Saiga antelope horn suppresses febrile seizures in rats by regulating neurotransmitters and the arachidonic acid pathway.

BACKGROUND: Saiga antelope horn (SAH) is a traditional Chinese medicine for treating febrile seizure (FS) with precise efficacy, but its mechanism of action and functional substances are still unclear. Given the need for further research on SAH, our group conducted studies to elucidate its mechanisms and active substances. METHODS: An FS rat pup model was constructed through intraperitoneal injection of LPS and hyperthermia induction. Behavioural indicators of seizures, hippocampal histopathological alterations, serum levels of inflammatory cytokines and hippocampal levels of neurotransmitters were observed and measured to investigate the effects of SAH on FS model rats. Hippocampal metabolomics and network pharmacology analyses were conducted to reveal the differential metabolites, key peptides and pathways involved in the suppression of FS by SAH. RESULTS: SAH suppressed FS, decreased the inflammatory response and regulated the Glu-GABA balance. Metabolomic analysis revealed 13 biomarkers of FS, of which SAH improved the levels of 8 differential metabolites. Combined with network pharmacology, a "biomarker-core target-key peptide" network was constructed. The peptides of SAH, such as YGQL and LTGGF, could exert therapeutic effects via the arachidonic acid pathway. Molecular docking and ELISA results indicated that functional peptides of SAH could bind to PTGS2 target, inhibiting the generation of AA and its metabolites in hippocampal samples. CONCLUSION: In summary, the functional peptides contained in SAH are the main material basis for the treatment of FS, potentially acting through neurotransmitter regulation and the arachidonic acid pathway.

Mechanism and functional substances of Saiga antelope horn in treating hypertension with liver-yang hyperactivity syndrome explored using network pharmacology and metabolomics.

ETHNOPHARMACOLOGICAL RELEVANCE: Saiga antelope horn (SAH) is a traditional Chinese medicine for treating hypertension with liver-yang hyperactivity syndrome (Gan-Yang-Shang-Kang, GYSK), that has a long history of clinical application and precise efficacy, but its mechanism and functional substances are still unknown. Based on the demand for alternative research on the rare and endangered SAH, the group designed and carried out the following studies. AIM OF THE STUDY: The purpose of this research was to demonstrate the functional substances and mechanisms of SAH in the treatment of GYSK hypertension. MATERIALS AND METHODS: The GYSK-SHR model was constructed by administering a decoction of aconite to spontaneously hypertensive rats (SHRs). Blood pressure (BP), behavioural tests related to GYSK, and pathological changes in the kidneys, heart and aorta were measured to investigate the effects of SAH on GYSK-SHRs. Proteomic analysis was used to identify the keratins and peptides of SAH. Moreover, network pharmacology and plasma metabolomics studies were carried out to reveal the mechanisms by which functional peptides in SAH regulate GYSK-hypertension. RESULTS: SAH has a significant antihypertensive effect on GYSK hypertensive animals. It has also been proven to be effective in protecting the function and structural integrity of the kidneys, heart and aorta. Moreover, SAH improved the abnormalities of 31 plasma biomarkers in rats. By constructing a "biomarker-target-peptide" network, 10 functional peptides and two key targets were screened for antihypertensive effects of SAH. The results indicated that SAH may exert a therapeutic effect by re-establishing the imbalance of renin-angiotensin (RAS) system. CONCLUSIONS: Functional peptides from keratin contained in SAH are the main material basis for the treatment of GYSK-hypertension and exhibited the protective effect on the GYSK-SHR model through the RAS system.

The state-of-art polyurethane nanoparticles for drug delivery applications.

Nowadays, polyurethanes (PUs) stand out as a promising option for drug delivery owing to their versatile properties. PUs have garnered significant attention in the biomedical sector and are extensively employed in diverse forms, including bulk devices, coatings, particles, and micelles. PUs are crucial in delivering various therapeutic agents such as antibiotics, anti-cancer medications, dermal treatments, and intravaginal rings. Effective drug release management is essential to ensure the intended therapeutic impact of PUs. Commercially available PU-based drug delivery products exemplify the adaptability of PUs in drug delivery, enabling researchers to tailor the polymer properties for specific drug release patterns. This review primarily focuses on the preparation of PU nanoparticles and their physiochemical properties for drug delivery applications, emphasizing how the formation of PUs affects the efficiency of drug delivery systems. Additionally, cutting-edge applications in drug delivery using PU nanoparticle systems, micelles, targeted, activatable, and fluorescence imaging-guided drug delivery applications are explored. Finally, the role of artificial intelligence and machine learning in drug design and delivery is discussed. The review concludes by addressing the challenges and providing perspectives on the future of PUs in drug delivery, aiming to inspire the design of more innovative solutions in this field.

