Copulatory plugs inhibit the reproductive success of rival males

Abstract

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 non-competitive 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 blocks fertilization by second males, even if first males are vasectomized. Taken together, our experiments demonstrate that first males lose almost all paternity if they never form 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.

Introduction

Characterizing the function of ejaculated proteins is critical to a comprehensive understanding of reproductive health and evolutionary fitness. In many sexually reproducing organisms, ejaculated proteins affect fertilization (Poiani, 2006, Price et al., 1999, Wigby et al., 2009), through a variety of functions that includes the balancing of pH (Arienti et al., 1999, Asari et al., 1996), protection and nutrition of sperm (Chen et al., 2002, Wai-Sum et al., 2006, Kawano et al., 2014), modification of sperm physiology (Zhu et al., 2006, Kawano & Yoshida, 2007, Peitz, 1988), and the induction of behavioral modifications in females (Wolfner, 2002, Chen et al., 1988, Heifetz et al., 2000).

A male's ejaculate coagulates to form a copulatory plug inside the female reproductive tract in many different taxa, including nematodes (Abele & Gilchrist, 1977, Barker, 1994, Palopoli et al., 2008), arachnids (Austad, 1984, Masumoto, 1993, Uhl et al., 2010), insects (Rogers et al., 2009, Orr & Rutowski, 1991, Parker, 1970, Dickinson & Rutowski, 1989), reptiles (Devine, 1975, Herman, 1994, Friesen et al., 2013), and mammals (Devine, 1975, Martan & Shepherd, 1976, Voss, 1979, Williams-Ashman, 1984, Dixson & Anderson, 2002). In rodents, this coagulation occurs when a prostate-derived transglutaminase, encoded by the gene transglutaminase IV (Tgm4), crosslinks seminal vesicle-derived secretions upon mixing in the female's reproductive tract (Notides & Williams-Ashman, 1967, Williams-Ashman et al., 1972, Kawano et al., 2014, Dean, 2013, Esposito et al., 1996, Fawell & Higgins, 1987, Lin et al., 2002, Lundwall, 1996, Lundwall et al., 1997, Tseng et al., 2008, Tseng et al., 2009).

Although the copulatory plug has been characterized in a diversity of taxa, its function remains incompletely understood. Schneider et al. (in review) recently reviewed experiments aimed at characterizing plug function, which generally fall into one of two categories. One category includes experimental removal or reduction of plugs soon after formation; these experiments generally demonstrate that first males lose paternity to second males when their plugs are compromised (Martan & Shepherd, 1976, Dickinson & Rutowski, 1989, Sutter & Lindholm, 2016, Sutter et al., 2016). Consistent with an interpretation that plugs evolved to block fertilization of competitor males, plugs are more prominent in species inferred to have higher levels of sperm competition (Hartung & Dewsbury, 1978, Dixson & Anderson, 2002) and in such species males develop relatively large seminal vesicles (Ramm et al., 2005). In gorillas, male dominance reduces the risk of sperm competition, and males have lost the ability to make plugs (Dixson, 1998, Jensen-Seaman & Li, 2003, Carnahan & Jensen-Seaman, 2008, Kingan et al., 2003). However, plugs do not always inhibit paternity of second males (Dewsbury, 1988), many plug-forming species show evidence of insemination by multiple males (Dean et al., 2006, Firman & Simmons, 2008, Searle, 1990, Birdsall & Nash, 1973, Eberle & Kappeler, 2004, Schwartz et al., 1989), and some monogamous species still form a copulatory plug (Ribble, 1991, Baumgardner et al., 1982, Gubernick, 1988, Foltz, 1981), suggesting the plug functions outside the context of mate-guarding.

Another category of studies relied upon surgical removal of accessory glands required for normal formation of copulatory plugs; these experiments generally demonstrate that plugs promote migration of the ejaculate through the female's reproductive tract and are necessary for normal fertility (Carballada & Esponda, 1992, Blandau, 1945a, Blandau, 1945b, Pang et al., 1979, Peitz, 1988, Lawlah, 1930, Cukierski et al., 1991). However, removal of accessory glands is likely to alter many features of the ejaculate, not just the ability of a male to form a plug.

