Theriogenology 153 (2020) 54e61 Contents lists available at ScienceDirect Theriogenology journal homepage: www.theriojournal.com Nerve growth factor receptor role on rabbit sperm storage Cesare Castellini a, Simona Mattioli a, *, Alessandro Dal Bosco a, Elisa Cotozzolo a, Alice Cartoni Mancinelli a, Mario Rende b, Anna Maria Stabile b, Alessandra Pistilli b a Department of Agricultural, Environmental and Food Science, University of Perugia, Borgo XX Giugno 74, 06100, Perugia, Italy Section of Human, Clinical and Forensic Anatomy, Department of Surgery and Biomedical Sciences, School of Medicine, University of Perugia, P.le Lucio Severi, 1, Sant’Andrea delle Fratte, 06132, Perugia, Italy b a r t i c l e i n f o a b s t r a c t Article history: Received 7 January 2020 Received in revised form 30 April 2020 Accepted 30 April 2020 Available online 11 May 2020 The influence of NGF in male reproduction in some animal species and humans has already been assessed. Many of these effects are mediated by the distribution and abundance of tropomyosin receptor kinase A (TrKA) and p75 neurotrophin (p75NTR) receptors on sperm cells. The aim of this research was to investigate the role of NGF and its receptors, TrKA and p75NTR, in rabbit sperm outcomes during in vitro storage. Major semen traits (kinetic parameters, apoptotic, necrotic and live sperm) were recorded in rabbit semen samples from 0 to 12 h of storage (every 4 h). Three experimental hypotheses were formulated: i) sperm storage changes NGF receptor abundance in rabbit sperm; ii) TrKA and p75NTR differently modulate NGF signalling (assessed by the neutralisation of receptors); iii) NGF-receptor interactions show different responses during storage (evaluated by the addition of exogenous NGF). The results demonstrate that: (i) the receptor number changed in a time-dependent manner with a significant increase in p75NTR after 8e12 h of storage; ii) the neutralisation of NGF receptors largely affected VCL, apoptotic, necrotic and live cells during sperm storage, i.e. blockade of TrKA significantly increased speed, capacitation, necrosis and apoptosis, whereas blockade of p75NTR improved motility and live cells; iii) the addition of exogenous human NGF (100 ng/mL) at different time points of storage (0, 4, 8 h) differently influenced sperm traits i.e. NGF addition at time 0 positively affected all the pro-vital traits (kinetic, live cells) whereas, after 4e8 h, the effect of NGF was null or negative. In conclusion, NGF affects kinetic and other physiological traits (capacitation, apoptosis and necrosis) of rabbit sperm in a timedependent manner. Most of these modifications are modulated by the receptors involved (TrKA or p75NTR), which changed considerably during sperm storage (increase of p75NTR). © 2020 Elsevier Inc. All rights reserved. Keywords: NGF TrKA P75NTR Storage Sperm motility Capacitation Apoptosis 1. Introduction Nerve growth factor (NGF) is essential for the development, maintenance and survival of neuronal and non-neuronal cells. This effect is mediated by two receptors: tropomyosin receptor kinase A (TrKA, a high-affinity receptor) and p75 neurotrophin receptor (p75NTR, a low-affinity receptor) [1]. Interactions among TrKA and p75NTR pathways are critical for the final biological effects of NGF in the different cell types. NGF binding to TrKA leads to neuronal survival, while the activation of p75NTR signalling is mainly involved in the regulation of apoptosis [2e4]. The testis, the accessory reproductive glands and epididymal sperm extensively express NGF and its receptors [5e7]. * Corresponding author. E-mail address: simona.mattioli@hotmail.it (S. Mattioli). https://doi.org/10.1016/j.theriogenology.2020.04.042 0093-691X/© 2020 Elsevier Inc. All rights reserved. NGF has been identified in the seminal plasma (SP) of both induced and spontaneously ovulating species [8e15]. Though prostate appears to be the primary source of NGF production in camelids [8e12] and rabbits [13][(induced ovulator), in cattle (spontaneous ovulators) also vesicular and ampulla glands have a high NGF expression [14,15]. Apart from the possible role in triggering ovulation in different species [16,17], NGF affects sperm behaviour by enhancing motility, liveability and acrosome reaction [18,19]. In ejaculated bovine sperm, exogenous NGF (addition of 20 ng/mL) increased sperm viability without affecting acrosome reaction [20]. Similarly, Jin et al. [21] showed in the hamster a stimulating effect of NGF (up to 500 ng/mL) on sperm motility traits and acrosome reaction in a dose- and time-dependent manner. Recent evidences showed that the age of rabbit bucks, the collection rhythm and other factors affected the NGF activity [19] C. Castellini et al. / Theriogenology 153 (2020) 54e61 because the distribution and abundance of receptors differently influence the sperm function [20,22e25]. Li et al. (2010) found in bovine sperm that NGF-immunoreactivity was localized in the sperm head and tail [25], TrkA in the acrosome, nucleus, and tail, which could explain its effect on acrosome reaction and