Is male reproductive senescence minimized in Mus species with high levels of sperm competition?

Is male reproductive senescence minimized in Mus species with high levels of sperm competition? Abstract Sperm competition, an evolutionary process in which the spermatozoa of two or more males compete for the fertilization of the same ovum, gives rise to several morphological and physiological adaptations. Generally, high levels of sperm competition enhance sperm function. In contrast, advanced age is known to lead to reproductive senescence, including a general decline in sperm function. Sperm competition and advanced age may thus have opposing effects on sperm function. Here, we tested the hypothesis that the increase in sperm function in species experiencing high levels of sperm competition will counteract the negative effects of advanced age. We measured a comprehensive set of reproductive traits in young and old males in three species of mice of the genus Mus, which differ greatly in their levels of sperm competition. Our prediction was that the expression of reproductive senescence would be highest in the species with low levels of sperm competition and lowest in the species with high levels of sperm competition. Surprisingly, we did not find a strong signal of reproductive senescence in any of the three Mus species. Overall, our results did not clearly support our hypothesis that high levels of sperm competition minimize the negative effects of ageing on sperm function. INTRODUCTION Sperm competition occurs when a female mates with two or more males and the spermatozoa of those males compete for the fertilization of the female’s ova (Parker, 1970; Birkhead & Møller, 1998). Sperm competition is a widespread phenomenon, and its occurrence leads to several evolutionary adaptations at the behavioural, morphological and physiological levels (Birkhead & Møller, 1998; delBarco-Trillo, Tourmente & Roldan, 2013). In many taxa, high levels of sperm competition are associated with an increase in the production (delBarco-Trillo et al., 2013), storage and allocation of spermatozoa (Parker & Pizzari, 2010; delBarco-Trillo, 2011), as well as with enhanced sperm function (Gomendio et al., 2006; Fitzpatrick et al., 2009; Kleven et al., 2009; Martín-Coello et al., 2009; Gómez Montoto et al., 2011a). For example, high levels of sperm competition in rodents lead to a higher proportion of spermatozoa that are morphologically normal, motile, and capable of reaching and fertilizing the ova (Gomendio et al., 2006; Gómez Montoto et al., 2011a), as well as to modifications in sperm dimensions (Gomendio & Roldan, 2008; Tourmente, Gomendio & Roldan, 2011) and sperm energy metabolism (Tourmente et al., 2013, 2015b) that may result in improvements in sperm motility. In contrast to the positive effect of sperm competition on sperm function, advanced age has been reported to lead to reproductive senescence (García-Palomares et al., 2009a, 2009b), having a negative impact on sperm function in particular (Pizzari et al., 2008). A decline in sperm function with age may be attributable to the accumulation of de novo mutations in the male germline that may occur during each cell division (Radwan, 2003) or to an increasingly impaired process of spermatogenesis with advancing age (Pizzari et al., 2008; Johnson & Gemmell, 2012). These processes may be driven or exacerbated by an accumulation of reactive oxygen species and an escalation of oxidative stress with age (Weir & Robaire, 2006; Johnson & Gemmell, 2012) or by reduced efficiency of DNA repair with advancing paternal age (Sloter et al., 2004; Paul, Nagano & Robaire, 2011). Indeed, across taxa there is strong evidence for a generalized decline in sperm function with age. This decline may involve a decline in the number of germinal cells and Sertoli cells in the seminiferous tubules (Dakouane et al., 2005), a reduction in the number of sperm ejaculated (Sasson, Johnson & Brockmann, 2012), an increase in sperm abnormalities (Syntin & Robaire, 2001), a decrease in sperm motility (Wolf et al., 2000; Møller et al., 2009), an increase in sperm DNA damage (Harris et al., 2011; Velando et al., 2011) or decreased reproductive success (Dean et al., 2010). Although the positive effect of sperm competition on sperm function is restricted to species experiencing high levels of sperm competition, the negative impacts of advanced age on sperm function can be considered to be similar across phylogenetically related species. Consequently, we hypothesized that the generalized increase in sperm function in species with high levels of sperm competition will diminish the negative impacts of senescence only in such species. That is, in species with high levels of sperm competition, selective pressures on sperm competitiveness may be strong throughout a male’s reproductive life and may reduce the incidence of sperm senescence. To test our hypothesis, we measured a comprehensive set of reproductive traits in young and old males in three species of mice of the genus Mus that differ in their levels of sperm competition based on relative testes size: Mus musculus, Mus spretus and Mus spicilegus (delBarco-Trillo et al., 2016). These reproductive traits included the number of spermatozoa in the caudae epididymides, sperm dimensions and morphology, the percentage of spermatozoa with morphological abnormalities, sperm motility and velocity, and adenosine triphosphate (ATP) content in spermatozoa. A decrease in sperm function can include a lower number of stored spermatozoa, shorter spermatozoa, a higher percentage of spermatozoa with morphological abnormalities, lower motility and velocity, and lower ATP content in spermatozoa. According to our hypothesis, we predicted that the decrease in sperm function in old males would be the highest in the species with low levels of sperm competition (M. musculus) and the lowest in the species with high levels of sperm competition (M. spicilegus). MATERIAL AND METHODS Animals We used adult males of three species from the genus Mus that differ greatly in their levels of sperm competition: M. musculus, M. spretus and M. spicilegus (N = 11 per species). These three species have been characterized as a good model for studies on sperm competition in rodents, representing low, intermediate and high levels of sperm competition, respectively (Gomendio et al., 2006; Gómez Montoto et al., 2011a). We selected