139. THE REGULATION OF TRP53 IN SPERM

2009 ◽  
Vol 21 (9) ◽  
pp. 58
Author(s):  
H. Mudaliar ◽  
C. O'Neill
Keyword(s):  
Protein Synthesis ◽  
Western Blot ◽  
Marked Reduction ◽  
Acrosome Reaction ◽  
Human Sperm ◽  
Cell Stress ◽  
Time Dependent ◽  
Mouse Sperm ◽  
Tumour Suppressor Protein ◽  
Acute Increase

TRP53 is a tumour suppressor protein that is a universal sensor of cell stress. Upon ejaculation, sperm undergo a process of capacitation which allows them to become fertile. We have previously shown that mouse and human sperm possess TRP53. In this study we analyzed the regulation TRP53 presence in sperm. Mouse sperm were collected from the epididymides and incubated for various times in fertilisation medium. TRP53 was detected by both Western blot analysis and immunolocalization. We found that sperm collected directly from the epididymis generally had little or no detectable TRP53. The level increased with time in culture over a period of 120 min. Most of the TRP53 was detected in the midpiece of sperm although it was also detected in the head of a small proportion of sperm. The increase in TRP53 with time accompanied the increase in the proportion of sperm undergoing the acrosome reaction. Yet, Trp53-null sperm still underwent the acrosome reaction at a normal rate. By contrast, sperm that were prevented from undergoing capacitation and the acrosome reaction (by the removal of albumin or calcium from media) showed a marked reduction in the amount of TRP53 detected. This shows that TRP53 may be dependent upon capacitation, but the reverse was not the case. Inhibition of protein synthesis by puromycin did not block the time-dependent increase in TRP53 in sperm. Canonically, the TRP53 level is controlled by its rate of degradation by MDM2-ubiquitin mediated proteolysis. We found that MDM2 was present in sperm and inhibition of MDM2 (by Nutlin-3) caused an acute increase in TRP53 detected in sperm. This study shows that TRP53 levels are acutely regulated in sperm during the time that sperm acquire the capacity to fertilise, yet sperm lacking TRP53 are capable of fertility. Identification of the role for this TRP53 in sperm is under investigation.

scholarly journals Soluble adenylyl cyclase inhibition prevents human sperm functions essential for fertilization

2021 ◽  
Author(s):  
Melanie Balbach ◽  
Lubna Ghanem ◽  
Thomas Rossetti ◽  
Navpreet Kaur ◽  
Carla Ritagliati ◽  
...  
Keyword(s):  
Adenylyl Cyclase ◽  
Acrosome Reaction ◽  
Human Sperm ◽  
Comprehensive Analysis ◽  
Multiple Points ◽  
Soluble Adenylyl Cyclase ◽  
Mouse Sperm ◽  
On Demand ◽  
Sperm Functions

AbstractSoluble adenylyl cyclase (sAC: ADCY10) is essential for activating dormant sperm. Studies of freshly dissected mouse sperm identified sAC as needed for initiating capacitation and activating motility. We now use an improved sAC inhibitor, TDI-10229, for a comprehensive analysis of sAC function in human sperm. Unlike dissected mouse sperm, human sperm are collected post-ejaculation, after sAC activity has already been stimulated. Even in ejaculated human sperm, TDI-10229 interrupts stimulated motility and capacitation, and it prevents acrosome reaction in capacitated sperm. At present, there are no non-hormonal, pharmacological methods for contraception. Because sAC activity is required post-ejaculation at multiple points during the sperm’s journey to fertilize the oocyte, sAC inhibitors define candidates for non-hormonal, on-demand contraceptives suitable for delivery via intravaginal devices in females.

scholarly journals Identification and spatial distribution of glycine receptor subunits in human sperm

Reproduction ◽  
2008 ◽  
Vol 136 (4) ◽  
pp. 387-390 ◽  
Author(s):  
Priyadarsini Kumar ◽  
Stanley Meizel
Keyword(s):  
Spatial Distribution ◽  
Western Blot ◽  
Blot Analysis ◽  
Acrosome Reaction ◽  
Glycine Receptor ◽  
Human Sperm ◽  
Equatorial Region ◽  
Mammalian Sperm ◽  
Principal Piece ◽  
Sperm Acrosome Reaction

The human sperm surface glycine receptor (GLR) plays a role in an important fertilization event, the sperm acrosome reaction. Here, by western blot analysis, we report the presence of GLRA1, GLRA2, GLRA3, and GLRB subunits in human sperm. Immunolocalization studies showed that the GLRA1 and GLRA2 subunits are present in the equatorial region, the GLRA3 subunit in the flagellar principal piece, and the GLRB subunit in the acrosomal region of sperm. This first demonstration of isoforms of the sperm GLRA subunit and of a differential spatial distribution of the α and β subunits on the surface of mammalian sperm suggests the possibility that human sperm GLRs have more than one function.