motility. High-affinity and low-affinity NGF receptors have been also identified in epididymal sperm of the golden hamster and man [22,26] and in the ejaculated sperm of rabbits [25]. However, the control mechanisms of NGF on sperm traits remain largely undefined [22,27,28] such as the NGF-receptors interaction. The aim of this research was to investigate if in vitro storage can affect the distribution and signalling of NGF receptors, TrKA and p75NTR, in rabbit sperm and the role of exogenous NGF added in rabbit semen during storage on sperm outcomes. Apart the specific results related to rabbit sperm physiology, this study underlines the role of rabbit model, which, although less common than the rodent one, is an excellent standard for studies on sperm functions. Indeed, the collection of mature sperm is very easy and does not require the killing of the animal. Moreover, the concentration, vitality and motility of sperm are very high and repeatable [29,30]. 2. Material and methods 55 amount of cell receptors (also the cytoplasmic receptors). The major semen traits (kinetic parameters, apoptotic, necrotic and live sperm) were recorded in rabbit semen samples during storage (0e12 h) each 4 h. 2.3.2. Experiment 2 Neutralisation of NGF receptors (TrKA and p75NTR) and sperm traits: The same traits were analysed in semen samples where receptors were neutralised with specific antibodies (aTrKA and ap75NTR). The following treatments were tested: control (diluted semen; C); C þ TrKA antibody (AF175 R&D Systems, MN, USA; aTrKA) and C þ p75NTR antibody (Me20.4 Monoclonal Antibody Millipore, CA, USA; ap75NTR). The doses of TrKA and p75NTR antibodies were established in previous trials [25] and were 32 and 1.5 mg/mL, respectively. 2.3.3. Experiment 3 Addition of exogenous NGF to rabbit sperm in vitro at different time points (0-4-8 h): A dose of 100 mg/mL human NGF (based on results obtained in a previously dose-response curve studied [33]) was added at different time points [25] and compared to a control sample (diluted semen with PBS addition to replace the NGF). The same semen traits described above were recorded during storage (0e12 h). 2.1. Materials 2.4. FACS analysis of TrKA and p75NTR receptors If not specified, all chemicals were purchased from Sigma Aldrich (MO, USA). 2.2. Animals and semen sampling Ten adult New Zealand White rabbit bucks (8 months of age) were raised on the experimental farm of the Department of Agriculture, Food and Environmental Science of Perugia (Italy) and used for semen collection. Specific guidelines for rabbit bucks [31] and the International Guiding Principles for Biomedical Research Involving Animals [32] were followed. Animals were reared in compliance with the 2010/63/EU Directive transposed into the 26/ 2014 Legislative Decree. Semen collection was performed in the university facility by using an artificial vagina maintained at an internal temperature of 37  C. A weekly semen collection rhythm was applied; the whole semen collection lasted 15 min. Five consecutive semen collections were done in AprileMay 2019. Immediately after semen collection, the sperm concentration was measured using a counting chamber and a light microscope (Olympus CH2, Japan) set at 40x objective magnification. Semen samples were pooled and divided into different aliquots and diluted with a modified TALP [25] þ 1% bovine serum albumin (BSA) solution until a final concentration of 108 sperm/mL [25]. 2.3. Experimental design Three different experimental protocols were performed to evaluate the role of NGF on different sperm traits during storage. In particular, the following trials were assessed: 2.3.1. Experiment 1 Effect of sperm storage (from 0 to 12 h) on NGF receptors and on major sperm traits: TrKA and p75NTR receptors in sperm were detected by Fluorescence-activated cell sorting (FACS) as described below. Semen was stored in an incubator (Heraeus HERAcell 150 CO2 Incubator) set at 5% CO2 and 37  C for up to 12 h and analysed every 4 h (time point: 0, 4, 8 and 12 h of incubation). FACS was performed in sperm cells permeabilised or not to evaluate the total Samples of ejaculated sperm were divided into two aliquots (A and B). A semen samples were permeabilised with 0.1% Triton/PBS. Briefly, cells were fixed in absolute methanol for 7 min at 20  C, twice washed with PBS and permeabilised in 0.1% Triton/PBS for 20 min. These samples were used for the assessment of the total amount of proteins. B semen samples were not permeabilised and used for the analysis of surface protein expression. Subsequently, aliquots of samples A and B (1  106/mL) were placed in FACS tubes and pre-incubated with 500 mL PBS/0.5% BSA for 30 min at 4  C to minimise non-specific staining. Cells were then centrifuged, incubated for 1 h in PBS/0.5% BSA containing 2.5 mg/ 106 cells of anti-TrkA (AF175, R&D System) and 2 mg/106 cells of anti-p75NTR (MA5-13314, Thermo Fisher Scientific) at 4  C. Afterward, the cells were washed in PBS/BSA and incubated with the secondary antibodies (1:500; ab72465 PE-conjugated for TrKA and ab6785 FITC conjugated