scholarly journals Antioxidant or Apoptosis Inhibitor Supplementation in Culture Media Improves Post-Thaw Recovery of Murine Spermatogonial Stem Cells

Antioxidants ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 754
Author(s):  
Sang-Eun Jung ◽  
Hui-Jo Oh ◽  
Jin-Seop Ahn ◽  
Yong-Hee Kim ◽  
Bang-Jin Kim ◽  
...  
Keyword(s):  
Stem Cells ◽  
Functional Activity ◽  
Culture Media ◽  
Quantitative Polymerase Chain Reaction ◽  
Spermatogonial Stem Cells ◽  
Ros Generation ◽  
Apoptosis Assay ◽  
Polymerase Chain ◽  
Apoptosis Inhibitor

We postulated that supplementation of antioxidant or apoptosis inhibitor in post-thaw culture media of spermatogonial stem cells (SSCs) alleviates reactive oxygen species (ROS) generation and apoptosis. Our aim was to develop an effective culture media for improving post-thaw recovery of SSCs. To determine the efficacy of supplementation with hypotaurine (HTU), α-tocopherol (α-TCP), and Z-DEVD-FMK (ZDF), we assessed the relative proliferation rate and SSC functional activity and performed a ROS generation assay, apoptosis assay, and western blotting for determination of the Bax/Bcl-xL ratio, as well as immunocytochemistry and real-time quantitative polymerase chain reaction (RT-qPCR) for SSC characterization. The relative proliferation rates with HTU 400 μM (133.7 ± 3.2%), α-TCP 400 μM (158.9 ± 3.6%), and ZDF 200 μM (133.1 ± 7.6%) supplementation were higher than that in the DMSO control (100 ± 3.6%). ROS generation was reduced with α-TCP 400 μM (0.8-fold) supplementation in comparison with the control (1.0-fold). Early apoptosis and Bax/Bcl-xL were lower with α-TCP 400 μM (2.4 ± 0.4% and 0.5-fold) and ZDF 200 μM (1.8 ± 0.4% and 0.3-fold) supplementation in comparison with the control (5.3 ± 1.4% and 1.0-fold) with normal characterization and functional activity. Supplementation of post-thaw culture media with α-TCP 400 μM and ZDF 200 μM improved post-thaw recovery of frozen SSCs via protection from ROS generation and apoptosis after cryo-thawing.

Effects of testicular interstitial fluid on the proliferation of the mouse spermatogonial stem cells in vitro

Zygote ◽  
2013 ◽  
Vol 22 (3) ◽  
pp. 395-403 ◽  
Author(s):  
Peng Wang ◽  
Yi Zheng ◽  
Ying Li ◽  
Hua Shang ◽  
Guang-Xuan Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Interstitial Fluid ◽  
Culture Medium ◽  
Physiological Role ◽  
Spermatogonial Stem Cells ◽  
Proliferation Rate ◽  
Pcr Analysis ◽  
Polymerase Chain

SummarySpermatogenesis is a process in adult male mammals supported by spermatogonial stem cells (SSCs). The cultivation of SSCs has potential value, for example for the treatment of male infertility or spermatogonial transplantation. Testicular interstitial fluid was added to culture medium to a final concentration of 5, 10, 20, 30 or 40%, in order to investigate its effects on proliferation of mouse SSCs in vitro, Alkaline phosphatase (AKP) assay, reverse transcription polymerase chain reaction (RT-PCR) analysis and indirect immunofluorescence of cells were performed to identify SSCs, and the proliferation rate and diameters of the SSCs colonies were measured. The results showed that the optimal addition of testicular interstitial fluid to culture medium was 30%. When medium supplemented with 30% testicular interstitial fluid was used to culture mouse SSCs, the optimum proliferation rate and diameter of the cell colonies were 72.53% and 249 μm, respectively, after 8 days in culture, values that were significant higher than those found for other groups (P < 0.05). In conclusion, proliferation of mouse SSCs could be promoted significantly by supplementation of the culture medium with 30% testicular interstitial fluid. More research is needed to evaluate and understand the precise physiological role of testicular interstitial fluid during cultivation of SSCs.

scholarly journals Technical Validation of a Reverse-Transcription Quantitative Polymerase Chain Reaction In Vitro Diagnostic Test for the Determination of MiR-31-3p Expression Levels in Formalin-Fixed Paraffin-Embedded Metastatic Colorectal Cancer Tumor Specimens

2018 ◽  
Vol 13 ◽  
pp. 117727191876335 ◽  
Author(s):  
Lucas Ramon ◽  
Catherine David ◽  
Karine Fontaine ◽  
Elodie Lallet ◽  
Charles Marcaillou ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Quantitative Polymerase Chain Reaction ◽  
Chain Reaction ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Polymerase Chain ◽  
Formalin Fixed

MiR-31-3p expression has been shown to be a predictive biomarker for response to anti-epithelial growth factor receptor therapy in patients with RAS wild-type metastatic colorectal cancer (mCRC). To aid in the quantification of miR-31-3p expression in formalin-fixed paraffin-embedded (FFPE) primary tumor samples from patients with mCRC, a reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assay was developed and validated. Assay development included the identification of a microRNA reference standard and the determination of an appropriate relative quantification cutoff for differentiating low versus high miR-31-3p expression. Sample specimens for the validation studies included both FFPE slides and shavings. Polymerase chain reaction (PCR) efficiency and linearity, analytical sensitivity and specificity, assay robustness, reproducibility, and accuracy were demonstrated across a number of test conditions and differing quantitative PCR platforms. The data from this study provide evidence as to the feasibility of quantifying the expression of miR-31-3p from FFPE tumor tissue using a standardized RT-qPCR assay.

scholarly journals Regulation of long non-coding RNAs and circular RNAs in spermatogonial stem cells

Reproduction ◽  
2019 ◽  
Vol 158 (1) ◽  
pp. R15-R25 ◽  
Author(s):  
Fan Zhou ◽  
Wei Chen ◽  
Yiqun Jiang ◽  
Zuping He
Keyword(s):  
Stem Cells ◽  
Biological Function ◽  
Spermatogonial Stem Cells ◽  
Circular Rnas ◽  
Biological Functions ◽  
Self Renewal ◽  
Coordinated Expression ◽  
Non Coding Rnas ◽  
Post Transcriptional Regulation

Spermatogonial stem cells (SSCs) are one of the most significant stem cells with the potentials of self-renewal, differentiation, transdifferentiation and dedifferentiation, and thus, they have important applications in reproductive and regenerative medicine. They can transmit the genetic and epigenetic information across generations, which highlights the importance of the correct establishment and maintenance of epigenetic marks. Accurate transcriptional and post-transcriptional regulation is required to support the highly coordinated expression of specific genes for each step of spermatogenesis. Increasing evidence indicates that non-coding RNAs (ncRNAs), including long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs), play essential roles in controlling gene expression and fate determination of male germ cells. These ncRNA molecules have distinct characteristics and biological functions, and they independently or cooperatively modulate the proliferation, apoptosis and differentiation of SSCs. In this review, we summarized the features, biological function and fate of mouse and human SSCs, and we compared the characteristics of lncRNAs and circRNAs. We also addressed the roles and mechanisms of lncRNAs and circRNAs in regulating mouse and human SSCs, which would add novel insights into the epigenetic mechanisms underlying mammalian spermatogenesis and provide new approaches to treat male infertility.

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