The Natural Flavonoid Naringenin Inhibits the Cell Growth of WilmsTumorinChildren by Suppressing TLR4/NF-κB Signaling

2020 ◽  
Vol 20 ◽  
Author(s):  
Hongtao Li ◽  
Peng Chen ◽  
Lei Chen ◽  
Xinning Wang
Keyword(s):  
Cell Growth ◽  
Western Blot ◽  
Wilms Tumor ◽  
Critical Role ◽  
Time Dependent ◽  
Luciferase Assay ◽  
Dependent Manner ◽  
Tlr4 Expression ◽  
Protein Levels

Background: Nuclear factor kappa B (NF-κB) is usually activated in Wilms tumor (WT) cells and plays a critical role in WT development. Objective: The study purpose was to screen a NF-κB inhibitor from natural product library and explore its effects on WT development. Methods: Luciferase assay was employed to assess the effects of natural chemical son NF-κB activity. CCK-8 assay was conducted to assess cell growth in response to naringenin. WT xenograft model was established to analyze the effect of naringenin in vivo. Quantitative real-time PCR and Western blot were performed to examine the mRNA and protein levels of relative genes, respectively. Results: Naringenin displayed significant inhibitory effect on NF-κB activation in SK-NEP-1 cells. In SK-NEP-1 and G-401 cells, naringenin inhibited p65 phosphorylation. Moreover, naringenin suppressed TNF-α-induced p65 phosphorylation in WT cells. Naringenin inhibited TLR4 expression at both mRNA and protein levels in WT cells. CCK-8 staining showed that naringenin inhibited cell growth of the two above WT cells in dose-and time-dependent manner, whereas Toll-like receptor 4 (TLR4) over expression partially reversed the above phenomena. Besides, naringenin suppressed WT tumor growth in dose-and time-dependent manner in vivo. Western blot found that naringenin inhibited TLR4 expression and p65 phosphorylation in WT xenograft tumors. Conclusion: Naringenin inhibits WT development viasuppressing TLR4/NF-κB signaling

TRPM8 in mouse sperm detects temperature changes and may influence the acrosome reaction

2011 ◽  
Vol 226 (6) ◽  
pp. 1620-1631 ◽  
Author(s):  
Pablo Martínez-López ◽  
Claudia L. Treviño ◽  
José Luis de la Vega-Beltrán ◽  
Gerardo De Blas ◽  
Esteban Monroy ◽  
...  
Keyword(s):  
Acrosome Reaction ◽  
Temperature Changes ◽  
Mouse Sperm

scholarly journals KLK11 promotes the activation of mTOR and protein synthesis to facilitate cardiac hypertrophy

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Yi Wang ◽  
Hongjuan Liao ◽  
Yueheng Wang ◽  
Jinlin Zhou ◽  
Feng Wang ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Angiotensin Ii ◽  
Cardiac Hypertrophy ◽  
Western Blot ◽  
Real Time ◽  
Real Time Pcr ◽  
Mtor Signaling ◽  
Heart Weight ◽  
Cardiomyocyte Hypertrophy ◽  
Quantitative Real Time Pcr

Abstract Background Cardiovascular diseases have become the leading cause of death worldwide, and cardiac hypertrophy is the core mechanism underlying cardiac defect and heart failure. However, the underlying mechanisms of cardiac hypertrophy are not fully understood. Here we investigated the roles of Kallikrein 11 (KLK11) in cardiac hypertrophy. Methods Human and mouse hypertrophic heart tissues were used to determine the expression of KLK11 with quantitative real-time PCR and western blot. Mouse cardiac hypertrophy was induced by transverse aortic constriction (TAC), and cardiomyocyte hypertrophy was induced by angiotensin II. Cardiac function was analyzed by echocardiography. The signaling pathway was analyzed by western blot. Protein synthesis was monitored by the incorporation of [3H]-leucine. Gene expression was analyzed by quantitative real-time PCR. Results The mRNA and protein levels of KLK11 were upregulated in human hypertrophic hearts. We also induced cardiac hypertrophy in mice and observed the upregulation of KLK11 in hypertrophic hearts. Our in vitro experiments demonstrated that KLK11 overexpression promoted whereas KLK11 knockdown repressed cardiomyocytes hypertrophy induced by angiotensin II, as evidenced by cardiomyocyte size and the expression of hypertrophy-related fetal genes. Besides, we knocked down KLK11 expression in mouse hearts with adeno-associated virus 9. Knockdown of KLK11 in mouse hearts inhibited TAC-induced decline in fraction shortening and ejection fraction, reduced the increase in heart weight, cardiomyocyte size, and expression of hypertrophic fetal genes. We also observed that KLK11 promoted protein synthesis, the key feature of cardiomyocyte hypertrophy, by regulating the pivotal machines S6K1 and 4EBP1. Mechanism study demonstrated that KLK11 promoted the activation of AKT-mTOR signaling to promote S6K1 and 4EBP1 pathway and protein synthesis. Repression of mTOR with rapamycin blocked the effects of KLK11 on S6K1 and 4EBP1 as well as protein synthesis. Besides, rapamycin treatment blocked the roles of KLK11 in the regulation of cardiomyocyte hypertrophy. Conclusions Our findings demonstrated that KLK11 promoted cardiomyocyte hypertrophy by activating AKT-mTOR signaling to promote protein synthesis.