The modern genetics era has enabled a third category of less invasive and more powerful approaches to be applied to studies of copulatory plugs (Dean, 2013, Kawano et al., 2014). Male mice missing a functional copy of transglumatinase IV (Tgm4) fail to form a copulatory plug but showed normal gross morphology, reproductive anatomy, sperm count, and sperm motility (Dean, 2013). In non-competitive matings, Tgm4 knockout males sired a litter in about 57% of crosses, compared to 82% of successful litters born to females mated to wild type males. This sub-fertility was correlated with a reduction in the amount of ejaculate that migrated past the cervix and into the uterus and oviducts (Dean, 2013). In spite of this reduced ejaculate migration, Tgm4 knockout males still fertilized an average of 6 oocytes, roughly equivalent to the average litter size sired by wild type males. This pattern suggested that the fertility defects of Tgm4 knockout males arise after fertilization, for example if their embryos were less likely to implant. Therefore, it remains unknown whether Tgm4 knockout males also show reduced fertility under competitive matings, where one female mates to two males in succesion. One reason they might show normal fertility is if the plug of the second male somehow rescues fertility, for example if second males provide copulatory stimulation required by the female to accept implantation of embryos.

Here we perform two main experiments to better understand the function of the copulatory plug. In Experiment 1, we showed that when first males could not form a copulatory plug, they lost almost all paternity to second males to mate. In contrast, when first males could form a plug, they fertilized almost all embryos. Thus, second males are able to fertilize oocytes before the first male when the first male fails to form a plug. In Experiment 2, we vasectomized first males to mate, and show that if first males could form a plug, they completely blocked second male paternity even though they contributed no sperm. Our study provides direct and non-invasive quantification that the plug biases paternity towards first males to mate, and this bias is not simply due to reduced sperm transport when first males cannot form a plug. We discuss these results in the context of natural mating ecology of house mice, where multiply inseminated females are surprisingly common.

Material and Methods

Study Organisms

All husbandry and experimental methods, as well as all personnel involved were approved by the University of Southern California's Institute for Animal Care and Use Committee, protocols #11394 and #11777.

Four strains of mice were used in this study: 1) wildtype males of the C57BL/6N strain (6N wildtype), 2) Tgm4 knockout (6N knockout) mice acquired from the Knockout Mouse Project (Austin et al., 2004, Testa et al., 2004), which are genetically identical to 6N wildtype except for a ~7kb “knockout first” cassette that spans exons 2-3 of Tgm4, 3) a closely related strain, C57BL/10J (10J), acquired from Jackson Labs (Bar Harbor, Maine), and 4) the highly fecund FVB/NJ (FVB).

We employed a serial mating design where one female mated in succession to two males. The first male to mate was always either a 6N wildtype male, which can form a copulatory plug, or a 6N knockout male, which cannot. The second male to mate was always a 10J male, and the females were always FVB. The second male (10J) was chosen because it is genetically similar to the first male (6N knockout or 6N wildtype), thus minimizing the Bruce effect, which occurs when females block implantation upon exposure to genetically distinct males (Bruce, 1960). We were still able to determine paternity since 10J is genetically distinguishable from 6N (McClive et al., 1994, Keane et al., 2011).

To breed experimental mice, sire and dam were paired for two weeks, then separated so that the dam could give birth in isolation. Males and females were weaned at 21-28 days postpartum. Females were weaned with up to three individuals per cage and were used in experiments at 6-8 weeks of age. Males were housed singly to avoid dominance interactions (Snyder, 1967), and used in experiments at 60-90 days of age. The colony was kept at 14:10 hours of dark:light and provided food ad libitum.

Experiment 1: Serial Mating

At 6-8 weeks of age, individual FVB virgin female mice were induced into estrus with an intraperitoneal injection of 5 U Pregnant Male Serum Gonadotropin (PMSG), followed approximately 48 hours later with an intraperitoneal injection of 5 U Human Chorionic Gonadotropin (hCG), which induces ovulation approximately 12 hours later (Nagy et al., 2003). Approximately 14 hours after the hCG injection, each female was placed in a randomly assigned male cage with either a 6N knockout or 6N wildtype for four hours. In vitro fertilization occurs within about four hours (Dean & Nachman, 2009), and in vivo fertilization is likely to take longer given the addition of behavioral and physiological interactions between male and female. Therefore, females were kept with first males for four hours, then removed and placed in a 10J male's cage for another four hours. When 6N wildtype males were first to mate, successful mating of the first male could be scored by visual inspection for a copulatory plug. Successful mating of the second male could not be confirmed because any plug deposited by the second male would be indistinguishable from the first. We note, however, that this does not compromise the interpretation of our experiment, as biasing paternity towards first males to mate could take the form of reduced mating attempts of the second male. When 6N knockout males were first to mate, we could not confirm successful mating visually since they do not form a copulatory plug. Therefore, for a subset of these crosses, we observed the first mating for the entire four-hour period. Ejaculation was confirmed from characteristic behaviors, including increasing intromissions which slow down closer to ejaculation, and a final shudder of the male and phase of immobility during which the pair often fall over onto their sides (McGill, 1962, McGill et al., 1978). The subset of confirmed ejaculations by 6N knockout males is presented in Table 1, and our conclusions remain unaltered if we confine the analyses to this subset of crosses.