Coordinated Priming of NKG2D Pathway by IL-15 Enhanced Functional Properties of Cytotoxic CD4+CD28- T Cells Expanded in Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disorder, and numerous aberrations of T cell responses have been reported and were implicated in its pathophysiology. Recently, CD4-positive T cells with cytotoxic potential were shown to be involved in autoimmune disease progression and tissue damage. However, the effector functions of this cell type and their potential molecular mechanisms in SLE patients remain to be elucidated. In this study, we find that cytotoxic CD4+CD28- T cells are expanded in SLE patients with flow cytometry analysis, and the percentage of CD4+CD28- T cells positively correlates with the Systemic Lupus International Collaborating Clinics/ACR Damage Index (SDI). Furthermore, our study suggests that interleukin-15 (IL-15) promotes the expansion, proliferation, and cytotoxic function of CD4+CD28- T cells in SLE patients through activation of the Janus kinase3-STAT5 pathway. Further study indicates that IL-15 not only mediates the upregulation of NKG2D, but also cooperates with the NKG2D pathway to regulate the activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway. Together, our study demonstrated that proinflammatory and cytolytic CD4+CD28- T cells expand in SLE patients. The pathogenic potential of these CD4+CD28- T cells is driven by the coupling of the IL-15/IL-15R signaling pathway and the NKG2D/DAP10 signaling pathway, which may open new avenues for therapeutic intervention to prevent SLE progression.

Differential expression of exosomal miRNAs and proteins in the plasma of systemic lupus erythematous patients.

Systemic lupus erythematous (SLE) is a complex chronic autoimmune disease with difficult early treatment and accurate diagnosis. Circulating exosomes containing proteins, lipids and nucleic acids can be ideal diagnostic biomarkers and disease management strategies for SLE. Our aim was to examine the unique expression profiles of circulating exosomal miRNAs and proteins in patients with SLE patients. Using RNA-sequencing and proteomic approaches, we compared the expression patterns of exosomal miRNAs and proteins in the plasma of SLE patients and healthy subjects, and discussed the underlying signaling network of circulating exosomes. We also summarize common molecules (miRNAs and proteins) and pathways shared by our plasma exosomes, as well as previously reported data (PBMC, T cells, B cells and plasma). We identified groups of differentially expressed exosomal miRNAs and proteins in the plasma of SLE patients and healthy controls. We obtained consensus molecules (39 miRNAs, 14 proteins) and 21 signaling pathways that are common in our current study and previous reports. Common molecules (miRNAs and proteins) and pathways shared by our plasma exosomes data and other circulating components data reported previously indicate their potential application in the clinical treatment and diagnosis of SLE disease.

Research update for the immune microenvironment of chronic endometritis.

Chronic endometritis (CE) is a persistent and subtle local inflammatory disease characterized by abnormal plasma cell infiltration in the endometrial stroma.The incidence of chronic endometritis is as high as 15-57.5% in women suffering from infertility, implantation failure of in vitro fertilization (IVF) and unexplained recurrent abortion. Many studies both at home and abroad have shown that CE can reduce the receptivity of endometrium and affect embryo implantation. According to the existing reproductive immunity research, the abnormality of immune cell subsets in endometrium is an important factors leading to pregnancy failure. The immune microenvironment in endometrium consists of immune cells and immune molecules, and their influence on embryo implantation can not be ignored. This review paper discusses the controversy of pathogenesis, diagnosis and treatment of CE from the perspective of immune microenvironment by referring to related literature at home and abroad, and investigates the possible ways to improve the diagnosis and treatment of CE.

Branched-chain amino acid supplementation impairs insulin sensitivity and promotes lipogenesis during exercise in diet-induced obese mice.

