Recent advances in isolation, identification, and culture of mammalian spermatogonial stem cells.

Continuous spermatogenesis depends on the self-renewal and differentiation of spermatogonial stem cells (SSCs). SSCs, the only male reproductive stem cells that transmit genetic material to subsequent generations, possess an inherent self-renewal ability, which allows the maintenance of a steady stem cell pool. SSCs eventually differentiate to produce sperm. However, in an in vitro culture system, SSCs can be induced to differentiate into various types of germ cells. Rodent SSCs are well defined, and a culture system has been successfully established for them. In contrast, available information on the biomolecular markers and a culture system for livestock SSCs is limited. This review summarizes the existing knowledge and research progress regarding mammalian SSCs to determine the mammalian spermatogenic process, the biology and niche of SSCs, the isolation and culture systems of SSCs, and the biomolecular markers and identification of SSCs. This information can be used for the effective utilization of SSCs in reproductive technologies for large livestock animals, enhancement of human male fertility, reproductive medicine, and protection of endangered species.

Rosmarinic acid improves boar sperm quality, antioxidant capacity and energy metabolism at 17°C via AMPK activation.

Boar sperm are susceptible to oxidative damage caused by reactive oxygen species (ROS) during storage. Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is an important therapeutic target, because it is a cellular metabolism energy sensor and key signalling kinase in spermatozoa. We evaluated the effects of rosmarinic acid (RA), an antioxidant, on boar sperm during liquid storage to determine whether it protects boar sperm via AMPK activation. Boar ejaculates were diluted with Modena extender with different concentrations of RA and stored at 17°C for 9 days. Sperm quality parameters, antioxidant capacity, energy metabolism, AMPK phosphorylation and fertility were analysed. Compared with the control, 40 µmol/L significantly improved sperm motility, plasma membrane integrity and acrosome integrity (p < .05). The effective storage time of boar sperm was up to 9 days. On the third and seventh days, the sperm with RA exhibited increased total antioxidant capacity (T-AOC), superoxide dismutase (SOD) activity, adenosine triphosphate (ATP) content, mitochondrial membrane potential (ΔΨm) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity, whereas malondialdehyde (MDA) content was significantly decreased (p < .05). Western blot showed that RA, as well as AICAR (AMPK activator), promoted AMPK phosphorylation, whereas Compound C (AMPK inhibitor) inhibited this effect. The sperm-zona pellucida binding experiment showed that 40 µmol/L RA increased the number of sperm attached to the zona pellucida (p < .05). These findings suggest meaningful methods for improved preservation of boar sperm in vitro and provide new insights into the mechanism by which RA protects sperm cells from oxidative damage via AMPK activation.

Melatonin Protects Goat Spermatogonial Stem Cells against Oxidative Damage during Cryopreservation by Improving Antioxidant Capacity and Inhibiting Mitochondrial Apoptosis Pathway.

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.

Effects of sulfanilamide on boar sperm quality, bacterial composition, and fertility during liquid storage at 17°C.

Sulfanilamide (SA) is an effective broad-spectrum antibacterial agent in human and veterinary medicine. The purpose of this study was to evaluate the effects of SA on boar sperm quality during liquid storage at 17°C and determine the optimal concentration of SA and its effects on bacterial growth, microbial composition, and maternal fertility. Boar ejaculates were diluted with a basic extender, containing different concentrations of SA, and stored in a 17°C incubator for 6 days. The sperm motility, plasma membrane integrity, and acrosome integrity were measured daily. The results showed that when the concentration of SA was 0.02 g/L, the sperm quality parameters were significantly higher than those of all other treatment groups (p < .05). We also monitored the bacterial growth and compared the differences in the microbial species between the 0.02 g/L SA group and the control by 16S rDNA sequencing. The results revealed that some bacteria, such as Staphylococcus and Pseudomonas, were considerably lower in the 0.02 g/L SA group than in the control group (p < .05). In addition, preserved semen was used for artificial insemination, and results showed that 0.02 g/L SA group had a higher litter size, and its pregnancy rate was 92.5%.