PARENT SESSION

(98) PHOSPHOTIDYLINOSITOL 3-KINASE (PI3 Kinase) IS A POTENTIAL SIGNAL TRANSDUCTION PATHWAY FOR VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) REGULATION OF EMBRYONIC TESTIS MORPHOGENESIS.

Bott, Rebecca¹, McFee, Ryann ¹, Baltes, Michelle¹, Clopton, Debra¹, Cupp, Andrea¹, ¹ University of Nebraska-Lincoln, Lincoln, NE

ABSTRACT- Development of seminiferous cords and sex-specific vascularization are the first hallmarks of testis morphogenesis in the male. We have previously established in rat testis organ cultures that inhibition of Vascular Endothelial Growth Factor (VEGF) signal transduction through treatment of VEGFR-TKI inhibits both cord formation and vascular development. To determine the mechanisms of VEGF regulation during testis morphogenesis we treated indifferent testis pairs from VEGFR2/LacZ mice with 8uM VEGFR-TKI or vehicle control beginning at embryonic day 11 (E11) or initial day (D0) through D4 of culture. On D0, cells expressing VEGFR-2 were abundant in the testis, with a small subset of these cells migrating into the developing testis. Endothelial cells expressing VEGFR-2 were able to migrate from the mesonephros into the testis of both control and treated organs throughout the culture period. However, the ability of these cells to form a densely organized vascular network within the testis and survive throughout the culture system was compromised in organs treated with VEGFR-TKI. The data from this experiment indicate that VEGFR-TKI treatment does not inhibit endothelial cell migration into the testis. Instead, a decline in cell survival throughout duration of the culture was observed as a result of VEGFR-TKI administration. We concluded that VEGF mediated cell survival pathways may be inhibited resulting in endothelial cell apoptosis. To further investigate this theory, E13 rat testis pairs were treated with 10 or 15 uM PI3 kinase inhibitor, LY294002 or vehicle control. Testes were subsequently analyzed after culture through whole mount immunohistochemistry and confocal analysis for PECAM expression, indicating presence of endothelial cells. No difference was detected in organ size for either dose of LY294002. Similarly there was no difference (p > 0.01) in PECAM density in 11 testes treated with 10 uM LY294002 (p > 0.01 vs paired controls). However, in preliminary trials utilizing 6 testis pairs, PECAM density in 15 uM LY294002 treated organs was reduced 75% when compared to controls (p <0.0001). These data indicate a potential mechanism for the PI3 kinase pathway in VEGF signal transduction necessary for cell survival during testis morphogenesis.

KEY WORDS: VEGF, testis morphogenesis, endothelial cell survival, signal transduction