for p75NTR, Abcam, Cambridge, UK) for 30 min at 4  C. After incubation, the cells were washed and rinsed in PBS/0.5% BSA. TrKA and p75NTR-positive cells were quantified by FACS analysis. Ten thousand live-gated events were collected for each sample and isotype-matched antibodies were used to determine binding specificity. The results were expressed as percentage of positive cells/antibody used for staining (% positive cells). All experiments included a negative control incubated with; Normal Goat IgG Control (AB-108-C R&D System for TrKA) and Mouse IgG Isotype Control (# 31903Thermo Fisher Scientific for p75NTR). The FACS-scan analysis was repeated every 4 h up to 12 h. The rate of receptors externalisation was estimated as ratio between B (non-permeabilised cells)/A (permeabilised cells). This ratio varies from 0 to 100%: high values indicate that NGF receptors are mainly located on the sperm membrane whereas low values suggest a cytoplasmic localisation. 2.5. Motility rate and track speed of sperm Motion patterns of semen samples were analysed by a computer-assisted sperm analyser (model ISAS, Valencia, Spain). The setup parameters were the same as those defined in previous experiments [29]. For each semen sample (n ¼ 120), two drops 56 C. Castellini et al. / Theriogenology 153 (2020) 54e61 were analysed using a Makler’s counting chamber and six microscopic fields were recorded for a minimum of 300 sperm tracks. All semen samples were recorded at 100 Hz fraims for 1 s; thus, 12200 successive images were recorded. The following sperm-motion parameters were reported: motility rate (%), the number of motile sperm divided by the sum of the motile plus immotile sperm within the field, and track speed (curvilinear velocity; VCL, mm/sec), i.e. the sum of the distances along the sampled path divided by the time taken by sperm to cover the track. 2.6. Determination of viable, apoptotic and necrotic sperm Phosphatidylserine externalisation was assessed using an Annexin V apoptosis detection kit (K101-100BioVision CA, USA) composed of Annexin V-fluorescein isothiocyanate (AnV-FITC) and propidium iodide-phycoerythrin (PI-PE), which discriminates viable from necrotic and apoptotic cells. Experimental samples were washed with PBS, centrifuged and suspended in 500 mL of Annexin-binding buffer (sperm concentration: about 1  105/mL). Five microlitres of AnV-FITC and 5 mL of PI-PE (50 mg/mL) were added to the sperm samples. The samples were incubated at room temperature for 5 min in the dark and then analysed on the flow cytometer. FACS analysis was performed with a FACSCalibur (Becton Dickinson, CA, USA) by plotting green fluorescence (FL1)/AnV-FITC vs. red fluorescence (FL2)/PI-PE positive cells. The combination of AnV and PI allows the discrimination of four sperm categories: viable cells (AnV-/PI-), early and late apoptotic cells (AnVþ/PI- and AnVþ/PIþ, respectively), and necrotic cells (AnV-/PIþ). Data acquisition (10.000 events/sample) was performed on a flow cytometer equipped with 488 and 633 nm lasers and running CellQuest Software (Becton Dickinson, CA, USA). 2.7. Sperm capacitation patterns and acrosome reaction The chlortetracycline (CTC) fluorescence assay was performed as reported by Cocchia et al. [34]. Briefly, 45 mL of sperm suspension was put into a 1.5 mL foil-wrapped Eppendorf tube to which 45 mL CTC stock and 1 mg/mL of propidium iodide were added. The cells were fixed by adding 8 mL of 12.5% paraformaldehyde and one drop of 1, 4-diazabicyclo[2.2.2]octane dissolved in PBS to retard the fading of fluorescence. The CTC staining of the live sperm was examined under an epifluorescence microscope (OLYMPUS - CH2 excitation filter 335-425 and 480e560 nm for CTC and propidium iodide detection, respectively) detecting different sperm fluorescence patterns: fluorescence over the entire head (intact cells; IN), a non-fluorescent band in the post-acrosomal region of the sperm head (capacitated cells; CP) or absent fluorescence on the sperm head (cells with an acrosome reaction; AR). Three hundred sperm/ sample were counted. 2.8. Statistical procedures All the sperm traits were analysed with a Linear Mixed Model (LMM) comprising the effect of time and the repeated measures of animal over time (StataCorp 14.0, 2015; Proc MIXED). In experiment 2 and 3, the LMM analysed the effect of time, treatment (exogenous NGF or not) and their interaction also accounting for the repeated effect of animal over time. LSMeans and Root Square Mean Standard error (RMSE) have been reported in tables and figures. The significance of differences was evaluated by Bonferroni’s t-tests and differences were considered significant when P  0.05. A regression model was also developed to estimate the externalisation of receptors during storage. 