Int J Stem Cells.  2020 Nov;13(3):353-363. 10.15283/ijsc20061.

Screening of Integrin Heterodimers Expressed Functionally on the Undifferentiated Spermatogonial Stem Cells in the Outbred ICR Mice

• Park HJ
• Yun JI
• Kim M
• Choi K
• Lee E
• Lee ST

• KMID: 2508909
• DOI: http://doi.org/10.15283/ijsc20061

Abstract

Background and Objectives
Outbred mice are widely used in toxicology, pharmacology, and fundamental biomedical research. However, there have been no reports of in vitro culture systems for spermatogonial stem cells (SSCs) derived from these mice.
Methods
As a step towards constructing a non-cellular niche supporting the in vitro maintenance of outbred mouse SSC self-renewal, we systematically investigated the types of integrin heterodimers that are expressed transcriptionally, translationally, and functionally in SSCs derived from Imprinting Control Region (ICR) mice.
Results
Among the genes encoding 25 integrin subunits, integrin α₁, α₅, α₆, α₉, α_V, and α_E, and integrin β₁ and β₅ had significantly higher transcriptional levels than the other subunits. Furthermore, at the translational level, integrin α₅, α₆, α₉, α_V, and α_E, and β₁ were localized on the surface of SSCs, but integrin α₁ and β₅ not. Moreover, significantly stronger translational expression than integrin α₉ and α_E was observed in integrin α₅, α₆, α_V, and β₁. SSCs showed significantly increased adhesion to fibronectin, laminin, tenascin C and vitronectin, and functional blocking of integrin α₅β₁, α₆β₁, α₉β₁ or α_Vβ₁ significantly inhibited adhesion to these molecules.
Conclusions
We confirmed that integrin α₅β₁, α₆β₁, α₉β₁ and α_Vβ₁ actively function on the surface of undifferentiated SSCs derived from outbred ICR mice.

Keyword

Spermatogonial stem cells; Undifferentiation; Integrin; Outbred; Mouse

Figure

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  Fig. 1 Transcriptional levels of integrin α and β subunit gene expression in SSC population including undifferentiated SSCs and peritubular myoid, sertoli and leydig cells derived from outbred ICR mouse testes. Testicular cells were retrieved enzymatically from testis derived from ICR mice, and the preparation of SSC population was conducted by sorting these testicular cells using a MACS technique based on anti-Thy1 antibody. Subsequently, mRNA levels of integrin α and β subunits in the isolated SSCs were examined quantitatively by real-time PCR. A total of 6 of 17 integrin α subunit genes (α1, α5, α6, α9, αV, and αE) (A) and 2 of 8 integrin β subunit genes (β1 and β5) showed significantly increased expression (B). All data shown are means±standard deviation of three independent experiments. a∼ep<0.05. ND: not detected.

• 

  Fig. 2 Identification of integrin α and β subunit proteins expressed on the surface of undifferentiated SSCs derived from outbred ICR mouse testes. To obtain testicular cells, testis derived from ICR mice was enzymatically treated and the preparation of SSC population which the proportion of SSCs in testicular cells was increased was conducted using a MACS technique based on anti-Thy1 antibody. Subsequently, the protein expression of integrin α and β subunits in the SSCs included in the sorted SSC population were identified by immunocytochemistry. As the results, integrin α5, α6, α9, αV, αE and β1 subunit proteins (arrow head; B∼G) were localized on the surface of SSCs expressing GFRα1 (arrow; a SSC-specific marker), whereas any localization of integrin α1, and β5 subunit proteins (A, H) was not detected on the surface of SSCs expressing GFRα1. All figures are representative immunocytochemistry images of integrin subunit proteins expressed on the surface of SSCs. Nuclear counterstaining was conducted using DAPI. n=3. Scale bars represent 10 μm.

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  Fig. 3 Translational levels of integrin α and β subunit gene expression in undifferentiated SSCs derived from outbred ICR mouse testes. In order to prepare SSC population which the proportion of SSCs in testicular cells was increased, testicular cells retrieved enzymatically from testis derived from ICR mice were sorted using a MACS technique based on anti-Thy1 antibody. Subsequently, the protein expression levels of integrin α and β subunits in the SSCs included in the sorted SSC population was analyzed by fluorescent immunoassays, and at the translational level, each integrin subunit was represented as the ratio of fluorescence intensity of stained cells to that of unstained cells. Among the six integrin subunits expressed on the surface of undifferentiated SSCs, significantly the strongest translational expression of integrin αV subunit gene was detected, whereas integrin α9 and αE subunit genes showed significantly the weakest expression at the translational level. Moreover, integrin α5, α6 and β1 subunit genes showed significantly intermediate translational expression. All of the data shown are means±standard deviation of three independent experi-ments. *-***p<0.05.

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  Fig. 4 Identification of the presence of integrin heterodimers interacting with fibronectin, laminin, tenascin C and vitronectin on the cell membrane of undifferentiated outbred ICR mouse SSCs. A 96-well tissue culture plates was coated with 0, 40, or 80 μg/ml fibronectin (A), 0, 200, or 400 μg/ml laminin (B), 0, 20, or 40 μg/ml tenascin C (C), and 0, 5, or 10 μg/ml vitronectin (D). Subsequently, SSC population were prepared by sorting testicular cells retrieved enzymatically from testis derived from ICR mice using a MACS technique based on anti-Thy1 antibody. Then, 1×104 cells in SSC population were resuspended in SSC culture medium and plated in each well. After incubation for 2 h at 37℃, adherent cells were stained with crystal violet, and the adhesion level was quantified using a microplate reader. The percentage of maximum adhesion is represented as the optical density of cells plated on ECM protein-free plates. Mouse SSCs cultured on fibronectin-, laminin-, tenascin C- and vitronectin-coated culture plates had significantly higher levels of adhesion than those on ECM protein-free culture plates. However, increasing concentrations of ECM on the culture plates did not induce a significant improvement of mouse SSC adhesion levels. All of the data shown are means±standard deviation of three independent experiments. *p<0.05.

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  Fig. 5 Functional analysis of integrin heterodimers suspected to function on the cell membrane of undifferentiated outbred ICR mouse SSCs. SSC population were prepared by sorting testicular cells retrieved enzymatically from testis derived from ICR mice using a MACS technique based on anti-Thy1 antibody. Subsequently, 1×104 cells in SSC population were incubated in the absence or presence of anti-integrin α5 (5H10–27 [MFR5]) (A), anti-integrin α6 (NKI-GoH3) (B), anti-integrin α9 (Y9A2) (C), or anti-integrin αV (RMV-7) (D) blocking antibody, plated on 40 μg/ml fibronectin-, 200 μg/ml laminin-, 20 μg/ml tenascin C, and 5 μg/ml vitronectin-coated wells, and incubated for 8 h at 37℃. After staining adherent cells with crystal violet, quantification of adhesion level was conducted using a microplate reader. As a parameter of functional blocking by antibodies, the percentage of maximum adhesion, which is represented by the optical density of cells plated on each ECM protein-coated well in the absence of any blocking antibodies, was determined. Mouse SSCs treated with integrin α5β1, α6β1, α9β1, and αVβ1 blocking antibodies showed significantly lower rates of attachment to each ECM component compared to mouse SSCs without blocking antibody. All of the data shown are means±standard deviation of three independent experiments. *p<0.05.

Reference

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