OBJECTIVE: Branched-chain amino acids (BCAAs) are popular dietary supplements for exercise. However, increased BCAA levels positively correlate with obesity and diabetes. The metabolic impact of BCAA supplementation on insulin sensitivity during exercise is less understood. METHODS: Male C57BL/6 mice were fed for 12 weeks with a high-fat diet, normal chow diet, or BCAA-restricted high-fat diet. They were subjected to running exercise with or without BCAA treatment for another 12 weeks. RESULTS: Exercise reduced body weight, improved insulin sensitivity, lowered BCAAs in plasma, and inhibited the upregulation of BCAAs and metabolites caused by BCAA supplementation in the subcutaneous white adipose tissue (sWAT) of obese mice. BCAA supplementation reversed insulin sensitivity ameliorated by exercise. The phosphorylation of protein kinase B (Ser473 and Ser474) was decreased by BCAAs in the sWAT of obese mice. However, BCAA supplementation had no such effects in lean mice. BCAAs also increased the expression of fatty acid synthase and other lipogenesis genes in the sWAT of exercised obese mice. BCAA restriction had no effect on body weight and insulin sensitivity in obese mice. CONCLUSIONS: BCAA supplementation impaired the beneficial effect of exercise on glycolipid metabolism in obese but not lean mice. Caution should be taken regarding the use of BCAAs for individuals with obesity who exercise.

Restoration of Autophagic Flux Improves Endothelial Function in Diabetes Through Lowering Mitochondrial ROS-Mediated eNOS Monomerization.

Endothelial nitric oxide synthase (eNOS) monomerization and uncoupling play crucial roles in mediating vascular dysfunction in diabetes, although the underlying mechanisms are still incompletely understood. Increasing evidence indicates that autophagic dysregulation is involved in the pathogenesis of diabetic endothelial dysfunction; however, whether autophagy regulates eNOS activity through controlling eNOS monomerization or dimerization remains elusive. In this study, autophagic flux was impaired in the endothelium of diabetic db/db mice and in human endothelial cells exposed to advanced glycation end products or oxidized low-density lipoprotein. Inhibition of autophagic flux by chloroquine or bafilomycin A1 were sufficient to induce eNOS monomerization and lower nitric oxide bioavailability by increasing mitochondrial reactive oxygen species (mtROS). Restoration of autophagic flux by overexpressing transcription factor EB (TFEB), a master regulator of autophagy and lysosomal biogenesis, decreased endothelial cell oxidative stress, increased eNOS dimerization, and improved endothelium-dependent relaxations (EDRs) in db/db mouse aortas. Inhibition of mammalian target of rapamycin kinase (mTOR) increased TFEB nuclear localization, reduced mtROS accumulation, facilitated eNOS dimerization, and enhanced EDR in db/db mice. Moreover, calorie restriction also increased TFEB expression, improved autophagic flux, and restored EDR in the aortas of db/db mice. Taken together, the findings of this study reveal that mtROS-induced eNOS monomerization is closely associated with the impaired TFEB-autophagic flux axis leading to endothelial dysfunction in diabetic mice.

Integrated analysis of competing endogenous RNA networks in peripheral blood mononuclear cells of systemic lupus erythematosus.

BACKGROUND: Systemic lupus erythematosus (SLE) is an autoimmune disease with a complicated pathogenesis, and its aetiology has not been clearly unveiled. The lack of effective diagnosis and treatment methods makes it necessary to explore the molecular mechanism of SLE. We aimed to identify some critical signalling pathways and key competing endogenous RNAs (ceRNAs) underlying the molecular mechanism of SLE and to map out the systematic signalling networks by integrating the data on different kinds of RNAs. METHODS: Peripheral blood mononuclear cells (PBMCs) were collected from both SLE patients and healthy subjects, RNA was extracted from the PBMCs, and RNA libraries including ribosomal RNA-depleted strand-specific libraries and small RNA libraries were built for deep RNA sequencing (RNA-seq). RNA-seq yielded differential expression profiles of lncRNAs/circRNAs/miRNAs/mRNAs related to SLE. The DAVID database (v. 6.8) was employed for Gene Ontology (GO) and KEGG pathway analysis. ceRNA networks (circRNA/lncRNA-miRNA-mRNA) were constructed and visualized using Cytoscape software (v. 3.5.0). The TargetScan and miRanda databases were used to predict target relationships in ceRNA networks. qRT-PCR was used to verify our data. RESULTS: Differential expression of ceRNAs related to SLE was detected in SLE patients' PBMCs: 644 mRNAs (384 upregulated, 260 downregulated), 326 miRNAs (223 upregulated, 103 downregulated), 221 lncRNAs (79 upregulated, 142 downregulated), and 31 circRNAs (21 upregulated, 10 downregulated). We drew ceRNA signalling networks made up of the differentially expressed mRNAs/miRNAs/lncRNAs/circRNAs mentioned above, and the hub genes included IRF5, IFNAR2, TLR7, IRAK4, STAT1, STAT2, C2, and Tyk2. These hub genes were involved in ceRNA signalling pathways, such as the IL-17 signalling pathway and type I interferon signalling pathway. CONCLUSIONS: We explored the differential expression profiles of various kinds of ceRNAs and integrated signalling networks constructed by ceRNAs. Our findings offer new insights into the pathogenesis of SLE and hint at therapeutic strategies.

