scholarly journals Regulation of Folliculogenesis by Growth Factors in Piglet Ovary Exposed Prenatally to β-Hydroxy-β-Methylbutyrate (HMB)

2020 ◽  
Vol 20 (3) ◽  
pp. 899-917
Author(s):  
Monika Hułas-Stasiak ◽  
Joanna Jakubowicz-Gil ◽  
Piotr Dobrowolski ◽  
Małgorzata Grzesiak ◽  
Siemowit Muszyński ◽  
...  
Keyword(s):  
Growth Factors ◽  
Western Blot ◽  
Follicular Development ◽  
Animal Husbandry ◽  
Dietary Treatment ◽  
Western Blot Assay ◽  
Primordial Follicles ◽  
Morphological Alterations ◽  
Newborn Piglets ◽  
Kit Ligand

AbstractΒ-hydroxy-β-methylbutyrate (HMB) is one of the leucine metabolites with protein anabolic effects which makes it very popular among athletes. Previously, it was shown that HMB administered during the prenatal period reduced the pool of primordial follicles and increased the proportion of developing follicles in newborn piglets. This work is a further step to understand these morphological alterations. Therefore, the aim of this study was to examine the effect of prenatal HMB treatment on the expression of the Kit ligand, BMP-4, bFGF, and the IGF-1/IGF-1R system which are the main growth factors controlling follicular development. Excised ovaries from 12 newborn piglets, originated from the control (n=6) and HMB-treated (n=6) sows were used for immunohistochemical and western-blot analysis. The tested proteins were localized within egg nests and ovarian follicles. Furthermore, the western-blot assay indicated higher BMP-4, Kit ligand, and IGF-1R expression, while the level of bFGF and IGF-1 proteins decreased after HMB dietary treatment. These findings show that HMB included into sow diet can modulate the expression of growth factors and thereby alter ovarian morphology in offspring. Therefore, this study opens a discussion about the benefits and risks of the diet supplemented with HMB and its potential application in medicine and animal husbandry, and further research is necessary in this area.

scholarly journals In vitro and in vivo regulation of follicular formation and activation in cattle

2011 ◽  
Vol 23 (1) ◽  
pp. 15 ◽  
Author(s):  
Joanne E. Fortune ◽  
Ming Y. Yang ◽  
Wanzirai Muruvi
Keyword(s):  
Follicular Development ◽  
First Trimester ◽  
Vascular Endothelial ◽  
Second Trimester ◽  
Primordial Follicles ◽  
Kit Ligand ◽  
Secondary Stage ◽  
Endothelial Growth Factor

The establishment of a stockpile of non-growing, primordial follicles and its gradual depletion through activation of primordial follicles are essential processes for female fertility. However, the mechanisms that regulate follicle formation, the activation of primordial follicles to begin growth and the primary-to-secondary follicle transition are poorly understood, especially in domestic animals and primates. The authors’ laboratory is engaged in studying early stages of follicular development in cattle and this review summarises the progress to date. Bovine follicles begin to form in fetal ovaries around the beginning of the second trimester of pregnancy (about Day 90), but the first activated, primary follicles do not appear until after Day 140. Bovine fetal ovaries produce steroids and production is highest during the first trimester. In vitro, oestradiol and progesterone inhibit follicle formation and acquisition by newly formed follicles of the capacity to activate. Meiotic arrest of the oocyte in the diplotene stage of first prophase does not occur until after follicle formation and is correlated with acquisition of the capacity to activate. This may explain the gap between follicle formation and appearance of the first activated follicles. Once capacity to activate has been acquired, it seems likely that activation in vivo is controlled by the balance between stimulators and inhibitors of activation. Insulin and kit ligand stimulate and anti-Müllerian hormone (AMH) inhibits activation in vitro. Few bovine follicles transition from the primary to the secondary stage in vitro, but this transition is increased by medium supplements, testosterone and vascular endothelial growth factor (VEGF).