Alteration of N6-methyladenosine epitranscriptome profile in unilateral ureteral obstructive nephropathy

Epigenomics ◽  
2020 ◽  
Vol 12 (14) ◽  
pp. 1157-1173
Author(s):  
Xueyan Li ◽  
Xu Fan ◽  
Xiaoming Yin ◽  
Huajian Liu ◽  
Yi Yang
Keyword(s):  
Western Blot ◽  
Interstitial Fibrosis ◽  
Unilateral Ureteral Obstruction ◽  
Time Dependent ◽  
Obstructive Nephropathy ◽  
Functional Importance ◽  
Renal Interstitial Fibrosis ◽  
Quantification Method ◽  
Promising Therapeutic Target ◽  
Rna Immunoprecipitation

Aim: To reveal the alterations of N6-methyladenosine (m6A) epitranscriptome profile in kidney after unilateral ureteral obstruction in mice. Materials & methods: Total renal m6A and expressions of methyltransferases and demethylases were detected by colorimetric quantification method, real-time PCR and western blot, respectively. Methylated RNA immunoprecipitation sequencing was performed to map epitranscriptome-wide m6A profile. Results: Total m6A levels were time-dependent decreased within 1 week, with the lowest level detected at day 7. A total of 823 differentially methylated transcripts in 507 genes were identified. Specifically, demethylated mRNAs selectively acted on multiple pathways, including TGF-β and WNT. Conclusion: m6A modification has a functional importance in renal interstitial fibrosis during obstructive nephropathy and might be a promising therapeutic target.

scholarly journals The Laminin-Induced Acrosome Reaction in Human Sperm Is Mediated by Src Kinases and the Proteasome1

2011 ◽  
Vol 85 (2) ◽  
pp. 357-366 ◽  
Author(s):  
Silvia Tapia ◽  
Marcelo Rojas ◽  
Patricio Morales ◽  
Marco A. Ramirez ◽  
Emilce S. Diaz
Keyword(s):  
Acrosome Reaction ◽  
Human Sperm ◽  
Src Kinases

Mitochondrial functionality modifies human sperm acrosin activity, acrosome reaction capability and chromatin integrity

2018 ◽  
Vol 34 (1) ◽  
pp. 3-11 ◽  
Author(s):  
Guowei Zhang ◽  
Wang Yang ◽  
Peng Zou ◽  
Fan Jiang ◽  
Yingfei Zeng ◽  
...  
Keyword(s):  
Acrosome Reaction ◽  
Human Sperm ◽  
Reaction Capability ◽  
Chromatin Integrity ◽  
Acrosin Activity

Mechanism of human sperm activation by extracellular ATP

1996 ◽  
Vol 270 (6) ◽  
pp. C1709-C1714 ◽  
Author(s):  
C. Foresta ◽  
M. Rossato ◽  
P. Chiozzi ◽  
F. Di Virgilio
Keyword(s):  
Plasma Membrane ◽  
Acrosome Reaction ◽  
Human Sperm ◽  
Extracellular Atp ◽  
Effective Concentration ◽  
Na Channel ◽  
Monovalent Cations ◽  
Sperm Activation ◽  
Gated Channel ◽  
Sperm Plasma Membrane

We have identified the mechanism whereby extracellular ATP (ATPe) triggers the acrosome reaction in human spermatozoa. This nucleotide opens a ligand-gated ion channel expressed on the sperm plasma membrane. ATPe threshold and 50% effective concentration calculated on the total added ATPe are 0.1 and 2 mM, respectively, corresponding to a free ATP concentration (ATP4-) of 3 and 200 microM, respectively. The ATPe-gated channel is selective for monovalent cations (Na+, choline, and methylglucamine), whereas on the contrary, permeability to Ca2+ is negligible. Isosmolar replacement of extracellular Na+ with sucrose fully blocked ATPe-dependent sperm activation, thus suggesting a mandatory role for Na+ influx. These results show that human sperm express an ATPe-gated Na+ channel that might have an important role in sperm activation before egg fertilization.

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