Ovarian aging is associated with declining numbers and quality of oocytes and follicles. Oxidative stress by reactive oxygen species (ROS) contributes to somatic aging in general, and also has been implicated in reproductive aging. Telomere shortening is also involved in aging, and telomeres are particularly susceptible to ROS-induced damage. Previously, we have shown that antioxidant N-acetyl-l-cysteine (NAC) effectively rescues oocytes and embryos from ROS-induced telomere shortening and apoptosis in vitro. Using mice as models, we tested the hypothesis that reducing oxidative stress by NAC might prevent or delay ovarian aging in vivo. Initially, young females were treated with NAC in drinking water for 2 months and the quality of fertilized oocytes and early embryo development were evaluated. Next, young mice 1–1½ months old were treated for 1 year with NAC added in drinking water, and their fertility was analyzed starting at 6 months, as indicated by litter size, oocyte number and quality. The ovaries were also examined for telomere activity and length and the expression of selected genes related to aging and DNA damage. Short-term treatment of mice for 2 months with NAC demonstrated that NAC improved the quality of fertilized oocytes and early embryo development. Mice treated with a long-term low concentration (0.1 mM) of NAC had increased litter sizes at the ages of 7–10 months compared with age-matched controls without NAC treatment. NAC also increased the quality of the oocytes from these older mice. Moreover, the expression of sirtuins was increased, telomerase activity was higher and telomere length was longer in the ovaries of mice treated with NAC compared with those of the control group. These data suggest that appropriate treatment with the antioxidant NAC postpones the process of oocyte aging in mice.

The relative tensile strengths of the sperm of seven mammalian species and sea urchins have been measured by determining the minimum shear necessary

Mouse embryo development is identically inhibited by raised osmolarity, whether produced by added NaCI or raffinose, demonstrating that high osmolari

Cells subjected to sustained high osmolarity almost universally respond by accumulating compatible organic osmolytes that, in contrast to inorganic ions, are not deleterious even at high intracellular concentrations. Their accumulation from the external environment by known organic osmolyte transporters, such as the four identified in mammals, occurs only slowly in response to sustained high osmolarity, by synthesis of new transporter proteins. Most cells, however, are not subject to high or varying osmolarity, and it is not clear whether organic osmolytes are generally required at normal osmolarities or how they are regulated. The fertilized egg of the mouse is protected in the oviduct from perturbations in osmolarity. However, deleterious effects of osmotic stress were evidentin vitroeven at normal oviductal osmolarity. Glycine was found to protect development, indicating that early mouse embryos may use glycine as an organic osmolyte at physiological osmolarity. We have now found that GLYT1, a glycine transporter of the neurotransmitter transporter gene family, functions as the organic osmolyte transporter that mediates the osmotically regulated accumulation of glycine and regulates cell volume in early embryos. Furthermore, osmotic stimulation of GLYT1 transport was immediate, without a requirement for protein synthesis, implying regulation different from known organic osmolyte transporters. Thus, GLYT1 appears to have a previously unidentified role as an organic osmolyte transporter that functions in acute organic osmolyte and volume homeostasis near normal osmolarity.

Preimplantation embryos are particularly susceptible to in vitro developmental blocks. These could be alleviated by lowering culture medium osmolarity. Because mammalian cells regulate their volumes by adjusting intracellular osmotic pressure, cell volume regulation could be critical to early embryos. We reviewed the literature on cell volume regulation in preimplantation embryos and the effects of increased osmolarity on embryo development, focusing also on the relation with improvements in embryo culture media. Embryos failed to develop from fertilized oocytes when osmolarity is increased. This could be alleviated by decreasing osmolarity or including certain compounds such as certain amino acids. Early preimplantation mouse embryos require intracellular accumulation of glycine to provide osmotic support and thus control cell volume. The glycine-specific transporter, GLYT1, mediates osmoregulated glycine accumulation in mouse embryos and likely in human embryos. GLYT1 is activated during meiotic maturation starting at ovulation. Prior to this, oocyte size is not independently controlled but instead is determined by strong adhesion between the oocyte plasma membrane and the inner surface of the zona pellucida. Early preimplantation embryos are particularly sensitive to increased osmolarity, and require the importation of glycine to regulate their cell volumes using a mechanism unique to early embryos. Cell volume regulation first appears when ovulation is triggered, oocyte zona pellucida adhesion is released, and glycine transport is activated. The requirement for supporting these physiological functions in oocytes and embryos should be taken into account when developing and improving systems for in vitro oocyte maturation and embryo culture.