The Anti-tumor Activity and Mechanisms of rLj-RGD3 on Human Laryngeal Squamous Carcinoma Hep2 Cells

2020 ◽  
Vol 19 (17) ◽  
pp. 2108-2119
Author(s):  
Yang Jin ◽  
Li Lv ◽  
Shu-Xiang Ning ◽  
Ji-Hong Wang ◽  
Rong Xiao
Keyword(s):  
Wound Healing ◽  
Western Blot ◽  
Morphological Changes ◽  
In Vivo Studies ◽  
Migration And Invasion ◽  
Tunel Staining ◽  
Dna Ladder ◽  
Western Blot Assay

Background: Laryngeal Squamous Cell Carcinoma (LSCC) is a malignant epithelial tumor with poor prognosis and its incidence rate increased recently. rLj-RGD3, a recombinant protein cloned from the buccal gland of Lampetra japonica, contains three RGD motifs that could bind to integrins on the tumor cells. Methods: MTT assay was used to detect the inhibitory rate of viability. Giemsa’s staining assay was used to observe the morphological changes of cells. Hoechst 33258 and TUNEL staining assay, DNA ladder assay were used to examine the apoptotic. Western blot assay was applied to detect the change of the integrin signal pathway. Wound-healing assay, migration, and invasion assay were used to detect the mobility of Hep2 cells. H&E staining assay was used to show the arrangement of the Hep2 cells in the solid tumor tissues. Results: In the present study, rLj-RGD3 was shown to inhibit the viability of LSCC Hep2 cells in vitro by inducing apoptosis with an IC50 of 1.23µM. Western blot showed that the apoptosis of Hep2 cells induced by rLj- RGD3 was dependent on the integrin-FAK-Akt pathway. Wound healing, transwells, and western blot assays in vitro showed that rLj-RGD3 suppressed the migration and invasion of Hep2 cells by integrin-FAKpaxillin/ PLC pathway which could also affect the cytoskeleton arrangement in Hep2 cells. In in vivo studies, rLj-RGD3 inhibited the growth, tumor volume, and weight, as well as disturbed the tissue structure of the solid tumors in xenograft models of BALB/c nude mice without reducing their body weights. Conclusion: hese results suggested that rLj-RGD3 is an effective and safe suppressor on the growth and metastasis of LSCC Hep2 cells from both in vitro and in vivo experiments. rLj-RGD3 might be expected to become a novel anti-tumor drug to treat LSCC patients in the near future.

scholarly journals Kisspeptin level in the aging ovary is regulated by the sympathetic nervous system

2017 ◽  
Vol 232 (1) ◽  
pp. 97-105 ◽  
Author(s):  
Daniela Fernandois ◽  
Gonzalo Cruz ◽  
Eun Kyung Na ◽  
Hernán E Lara ◽  
Alfonso H Paredes
Keyword(s):  
Adrenergic Receptor ◽  
Follicular Development ◽  
Sympathetic Nerves ◽  
Female Rats ◽  
Reproductive Aging ◽  
Beta Adrenergic Receptor ◽  
Beta Adrenergic ◽  
Primordial Follicles ◽  
Follicular Cyst

Previous work has demonstrated that the increase in the activity of sympathetic nerves, which occurs during the subfertility period in female rats, causes an increase in follicular cyst development and impairs follicular development. In addition, the increase in ovarian sympathetic activity of aged rats correlates with an increased expression of kisspeptin (KISS1) in the ovary. This increase in KISS1 could participate in the decrease in follicular development that occurs during the subfertility period. We aimed to determine whether the blockade of ovarian sympathetic tone prevents the increase in KISS1 expression during reproductive aging and improves follicular development. We performed 2 experiments in rats: (1) an in vivo blockade of beta-adrenergic receptor with propranolol (5.0 mg/kg) and (2) an ovarian surgical denervation to modulate the sympathetic system at these ages. We measured Kisspeptin and follicle-stimulating hormone receptor (FSHR) mRNA and protein levels by qRT-PCR and western blot and counted primordial, primary and secondary follicles at 8, 10 and 12 months of age. The results showed that ovarian KISS1 decreased but FSHR increased after both propranolol administration and the surgical denervation in rats of 8, 10 and 12 months of age. An increase in FSHR was related to an increase in the number of smaller secondary follicles and a decreased number of primordial follicles at 8, 10 and 12 months of age. These results suggest that intraovarian KISS1 is regulated by sympathetic nerves via a beta-adrenergic receptor and participates locally in ovarian follicular development in reproductive aging.