males of two age classes, hereafter referred as ‘young’ and ‘old’ for simplicity. Young males (N = 6 per species) were 4–6 months of age. At this age, mice are no longer juveniles but they are not old enough to be affected by reproductive senescence. Old males (N = 5 per species) were 24–28 months of age. Males were selected so that ages of young (155.11 ± 37.31 days; mean ± SD) and old animals (769.93 ± 43.96 days) were similar across species. Old males in our study were older than males considered to be senescent in other studies of mice (Biddle et al., 1997; Anjum et al., 2012; Tognetti et al., 2017). We were not able to measure all reproductive traits for all individuals. However, N ≥ 5 for any species and age class combination. Adult males were close descendants of animals acquired from the Institut des Sciences de l’Evolution, CNRS – Université Montpellier 2, France, belonging to the following wild-derived strains: M. musculus, strain MPB (from Bialowieza, Poland); M. spretus, strain SEB (from Barcelona, Spain); and M. spicilegus, strain ZRU (from Kalomoyevka, Ukraine). Crossings in our colony were arranged to minimize inbreeding. All males were maintained in standard conditions (14 h light–10 h darkness, 22–24 °C, 55–60% relative humidity), with food (rodent chow, Harlan Laboratories; seeds and fresh apple) and water provided ad libitum. All males used in this study were housed individually for at least a month before sampling to eliminate the possibility that males had a different perceived risk of sperm competition. The research protocol was approved by the Ethics Committee of the Spanish Research Council (CSIC). All procedures were carried out following Spanish Animal Protection Regulation RD53/2013, which conforms to European Union Regulation 2010/63. Morphological measurements Males were sacrificed by cervical dislocation, weighed (in grams) and measured (body length and tail length; in millimetres). To evaluate body condition, we calculated a body mass index as weight (in grams)/length squared (in millimetres squared) (Labocha, Schutz & Hayes, 2014). Testes were removed and weighed (in grams). Relative testes mass (RTS) has been shown to reflect sperm competition levels in rodents (Ramm, Parker & Stockley, 2005; Long & Montgomerie, 2006; Bryja et al., 2008; Firman & Simmons, 2008; Soulsbury, 2010). The RTS was calculated using Kenagy and Trombulak’s rodent-specific regression equation: RTS = testes mass/0.031 × body mass0.77 (Kenagy & Trombulak, 1986). Compared with young mice, old mice had higher body weights (two-way ANOVA: F1,27 = 23.04, P < 0.0001), longer bodies (F1,27 = 7.28, P = 0.012) and longer tails (F1,27 = 30.08, P < 0.0001). Body mass index (used as a measure of body condition) was also higher in old mice than in young mice (F1,27 = 8.83, P = 0.006). Relative testes size differed among the three species, following the predicted pattern, with lowest values in M. musculus and highest in M. spicilegus (two-way ANOVA: F2,27 = 221.75, P < 0.0001; Table S1; Supporting Information). Relative testes size, however, did not differ between young and old males (F1,27 = 2.51, P = 0.13). Preparation of sperm suspension and sperm measurements Mature spermatozoa were collected from the caudae epididymides and vasa deferentia, by placing the tissue in a Petri dish containing Hepes-buffered modified Tyrode’s medium (mT-H; for details, see Supporting Information, Methods) prewarmed to 37 °C, making several cuts and allowing spermatozoa to swim out for a period of 5 min. After the 5 min swim-out incubation, the sperm suspension was transferred to a prewarmed Eppendorf tube. Each sperm suspension was maintained at 37 °C until processing. Some samples were assessed immediately (we refer to this time as ‘0 h’). Sperm suspensions were also incubated for 3 h at 37 °C in mT-H under air, after which samples were taken and some of the sperm parameters assessed again (we refer to this time as ‘3 h’). The duration of incubation (3 h) was selected based on the maintenance of sperm motility in vitro in a subset of rodent species, including the three species studied here (Tourmente et al., 2015b). This period of incubation does not result in complete sperm immobilization in the species with low sperm survival. Moreover, because fertilization takes place a few hours after copulation in the muroid species for which data are available (Suarez et al., 1990), our selected incubation time is within physiological time frames. We used a haemocytometer (modified Neubauer chamber) to estimate the total number of spermatozoa stored in the caudae epididymides. To measure sperm linear dimensions, 5 µl of the sperm suspension was smeared onto a slide, fixed with formaldehyde in a phosphate buffer, stained with Giemsa as previously described (Gómez Montoto et al., 2011a) and examined using bright field microscopy. All samples were evaluated and photographed at ×1000 magnification for subsequent digitalization using an Eclipse E-600 microscope (Nikon, Tokyo, Japan) with Pan-Fluor optics and a DS5 camera (Nikon, Tokyo, Japan). Spermatozoa were photographed by using the software NIS-Elements v.3.0 (Nikon, Tokyo, Japan). For each individual, we measured 25 different spermatozoa. Linear dimensions were obtained by measuring captured sperm images using ImageJ software v.1.41 (National Institutes of Health, Bethesda, MD, USA) (Gómez Montoto et al., 2011b). Measurements included head length, head width, head area, total flagellum length and total sperm length. Head length was measured as the linear distance between the most basal point and the most apical one of the sperm head. Head width was taken as a straight line between the dorsal and ventral regions in the wider region of the sperm head. Head area was measured considering the entire sperm head, including the apical hook. To quantify differences in sperm head morphology, we used a geometric morphometric approach described previously (Varea Sánchez, Bastir & Roldan, 2013; for details, see Supporting Information, Methods). To assess sperm abnormalities, we used sperm smears stained first with Eosin and Nigrosin and subsequently with Giemsa (Gómez Montoto et al., 2011a). Briefly, 5 µl sperm suspension and 10 µl Eosin and Nigrosin solution were mixed on a glass slide placed on a stage at 37 °C, and 30 s later the mix was smeared and allowed to air dry. Smears were stained with Giemsa solution and