DISCUSSION
Reproductive traits are among the most rapidly evolving traits in nature and are driven by many evolutionary processes, including post-copulatory sexual selection (e.g., Eberhard 2004; Clark et al. 2006; Martin-Coello et al. 2009; Ramm et al. 2009). In polyandrous systems, sperm from multiple partners are expected to interact and compete for fertilization of the available ova, and females are predicted to evolve traits that allow them to exert control over the outcome of this competition (reviewed in Eberhard 1996; Firman et al. 2017). In this study, we asked whether female reproductive traits that impact the successful migration of sperm to the site of fertilization differ between polyandrous and monogamous species. We examined the composition of fluids collected from the two reproductive organs closest to the fertilization site – the oviduct and the uterus – using Peromyscus mice with naturally variable mating systems among closely related species. Our results show that (1) polyandrous species have significantly more viscous fluid in the uterus but less viscous fluid in the oviduct than monogamous species, and a viscosity gradient from the uterus to the oviduct increases in monogamous species but decreases in polyandrous species; (2) the reproductive fluid pH is significantly higher in the uterus and oviduct of the polyandrous P. maniculatus compared to its monogamous congener, P. polionotus , but both species have a more alkaline environment in the uterus than oviduct overall; and (3) there are no differences in the calcium content between species, but P. maniculatus has a calcium gradient that decreases at the distal end of the reproductive tract. Given that these traits and their interactions are likely to affect sperm motility and migration toward the site of fertilization, these fluidic properties warrant further study to determine the extent to which they provide females control of sperm use within these mice.
Using a highly sensitive method (Duncan et al. 2016), we found that polyandrous species (P. maniculatus , P. leucopus , andP. gossypinus ) have significantly more viscous fluid in the uterus but less viscous fluid in the oviduct than their closely related monogamous congeners (P. californicus , P. eremicus , andP. polionotus ). In vitro experiments in Peromyscushave shown that increasing the viscosity of the microenvironment leads to reduced sperm velocity (Hook et al. 2022), consistent with other systems (Smith et al. 2009; Miki and Clapham 2013). Our findings suggest that in polyandrous species, sperm motility is hindered more in the uterus, which is closest to the sperm entry site and therefore provides an initially more competitive environment that likely selects for the most motile sperm prior to ever reaching the oviduct (Holt and Fazeli 2016; Suarez 2016). The opposite pattern was observed in the monogamous species, in which sperm motility would be enhanced in the uterus but hindered in the oviduct. In these monogamous species, it is possible that the UTJ – a constricted passageway that separates these two tract regions and contains highly viscous fluid in other species (reviewed in Hunter 1995) – provides an adequately effective barrier to remove morphologically abnormal and slower sperm (Chatdarong et al. 2004; Druart 2012), as has been observed in a previous study examining their collective sperm groups (Hook et al. 2022), and that the higher oviductal fluid viscosity is effective in reducing sperm motility and, thus, the possibility of polyspermy (Kim et al. 1996; Firman and Simmons 2013; Firman 2018). Overall, the significant differences in fluid viscosity based on mating systems is suggestive of an association with post-copulatory sexual selection. Further studies are warranted in these species to examine how sperm interact with uterine and oviductal fluids, as well as seminal fluids after mating (Miki and Clapham 2013), to transverse the UTJ and reach the fertilization site given these observed differences in female fluidic viscosity.
We also found that the reproductive fluid pH is significantly higher in the uterus and oviduct of the polyandrous deer mouse (P. maniculatus ) during estrus compared to its monogamous congener,P. polionotus , but that both species have a more alkaline environment in the uterus than oviduct. This result is surprising given that the opposite has been observed in other studies (López-Albors et al. 2021, but see Ng et al. 2018). Such alkaline environments have been shown to enhance sperm motility in birds (Gallus domesticus, Coturnix coturnix, Meleagris gallopavo ; Holm and Wishart 1998) and humans (Saito et al. 1996; Zhou et al. 2015), and to activate sperm-specific CatSper channels in mice (Mus musculus ; Kirichok et al. 2006) and humans (Lishko et al. 2010), thereby inducing sperm hyperactivation (Suarez et al. 1993). Sperm hyperactivation, which takes place in the lower oviduct in rabbits (Overstreet and Cooper 1979) and mice (Suárez and Osman 1987), is an important sperm movement pattern characterized by a deep flagellar bend (reviewed in Suarez and Ho 2003). In bulls, sperm cells exhibit deep asymmetrical bends in pH 7.9-8.5 solutions and shallow asymmetrical bends in pH 7.0-7.5 solutions (Ho et al. 2002). We found that the average pH in upper oviduct fluid was 7.44 in P. maniculatus and 7.28 in P. polionotus , which is consistent with shallow asymmetrical bends in hyperactivated sperm in both species. Our finding that this trait differs between species based on mating system suggests it might be a trait that is driven by post-copulatory sexual selection, although the greater pH of fluids in the polyandrous species suggests this trait is enabling them to have greater sperm motility, rather than hindering their movement or serving as a barrier for movement. Further studies are warranted to verify the effects of pH on Peromyscus sperm movements in vivo and how this trait synergistically interacts with the viscosity or calcemic contents of female reproductive fluids or impacts the ability of sperm to capacitate (Stival et al. 2016) or form collective groups that swim together (Fisher and Hoekstra 2010; Hook et al. 2022).