scholarly journals Salidroside promoted osteogenic differentiation of adipose-derived stromal cells through Wnt/β-catenin signaling pathway

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Xiao-hua Li ◽  
Fu-ling Chen ◽  
Hong-lin Shen
Keyword(s):  
Western Blot ◽  
Osteogenic Differentiation ◽  
Signaling Pathway ◽  
Differentially Expressed Genes ◽  
Stromal Cells ◽  
Differentially Expressed ◽  
Calcium Deposition ◽  
Western Blot Assay ◽  
Adipose Derived Stromal Cells ◽  
Interfering Rna

Abstract Background Bone disease causes short-term or long-term physical pain and disability. It is necessary to explore new drug for bone-related disease. This study aimed to explore the role and mechanism of Salidroside in promoting osteogenic differentiation of adipose-derived stromal cells (ADSCs). Methods ADSCs were isolated and treated with different dose of Salidroside. Cell count kit-8 (CCK-8) assay was performed to assess the cell viability of ADSCs. Then, ALP and ARS staining were conducted to assess the early and late osteogenic capacity of ADSCs, respectively. Then, differentially expressed genes were obtained by R software. Then, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the differentially expressed genes were further analyzed. The expression of OCN, COL1A1, RUNX2, WNT3A, and β-catenin were measured by real-time PCR and Western blot analysis. Last, β-catenin was silenced by small interfering RNA. Results Salidroside significantly increased the ADSCs viability at a dose-response manner. Moreover, Salidroside enhanced osteogenic capacity of ADSCs, which are identified by enhanced ALP activity and calcium deposition. A total of 543 differentially expressed genes were identified between normal and Salidroside-treated ADSCs. Among these differentially expressed genes, 345 genes were upregulated and 198 genes were downregulated. Differentially expressed genes enriched in the Wnt/β-catenin signaling pathway. Western blot assay indicated that Salidroside enhanced the WNT3A and β-catenin expression. Silencing β-catenin partially reversed the promotion effects of Salidroside. PCR and Western blot results further confirmed these results. Conclusion Salidroside promoted osteogenic differentiation of ADSCs through Wnt/β-catenin signaling pathway.

Ginkgolic acid induces apoptosis and autophagy of endometrial carcinoma cells via inhibiting PI3K/Akt/mTOR pathway in vivo and in vitro

2021 ◽  
pp. 096032712110237
Author(s):  
L Zhou ◽  
S Li ◽  
J Sun
Keyword(s):  
Endometrial Cancer ◽  
B Cells ◽  
Western Blot ◽  
Signal Pathway ◽  
Mtor Pathway ◽  
Related Protein ◽  
Western Blot Assay ◽  
Ginkgolic Acid

