LncRNA5251 inhibits spermatogenesis via modification of cell-cell junctions.

BACKGROUND: Male factors-caused decline in total fertility has raised significant concern worldwide. LncRNAs have been identified to play various roles in biological systems, including spermatogenesis. This study aimed to explore the role of lncRNA5251 in mouse spermatogenesis. METHODS: The expression of lncRNA5251 was modulated in mouse testes in vivo or spermatogonial stem cells (C18-4 cells) in vitro by shRNA. RESULTS: The sperm motility in two generations mice after modulation of lncRNA5251 (muF0 and muF1) was decreased significantly after overexpression of lncRNA5251. GO enrichment analysis found that knockdown lncRNA5251 increased the expression of genes related to cell junctions, and genes important for spermatogenesis in mouse testes. Meanwhile, overexpressing lncRNA5251 decreased the gene and/or protein expression of important genes for spermatogenesis and immune pathways in mouse testes. In vitro, knockdown lncRNA5251 increased the expression of genes for cell junction, and the protein levels of some cell junction proteins such as CX37, OCLN, JAM1, VCAM1 and CADM2 in C18-4 cells. LncRNA5251 is involved in spermatogenesis by modulation of cell junctions. CONCLUSION: This will provide a theoretical basis for improving male reproductive ability via lncRNA.

Taxifolin increased semen quality of Duroc boars by improving gut microbes and blood metabolites.

Taxifolin (TAX), as a natural flavonoid, has been widely focused on due to its strong anti-oxidation, anti-inflammation, anti-virus, and even anti-tumor activity. However, the effect of TAX on semen quality was unknown. The purpose of this study was to analyze the beneficial influences of adding feed additive TAX to boar semen in terms of its quality and potential mechanisms. We discovered that TAX increased sperm motility significantly in Duroc boars by the elevation of the protein levels such as ZAG, PKA, CatSper, and p-ERK for sperm quality. TAX increased the blood concentration of testosterone derivatives, antioxidants such as melatonin and betaine, unsaturated fatty acids such as DHA, and beneficial amino acids such as proline. Conversely, TAX decreased 10 different kinds of bile acids in the plasma. Moreover, TAX increased "beneficial" microbes such as Intestinimonas, Coprococcus, Butyrivibrio, and Clostridium_XlVa at the Genus level. However, TAX reduced the "harmful" intestinal bacteria such as Prevotella, Howardella, Mogibacterium, and Enterococcus. There was a very close correlation between fecal microbes, plasma metabolites, and semen parameters by the spearman correlation analysis. Therefore, the data suggest that TAX increases the semen quality of Duroc boars by benefiting the gut microbes and blood metabolites. It is supposed that TAX could be used as a kind of feed additive to increase the semen quality of boars to enhance production performance.

LncRNA8276 primes cell-cell adhesion for regulation of spermatogenesis.

BACKGROUND: Human sperm concentration and motility have dropped dramatically (50%) in the past few decades, and environmental factors are involved in this decline. Long non-coding RNAs (lncRNA) have been discovered to be involved in many cellular processes including spermatogenesis. OBJECTIVE: This investigation aimed to explore the role of lncRNA8276 in murine spermatogenesis. MATERIALS AND METHODS: The expression of lncRNA8276 was modified by knockdown or overexpression in mouse testes and spermatogonial stem cells (C18-4 cell line). Sperm quality was determined in the F0 and F1 generations of mice. Furthermore, the underlying mechanisms were studied through gene expression and/or protein expression of spermatogenesis-related genes and cell junction-related genes by different methods. RESULTS: In the current investigation, we discovered that sperm lncRNA8276 was decreased by NH3 /H2 S in three generations (F0, F1, and F2) of mouse sperm. In vivo testicular knockdown of lncRNA8276 led to a decline in sperm concentration and motility in both F0 (muF0) and F1 (muF1) generations Moreover, knockdown lncRNA8276 decreased the gene and protein levels of important genes related to cell-cell junctions and spermatogenesis. The data were further confirmed in mouse spermatogonia stem cell line C18-4 cells through knockdown of lncRNA8276. DISCUSSION AND CONCLUSION: Our study suggests that lncRNA8276 may be involved in cell-cell junction formation in the mouse testis to regulate spermatogenesis. It may be a target for the modification of spermatogenesis and male fertility, or male contraception. This investigation offers a potential therapeutic strategy for male infertility.

Alginate oligosaccharides increase boar semen quality by affecting gut microbiota and metabolites in blood and sperm.

