Abstract
BackgroundMelatonin is a pleiotropic hormone with powerful antioxidant activity both in vivo and in vitro. The present study aimed to investigate the effects of melatonin on the proliferation efficiency of neonatal mouse spermatogonial stem cells (SSCs) using a three-dimensional soft agar culture system (SACS) which has the capacity to induce development of SSCs similar to in vivo conditions.MethodsSSCs were isolated from testes of neonate mice and their purities were assessed by flow cytometry using PLZF antibody. Isolated testicular cells were cultured in the upper layer of the SACS in αMEM medium in the absence or presence of melatonin extract for 4 weeks.ResultsThe identity of colonies was confirmed by alkaline phosphatase staining and immunocytochemistry using PLZF and α6 integrin antibodies. The number and diameter of colonies of SSCs in the upper layer were evaluated at days 14 and 28 of culture. The number and diameter of colonies of SSCs were significantly higher in the melatonin group compared with the control group. The levels of expression of ID-4 and Plzf, unlike c-kit, were significantly higher in the melatonin group than in the control group.ConclusionsResults of the present study show that supplementation of the culture medium (SACS) with 100 μM melatonin significantly decreased reactive oxygen species (ROS) production in the treated group compared with the control group, and increased SSC proliferation.
Highlights
Melatonin is a pleiotropic hormone with powerful antioxidant activity both in vivo and in vitro
Our findings show that soft agar culture system (SACS), along with the novel innovative medium used in this study, inhibits the release of free radicals during in vitro culture of Spermatogonial stem cell (SSC) and provides a promising strategy for the expansion of SSCs; the survival rate of SSCs was enhanced during a 4-week culture period using melatonin as an important antioxidant in the culture medium
The results show that the majority of the cells (91.32 ± 4.2%) were viable immediately after the two-step enzymatic digestion (P ≤ 0.05)
Summary
Melatonin is a pleiotropic hormone with powerful antioxidant activity both in vivo and in vitro. The present study aimed to investigate the effects of melatonin on the proliferation efficiency of neonatal mouse spermatogonial stem cells (SSCs) using a three-dimensional soft agar culture system (SACS) which has the capacity to induce development of SSCs similar to in vivo conditions. The goal of the present study was to investigate the effects of melatonin (antioxidant) supplementation to the SACS culture medium containing LIF and GDNF on the proliferation of neonate mouse SSCs. Our findings show that SACS, along with the novel innovative medium used in this study, inhibits the release of free radicals during in vitro culture of SSCs and provides a promising strategy for the expansion of SSCs; the survival rate of SSCs was enhanced during a 4-week culture period using melatonin as an important antioxidant in the culture medium
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