Being pluripotent VSELs express nuclear OCT-4 which is usually no longer required to maintain the pluripotent state, shifts to the cytoplasm in the immediate descendants SSCs and eventually disappears upon further differentiation
Being pluripotent VSELs express nuclear OCT-4 which is usually no longer required to maintain the pluripotent state, shifts to the cytoplasm in the immediate descendants SSCs and eventually disappears upon further differentiation. Correct meiotic progression has remained the major bottle-neck and various groups have used chemicals like retinoic acid for meiotic entry of germ cells . of healthy Sertoli or mesenchymal cells [observe Additional file 1 for further details]. The increase in quantity of testicular VSELs in busulphan treated testis was much like an earlier statement where Ratajczak et al  reported that VSELs survive total body irradiation in mouse bone marrow and are increased in figures as obvious by increased BrdU uptake by circulation cytometry. A significant depletion of hematopoietic stem cells Piroxicam (Feldene) (HSCs) was observed by them after radiotherapy much like a loss of SSCs in testis observed by us after busulphan treatment. Possibly the chemo- and radiotherapy destroys the micro-environment (niche supporting the stem cells) in both the bone marrow and testis, and as a result the surviving VSELs are able to proliferate and increase in figures but cannot differentiate (since the growth factors/cytokines required for differentiation are not available due to the compromised nature of the niche). Recently it was reported by our group that Piroxicam (Feldene) busulphan and cyclophosphamide treatment depletes mice ovaries of follicular reserve but VSELs survive, increase in figures in response to follicle stimulating hormone treatment and also undergo spontaneous differentiation into oocyte-like structures . Our group has also earlier reported that culture of VSELs enriched from adult mammalian (human, sheep, monkey and rabbit) ovary surface epithelium spontaneously differentiate into oocyte-like structures in a very simple culture medium (with no additional cocktail of growth factors) within three weeks; whereas the epithelial cells differentiate into mesenchymal-like fibroblasts and take action essentially as a source of growth factors and cytokines required for the differentiation of oocyte-like structures [27,28]. The aim of the present study was to study the differentiation potential of surviving stem cells collected from busulphan treated mouse testis. For this, cells collected by enzymatic digestion of seminiferous tubules (VSELs, possibly few spermatogonial stem cells and Sertoli cells) were used to establish primary cultures. Results show that the whole process of spermatogenesis gets replicated in basic culture medium. Methods The study was Piroxicam (Feldene) approved by the Institutes Animal Ethics Committee (IAEC). Adult male Swiss mice managed in the Institute experimental animal facility were utilized for the study. They were housed in a heat and humidity controlled room on a 12?hour light/12?hour darkness cycle with free access to food and water. Eight weeks aged Swiss mice were treated with busulphan (25?mg/Kg body weight through intraperitoneal route; body weight; Sigma-Aldrich, USA). One month after the treatment, mice were sacrificed by cervical dislocation; testes were collected and further processed for the study. As reported earlier by our group, this dose of busulphan caused effective loss of SSCs and germ cell aplasia evidenced by histology, significant reduction of transcripts specific for SSCs (Gfra) and germ cells (and also at protein level for DAZL. Besides the Sertoli cells, relatively quiescent VSELs were found to persist and increase in figures as confirmed by circulation cytometry and higher expression of specific transcripts and Sca-1 by qRT-PCR studies [14, Additional file 1]. Preparation of testicular cell suspension Testes were washed with phosphate buffer saline (PBS) supplemented with penicillin 100 U/ml and streptomycin 100 mg/ml. All reagents were from Invitrogen (USA) unless normally specified. Single cell suspension of testicular cells was obtained by a two-step enzymatic process as described earlier . Briefly, this involved detunication of testes, washing the tubules in PBS followed by sequential enzymatic digestion with 1mg/ml collagenase IV and 0.25% trypsin EDTA and pipetting. The cell suspension was filtered through 40?m cell strainer (BD Falcon; USA). This single cell suspension was washed twice again with PBS by centrifugation at 1000 g at 4C for 10 minutes and cells were used to determine viability and for culture. Initial cell viability was usually found to be greater than 95% by trypan blue exclusion method. The cells suspension obtained by digesting the testicular tissue was heterogeneous in nature and different strategies were used to further enrich the surviving VSELs including (i) differential plating i.e. cells suspension was plated in 35 mm culture dish, Sertoli Rabbit Polyclonal to RBM34 cells attached overnight and unattached floating cells were collected next day for the study (ii) immuno-magnetic enrichment of SCA-1 sorted cells (explained below) (iii) differential centrifugation, which involved initial centrifugation at 500 rpm for 10 minutes that allowed larger cells to.