Alginate oligosaccharides (AOS), natural polymers from brown seaweeds (such as Laminaria japonica, Undaria pinnatifida, and Sargassum fusiforme), have been reported to possess many beneficial advantages for health. In the current study, after 9 weeks of dietary supplementation, AOS 10 mg/kg group (AOS 10) group increased boar sperm motility from 87.8% to 93.5%, p < 0.05. Moreover, AOS10 increased the relative abundances of Bifidobacterium, Coprococcus, Butyricicoccus (1.3-2.3-fold; p < 0.05) to increase the beneficial blood and sperm metabolites (1.2-1.6-fold; p < 0.05), and important sperm proteins such as gelsolin, Zn-alpha2 glycoprotein, Cation Channel Sperm-Associated Protein, outer dense fiber of sperm tails, etc. (1.5-2.2-fold; p < 0.05). AOS had a long-term beneficial advantage on boar semen quality by the increase in semen volume (175 vs. 160 ml/ejaculation, p < 0.05). AOS may be used as dietary additives for improving semen quality.

Single-Cell Transcriptome Sequencing and Proteomics Reveal Neonatal Ileum Dynamic Developmental Potentials.

The neonatal period is a crucial time during development of the mammalian small intestine. Moreover, neonatal development and maturation of the small intestine are exceptionally important for early growth, successful weaning, and postweaning growth and development, in order to achieve species-specific milestones. Although several publications recently characterized intestinal epithelial cell diversity at the single-cell level, it remains unclear how differentiation and molecular interactions take place between types and subtypes of epithelial cells during the neonatal period. A single-cell RNA sequencing (scRNA-seq) survey of 40,186 ileal epithelial cells and proteomics analysis of ileal samples at 6 time points in the swine neonatal period were performed. The results revealed previously unknown developmental changes: specific increases in undifferentiated cells, unique enterocyte differentiation, and time-dependent reduction in secretory cells. Moreover, we observed specific transcriptional factors, ligand-receptor pairs, G protein-coupled receptors, transforming growth factor ß, bone morphogenetic protein signaling pathways, and gut mucosal microbiota playing vital roles in ileal development during the neonatal window. This work offers new comprehensive information regarding ileal development throughout the neonatal period. Reference to this data set may assist in the creation of novel interventions for inflammation-, metabolism-, and proliferation-related gut pathologies. IMPORTANCE We found previously unknown neonatal ileum developmental potentials: specific increases in undifferentiated cells, unique enterocyte differentiation, and time dependent reduction in secretory cells. Specific transcriptional factors (TFs), ligand-receptor pairs, G protein-coupled receptors, transforming growth factor ß, bone morphogenetic protein signaling pathways, and the gut mucosal microbiota are involved in this process. Our results may assist in the creation of novel interventions for inflammation-, metabolism-, and proliferation-related gut pathologies.

Muscarinic acetylcholine receptor M5 is involved in spermatogenesis through the modification of cell-cell junctions.

Muscarinic acetylcholine receptor (mAChR) antagonists have been reported to decrease male fertility; however, the roles of mAChRs in spermatogenesis and the underlying mechanisms are not understood yet. During spermatogenesis, extensive remodeling between Sertoli cells and/or germ cells interfaces takes place to accommodate the transport of developing germ cells across the blood-testis barrier (BTB) and adluminal compartment. The cell-cell junctions play a vital role in the spermatogenesis process. This study used ICR male mice and spermatogonial cells (C18-4) and Sertoli cells (TM-4). shRNA of control or M5 gene was injected into 5-week-old ICR mice testes. Ten days post-viral grafting, mice were deeply anesthetized with pentobarbital and the testes were collected. One testicle was fresh frozen for RNA-seq analysis or Western blotting (WB). The second testicle was fixed for immunofluorescence staining (IHF). C18-4 or TM-4 cells were treated with shRNA of control or M5 gene. Then, the cells were collected for RNA-seq analysis, WB, or IHF. Knockdown of mAChR M5 disrupted mouse spermatogenesis and damaged the actin-based cytoskeleton and many types of junction proteins in both Sertoli cells and germ cells. M5 knockdown decreased Phldb2 expression in both germ cells and Sertoli cells which suggested that Phldb2 may be involved in cytoskeleton and cell-cell junction formation to regulate spermatogenesis. Our investigation has elucidated a novel role for mAChR M5 in the regulation of spermatogenesis through the interactions of Phldb2 and cell-cell junctions. M5 may be an attractive future therapeutic target in the treatment of male reproductive disorders.

Hydroxytyrosol Benefits Boar Semen Quality via Improving Gut Microbiota and Blood Metabolome.

Semen quality is one of the most important factors for the success of artificial insemination which has been widely applied in swine industry to take the advantages of the superior genetic background and higher fertility capability of boars. Hydroxytyrosol (HT), a polyphenol, has attracted broad interest due to its strong antioxidant, anti-inflammatory, and antibacterial activities. Sperm plasma membrane contains a large proportion of polyunsaturated fatty acids which is easily impaired by oxidative stress and thus to diminish semen quality. In current investigation, we aimed to explore the effects of dietary supplementation of HT on boar semen quality and the underlying mechanisms. Dietary supplementation of HT tended to increase sperm motility and semen volume/ejaculation. And the follow-up 2 months (without HT, just basal diet), the semen volume was significantly more while the abnormal sperm was less in HT group than that in control group. HT increased the "beneficial microbes" Bifidobacterium, Lactobacillus, Eubacterium, Intestinimonas, Coprococcus, and Butyricicoccus, however, decreased the relative abundance of "harmful microbes" Streptococcus, Oscillibacter, Clostridium_sensu_stricto, Escherichia, Phascolarctobacterium, and Barnesiella. Furthermore, HT increased plamsa steroid hormones such as testosterone and its derivatives, and antioxidant molecules while decreased bile acids and the derivatives. All the data suggest that HT improves gut microbiota to benefit plasma metabolites then to enhance spermatogenesis and semen quality. HT may be used as dietary additive to enhance boar semen quality in swine industry.

