Theriogenology 126 (2019) 206e213 Contents lists available at ScienceDirect Theriogenology journal homepage: www.theriojournal.com Characterization of b-Nerve Growth Factor-TrkA system in male reproductive tract of rabbit and the relationship between b-NGF and testosterone levels with seminal quality during sexual maturation Ana Sanchez-Rodriguez a, Maria Arias-Alvarez b, Patricia Timon a, Jose M. Bautista c, * Pilar G. Rebollar d, Pedro L. Lorenzo a, Rosa M. Garcia-Garcia a, a Department of Physiology, Faculty of Veterinary Sciences, Complutense University of Madrid, Madrid, Spain b Department of Animal Production, Faculty of Veterinary Sciences, Complutense University of Madrid, Madrid, Spain c Department of Biochemistry and Molecular Biology, Faculty of Veterinary Sciences, Complutense University of Madrid, Madrid, Spain d Department of Agrarian Production, ETSIAAB, Polytechnic University of Madrid, Madrid, Spain article info abstract Article history: b-Nerve Growth Factor (b-NGF) is a neurotrophin which acts through its receptors TrkA and p75, per- Received 6 July 2018 forming important actions in male reproductive physiology and its presence in seminal plasma (SP) has Received in revised form been related to male fertility. The aim of the present study was to evaluate the gene expression profile 3 December 2018 and the immunolocalization of b-NGF and its high-affinity receptor TrkA in sex organs in rabbits during Accepted 4 December 2018 sexual maturation period. b-NGF concentration for both SP and blood plasma (BP) and BP testosterone Available online 10 December 2018 levels were determined as well as the seminal parameters during such period. Ten New Zealand White x California young rabbits were trained to semen collection since 20 weeks of age and routinely done once Keywords: b-NGF a week with two ejaculations per session. At 22 and 37 weeks of age, semen collection was carried out TrkA receptor three times a week and seminal parameters were evaluated. Four males were randomly assigned and Seminal plasma slaughtered in each age (n ¼ 8); sex organs (prostate, bulbourethral glands and epididymis) were Sex organs dissected and collected to determine b-NGF and TrkA gene expression and immunolocalization. SP and Testosterone levels BP were also taken at each semen collection session to evaluate b-NGF concentration, and testosterone Male rabbits levels were also assessed in BP. The highest b-NGF mRNA expression was observed in prostate compared to bulbourethral glands and epididymis. These two last tissues showed residual b-NGF mRNA expression and limited localization of the neurotrophin. The prostate epithelial cells and lumen were strongly stained with regard to the other sex organs indicating that immunolocalization of b-NGF rely mainly in the prostate. TrkA gene expression was lower but constant and differentially immunolocalized in the sex organ tissues. Finally, b-NGF concentration in SP and BP remained unchanged in accordance to age, while some seminal characteristics such as sperm concentration, percentage of live sperm and mass and progressive motility were enhanced as endowed by BP testosterone variation. b-NGF and its cognate TrkA receptor are expressed and immunolocalized in the male reproductive tract in the two ages studied, independently of the circulating levels of testosterone and b-NGF. © 2018 Elsevier Inc. All rights reserved. 1. Introduction function is mediated by two receptors: a specific high-affinity transmembrane receptor, named TrkA, and a low-affinity and b-Nerve Growth Factor (b-NGF) is a neurotrophic factor essential non-selective receptor member of the death-promoting Tumor for survival, development and maintenance of sympathetic and Necrosis Factor family, called p75. b-NGF/TrkA/p75 system is pre- sensory neurons of the peripheral nervous system [1]. Its biological sent in numerous organs apart from the nervous system, such as the reproductive tract both in males [2e10] and females [11e14], where has been demonstrated to have relevant physiological functions. Thus, b-NGF and its receptors have been associated with * Corresponding author. Dept. Physiology, Faculty of Veterinary Science, Com- plutense University of Madrid, Avda. Puerta de Hierro s/n, 28042, Madrid, Spain. the morphological sex determination, testis morphogenesis and E-mail address: [email protected] (R.M. Garcia-Garcia). https://doi.org/10.1016/j.theriogenology.2018.12.013 0093-691X/© 2018 Elsevier Inc. All rights reserved. A. Sanchez-Rodriguez et al. / Theriogenology 126 (2019) 206e213 207 development, and sperm maturation [15e19], suggesting the the experiment (37 weeks old), where four were sacrificied for involvement of b-NGF system in the reproductive tract maturation. tissue recovery. The two remaining rabbits were kept in the farm as In addition, it is hypothesized that b-NGF may intervene in sper- breeding males. b-NGF in SP and BP and testosterone BP levels were matogenesis process by mediating the effects of testosterone [19]. determined in these rabbits at 22 and 37 weeks