Observational study on morphology, growth rate and germ cell attachment of human sertoli cells

Abstract

he aim of this study was to investigate the morphology and germ cell attachment of Sertoli cells isolated from human testicular biopsy samples and compare the growth rate of Sertoli cells using two distinct culture conditions: normal culture media and culture media supplemented with FBS. Sertoli cells were isolated from testicular biopsy samples (n=2) obtained for extracting sperm for intra-cytoplasmic sperm injection (ICSI), after obtaining their informed consent. Sertoli cells were isolated by two enzyme digestion methods using collagenase 1 and trypsin-EDTA. The cultured cells were characterized by morphological observations. The growth rate of Sertoli cells was examined by introducing 5 x 103 number of cells into separate wells of a 24-well culture plate. One group of wells was treated with normal DMEM F12 media supplemented with 10% fetal bovine serum (FBS), while the other group was treated with follicle-stimulating hormone (FSH, 100 IU/mg) added DMEM F12 media supplemented with 10% FBS. The cell doubling time was calculated after 24 hours of culture. Interaction between cells was evaluated by mixing Sertoli cells with germ cells which were isolated from semen using the density gradient centrifugation method. After 24 hours of incubation at 33 C̊, the number of germ cells attached to a single Sertoli cell was counted using a light microscope. A sample consisting of Sertoli cells only was maintained as a negative control. Three replicates were done from each sample. Morphological observations revealed multinucleated and irregularly shaped cells with cytoplasmic extensions for intercellular connections. The average doubling time for cells treated with normal media was 23.1 hours, whereas cells treated with FSH-containing media exhibited a shorter doubling time of 16.9 hours. The average number of germ cells attached to a single Sertoli cell was found to be eight (8), while in the negative control, small cells surrounding the Sertoli cells were observed and counted as three (3). This study provides valuable insights into the morphological characteristics of Sertoli cells and their ability to support germ-cell attachment. Additionally, the growth rate of Sertoli cells was influenced by the presence of FSH, suggesting its potential role in regulating cellular proliferation.