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Proc Natl Acad Sci U S A. Oct 10, 2006; 103(41): 15113–15117.
Published online Sep 21, 2006. doi:  10.1073/pnas.0605795103
PMCID: PMC1570616
From the Cover
Evolution

Sperm competition enhances functional capacity of mammalian spermatozoa

Abstract

When females mate promiscuously, sperm from rival males compete within the female reproductive tract to fertilize ova. Sperm competition is a powerful selective force that has shaped sexual behavior, sperm production, and sperm morphology. However, nothing is known about the influence of sperm competition on fertilization-related processes, because it has been assumed that sperm competition only involves a race to reach the site of fertilization. We compared four closely related rodent species with different levels of sperm competition to examine whether there are differences in the proportion of spermatozoa that become ready to interact with the ovum (“capacitated”) and in the proportion of spermatozoa that experience the acrosome reaction in response to a natural stimulant. Our results show that differences between species in levels of sperm competition were associated with the proportion of spermatozoa that undergo capacitation and with the proportion of spermatozoa that respond to progesterone, an ovum-associated signal. Sperm competition thus favors a larger population of spermatozoa that are competent to fertilize, and spermatozoa that are more sensitive to the signals emitted by the ovum and that may penetrate the ova vestments more rapidly. These results suggest that, contrary to previous assumptions, competition between spermatozoa from rival males continues at the site of fertilization. These findings may have further evolutionary implications because the enhanced competitiveness of spermatozoa during fertilization may increase the risk of polyspermy to females. This could lead to antagonistic coevolution between the sexes and may contribute to the explanation of the rapid divergence observed in fertilization-related traits.

Keywords: sperm function, capacitation, acrosome reaction fertilization, antagonistic coevolution

Postcopulatory sexual selection occurs when females mate with more than one male, creating the potential for competition between rival ejaculates to fertilize the available ova (1) and for cryptic female choice (2). Among internal fertilizers, the features of the female reproductive tract set the rules of the competition and thus determine which ejaculate features will improve their competitiveness.

Comparative studies between species have shown that sperm competition has favored an increase in testes size and enhanced sperm production in taxa as diverse as mammals (reviewed in ref. 3), birds (4), butterflies (5), fishes (6), and amphibians (7). Microevolutionary manipulations have induced changes in testes size and sperm production by modifying the intensity of sperm competition (810), providing strong support for a causal relationship. There is ample evidence that in competitive contexts males with greater sperm numbers father more offspring (see reviews in ref. 11).

The competitiveness of an ejaculate is also largely determined by sperm motility and sperm swimming velocity. In birds, males with high sperm “mobility” (as measured by an in vitro motility assay) fathered the majority of offspring in sperm competition experiments (12), because sperm mobility determines the rate at which sperm are released from female storage sites (13). In the Atlantic salmon (Salmo salar), sperm velocity is the key determinant of sperm competition success (14).

Among mammals, evidence of longer spermatozoa in polyandrous species suggests that improved sperm swimming velocity under sperm competition could be achieved by an increase in sperm size (15). Sperm competition can also select for unique morphological traits that improve swimming velocity, as is the case in the male common wood mouse (Apodemus sylvaticus), which has spermatozoa with extremely long apical hooks by which they intertwine, forming “trains” of spermatozoa (16). These sperm associations swim nearly twice as fast as nonassociated sperm toward the site of fertilization. Evidence of an association between sperm competition and increased sperm size comes from diverse taxa [birds (4), butterflies (17), fish (18), and frogs (19)], although other studies have found no relationship (20) or an inverse relationship [fish (6)]. The finding that there is coevolution between the size of spermatozoa and the size of the female sperm storage organs (21) suggests that some inconsistencies may be due to the need to consider both male and female traits simultaneously. In groups with ameboid sperm, sperm competition does favor larger sperm (22, 23), which crawl faster and displace smaller sperm from the spermathecae (24). Experimental evidence shows that increased sperm competition leads to an increase in sperm size (25).

Sperm competition may also improve other aspects of ejaculate quality, such as an increase in the proportion of viable spermatozoa in the ejaculate (26). Experiments involving sperm competition trials have shown that, among insects, paternity success is determined by the proportion of live spermatozoa in a male's ejaculate (27).

