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.