Copulatory plugs inhibit the reproductive success of rival males.

Ejaculated proteins play important roles in reproductive fitness. In many species, seminal fluid coagulates and forms what has been referred to as a copulatory plug in the female's reproductive tract. In mice, previous work demonstrated that knockout males missing a key seminal fluid protein were unable to form a plug and less successful at siring litters in noncompetitive matings (one female, one male), probably the result of reduced sperm transport or insufficient stimulation of the female. Here, we extend these previous studies to competitive matings (one female, two males) and make two key insights. First, when first males were unable to form a plug, they lost almost all paternity to second males to mate. Thus, the copulatory plugs of second males could not rescue the reduced fertility of first males. Second, we showed that the copulatory plug of first males effectively blocked fertilization by second males, even if first males were vasectomized. Taken together, our experiments demonstrated that first males lost almost all paternity if they never formed a plug. We discuss our results in the context of natural populations, where in spite of the strong effects seen here, pregnant female mice regularly carry litters fertilized by more than one male.

Mangels R ，Tsung K ，Kwan K ，Dean MD ... -  《-》

The copulatory plug delays ejaculation by rival males and affects sperm competition outcome in house mice.

Females of many species mate with multiple males (polyandry), resulting in male-male competition extending to post-copulation (sperm competition). Males adapt to such post-copulatory sexual selection by altering features of their ejaculate that increase its competitiveness and/or by decreasing the risk of sperm competition through female manipulation or interference with rival male behaviour. At ejaculation, males of many species deposit copulatory plugs, which are commonly interpreted as a male adaptation to post-copulatory competition and are thought to reduce or delay female remating. Here, we used a vertebrate model species, the house mouse, to study the consequences of copulatory plugs for post-copulatory competition. We experimentally manipulated plugs after a female's first mating and investigated the consequences for rival male behaviour and paternity outcome. We found that even intact copulatory plugs were ineffective at preventing female remating, but that plugs influenced the rival male copulatory behaviour. Rivals facing intact copulatory plugs performed more but shorter copulations and ejaculated later than when the plug had been fully or partially removed. This suggests that the copulatory plug represents a considerable physical barrier to rival males. The paternity share of first males increased with a longer delay between the first and second males' ejaculations, indicative of fitness consequences of copulatory plugs. However, when males provided little copulatory stimulation, the incidence of pregnancy failure increased, representing a potential benefit of intense and repeated copulation besides plug removal. We discuss the potential mechanisms of how plugs influence sperm competition outcome and consequences for male copulatory behaviour.

Sutter A ，Lindholm AK 《-》

Seminal vesicle secretory protein 7, PATE4, is not required for sperm function but for copulatory plug formation to ensure fecundity†.

Seminal vesicle secretions (SVSs), together with spermatozoa, are ejaculated into the female reproductive tract. SVS7, also known as PATE4, is one of the major SVS proteins found in the seminal vesicle, copulatory plug, and uterine fluid after copulation. Here, we generated Pate4 knockout (-/-) mice and examined the detailed function of PATE4 on male fecundity. The morphology and weight of Pate4-/- seminal vesicles were comparable to the control. Although Pate4-/- cauda epididymal spermatozoa have no overt defects during in vitro fertilization, Pate4-/- males were subfertile. We found that the copulatory plugs were smaller in the vagina of females mated with Pate4-/- males, leading to semen leakage and a decreased sperm count in the uterus. When the females mated with Pate4-/- males were immediately re-caged with Pate4+/+ males, the females had subsequent productive matings. When the cauda epididymal spermatozoa were injected into the uterus and plugged artificially [artificial insemination (AI)], Pate4-/- spermatozoa could efficiently fertilize eggs as compared to wild-type spermatozoa. We finally examined the effect of SVSs on AI, and observed no difference in fertilization rates between Pate4+/+ and Pate4-/- SVSs. In conclusion, PATE4 is a novel factor in forming the copulatory plug that inhibits sequential matings and maintains spermatozoa in the uterus to ensure male fecundity.