Ang II Promotes Cardiac Autophagy and Hypertrophy via Orai1/STIM1.

The pathophysiology of cardiac hypertrophy is complex and multifactorial. Both the store-operated Ca2+ entry (SOCE) and excessive autophagy are the major causative factors for pathological cardiac hypertrophy. However, it is unclear whether these two causative factors are interdependent. In this study, we examined the functional role of SOCE and Orai1 in angiotensin II (Ang II)-induced autophagy and hypertrophy using in vitro neonatal rat cardiomyocytes (NRCMs) and in vivo mouse model, respectively. We show that YM-58483 or SKF-96365 mediated pharmacological inhibition of SOCE, or silencing of Orai1 with Orail-siRNA inhibited Ang II-induced cardiomyocyte autophagy both in vitro and in vivo. Also, the knockdown of Orai1 attenuated Ang II-induced pathological cardiac hypertrophy. Together, these data suggest that Ang II promotes excessive cardiomyocyte autophagy through SOCE/Orai1 which can be the prime contributing factors in cardiac hypertrophy.

Advances in applying of multi-omics approaches in the research of systemic lupus erythematosus.

Systemic lupus erythematosus (SLE), an autoimmune disease that causes multiorgan injury, has an unclear etiology and complex pathogenesis. Numerous studies have found abnormal alterations in mRNAs, proteins and/or metabolites in SLE patients. These findings have extended our understanding of the pathogenesis of SLE. Novel omics techniques, such as transcriptome, proteome and metabolome profiling, can identify and quantify large numbers of biomarkers of human diseases. However, in most cases, biological reactions are the consequences of interactions among genes, proteomes, and metabolites. Single biomolecules or signaling pathways cannot fully explain biological traits or functions. Therefore, integrative multi-omics analysis can help us systematically comprehend the intrinsic molecular mechanisms underlying biological function and pathogenesis. Integrating transcriptome, proteome, and metabolome KEGG enrichment analysis data will expand our knowledge of the pathogenesis of SLE. This review discusses the application, research progress and outlook on integrative multi-omics analysis in SLE research.

Focal TLR4 activation mediates disturbed flow-induced endothelial inflammation.

AIMS: Disturbed blood flow at arterial branches and curvatures modulates endothelial function and predisposes the region to endothelial inflammation and subsequent development of atherosclerotic lesions. Activation of the endothelial Toll-like receptors (TLRs), in particular TLR4, contributes to vascular inflammation. Therefore, we investigate whether TLR4 can sense disturbed flow (DF) to mediate the subsequent endothelial inflammation. METHODS AND RESULTS: En face staining of endothelium revealed that TLR4 expression, activation, and its downstream inflammatory markers were elevated in mouse aortic arch compared with thoracic aorta, which were absent in Tlr4mut mice. Similar results were observed in the partial carotid ligation model where TLR4 signalling was activated in response to ligation-induced flow disturbance in mouse carotid arteries, and such effect was attenuated in Tlr4mut mice. DF in vitro increased TLR4 expression and activation in human endothelial cells (ECs) and promoted monocyte-EC adhesion, which were inhibited in TLR4-knockdown ECs. Among endogenous TLR4 ligands examined as candidate mediators of DF-induced TLR4 activation, fibronectin containing the extra domain A (FN-EDA) expressed by ECs was increased by DF and was revealed to directly interact with and activate TLR4. CONCLUSION: Our findings demonstrate the indispensable role of TLR4 in DF-induced endothelial inflammation and pinpoint FN-EDA as the endogenous TLR4 activator in this scenario. This novel mechanism of vascular inflammation under DF condition may serve as a critical initiating step in atherogenesis.

Endothelial TFEB (Transcription Factor EB) Restrains IKK (IκB Kinase)-p65 Pathway to Attenuate Vascular Inflammation in Diabetic db/db Mice.

