The effects of melatonin on colonization of neonate spermatogonial mouse stem cells in a three-dimensional soft agar culture system

Abstract

Background: 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.

Methods: SSCs 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.

Results: The 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.

Conclusions: Results 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.

Keywords: Colonization; Melatonin; Proliferation; Spermatogonial stem cell; Three-dimensional soft agar culture system.

Fig. 1

Flow cytometry analysis for the detection of the percentage of purity of SSCs with Plzf marker after the placement of the dish coated with gelatin. M1: PLZF-negative cells, M2: PLZF-positive cells

Fig. 2

MTT analysis for assessment of SSC viability in different treatment groups. Data are expressed as means ± SD. **P ≤ 0.01

Fig. 3

Microscopic morphology of SSCs derived from neonatal male mice (five mice at a time in each group). Each type of experiment has three replicates. The sizes of the colonies are shown at the end of the second week (a, c) and the end of the fourth week (b, d) of SACS, as well as being positive for alkaline phosphatase activity (e). After cultivation of SSCs, the diameter and number of colonies increased in both groups, particularly in the experimental (melatonin) group. All the tests performed at least five times

Fig. 4

Immunofluorescent staining of SSC colonies. SSCs were positive for Plzf in the nuclei and α6 integrin in the cytoplasm (green) (b, e), and nuclei were stained with DAPI (blue) (a, d). c, f A merge of Plzf and α6 integrin DAPI. A single SSC cell (arrowhead) proliferated and created a colony of SSCs (arrow). Scale bars = 50 μm

Fig. 5

Comparison of colony numbers (a) and diameter (b) between the control and antioxidant groups. Data are expressed as means ± SD. ***P ≤ 0.001

Fig. 6

Expression pattern of c-kit, Plzf, and ID4 genes after culture (with antioxidant) analyzed by real-time PCR. Levels of ID4 and Plzf in the antioxidant group had an increased value compared with the control group, but the level of c-kit in the antioxidant group had no significant differences compared with the control group. Data are expressed as means ± SD. **P ≤ 0.01, ***p ≤ 0.001. ns not significant

Fig. 7

Flow cytometry analysis for the detection of reactive oxygen species (ROS) in different groups. a Fresh group, b control group (without melatonin), c experimental group (melatonin 100 μM). M2: DCFDA-negative cells, M1: DCFDA-positive cells. d ROS production of SSCs before and after SACS analyzed by flow cytometry. Note the significantly lower production of ROS after SACS with 100 μM melatonin compared to the control group. Data are expressed as means ± SD. ***P ≤ 0.0001