old. For sperm This fact has been studied in some species but the regulation of b- analyses, semen collection was performed three times a week with NGF/TrkA system by androgens [20] or vice versa [21] is unclear. In two ejaculations per session at the same ages. For each male in one this respect, the characterization of this system in the reproductive session, first and second ejaculate were mixed to determine semen tract during sexual maturity and the relationship with sexual hor- quality and to separate SP. mones could be interesting to determine its function in this All the procedures were performed in accordance with the process. policies of the University Scientific Ethics Spanish Policy for Animal b-NGF has been found in the seminal plasma (SP) [9,10,22e26], Protection RD53/2013 [34], which meets the European Union which is a mixture of secretions from testes, epididymis and male Directive about the protection of animals used in experimentation. accessory glands [27]. Proteins present in SP have been demon- strated to perform important functions in protection, sperm 2.2. Housing and feeding transport, capacitation and motility of sperm within the female reproductive tract [28,29]. In this way, b-NGF expression levels in Male rabbits (Oryctolagus cuniculus) were held on the experi- bovine sperm had positive influence on some sperm velocity pa- mental farm at the Agrarian Production Department, Polytechnic rameters, which suggest an important role of b-NGF on fertility University of Madrid (UPM, Spain). They were housed in individual [30]. In addition, this neurotrophin in SP has been revealed as an cages (700 mm  500 mm  320 mm) under controlled conditions ovulation trigger factor in induced ovulating species, such as of constant photoperiod (16L:8D), 20 to 25 C of temperature and a camelids [22e25]. In rabbits, despite being reflex ovulators, neither relative humidity of 60e75% maintained by a forced ventilation rabbit SP [24,31] nor murine b-NGF [10] have been able to provoke system. Each animal had free access to food and water. the ovulation, thus the role of b-NGF as an ovulation-inducing factor is still inconclusive. Nonetheless, a recent study in this spe- 2.3. Collection and evaluation of seminal samples cies proposes that this neurotrophin participates in ovulation in- duction by both endocrine and nervous pathways [32] with some The semen samples recovered at 22 and 37 weeks of age were unrevealed function/s in the female reproduction. analyzed as follow. After semen collection, the gel fraction was In male rabbits, b-NGF were detected in testes, prostate and removed and the ejaculates were kept warmed in an incubator at seminal vesicles [2,9,10]. Although we have recently described the 37 C after being macroscopically analyzed, taking into account gene expression and distribution of low-affinity receptor p75 in their color, their volume (assessed by a graduated plastic pipette) epididymis and bulbourethral glands in rabbits [33], the charac- and the presence of sedimentation (presence of desquamation terization of b-NGF and its specific receptor TrkA in some male cells, droplets and vesicles in the ejaculate). Only those ejaculates reproductive organs remains unknown. To the best of our knowl- with white color, with a volume greater than 0.1 ml and without edge, there are no evidences of their presence in epididymis in sedimentation were selected. Afterwards, each single ejaculate rabbits, whereas it is described in other mammals [5,15], and there from the same male (two ejaculates per male at the same day) was is only one work which studies b-NGF by radioimmunoassay in pooled together. Then, we first estimated the mass motility of raw bulbourethral glands [2]. Nothing is known about their expression semen rating from 1 to 4 in an optical microscope (x 20 magnifi- and distribution during sexual maturity in males or their correla- cation), considering 1: no sperm cells with motion, 2: sperm cells tion to seminal characteristics and testosterone levels. moving slowly, 3: nearly all sperm cells moving with high and Therefore, in the present study, to further understanding of the homogeneous motility, 4: strong waves and whirlwind appearance role of b-NGF/TrkA system in male rabbit reproduction, we exam- (based on Brun et al. [35]). Then, aliquots of semen were fixed in ined the distribution and gene expression of b-NGF and its high- 2.5% glutaraldehyde to determine the semen concentration (mil- affinity receptor (TrkA) in prostate, bulbourethral glands and lions of sperm cells/ml) with a Neubauer chamber and to evaluate epididymis during the sexual maturation period. In addition, we the integrity of the acrosome in a phase-contrast microscope. To investigated the variation of b-NGF expression in SP in relation to b- measure the percentage of live sperm and the percentage of ab- NGF and testosterone levels in blood plasma (BP) and SP, as well as normalities, the nigrosin staining technique was made for each their relationship with seminal parameters to determine whether sample.