This study aimed to investigate whether Artificial Seminiferous Tubules (AST) coated with testicular extra cellular matrix (ECM) support maturation of spermatogenic cells from immature testes.
Materials and methods
Medical grade tubes coated with testicular ECM were used. Testes from immature mice were loaded in the concentration of 10000- 20000/ml. Tubes were then immersed in germ cell culture medium supplemented with a mixture of growth factors and cultured at 32oC for 30 days. After 7 days in culture maturation inducing factors including FSH, SCF and Retinoic Acid were added. After 14 days in culture, the contents of some tubes were collected and samples were taken for histology, DNA content and gene expression analyses. Also some cells were co-cultured with the Sertoli cell monolayer. After 3-4 weeks, cells with the morphology of spermatids and sperm were collected and their ability to fertilize eggs was determined by ICSI and IVF. Furthermore, the ability of the fertilized eggs to develop to embryos was investigated.
After 7 and 14 days, small cells similar to the morphology of round and elongated spermatids were present. Localization of the Peanut Agglutinin (PNA) as well as PAS staining revealed the presence of acrosomal structure in these cells. Cells matured in AST expressed meiotic and post meiotic markers and on average 75% of the cells reached to haploid stage after two weeks of maturation. After 18 days in culture more elongated spermatids and cells with the appearance of mature sperm containing head, acrosome, mid piece and tail were formed. Injection of round spermatids developed in AST into MII eggs resulted in GFP embryos.
AST coated with testes ECM created a micro milieu similar to testicular seminiferous tubules and supported maturation of spermatogenic cells.