Journal of Animal Reproduction and Biotechnology 2024; 39(3): 194-200
Published online September 30, 2024
https://doi.org/10.12750/JARB.39.3.194
Copyright © The Korean Society of Animal Reproduction and Biotechnology.
Jaewoo Choi1 , Heejun Jung1 , Yubin Song1 and Minjung Yoon1,2,3,*
1Department of Animal Science and Biotechnology, Kyungpook National University, Sangju 37224, Korea
2Department of Horse, Companion and Wild Animal Science, Kyungpook National University, Sangju 37224, Korea
3Research Institute for Innovative Animal Science, Kyungpook National University, Sangju 37224, Korea
Correspondence to: Minjung Yoon
E-mail: mjyoonemail@gmail.com
This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background: Brain-derived neurotrophic factor (BDNF) and its receptor, neurotrophic tyrosine receptor kinase-2 (NTRK2), are well known for their roles in the central nervous and animal reproductive systems. Several studies have observed the extensive expression of BDNF and NTRK2 in non-neuronal tissues, especially reproductive organs. However, most of these studies focused on ovarian development and regulation; thus, scientific research on BDNF and NTRK2 in males is required to determine their roles in the male reproductive system. Therefore, this study aimed to investigate BDNF and NTRK2 expression in bovine testes.
Methods: Testes were collected from six Hanwoo bulls (6-8 months old). Reverse transcription-polymerase chain reaction (RT-PCR) analysis was performed to investigate the mRNA expression of BDNF and NTRK2 in the testes. Western blot analysis was performed to verify the cross-reactivity of BDNF and NTRK2 antibodies with bovine testicular tissues. Immunohistochemistry was conducted to determine BDNF and NTRK2 protein expression in the testes.
Results: RT-PCR analysis revealed BDNF and NTRK2 mRNA expression in bovine testes. In Western blotting, BDNF and NTRK2 protein bands were observed at 32 and 45 kDa, respectively. Immunofluorescence demonstrated BDNF expression in the nuclei of spermatogonia and Sertoli cells as well as in the cytoplasm of Leydig cells. NTRK2 was exclusively expressed in Sertoli cells. These results suggest that BDNF plays a potential role in spermatogenesis via BDNF and NTRK2 signaling in bovine testes, a finding supported by previous results in different animal species.
Conclusions: The expression patterns of BDNF and NTRK2 indicate their functional importance in the bovine reproductive system.
Keywords: BDNF, bull, NTRK2, reproduction, testis
Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family. It is the most extensively distributed neurotrophin member in the brain (Rios, 2013), regulating neuronal proliferation, differentiation, and maturation by binding to its high-affinity receptor neurotrophic tyrosine receptor kinase-2 (NTRK2) (Wang et al., 1995; Simpson et al., 2002). Although the role of BDNF in the nervous system is well established, its importance in non-neuronal tissues is an emerging research hotspot. For example, in adult mouse tissues, the administration of BDNF to non-ischemic ears and ischemic limbs reportedly induced neo-angiogenesis (Kermani et al., 2005). Treatment with a soluble NTRK2 ectodomain or the Trk receptor inhibitor K252a suppressed cell proliferation in cultured human choriocarcinoma cells (Kawamura et al., 2010). BDNF–NTRK2 signaling has also been shown to induce cellular adhesion and resistance to apoptosis (Geiger and Peeper, 2007). These findings suggest that BDNF–NTRK2 signaling potentially serves an important role in non-neuronal tissues.
Several studies have observed BDNF and NTRK2 expression in the testes of various mammalian species, such as humans, mice, and rats (Mutter et al., 1999; Levine et al., 2000; Robinson et al., 2003; Müller et al., 2006; Gao et al., 2019). BDNF and NTRK2 proteins were predominantly expressed in the Leydig cells of human testes. In fetal testes, significant changes in immunostaining were observed with age, suggesting that BDNF exerts morphogenetic effects during testicular organogenesis (Müller et al., 2006). BDNF and NTRK2 mRNA expression was detected in human fetal testis at 14-19 weeks of gestation (Robinson et al., 2003). They demonstrated their possible roles in regulating the proliferation and survival of germ and peritubular cells. In rat testis cell culture, BDNF gene expression was also detected in embryonic rat testes (Levine et al., 2000). Another study treated mouse TM3 Leydig cells with exogenous BDNF, demonstrating that BDNF induces the proliferation and testosterone biosynthesis of Leydig cells (Gao et al., 2019). These studies, which localized BDNF and NTRK2 mRNA expression in the fetal testes of numerous mammalian species, suggest their participation in testicular development (Chow et al., 2020) and show that BDNF may have an apparent function in testicular cells.
Therefore, this study primarily aimed to elucidate BDNF and NTRK2 expression in bovine testes. Its results may provide cues to the roles of BDNF and NTRK2 in testicular function.