Table 1.

Distribution of pregnancies sired by first or second males. Numbers in parentheses indicate confirmed ejaculations (see text). Multiply sired pregnancies are assigned to the male that sired the minority of the embryos.

1st Male to Mate	Successful matings	1st Male Paternity	2nd Male Paternity
6N wildtype	26	19	1
6N knockout	40 (18)	0 (0)	25 (13)

Experiment 2: Serial Mating, First Male Vasectomized

The above experiment showed that first males achieved nearly all paternity when they could form a plug (see below). It remained unclear whether this paternity bias occurred because first males fertilized all the oocytes by the time the second male mated, or if the plug per se somehow blocked access of the second male. To investigate this question, we repeated the above experiment with vasectomized 6N knockout and 6N wildtype males. Vasectomized males do not transfer sperm but still transfer seminal fluid, and vasectomized 6N wildtype males produce normal copulatory plugs. If the plugs themselves blocked access, then we predict that second males cannot impregnate females if vasectomized 6N wildtype males mate first, but will impregnate females if vasectomized 6N knockout males mate first.

Mice were vasectomized using the “scrotal entry” technique, initially anesthetizing them with 4-5% mg/kg isoflurane followed by~1.5% for maintenance during surgery (Nagy et al., 2003). Briefly, once the mouse was anesthetized, a small incision was made in the scrotum, and approximately 3 mm of the vas deferens was removed. Second male paternity was scored as the proportion of times that a female became pregnant, and confirmed with genetic assays as described next.

Paternity Assignment

Females were euthanized at 12-14 days post-coitum (gestation in mice is generally 21 days) and embryos were dissected for genetic assignment of paternity. DNA was extracted from a small piece of embryonic tissue, purified using a Master-Pure complete DNA extraction kit (Epicentre, Madison, WI). We only performed paternity analysis on fully formed embryos to avoid resorption sites (as shown below, there was no difference in the frequency of resorption across treatments). Each embryo had a known mother (FVB), and was sired by either the first male (either 6N knockout or 6N wildtype) or the second male (10J). Using published genomes of these three strains (Wong et al., 2012, Keane et al., 2011, McClive et al., 1994), we designed genotype-specific PCR reactions to distinguish between the 3 genotypes. A 6N-specific forward primer (5’-CCACAGACATTGAGAGTGTCAGCA-3’) amplified a 346 bp fragment, or a 10J-specific forward primer (5’-AGACACCAGGAGAGCCAACAGTCCC-3’) amplified a 230 bp fragment, when used with a common reverse primer (5’-CAGCAGAATGTTCCCAGATACCCT-3’). The latter primer pair also amplified a fragment from the maternal (FVB) DNA. Therefore, paternity was assigned to the 6N male if two bands were observed (one band indicating 6N, the other indicating FVB) while paternity was assigned to the 10J male if one band was observed (10J and FVB fragments of the same length). All three primers were added to each PCR reaction.

Since the band produced by FVB and 10J genotype were indistinguishable, we designed a 10J-specific forward primer (5’-GCTAGAGAGGCCCCATGGGAG -3’) that amplified a 116 bp fragment, and a FVB-specific forward primer (5’-AAGGACAGGGAGAAGGGCCC -3’) that amplified a 220 bp fragment, when used in combination with a common reverse primer (5’-CCTGACTTTGCTCTGTCCTTC-3’). All three primers were added to each PCR reaction. We tested a subset of reactions where we observed a single band in the first PCR reaction, and always observed two bands in this second PCR reaction as expected.

DNA was amplified with the same parameters for all primer sets: 11 cycles of denaturation (94 C, 30 seconds), annealing (65 C, 30 seconds, lowered 1 C every cycle) and extension (72 C and 60 seconds) followed by 19 cycles of denaturation (94 C, 30 seconds), annealing (52 C, 30 seconds) and extension (72 C and 60 seconds). All PCR reactions used Fermentas 2X master mix. PCR products were scored on a 3% agarose gel.