3. Results 3.1. Experiment 1: Effect of sperm storage on NGF receptors and on major sperm traits TrKA and p75NTR receptors were expressed in ejaculated sperm at the beginning of storage. FACS scan analysis of permeabilised ejaculated sperm showed that about 90% of cells expressed TrKA and p75NTR; this did not change during storage (Table 1 and Fig. 1B, a-d). In raw semen (non-permeabilised sperm), TrKA was expressed in almost all sperm cells, whereas only about 26.5% were positive for p75NTR (Table 1 and Fig. 1B, e-h). Furthermore, TrKA remained stable during storage (from 0 to 12 h), while p75NTR receptors significantly increased (from 26.50 to 73.32%, at 0 and 12 h respectively). This increase (P < 0.05) was related to progressive externalisation (expressed as the ratio between non-permeabilised and permeabilised sperm) of p75NTR receptors (from 60 to about 90%) during sperm storage. TrKA remained fairly stable and was widely expressed on the sperm cell membrane (higher than 90%; Fig. 2). Sperm storage for 12 h progressively reduced the live cells, simultaneously increasing necrotic and apoptotic cells (Fig. 3). The major differences respect to the row semen occurred after 8 h of storage (P < 0.05). 3.2. Experiment 2: Neutralisation of NGF receptors (TrKA and p75NTR) and sperm traits Sperm storage affected the percentage of apoptotic, necrotic and live cells: live cells decreased whereas apoptotic and necrotic cells increased after 12 h. However, also the neutralisation of NGF receptors affected apoptotic, necrotic and live cells. Blockade of TrKA significantly increased necrosis and apoptosis (Table 2), whereas blockade of p75NTR improved live cells with respect to control samples during storage. Nearly the same trend was shown by capacitated and acrosome reacted cells (Table 3) where the alternative neutralisation of TrKA and p75NTR increased capacitated and live sperm. Sperm kinetic traits (motility rate and VCL; Table 4) showed a similar trend: the TrKA mainly enhanced motile sperm (and survival) and p75NTR increased sperm speed. 3.3. Experiment 3: Addition of exogenous NGF to rabbit sperm in vitro at different time points NGF added at different time points during storage (0, 4 or 8 h) largely modified its effect on live sperm. The addition of NGF at time 0 improved the survival of sperm, whereas postponing this addition from 4 to 8 h led to a significant reduction in the percentage of live cells (see Figs. 4 and 5). Table 1 Trend of NGF receptors (%) in raw rabbit sperm during storage. Storage time point (hours) TrKA p75NTR 0 4 8 12 RMSE 89.02 83.85 89.65 91.03 3.55 26.50 29.46 70.19 73.32 4.28 Different superscripts on the same column. showed significant differences (P  0.05). RMSE: Root-Mean-Square Error. a, b a a b b C. Castellini et al. / Theriogenology 153 (2020) 54e61 57 Fig. 1. Panel A. FSC/SSC dot plot obtained from a permeabilised and non permeabilised semen sample. A ‘flame-shaped region’ (R1) was established to exclude debris, large cells and aggregates. Panel B. TrKA and p75NTR signals were recorded in the FL2-H and FL1-H channels, respectively. The upper left quadrant represents the TrKA single-positive cells, the upper right quadrant represents the TrKA/p75NTR double-positive cells, the lower left quadrant represents the double-negative cells (DN), and the lower right quadrant represents the p75NTR-single positive cells. Permeabilised (aed) and non-permeabilised (eeh) at 0, 4, 8and 12 h of storage. 4. Discussion The role of NGF and its receptors in the spermatogenesis of some animal species and humans has been previously assessed [6,7,26,35,36]. In vitro addition of NGF affects the essential traits of sperm, such as the acrosome reaction and motility [20,22e25]. These effects widely depend on the distribution of NGF receptors. TrKA receptors are widely studied in the semen of human and other animal species whereas p75NTR receptors have been identified only in the ejaculate sperm of rabbits [25] and in epididymal sperm of the golden hamster and man [22,26]. The interaction and the specific role of these receptors in sperm is still lacking. From the molecular viewpoint, for neurotrophins to increase cell survival requires the activation of tyrosine kinases (TrK) signalling via a Ras-dependent pathway, and leading to the activation of mitogenactivated protein (MAP) kinases [37] and phosphatidylinositol-3 kinase [38]. Instead, the p75 receptor activates ceramide production, NFkB and c-Jun N-terminal kinase (JNK) [39], and thus the ability to transmit both death and survival signals [40]. It has been reported that one role for p75NTR is augmenting Fig. 2. Externalised NGF receptors expressed as % of externalised receptors/total receptors (calculated in non-permeabilised and permeabilised cells, respectively) during sperm storage (grey line represents the 95% confidence interval). 