Noda T ，Fujihara Y ，Matsumura T ，Oura S ，Kobayashi S ，Ikawa M ... -  《-》

The baculum affects paternity success of first but not second males in house mouse sperm competition.

The vast variation observed in genital morphology is a longstanding puzzle in evolutionary biology. Studies showing that the morphology of the mammalian baculum (penis bone) can covary with a male's paternity success indicate a potential impact of baculum morphology on male fitness, likely through influencing sperm competition outcomes. We therefore measured the size (measurements of length and width) and shape (geometric morphometric measurements) of the bacula of male house mice used in previously published sperm competition experiments, in which two males mated successively with the same female in staged matings. This enabled us to correlate baculum morphology with sperm competition success, incorporating potential explanatory variables related to copulatory plugs, male mating behavior and a selfish genetic element that influences sperm motility. We found that a wider baculum shaft increased a male's paternity share when mating first, but not when mating second with a multiply-mating female. Geometric morphometric shape measurements were not clearly associated with fertilization success for either male. We found limited evidence that the effect of baculum morphology on male fertilization success was altered by experimental removal of the copulatory plug. Furthermore, neither genetic differences in sperm motility, nor covariation with male mating behavior mediated the effect of baculum morphology on male fertilization success. Taken together with previous findings, the mating-order effects we found here suggest that baculum-mediated stimulation by the first male might be particularly important for fertilization.

Winkler L ，Lindholm AK ，Ramm SA ，Sutter A ... -  《-》

An integrative view of sexual selection in Tribolium flour beetles.

Sexual selection is a major force driving the evolution of diverse reproductive traits. This evolutionary process is based on individual reproductive advantages that arise either through intrasexual competition or through intersexual choice and conflict. While classical studies of sexual selection focused mainly on differences in male mating success, more recent work has focused on the differences in paternity share that may arise through sperm competition or cryptic female choice whenever females mate with multiple males. Thus, an integrative view of sexual selection needs to encompass processes that occur not only before copulation (pre-mating), but also during copulation (peri-mating), as well as after copulation (post-mating), all of which can generate differences in reproductive success. By encompassing mechanisms of sexual selection across all of these sequential reproductive stages this review takes an integrative approach to sexual selection in Tribolium flour beetles (Coleoptera: Tenebrionidae), a particularly well-studied and economically important model organism. Tribolium flour beetles colonize patchily distributed grain stores, and juvenile and adult stages share the same food resources. Adults are highly promiscuous and female reproduction is distributed across an adult lifespan lasting approximately 1 year. While Tribolium males produce an aggregation pheromone that attracts both sexes, there appears to be little pre-mating discrimination among potential mates by either sex. However, recent work has revealed several peri-mating and post-mating mechanisms that determine how offspring paternity is apportioned among a female's mates. During mating, Tribolium females reject spermatophore transfer and limit sperm numbers transferred by males with low phenotypic quality. Although there is some conflicting evidence, male copulatory leg-rubbing appears to be associated with overcoming female resistance to insemination and does not influence a male's subsequent paternity share. Evidence suggests that Tribolium beetles have several possible post-mating mechanisms that they may use to bias paternity. Male sperm precedence has been extensively studied in Tribolium spp. and the related Tenebrio molitor, and several factors influencing male paternity share among a female's progeny have been identified. These include oviposition time, inter-mating interval, male strain/genotype, the mating regimen of a male's mother, male starvation, and tapeworm infection. Females exert muscular control over sperm storage, although there is no evidence to date that females use this to differentiate among mates. Females could also influence offspring paternity by re-mating with additional males, and T. castaneum females more readily accept spermatophores when they are re-mating with more attractive males. Additional work is needed to examine the possible roles played by both male and female accessory gland products in determining male paternity share. Sexual selection during pre-mating episodes may be reinforced or counteracted by peri- and post-copulatory selection, and antagonistic coevolution between the sexes may be played out across reproductive stages. In Tribolium, males' olfactory attractiveness is positively correlated with both insemination success and paternity share, suggesting consistent selection across different reproductive stages. Similar studies across sequential reproductive stages are needed in other taxa to provide a more integrative view of sexual selection.

Fedina TY ，Lewis SM 《-》