Abstract
Spermatogonial stem cells (SSCs) are the only adult stem cells that pass genes to the next generation and can be used in assisted reproductive technology and stem cell therapy. SSC cryopreservation is an important method for the preservation of immature male fertility. However, freezing increases the production of intracellular reactive oxygen species (ROS) and causes oxidative damage to SSCs. The aim of this study was to investigate the effect of melatonin on goat SSCs during cryopreservation and to explore its protective mechanism. We obtained SSCs from dairy goat testes by two-step enzymatic digestion and differential plating. The SSCs were cryopreserved with freezing media containing different melatonin concentrations. The results showed that 10−6 M of melatonin increased significantly the viability, total antioxidant capacity (T-AOC), and mitochondrial membrane potential of frozen-thawed SSCs, while it reduced significantly the ROS level and malondialdehyde (MDA) content (P < 0.05). Further analysis was performed by western blotting, flow cytometry, and transmission electron microscopy (TEM). Melatonin improved significantly the enzyme activity and protein expression of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) (P < 0.05), thereby activating the antioxidant defense system of SSCs. Furthermore, melatonin inhibited significantly the expression of proapoptotic protein (Bax) and increased the expression of antiapoptotic proteins (Bcl-2 and Bcl-XL) (P < 0.05). The mitochondrial apoptosis pathway analysis showed that the addition of melatonin reduced significantly the mitochondrial swelling and vacuolation, and inhibited the release of cytochrome C from mitochondria into the cytoplasm, thereby preventing the activation of caspase-3 (P < 0.05) and inhibiting SSC apoptosis. In addition, melatonin reduced significantly the autophagosome formation and regulated the expression of autophagy-related proteins (LC3-I, LC3-II, P62, Beclin1, and ATG7) (P < 0.05), thereby reversing the freeze-induced excessive autophagy. In summary, melatonin protected goat SSCs during cryopreservation via antioxidant, antiapoptotic, and autophagic regulation.
Highlights
Spermatogonial stem cells (SSCs), which are types of primitive spermatogonia in the male reproductive system, are located in the basement membrane of the seminiferous tubules [1, 2]
Immunofluorescence staining of SSC markers promyelocytic leukemia zinc finger (PLZF) and Thy-1 cell surface antigen (THY1) was used to identify cells
This study investigated for the first time the effect of melatonin on goat SSCs during cryopreservation and explored its protective mechanisms in terms of oxidation, apoptosis, and autophagy
Summary
Spermatogonial stem cells (SSCs), which are types of primitive spermatogonia in the male reproductive system, are located in the basement membrane of the seminiferous tubules [1, 2]. SSCs both self-renew to keep the stability of the stem cell pool and differentiate to contribute to spermatogenesis, thereby maintaining the homeostasis of testicular cells [3]. As adult stem cells that produce spermatocytes, SSCs transmit genetic information to the generation [4, 5]. Studies have shown that SSCs can be cultured and passaged in vitro, and differentiate into sperm after transplantation [6]. The study of SSCs is significant in terms of understanding the male germ cell differentiation mechanism, and has clinical application value in the treatment of male infertility [7, 8]. The number of SSCs is low compared to other testicular cells, as, for example, these account for only 0.02–0.03% of the mouse testicular cells [9]
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