Testicular tissues were collected from six Hanwoo bulls via routine castration performed under field conditions by a local veterinary surgeon in the Republic of Korea. The age of the bulls was 7 ± 1 months. The testicular samples were carefully assessed during testicular dissection and histological observation. No animal ethics or welfare statement approval was required for this study, as the castration was conducted during a routine field visit by the local veterinary surgeon.
Testicular tissue samples were fixed and preserved as previously described, with minor modifications (Rawlings et al., 2008). Briefly, immediately succeeding the routine castration, the testicular tissues were placed in an ice box to preserve them at 4℃ and kept in a refrigerator until use. They were subsequently sliced into 0.5- and 1.0-cm3 pieces. For reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analyses, the tissue (0.5 cm3) was snap-frozen and maintained at -80℃. For immunohistochemistry, the tissue (1.0 cm3) was submerged in 4% paraformaldehyde for ≥ 24 h at room temperature. Substantial washing was conducted with phosphate-buffered saline (PBS) for 24 h, and the tissue was subsequently dehydrated via successive dilution with ethanol and fixed in paraffin blocks.
Three RNA samples isolated from bovine testes (7 ± 1 months old) were used to determine BDNF and NTRK2 mRNA expression in bovine testes. RT-PCR was conducted as previously described (Lee et al., 2016), with slight modifications. Frozen testicular tissue samples were thawed at room temperature and homogenized for total RNA extraction for 5 min using a homogenizer (HG-15A; DAIHAN®, Republic of Korea). The extracted RNA was quantified using a BioTek NanoDrop spectrophotometer (BioTek Instruments, USA). The BDNF primers FW. 5’-TCCGCCATGCAATTTCCACT-3’ and Rev. 5’-AAAGTGTCAGCCAACGACGA-3’ (Macrogen, Republic of Korea) and NTRK2 primers FW. 5’- GGGACACCAATGCAGAGACA-3’ and Rev. 5’- ACAGCCCCCTACCAATTAGC-3’ (Macrogen) were used. RT-PCR was performed using a SimpliAmpTM Thermal Cycler (Life Technologies, USA), and the following cycling conditions were applied: cDNA synthesis at 50℃ for 30 min, activation at 95℃ for 10 min, denaturation for 40 cycles at 95℃ for 20 s, annealing at 60℃ for 20 s, extension at 72℃ for 40 s, and a final extension at 72℃ for 5 min. Thereafter, the samples were loaded onto 1.5% agarose gel, and electrophoresis was performed for 30 min. The BioDoc-ItTM Imaging System (UVP, USA) was used to capture the RT-PCR results.
Western blot analysis was conducted using a previously reported protocol, with minor modifications (Rawlings et al., 2008). Protein was extracted from each testicular tissue sample and diluted with radioimmunoprecipitation assay buffer (WE324435; Thermo Fisher Scientific, Massachusetts, USA). Samples were loaded onto 10% sodium dodecyl sulfate–polyacrylamide gel and separated using the Mini-PROTEAN electrophoresis system (Bio-Rad, Hercules, CA, USA) for 1 h. The membrane was blocked with 5% blotto (2.5 g nonfat milk, 50 mL PBS, and 0.1% Tween 20) and subsequently incubated with primary antibodies: BDNF (1:200 dilution, rabbit anti-horse, LS-C293050; LSBio, USA) and NTRK2 (1:200 dilution, rabbit anti-human, LS-C135253; LSBio, USA), which were diluted with BLOTTO milk for overnight incubation at 4℃. For the negative control, normal rabbit immunoglobulin G (IgG) (2729S; Cell Signaling Technology, USA) was administered to the membrane at the same concentrations as those of the primary antibodies. Secondary antibody incubation was performed for 1 h using anti-rabbit IgG (7074, 1:10,000; Cell Signaling Technology). The iBright CL1000 imaging system (Thermo Fisher Scientific) was used to develop a film.
Immunofluorescence imaging was performed using a previously reported protocol, with slight modifications (Kim et al., 2015). Testicular tissue slides were treated with xylene (Duksan Pure Chemicals Co. Ltd., Asan, Korea) to remove paraffin, rehydrated using a graded series of ethanol washes (100, 95, 80, 70, 50, and 25%), and subsequently treated with antigen retrieval buffer (100× citrate buffer, pH 6.0; Abcam, UK) at 97.5℃ for 30 min. After antigen retrieval, the slides were blocked with 5% donkey buffer (Sigma, St. Louis, MO, USA) for 30 min. Rabbit anti-BDNF (LSBio) and anti-NTRK2 rabbit (LSBio) antibodies were separately diluted at 1:200 in a blocking buffer and incubated in a humid chamber for 1.5 h. For the negative control, normal rabbit IgG (2729S, Cell Signaling) was used at the same concentration rate as that of the primary antibodies. Thereafter, tissue sections were incubated in donkey anti-rabbit IgG (1:1,000 dilution; Alexa FluorTM 488, Thermo Fisher Scientific, USA) for 45 min. BDNF and NTRK2 expression in testicular tissues was examined using a Leica DM2500 fluorescence microscope (Leica, Germany). Testicular tissue images were captured using a Leica DFC 450 C camera. Cells with BDNF and NTRK2 emitting green fluorescence were considered positive, whereas those that did not stain green were considered negative.