Objective- TFEB (transcription factor EB) was recently reported to be induced by atheroprotective laminar flow and play an anti-atherosclerotic role by inhibiting inflammation in endothelial cells (ECs). This study aims to investigate whether TFEB regulates endothelial inflammation in diabetic db/db mice and the molecular mechanisms involved. Approach and Results- Endothelial denudation shows that TFEB is mainly expressed in ECs in mouse aortas. Western blotting shows TFEB total protein level decreases whereas the p-TFEB S142 (phosphorylated form of TFEB) increases in db/db mouse aortas, suggesting a decreased TFEB activity. Adenoviral TFEB overexpression reduces endothelial inflammation as evidenced by decreased expression of vascular inflammatory markers in db/db mouse aortas, and reduced expression of a wide range of adhesion molecules and chemokines in human umbilical vein ECs. Monocyte attachment assay shows TFEB suppresses monocyte adhesion to human umbilical vein ECs. RNA sequencing of TFEB-overexpressed human umbilical vein ECs suggested TFEB inhibits NF-κB (nuclear factor-kappa B) signaling. Indeed, luciferase assay shows TFEB suppresses NF-κB transcriptional activity. Mechanistically, TFEB suppresses IKK (IκB kinase) activity to protect IκB-α from degradation, leading to reduced p65 nuclear translocation. Inhibition of IKK by PS-1145 abolished TFEB silencing-induced inflammation in human umbilical vein ECs. Lastly, we identified KLF2 (Krüppel-like factor 2) upregulates TFEB expression and promoter activity. Laminar flow experiment showed that KLF2 is required for TFEB induction by laminar flow and TFEB is an anti-inflammatory effector downstream of laminar flow-KLF2 signaling in ECs. Conclusions- These findings suggest that TFEB exerts anti-inflammatory effects in diabetic mice and such function in ECs is achieved by inhibiting IKK activity and increasing IκBα level to suppress NF-κB activity. KLF2 mediates TFEB upregulation in response to laminar flow.

Inhibition of miR-92a Suppresses Oxidative Stress and Improves Endothelial Function by Upregulating Heme Oxygenase-1 in db/db Mice.

AIMS: Inhibition of microRNA-92a (miR-92a) is reported to suppress endothelial inflammation and delay atherogenesis. We hypothesize that miR-92a inhibition protects endothelial function through suppressing oxidative stress in diabetic db/db mice. RESULTS: In this study, we found elevated expression of miR-92a in aortic endothelium from db/db mice and in renal arteries from diabetic subjects. Endothelial cells (ECs) exposed to advanced glycation end products (AGEs) and oxidized low-density lipoprotein express higher level of miR-92a. Overexpression of miR-92a impairs endothelium-dependent relaxations (EDRs) in C57BL/6 mouse aortas. Overexpression of miR-92a suppresses expression of heme oxygenase-1 (HO-1), a critical cytoprotective enzyme, whereas inhibition of miR-92a increases HO-1 expression in human umbilical vein ECs (HUVECs) and db/db mouse aortas. Importantly, miR-92a inhibition by Ad-anti-miR-92a improved EDRs and reduced reactive oxygen species (ROS) production in db/db mouse aortas. HO-1 inhibition by SnMP or HO-1 knockdown by shHO-1 reversed the suppressive effect of miR-92a inhibition on ROS production induced by AGE treatment in C57BL/6 mouse aortas. In addition, SnMP reversed miR-92a inhibition-induced improvement of EDRs in AGE-treated C57BL/6 mouse aortas and in db/db mouse aortas. INNOVATION: Expression of miR-92a is increased in diabetic aortic endothelium and inhibition of miR-92a exerts vasoprotective effect in diabetic mice through HO-1 upregulation in ECs. CONCLUSION: MiR-92a expression is elevated in diabetic ECs. MiR-92a overexpression impairs endothelial function and suppresses HO-1 expression in ECs. Inhibition of miR-92a attenuates oxidative stress and improves endothelial function through enhancing HO-1 expression and activity in db/db mouse aortas. Antioxid. Redox Signal. 28, 358-370.

CD49f promotes proliferation of male dairy goat germline stem cells.

