INTRODUCTION
When females mate with more than one male within a single reproductive
cycle (i.e., polyandry), post-copulatory sexual selection is
hypothesized to favor male reproductive traits that allow them to
outcompete rivals in their race to fertilize a females’ ova (i.e., sperm
competition; Parker 1970) and female traits that allow them to
preferentially bias sperm use in favor of certain males over others
(i.e., cryptic female choice; Thornhill 1983; Eberhard 1996) and to
prevent polyspermy (Kim et al. 1996; Firman and Simmons 2013; Firman
2018). Consequently, female reproductive traits that enable
post-copulatory control and selective fertilization of ova are predicted
to occur in polyandrous – but not monogamous – species. This
prediction has been poorly tested, however, likely due to lack of
attention to female reproductive traits compared to male reproductive
traits in post-copulatory sexual selection studies (Orr et al. 2020),
the technical challenges of examining covert mechanisms of ‘cryptic
female choice’ for internally fertilizing species (reviewed in Firman et
al. 2017; Ng et al. 2018), and the difficulty of disentangling these
female mechanisms from sperm competition and sexual conflict (Simmons
and Wedell 2020).
Despite the challenges with demonstrating cryptic female choice, several
studies across diverse taxa have provided empirical support that females
are capable of preferentially biasing and controlling sperm use
(reviewed in Firman et al. 2017; Firman 2020). For example, in feral
fowl (Gallus domesticus ), females use muscular contractions to
eject sperm from socially subdominant males to prevent insemination and
fertilization of their ova (Pizzari and Birkhead 2000); in Japanese
macaques (Macaca fuscata ), females increase orgasm-like muscular
contractions after mating with a socially dominant male (Troisi and
Carosi 1998), which increases sperm retention within their reproductive
tract (Baker and Bellis 1993); and in red flour beetles (Tribolium
castaneum ), females appear to be in control of the observed sperm
precedence patterns based on male copulatory behavior (Edvardsson and
Göran 2000). There is also evidence that physical structures within the
female reproductive tract enables female control of sperm use across
taxa. For instance, in fruit flies (Drosophila melanogaster ),
female sperm storage organs allow them to control the timing and use of
sperm stored after copulation with multiple males (Manier et al. 2010).
Moreover, many female vertebrates possess a tube-like passageway to
their ovaries (i.e., the oviduct), and there is evidence in birds and
mammals that features of this structure (Holt and Fazeli 2016), such as
its length (Gomendio and Roldan 1993; Anderson et al. 2006), positively
correlate with relative testis size, a proxy for sperm competition level
(reviewed in Simmons and Fitzpatrick 2012; Vahed and Parker 2012; Lüpold
et al. 2020). These findings suggest that the variable structural
architecture of the female reproductive tract may have evolved to
regulate sperm uptake (Suarez 2008; Tung and Suarez 2021) by selecting
for only those sperm cells that are able to bypass its challenging
features (Holt and Fazeli 2016; Suarez 2016) while excluding pathogens
or microbes (Tung et al. 2015; Holt and Fazeli 2016; Rowe et al. 2020).
The composition of fluids within the female reproductive tract may
provide yet another potential mechanism of female control within
internally fertilizing species, given that their biochemical properties
have been shown to change after insemination, vary throughout the tract,
and modulate sperm motility and migration to the ova and, thus, the
outcomes of fertilization (reviewed in Holm and Ridderstråale 1998;
Hunter et al. 2011; Kirkman-Brown and Smith 2011; Holt and Fazeli 2016;
Ng et al. 2018; Gasparini et al. 2020). For example, fluids within the
reproductive tract can vary in their viscoelastic properties (Johansson
et al. 2000; Rodríguez-Martínez et al. 2005; Suarez 2016), which can
influence sperm motility patterns and trajectory (Tung et al. 2015; Holt
and Fazeli 2016; Tung and Suarez 2021). In humans, mucus coats the
entire female reproductive tract, and sperm must swim through
viscoelastic cervical mucous as well as the cumulus mass en route to the
oocyte (Kirkman-Brown and Smith 2011); a previous study used artificial
insemination to demonstrate that this change in fluidic properties
effectively serves as a barrier, allowing only more motile and
morphologically normal sperm to pass through to the oviduct (Hanson and
Overstreet 1981). Moreover, a pH gradient has been demonstrated
throughout the female reproductive tract of different mammals, with the
uterine environment being more acidic (i.e., less alkaline) than the
oviductal environment (reviewed in Ng et al. 2018). Alkaline
environments have been shown to increase sperm velocity and induce sperm
hyperactivation in mammals, in part through the activation of essential
sperm-specific CatSper protein channels (Kirichok et al. 2006; Lishko et
al. 2010) that increases sperm intracellular calcium concentrations (Ho
and Suarez 2001; Suarez 2008) and subsequently increases their flagella
beat frequency and velocity (Brokaw et al. 1974; Suarez et al. 1993). In
boars (Sus scrofa ), high calcium environments lead to greater
sperm motility, whereas low calcium environments cause sperm cells to
stick to oviductal epithelium and be less motile (Petrunkina et al.
2001). Together these studies suggest that the chemical composition of
female reproductive fluids provides a promising mechanism for female
sperm control driven by post-copulatory sexual selection, but whether
these fluidic properties differ between polyandrous and monogamous
species remains unknown.
In this study, we test whether the composition of female reproductive
tract fluids diverge between species that have evolved under divergent
mating systems in Peromyscus mice. More specifically, we
collected fluids from two distinct regions of the reproductive tract –
the uterus and the oviduct – for three polyandrous species (P.
maniculatus , P. leucopus , and P. gossypinus ) and their
closely related monogamous congeners (P. californicus, P.
eremicus, and P. polionotus ; Turner et al. 2010; Bedford and Hoekstra
2015). From these fluids, we measured viscosity, pH, and calcium
concentration, all of which have been shown to significantly impact
sperm movements in other taxa. We compared these physiological
properties between species that evolved under polyandry to those that
evolved under monogamy to examine associations between mating strategy
and potential mechanisms of post-copulatory female control. From these
data, we were also able to establish for each species whether a gradient
for each of these properties exists within the reproductive tract and
indirectly assess how that might impact sperm motility and their unique
ability to form collective groups within these mice (Hook et al. 2022).