RT-PCR was performed to detect the expression of BDNF and NTRK2 genes in bovine testes. The mRNA bands of BDNF and NTRK2 were detected at expected sites, approximately 430 and 546 bp, respectively (Fig. 1A and 1B). The bands were not observed in the negative control treated without template (Fig. 1A and 1B).
Western blotting was conducted to examine the cross-reactivity of the bovine testicular tissues (n = 3) with BDNF and NTRK2 antibodies. The BDNF protein band yielded an approximate weight of 32-58 kDa (Fig. 2). Thereafter, the NTRK2 protein band was observed at an approximate weight of 45 kDa. The β-actin protein band was observed at 46 kDa. The negative controls, treated with the same concentration of rabbit IgG as a primary antibody, did not exhibit any protein band (Fig. 2). These results suggest that the antibodies cross-reacted with the proteins in bovine testes.
Immunofluorescence imaging was performed to observe the localization of BDNF and NTRK2 in bovine testicular tissues. BDNF immunostaining (green fluorescence) was observed in Leydig cells, Sertoli cells, and spermatogonia (Fig. 3A-D). In Leydig cells, BDNF expression was primarily observed in the cytoplasm, while in Sertoli cells, it was localized in the nuclei (Fig. 3A-D). BDNF expression was also localized in the round-shaped nuclei of the spermatogonia (Fig. 3A-D), albeit at lower expression levels than those in Leydig and Sertoli cells. NTRK2 immunostaining (green fluorescence) was exclusively observed in Sertoli cells (Fig. 3F-I). Specifically, it was visualized as a dot in the nucleus of each Sertoli cell, and most Sertoli cells were immunolabeled with NTRK2 (Fig. 3F-I). No NTRK2 immunolabeling was observed in Leydig and germ cells. No immunolabeling of testicular cells was observed in the negative control tissue (Fig. 3E and 3J).
BDNF and NTRK2 are well recognized for their significant roles in the nervous system. Furthermore, several studies have discovered their involvement in female reproductive physiology, including follicle recruitment, oogenesis (Harel et al., 2006), germ cell survival (Paredes et al., 2004), and oocyte maturation (Lee et al., 2007). Therefore, studying the localization of BDNF and NTRK2 is requisite to improving our understanding of male reproductive physiology, especially spermatogenesis. Our present study investigated BDNF and NTRK2 expression in the Leydig cells, Sertoli cells, and spermatogonia of bovine testes.
Recent studies suggest that BDNF and NTRK2 are critical regulatory factors for ovarian development and function. In mice, NTRK2 deficiency blocked oocyte growth and granulosa cell proliferation, leading to arrested follicle development at the primary stage (Paredes et al., 2004). BDNF–NTRK2 signaling also plays a key role in oocyte survival, facilitated by the PI3K/AKT–mediated survival pathway (Dorfman et al., 2014). Several studies have detected BDNF and NTRK2 expression in the testes, suggesting a potential role for BDNF–NTRK2 signaling in testicular function. However, the mechanism by which this signaling pathway regulates testicular functions remains unclear. Therefore, further research is needed to clarify the precise role and mechanism of BDNF in the testes.
Leydig cells play an essential role in producing testosterone, a crucial hormone for normal reproductive function in the male reproductive tract (Ge et al., 2021). According to immunohistochemistry, BDNF immunolabeling was observed in the cytoplasm of Leydig cells. BDNF expression has also been described in human (Müller et al., 2006) and mouse (Gao et al., 2019) Leydig cells. Moreover, it has been detected in the Leydig cells of both fetal and adult human testes. In adult males, BDNF is predominantly localized in the Leydig cells of human testes. In a mouse Leydig cell culture study (Gao et al., 2019), treatment with exogenous BDNF increased testosterone levels in primary Leydig and TM3 Leydig cells. The researchers also detected an elevated level of enzymes, which are involved in generating testosterone. Conversely, testosterone and enzyme levels were suppressed in BDNF-knockdown TM3 Leydig cells. This study provides evidence that BDNF affects testosterone biosynthesis in Leydig cells. Normally, in bovine testes, the number of mature Leydig cells begins to rapidly increase through mitosis from 4 weeks of age, and by week 30, the rate of increase decelerates as mitosis becomes limited (Wrobel, 1990). As mature Leydig cell production nears completion, serum testosterone levels subsequently increase rapidly at approximately 20-35 weeks of age (Rawlings et al., 2008). The bulls used in our study were 6-8 months of age, displaying the active differentiation of Leydig cells and an increase in testosterone levels. Therefore, our study, which observed BDNF immunolabeling in Leydig cells, suggests that BDNF may be involved in the functioning of Leydig cells during steroidogenesis and differentiation.
