking down OR2C1 significantly inhibits sperm capacitation. In conclusion, we highlighted a novel olfactory receptor, OR2C1, in boar sperm and disclosed the potential binding of heparin to Pro287, a conserved residue in the transmembrane helices region 7 (TMH7). Our findings will benefit the further understanding of ORs involved in sperm capacitation and fertilization.

P16 I NK 4a Deletion Ameliorates Damage of Intestinal Epithelial Barrier and Microbial Dysbiosis in a Stress-Induced Premature Senescence Model of Bmi-1 Deficiency.

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 p16 INK4a deletion could reverse the damage of intestinal epithelial barrier and microbial dysbiosis. Intestines from Bmi-1-deficient (Bmi-1-/- ), Bmi-1 and p16 INK4a double-knockout (Bmi-1-/-p16 INK4a-/- ), and wild-type mice were observed for aging and inflammation. Duolink Proximity Ligation Assay, immunoprecipitation, and construction of p16 INK4a overexpressed adenovirus and the overexpressed plasmids of full-length, mutant, or truncated fragments for occludin were used for analyzing the interaction between p16 INK4a 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-/-p16 INK4a-/- 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 p16 INK4a 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. P16 INK4a 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 p16 INK4a accumulation. The clearance of p16 INK4a -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 p16 INK4a with occludin for protecting TJ.

Parathyroid hormone promotes the osteogenesis of lipopolysaccharide-induced human bone marrow mesenchymal stem cells through the JNK MAPK pathway.

Periodontitis is a series of inflammatory processes caused by bacterial infection. Parathyroid hormone (PTH) plays a critical role in bone remodeling. The present study aimed to investigate the influences of PTH on human bone marrow mesenchymal stem cells (HBMSCs) pretreated with lipopolysaccharide (LPS). The proliferative ability was measured using cell counting kit-8 (CCK-8) and flow cytometry. The optimal concentrations of PTH and LPS were determined using alkaline phosphatase (ALP) activity assay, ALP staining, and Alizarin Red staining. Osteogenic differentiation was further assessed by quantitative reverse-transcription polymerase chain reaction (RT-qPCR), Western blot analysis, and immunofluorescence staining. PTH had no effects on the proliferation of HBMSCs. Also, 100 ng/ml LPS significantly inhibited HBMSC osteogenesis, while 10-9 mol/l PTH was considered as the optimal concentration to reverse the adverse effects. Mechanistically, c-Jun N-terminal kinase (JNK) phosphorylation was activated by PTH in LPS-induced HBMSCs. SP600125, a selective inhibitor targeting JNK mitogen-activated protein kinase (MAPK) signaling, weakened the effects of PTH. Taken together, the findings revealed the role and mechanism of PTH and JNK pathway in promoting the osteogenic differentiation of LPS-induced HBMSCs, which offered an alternative for treating periodontal diseases.

Transcriptome-wide m6A profiling reveals mRNA post-transcriptional modification of boar sperm during cryopreservation.

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.

Comparative Analysis of piRNA Profiles Helps to Elucidate Cryoinjury Between Giant Panda and Boar Sperm During Cryopreservation.

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.

Odorant and Taste Receptors in Sperm Chemotaxis and Cryopreservation: Roles and Implications in Sperm Capacitation, Motility and Fertility.

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.

Parathyroid hormone ameliorates osteogenesis of human bone marrow mesenchymal stem cells against glucolipotoxicity through p38 MAPK signaling.

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. PTH had no effects on HBMSC proliferation, with or without GP treatment. PTH reversed inadequate osteogenesis and excessive oxidative stress in GP-treated HBMSCs. Mechanistically, PTH activated p38 MAPK signaling, while inhibiting p38 MAPK-suppressed PTH's beneficial impacts on HBMSCs. Collectively, PTH promotes osteogenic differentiation in HBMSCs against glucolipotoxicity via p38 MAPK signaling.

piR-121380 Is Involved in Cryo-Capacitation and Regulates Post-Thawed Boar Sperm Quality Through Phosphorylation of ERK2 via Targeting PTPN7.

