Lakpour Mohammad Reza, Koruji Morteza, Shahverdi Abdolhossein, Aghajanpour Samaneh, Rajabian Naghandar Majid, Sadighi Gilani Mohammad Ali, Sabbaghian Marjan, Aflatoonian Reza
Department of Andrology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Department of Biology, Payam Noor University, Tehran, Iran.
Cell J. 2017 Oct;19(3):375-385. doi: 10.22074/cellj.2017.4300. Epub 2017 Aug 19.
Toll-like receptors (TLRs) on Sertoli cells are thought to have essential roles in sperm protection. This study was conducted to investigate the expression of TLR2 and TLR3 in Sertoli cells of men with azoospermia.
In this experimental study, testicular biopsies were taken from ten azoospermic men. Following enzymatic dissociation, the samples were moved to lectin coated petri dishes. After a few passages, all cells were cultivated and Seroli cells were sorted by flow cytometry. To confirm Sertoli cell purification, alkaline phosphatase activity (ALP) and immunohistochemistry assays were employed. The expression of TLR2 and TLR3 at the transcript and protein levels was examined with real-time quantitative reverse transcription-polymerase chain reaction (RT-QPCR) and western blot, respectively.
Isolation, purification and cultivation of human Sertoli cells were performed successfully. Efficacy of purification of Sertoli cells by fluorescence-activated cell sorting (FACS) sorter was ~97%. The type of cultured cells was confirmed by vimentin and follicle-stimulating hormone (FSH) receptor markers. Furthermore, the existence of anti- Müllerian hormone in culture was confirmed. RT-PCR showed that both genes were expressed in Sertoli cells. Consistently, proteins of both were also expressed in Sertoli cells. Moreover, QPCR showed that the relative expression of TLR3 transcripts was significantly higher than TLR2 in Sertoli cells. Although both genes are expressed in fibroblast cells, their level of expression was significantly lower than in Sertoli cells.
This study confirmed expression of TLR2 and TLR3 in human Sertoli cells. This may be an indicator of their roles in developing immunity against pathogens as well as allo- and auto-antigens or viral antigens in seminiferous tubules.
支持细胞上的Toll样受体（TLRs）被认为在精子保护中起重要作用。本研究旨在调查无精子症男性支持细胞中TLR2和TLR3的表达情况。
在本实验研究中，从10名无精子症男性中获取睾丸活检组织。酶解后，将样本转移至凝集素包被的培养皿中。经过几次传代后，培养所有细胞，并通过流式细胞术分选支持细胞。为确认支持细胞的纯化，采用了碱性磷酸酶活性（ALP）检测和免疫组织化学分析。分别通过实时定量逆转录-聚合酶链反应（RT-QPCR）和蛋白质印迹法检测TLR2和TLR3在转录水平和蛋白质水平的表达。
成功进行了人支持细胞的分离、纯化和培养。通过荧光激活细胞分选（FACS）仪纯化支持细胞的效率约为97%。通过波形蛋白和促卵泡激素（FSH）受体标志物确认了培养细胞的类型。此外，还证实了培养物中存在抗苗勒管激素。RT-PCR显示这两个基因均在支持细胞中表达。一致地，这两种蛋白也在支持细胞中表达。此外，QPCR显示支持细胞中TLR3转录本的相对表达明显高于TLR2。虽然这两个基因在成纤维细胞中均有表达，但其表达水平明显低于支持细胞。
本研究证实了TLR2和TLR3在人支持细胞中的表达。这可能表明它们在生精小管中对抗病原体以及同种和自身抗原或病毒抗原产生免疫方面发挥作用。