Sertoli cells are essential for proper testicular function, particularly in spermatogenesis (Griswold, 1998). They produce diverse factors that play a vital role in maintaining the balance among maturation, differentiation, and germ cell self-renewal, suggesting that germ and Sertoli cells are correlated in their physiological functions and mutual control (Tripathi et al., 2014). Immature Sertoli cells in bovine testes proliferate from 4 to 20 weeks of age and begin to differentiate into mature Sertoli cells (Sinowatz and Amselgruber, 1986). The differentiation commences at approximately 20 weeks of age and generally reaches completion between 30 and 40 weeks of age (Sinowatz and Amselgruber, 1986). Therefore, in our study, testicular tissues collected from 6-8-month-old bovine testes indicated that they were likely in the middle of Sertoli cell differentiation. Immunohistochemistry revealed BDNF immunolabeling in the nuclei of Sertoli cells. BDNF protein expression in Sertoli cells has also been reported in several studies (Park et al., 2001; Müller et al., 2006; Cacialli et al., 2018). In zebrafish testes, BDNF expression was observed in the Sertoli cells, suggesting that BDNF is involved in the differentiation and proliferation of germ cells (Cacialli et al., 2018). They further proposed that Sertoli cells are possibly a key target for BDNF. However, owing to the absence of NTRK2 expression in zebrafish testes, they could only speculate on the specific roles of BDNF in these cells. Our present results, wherein NTRK2 expression was detected in the Sertoli cells of bovine testes, corroborate their hypothesis. Consequently, our findings suggest that Sertoli cells constitute a key target for BDNF, which may serve a critical role in the differentiation and proliferation of germ cells during spermatogenesis and may also be involved in the differentiation of Sertoli cells in bovine testes.
In our study, immunohistochemistry revealed BDNF immunolabeling in the nuclei of spermatogonia from the testes of 6-8-month-old bulls. Along with rapid testicular growth, spermatogenic development occurs after 20-25 weeks of age. Primary spermatocytes appear at approximately 20 weeks, secondary spermatocytes at 20-30 weeks, spermatids at 25-35 weeks, and mature spermatozoa at 32-40 weeks of age (Rawlings et al., 2008). These findings suggest that spermatogonia and primary spermatocytes are predominantly present in 6-8-month-old bovine testes. Therefore, the presence of BDNF in spermatogonia suggests that BDNF may play a role in spermatogenesis.
In this study, we observed a dot-like immunostaining pattern of NTRK2 in the nuclei of Sertoli cells. This localization of NTRK2 differs significantly from that found in other species. For instance, in humans, while less intense immunoreactivity was observed in Sertoli cells, most NTRK2 immunoreactivity was concentrated within the cytoplasm of Leydig cells (Mutter et al., 1999; Müller et al., 2006). Similarly, in mice, NTRK2 immunostaining was localized in Sertoli, peritubular myoid, and Leydig cells, but predominantly in spermatogonia A and B (Park et al., 2001). Furthermore, in rat testes, no NTRK2 immunostaining was noted in Sertoli and spermatogenic cells; it was mainly localized in interstitial cells (Moon et al., 2005). These findings indicate the variation in NTRK2 expression across species, suggesting species-specific roles and mechanisms in testicular function.
In this study, BDNF protein bands were observed at 32 and 58 kDa. A band was observed at the adequate molecular size (32 kDa); nevertheless, the band also appeared at an unexpected molecular size (58 kDa). Even though we detected an unexpected band at 58 kDa, the LS-C293050 antibody-based BDNF staining pattern in the testicular cells in this study is similar to the BDNF staining pattern observed in the testicular cells of other species using different BDNF antibodies (Park et al., 2001; Müller et al., 2006). Therefore, the band at 58 kDa is apparently a non-specific band.
In our study, we used testicular tissue obtained from 6-8-month-old Hanwoo bulls. Gathering testes from various reproductive stages is vital for studying different phases of reproductive development. However, Hanwoo is a beef cattle breed mainly castrated before 8 months of age, before the onset of significant hormone secretion and sexual maturation (Ahn et al., 2023). Therefore, we could not obtain testes from other reproductive stages. Future studies should explore the differences in bovine testes across various reproductive stages.
BDNF and NTRK2 expression in testicular cells suggests that BDNF–NTRK2 signaling may be a vital factor for the functions of bovine testes. Further studies are warranted to define the function of BDNF in individual NTRK2-expressing cells in the bovine reproductive system.
For their assistance in the experiment, the authors are grateful to Muhammad Shakeel, Jun-Young Kim, Youngwook Jung, Yeonju Choi, and Geumhee Lee (Kyungpook National University, the Republic of Korea).
Conceptualization, J.C., M.Y.; methodology, J.C., M.Y.; investigation, J.C., Y.S.; data curation, J.C., M.Y.; writing - original draft preparation, J.C.; writing - review and editing, M.Y., H.J.; supervision, M.Y.; project administration, M.Y., H.J.; funding acquisition, M.Y.
This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (RS-2024-00400429).
Not applicable.
Not applicable.
Not applicable.
Not applicable.