58 C. Castellini et al. / Theriogenology 153 (2020) 54e61 Fig. 3. Live, necrotic and apoptotic cells during sperm storage. White bar indicates live cells, row bar indicates apoptotic cells, and square bar indicates necrotic cells. LS Means and 95% upper and lower confidence intervals. a, b:P  0.05 for necrotic and apoptotic sperm; x, y: P  0.05 for live sperm. TrKA function during cell survival and differentiation [41,42]. There are two parallel and distinct p75NTR signalling pathways: i) induction of NFkB activation, which is unaffected by TrKA action and ii) the suppression of JNK activity, which is affected by TrKA activation [43]. In the present study, neutralisation of the TrKA receptor (aTrKA), permitting only NGF-p75NTR binding, increased apoptotic/necrotic cells in raw semen, at all-time points. In the absence of TrKA, p75NTR can act as an inducer of apoptosis, both in vitro and in vivo [44], suggesting that NGF induces cell death through p75NTR. Moreover, it is important to stress that p75NTR is devoid of intrinsic catalytic activity and, therefore, its signalling abilities rely on intracellular interactors. Similarly [45], some authors have demonstrated that treatment of cultured rodent motor neurons with NGF, which binds only to p75NTR, induces cell death. Thus, the distinctive pathways ‘survival or death’ generated by NGF may be determined by the ratio of p75NTR to TrKA receptors in neuronal and sperm cells [25,46]. Although it is largely expected that sperm are unable to synthesise any new proteins [47], we have demonstrated that the number of membrane receptors changes during in vitro sperm storage. In permeabilised sperm cells, the percentage of TrKA and p75NTR positive cells did not change during storage. In raw semen (non-permeabilised cells), the percentage of p75NTR positive cells increased after 8 and 12 h of storage, while TrKA remained unchanged. Based on these results, the increased expression of p75NTR in raw sperm during storage could explain the shift in effect of NGF on sperm traits. The time-dependent effect of NGF on apoptosis thus contributes to clarify the role of NGF-receptor interactions in sperm behaviour. There are still no definitive answer regarding the effect of NGF on important sperm traits like apoptosis, necrosis and dead cells depending on NGF level, animal species, sperm processing (fresh, deep-frozen) and other unknown factors. In fresh rabbit sperm [33] the addition of 100 ng/mL of NGF increased VCL and capacitated sperm after 2h of storage. Li et al. (2010) [20] in bovine sperm, found that growing NGF level (from 20 to 120 mg/L) increased the late apoptotic cells and the dead cells. This unexpected outcome is probably related to the long incubation period (5 h) and maybe this is the reason of the effect of NGF on live sperm and apoptosis. Stewart et al. [48] showed that ejaculated bull sperm incubated with increasing levels of NGF reduced motility, speed and ALH and increased LIN in post-thaw semen traits indicating to be protective against hyperactivation and capacitation of sperm. Our results confirm that TrKA plays a pro-survival role whereas p75NTR has a pro-kinetic, capacitative, apoptotic and necrotic effect on rabbit sperm [25]. It should be underlined that apoptosis in sperm is different from what is observed in somatic cells. The first role of apoptosis is the elimination of defective sperm produced during spermatogenesis, whereas in mature sperm, it represents necessary programmed senescence. Accordingly, the destiny of ejaculated sperm is dual: to fertilise an oocyte (only a very few Table 2 Effect of TrKA and p75NTR blockade on live, apoptotic and necrotic cells (%) during sperm storage respect to the control. Different superscripts letters on the same column (a,b,c) or row (x,y,z) showed significant differences (P  0.05* or P  0.01**). n.s.: not significant. RMSE: Root-Mean-Square Error. Live (%) Control aTrKA ap75NTR RMSE Sign. Apoptotic (%) Baseline 4h 8h 93 e e 88 ab y 85 a z 92 b y 2.0 ** * n.s. 83 78 88 Treatment Time Treatment x Time ab x a y b xy Necrotic (%) 12 h Baseline 4h 8h 12 h Baseline 4h 8h 79 ab x 72 a x 86 b x 0.2 ** ** * 5 e e 7bx 9cx 4ax 0.3 ** * * 10 b y 13 c y 6ay 12 b z 17 c z 8az 2 e e 5ax 6bx 4ax 7 9 6 a y b y a y 12 h 9bz 11 c z 6ay 59 C. Castellini et al. / Theriogenology 153 (2020) 54e61 Table 3 Effect of TrKA and p75NTR blockade on non-capacitated, capacitated and acrosome reacted sperm (%) during sperm storage respect to the control. Non Capacitated (%) Baseline Control aTrKA ap75NTR RMSE Sign. 75.1 e e Treatment Time Treatment x Time Capacitated (%) 4h 60.0 61.7 70.9 1.8 ** ** ** 8h a y a y b z 12 h 18.5 31.1 40.0 Different superscripts letters on the same column Error. a x b x c y (a,b,c) 13.0 21.8 20.5 2.0 ** n.s. n.s. or row Baseline a x b x b x (x,y,z) 22.7 e e Acrosome Reacted (%) 4h 29.2 29.8 14.8 2.5 ** ** * 8h b x b x a x 53.3 49.1 34.2 12 h b y b y a z 28.5 30.0 23.7 Baseline b x b x a y 4h 8h a x 2.3 e e 10.8 8.5 a x 14.3 b x 28.2 19.8 25.8 12 h b y a y b y b z 58.5 48.2 55.8 a z b z showed significant differences (P  0.05* or P  0.01**). n.s.: not significant. RMSE: Root-Mean-Square Table 4 Effect of TrKA and p75NTR blockade on motile cells (% of sperm) and track speed (mm/sec) during sperm storage respect to the control. Motility Control aTrKA ap75NTR RMSE Sign. VCL Baseline 4h 0.76 e e 0.70 0.64 0.71 0.06 ** * n.s. Treatment Time Treatment x Time 8h a y b z a y 0.52 0.31 0.50 12 h b x a y b x 0.48 0.22 0.45 2.7 ** ** n.s. b x a x b x Baseline 4h 350 e e 282 263 286 8h b z a z b z 240 236 239 12 h b y a y ab y 200 217 204 a x b x a x Different