Limbal epithelial stem cell activity and corneal epithelial cell cycle parameters in adult and aging mice.

Limbal epithelial stem cells (LESCs) are believed to be responsible for corneal epithelial maintenance and repair after injury, but their activity has never been properly quantified in aging or wounded eyes. In this study, labelling with thymidine analogues, 5-iodo-2'-deoxyuridine (IdU), 5-chloro-2'-deoxyuridine (CldU) and 5-ethynyl-2'-deoxyuridine (EdU), was used to estimate cell-cycle time of the corneal and limbal epithelia in wild-type eyes, comparing aging (12 months) and young adult (8 week) mice. In C57BL/6 mice, cells cycled significantly faster in the central corneal epithelium of aging eyes (3.24 ±â€¯0.2 days) compared to 10 week old mice (4.97 ±â€¯0.5 days). Long-term labelling with IdU was used to detect slow-cycling stem cells, followed by CldU or EdU labelling to quantify the proliferative dynamics of LESCs during corneal wound healing. In unwounded eyes, 4.52 ±â€¯1.4% of LESCs were shown to enter S phase in a 24 h period and were estimated to divide every 2-3 weeks. Within 24 h of corneal injury this rose significantly to 32.8 ±â€¯10.0% of stem cells indicating a seven-fold increase in activation. In contrast, no comparable increase in LESC activation was observed in aging mice after wounding. In the 24-48 h period after wounding in young adults, LESC activation continued to increase (86.5 ±â€¯8.2% of label-retaining cells in wounded eye were in S-phase) but surprisingly, 46.0 ±â€¯9.4% of LESCs were observed to reenter S-phase in the contralateral unwounded eye. These data imply an unsuspected systemic effect of corneal wounding on LESC activation suggesting that injury to one eye elicits a regenerative response in both.

Male-Female Interactions and the Evolution of Postmating Prezygotic Reproductive Isolation among Species of the Virilis Subgroup.

Reproductive isolation reduces breeding between species. Traditionally, prezygotic and postzygotic barriers to reproduction have been broadly studied, but in recent years, attention has been brought to the existence of barriers that act after copulation but before fertilization. Here, we show that when D. virilis females from different geographic locations mate with D. novamexicana males, egg laying is normal, but fertilization rates are severely reduced, despite normal rates of sperm transfer. This reduction in fertilization is probably due to lower retention of heterospecific sperm in female storage organs one-to-two days after copulation. An inspection of egg hatchability in crosses between females and males from other virilis subgroup species reveals that isolation due to poor egg hatchability likely evolved during the diversification of D. virilis/D. lummei from species of the novamexicana-americana clade. Interestingly, the number of eggs laid by D. virilis females in heterospecific crosses was not different from the numbers of eggs laid in conspecific crosses, suggesting that females exert some form of cryptic control over the heterospecific ejaculate and that future studies should focus on how female and female-sperm interactions contribute to the loss or active exclusion of heterospecific sperm from storage.