PARENT SESSION
Epididymis and Sperm Maturation

STUDIES OF VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) AND ITS RECEPTOR EXPRESSION AND LOCALIZATION IN THE RAT EPIDIDYMIS.

Au, Chak¹, Lun, Wei¹, Yeung, Lam¹, Chan, Lai¹, ¹

ABSTRACT- VEGF is a potent angiogenic factor with direct mitogenic action specifically on endothelial cells. Recent studies indicate that it also acts on non-endothelial cells. Transgenic mice overexpressing VEGF in the epididymis have dilated ductus epididymis containing areas of epithelial hyperplasia, implying a VEGF action on the epididymal epithelium. In the present study, the expression and localization of VEGF and its receptors (Flt-1 and Flk-1) were examined in intact and castrated adult rats with and without receiving testosterone (T) replacement. Using RT-PCR, mRNAs of VEGF, Flt-1 and Flk-1 were demonstrated in the head (initial segment and caput), body (corpus) and tail (cauda) region of the rat epididymis. Three spice variants of VEGF mRNA were found corresponding to VEGF₁₂₀, VEGF₁₆₄ and VEGF₁₈₈. These were confirmed by Western blot showing three major bands of 27, 21 and 14 kd. Both the full-length and truncated forms of Flt-1 were demonstrated by RT-PCR. In Western blot, the full-length Flt-1 occurred at the expected size of 164 kd and Flk-1 at 170 kd. Immunostaining showed specific localization of VEGF, Flt-1 and Flk-1 along the entire length of the epididymis. VEGF immunoreactivity occurred mainly in principal cells and peritubular cells. In the caput where VEGF was most abundant, intense staining was found above and below the nuclei of principal cells. Flt-1 showed diffuse and variable staining around the nuclei and under the apical borders of principal cells. Some macrophages found within and around the epididymal ducts were also Flt-1 positive. Flk-1 staining of epididymal epithelium was weak except for some narrow cells and basal cells. Two weeks after castration, VEGF immunoreactivity remained in the epididymal epithelium, though the pattern changed with the collapse of the ducts. In Western blot, the signal intensities for the three VEGF isoforms showed little changes relative to the same amount of total protein, whereas the ones for Flk-1 and Flt-1 decreased. On the whole, T replacement restored the patterns of VEGF, Flt-1 and Flk-1 immunostaining in the epididymis, and their signal intensities in Western blot. In conclusion, the present findings are in line with a direct VEGF action on the epididymal epithelium, which is likely to be androgen-dependent.

KEY WORDS: vascular endothelial growth factor, epididymis, castration