Last, we found that the concentration of calcium in the uterine and oviductal fluids extracted from the polyandrous P. maniculatusand monogamous P. polionotus did not significantly differ. If this fluidic component enables female control of sperm use, we would expect a difference between these species that differ by mating system. However, it is interesting to note that only in P. maniculatusdid we observe a gradient in which calcium concentration decreases moving up the female reproductive tract from the uterus to the oviduct. There is a positive association between extracellular calcium and sperm velocity in humans (Zhou et al. 2015), rats (Lindemann and Goltz 1988), and hamsters (Suarez and Dai 1995), which suggests that the reduction in calcemic contents in the oviductal fluids of the polyandrous P. maniculatus may impose a barrier on sperm motility. Alternatively, it may indicate that sperm hyperactivate much earlier in P. maniculatus , given that calcium is essential for sperm hyperactivation (Yanagimachi 1982), and increases in extracellular calcium levels increase calcium entry through sperm-specific CatSper channels (Marquez and Suarez 2007). We cannot rule out that this property interacts with other fluidic properties or with seminal fluids to control sperm migration, so future studies that examine these effects – specifically through in vivo experiments and accounting for potential interspecies variation in these fluidic properties – are warranted.
Our results demonstrate some important differences in the fluid collected from different regions of the female reproductive tract, however finer-scale changes in more localized areas of the reproductive tract and in response to seminal fluids will further enhance our understanding of how selection has shaped the fertilization environment in Peromyscus . Our understanding of the physiological mechanisms required for mammalian fertilization remain obscure without the ability to measure conditions in vivo in real-time (Ng et al. 2018), especially in small animals, but our results suggest that even closely-related species may exhibit striking differences similar in magnitude to differences in highly divergent taxa (López-Albors et al. 2021). In other mammals, evidence suggests that uterine fluid near the cervix is more viscous than more proximal regions of the uterus, and oviductal fluid in the ampullary region contains viscous compounds produced from ovulating follicles and peritoneal fluid during estrus (reviewed in Hunter et al. 2011). In bovine, for example, the greater amount mucus in the ampulla compared to lower regions of the oviduct is associated with reduced sperm numbers near the fertilization site (Suarez et al. 1997). Our study was limited by the small quantity of fluid we could extract from Peromyscus oviducts – although we aimed to maximize this with our approach. The comparisons we were able to make between fluid obtained from the isthmus and ampullary regions suggest that fluidic properties in the oviduct may be equally as dynamic as the structural features (e.g., Yániz et al. 2000; Suarez 2016; Miller 2018).
Taken together, our results support the prediction that female reproductive fluids can vary by the species’ mating system, and thus level of post-copulatory sexual selection, yet the directionality of the differences make their functional significance less clear. We found that female reproductive fluids in polyandrous species is more acidic with differing viscosities throughout the tract compared to monogamousPeromyscus females. We also found variation between distinct regions of the reproductive tract, providing indirect evidence for how these properties might impact sperm cells as they migrate up toward the site of fertilization. Our results suggest that fluid viscosity and pH may provide promising avenues for investigating a female reproductive trait that is driven by cryptic female choice, although follow-up experiments are needed to assess their impacts on sperm motilityin vivo and on male fertilization success within a competitive context.
Data Availability Statement : All data: Dryad doi:TBA.
Competing Interests : We declare we have no competing interests.
Author Contributions: KAH, HSF, and CL conceived of the study, designed experiments, and interpreted results; CL, KAH, and KAJ collected the data, KAJ analyzed video data, KAH carried out the statistical analyses; all authors wrote the manuscript and all authors gave final approval for publication.
Acknowledgements: We are grateful to Hopi Hoekstra for providing P. gossypinus males and to Erica Glasper for providingP. californicus males and providing use of a microplate reader. Thanks to W. David Weber for help in determining estrous phase of female mice, maintaining the mouse colony, and collecting many of the reproductive tracts analyzed for this study. We thank Mollie Manier, Halli Weiner, and Patricia Martin-DeLeon for advice on methods for measuring calcium and pH and Shelby Wilson for statistical advice. Thanks to Harrison Arsis, Madeline-Sophie Dang, Catherine Liu, and Audrey Mvemba for their assistance with video analyses. Funding was provided by a National Science Foundation Postdoctoral Research Fellowship [1711817] to KAH, a University of Maryland Honors College Research Grant to CL, the Cystic Fibrosis Foundation (JOYNER18FO) to KAJ, a Burroughs Wellcome Fund Career Award at the Scientific Interface to GAD, and a Eunice Kennedy Shriver National Institute of Child Health and Human Development K99/R00 Pathway to Independence Award [R00HD071972] to HSF.