Although there is ample evidence of the effects of sperm competition on sperm numbers and quality, the possibility that sperm competition has also influenced processes that take place around the time of fertilization has not been explored. Yet the race to fertilize the available ova does not only imply greater motility to reach the site of fertilization first. Mammalian spermatozoa need to undergo a process known as “capacitation” to be able to fertilize the ova (2830). Capacitation can take up to several hours, and indirect experimental evidence suggests that when spermatozoa are placed in direct competition, those that capacitate faster are more successful at fertilizing the ova (31). In addition, spermatozoa must experience the “acrosome reaction” to release the enzymes needed to penetrate the ova vestments and to be able to fuse with the ovum's plasma membrane (28, 32). The acrosome reaction must be carefully synchronized with the ovum, because sperm that undergo the acrosome reaction too early cannot penetrate the cumulus oophorus (33) or, once in the cumulus, cannot establish high-avidity adhesion to the zona pellucida (ZP) and thus fail to achieve fertilization (32). The acrosome reaction probably begins when the spermatozoon interacts with the progesterone secreted by the cumulus oophorus (or trapped in the cumulus matrix), resulting in the activation of some initial molecular signaling events. The acrosome reaction is then completed on the surface of the ZP in response to ZP glycoprotein(s) that elicit further sperm signaling processes ending in fusion of the sperm plasma membrane with the underlying outer acrosomal membrane (34).

When spermatozoa compete with rival ejaculates, males that have a higher proportion of spermatozoa ready to interact with the ovum, and whose spermatozoa respond more efficiently to the signals emitted by the ova, will be more likely to win the final stages of the competition. In this study, we test these predictions by comparing four species of rodents with different levels of sperm competition.

Results

We have examined four species of murid rodents (Mus musculus, Mus pahari, Mus spicilegus, and Mus spretus) for which we have measured relative testes weight, a reliable indicator of the level of sperm competition. To place this group of related species in a broader context, we show in Fig. 1 that when we compare 31 murid rodent species for which data are available (ref. 35 and our unpublished results), both M. spicilegus and M. spretus have large testes in relation to their body size, with M. spicilegus showing higher values in terms of relative testes size. Both M. musculus and M. pahari have small testes in relation to their body size, with M. pahari having considerably smaller testes. Thus, by choosing these four species, we have been able to cover a broad range including species with high, high-intermediate, low-intermediate, and low levels of sperm competition to compare sperm function and the degree of sensitivity in response to signals released by the ovum.

Fig. 1.
Relation between body weight and testes weight in murid rodents (r2 = 0.4587, n = 31, P < 0.0001). Filled circles: Apodemus agrarius, Apodemus flavicollis, Apodemus microps, A. sylvaticus, Micromys minutus, Mus bactrianus, Mus castaneus, Mus cookii ...

For the four species examined, we have calculated differences in relative testes weight and in the relative number of spermatozoa produced (Table 1). Of the four species examined, Mus spicilegus was found to have the highest relative testes weight, followed by M. spretus, M. musculus, and, finally, M. pahari with the lowest values, being the differences in relative testes weight between these species statistically significant (ANOVA, F3,12 = 1987.77, P < 0.0001; all post hoc comparisons between pairs of species, P < 0.0001) (Fig. 2a). The differences in relative testes weight were clearly associated with differences between species in the rate of sperm production, so that it was more than 10 times greater in M. spicilegus than in M. pahari, the two species with extreme values for both traits. Differences between species in relative number of spermatozoa were also statistically significant (ANOVA, F3,12 = 110.37, P < 0.0001; all post hoc comparisons between pairs of species, P < 0.002).

Table 1.
Relative testes weight and relative number of spermatozoa in four species of murid rodents
Fig. 2.
Relative testes size, proportion of capacitated spermatozoa, and acrosome reaction in response to progesterone in four murid species. (a) Relative testes size. (b) Proportion of spermatozoa showing the “B” pattern, indicative of capacitated, ...