mounted with DPX. Smears were examined at ×1000 under bright field illumination, and 200 spermatozoa per male were examined to evaluate the percentage of morphologically normal spermatozoa (i.e. without abnormal head, midpiece or principal piece and without a cytoplasmic droplet or coiled flagellum). The percentage of motile spermatozoa (MOT) was evaluated by examining 10 µl of the sperm suspension that was placed between a pre-warmed slide and a coverslip at ×100 magnification under phase contrast optics. We also estimated the percentage of spermatozoa exhibiting forward progression. To assess sperm swimming patterns, an aliquot of sperm suspension was diluted to ~5 × 106 spermatozoa ml−1, placed in a pre-warmed microscopy chamber with a depth of 20 µm (Leja, Nieuw-Vennep, The Netherlands), and filmed at ×40 using a phase contrast microscope connected to a digital video camera (Basler A312fc; Vision Technologies, Glen Burnie, MD, USA). A minimum of 150 sperm trajectories were assessed per male using a computer-assisted sperm analyser (CASA; Sperm Class Analyzer version 4.0; Microptic, Barcelona, Spain), and the following swimming parameters were estimated for each trajectory: curvilinear velocity (VCL; in micrometres per second), straight line velocity (VSL; in micrometres per second), average path velocity (VAP; in micrometres per second), wobble (WOB = VAP/VCL), linearity (LIN = VSL/VCL), straightness (STR = VSL/VAP), amplitude of lateral head displacement (ALH; in micrometres) and beat-cross frequency (BCF; in herz). Sperm ATP content was measured using a luciferase-based ATP bioluminescence assay kit (HS II; Roche Applied Science; Tourmente et al., 2015a; for details, see Supporting Information, Methods). Statistical analyses All statistical analyses were conducted using R version 3.1.0 (R Core Team, 2014) unless otherwise specified. Normality was checked with the Shapiro–Wilk normality test. If normality was not met, we used logarithmic and arcsine transformations as required. Average values are reported as the mean ± SD. Significance level (α) was set at 0.05 for all the tests. We used principal component analysis (PCA) to reduce potentially correlated variables and obtain measures of ‘overall sperm morphology’, ‘overall sperm velocity’ and ‘overall trajectory shape’ (for details, see Supporting Information, Methods). We implemented two-way ANOVAs and two-way ANCOVAs fitted using the function aov. The two factors were species (three levels) and age (young and old). The covariate in the ANCOVAs was body mass. We also considered the interaction between species and age to determine whether any significant difference between young and old mice differed across species, and whether such a species effect paralleled the different levels of sperm competition among those species. Geometric morphometrics statistical analyses were conducted with MorphoJ v1.06d (Klingenberg, 2011). Differences in sperm head shape between young and old individuals were quantified by examining the distance between the mean of both groups by conducting a discriminant analysis (Timm, 2002). RESULTS The number of spermatozoa stored in the cauda epididymides differed between species, following the predicted pattern (two-way ANCOVA: F2,26 = 50.36, P < 0.0001; Table S1; Supporting Information), but there was no difference between young and old males (F1,26 = 0.4, P = 0.54). The overall sperm morphology differed between species (F2,24 = 168.42, P > 0.0001) and between young and old males (F1,24 = 6.15, P = 0.02), but there was no significant interaction between species and age (F2,24 = 0.1, P = 0.91). The shape of the sperm head differed between species (discriminant analyses: P < 0.0001), but not between young and old mice (P = 0.2; Fig. 1). Figure 1. View largeDownload slide Sperm head shape in young and old males of three Mus species. Dots indicate the landmarks used for geometric morphometrics analyses. Figure 1. View largeDownload slide Sperm head shape in young and old males of three Mus species. Dots indicate the landmarks used for geometric morphometrics analyses. The percentage of normal spermatozoa differed between species (F2,27 = 7.44, P = 0.003); even though there was not an overall difference between young and old males (F1,27 = 0.99, P = 0.33), we found a statistically significant interaction between species and age (F2,27 = 4.07, P = 0.029). Subanalyses by species showed no differences between young and old mice in M. spicilegus and M. spretus (P > 0.05), but a higher percentage of normal spermatozoa in young males than in old males in M. musculus (P = 0.008; Fig. 2), the species with the lowest level of sperm competition. Figure 2. View largeDownload slide Percentage of morphologically normal spermatozoa in young and old males of three Mus species. For each boxplot, the bar within each box represents the sample median, each box represents 50% of the data around the median, and the two whiskers represent the 95% confidence interval. **P < 0.001; NS denotes P > 0.05. Figure 2. View largeDownload slide Percentage of morphologically normal spermatozoa in young and old males of three Mus species. For each boxplot, the bar within each box represents the sample median, each box represents 50% of the data around the median, and the two whiskers represent the 95% confidence interval. **P < 0.001; NS denotes P > 0.05. There were differences in sperm motility across species (two-way ANCOVA: F2,26 = 36.47, P < 0.0001), with this trait being higher in young males than in old males (F1,26 = 7.25, P = 0.01). However, all species were affected similarly by age (interaction: F2,26 = 0.1, P = 0.9). After 3 h of incubation, significant differences among species remained in sperm motility (F2,26 = 25.05, P < 0.0001), but there were no longer differences between age classes (F1,26 = 0.41, P = 0.53). Sperm forward progression also differed between species, both at 0 h (F2,26 = 6.22, P = 0.006) and after 3 h of incubation (F2,26 = 17.71, P < 0.0001), and although it was similar in young and old males at 0 h (F1,26 = 0.25, P = 0.62), after 3 h of incubation it was higher in old males than in young males (F1,26 = 7.38, P = 0.01). Overall sperm velocity and overall trajectory shape differed between species (P < 0.0001 in both analyses) and ages (P < 0.01), but there was not a significant interaction between species and age (P > 0.05). There were significant differences