Electrospun nanofiber matrices sufficiently mimic the structural morphology of natural extracellular matrix. In this study, we aimed to examine the effects of agar/polyvinyl alcohol nanofiber (PVA) scaffold on the proliferation efficiency and differentiation potential of neonate mouse spermatogonial stem cells (SCCs). Testicular cells were isolated from testes of 40 mouse pups and were seeded in: 1) 2D cell culture plates in the absence (2D/−GF) or presence (2D/+GF) of growth factors and 2) onto agar/PVA scaffold in the absence (3D/−GF) or presence (3D/+GF) of growth factors. The cells were subsequently cultured for 4 weeks. First 2 weeks were dedicated to proliferative phase, whereas the next 2 weeks emphasized the differentiation phase. The identity of the SCCs was investigated at different time-points by flow cytometry and quantitative reverse transcription PCR (qRT-PCR) analyses against the germ cell markers, including PLZF, Id-4, Gfrα-1, Tekt-1, and Sycp-3. After 2 weeks of culture, the 3D/+GF group showed the highest percentage of PLZF-positive cells among culture systems (P < 0.05). The expression levels of pre-meiotic markers (Id-4 and Gfrα-1) decreased significantly in all groups, particularly in 3D/+GF group after 28 days of culture. Additionally, the cells in the 3D/+GF group displayed the highest expression of meiotic (Sycp-3) and post-meiotic markers (Tekt-1) 14 days after differentiation induction. Seemingly, the combination of the agar/PVA scaffold and growth factor-supplemented medium synergistically increased the differentiation rate of mouse SSCs into meiotic and post-meiotic cells. Thus, agar/PVA nanofiber scaffolds may have the potential for applications in the restoration of infertility, especially in azoospermic males.

Abbreviations

2D: two dimentional; 3D: three dimentional; bFGF: basic fibroblast growth factor; BMP-4: bone morphogenetic protein 4; DMEM: Dulbecco’s modified Eagle’s medium; ECM: extracellular matrix; FCS: fetal calf serum; FTIR: Fourier-transform infrared spectroscopy; GDNF: glial cell line-derived neurotrophic factor; GF: growth factors; Gfrα-1, GDNF family co-receptor α1; Id-4, Inhibitor of DNA Binding 4; MTT: methylthiazoltetrazolium; PLZF: promyelocytic leukemia zinc finger; PVA: polyvinyl alcohol; qRT-PCR: quantitative reverse transcription PCR; RA: retinoic acid; SACS: soft agar culture system; SD: standard deviation; SEM: scanning electron microscope; SSCs: spermatogonial stem cells; Sycp-3, Synaptonemal complex protein 3; Tekt-1, Tektin 1.

中文翻译：

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在三维琼脂/聚乙烯醇纳米纤维支架上分化新生小鼠精原干细胞。

电纺纳米纤维基质足以模拟天然细胞外基质的结构形态。在这项研究中，我们旨在检查琼脂/聚乙烯醇纳米纤维（PVA）支架对新生小鼠精原干细胞（SCC）的增殖效率和分化潜能的影响。从40只小鼠幼崽的睾丸中分离出睾丸细胞，并将其接种在：1）在不存在（2D / -GF）或存在（2D / + GF）生长因子的2D细胞培养板上，以及2）在琼脂/ PVA支架中生长因子的缺失（3D / -GF）或存在（3D / + GF）。随后将细胞培养4周。前2周专用于增殖期，而后2周则强调分化期。Id-4，Gfrα-1，Tekt-1和Sycp-3。培养2周后，3D / + GF组在培养系统中显示出最高的PLZF阳性细胞百分比（P <0.05）。培养28天后，所有组中减数分裂前标志物（Id-4和Gfrα-1）的表达水平均显着降低。此外，3D / + GF组中的细胞显示出减数分裂（Sycp-3）和减数分裂后标记（Tekt-1）的最高表达）分化诱导后14天。似乎，琼脂/ PVA支架和生长因子补充的培养基的组合协同增加了小鼠SSCs向减数分裂和减数分裂后细胞的分化率。因此，琼脂/ PVA纳米纤维支架可能具有恢复不育的潜力，尤其是在无精子症的男性中。

缩略语

2D：二维；3D：3维；bFGF：碱性成纤维细胞生长因子；BMP-4：骨形态发生蛋白4；DMEM：Dulbecco改良的Eagle培养基；ECM：细胞外基质；FCS：胎牛血清；FTIR：傅里叶变换红外光谱；GDNF：神经胶质细胞源性神经营养因子；GF：生长因子；Gfrα-1，GDNF家族共受体α1；Id-4，DNA结合抑制剂4；MTT：甲基噻唑四唑；PLZF：早幼粒细胞白血病锌指；PVA：聚乙烯醇；qRT-PCR：定量逆转录PCR；RA：视黄酸；SACS：软琼脂培养系统；SD：标准偏差；SEM：扫描电子显微镜；SSC：精原干细胞；Sycp-3，突触复合蛋白3；Tektin Tekt-1 1。