In the vertebrate lens, nuclei and other cytoplasmic organelles are degraded in fiber cells situated in the center of the tissue. This is believed to ensure the transparency of the tissue. The mechanism that triggers this process is unknown. We hypothesized that standing gradients of oxygen generated within the tissue may serve as a spatial cue for organelle degradation. To examine this possibility, we incubated fertilized chicken eggs under hyperoxic (50% O2) or normoxic (21% O2) conditions. Hyperoxic treatment was initiated on the seventh day of embryonic development (E7), five days before organelle degradation normally commences in the lens core. Hyperoxia was maintained until E17. Under normoxic conditions, the partial pressure of oxygen (PO2) within the vitreous compartment was low. Direct measurement of PO2 using an optode oxygen sensor indicated values of 1.3 kPa and 0.4 kPa for the mid- and anterior vitreous, respectively. Similarly, treatment with pimonidazole, a bio-reductive hypoxia marker, led to the formation of immuno-positive protein adducts within the lens, suggesting that the embryonic lens is chronically hypoxic in situ. Following hyperoxic treatment, vitreous PO2 significantly increased, although pimonidazole staining in the lens was not markedly affected. Confocal microscopy of slices prepared from hyperoxic lenses revealed a significant increase in the size of the lens relative to age-matched normoxic controls. By E13, an organelle-free zone (OFZ) was present in the center of normoxic and hyperoxic lenses. However, in hyperoxic lenses, the OFZ was consistently smaller, and the distance from the lens surface to the border of the OFZ significantly larger, than in normoxic controls. These observations suggest that hyperoxia delays organelle breakdown and are consistent with a model in which hypoxia in the deep cortical layers of the normal lens serves as a trigger for the organelle loss process.
- Confocal microscopy