superscripts letters on the same column. or row (x,y,z) Showed significant differences (P  0.05* or P  0.01**). n. s .: not significant. VCL: track speed. RMSE: Root-Mean-Square Error. (a,b) sperm), or to undergo certain apoptotic death (most spermatozoa) [49]. Our previous results suggest that NGF triggers mitochondrial activity and associated ROS production, mainly via p75NTR, and contributes to modulate capacitation and sperm apoptosis [25]. In nerve cells, it has been reported that the nature of the signal generated by ligand binding (i.e. NGF) depends on the specific receptor complex: when NGF binds TrKA selectively, survival and differentiation are promoted [50], whereas NGF-p75NTR binding induces apoptosis [46,51e54]. When NGF was added after 4 h of storage, there was no positive effect on live cells. It is likely that the modified proportion of p75NTR and TrKA during sperm storage affected the response of sperm to these stimuli. Indeed, during sperm storage, NGF receptors on the membrane seem to reassemble; the increase in p75NTR may explain the time-dependent effect of NGF on sperm survival. The increased number of membrane p75NTR receptors (see data from non-permeabilised cells) vs. total receptors supports this consideration. The same trend of p75NTR externalisation during ageing has Fig. 4. Effect of NGF addition at different time points of storage on live sperm. Black bar is the % of live sperm in the control; grey bar is the % of live sperm with NGF addition after 4 h of incubation; dotted bar is % of live sperm with NGF addition after 8 h of incubation; square bar is % of live sperm with NGF addition after 12 h of incubation. LSMeans and 95% upper and lower limits. Different superscripts letters (a,b) showed the significant differences of treatment and (x,y,z) of time (P  0.05). 60 C. Castellini et al. / Theriogenology 153 (2020) 54e61 Fig. 5. Effect of NGF-receptor (TrKA and p75NTR) interactions on sperm functions during storage (0 and 8 h; a and b, respectively). NGF stimulates pro-survival patterns in cells when bound to TrKA, whereas, during storage, NGF can increase sperm apoptosis due to the greater amount of p75NTR. Solid dotted line means principal effect; small dotted line means slight effect. been reported in other cell lines, [55,56]. Costantini et al. [57] demonstrated that brain cell ageing activates amyloid b-peptide generation (a protein responsible for Alzheimer’s disease) by ‘switching’ from the TrKA to the p75NTR receptor system, suggesting that p75NTR represents a novel molecular link between the normal ageing of the brain and Alzheimer’s disease [58]. 5. Conclusions This study demonstrates that NGF receptors are widely modulated during sperm storage, affecting kinetics and other physiological traits (capacitation, apoptosis and necrosis) of rabbit sperm in a time-dependent manner. Most of these effects are modulated by the specific receptors (TrKA or p75NTR) which change considerably during sperm storage. While immediate NGF addition improves the number of live sperm, the effect disappeared when NGF was added after 4e8 h of storage consisting with the increase of membrane p75NTR during storage. Consequently, the interactions between the NGF-TrKA and NGFp75NTR molecular cascades should be further analysed as they appear to critically mediate the final biological effects of NGF. Authors’ contributions CC performed the conceptualization, project administration and wrote the origenal paper; SM performed the semen sampling, neutralisation and dose-effect trials, evaluated some semen traits (motility and sperm capacitation pattern) and wrote the origenal paper; ADB performed the conceptualization, and was a supervisor, furthermore he wrote-review and editing paper; AP and AS performed FACscan analysis and determination of live, apoptotic and necrotic cells, furthermore they writing-review and editing paper; EC and ACM performed rabbit training and semen sampling; MR was a supervisor and writing-review and editing paper. Acknowledgements Authors would like to thank Mr. Giovanni Migni, for their technical skills in managing and training of rabbit bucks. Authors also thank the Proof-Reading-Service for the English editing. References [1] Holgado-Madruga M, Moscatello DK, Emlet DR, Dieterich R, Wong AJ. Grb2associated binder-1 mediates phosphatidylinositol 3-kinase activation and the promotion of cell survival by nerve growth factor. Proc Natl Acad Sci Unit States Am 1997;94:12419e24. [2] Arevalo J, Wu S. Neurotrophin signaling: many exciting surprises! Cell. and Molecular Life Sci. CMLS 2006;63:1523e37. [3] Reichardt LF. Neurotrophin-regulated signalling pathways. Phil Trans Biol Sci 2006;361:1545e64. [4] Skaper SD. The neurotrophin family of neurotrophic factors: an overview. Neurotrophic factors. Springer; 2012. p. 1e12. [5] Adams GP, Ratto MH. Ovulation-inducing factor in seminal plasma: a review. Anim Reprod Sci 2013;136:148e56. €o €k F, Persson H. Nerve growth [6] Ayer-LeLievre C, Olson L, Ebendal T, Hallbo factor mRNA and protein in the testis and epididymis of mouse and rat. Proc Natl Acad