No potential conflict of interest relevant to this article was reported.
Journal of Animal Reproduction and Biotechnology 2024; 39(3): 194-200
Published online September 30, 2024 https://doi.org/10.12750/JARB.39.3.194
Copyright © The Korean Society of Animal Reproduction and Biotechnology.
Jaewoo Choi1 , Heejun Jung1 , Yubin Song1 and Minjung Yoon1,2,3,*
1Department of Animal Science and Biotechnology, Kyungpook National University, Sangju 37224, Korea
2Department of Horse, Companion and Wild Animal Science, Kyungpook National University, Sangju 37224, Korea
3Research Institute for Innovative Animal Science, Kyungpook National University, Sangju 37224, Korea
Correspondence to:Minjung Yoon
E-mail: mjyoonemail@gmail.com
This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background: Brain-derived neurotrophic factor (BDNF) and its receptor, neurotrophic tyrosine receptor kinase-2 (NTRK2), are well known for their roles in the central nervous and animal reproductive systems. Several studies have observed the extensive expression of BDNF and NTRK2 in non-neuronal tissues, especially reproductive organs. However, most of these studies focused on ovarian development and regulation; thus, scientific research on BDNF and NTRK2 in males is required to determine their roles in the male reproductive system. Therefore, this study aimed to investigate BDNF and NTRK2 expression in bovine testes.
Methods: Testes were collected from six Hanwoo bulls (6-8 months old). Reverse transcription-polymerase chain reaction (RT-PCR) analysis was performed to investigate the mRNA expression of BDNF and NTRK2 in the testes. Western blot analysis was performed to verify the cross-reactivity of BDNF and NTRK2 antibodies with bovine testicular tissues. Immunohistochemistry was conducted to determine BDNF and NTRK2 protein expression in the testes.
Results: RT-PCR analysis revealed BDNF and NTRK2 mRNA expression in bovine testes. In Western blotting, BDNF and NTRK2 protein bands were observed at 32 and 45 kDa, respectively. Immunofluorescence demonstrated BDNF expression in the nuclei of spermatogonia and Sertoli cells as well as in the cytoplasm of Leydig cells. NTRK2 was exclusively expressed in Sertoli cells. These results suggest that BDNF plays a potential role in spermatogenesis via BDNF and NTRK2 signaling in bovine testes, a finding supported by previous results in different animal species.
Conclusions: The expression patterns of BDNF and NTRK2 indicate their functional importance in the bovine reproductive system.
Keywords: BDNF, bull, NTRK2, reproduction, testis
Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family. It is the most extensively distributed neurotrophin member in the brain (Rios, 2013), regulating neuronal proliferation, differentiation, and maturation by binding to its high-affinity receptor neurotrophic tyrosine receptor kinase-2 (NTRK2) (Wang et al., 1995; Simpson et al., 2002). Although the role of BDNF in the nervous system is well established, its importance in non-neuronal tissues is an emerging research hotspot. For example, in adult mouse tissues, the administration of BDNF to non-ischemic ears and ischemic limbs reportedly induced neo-angiogenesis (Kermani et al., 2005). Treatment with a soluble NTRK2 ectodomain or the Trk receptor inhibitor K252a suppressed cell proliferation in cultured human choriocarcinoma cells (Kawamura et al., 2010). BDNF–NTRK2 signaling has also been shown to induce cellular adhesion and resistance to apoptosis (Geiger and Peeper, 2007). These findings suggest that BDNF–NTRK2 signaling potentially serves an important role in non-neuronal tissues.
Several studies have observed BDNF and NTRK2 expression in the testes of various mammalian species, such as humans, mice, and rats (Mutter et al., 1999; Levine et al., 2000; Robinson et al., 2003; Müller et al., 2006; Gao et al., 2019). BDNF and NTRK2 proteins were predominantly expressed in the Leydig cells of human testes. In fetal testes, significant changes in immunostaining were observed with age, suggesting that BDNF exerts morphogenetic effects during testicular organogenesis (Müller et al., 2006). BDNF and NTRK2 mRNA expression was detected in human fetal testis at 14-19 weeks of gestation (Robinson et al., 2003). They demonstrated their possible roles in regulating the proliferation and survival of germ and peritubular cells. In rat testis cell culture, BDNF gene expression was also detected in embryonic rat testes (Levine et al., 2000). Another study treated mouse TM3 Leydig cells with exogenous BDNF, demonstrating that BDNF induces the proliferation and testosterone biosynthesis of Leydig cells (Gao et al., 2019). These studies, which localized BDNF and NTRK2 mRNA expression in the fetal testes of numerous mammalian species, suggest their participation in testicular development (Chow et al., 2020) and show that BDNF may have an apparent function in testicular cells.
Therefore, this study primarily aimed to elucidate BDNF and NTRK2 expression in bovine testes. Its results may provide cues to the roles of BDNF and NTRK2 in testicular function.