OBJECTIVES: CD49f enhances multipotency and maintains stemness in embryonic stem cells (ESCs), however, whether it would be effective in mGSCs has remained unclear. Moreover, better standards for mGSC enrichment and purification are necessary. The present study was conducted to determine roles of CD49f in mGSC enrichment and regulation. MATERIALS AND METHODS: CD49f expression patterns were investigated in dairy goats. CD49f positive cells were purified and enriched using magnetic-activated cell sorting (MACS), and characteristics of the cultured cells were assayed using alkaline phosphatase (AP) analysis, quantitative real-time PCR (QRT-PCR) and immunofluorescence analysis. Furthermore, the exogenous CD49f gene was transfected into mGSCs and its effects were analysed. RESULTS: CD49f was found to be conserved in both mRNA and amino acid sequences and that it was an efficient marker for dairy goat mGSC identification, enrichment and purification. CD49f positive cells expressed higher levels of mGSC-specific markers, and proliferated faster than CD49f negative cells. Overexpression CD49f promoted proliferation of dairy goat mGSCs, and Oct4 expression was upregulated; histone H3-lysine 9 dimethylation (H3K9me2) was reduced. CONCLUSIONS: Taken together, our data suggest that CD49f plays novel and dynamic roles in regulating maintenance of pluripotency in mGSCs via Oct4 crosstalk and histone methylation dynamics,which may provide new solutions for mGSCs stability in vitro.

PLZF-Induced Upregulation of CXCR4 Promotes Dairy Goat Male Germline Stem Cell Proliferation by Targeting Mir146a.

Previous studies have shown that promyelocytic leukemia zinc finger (PLZF), chemokine (C-X-C motif) receptor 4 (CXCR4) and mir146a were associated with the self-renewal of mouse spermatogonial stem cells (SSCs); however, there is little information on their effects on the fate of livestock SSCs. Here, we have identified a regulatory pathway in dairy goat mGSCs, involving PLZF, mir146a and the SDF-1 receptor CXCR4. PLZF overexpression downregulated mir146a and simultaneously upregulated the expression of CXCR4 protein, whereas PLZF knockdown (siPLZF) induced the specifically opposite effects. The in vitro assays demonstrated that PLZF specifically interacts with and suppresses the mir146a promoter, and mir146a targets CXCR4 to impede its translation. The levels of ERK1/2 phosphorylation in the mGSCs overexpressed CXCR4 and PLZF were upregulated, respectively, whereas mir146a expression was decreased and CXCR4 protein was increased. Mir146a overexpression and siPLZF impaired mGSC proliferation and differentiation, however, Mir146a knockdown induced the opposite effects. The effects of PLZF and mir146a were mediated regulation by mir146a and CXCR4, respectively. Overexpression of CXCR4 or addition of CXCL12 in cultures of dairy goat mGSCs resulted in the upregulation of their signaling, and the phosphorylation of ERK1/2 was increased. Collectively, these findings indicate that PLZF is an important transcription factor in the regulation of the expression of CXCR4 to promote dairy goat mGSC proliferation by targeting mir146a.

Ras/ERK1/2 pathway regulates the self-renewal of dairy goat spermatogonia stem cells.

Spermatogonia stem cells (SSCs), also named the male germline stem cells (mGSCs), which is located at the base of the seminiferous tubules of testis, is the basis for generating sperm steadily in male animals. Currently, there are some preliminary study on the self-renewal and differentiation of SSCs, but further mechanism, especially in large animals, has not been clearly understood. Ras/ERK1/2 pathway is widely distributed in multiple cells in vivo. It plays an important role in cell proliferation, differentiation and so on. However, the study on the function for the self-renewal of dairy goats SSCs has not been investigated. In this study, the dairy goat SSCs characterization were evaluated by semi-RT-PCR, alkaline phosphatase (AP) staining, and immunofluorescence staining. Then, Ras/ERK1/2 pathway was blocked by specific MEK1/2 inhibitor PD0325901. We analyzed the proliferation by cell number, cell growth curve, Brdu incorporation assay, and cell cycle analysis. The results showed that the proliferation was significantly inhibited by PD0325901. Cell apoptosis induced by blocking the Ras/ERK1/2 pathway was analyzed by TUNEL. The expression of ETV5 and BCL6B, the downstream gene of Ras/ERK1/2 pathway, was downregulated. This study suggest that the Ras/ERK1/2 pathway plays a critical role in maintaining the self-renewal of dairy goat SSCs via regulation of ETV5 and BCL6B. This study laid a foundation for insights into the mechanism of SSCs self-renewal comprehensively.

miR-544 Regulates Dairy Goat Male Germline Stem Cell Self-Renewal via Targeting PLZF.