Statistical Analysis

Across all experiments, 54 pregnancies were collected. Fifty-one of these were sired entirely by one male. For the other three, we either 1) excluded them, 2) assigned them to the male that sired more individual embryos in those pregnancies, or 3) assigned them to the male that sired the minority of the embryos (which increased our chance of losing statistical significance). Our conclusions remained the same regardless of which strategy we employed; we present the third strategy since it is the most conservative. We performed a Fisher's Exact Test (FET) to determine if the number of pregnancies sired by the second male depended on the first male's ability to form a copulatory plug. In Experiment 1, we excluded all resorption sites from tests of paternity, but this is only justified if resorption sites are randomly distributed among treatments. To test this, we tested the distribution of resorption sites using a generalized linear model, with first male as fixed effect, and the number of embryos vs. number of resorption sites per litter as the two-vector response vaiable, using the function glm of the R package mass (Ripley, 2002).

Results

We performed 96 successful crosses across both experiments (Supplementary Table 1).

Experiment 1: First males achieved nearly all paternity when they could form a plug

Twenty-six females successfully mated with 15 6N wildtype males and were subsequently paired with a 10J male, resulting in 20 pregnancies (Table 1). Of these 20 pregnancies (average number of embryos= 12.55 +/−7.19), 19 were sired exclusively by the 6N wildtype male. One pregnancy included nine embryos sired by the 6N wildtype male and one embryo sired by the 10J male. These 20 pregnancies were sired by 13 different individual males (some males were used more than once, Supplementary Table 1).

Forty females were paired with 6N knockout males first then successfully mated with a 10J male. Out of these 40 females, 25 resulted in pregnancies, 23 of which were sired by the second male (average number of embryos=10.53±6.55). Two pregnancies were multiply sired; one had 17 embryos sired by 6N and one sired by 10J, the other had 7 sired by 6N and 5 sired by 10J. First males sired significantly more litters when they could form a plug (Table 1, FET=10⁻¹²).

There was no statistical difference in the number of embryos per pregnancy between the two treatments (Wilcoxon Rank Sum Test, p=0.42), nor a difference in the proportion of crosses that resulted in pregnancy among treatments (χ²=0.92, df=1, p=0.34). Furthermore, the number of resorption sites was randomly distributed among treatment (p=0.80). In sum, only the distribution of paternity, and no other characteristics of litter size or fertilization success, varied according to the first male to mate.

It has been reported previously that 6N knockout male mating behavior does not differ from 6N wildtype, they have normal sperm count and sperm motility, and they are able to fertilize oocytes (Dean, 2013). Nevertheless, we repeated our analysis after only including crosses where ejaculation by 6N knockout males was visually confirmed. Eighteen females were paired with 6N knockout males first, where ejaculation was visually confirmed. Out of these 18 females, 13 resulted in pregnancies, 11 of which were sired by the second male (average number of embryos=10.3±6.0). The two multiply sired pregnancies were from this subset of data. There was no statistical difference in the number of embryos per pregnancy between the two cross types (Wilcoxon Rank Sum Test=0.56), nor a difference in the proportion of crosses that resulted in pregnancy (χ²=0, df=1, p=1). First males sired significantly more litters when they could form a plug (numbers in parentheses of Table 1, FET=10⁻⁸). In sum, first males achieved nearly 100% paternity if they could form a plug, but nearly 0% paternity if they were unable to form a plug, even when ejaculation was visually confirmed in the latter case.

Experiment 2: Paternity skew remained even when first males were vasectomized

Thirteen females successfully mated with a vasectomized 6N wildtype male, as confirmed by the presence of a copulatory plug, and were subsequently placed with a 10J male. None of these females were pregnant 14 days post-coitum. Seventeen females were paired with vasectomized 6N knockout males then subsequently placed with a 10J male, of which 9 resulted in a pregnancy (average number of embryos= 8.11±5.06), all of which were sired by the second male (as expected since the first male was vasectomized). The ability of a second male to impregnate a female was significantly correlated with the first male's ability to form a plug (FET=0.003).

Discussion

Here we employed a knockout mouse model to further characterize the functions of the copulatory plug in mice. First males sired almost all embryos if they could form a plug, but sired almost none if they could not. Therefore, the previously observed reduction in ability of Tgm4 knockout males to sire litters (Dean, 2013) cannot be rescued by second male plugs. Perhaps most importantly, the skew in paternity remains even if first males were vasectomized, indicating the absence of the plug (and not just reduced fertility of Tgm4 knockout males) allowed second males to gain paternity.

The blocking effect of the copulatory plug could arise through physical prevention of mating (Parker, 1970, Devine, 1975, Voss, 1979, Shine et al., 2000), or by discouraging second males from mating through visual or olfactory cues (Ramm & Stockley, 2014). In mice, the first male to mate has an advantage (Levine, 1967), which may select for males to avoid mating with females that have already mated (Ramm & Stockley, 2014). Along with indirect evidence that mating and ejaculation is energetically costly for males (Pizzari et al., 2003, Lüpold et al., 2010, Friesen et al., 2015, Drickamer et al., 2003, Gowaty et al., 2003, Drickamer et al., 2000), males may be selected to conserve ejaculates when possible (Ramm & Stockley, 2007, Parker, 1998).