between species in the amount of ATP per sperm cell, both at 0 h (F2,26 = 6.85, P = 0.004) and after 3 h of incubation (F2,26 = 9.59, P = 0.0008; Table S1; Supporting Information). However, there was not a significant difference in ATP concentration between young and old males, nor a significant interaction between species and age at either time (P > 0.05 for all analyses). DISCUSSION Overall, our results do not support the hypothesis that males in species with high levels of sperm competition suffer less reproductive senescence than in species with low levels of sperm competition. We found that many reproductive traits were unaffected by age, whereas others were either enhanced or lessened in old males compared with young males, but the level of sperm competition did not have an influence in most of these traits. Only the percentage of normal spermatozoa matched our prediction, with a decline in old males in M. musculus (i.e. the species with low levels of sperm competition), but not in the other two species, which experience higher levels of sperm competition (M. spretus and M. spicilegus). This result may be driven by an enhanced process of spermatogenesis in species with high levels of sperm competition, which would either directly or indirectly minimize the occurrence of sperm abnormalities in old males, but this is an area of research that requires further investigation. Even though we considered many reproductive traits that could be affected by senescence, there are many other traits that could be affected in a different manner in young and old males of a species, depending on the level of sperm competition normally experienced in that species. These traits include chromosomal abnormalities, DNA damage in spermatozoa and any traits that regulate or determine the success of the capacitation and fertilization processes (Gogol, Bochenek & Smorag, 2002; Momand, Xu & Walter, 2013). For example, in brown rats, the spermatozoa of old males are more susceptible to oxidative damage and DNA fragmentation (Zubkova, Wade & Robaire, 2005), as well as having a decreased antioxidant capacity and an increased production of reactive oxygen species (Weir & Robaire, 2006). It is important to notice that the genomic damage in spermatozoa driven by ageing may be independent of sperm function. Despite a normal expression of sperm function in old males, any genomic damage in their spermatozoa will increase the risk of transmission of multiple genetic and chromosomal defects to offspring (Wyrobek et al., 2006). Surprisingly, we did not find a strong signal of reproductive senescence in the three species of mice that we studied. Reproductive senescence may thus not play an important role in the natural populations of the three Mus species under study. Indeed, given the high predation rates suffered by rodents, most males will normally die before the inception of any signs of senescence. Another study using wild-captured Mus musculus domesticus found that epididymal sperm counts declined with age, although only a range of relatively advanced ages (21–32 months) were studied (Garratt et al., 2011). It must be noted that most of the available knowledge on reproductive senescence in rodents is based on laboratory strains (Parkening, 1989; Katz-Jaffe et al., 2013; Lucio et al., 2013). This may be a shortcoming, as in a benign captive environment the negative effects of ageing can be minimized and thus differences between age classes might be obscured. Even though there are many studies describing the timing and incidence of reproductive senescence, there are also many studies in which reproductive condition remains unchanged or is even enhanced in old individuals (Gasparini et al., 2010; Kanuga et al., 2011; Johnson & Gemmell, 2012). It is still unclear why reproductive senescence is pronounced in some species but not in others. It is important to consider whether any differences between young and old males lead to a fertilizing advantage for one age class or the other. For example, young male guppies produce faster swimming spermatozoa compared with old males; however, young males do not have a fertilizing advantage in sperm competition scenarios (Gasparini et al., 2010). It is equally possible that despite a lack of consistent differences in sperm function between young and old males, as we found in our three species of mice, undetected differences between their spermatozoa could result in a lower reproductive potential in older individuals. We can thus conclude that even though our results did not support our hypothesis that high levels of sperm competition can minimize the impacts of senescence, more reproductive measurements, including sperm competition tests and measurements of fertilizing ability and offspring health, and possibly higher sample sizes than we used, are required to support or disprove our hypothesis completely. SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Table S1. Reproductive parameters in young and old males from three Mus species with different levels of sperm competition. Methods.pdf. Specific methods. ACKNOWLEDGEMENTS This work was supported by a Ramón y Cajal fellowship from the Spanish Ministry of Economy and Competitiveness (RYC-2011-07943) and a Marie Curie Career Integration Grant (PCIG11-GA-2012-321888) to J.d.-T., a Juan de la Cierva fellowship from the Spanish Ministry of Economy and Competitiveness to M.T. (JCI-2011-10381) and grants from the Spanish Ministry of Economy and Competitiveness (CGL2011-26341 and CGL2016-80577-P to E.R.S.R. and CGL2012-37423 to J.d.-T.). We are grateful to Annie Orth and François Bonhomme (Institut des Sciences de l’Evolution de Montpellier, CNRS-Université Montpellier 2, France) for facilitating access to animals, and Juan Antonio Rielo for managing the animal facilities and Esperanza Navarro for animal care at the Museo Nacional de Ciencias Naturales (CSIC). We thank Alberto Vicens, Pilar Villar and Ester Sansegundo for their help with processing spermatozoa in the laboratory. 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Google Scholar CrossRef Search ADS PubMed  © 2017 The Linnean Society of London, Biological Journal of the Linnean Society http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Biological Journal of the Linnean Society Oxford University Press

Is male reproductive senescence minimized in Mus species with high levels of sperm competition?