To test whether capacitation and the acrosome reaction differ between species, we incubated spermatozoa under capacitating conditions [in a bicarbonate/CO2-buffered modified Tyrode's medium (mT-B25) under 5% CO2/air at 37°C] and evaluated chlortetracycline (CTC) staining patterns (which are indicative of functional changes) at different time points (36). At the point of maximum capacitation, the four species differed in the proportion of sperm that were capacitated (“B” pattern), and these differences were associated with the level of sperm competition for each species. Thus, the species with low levels of sperm competition (M. pahari) had only ≈30% capacitated spermatozoa, and this proportion increased for M. musculus, followed by M. spretus, and was highest for M. spicilegus (>60% of capacitated spermatozoa) (Fig. 2b) (ANOVA, F3,8 = 24.119, P = 0.0002; all post hoc comparisons between pairs of species, P < 0.02). This finding implies that as sperm competition increases, so does the population of spermatozoa that are competent to interact with the ovum.

To test how efficiently spermatozoa respond to signals from the ovum, we measured the response to stimulation with progesterone, a physiological inducer of the acrosome reaction (34). Spermatozoa from the four species were incubated until they reached the time point of maximal capacitation (as revealed in the previous experiment) and were then exposed to progesterone or to its solvent as control. As shown in Fig. 2c, the response in acrosome reactions over the background levels of acrosome loss seen in untreated, control spermatozoa differed between the four species. Spermatozoa were increasingly responsive to progesterone as the levels of sperm competition increased, and the differences between the four species were statistically significant (ANOVA, F3,11 = 19.04, P = 0.0001; all post hoc comparisons between pairs of species, P < 0.025).

To avoid the possibility that the proportion of spermatozoa that experience the acrosome reaction in response to progesterone is merely a consequence of the proportion of spermatozoa that become capacitated, we calculated the proportion of spermatozoa that underwent the acrosome reaction in relation to the proportion of capacitated spermatozoa (Fig. 2d). We found that, as sperm competition increases, so does the proportion of spermatozoa that experience the acrosome reaction in relation to the proportion of capacitated spermatozoa (ANOVA, F3,11 = 19.6737, P < 0.0001; all post hoc comparisons between pairs of species, P < 0.035, with the exception of M. musculus versus M. spretus, which is nonsignificant).

Discussion

Our findings show that sperm competition influences sperm function by increasing the proportion of the sperm population that undergoes capacitation. In addition, sperm competition selects for spermatozoa that are more sensitive to the signals released by the ovum and undergo the acrosome reaction during fertilization. Thus, sperm competition selects a larger population of spermatozoa that are competent to fertilize the ovum. These results show that sperm competition influences individual spermatozoa beyond the morphological and structural level, shaping biochemical and cellular processes that are crucial for fertilization success.

Indirect evidence relating the speed of attachment to and penetration of the ovum by spermatozoa to fertilization success in competitive contexts comes from studies of heterospermic insemination which showed that the time of insemination relative to ovulation was crucial for the success of certain types of males (reviewed in ref. 37). Eutherian spermatozoa are unique in that they need to become capacitated before they can fertilize the ova, and this process can take several hours (28). Thus, spermatozoa that capacitate rapidly will be ready to fertilize before rival spermatozoa; but if mating has taken place too early before ovulation, they will lose their fertilizing capacity before the ova are ready for fertilization (38). This cost of producing short-lived sperm has probably contributed to the evolution of multiple copulation in polyandrous mammals (3). When there is sperm competition, males tend to ejaculate many times, and in this way they ensure that the female tract has sperm populations ready to fertilize at different times, thus maximizing the chances that spermatozoa will be ready when ovulation occurs. In addition, ejaculates contain subpopulations of spermatozoa that behave differently in terms of timing of capacitation (30), and this could be a means to ensure that when ovulation occurs, there will be a proportion of spermatozoa ready to fertilize. Using aggregation chimeras, Krzanowska (31) found that when spermatozoa from two strains were placed in competition, spermatozoa from one strain were more successful, but this advantage decreased if females were inseminated several hours before ovulation, suggesting that more rapid capacitation was responsible for the competitive advantage of spermatozoa. Our findings provide direct evidence suggesting that sperm competition favors an increase in the proportion of spermatozoa that undergo capacitation, thus generating a larger population of spermatozoa ready to interact with the ovum.