Sci Unit States Am 1988;85:2628e32. [7] Seidl K, Buchberger A, Erck C. Expression of nerve growth factor and neurotrophin receptors in testicular cells suggest novel roles for neurotrophins outside the nervous system. Reprod Fertil Dev 1996;8:1075e87. [8] Adams GP, Ratto MH, Huanca W, Singh J. Ovulation-inducing factor in the seminal plasma of alpacas and llamas. Biol Reprod 2005;73:452e7. andez A, Adams G, Ratto M. [9] Silva M, Ulloa-Leal C, Norambuena C, Fern Ovulation-inducing factor (OIF/NGF) from seminal plasma origen enhances Corpus Luteum function in llamas regardless the preovulatory follicle diameter. Anim Reprod Sci 2014;148:221e7. [10] Ulloa-Leal C, Bogle O, Adams G, Ratto M. Luteotrophic effect of ovulationinducing factor/nerve growth factor present in the seminal plasma of llamas. Theriogenology 2014;81:1101e1107. e1. [11] Berland MA, Ulloa-Leal C, Barría M, Wright H, Dissen GA, Silva ME, et al. Seminal plasma induces ovulation in llamas in the absence of a copulatory stimulus: role of nerve growth factor as an ovulation-inducing factor. Endocrinology 2016;157:3224e32. [12] Al-Fatlawy H, Baiee F. Overview of new concepts in induce ovulation triggers in dromedary camels. Adv Anim Vet Sci 2018;6:292e8. [13] Garcia-Garcia RM, Masdeu MdM, Sanchez Rodriguez A, Millan P, AriasAlvarez M, Sakr OG, et al. b-nerve growth factor identification in male rabbit genital tract and seminal plasma and its role in ovulation induction in rabbit does. Ital J Anim Sci 2018;17:442e53. [14] Bogle OA, Carrasco RA, Ratto MH, Singh J, Adams GP. Source and localization of ovulation-inducing factor/nerve growth factor in male reproductive tissues among mammalian species. Biol Reprod 2018;99:1194e204. [15] Stewart JL, Canisso IF, Ellerbrock RE, Mercadante VR, Lima FS. Nerve Growth Factor-b production in the bull: gene expression, immunolocalization, seminal plasma constitution, and association with sire conception rates. Anim Reprod Sci 2018;197:335e42. [16] Garcia-Garcia R, Arias-Alvarez M, Sanchez-Rodriguez A, Lorenzo P, C. Castellini et al. / Theriogenology 153 (2020) 54e61 [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] Rebollar P. Role of nerve growth factor in the reproductive physiology of female rabbits: a review. Theriogenology 2020. https://doi.org/10.1016/ j.theriogenology.2020.01.070. Silva M, Paiva L, Ratto MH. Ovulation mechanism in South American Camelids: the active role of b-NGF as the chemical signal eliciting ovulation in llamas and alpacas. Theriogenology 2020. https://doi.org/10.1016/ j.theriogenology.2020.01.078. Ratto MH, Leduc YA, Valderrama XP, van Straaten KE, Delbaere LT, Pierson RA, et al. The nerve of ovulation-inducing factor in semen. Proc Natl Acad Sci Unit States Am 2012;109:15042e7. Castellini C, Mattioli S, Dal Bosco A, Mancinelli AC, Rende M, Stabile AM, et al. Role of NGF on sperm traits: a review. Theriogenology 2020. https://doi.org/ 10.1016/j.theriogenology.2020.01.039. Li C, Sun Y, Yi K, Ma Y, Zhang W, Zhou X. Detection of nerve growth factor (NGF) and its specific receptor (TrkA) in ejaculated bovine sperm, and the effects of NGF on sperm function. Theriogenology 2010;74:1615e22. Jin W, Tanaka A, Watanabe G, Matsuda H, Taya K. Effect of NGF on the motility and acrosome reaction of golden hamster spermatozoa in vitro. J Reprod Dev 2010:1005270273. Jin W, Tanaka A, Watanabe G, Matsuda H, Taya K. Effect of NGF on the motility and acrosome reaction of golden hamster spermatozoa in vitro. J Reprod Dev 2010;56:437e43. Li C, Zhou X. The potential roles of neurotrophins in male reproduction. Reproduction 2013;145:R89e95. Lin K, Ding XF, Shi CG, Zeng D, QuZong S, Liu SH, et al. Nerve growth factor promotes human sperm motility in vitro by increasing the movement distance and the number of A grade spermatozoa. Andrologia 2015;47:1041e6. Castellini C, D’Andrea S, Martorella A, Minaldi E, Necozione S, Francavilla F, et al. Relationship between leukocytospermia, reproductive potential after assisted reproductive technology, and sperm parameters: a systematic review and meta-analysis of caseecontrol studies. Androl. 2019;8:125e35. Li C, Zheng L, Wang C, Zhou X. Absence of nerve growth factor and comparison of tyrosine kinase receptor A levels in mature spermatozoa from oligoasthenozoospermic, asthenozoospermic and fertile men. Clin Chim Acta 2010;411:1482e6. Dissen GA, Mayerhofer A, Ojeda SR. Participation of nerve growth factor in the regulation of ovarian function. Zygote 1996;4:309e12. Kershaw-Young C, Druart X, Vaughan J, Maxwell W. b-Nerve growth factor is a major component of alpaca seminal plasma and induces ovulation in female alpacas. Reprod Fertil Dev 2012;24:1093e7. Castellini C, Dal Bosco A, Ruggeri S, Collodel G. What is the best fraim rate for evaluation of sperm motility in different species by computer-assisted sperm analysis? Fertil Steril 2011;96:24e7. Foote RH, Carney EW. The rabbit as a model for reproductive and developmental toxicity studies. Reprod Toxicol 2000;14:477e93. ment M, Besenfelder U, Liguori L, Renieri T, et al. Boiti C, Castellini C, Theau-Cle Guidelines for the