β-Nerve growth factor (β-NGF) is a neurotrophin with different roles in reproduction that could be regulated by sexual hormone concentrations. The objective of the present study was to characterise β-NGF in the accessory glands and epididymis of male rabbit and to determine whether its expression differentially changed near puberty (week 22) and adulthood (week 37) according to serum testosterone levels and NGF levels in both serum and seminal plasma (SP). Semen and blood were collected every week from 6 males during 3 months and analysed at 3 time points (weeks 22, 30, and 37) by ELISA. The SP was separated from sperm cells by centrifugation (3,000 × g for 15 min at 4°C) and stored at –20°C; blood was collected in EDTA tubes, centrifuged 15 min at 700 × g at 4°C and stored at –20°C too. Reproductive tissues (prostate, bulbouretral gland, and caput and cauda of epididymis) were collected at the beginning (week 22; n = 4) and at the end (week 37; n = 4) of the experiment. For tissue recovery, males were killed and glands and epididymis were dissected, fixed in modified Bouin’s fluid, and mounted in paraffin. The ELISA for β-NGF (abx259154, Rabbit NGF ELISA kit, Abbexa, Cambridge, UK) and testosterone (DE1559, Demeditec Diagnostics, Kiel, Germany) were performed following the kit protocols. For immunohistochemistry (IHC), goat polyclonal anti-NGF antibody was used in a dilution 1:100 (N8773, Sigma Aldrich, St. Louis, MO, USA). The avidin-biotin-peroxidase complex (ABC) method was performed with the Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA) and then slides were contrasted with hematoxylin. Results of serum testosterone levels revealed a significant decrease (P < 0.05) at week 37 (0.57 ± 0.18 ng mL−1) from week 30 (4.68 ± 0.86 ng mL−1) and week 22 (3.38 ± 0.99 ng mL−1). However, β-NGF levels at week 37 in serum (299.32 ± 60.22 pg mL−1) and in SP (947.29 ± 249.45 pg mL−1) also decreased but there were no significant differences. Also, we found no correlation between β-NGF in serum and SP and testosterone levels. β-Nerve growth factor was immunolocated in prostate of both ages where epithelial cells were highly stained. At 22 weeks old, stain was located mostly in the apical zone of the cytoplasm, whereas at 37 weeks old, the protein was localised in the entire cytoplasm of the cell. Furthermore, the interstitial tissue in adult males had a moderate stain in contrast with younger males, which did not show signal in that tissue. The content in the prostate lumen was stained too. Bulbourethral glands had a low signal in interstitial tissue that seems to be greater at week 37. Caput and cauda of epididymis of both ages were not stained. These results suggest that β-NGF has different immunolocation in the rabbit male accessory glands depending on the age of animal that could be related to changes in serum testosterone levels. However, these differences were not correlated with β-NGF levels neither in blood serum nor in SP.
Research funded by AGL2015-65572-C2-2-R Grant and Predoctoral Contract UCM-Santander.
Characterization of β-Nerve Growth Factor-TrkA system in male reproductive tract of rabbit and the relationship between β-NGF and testosterone levels with seminal quality during sexual maturation
