Brain-derived neurotrophic factor and neurotrophic tyrosine receptor kinase-2 in stallion testes: insights into seasonal changes and potential roles in spermatogenesis

Abstract

Brain-derived neurotrophic factor (BDNF) and its receptor neurotrophic tyrosine receptor kinase-2 (NTRK2) have known important roles in the central nervous system for neurite growth, survival, and differentiation. Nevertheless, the significance of BDNF in spermatogenesis remains unclear in stallions. Therefore, the present study was designed 1) to investigate the expression of BDNF and its receptor NTRK2 and 2) the seasonal variation in the expression patterns of BDNF and NTRK2 in stallions' testes. We used testes from eight postpubertal Thoroughbred stallions collected after a field castration during two different seasons of the year (breeding season [BS] and nonbreeding season [NBS]). Reverse transcription-quantitative polymerase chain reaction (RT-qPCR), Western blotting (WB), and immunofluorescence were performed. RT-qPCR results showed upregulation of mRNA levels of BDNF and NTRK2 in the testes collected during the NBS. The quantification of the protein bands obtained after WB displayed significantly higher relative intensity in NBS. The immunofluorescence assay identified the localization of BDNF in the cytoplasm of Sertoli and Leydig cells in BS. The cytoplasm of germs cells and Leydig cells were stained with BDNF in NBS. NTRK2 was observed in the cytoplasm of Leydig cells of BS and NBS. Moreover, different stages of germ cells including undifferentiated spermatogonia and spermatocytes were immune labeled with NTRK2 in the NBS. These findings provided the first evidence of the localization of BDNF and NTRK2 in the testicular cells of stallions, suggesting the potential role of BDNF signaling in testes development and spermatogenesis. Further investigation is necessary to explore the functional implications of BDNF signaling on spermatogenesis, focusing on the regulatory mechanisms that govern the seasonal expression patterns observed. This will help confirm the paracrine/autocrine importance of this neurotrophin in the stallions testes.

Keywords: Brain-derived neurotrophic factor; Leydig cells; Neurotrophic tyrosine receptor kinase-2; Season; Sertoli cells; Spermatogenesis.

Fig. 1.. Relative BDNF and NTRK2 mRNA abundance in the testicular tissues of the breeding and nonbreeding stallions.

Both BDNF and its receptor NTRK2 mRNA were upregulated in the nonbreeding season’s stallion testes than in the breeding season’s stallion testes. The mRNA transcript abundance of the target genes BDNF and NTRK2 was evaluated with reference to that of GAPDH mRNA transcript abundance. Data are represented as ± SEM of four individuals per group. * p < 0.05. BDNF, brain-derived neurotrophic factor; NTRK2, neurotrophic tyrosine receptor kinase-2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Fig. 2.. Cross-reactivity of rabbit anti-horse polyclonal BDNF antibody and rabbit anti-human polyclonal NTRK2 with a stallion.

The protein band in the stallion testes tissues obtained after Western blotting for BDNF was observed at 26 kDa. The protein band in the stallion testes obtained after Western blotting for NTRK2 was observed at 68 kDa. The protein band of control positive β-actin was observed at 43 kDa. In the negative control lane that was probed with rabbit IgG rather than with a primary antibody, the protein band was absent. IgG, immunoglobulin G; NTRK2, neurotrophic tyrosine receptor kinase-2; BDNF, brain-derived neurotrophic factor.

Fig. 3.. The testes of stallions were examined for season-dependent BDNF expression.

The analysis of the relative intensity of BDNF in stallion testes was performed using the ImageJ software by normalizing with β-actin. When compared to testes obtained during the breeding season, the intensity was greater for testes obtained during the non-breeding season. Data are represented as ± SEM of four individuals per group. ** p < 0.01. BDNF, brain-derived neurotrophic factor.

Fig. 4.. The testes of stallions were examined for season-dependent NTRK2 expression.

The analysis of the relative intensity of NTRK2 in stallion testes was performed using the ImageJ software by normalizing with β-actin. Compared to testes obtained during the breeding season, the intensity was greater for the testes obtained during the non-breeding season. Data are represented as ± SEM of four individuals per group. ** p < 0.01. NTRK2, neurotrophic tyrosine receptor kinase-2.

Fig. 5.. Brain-derived neurotrophic factor (BDNF) immunostaining in breeding (A−D) and nonbreeding (F−I) stallion testes.

The cytoplasm of Sertoli and Leydig cells were stained with BDNF antibody. Sertoli cells were immunolabeled with BDNF in the breeding season (A and C). Germ cells were not immunolabeled with BDNF in the breeding season. The cytoplasm of a few spermatogonia was stained in the NBS (F and H). The normal rabbit IgG stained with the same dilution as BDNF revealed no immunolocalization in any type of testes cells in both the groups (E and J). The regions (C and H) enclosed by white broken-line boxes were expanded (D and I) respectively. Red arrowhead, BDNF-positive Sertoli cells, yellow arrowheads, BDNF-positive Leydig cells, green arrowheads, BDNF-positive germ cells. Scale bar = 25 µm. DAPI, 4′,6-diamidino-2-phenylindole; IgG, immunoglobulin G.

Fig. 6.. Neurotrophic tyrosine receptor kinase-2 (NTRK2) immunostaining in breeding (A−D) and nonbreeding (F−I) stallion testes.

The Leydig cells were stained with NTRK2 antibody, but no Sertoli or germ cells were stained in the breeding season (A–D). In the non-breeding season, the localization was identified in the cytoplasm of Leydig cells and at different stages of spermatogonia (F–I). The normal rabbit IgG stained with the same concentration as NTRK2 showed no immunolocalization in any type of testes cells (E and J). The regions (C and H) enclosed by white broken-line boxes were expanded (D and I) respectively. Red arrowheads, NTRK2-positive germ cells, yellow arrowheads, NTRK2-positive Leydig cells, green arrowheads, NTRK2-positive primary spermatocytes, purple arrowheads, NTRK2-positive secondary spermatocytes. Scale bar = 25 µm. DAPI, 4′,6-diamidino-2-phenylindole; IgG, immunoglobulin G.