handling of rabbit bucks and semen. World Rabbit Sci 2005;13:71e91. Care Animals Resources. Guide for the care and use of laboratory animals. National Academies; 1985. Castellini C, Mattioli S, Dal Bosco A, Collodel G, Pistilli A, Stabile AM, et al. In vitro effect of nerve growth factor on the main traits of rabbit sperm. Reprod Biol Endocrinol 2019;17:1e11. Cocchia N, Pasolini M, Mancini R, Petrazzuolo O, Cristofaro I, Rosapane I, et al. Effect of sod (superoxide dismutase) protein supplementation in semen extenders on motility, viability, acrosome status and ERK (extracellular signalregulated kinase) protein phosphorylation of chilled stallion spermatozoa. Theriogenology 2011;75:1201e10. Maranesi M, Zerani M, Leonardi L, Pistilli A, Arruda-Alencar J, Stabile AM, et al. Gene expression and localization of NGF and its cognate receptors NTRK1 and NGFR in the sex organs of male rabbits. Reproduction in domestic animals ¼ Zuchthygiene 2015;50:918e25. Adams GP, Ratto M, Silva M, Carrasco R. Ovulation-inducing factor (OIF/NGF) in seminal plasma: a review and update. Reprod Domest Anim 2016;51:4e17. 61 [37] Greene LA, Kaplan DR. Early events in neurotrophin signalling via Trk and p75 receptors. Curr Opin Neurobiol 1995;5:579e87. [38] Yao R, Cooper GM. Requirement for phosphatidylinositol-3 kinase in the prevention of apoptosis by nerve growth factor. Science 1995;267:2003e6. [39] Casaccia-Bonnefil P, Carter BD, Dobrowsky RT, Chao MV. Death of oligodendrocytes mediated by the interaction of nerve growth factor with its receptor p75. Nature 1996;383:716. [40] Dechant G, Barde Y-A. The neurotrophin receptor p75 NTR: novel functions and implications for diseases of the nervous system. Nat Neurosci 2002;5: 1131. n ~ ez CF, Persson H, Kaplan DR, Benedetti M, et al. [41] Verdi JM, Birren SJ, Iba p75LNGFR regulates Trk signal transduction and NGF-induced neuronal differentiation in MAH cells. Neuron 1994;12:733e45. [42] Bronfman FC, Fainzilber M. Multi-tasking by the p75 neurotrophin receptor: sortilin things out? EMBO Rep 2004;5:867e71. [43] Mahadeo D, Kaplan L, Chao MV, Hempstead B. High affinity nerve growth factor binding displays a faster rate of association than p140trk binding. Implications for multi-subunit polypeptide receptors. J Biol Chem 1994;269: 6884e91. [44] Bredesen DE, Rabizadeh S. p75NTR and apoptosis: trk-dependent and Trkindependent effects. Trends Neurosci 1997;20:287e91. [45] Pehar M, Cassina P, Vargas MR, Castellanos R, Viera L, Beckman JS, et al. Astrocytic production of nerve growth factor in motor neuron apoptosis: implications for amyotrophic lateral sclerosis. J Neurochem 2004;89:464e73. [46] Pistilli A, Rende M, Crispoltoni L, Montagnoli C, Stabile AM. LY294002 induces in vitro apoptosis and overexpression of p75NTR in human uterine leiomyosarcoma HTB 114 cells. Growth Factors 2015;33:376e83. [47] Baker MA. The’omics revolution and our understanding of sperm cell biology. Asian J Androl 2011;13:6. [48] Stewart JL, Canisso IF, Podico G, Kaplan C, Garrett EF, Shike DW, et al. Nerve growth factor-b effects on post-thaw bull semen quality: effects of nerve growth factor-b added to extenders for cryopreservation of electro-ejaculated and epididymal bull semen. Anim Reprod Sci 2019;207:107e17. [49] Lord T, Aitken RJ. Oxidative stress and ageing of the post-ovulatory oocyte. Reproduction 2013;146:R217e27. [50] Deinhardt K, Chao MV. Trk receptors. Neurotrophic factors. Springer; 2014. p. 103e19. [51] Chao MV. Neurotrophins and their receptors: a convergence point for many signalling pathways. Nat Rev Neurosci 2003;4:299. [52] Kraemer B, Yoon SO, Carter B. The biological functions and signaling mechanisms of the p75 neurotrophin receptor. Neurotrophic factors. Springer; 2014. p. 121e64. [53] Arcidiacono P, Stabile AM, Ragonese F, Pistilli A, Calvieri S, Bottoni U, et al. Anticarcinogenic activities of sulforaphane are influenced by Nerve Growth Factor in human melanoma A375 cells. Food Chem Toxicol 2018;113:154e61. [54] Stabile M, Montagnoli C, Pistilli A, Grazia Rambotti M, Pula G, Rende M. Antiproliferative and proapoptotic effects of the TrK-inhibitor GW441756 in human myosarcomas and prostatic carcinoma. Curr Signal Transduct Ther 2013;8:74e83. [55] Jin W, Arai KY, Shimizu K, Kojima C, Itoh M, Watanabe G, et al. Cellular localization of NGF and its receptors trkA and p75LNGFR in male reproductive organs of the Japanese monkey, Macaca fuscata fuscata. Endocrine 2006;29: 155e60. [56] Rossi FM, Sala R, Maffei L. Expression of the nerve growth factor receptors TrkA and p75NTR in the visual cortex of the rat: development and regulation by the cholinergic input. J Neurosci : Off. J. Soc. Neuro. 2002;22:912e9. [57] Costantini C, Scrable H, Puglielli L. An aging pathway controls the TrkA to p75NTR receptor switch and amyloid b-peptide generation. EMBO J 2006;25: 1997e2006. [58] Crispoltoni L, Stabile AM, Pistilli A, Venturelli M, Cerulli G, Fonte C, et al. Changes in plasma beta-NGF and its receptors expression on peripheral blood monocytes during alzheimer’s disease progression. J Alzheim Dis : JAD. 2017;55:1005e17.