Once the spermatozoon has become capacitated, but still with its acrosome intact, it penetrates the cumulus oophorus and reaches the surface of the extracellular coat, or zona pellucida (ZP), of the ovum (39). Spermatozoa must undergo the acrosome reaction that probably begins (or is primed) when the spermatozoon interacts with the progesterone secreted by or present in the cumulus oophorus (34). Although progesterone may prime the spermatozoon (initiating a series of early molecular events), it is possible that the sperm cell would bind to the zona surface with its plasma membrane still intact to allow for sperm–ovum recognition. The subsequent action of the ZP glycoprotein ZP3 triggers further sperm intracellular signaling events ending in membrane fusion and the release of enzymes contained in the acrosome.

Our results show that sperm competition selects a higher proportion of spermatozoa that undergo the acrosome reaction and spermatozoa that are more sensitive to the signals emitted by the ovum. The importance of the competitiveness of spermatozoa at this stage of the race has been largely overlooked, but it is a crucial determinant of fertilization success. Until the spermatozoon penetrates the ovum's ZP and fuses with the plasma membrane, further sperm that may have penetrated the ZP are not prevented from fusing with the plasma membrane; and only when the ovum is fertilized are free-swimming spermatozoa no longer able to bind to the ZP (39). Thus, the ability to respond efficiently to the signals released by the ovum when it is ready for fertilization, and the speed of attachment and penetration of the ova vestments, will be the ultimate determinants of a male's success in sperm competition. Thus, competition between rival ejaculates continues until the point of fertilization.

The influence of sperm competition in favoring a larger population of competent spermatozoa and in selecting spermatozoa that respond more efficiently to the ovum may have a cost for females in terms of increased chances of polyspermy, which results in nonviable embryos (40). Recent models have suggested that, under most conditions of sperm competition, selection favors maximal speed of penetration by spermatozoa despite the significant mortality imposed on both spermatozoa and ova (41). As ejaculates become more efficient, females may respond to the increased risk of polyspermy by increasing and diversifying the vestments that protect the ovum. This conflict of interests between the sexes would lead to antagonistic coevolution, a phenomenon that has been proposed to explain the high level of divergence found in female and sperm proteins involved in fertilization among mammals (42). The proposed link between conflicts between competitive males and defensive females at the site of fertilization and divergence in fertilization-related proteins deserves further study.

Materials and Methods

Animals and Phenotypic Traits.

Adult males (12–20 weeks old) of four species of murid rodents (M. musculus, M. pahari, M. spicilegus, and M. spretus) were purchased from the Laboratoire Génome et Populations of the Université de Montpellier II (Montpellier, France) and kept in individual cages under standard laboratory mouse conditions in an environmentally controlled room on a 14 h light:10 h darkness photoperiod. Animals were provided with food and water ad libitum. Animals (n = 5 for each species) were killed by cervical dislocation and weighed. Testes were removed and weighed. Spermatozoa were collected from epididymides and vasa deferentia into a Hepes-buffered modified Tyrode's medium (mT-H) (36). The sperm concentration in the resulting suspension was estimated by using a hemocytometer, and the total number of spermatozoa was calculated.

Collection, Incubation, and Treatment of Spermatozoa.

Spermatozoa from epididymides and vasa deferentia from a different set of males of the four species (n = 3 for each species) were released into a bicarbonate/CO2-buffered modified Tyrode's medium (mT-B25) (36). Sperm suspensions (concentration of 3–5 × 107 cells per ml) were placed in 35-mm plastic culture dishes (Falcon; Becton-Dickinson, Madrid, Spain), covered with mineral oil (Sigma, Madrid, Spain), and incubated at 37°C under 5%CO2/air for up to 150 min. Subsamples were taken every 30 min until the end of incubation, and spermatozoa were stained with CTC to assess their functional status (see below). There were no differences between species in the proportion of live spermatozoa during incubation, as assessed by staining with Hoechst 33258 (see below).