Testicular tissues were collected from six Hanwoo bulls via routine castration performed under field conditions by a local veterinary surgeon in the Republic of Korea. The age of the bulls was 7 ± 1 months. The testicular samples were carefully assessed during testicular dissection and histological observation. No animal ethics or welfare statement approval was required for this study, as the castration was conducted during a routine field visit by the local veterinary surgeon.
Testicular tissue samples were fixed and preserved as previously described, with minor modifications (Rawlings et al., 2008). Briefly, immediately succeeding the routine castration, the testicular tissues were placed in an ice box to preserve them at 4℃ and kept in a refrigerator until use. They were subsequently sliced into 0.5- and 1.0-cm3 pieces. For reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analyses, the tissue (0.5 cm3) was snap-frozen and maintained at -80℃. For immunohistochemistry, the tissue (1.0 cm3) was submerged in 4% paraformaldehyde for ≥ 24 h at room temperature. Substantial washing was conducted with phosphate-buffered saline (PBS) for 24 h, and the tissue was subsequently dehydrated via successive dilution with ethanol and fixed in paraffin blocks.
Three RNA samples isolated from bovine testes (7 ± 1 months old) were used to determine BDNF and NTRK2 mRNA expression in bovine testes. RT-PCR was conducted as previously described (Lee et al., 2016), with slight modifications. Frozen testicular tissue samples were thawed at room temperature and homogenized for total RNA extraction for 5 min using a homogenizer (HG-15A; DAIHAN®, Republic of Korea). The extracted RNA was quantified using a BioTek NanoDrop spectrophotometer (BioTek Instruments, USA). The BDNF primers FW. 5’-TCCGCCATGCAATTTCCACT-3’ and Rev. 5’-AAAGTGTCAGCCAACGACGA-3’ (Macrogen, Republic of Korea) and NTRK2 primers FW. 5’- GGGACACCAATGCAGAGACA-3’ and Rev. 5’- ACAGCCCCCTACCAATTAGC-3’ (Macrogen) were used. RT-PCR was performed using a SimpliAmpTM Thermal Cycler (Life Technologies, USA), and the following cycling conditions were applied: cDNA synthesis at 50℃ for 30 min, activation at 95℃ for 10 min, denaturation for 40 cycles at 95℃ for 20 s, annealing at 60℃ for 20 s, extension at 72℃ for 40 s, and a final extension at 72℃ for 5 min. Thereafter, the samples were loaded onto 1.5% agarose gel, and electrophoresis was performed for 30 min. The BioDoc-ItTM Imaging System (UVP, USA) was used to capture the RT-PCR results.
Western blot analysis was conducted using a previously reported protocol, with minor modifications (Rawlings et al., 2008). Protein was extracted from each testicular tissue sample and diluted with radioimmunoprecipitation assay buffer (WE324435; Thermo Fisher Scientific, Massachusetts, USA). Samples were loaded onto 10% sodium dodecyl sulfate–polyacrylamide gel and separated using the Mini-PROTEAN electrophoresis system (Bio-Rad, Hercules, CA, USA) for 1 h. The membrane was blocked with 5% blotto (2.5 g nonfat milk, 50 mL PBS, and 0.1% Tween 20) and subsequently incubated with primary antibodies: BDNF (1:200 dilution, rabbit anti-horse, LS-C293050; LSBio, USA) and NTRK2 (1:200 dilution, rabbit anti-human, LS-C135253; LSBio, USA), which were diluted with BLOTTO milk for overnight incubation at 4℃. For the negative control, normal rabbit immunoglobulin G (IgG) (2729S; Cell Signaling Technology, USA) was administered to the membrane at the same concentrations as those of the primary antibodies. Secondary antibody incubation was performed for 1 h using anti-rabbit IgG (7074, 1:10,000; Cell Signaling Technology). The iBright CL1000 imaging system (Thermo Fisher Scientific) was used to develop a film.
Immunofluorescence imaging was performed using a previously reported protocol, with slight modifications (Kim et al., 2015). Testicular tissue slides were treated with xylene (Duksan Pure Chemicals Co. Ltd., Asan, Korea) to remove paraffin, rehydrated using a graded series of ethanol washes (100, 95, 80, 70, 50, and 25%), and subsequently treated with antigen retrieval buffer (100× citrate buffer, pH 6.0; Abcam, UK) at 97.5℃ for 30 min. After antigen retrieval, the slides were blocked with 5% donkey buffer (Sigma, St. Louis, MO, USA) for 30 min. Rabbit anti-BDNF (LSBio) and anti-NTRK2 rabbit (LSBio) antibodies were separately diluted at 1:200 in a blocking buffer and incubated in a humid chamber for 1.5 h. For the negative control, normal rabbit IgG (2729S, Cell Signaling) was used at the same concentration rate as that of the primary antibodies. Thereafter, tissue sections were incubated in donkey anti-rabbit IgG (1:1,000 dilution; Alexa FluorTM 488, Thermo Fisher Scientific, USA) for 45 min. BDNF and NTRK2 expression in testicular tissues was examined using a Leica DM2500 fluorescence microscope (Leica, Germany). Testicular tissue images were captured using a Leica DFC 450 C camera. Cells with BDNF and NTRK2 emitting green fluorescence were considered positive, whereas those that did not stain green were considered negative.