The balance between the self-renewal and differentiation of male germline stem cells (mGSCs) is critical for the initiation and maintenance of mammalian spermatogenesis. The promyelocytic leukemia zinc finger (PLZF), a zinc finger protein, is a critical factor for maintaining the self-renewal of mGSCs, so, evaluation of the PLZF pathway in mGSCs may provide a deeper insight into mammalian spermatogenesis. miRNA was also an important regulating factor for the self-renewal and differentiation of mGSCs; however, there is currently no data indicating that which miRNA regulate the self-renewal and differentiation of mGSCs via PLZF. Here, we predicted the prospective miRNA targeting to PLZF using the online Bioinformatics database-Targetscan, and performed an analysis of the dual-luciferase recombinant vector, psiCHCEKTM-2-PLZF-3'UTR. miR-544 mimics (miR-544m), miR-544 inhibitors (miR-544i), Control (NC, scrambled oligonucleotides transfection), pPLZF-IRES2-EGFP or PLZF siRNA were transfected into mGSCs; the cells proliferation was evaluated by BRDU incorporation assay and flow cytometry, and the mGSC marker, GFRa1, PLZF, KIT, DAZL, and VASA expression were analyzed by RT-qPCR, immunofluorescence and Western blot. The results showed that miR-544 regulates dairy goat male germline stem cell self-renewal via targeting PLZF. Our study identifies a new regulatory pathway for PLZF and expands upon the PLZF regulatory network in mGSCs.

CD49f-positive testicular cells in Saanen dairy goat were identified as spermatogonia-like cells by miRNA profiling analysis.

miRNAs, a type of small RNA, play critical roles in mammalian spermatogenesis. Spermatogonia are the foundation of spermatogenesis and are valuable for the study of spermatogenesis. However, the expression profiling of the miRNAs in spermatogonia of dairy goats remains unclear. CD49f has been one of the surface markers used for spermatogonia enrichment by magnetic activated cell sorting (MACS). Therefore, we used a CD49f microbead antibody to purify CD49f-positive and -negative cells of dairy goat testicular cells by MACS and then analysed the miRNA expression in these cells in depth using Illumina sequencing technology. The results of miRNA expression profiling in purified CD49f-positive and -negative testicular cells showed that 933 miRNAs were upregulated in CD49f-positive cells and 916 miRNAs were upregulated in CD49f-negative cells with a twofold increase, respectively; several miRNAs and marker genes specific for spermatogonial stem cells (SSCs) in testis had a higher expression level in CD49f-positive testicular cells, including miR-221, miR-23a, miR-29b, miR-24, miR-29a, miR-199b, miR-199a, miR-27a, and miR-21 and CD90, Gfra1, and Plzf. The bioinformatics analysis of differently expressed miRNAs indicated that the target genes of these miRNAs in CD49f-positive cells were involved in cell-cycle biological processes and the cell-cycle KEGG pathway. In conclusion, our comparative miRNAome data provide useful miRNA profiling data of dairy goat spermatogonia cells and suggest that CD49f could be used to enrich dairy goat spermatogonia-like cells, including SSCs.

Characterization of immortalized dairy goat male germline stem cells (mGSCs).

Male germline stem cells (mGSCs), in charge for the fertility in male testis, are the only kind of adult stem cells that transmit genetic information to next generation, with promising prospects in germplasm resources preservation and optimization, and production of transgenic animals. Mouse male germline stem cell lines have been established and are valuable for studying the mechanisms of spermatogenesis. However, there is a lack of stable mGSC cell lines in livestock, which restricts the progress of transgenic research and related biotechnology. Here, we firstly established an immortalized dairy goat mGSC cell line to study the biological properties and the signaling pathways associated with mGSCs self-renewal and differentiation. The ectopic factors SV40 large T antigen and Bmi1 genes were transduced into dairy goat mGSCs, and the results showed that the proliferation of these cells that were named mGSCs-I-SB was improved significantly. They maintained the typical characteristics including the expression of mGSC markers, and the potential to differentiate into all three germ layers, sperm-like cells in vitro. Additionally, mGSCs-I-SB survived and differentiated into three germ layer cell types when they were transplanted into chicken embryos. Importantly, the cells also survived in mouse spermatogenesis deficiency model testis which seemed to be the golden standard to examine mGSCs. Conclusively, our results demonstrate that mGSCs-I-SB present the characteristics of mGSCs and may promote the future study on goat mGSCs.