Collecting fluid and particle tracking microrheology (PTM)
To collect fluid for viscosity measurements, we removed the female
reproductive tract and submersed it in mineral oil at 4ºC until fluid
extraction, which took place within 24 hours. To visually distinguish
the mineral oil from biological fluid, we dyed the oil blue using a
colored gel dye (Wilton Candy Colors, USA) in a 1:75 dye:oil ratio.
Under 0.63x magnification (Zeiss Stemi 508, USA), we trimmed the fat
surrounding the reproductive tract, unraveled the coiled oviducts,
severed the uterus from the oviduct at the utero-tubal junction (UTJ),
divided the oviduct in half to separate the lower and upper regions, and
submersed in dyed mineral oil. We used glass Pasteur pipettes bent into
a u-shape under a flame to push down from one end of the tissue to the
other to squeeze fluid within the tissue out into the oil, then
collected and centrifuged the samples, removed the oil supernatant and
stored fluids at -80ºC (Yuana et al. 2015; Patczai et al. 2017).
To obtain sufficient volume for downstream methods, we pooled the
samples for each region from at least ten individuals per species, then
warmed pools to 37ºC to simulate natural physiological values, and again
centrifuged at 3000 rpm for 3 min to ensure full separation of the
mineral oil from the reproductive fluid. We then combined 2µL of fluid
with 0.5µL of ~0.002% w/v suspension of fluorescent
nanoparticles (PEG-coated polystyrene particles, PS-PEG). PS-PEG were
prepared by coating red fluorescent carboxylate-modified PS spheres
(PS-COOH), 500 nm in diameter (ThermoFisher FluoSpheres
Carboxylate-Modified Microspheres, 0.5 µm, red fluorescent
(580ex/605em), 2% solids, USA) with 5-kDa methoxy-PEG-amine (Creative
PEG-Works, USA) via NHS-ester chemistry as previously described (Joyner
et al. 2019). We gently reverse-pipetted the mixture to make sure the
nanoparticles were homogeneously scattered throughout and pipetted 2.0µL
into a 1-mm ID Viton O-ring microscopy chamber (McMaster Carr, USA) and
covered with a small circular glass coverslip, both of which were sealed
with vacuum grease (Dow Corning, USA) to prevent fluid flow and
evaporation, and equilibrated for 30 minutes prior to imaging to reduce
dynamic error.
To measure viscosity, we recorded a minimum of three videos of the
suspended fluorescent nanoparticles within each fluid sample at a frame
rate of 33.33 Hz for 300 frames (10 sec) using an EMCCD camera (Axiocam
702; Zeiss, Germany) attached to an Zeiss 800 LSM inverted microscope
and x63/1.20 W Korr UV VIS IR water-immersion objective with image
resolution of 0.093μ per pixel. To avoid edge effects on nanoparticle
movement, we randomly selected central locations within the chamber for
our video recordings. All samples remained at 37ºC during imaging using
a stage incubator (PM 2000 Rapid Balanced Temperature, PeCon, Germany).
To track the diffusion of PS-PEG nanoparticles in each sample, we used
particle tracking data analysis using automated software custom-written
in MATLAB (Mathworks, USA). Based on a previously developed algorithm
(Crocker and Grier 1996), the program determined the x andy positions of nanoparticle centers based on an intensity
threshold and then constructed particle trajectories by connecting
particle centers between sequential images given an input maximum moving
distance between frames. Finally, the program calculated the
time-averaged mean squared displacement [MSD (τ)] as
〈Δr2( τ)〉 = 〈x (t+ τ) –
x (t )]2 + [t (t + τ )+ y (t )]2〉
where τ is the time lag between frames and angle brackets denote the
average over the time points. The MSD of PS-PEG nanoparticles is
directly proportional the viscosity of the surrounding fluid. A
fast-moving particle (high MSD) reflects a low viscosity fluid whereas a
slow-moving particle (low MSD) reflects a high viscosity fluid. Using
the generalized Stokes–Einstein relation, measured MSD values were used
to compute viscoelastic properties of the hydrogels (Joyner et al.
2020). The Laplace transform of
〈Δr 2(τ )〉,
〈Δr 2(s )〉, is related to viscoelastic
spectrum G (s ) using the equation G (s ) =
2k BT /[πas 〈Δr 2(s )
〉], where k BT is the thermal
energy, a is the particle radius, s is the complex Laplace
frequency. The complex modulus can be calculated as G *(ω )
= G ′(ω ) + G ′′(iω ), with iω being
substituted for s , where i is a complex number
and ω is frequency. We pooled these data across all particles to
characterize female reproductive fluid viscosity per species. Due to
technical difficulties, we were unable to collect viscosity data from
the upper oviduct for two of the polyandrous species (P.
maniculatus and P. gossypinus ). For this reason, we combined
viscosity data for both the lower and upper oviducts into a single
measure for every focal species.