title: Role of Somatic Testicular Cells during Mouse Spermatogenesis in Three-Dimensional Collagen Gel Culture System.
abstract: OBJECTIVE
  Spermatogonial stem cells (SSCs) are the only cell type that can restore fertility to an infertile recipient following transplantation. Much effort has been made to develop a protocol for differentiating isolated SSCs in vitro. Recently, three-dimensional (3D) culture system has been introduced as an appropriate microenvironment for clonal expansion and differentiation of SSCs. This system provides structural support and multiple options for several manipulation such as addition of different cells. Somatic cells have a critical role in stimulating spermatogenesis. They provide complex cell to cell interaction, transport proteins and produce enzymes and regulatory factors. This study aimed to optimize the culture condition by adding somatic testicular cells to the collagen gel culture system in order to induce spermatogenesis progression.
  
  
  METHODS
  In this experimental study, the disassociation of SSCs was performed by using a two-step enzymatic digestion of type I collagenase, hyaluronidase and DNase. Somatic testicular cells including Sertoli cells and peritubular cells were obtained after the second digestion. SSCs were isolated by Magnetic Activated Cell Sorting (MACS) using GDNF family receptor alpha-1 (Gfrα-1) antibody. Two experimental designs were investigated. 1. Gfrα-1 positive SSCs were cultured in a collagen solution. 2. Somatic testicular cells were added to the Gfrα-1 positive SSCs in a collagen solution. Spermatogenesis progression was determined after three weeks by staining of synaptonemal complex protein 3 (SCP3)-positive cells. Semi-quantitative Reverse Transcription PCR was undertaken for SCP3 as a meiotic marker and, Crem and Thyroid transcription factor-1 (TTF1) as post meiotic markers. For statistical analysis student t test was performed.
  
  
  RESULTS
  Testicular supporter cells increased the expression of meiotic and post meiotic markers and had a positive effect on extensive colony formation.
  
  
  CONCLUSIONS
  Collagen gel culture system supported by somatic testicular cells provides a microenvironment that mimics seminiferous epithelium and induces spermatogenesis in vitro.
authors: Department of Anatomical Sciences, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
authors: Noushafarin
authors: N
authors: Khajavi
authors: Department of Anatomical Sciences, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
authors: Mohammad
authors: M
authors: Akbari
authors: Department of Oral Medicine, Faculty of Dentistry, Hamadan University of Medical Sciences, Hamadan, Iran.
authors: Hamid Reza
authors: HR
authors: Abdolsamadi
authors: Department of Anatomical Sciences, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
authors: Farid
authors: F
authors: Abolhassani
authors: Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
authors: Ahmad Reza
authors: AR
authors: Dehpour
authors: Department of Anatomical Sciences, School of Medicine, Iran University of Medical Sciences, Tehran, Iran.
authors: Morteza
authors: M
authors: Koruji
authors: Research Center, Iranian Blood Transfusion Organization, Tehran, Iran.
authors: Mehryar
authors: M
authors: Habibi Roudkenar
keywords: Cell Culture Technique; Mouse; Spermatogenesis; Spermatogonia