RT-PCR was performed to detect the expression of BDNF and NTRK2 genes in bovine testes. The mRNA bands of BDNF and NTRK2 were detected at expected sites, approximately 430 and 546 bp, respectively (Fig. 1A and 1B). The bands were not observed in the negative control treated without template (Fig. 1A and 1B).
Western blotting was conducted to examine the cross-reactivity of the bovine testicular tissues (n = 3) with BDNF and NTRK2 antibodies. The BDNF protein band yielded an approximate weight of 32-58 kDa (Fig. 2). Thereafter, the NTRK2 protein band was observed at an approximate weight of 45 kDa. The β-actin protein band was observed at 46 kDa. The negative controls, treated with the same concentration of rabbit IgG as a primary antibody, did not exhibit any protein band (Fig. 2). These results suggest that the antibodies cross-reacted with the proteins in bovine testes.
Immunofluorescence imaging was performed to observe the localization of BDNF and NTRK2 in bovine testicular tissues. BDNF immunostaining (green fluorescence) was observed in Leydig cells, Sertoli cells, and spermatogonia (Fig. 3A-D). In Leydig cells, BDNF expression was primarily observed in the cytoplasm, while in Sertoli cells, it was localized in the nuclei (Fig. 3A-D). BDNF expression was also localized in the round-shaped nuclei of the spermatogonia (Fig. 3A-D), albeit at lower expression levels than those in Leydig and Sertoli cells. NTRK2 immunostaining (green fluorescence) was exclusively observed in Sertoli cells (Fig. 3F-I). Specifically, it was visualized as a dot in the nucleus of each Sertoli cell, and most Sertoli cells were immunolabeled with NTRK2 (Fig. 3F-I). No NTRK2 immunolabeling was observed in Leydig and germ cells. No immunolabeling of testicular cells was observed in the negative control tissue (Fig. 3E and 3J).
BDNF and NTRK2 are well recognized for their significant roles in the nervous system. Furthermore, several studies have discovered their involvement in female reproductive physiology, including follicle recruitment, oogenesis (Harel et al., 2006), germ cell survival (Paredes et al., 2004), and oocyte maturation (Lee et al., 2007). Therefore, studying the localization of BDNF and NTRK2 is requisite to improving our understanding of male reproductive physiology, especially spermatogenesis. Our present study investigated BDNF and NTRK2 expression in the Leydig cells, Sertoli cells, and spermatogonia of bovine testes.
Recent studies suggest that BDNF and NTRK2 are critical regulatory factors for ovarian development and function. In mice, NTRK2 deficiency blocked oocyte growth and granulosa cell proliferation, leading to arrested follicle development at the primary stage (Paredes et al., 2004). BDNF–NTRK2 signaling also plays a key role in oocyte survival, facilitated by the PI3K/AKT–mediated survival pathway (Dorfman et al., 2014). Several studies have detected BDNF and NTRK2 expression in the testes, suggesting a potential role for BDNF–NTRK2 signaling in testicular function. However, the mechanism by which this signaling pathway regulates testicular functions remains unclear. Therefore, further research is needed to clarify the precise role and mechanism of BDNF in the testes.
Leydig cells play an essential role in producing testosterone, a crucial hormone for normal reproductive function in the male reproductive tract (Ge et al., 2021). According to immunohistochemistry, BDNF immunolabeling was observed in the cytoplasm of Leydig cells. BDNF expression has also been described in human (Müller et al., 2006) and mouse (Gao et al., 2019) Leydig cells. Moreover, it has been detected in the Leydig cells of both fetal and adult human testes. In adult males, BDNF is predominantly localized in the Leydig cells of human testes. In a mouse Leydig cell culture study (Gao et al., 2019), treatment with exogenous BDNF increased testosterone levels in primary Leydig and TM3 Leydig cells. The researchers also detected an elevated level of enzymes, which are involved in generating testosterone. Conversely, testosterone and enzyme levels were suppressed in BDNF-knockdown TM3 Leydig cells. This study provides evidence that BDNF affects testosterone biosynthesis in Leydig cells. Normally, in bovine testes, the number of mature Leydig cells begins to rapidly increase through mitosis from 4 weeks of age, and by week 30, the rate of increase decelerates as mitosis becomes limited (Wrobel, 1990). As mature Leydig cell production nears completion, serum testosterone levels subsequently increase rapidly at approximately 20-35 weeks of age (Rawlings et al., 2008). The bulls used in our study were 6-8 months of age, displaying the active differentiation of Leydig cells and an increase in testosterone levels. Therefore, our study, which observed BDNF immunolabeling in Leydig cells, suggests that BDNF may be involved in the functioning of Leydig cells during steroidogenesis and differentiation.
