Female reproductive fluid composition differs based on mating system in Peromyscus mice

Post-copulatory sexual selection is theorized to favor female traits that allow them to control sperm use and fertilization, leading to the prediction that female reproductive traits that influence sperm migration should differ between polyandrous and monogamous species. Here we exploit natural variation in the female mating strategies of closely related Peromyscus mice to compare female traits that influence sperm motility – the viscosity, pH, and calcium concentration of fluids in the reproductive tract – between polyandrous and monogamous species. We find that the viscosity and pH, but not calcium concentration, of fluids collected from both the uterus and the oviduct significantly differ between species based on mating system. Our results demonstrate the existence of a viscosity gradient within the female reproductive tract that increases in monogamous species but decreases in polyandrous species. Both species have a more alkaline environment in the uterus than oviduct, but only in the polyandrous species did we observe a decrease in calcium in the distal end of the tract. These results suggest that fluid viscosity and pH in the female reproductive tracts of these mice may be influenced by post-copulatory sexual selection and provide a promising potential mechanism for female sperm control given their importance in modulating sperm behavior.

To obtain sufficient volume for downstream methods, we pooled the samples for each region from at 150 least ten individuals per species, then warmed pools to 37ºC to simulate natural physiological values, and 151 again centrifuged at 3000 rpm for 3 min to ensure full separation of the mineral oil from the reproductive 179 reflects a high viscosity fluid. Using the generalized Stokes-Einstein relation, measured MSD values were 180 used to compute viscoelastic properties of the hydrogels (Joyner et al. 2020). The Laplace transform of 〈 181 Δr 2 (τ), 〈Δr 2 (s), is related to viscoelastic spectrum G(s) using the equation G(s) = 2k B T/[πasΔr 2 (s) ], 182 where k B T is the thermal energy, a is the particle radius, s is the complex Laplace frequency. The complex 183 modulus can be calculated as G*(ω) = G′(ω) + G′′(iω), with iω being substituted for s, where i is a 184 complex number and ω is frequency. We pooled these data across all particles to characterize female 185 reproductive fluid viscosity per species. Due to technical difficulties, we were unable to collect viscosity 186 data from the upper oviduct for two of the polyandrous species (P. maniculatus and P. gossypinus). For 187 this reason, we combined viscosity data for both the lower and upper oviducts into a single measure for 188 every focal species.

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190 Collecting and measuring fluid pH and calcium 191 Due to the limited quantity of fluids collected from each female reproductive tract for our viscosity 192 measurements, we were not able to also measure pH and calcium using these same individuals. Because 193 we were further limited by the number of available animals in our lab colony, we focused on two species 194 in which ten females were available for this study -the polyandrous P. maniculatus and its monogamous 195 sister-species P. polionotus. To extract reproductive fluid for these measurements, we dissected the 196 reproductive tract, reserved the right side of each tract for the pH measurements and submersed the left 197 side of each tract into phosphate buffer solution (PBS) at 4ºC for the calcium measurements.

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To assess differences in the viscosity of female reproductive fluids between polyandrous and 224 monogamous species, we used our dataset of individually tracked particles and pooled these data across 225 species with shared mating systems (categorized as either monogamous or polyandrous). We excluded 226 data that were ±2SD of the mean within each sample because they represented clear outliers. All viscosity 227 values were log-transformed to improve model assumptions of normality. We assessed differences in the 228 viscosity of female reproductive fluids in the uterus and in the oviduct using separate linear models (LM), 229 with log viscosity as the response variable and mating system as the predictor variable. We also analyzed 230 fluidic differences in each tract region within each species using separate linear models, with log viscosity 231 as the response variable and the region of the female reproductive tract as the predictor variable. Last, we 232 used the data sets from three focal samples (P. eremicus, P. gossypinus, and P. polionotus) to statistically 233 compare viscosity measurements to their dyed mineral oil controls using separate linear models, with log 234 viscosity as the response variable and the sample type as the predictor variable. Post-hoc pairwise 235 comparisons were made using Tukey HSD adjustments for multiple comparisons using the 'LSmeans' R 236 package (Lenth 2016).

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To assess differences in the pH and calcium levels of female reproductive fluids between polyandrous 238 and monogamous species, we used separate linear models for each region of the tract, with either pH or 239 the log calcium measurements included as the response variable and mating system included as the 240 explanatory variable. Last, we used paired t-tests to conduct pairwise comparisons for pH and calcium 241 measurements from each region of the tract within each species to control for differences among 242 individual females.  Table 1. We found that the viscosity of fluids in both the uterus and the oviduct 248 significantly differed based on mating system in Peromyscus mice. More specifically, polyandrous 249 species have significantly more viscous fluid in the uterus (LM: F 1,1255 = 27.09, p < 0.001) but 250 significantly less viscous fluid in the oviduct (LM: F 1,1755 = 24.7, p < 0.001) than monogamous species 251 ( Figure 1). Within-species analyses revealed that fluids were significantly more viscous when collected 252 from the uterus compared to the oviduct in P. maniculatus, P. leucopus, and P. californicus, but the 253 opposite was true in P. eremicus and P. polionotus; no difference was observed in the viscosity of uterine 254 fluid or oviductal fluid in P. gossypinus, however (Table 1).

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We also found that the reproductive fluid pH is significantly higher in the uterus and oviduct of the 309 polyandrous deer mouse (P. maniculatus) during estrus compared to its monogamous congener, P.

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Last, we found that the concentration of calcium in the uterine and oviductal fluids extracted from the 332 component enables female control of sperm use, we would expect a difference between these species that 333 differ by mating system. However, it is interesting to note that only in P. maniculatus did we observe a 334 gradient in which calcium concentration decreases moving up the female reproductive tract from the 335 uterus to the oviduct. There is a positive association between extracellular calcium and sperm velocity in 336 humans (Zhou et al. 2015), rats (Lindemann and Goltz 1988), and hamsters (Suarez and Dai 1995), which 337 suggests that the reduction in calcemic contents in the oviductal fluids of the polyandrous P. maniculatus 338 may impose a barrier on sperm motility. Alternatively, it may indicate that sperm hyperactivate much 339 earlier in P. maniculatus, given that calcium is essential for sperm hyperactivation (Yanagimachi 1982 1. Violin plots of the log-transformed viscosity measurements obtained from fluids collected from two regions of the female reproductive tract within Peromyscus mice species with naturally varying mating systems. Blue dots represent particle tracking data obtained from three monogamous species (P. californicus, P. eremicus, and P. polionotus), red dots represent particle tracking data obtained from three polyandrous species (P. maniculatus, P. leucopus, and P. gossypinus). Asterisks denote differences within each reproductive region between the mating systems (*** P < 0.001).  The calcemic content of reproductive fluids collected from the uterus and oviduct did not significantly differ between the polyandrous P. maniculatus (red dots) and its monogamous sister species, P. polionotus (blue dots). The calcium concentration between these regions significantly differed only for P. maniculatus. Small dots denote values measured for individual females; large dots represent the mean values for each species.