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© 2017 The Linnean Society of London, Biological Journal of the Linnean Society
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Abstract

Abstract Sperm competition, an evolutionary process in which the spermatozoa of two or more males compete for the fertilization of the same ovum, gives rise to several morphological and physiological adaptations. Generally, high levels of sperm competition enhance sperm function. In contrast, advanced age is known to lead to reproductive senescence, including a general decline in sperm function. Sperm competition and advanced age may thus have opposing effects on sperm function. Here, we tested the hypothesis that the increase in sperm function in species experiencing high levels of sperm competition will counteract the negative effects of advanced age. We measured a comprehensive set of reproductive traits in young and old males in three species of mice of the genus Mus, which differ greatly in their levels of sperm competition. Our prediction was that the expression of reproductive senescence would be highest in the species with low levels of sperm competition and lowest in the species with high levels of sperm competition. Surprisingly, we did not find a strong signal of reproductive senescence in any of the three Mus species. Overall, our results did not clearly support our hypothesis that high levels of sperm competition minimize the negative effects of ageing on sperm function. INTRODUCTION Sperm competition occurs when a female mates with two or more males and the spermatozoa of those males compete for the fertilization of the female’s ova (Parker, 1970; Birkhead & Møller, 1998). Sperm competition is a widespread phenomenon, and its occurrence leads to several evolutionary adaptations at the behavioural, morphological and physiological levels (Birkhead & Møller, 1998; delBarco-Trillo, Tourmente & Roldan, 2013). In many taxa, high levels of sperm competition are associated with an increase in the production (delBarco-Trillo et al., 2013), storage and allocation of spermatozoa (Parker & Pizzari, 2010; delBarco-Trillo, 2011), as well as with enhanced sperm function (Gomendio et al., 2006; Fitzpatrick et al., 2009; Kleven et al., 2009; Martín-Coello et al., 2009; Gómez Montoto et al., 2011a). For example, high levels of sperm competition in rodents lead to a higher proportion of spermatozoa that are morphologically normal, motile, and capable of reaching and fertilizing the ova (Gomendio et al., 2006; Gómez Montoto et al., 2011a), as well as to modifications in sperm dimensions (Gomendio & Roldan, 2008; Tourmente, Gomendio & Roldan, 2011) and sperm energy metabolism (Tourmente et al., 2013, 2015b) that may result in improvements in sperm motility. In contrast to the positive effect of sperm competition on sperm function, advanced age has been reported to lead to reproductive senescence (García-Palomares et al., 2009a, 2009b), having a negative impact on sperm function in particular (Pizzari et al., 2008). A decline in sperm function with age may be attributable to the accumulation of de novo mutations in the male germline that may occur during each cell division (Radwan, 2003) or to an increasingly impaired process of spermatogenesis with advancing age (Pizzari et al., 2008; Johnson & Gemmell, 2012). These processes may be driven or exacerbated by an accumulation of reactive oxygen species and an escalation of oxidative stress with age (Weir & Robaire, 2006; Johnson & Gemmell, 2012) or by reduced efficiency of DNA repair with advancing paternal age (Sloter et al., 2004; Paul, Nagano & Robaire, 2011). Indeed, across taxa there is strong evidence for a generalized decline in sperm function with age. This decline may involve a decline in the number of germinal cells and Sertoli cells in the seminiferous tubules (Dakouane et al., 2005), a reduction in the number of sperm ejaculated (Sasson, Johnson & Brockmann, 2012), an increase in sperm abnormalities (Syntin & Robaire, 2001), a decrease in sperm motility (Wolf et al., 2000; Møller et al., 2009), an increase in sperm DNA damage (Harris et al., 2011; Velando et al., 2011) or decreased reproductive success (Dean et al., 2010). Although the positive effect of sperm competition on sperm function is restricted to species experiencing high levels of sperm competition, the negative impacts of advanced age on sperm function can be considered to be similar across phylogenetically related species. Consequently, we hypothesized that the generalized increase in sperm function in species with high levels of sperm competition will diminish the negative impacts of senescence only in such species. That is, in species with high levels of sperm competition, selective pressures on sperm competitiveness may be strong throughout a male’s reproductive life and may reduce the incidence of sperm senescence. To test our hypothesis, we measured a comprehensive set of reproductive traits in young and old males in three species of mice of the genus Mus that differ in their levels of sperm competition based on relative testes size: Mus musculus, Mus spretus and Mus spicilegus (delBarco-Trillo et al., 2016). These reproductive traits included the number of spermatozoa in the caudae epididymides, sperm dimensions and morphology, the percentage of spermatozoa with morphological abnormalities, sperm motility and velocity, and adenosine triphosphate (ATP) content in spermatozoa. A decrease in sperm function can include a lower number of stored spermatozoa, shorter spermatozoa, a higher percentage of spermatozoa with morphological abnormalities, lower motility and velocity, and lower ATP content in spermatozoa. According to our hypothesis, we predicted that the decrease in sperm function in old males would be the highest in the species with low levels of sperm competition (M. musculus) and the lowest in the species with high levels of sperm competition (M. spicilegus). MATERIAL AND METHODS Animals We used adult males of three species from the genus Mus that differ greatly in their levels of sperm competition: M. musculus, M. spretus and M. spicilegus (N = 11 per species). These three species have been characterized as a good model for studies on sperm competition in rodents, representing low, intermediate and high levels of sperm