Metformin improves sheep sperm cryopreservation via vitalizing the AMPK pathway.

Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) is a key regulator of sperm function and physiological metabolism. Metformin, an inexpensive and effective antioxidant, is known to play an important role in the activation of AMPK. Therefore metformin has potential to improve sperm cryopreservation. The aim of this study was to investigate the effect of metformin during semen cryopreservation of sheep and to find the most effective concentration in freezing extender. Semen were cryopreserved with extender containing different concentrations of metformin (0, 0.25, 0.5, 1.0, 2.0 and 4.0 mmol/L). Sperm motility, acrosome integrity and plasma membrane integrity were measured after semen freezing and thawing. All results showed that sperm quality was significantly increased in the 1.0 mmol/L metformin-treated group compared with the control group (P < 0.05). In addition, the study showed that metformin effectively reduced the content of malondialdehyde (MDA) and reactive oxygen species (ROS), and increased the activity of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), catalase (CAT) and total antioxidant capacity (T-AOC) of freeze-thawed sperm (P < 0.05). The optimal concentration of metformin was 1.0 mmol/L. Moreover, the results showed that AMPK was localized in the acrosome region, junction and midsection of sperm, and p-AMPK was distributed in the post-acrosomal region, junction and midsection. Western blot analysis indicated that 1.0 mmol/L metformin stimulated the phosphorylation of AMPK in sperm. Further results showed that 1.0 mmol/L metformin significantly increased the mitochondrial membrane potential (ΔΨm), ATP content, glucose uptake and lactate efflux of post-thawed sperm through the AMPK pathway, improved sperm quality, and increased the cleavage rate of in vitro fertilization (P < 0.05).

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.