For the induction of acrosome reactions, spermatozoa were preincubated in mT-B25 until the time point of maximum capacitation, as established in the previous experimental series; exposed to 15 μM progesterone or its solvent (DMSO; maximal concentration of 1%) as control (34, 36); and further incubated for 30 min at 37°C under 5%CO2/air. The concentrations of progesterone or DMSO did not affect motility or cell viability (as assessed by staining spermatozoa with Hoechst 33258; see below). At the end of the incubations, subsamples were stained with CTC to assess sperm functional status (see below). To calculate the proportion of cells that responded to progesterone stimulation, values of spontaneous acrosome reactions in controls were subtracted from values of spermatozoa without acrosomes after exposure to progesterone.

Assessment of Capacitation and the Acrosome Reaction.

For the assessment of capacitation, spermatozoa were stained with CTC. Staining with CTC was carried out as described previously (43, 44) but with some modifications (36). The CTC staining solution was prepared by dissolving CTC-HCl at a concentration of 250 μM in TN buffer (20 mM Tris/130 mM NaCl) with 5 mM cysteine (pH 7.8). The CTC solution was wrapped with foil and kept on ice until use; fresh CTC stock was prepared daily. Before use, aliquots of 20 μl of CTC solution were placed in Eppendorf tubes, prewarmed to 37°C in a heating block, and kept in the dark. Sperm suspensions (20 μl) were added, and this was followed immediately by the addition of 3.5 μl of 12.5% glutaraldehyde in 1.25 M Tris buffer (pH 7.8) and gentle mixing. Fixed samples were kept in a dark box. Slides were prepared 1–3 h after fixation and examined at ×1,000 magnification under phase-contrast and fluorescence microscopy, using for the latter an mercury excitation beam passed through a 405-nm filter and fluorescence emission with a DM 455 dichroic mirror. A minimum of 100 spermatozoa were counted to assess the different CTC staining patterns: F, characteristic of uncapacitated, acrosome-intact spermatozoa; B, representing capacitated, acrosome-intact spermatozoa; and AR, corresponding to spermatozoa that have undergone acrosomal exocytosis.

For the assessment of acrosome reactions, spermatozoa were also stained with a vital stain, to discriminate live and dead cells, in addition to staining with CTC to assess their acrosomal status. Briefly, sperm subsamples (20 μl) were fixed in an equal volume of prewarmed 2% glutaraldehyde in 0.165 M cacodylate/HCl buffer. They were then stained with CTC solution, prepared as described above, by diluting 20 μl of fixed sperm suspension with 20 μl of 250 μM CTC solution and incubating in the dark for 5 min. Spermatozoa were then stained with a Hoechst 33258 solution (concentration of 6 μg/ml) prepared in mT-H medium without BSA and CaCl2. For staining, 8 μl of the Hoechst solution was added to fixed sperm samples stained with CTC (final Hoechst 33258 concentration of 1 μg/ml). Samples were incubated in the dark for 3 min and immediately examined at ×1,000 by using phase-contrast and fluorescence microscopy. For CTC, we used a mercury excitation beam passed through a 405-nm filter and fluorescence emission with a DM 455 dichroic mirror (Nikon BV-2A filter). For Hoechst 33258, we used a 330-nm filter and fluorescence emission via a DM 400 dichroic mirror (Nikon UV-2A filter). A minimum of 100 spermatozoa were counted per sample, discriminating dead spermatozoa (those staining with Hoechst 33258) and, among those that did not stain with Hoechst 33258 (i.e., live spermatozoa), the three patterns of CTC staining described above: F, uncapacitated; B, capacitated, acrosome-intact; and AR, acrosome-reacted spermatozoa. Thus, functional status was assessed among live spermatozoa.

Variables were log-transformed or arcsine-transformed to fulfill parametric assumptions. Differences between species were tested by using ANOVA.

Acknowledgments

We thank Ana del Olmo for help with experiments and Marili Calduch for help with mouse husbandry. Funding was provided by the Spanish Ministry of Science and Technology and by the Ministry of Education and Science. J.M.C. is supported by a studentship from the Ministry of Science and Education, and C.C. is supported by a studentship from the I3P-CSIC Program.

Abbreviations

CTC
chlortetracycline
ZP
zona pellucida.

Footnotes

The authors declare no conflict of interest.

This paper was submitted directly (Track II) to the PNAS office.

See Commentary on page 14983.

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