Endometrial cancer (EC) is the fourth most common malignancy in women in developed countries. The prognosis of EC is extremely poor, and it is an important factor that contributes to the death of patients. Therefore, studying EC pathogenesis and therapeutic targets, and exploring effective drugs are the primary tasks to improve the prognosis of EC. In the present study, we aimed to explore the function of ginkgolic acid (GA) in EC cell apoptosis and autophagy through PI3K/Akt/mTOR signal pathway in vitro and in vivo. Firstly, MTT assay and clone formation assay were employed to analyze the Ishikawa and HEC-1-B cell viabilities and proliferation after treatment with GA. The results showed that GA inhibited endometrial cancer cell survival. Flow cytometry assay and western blot assay were applied to examine the apoptosis and apoptosis related protein Bcl-2, Bax, Cleaved caspase-3 expression levels of Ishikawa and HEC-1-B cells after treatment with GA. Next, we applied western blot assay to analyze the autophagy associated proteins LC3I, LC3II, p62 and Beclin-1 in GA treated Ishikawa and HEC-1-B cells. We found that GA promoted apoptosis and induced autophagy of endometrial cancer cells. Meanwhile, western blot assay was also used to determine the expression levels of the PI3K/Akt/mTOR signal pathway related protein and the results revealed that GA inhibited the activity of PI3K/Akt/mTOR pathway. Finally, we found that GA inhibited tumor growth in vivo through immunohistochemistry assay. In conclusion, GA induces apoptosis and autophagy of EC cells via inhibiting PI3K/Akt/mTOR pathway in vivo and vitro.

scholarly journals Gambogic acid protects LPS-induced apoptosis and inflammation in a cell model of neonatal pneumonia through the regulation of TrkA/Akt signaling pathway

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Xu Gao ◽  
Jingya Dai ◽  
Guifang Li ◽  
Xinya Dai
Keyword(s):  
Western Blot ◽  
Signaling Pathway ◽  
Cell Model ◽  
Akt Signaling ◽  
Gambogic Acid ◽  
Akt Signaling Pathway ◽  
Western Blot Assay ◽  
A Cell ◽  
Induced Apoptosis ◽  
Apoptosis And Inflammation

Abstract Objective In this work, we investigated the effects of gambogic acid (GA) on lipopolysaccharide (LPS)-induced apoptosis and inflammation in a cell model of neonatal pneumonia. Method Human WI-38 cells were maintained in vitro and incubated with various concentrations of GA to examine WI-38 survival. GA-preincubated WI-38 cells were then treated with LPS to investigate the protective effects of GA on LPS-induced death, apoptosis and inflammation. Western blot assay was utilized to analyze the effect of GA on tropomyosin receptor kinase A (TrkA) signaling pathway in LPS-treated WI-38 cells. In addition, human AKT serine/threonine kinase 1 (Akt) gene was knocked down in WI-38 cells to further investigate the associated genetic mechanisms of GA in protecting LPS-induced inflammation and apoptosis. Results Pre-incubating WI-38 cells with low and medium concentrations GA protected LPS-induced cell death, apoptosis and inflammatory protein productions of IL-6 and MCP-1. Using western blot assay, it was demonstrated that GA promoted TrkA phosphorylation and Akt activation in LPS-treated WI-38 cells. Knocking down Akt gene in WI-38 cells showed that GA-associated protections against LPS-induced apoptosis and inflammation were significantly reduced. Conclusions GA protected LPS-induced apoptosis and inflammation, possibly through the activations of TrkA and Akt signaling pathway. This work may broaden our understanding on the molecular mechanisms of human neonatal pneumonia.

scholarly journals NEAT1 aggravates sepsis-induced acute kidney injury by sponging miR-22-3p

Open Medicine ◽  
2020 ◽  
Vol 15 (1) ◽  
pp. 333-342
Author(s):  
Yawei Feng ◽  
Jun Liu ◽  
Ranliang Wu ◽  
Peng Yang ◽  
Zhiqiang Ye ◽  
...  
Keyword(s):  
Acute Kidney Injury ◽  
Western Blot ◽  
Cell Apoptosis ◽  
Noncoding Rna ◽  
Cell Injury ◽  
Kidney Injury ◽  
Inflammatory Factors ◽  
Luciferase Reporter ◽  
Enzyme Linked Immunosorbent Assay ◽  
Western Blot Assay

