Starvation is more efficient than the washing technique for purification of rat Sertoli cells.
Sertoli cells (SCs), one of the most important components of seminiferous tubules, are vital for normal spermatogenesis and male fertility. In recent years, numerous in vitro studies have shown the potential and actual activities of SCs. However, pure SCs are necessary for various in vitro studies. In this study, we have evaluated the efficiency of the starvation method for SC purification as compared with the washing method. Seminiferous tubule-derived cells (STDCs) of rats' testes underwent two different techniques for SC purification. In the first group (washing group), the medium was changed every 3-4 d, and cells were washed twice with phosphate-buffered saline that lacked CaC12 and MgSO4 (PBS(-)) before the addition of fresh medium. In the second group (starvation), the medium was changed every 7-8 d. Primary culture (P0), passage 1 (P1), and passage 2 (P2) cells were analyzed for the expression of SC-specific genes, vimentin, Wilm's tumor 1 (WT1), germ cell gene (vasa), Leydig cell marker, 17beta-hydroxysteroid dehydrogenase type 3 (Hsd17b3), and a marker of peritubular myoid cells, alpha smooth muscle actin (αSma), by reverse transcriptase polymerase chain reaction (RT-PCR) and real-time RT-PCR. Gene expression analysis showed that P0 cells expressed all tested genes except Hsd17b3. The starvation method caused significant downregulation of vasa and αSma expression in P0, P1, and P2 cells, whereas vimentin and WT1 were upregulated. In contrast, the washing method was less effective than the starvation method for the removal of germ and pretubular myoid cells (p < 0.001). Totally, the results have revealed that although washing is the only common technique for elimination of contaminant cells in SC cultures, starvation has a stronger effect and is a suitable, affordable technique for SC purification. We propose that starvation is an efficient, inexpensive method that can be used for purification of SCs in animal species.
Fatemeh-Zahra Infertility and Reproductive Health Research Center, Babol University of Medical Sciences, P.O. Box: 47135-547, Babol, Iran, ghasemzadeh78@gmail.com.
Mohammad
M
Ghasemzadeh-Hasankolaei
Mohamadreza Baghaban
MB
Eslaminejad
Mohammadali
M
Sedighi-Gilani
Aram
A
Mokarizadeh
D000818:Animals; D001391:Azo Compounds; D002478:Cells, Cultured; D004396:Coloring Agents; D003584:Cytological Techniques / Q000379:methods; D008297:Male; D012313:RNA / Q000302:isolation & purification; D051381:Rats; D017208:Rats, Wistar; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012708:Sertoli Cells / Q000166:cytology* / Q000648:ultrastructure
D001391:Azo Compounds; D004396:Coloring Agents; D012313:RNA; C011049:oil red O