Evidence that plug-forming species show multiple paternity

In apparent contrast to the very strong effects we observed in this study, 10-70% of pregnant female house mice carry offspring sired by more than one male in natural populations (Dean et al., 2006, Firman & Simmons, 2008). Many other plug-forming species also show high rates of multiple paternity (Eberle & Kappeler, 2004, Schwartz et al., 1989, Searle, 1990, Birdsall & Nash, 1973, Møller, 1998, Uhl et al., 2010). Several hypotheses could reconcile the strong inhibitory effects of the plug observed here with the common observation of multiple paternity in nature.

We did not vary several aspects of mating ecology that might influence paternity, such as the number of males a female encountered during a single fertile period. The opportunities for multiple mating may not be confined to two males presented in succession, as occurred in our experiments, if the females actively pursue multiple mating in natural populations. For example, multiple paternity is more common in relatively dense populations (Dean, 2013), and we would not have captured such variation in our serial mating design. Furthermore, we did not vary the time each male spent with the female. Here, the first male was left with the female for 4 hours. Based on our watched subset, mating occurred on average of 2.5 hours after crossing (Supplementary Table 1). The sooner a second male is able to dislodge a first male's plug, the more effective he is at gaining paternity (Wallach & Hart, 1983, Sutter & Lindholm, 2016, Sutter et al., 2016) since the copulatory plug promotes sperm transport (Toner et al., 1987, Matthews Jr & Adler, 1978, Dean, 2013). It is possible that our four-hour time window biased paternity towards the first male, and this was amplified when the first male could form a plug.

In the present study, ovulation and behavioral estrus were experimentally controlled through hormonal manipulation, and copulation was likely to have occurred very close to ovulation. In nature, the time between copulation and estrus is likely to be more variable. For example, if first males mate “too early” relative to a female's ovulation, their plug might not survive long enough to inhibit later, better-timed matings of second males (Firman & Simmons, 2009, Breedveld & Fitze, 2016, Coria-Avila et al., 2004). Copulatory plugs get smaller and less adhered to the vaginal-cervical canal over time (Mangels et al., 2015), suggesting that the efficacy of the plug decreases over time. Furthermore, not all matings result in a well-formed and properly seated copulatory plug (Matthews Jr & Adler, 1978, Masumoto, 1993, Hartung & Dewsbury, 1978). In fact, females may exert some control in this context, by not allowing proper orientation of the copulating male (Matthews Jr & Adler, 1978) or by removing plugs (Koprowski, 1992, Parga, 2003).

Females may prevent proper orientation of the copulating male (Matthews Jr & Adler, 1978) or remove deposited plugs (Koprowski, 1992, Parga, 2003). In our study, such females may have been excluded, because crossing to 6N wildtype males was deemed successful only after observing a well-formed plug. By potentially excluding females that had already removed plugs, we might be overestimating success of 6N wildtype males.

Subsequent males may also affect the efficiency of the plug by actively removing the copulatory plug before copulation (Parga, 2003, Parga et al., 2006) or dislodging the copulatory plug through multiple intromissions before ejaculation (Dewsbury, 1981, Wallach & Hart, 1983, Milligan, 1979, Mosig & Dewsbury, 1970, Sutter & Lindholm, 2016, Sutter et al., 2016). We observed instances where the second male removed the copulatory plug of the first male, which may be interpreted as the second male attempting to gain fertilization. However, these trials did not result in the second male paternity.

Lastly, we did not vary male or female genotypes in the current study. It is possible that the 6N wildtype males used here make especially strong plugs, that the 10J males (always second to mate) were simply unable to remove previously deposited plugs, or that the FVB females were especially resistant to remating if a plug-forming male was first to mate. Mangels et al. (2015) showed that different male genotypes vary in both the size and survival of copulatory plugs they form.

Conclusions

Our study provides direct, non-invasive quantification of the copulatory plug's role in inhibiting second male paternity, at least in a laboratory setting. When first males cannot form a plug, they lose almost all paternity to second males, and this cannot simply be due to reduced sperm transport of the first male. It is important to recognize that our study does not reject the role of the copulatory plug in other aspects of fertility. Future studies should place lessons learned here in the context of the many variables encountered in nature, to better understand the ecology and evolution of the copulatory plug.