Modulation of TGFbeta1 and VEGF-A expression in the testes of transgenic mice overexpressing hCG.

GONZALEZ C, RULLI SB, GONZALEZ B, HUHTANIEMI I, CALANDRA RS, GONZALEZ-CALVAR SI.

Belo Horizonte, Brasil

Simposio; International Symposium on Animal Behaviour of Reproduction; 2006

Brazilian College of Animal Reproduction

INTRODUCTION Transgenic male mice (TG) over-expressing a and b subunits of hCG are infertile and show enhanced steroidogenesis. The chronic hCG hyper-stimulation leads to Leydig cell hyperplasia and hypertrophy in prepubertal mice, being reduced at adulthood (1,2). Transforming growth factor-b1 (TGF-b1) belongs to a superfamily of growth factors that regulates a variety of cellular processes, including cell cycle progression, cell differentiation, reproductive function and development. Several reports indicate that TGF-b1 is involved in vascular endothelial growth factor (VEGF) expression. The aim of this study was to analyze the expression of TGF-b1, its co-receptor endoglin and VEGF-A isoforms (VEGF-A 120, 164 and 188) and the receptors VEGF-R1 and VEGF-R2 in the testes of TG at 3 and 8 weeks of age. In order to correlate the “in vivo” studies, we analyzed “in vitro” effect of TGF-b1 on testicular VEGF-A expression. MATERIALS AND METHODS Testes from TG and wild type FVB/n (WT) (3 and 8 weeks of age) were removed and frozen at –80ºC. Total RNA was isolated with Trizol reagent and used for RT-PCR. The expression of TGF-b1, endoglin, VEGF-A and their isoforms and receptors (VEGF-R1 and R2) were evaluated. Actin was used as house-keeping gene. Testes were also fixed in para-formaldehyde and embedded in paraffin for VEGF immunohistochemistry. Whole testes from WT (8 weeks of age) were incubated “in vitro” in 199 Medium with or without TGF-b1 (1-10 ng/ml) for 15 and 30 min. RESULTS AND DISCUSSION TG showed an increase in the expression of: 1) TGF-b1 at 8 weeks of age (p<0,05)(Fig. 1); 2) VEGF-A (Fig 1) and its isoforms as well as VEGF-R2 in TG at both ages (p<0,05). Endoglin presented a non-significant increase at 3 weeks of age. “In vitro” incubation (15 or 30 min.) with 1 ng/ml of TGF-b1 induced an increase in testicular VEGF-A expression (Fig 2) whereas a decrease on this parameter is observed at 10 ng/ml of TGF-b1 (p<0,05). The level of expression of VEGF-R1 and R2 remained unchanged.  Immunohistochemistry showed that VEGF-A is localized in Leydig cells. In summary, high and chronic levels of hCG induced testicular TGF-b1 expression on TG at 8 weeks of age. Furthermore, TG showed an increase on testicular VEGF-A expression, its isoforms and VEGF-R2. “In vitro” studies showed that TGF-b1 exerted a dose-dependent biphasic effect on VEGF-A expression. The temporal expression of these factors might influence the growth and development of the testes. REFERENCES (1)Rulli S, Ahtianen P, Makela S, Toppari J, Poutanen M, Huhtaniemi I. Endocrinology 144:4980, 2003. (2) Ahtianen P, Rulli S, Shariatmadari R, Pelliniemi L, Toppari J, Poutanen M, Huhtaniemi I. Oncogene 24:7301, 2005.