Cryopreservation induces capacitation-like (cryo-capacitation) changes, similar to natural capacitation, and affects the fertility potential of post-thawed sperm. The molecular mechanism of sperm cryo-capacitation during cryopreservation remains unknown. PIWI-interacting RNAs (piRNAs) have been reported to be involved in cryo-capacitation of post-thawed sperm and regulation of sperm motility, capacitation, and chemotaxis. In this study, protein tyrosine phosphatase nonreceptor type 7 (PTPN7) was positively targeted by piR-121380 after a dual luciferase assay. The mRNA expression of PTPN7 and piR-121380 was significantly decreased (p < 0.01); however, PTPN7 protein was significantly increased (p < 0.01) in post-thawed boar sperm. Furthermore, E1RK1/2 phosphorylation was reduced during cryopreservation. Six hours after transfection with piR-121380 mimic and inhibitor, the phosphorylation of ERK2 was significantly increased and decreased (p < 0.01), respectively. Furthermore, the highest and lowest total sperm motility, forward motility, and capacitation rate were observed after piR-121380 mimic and inhibitor treatments, respectively. The concentration of intracellular calcium ([Ca2+]i) showed no significant difference after transfection with either piR-121380 mimic or inhibitor at 1, 3, and 6 h. In conclusion, we demonstrated that piR-121380 modulates ERK2 phosphorylation by targeting PTPN7, which induces sperm cryo-capacitation, and eventually affects the motility and fertility potential of post-thawed sperm.

Toll­like receptor 4 activates the NLRP3 inflammasome pathway and periodontal inflammaging by inhibiting Bmi­1 expression.

Overproduction of pro­inflammatory cytokines in the aged, which is called inflammaging, leads to the deterioration of periodontitis. Toll­like 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, wild­type and TLR4 gene knockout mice were used to investigate the activation of the TLR4 pathway in mouse periodontitis and the expression of the nucleotide­binding and oligomerization domain­like 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 senescence­associated secretory phenotype (SASP), which boosted the inflammaging process. Conversely, TLR4 activation downregulated the expression of B cell­specific Moloney murine leukemia virus integration site 1 (Bmi­1) and promoted the priming of NLRP3 inflammasome, both of which are regulators of SASP. Treating gingival fibroblasts with Bmi­1 inhibitor PTC209, it was demonstrated that TLR4 activated the NLRP3 pathway and the inflammaging process by suppressing Bmi­1. In addition, there was a significant reduction in the expression of Bmi­1 expression in the gingiva of patients with periodontitis compared with healthy controls. In conclusion, the present study demonstrated that TLR4 acted by inhibiting Bmi­1 to enhance the NLRP3 pathway and SASP factors. This cascade of reactions may contribute to the senescence of the periodontium.

P16INK4a played a critical role in exacerbating acute tubular necrosis in acute kidney injury.

Acute kidney injury (AKI) is a common clinical syndrome with high morbidity and mortality, which is mostly caused by acute tubular necrosis (ATN). AKI is associated with many factors, including cell senescence, inflammatory infiltration, apoptosis and excessive accumulation of reactive oxygen species (ROS). P16INK4a (hereafter termed p16) inhibits cell cycle, and the absence of p16 can significantly slow the progression of cell senescence. We found that the expression of p16 was significantly increased after ATN. To determine whether p16 could exacerbate ATN degree and whether p16 deletion had protective effects against the ATN and renal dysfunction in AKI progression, glycerol-rhabdomyolysis-induced ATN was performed in eight-week-old p16 knockout and wild-type (WT) littermates. Their ATN phenotypes were analyzed; the levels of serum creatinine and serum urea nitrogen were detected; inflammation, cell apoptosis, ROS level and ROS signaling pathway molecules were examined using histopathological and molecular techniques. We found that compared to WT mice, p16 deletion has protective effects against the ATN phenotype and renal dysfunction in AKI progression through ameliorating inflammatory infiltration and proinflammatory factor expression by inhibiting NF-κB proinflammatory pathway, decreasing cell apoptosis by balancing the expressions between pro-apoptotic and anti-apoptotic molecules, and reducing ROS levels and downregulating ROS signaling pathway molecules including AIF, PGAM5 and KEAP1. Thus, p16 deletion or inhibition and p16 positive cell clearance would be a novel strategy for preventing ATN in AKI progression.