Sertoli cells are essential for proper testicular function, particularly in spermatogenesis (Griswold, 1998). They produce diverse factors that play a vital role in maintaining the balance among maturation, differentiation, and germ cell self-renewal, suggesting that germ and Sertoli cells are correlated in their physiological functions and mutual control (Tripathi et al., 2014). Immature Sertoli cells in bovine testes proliferate from 4 to 20 weeks of age and begin to differentiate into mature Sertoli cells (Sinowatz and Amselgruber, 1986). The differentiation commences at approximately 20 weeks of age and generally reaches completion between 30 and 40 weeks of age (Sinowatz and Amselgruber, 1986). Therefore, in our study, testicular tissues collected from 6-8-month-old bovine testes indicated that they were likely in the middle of Sertoli cell differentiation. Immunohistochemistry revealed BDNF immunolabeling in the nuclei of Sertoli cells. BDNF protein expression in Sertoli cells has also been reported in several studies (Park et al., 2001; Müller et al., 2006; Cacialli et al., 2018). In zebrafish testes, BDNF expression was observed in the Sertoli cells, suggesting that BDNF is involved in the differentiation and proliferation of germ cells (Cacialli et al., 2018). They further proposed that Sertoli cells are possibly a key target for BDNF. However, owing to the absence of NTRK2 expression in zebrafish testes, they could only speculate on the specific roles of BDNF in these cells. Our present results, wherein NTRK2 expression was detected in the Sertoli cells of bovine testes, corroborate their hypothesis. Consequently, our findings suggest that Sertoli cells constitute a key target for BDNF, which may serve a critical role in the differentiation and proliferation of germ cells during spermatogenesis and may also be involved in the differentiation of Sertoli cells in bovine testes.
In our study, immunohistochemistry revealed BDNF immunolabeling in the nuclei of spermatogonia from the testes of 6-8-month-old bulls. Along with rapid testicular growth, spermatogenic development occurs after 20-25 weeks of age. Primary spermatocytes appear at approximately 20 weeks, secondary spermatocytes at 20-30 weeks, spermatids at 25-35 weeks, and mature spermatozoa at 32-40 weeks of age (Rawlings et al., 2008). These findings suggest that spermatogonia and primary spermatocytes are predominantly present in 6-8-month-old bovine testes. Therefore, the presence of BDNF in spermatogonia suggests that BDNF may play a role in spermatogenesis.
In this study, we observed a dot-like immunostaining pattern of NTRK2 in the nuclei of Sertoli cells. This localization of NTRK2 differs significantly from that found in other species. For instance, in humans, while less intense immunoreactivity was observed in Sertoli cells, most NTRK2 immunoreactivity was concentrated within the cytoplasm of Leydig cells (Mutter et al., 1999; Müller et al., 2006). Similarly, in mice, NTRK2 immunostaining was localized in Sertoli, peritubular myoid, and Leydig cells, but predominantly in spermatogonia A and B (Park et al., 2001). Furthermore, in rat testes, no NTRK2 immunostaining was noted in Sertoli and spermatogenic cells; it was mainly localized in interstitial cells (Moon et al., 2005). These findings indicate the variation in NTRK2 expression across species, suggesting species-specific roles and mechanisms in testicular function.
In this study, BDNF protein bands were observed at 32 and 58 kDa. A band was observed at the adequate molecular size (32 kDa); nevertheless, the band also appeared at an unexpected molecular size (58 kDa). Even though we detected an unexpected band at 58 kDa, the LS-C293050 antibody-based BDNF staining pattern in the testicular cells in this study is similar to the BDNF staining pattern observed in the testicular cells of other species using different BDNF antibodies (Park et al., 2001; Müller et al., 2006). Therefore, the band at 58 kDa is apparently a non-specific band.
In our study, we used testicular tissue obtained from 6-8-month-old Hanwoo bulls. Gathering testes from various reproductive stages is vital for studying different phases of reproductive development. However, Hanwoo is a beef cattle breed mainly castrated before 8 months of age, before the onset of significant hormone secretion and sexual maturation (Ahn et al., 2023). Therefore, we could not obtain testes from other reproductive stages. Future studies should explore the differences in bovine testes across various reproductive stages.
BDNF and NTRK2 expression in testicular cells suggests that BDNF–NTRK2 signaling may be a vital factor for the functions of bovine testes. Further studies are warranted to define the function of BDNF in individual NTRK2-expressing cells in the bovine reproductive system.
For their assistance in the experiment, the authors are grateful to Muhammad Shakeel, Jun-Young Kim, Youngwook Jung, Yeonju Choi, and Geumhee Lee (Kyungpook National University, the Republic of Korea).
Conceptualization, J.C., M.Y.; methodology, J.C., M.Y.; investigation, J.C., Y.S.; data curation, J.C., M.Y.; writing - original draft preparation, J.C.; writing - review and editing, M.Y., H.J.; supervision, M.Y.; project administration, M.Y., H.J.; funding acquisition, M.Y.
This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (RS-2024-00400429).
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No potential conflict of interest relevant to this article was reported.
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