competition, respectively (Gomendio et al., 2006; Gómez Montoto et al., 2011a). We selected males of two age classes, hereafter referred as ‘young’ and ‘old’ for simplicity. Young males (N = 6 per species) were 4–6 months of age. At this age, mice are no longer juveniles but they are not old enough to be affected by reproductive senescence. Old males (N = 5 per species) were 24–28 months of age. Males were selected so that ages of young (155.11 ± 37.31 days; mean ± SD) and old animals (769.93 ± 43.96 days) were similar across species. Old males in our study were older than males considered to be senescent in other studies of mice (Biddle et al., 1997; Anjum et al., 2012; Tognetti et al., 2017). We were not able to measure all reproductive traits for all individuals. However, N ≥ 5 for any species and age class combination. Adult males were close descendants of animals acquired from the Institut des Sciences de l’Evolution, CNRS – Université Montpellier 2, France, belonging to the following wild-derived strains: M. musculus, strain MPB (from Bialowieza, Poland); M. spretus, strain SEB (from Barcelona, Spain); and M. spicilegus, strain ZRU (from Kalomoyevka, Ukraine). Crossings in our colony were arranged to minimize inbreeding. All males were maintained in standard conditions (14 h light–10 h darkness, 22–24 °C, 55–60% relative humidity), with food (rodent chow, Harlan Laboratories; seeds and fresh apple) and water provided ad libitum. All males used in this study were housed individually for at least a month before sampling to eliminate the possibility that males had a different perceived risk of sperm competition. The research protocol was approved by the Ethics Committee of the Spanish Research Council (CSIC). All procedures were carried out following Spanish Animal Protection Regulation RD53/2013, which conforms to European Union Regulation 2010/63. Morphological measurements Males were sacrificed by cervical dislocation, weighed (in grams) and measured (body length and tail length; in millimetres). To evaluate body condition, we calculated a body mass index as weight (in grams)/length squared (in millimetres squared) (Labocha, Schutz & Hayes, 2014). Testes were removed and weighed (in grams). Relative testes mass (RTS) has been shown to reflect sperm competition levels in rodents (Ramm, Parker & Stockley, 2005; Long & Montgomerie, 2006; Bryja et al., 2008; Firman & Simmons, 2008; Soulsbury, 2010). The RTS was calculated using Kenagy and Trombulak’s rodent-specific regression equation: RTS = testes mass/0.031 × body mass0.77 (Kenagy & Trombulak, 1986). Compared with young mice, old mice had higher body weights (two-way ANOVA: F1,27 = 23.04, P < 0.0001), longer bodies (F1,27 = 7.28, P = 0.012) and longer tails (F1,27 = 30.08, P < 0.0001). Body mass index (used as a measure of body condition) was also higher in old mice than in young mice (F1,27 = 8.83, P = 0.006). Relative testes size differed among the three species, following the predicted pattern, with lowest values in M. musculus and highest in M. spicilegus (two-way ANOVA: F2,27 = 221.75, P < 0.0001; Table S1; Supporting Information). Relative testes size, however, did not differ between young and old males (F1,27 = 2.51, P = 0.13). Preparation of sperm suspension and sperm measurements Mature spermatozoa were collected from the caudae epididymides and vasa deferentia, by placing the tissue in a Petri dish containing Hepes-buffered modified Tyrode’s medium (mT-H; for details, see Supporting Information, Methods) prewarmed to 37 °C, making several cuts and allowing spermatozoa to swim out for a period of 5 min. After the 5 min swim-out incubation, the sperm suspension was transferred to a prewarmed Eppendorf tube. Each sperm suspension was maintained at 37 °C until processing. Some samples were assessed immediately (we refer to this time as ‘0 h’). Sperm suspensions were also incubated for 3 h at 37 °C in mT-H under air, after which samples were taken and some of the sperm parameters assessed again (we refer to this time as ‘3 h’). The duration of incubation (3 h) was selected based on the maintenance of sperm motility in vitro in a subset of rodent species, including the three species studied here (Tourmente et al., 2015b). This period of incubation does not result in complete sperm immobilization in the species with low sperm survival. Moreover, because fertilization takes place a few hours after copulation in the muroid species for which data are available (Suarez et al., 1990), our selected incubation time is within physiological time frames. We used a haemocytometer (modified Neubauer chamber) to estimate the total number of spermatozoa stored in the caudae epididymides. To measure sperm linear dimensions, 5 µl of the sperm suspension was smeared onto a slide, fixed with formaldehyde in a phosphate buffer, stained with Giemsa as previously described (Gómez Montoto et al., 2011a) and examined using bright field microscopy. All samples were evaluated and photographed at ×1000 magnification for subsequent digitalization using an Eclipse E-600 microscope (Nikon, Tokyo, Japan) with Pan-Fluor optics and a DS5 camera (Nikon, Tokyo, Japan). Spermatozoa were photographed by using the software NIS-Elements v.3.0 (Nikon, Tokyo, Japan). For each individual, we measured 25 different spermatozoa. Linear dimensions were obtained by measuring captured sperm images using ImageJ software v.1.41 (National Institutes of Health, Bethesda, MD, USA) (Gómez Montoto et al., 2011b). Measurements included head length, head width, head area, total flagellum length and total sperm length. Head length was measured as the linear distance between the most basal point and the most apical one of the sperm head. Head width was taken as a straight line between the dorsal and ventral regions in the wider region of the sperm head. Head area was measured considering the entire sperm head, including the apical hook. To quantify differences in sperm head morphology, we used a geometric morphometric approach described previously (Varea Sánchez, Bastir & Roldan, 2013; for details, see Supporting Information, Methods). To assess sperm abnormalities, we used sperm smears stained first with Eosin and Nigrosin and subsequently with Giemsa (Gómez Montoto et al., 2011a). Briefly, 5 µl sperm suspension and 10 µl Eosin and Nigrosin