Abstract
The access to sufficient numbers of spermatogonial stem cells (SSCs) is a prerequisite for the study of their regulation and further biomanipulation. Rho kinase (ROCK) belongs to a family of serine/threonine kinases and involves in a wide range of fundamental cellular functions. The aim of the present study was to study the effect of ROCK inhibitor, Y-27632 (0.1-40 µM), during the primary culture of ovine SSCs. SSCs were collected from 3-5-month-old’s lamb testes. The viability of SSCs, the apoptosis assay of SSCs, the intracellular reactive oxygen species (ROS) analysis, and the SSCs markers and apoptosis-related gene expressions were detected by MTT reduction assay, Annexin V–FITC/ Propidium Iodide (PI) dual staining, flow cytometry and real-time-PCR studies, respectively. Morphological analyses indicated that the 5-10 µM Y-27632 had an optimal effect on the number of presumptive SSCs colonies and the area covered by them after a 10 days culture. The cell viability, apoptosis and necrosis of SSCs after 10 days’ culture were not affected in comparison with the control group, and the 20 µM of Y-27632 resulted in significantly decreased cell viability (P<0.05) and an increased necrosis of cells. On day 10 after culture, the expression of P53 was decreased with an increase from 0 to 10 µM in the Y-27632 dose. In the 20 µM Y-27632 group, the expressions of P53 and Bax were higher and the Bcl-2 was lower than other groups and these values were significantly different from 5 and 10 µM Y-27632 groups (P<0.05). The level of intracellular ROS was decreased with an increase in the Y-27632 dose from 5 to 20 µM in comparison with the control group. In conclusion, the present study demonstrated that Y-27632 at a concentration of 5-10 µM provided optimal culture conditions for the primary culture of ovine SSCs.
Highlights
Spermatogonial stem cells in the mammalian testis are unipotent stem cells, which demonstrate distinctive cell features as stem and germ cells after being separated from the testis and cultured in vitro (Sahare et al, 2018)
This study aims at improving primary culture of spermatogonial stem cells (SSCs) by adding different concentrations of Y-27632 on viability, colony formation, proliferation and apoptosis of SSCs
Viability of SSCs after 48h culture As shown in Figure 1, the viability of SSCs was not affected by 0.1 -10 μM Y-27632; higher concentration of Y-27632 (20 and 40 μM) significantly decreased the viability of SSCs (P
Summary
Spermatogonial stem cells in the mammalian testis are unipotent stem cells, which demonstrate distinctive cell features as stem and germ cells after being separated from the testis and cultured in vitro (Sahare et al, 2018). Among the stem cells in an adult male body, SSCs are very unique (Heidari et al, 2012) since they can transmit genetic information from one generation to another, and it is of significant importance (Shams et al, 2017). The fact mentioned above makes it possible for in vitro culture of SSCs (Dobrinski, 2006). Studies on SSCs have been initiated a few decades ago, concentrating on the production of goat and sheep offspring (Binsila et al, 2018). Using this technology in sheep is of great importance
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