Microbiota from alginate oligosaccharide-dosed mice successfully mitigated small intestinal mucositis.

BACKGROUND: The increasing incidence of cancer and intestinal mucositis induced by chemotherapeutics are causing worldwide concern. Many approaches such as fecal microbiota transplantation (FMT) have been used to minimize mucositis. However, it is still unknown whether FMT from a donor with beneficial gut microbiota results in more effective intestinal function in the recipient. Recently, we found that alginate oligosaccharides (AOS) benefit murine gut microbiota through increasing "beneficial" microbes to rescue busulfan induced mucositis. RESULTS: In the current investigation, FMT from AOS-dosed mice improved small intestine function over FMT from control mice through the recovery of gene expression and an increase in the levels of cell junction proteins. FMT from AOS-dosed mice showed superior benefits over FMT from control mice on recipient gut microbiotas through an increase in "beneficial" microbes such as Leuconostocaceae and recovery in blood metabolome. Furthermore, the correlation of gut microbiota and blood metabolites suggested that the "beneficial" microbe Lactobacillales helped with the recovery of blood metabolites, while the "harmful" microbe Mycoplasmatales did not. CONCLUSION: The data confirm our hypothesis that FMT from a donor with superior microbes leads to a more profound recovery of small intestinal function. We propose that gut microbiota from naturally produced AOS-treated donor may be used to prevent small intestinal mucositis induced by chemotherapeutics or other factors in recipients. Video Abstract.

Alginate oligosaccharides enhance small intestine cell integrity and migration ability.

BACKGROUND: An integral intestinal barrier is essential for intestinal homeostasis. Yet, as a side effect of cancer treatment, chemotherapeutic drugs have been reported to cause mucositis. In a recent study, we found that alginate oligosaccharides (AOS) prevent busulfan induced intestinal mucositis. However, it is not known if AOS improves small intestine epithelial cell integrity and migration, which are two essential processes for maintaining the mechanical barrier function of the small intestine. In the current investigation, we aimed to explore the effects of AOS on the integrity and migration of small intestine cells using swine intestinal epithelial IPEC-J2 cells. METHODS: Cell integrity was determined using the TEER assay. Cell migration capability was detected using a wound healing experiment. Small interfering RNA (siRNA) was used to inhibit mannose receptor (MR) expression. Western blotting and immunofluorescence staining were used to determine protein expression. RESULTS: Increasing levels of AOS improved cell integrity as measure by TEER. At the same time, AOS improved IPEC-J2 cell migration capacity as shown in the wound closure assay. It is interesting to note that AOS increased the expression of intestinal microvillus proteins and junction proteins to benefit cell integrity. MR siRNA blocked the action of AOS on cell integrity and cell migration and inhibited the expression of microvillus and cell junction proteins. CONCLUSION: We identified the underlying mechanisms by which AOS improved small intestinal mucositis. As a novel, natural food additive, AOS may be administered to prevent intestinal mucositis induced by chemotherapy or other issues.

Studying drug-plasma protein interactions by two-injector microchip electrophoresis frontal analysis.

We developed a simple, rapid, and sensitive two-injector microchip electrophoresis frontal analysis (MCE-FA) method for studying drug-plasma protein interactions. In this method, large volumes of a reference sample and drug-plasma protein mixture were simultaneously introduced into the respective sections of the microchannel through the separated injectors and then electrophoresed. Since the reference sample did not meet with the interacting species during migration, it could be used as an external standard. The interaction between heparin and HSA was quantitatively characterized as a model system. The binding constant was found to be (1.53 +/- 0.01) x 10(4) M(-1).

Studying protein-drug interaction by microfluidic chip affinity capillary electrophoresis with indirect laser-induced fluorescence detection.

We developed a microfluidic chip-affinity CE method based on indirect LIF detection to study protein-drug interactions. The interaction between heparin and BSA was quantitatively studied, as a model system. In our method, sodium fluorescein was chosen as background, and redistilled water as marker to monitor EOF. The electrophoretic mobility changes of BSA were measured, with various concentrations of heparin added to the running buffer. Each run was completed within 80 s. The binding constant was determined to be (1.24 +/- 0.05) x 10(3) M(-1), which was in good agreement with that reported in the literature.