AbstractBackground and aimAcute kidney injury (AKI) is a common complication of sepsis. Long noncoding RNA nuclear-enriched abundant transcript 1 (NEAT1) plays a vital role in various diseases, including AKI. This study aimed to investigate the function and mechanism of NEAT1 in sepsis-induced AKI.Materials and methodsA septic AKI model was established by treating HK-2 cells with lipopolysaccharide (LPS). The levels of NEAT1 and miR-22-3p were measured by quantitative real-time PCR. Cell apoptosis was assessed by flow cytometry. The levels of apoptosis-related protein and autophagy-related factors were examined by the western blot assay. An enzyme-linked immunosorbent assay was used to calculate the contents of inflammatory factors. The interaction between NEAT1 and miR-22-3p was validated by dual-luciferase reporter assay, RNA immunoprecipitation assay, and RNA pull-down assay. The levels of nuclear factor (NF)-κB pathway-related proteins were evaluated by the western blot assay.ResultsNEAT1 was upregulated, while miR-22-3p was downregulated in patients with sepsis and in LPS-stimulated HK-2 cells. LPS treatment triggered cell apoptosis, autophagy, and inflammatory response in HK-2 cells. NEAT1 knockdown attenuated LPS-induced cell injury. NEAT1 modulated LPS-triggered cell injury by targeting miR-22-3p. Furthermore, NEAT1 regulated the NF-κB pathway by modulating miR-22-3p.ConclusionDepletion of NEAT1 alleviated sepsis-induced AKI via regulating the miR-22-3p/NF-κB pathway.

scholarly journals Interlaboratory Optimization and Evaluation of a Serological Assay for Diagnosis of Human Baylisascariasis

2013 ◽  
Vol 20 (11) ◽  
pp. 1758-1763 ◽  
Author(s):  
Lisa N. Rascoe ◽  
Cynthia Santamaria ◽  
Sukwan Handali ◽  
Sriveny Dangoudoubiyam ◽  
Kevin R. Kazacos ◽  
...  
Keyword(s):  
Western Blot ◽  
Cross Reactivity ◽  
Western Blot Assay ◽  
Content Type ◽  
National Reference ◽  
Baylisascaris Procyonis ◽  
Reference Centre ◽  
Assay Performance ◽  
Positive Serum ◽  
Control And Prevention

ABSTRACTA Western blot assay using a recombinant protein, recombinantBaylisascaris procyonisRAG1 protein (rBpRAG1), was developed for the diagnosis of human baylisascariasis concurrently by the Centers for Disease Control and Prevention (CDC) in Atlanta, Georgia, and the National Reference Centre for Parasitology (NRCP) in Montreal, Canada. Assay performance was assessed by testing 275 specimens at the CDC and 405 specimens at the NRCP. Twenty specimens from 16 cases of baylisascariasis were evaluated. Eighteen were positive, with the assay correctly identifying 14 of 16 patients. The rBpRAG1 Western blot assay showed no cross-reactivity withToxocara-positive serum and had an overall sensitivity of 88% and a specificity of 98%.

scholarly journals Awakening the oocyte: controlling primordial follicle development

Reproduction ◽  
2009 ◽  
Vol 137 (1) ◽  
pp. 1-11 ◽  
Author(s):  
Eileen A McLaughlin ◽  
Skye C McIver
Keyword(s):  
Population Control ◽  
Reproductive Potential ◽  
Animal Husbandry ◽  
Primordial Follicle ◽  
Domestic Animal ◽  
Follicle Growth ◽  
Cellular Mechanisms ◽  
Primordial Follicles ◽  
Control Oocyte ◽  
Signalling Systems

Oocytes are sequestered in primordial follicles before birth and remain quiescent in the ovary, often for decades, until recruited into the growing pool throughout the reproductive years. Therefore, activation of follicle growth is a major biological checkpoint that controls female reproductive potential. However, we are only just beginning to elucidate the cellular mechanisms required for either maintenance of the quiescent primordial follicle pool or initiation of follicle growth. Understanding the intracellular signalling systems that control oocyte maintenance and activation has significant implications for improving female reproductive productivity and longevity in mammals, and has application in domestic animal husbandry, feral animal population control and infertility in women.

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