solution were mixed on a glass slide placed on a stage at 37 °C, and 30 s later the mix was smeared and allowed to air dry. Smears were stained with Giemsa solution and mounted with DPX. Smears were examined at ×1000 under bright field illumination, and 200 spermatozoa per male were examined to evaluate the percentage of morphologically normal spermatozoa (i.e. without abnormal head, midpiece or principal piece and without a cytoplasmic droplet or coiled flagellum). The percentage of motile spermatozoa (MOT) was evaluated by examining 10 µl of the sperm suspension that was placed between a pre-warmed slide and a coverslip at ×100 magnification under phase contrast optics. We also estimated the percentage of spermatozoa exhibiting forward progression. To assess sperm swimming patterns, an aliquot of sperm suspension was diluted to ~5 × 106 spermatozoa ml−1, placed in a pre-warmed microscopy chamber with a depth of 20 µm (Leja, Nieuw-Vennep, The Netherlands), and filmed at ×40 using a phase contrast microscope connected to a digital video camera (Basler A312fc; Vision Technologies, Glen Burnie, MD, USA). A minimum of 150 sperm trajectories were assessed per male using a computer-assisted sperm analyser (CASA; Sperm Class Analyzer version 4.0; Microptic, Barcelona, Spain), and the following swimming parameters were estimated for each trajectory: curvilinear velocity (VCL; in micrometres per second), straight line velocity (VSL; in micrometres per second), average path velocity (VAP; in micrometres per second), wobble (WOB = VAP/VCL), linearity (LIN = VSL/VCL), straightness (STR = VSL/VAP), amplitude of lateral head displacement (ALH; in micrometres) and beat-cross frequency (BCF; in herz). Sperm ATP content was measured using a luciferase-based ATP bioluminescence assay kit (HS II; Roche Applied Science; Tourmente et al., 2015a; for details, see Supporting Information, Methods). Statistical analyses All statistical analyses were conducted using R version 3.1.0 (R Core Team, 2014) unless otherwise specified. Normality was checked with the Shapiro–Wilk normality test. If normality was not met, we used logarithmic and arcsine transformations as required. Average values are reported as the mean ± SD. Significance level (α) was set at 0.05 for all the tests. We used principal component analysis (PCA) to reduce potentially correlated variables and obtain measures of ‘overall sperm morphology’, ‘overall sperm velocity’ and ‘overall trajectory shape’ (for details, see Supporting Information, Methods). We implemented two-way ANOVAs and two-way ANCOVAs fitted using the function aov. The two factors were species (three levels) and age (young and old). The covariate in the ANCOVAs was body mass. We also considered the interaction between species and age to determine whether any significant difference between young and old mice differed across species, and whether such a species effect paralleled the different levels of sperm competition among those species. Geometric morphometrics statistical analyses were conducted with MorphoJ v1.06d (Klingenberg, 2011). Differences in sperm head shape between young and old individuals were quantified by examining the distance between the mean of both groups by conducting a discriminant analysis (Timm, 2002). RESULTS The number of spermatozoa stored in the cauda epididymides differed between species, following the predicted pattern (two-way ANCOVA: F2,26 = 50.36, P < 0.0001; Table S1; Supporting Information), but there was no difference between young and old males (F1,26 = 0.4, P = 0.54). The overall sperm morphology differed between species (F2,24 = 168.42, P > 0.0001) and between young and old males (F1,24 = 6.15, P = 0.02), but there was no significant interaction between species and age (F2,24 = 0.1, P = 0.91). The shape of the sperm head differed between species (discriminant analyses: P < 0.0001), but not between young and old mice (P = 0.2; Fig. 1). Figure 1. View largeDownload slide Sperm head shape in young and old males of three Mus species. Dots indicate the landmarks used for geometric morphometrics analyses. Figure 1. View largeDownload slide Sperm head shape in young and old males of three Mus species. Dots indicate the landmarks used for geometric morphometrics analyses. The percentage of normal spermatozoa differed between species (F2,27 = 7.44, P = 0.003); even though there was not an overall difference between young and old males (F1,27 = 0.99, P = 0.33), we found a statistically significant interaction between species and age (F2,27 = 4.07, P = 0.029). Subanalyses by species showed no differences between young and old mice in M. spicilegus and M. spretus (P > 0.05), but a higher percentage of normal spermatozoa in young males than in old males in M. musculus (P = 0.008; Fig. 2), the species with the lowest level of sperm competition. Figure 2. View largeDownload slide Percentage of morphologically normal spermatozoa in young and old males of three Mus species. For each boxplot, the bar within each box represents the sample median, each box represents 50% of the data around the median, and the two whiskers represent the 95% confidence interval. **P < 0.001; NS denotes P > 0.05. Figure 2. View largeDownload slide Percentage of morphologically normal spermatozoa in young and old males of three Mus species. For each boxplot, the bar within each box represents the sample median, each box represents 50% of the data around the median, and the two whiskers represent the 95% confidence interval. **P < 0.001; NS denotes P > 0.05. There were differences in sperm motility across species (two-way ANCOVA: F2,26 = 36.47, P < 0.0001), with this trait being higher in young males than in old males (F1,26 = 7.25, P = 0.01). However, all species were affected similarly by age (interaction: F2,26 = 0.1, P = 0.9). After 3 h of incubation, significant differences among species remained in sperm motility (F2,26 = 25.05, P < 0.0001), but there were no longer differences between age classes (F1,26 = 0.41, P = 0.53). Sperm forward progression also differed between species, both at 0 h (F2,26 = 6.22, P = 0.006) and after 3 h of incubation (F2,26 = 17.71, P < 0.0001), and although it was similar in young and old males at 0 h (F1,26 = 0.25, P = 0.62), after 3 h of incubation it was higher in old males than in young males (F1,26 = 7.38, P = 0.01). Overall sperm velocity and overall trajectory shape differed between species (P < 0.0001 in both analyses) and ages (P < 0.01), but there was not a significant interaction between species and age (P > 0.05). There were significant differences between species in the amount of ATP per sperm cell, both at 0 h (F2,26 = 6.85, P = 0.004) and after 3 h of incubation (F2,26 = 9.59, P = 0.0008; Table S1; Supporting Information). However, there was not a significant difference in ATP concentration between young and old males, nor a significant interaction between species and age at either time (P > 0.05 for all analyses). DISCUSSION Overall, our results do not support the hypothesis that males in species with high levels of sperm competition suffer less reproductive senescence than in species with low levels of sperm competition. We found that many reproductive traits were unaffected by age, whereas others were either enhanced or lessened in old males compared with young males, but the level of sperm competition did not have an influence in most of these traits. Only the percentage of normal spermatozoa matched our prediction, with a decline in old males in M. musculus (i.e. the species with low levels of sperm competition), but not in the other two species, which experience higher levels of sperm competition (M. spretus and M. spicilegus). This result may be driven by an enhanced process of spermatogenesis in species with high levels of sperm competition, which would either directly or indirectly minimize the occurrence of sperm abnormalities in old males, but this is an area of research that requires further investigation. Even though we considered many reproductive traits that could be affected by senescence, there are many other traits that could be affected in a different manner in young and old males of a species, depending on the level of sperm competition normally experienced in that species. These traits include chromosomal abnormalities, DNA damage in spermatozoa and any traits that regulate or determine the success of the capacitation and fertilization processes (Gogol, Bochenek & Smorag, 2002; Momand, Xu & Walter, 2013). For example, in brown rats, the spermatozoa of old males are more susceptible to oxidative damage and DNA fragmentation (Zubkova, Wade & Robaire, 2005), as well as having a decreased antioxidant capacity and an increased production of reactive oxygen species (Weir & Robaire, 2006). It is important to notice that the genomic damage in spermatozoa driven by ageing may be independent of sperm function. Despite a normal expression of sperm function in old males, any genomic damage in their spermatozoa will increase the risk of transmission of multiple genetic and chromosomal defects to offspring (Wyrobek et al., 2006). Surprisingly, we did not find a strong signal of reproductive senescence in the three species of mice that we studied. Reproductive senescence may thus not play an important role in the natural populations of the three Mus species under study. Indeed, given the high predation rates suffered by rodents, most males will normally die before the inception of any signs of senescence. Another study using wild-captured Mus musculus domesticus found that epididymal sperm counts declined with age, although only a range of relatively advanced ages (21–32 months) were studied (Garratt et al., 2011). It must be noted that most of the available knowledge on reproductive senescence in rodents is based on laboratory strains (Parkening, 1989; Katz-Jaffe et al., 2013; Lucio et al., 2013). This may be a shortcoming, as in a benign captive environment the negative effects of ageing can be minimized and thus differences between age classes might be obscured. Even though there are many studies describing the timing and incidence of reproductive senescence, there are also many studies in which reproductive condition remains unchanged or is even enhanced in old individuals (Gasparini et al., 2010; Kanuga et al., 2011; Johnson & Gemmell, 2012). It is still unclear why reproductive senescence is pronounced in some species but not in others. It is important to consider whether any differences between young and old males lead to a fertilizing advantage for one age class or the other. For example, young male guppies produce faster swimming spermatozoa compared with old males; however, young males do not have a fertilizing advantage in sperm competition scenarios (Gasparini et al., 2010). It is equally possible that despite a lack of consistent differences in sperm function between young and old males, as we found in our three species of mice, undetected differences between their spermatozoa could result in a lower reproductive potential in older individuals. We can thus conclude that even though our results did not support our hypothesis that high levels of sperm competition can minimize the impacts of senescence, more reproductive measurements, including sperm competition tests and measurements of fertilizing ability and offspring health, and possibly higher sample sizes than we used, are required to support or disprove our hypothesis completely. SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Table S1. Reproductive parameters in young and old males from three Mus species with different levels of sperm competition. Methods.pdf. Specific methods. ACKNOWLEDGEMENTS This work was supported by a Ramón y Cajal fellowship from the Spanish Ministry of Economy and Competitiveness (RYC-2011-07943) and a Marie Curie Career Integration Grant (PCIG11-GA-2012-321888) to J.d.-T., a Juan de la Cierva fellowship from the Spanish Ministry of Economy and Competitiveness to M.T. (JCI-2011-10381) and grants from the Spanish Ministry of Economy and Competitiveness (CGL2011-26341 and CGL2016-80577-P to E.R.S.R. and CGL2012-37423 to J.d.-T.). We are grateful to Annie Orth and François Bonhomme (Institut des Sciences de l’Evolution de Montpellier, CNRS-Université Montpellier 2, France) for facilitating access to animals, and Juan Antonio Rielo for managing the animal facilities and Esperanza Navarro for animal care at the Museo Nacional de Ciencias Naturales (CSIC). We thank Alberto Vicens, Pilar Villar and Ester Sansegundo for their help with processing spermatozoa in the laboratory. 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