Neutralization of vascular endothelial growth factor antiangiogenic isoforms or administration of proangiogenic isoforms stimulates vascular development in the rat testis

Abstract

Vascular endothelial growth factor A (VEGFA) plays a role in both angiogenesis and seminiferous cord formation, and alternative splicing of the Vegfa gene produces both proangiogenic isoforms and antiangiogenic isoforms (B-isoforms). The objectives of this study were to evaluate the expression of pro- and antiangiogenic isoforms during testis development and to determine the role of VEGFA isoforms in testis morphogenesis. Quantitative RT-PCR determined that Vegfa_165b mRNA was most abundant between embryonic days 13.5 and 16 (E13.5 and 16; P<0.05). Compared with ovarian mRNA levels, Vegfa_120 was more abundant at E13-14 (P<0.05), Vegfa_164 was less abundant at E13 (P<0.05), and Vegfa_165b tended to be less abundant at E13 (P<0.09) in testes. Immunohistochemical staining localized antiangiogenic isoforms to subsets of germ cells at E14-16, and western blot analysis revealed similar protein levels for VEGFA_165B, VEGFA_189B, and VEGFA_206B at this time point. Treatment of E13 organ culture testes with VEGFA_120, VEGFA_164, and an antibody to antiangiogenic isoforms (anti-VEGFAxxxB) resulted in less organized and defined seminiferous cords compared with paired controls. In addition, 50 ng/ml VEGFA_120 and VEGFA_164 treatments increased vascular density in cultured testes by 60 and 48% respectively, and treatment with VEGFAxxxB antibody increased vascular density by 76% in testes (0.5 ng/ml) and 81% in ovaries (5 ng/ml) compared with controls (P<0.05). In conclusion, both pro- and antiangiogenic VEGFA isoforms are involved in the development of vasculature and seminiferous cords in rat testes, and differential expression of these isoforms may be important for normal gonadal development.

Figure 1

Quantitative RT-PCR for Vegfa_120 (A), Vegfa_164 (B), and Vegfa_165b (C) from E13 through P3 of testis development. Gapdh was used as an endogenous control to account for differences in starting material. These data are the result of at least 3 different pools of each age tissue. The mean ± SEM normalized QRT-PCR values are presented for each developmental age. Developmental ages are labeled with letters to represent statistical comparisons: ages labeled with a common letter are not different while ages without a common letter are significantly different (P < 0.05).

Figure 2

Comparison of quantitative RT-PCR values between E13 and E14 testes and ovaries for Vegfa_120 (A), Vegfa_164 (B), and Vegfa_165b (C). Gapdh was used as an endogenous control to account for differences in starting material. These data are the result of at least 3 different pools of each age tissue. The mean ± SEM normalized QRT-PCR values are presented for each developmental age. Asterisks represent a statistically significant difference between testes and ovaries at each age (*P < 0.0001, **P < 0.05). The plus sign indicates a tendency toward different means (+P < 0.09).

Figure 3

(A) Western blot analysis for VEGFA isoforms from E13 to P4 of testis development. Black lines indicate where different blot images have been spliced together. Recombinant VEGFA_164 served as a positive control (data not shown). Quantitative analysis of western blot bands for VEGFA_120/121B (B), VEGFA_164/165B (C), VEGFA_188/189B (D), and VEGFA_205/206B (E) based upon a standard curve created from recombinant VEGFA_164. These data are the result of at least 3 different pools of each age tissue. The mean ± SEM densitometry values are presented for each developmental age. Developmental ages are labeled with letters to represent statistical comparisons: ages labeled with a common letter are not different while ages without a common letter are significantly different (P < 0.05).

Figure 4

(A) Western blot analysis for antiangiogenic VEGFA isoforms from E13 to P4 of testis development. Recombinant VEGFA_165B served as a positive control (data not shown). Quantitative analysis of western blot bands for VEGFA_121B (B), VEGFA_165B (C), VEGFA_189B (D), and VEGFA_206B (E) based upon a standard curve created from recombinant VEGFA_165B. These data are the result of at least 3 different pools of each age tissue. The mean ± SEM densitometry values are presented for each developmental age. Developmental ages are labeled with letters to represent statistical comparisons: ages labeled with a common letter are not different while ages without a common letter are significantly different (P < 0.05).

Figure 5

Immunohistochemistry for VEGFA antiangiogenic isoforms in E14 (B), E16 (C), and P0 (D) testes. D was counterstained with hemotoxylin. (A) E14 testis sections with no primary antibody served as negative controls. These data are the result of at least 3 different testes from each developmental time point. Scale bar represents 50 µm. T, testis; M, mesonephros; c, seminiferous cord; g, germ cell; i, interstitium.

Figure 6

Embryonic Day 13 testis organ cultures without treatment (A–C) or treatment with 50 ng/ml of recombinant VEGFA_120 (D–F), 50 ng/ml of recombinant VEGFA_164 (G–I), 0.5 ng/ml of VEGFAxxxB antibody (J–L), and 5 ng/ml of VEGFAxxxB antibody (M–O). (A, D, G, J, and M) Bright-field images. (B, C, E, F, H, I, K, L, N, and O) Confocal images of whole mount immunohistochemistry staining for PECAM1 (red indicates endothelial cell marker) to localize vasculature. Scale bar represents 200 µm. T, testis; M, mesonephros; c, cord.

Figure 7

Effect of VEGFA_120, VEGFA_164, 0.5 ng/ml VEGFAxxxB antibody, and 5 ng/ml VEGFAxxxB antibody on vascular density in cultured testes expressed as a percentage of control organs. Data are the result of 21 (A [VEGFA_120 and VEGFA_164]), 24 (A [0.5 ng/ml VEGFAxxxB antibody]), and 22 (A [5 ng/ml VEGFAxxxB antibody]) testis pairs. The mean ± SEM stained areas for PECAM1 (vascular density) are presented for each organ culture treatment. Asterisks represent a statistically significant difference between treated and control groups (*P < 0.008, **P < 0.05). The plus sign indicates a tendency toward different means (+P < 0.09). Ab, antibody.