Isolation characterization and morpho biometric evaluation of pre pubertal porcine spermatogonial stem cells in different culture media

Abstract

Testes samples were collected from 7-15 days old pre-pubertal male crossbred piglets (Local × Hampshire) for isolation, enrichment and in vitro culture of porcine spermatogonial stem cells (SSCs). Isolation of spermatogonial stem cells like cells was performed by double enzymatic digestion using four enzymes viz., collagenase, DNase I, hyaluronidase type II and trypsin-EDTA. Isolated cells were further enriched by differential plating and percoll density gradient centrifugation method. Enriched cells were cultured on Sertoli cell feeder layer in three different culture media. All the three media consisted of same concentration of DMEM, NEEA, L-glutamine, FBS, EGF and FGF, however in addition to these, LIF was added to media I, GDNF was added to media II and both LIF and GDNF were added to media III. Characterization of SSCs was done by alkaline phosphatase and immunoflourescence staining. Expression of SSC specific pluripotent marker genes by putative SSCs was also studied by RT-PCR study. Porcine SSCs were observed as dome shaped round or oval bodies on 5th -6th day of culture in all the three media. Clustering of cell was observed from 4th -5th day of culture and single, paired or multiple colonies were observed from 8-10th day of culture. The SSCs colonies appeared as mulberry, grape or rosette shaped with irregular distinct boundary from feeder layer on 15th - 19th day of culture in all the three media. However, the shape of the SSCs was found to be distorted with increase in the days of culture. The morphology of the SSC colonies was maintained best up to 30th day of culture in media III. The SSC colony number was recorded as 82.14 ± 2.91, 60.07 ± 2. 78 and 48.43 ± 1.96 on 5th, 15th and 30th day of culture respectively in media I. The corresponding numbers were 91.71 ± 2.62, 67.00 ± 2.05 and 57.29 ± 2.17 in media II and 105.93 ± 2.82, 80.21 ± 2.45 and 62.50 ± 2.09 in media III respectively. The SSC colony diameter was found to be 64.26 ± 0.85, 125.30 ± 1.88 and 123.01 ± 5.49and#956;m on 5th, 15th and 30th day of cu