Transport processes in equine oocytes and ovarian tissue during loading with cryoprotective solutions
Graphical abstract
Introduction
Female fertility can be preserved by cryopreserving oocytes or ovarian tissue [[1], [2], [3]]. In order to withstand the damaging effects of cooling, cryogenic storage, thawing and warming, specimens need to be loaded with cryoprotective agents (CPAs). Cryopreservation can be done by either slow-freezing or vitrification. With slow-freezing, low to moderate cooling rates and relatively low concentrations of permeating agents (<2 M), such as dimethyl sulfoxide (DMSO) or propylene glycol (PG) are used to control the rate of cellular dehydration during freezing while minimizing the occurrence of intracellular ice formation [4,5]. The aim of vitrification is to avoid ice formation, by transitioning the entire sample and its surrounding into a glassy state, which can be done using both high concentrations (4–8 M) of mixtures of CPAs and high cooling rates (>100 °C min−1) [6]. Slow-freezing cryopreservation requires knowledge of the optimal cooling rate for a given cell type, where survival after freezing-and-thawing is maximal, whereas the cooling rate used in ice-free cryopreservation approaches is not cell-specific, making it particularly suitable for tissues consisting of different cell types.
Rational design of cryopreservation protocols requires insights in the diffusion kinetics of CPAs into cells or tissues to ensure homogeneous distribution, while minimizing the exposure time and toxicity effects [[7], [8], [9]]. Cellular membranes and the extracellular tissue matrix both present barriers that need to be passed by CPAs, in order to protect the tissue matrix and to provide intracellular protection. Cellular membranes have specific permeability characteristics, which define the rate at which transport processes (i.e., movement of water and solutes) occur. Once cell-specific membrane transport parameters are known they can be used to design CPA loading protocols, taking the tolerance limits for cell volume excursion into account [[10], [11], [12]]. However, little information is available on cell membrane permeability properties of cells in tissues, and how connective tissue affects CPA permeation. The extracellular matrix likely forms a fundamentally different type of diffusion barrier for solutes compared to the lipid bilayer of membranes.
Cells respond to changes in the external medium by transporting water and solutes across the cellular membrane to maintain equilibrium between the intra- and extracellular solute concentrations [13]. If cells are exposed to hyper- or hypotonic media, they shrink or swell, respectively, mainly due to water movement into or out of the cell. Permeating CPAs can easily pass the cellular membrane. However, since water moves substantially faster across the cellular membrane, exposing cells to a CPA solution first causes cellular dehydration due to water movement out of the cell. Thereafter, both CPAs and water move into the cell until equilibrium is reached between the extra- and intracellular milieu. Cell volume responses can be captured microscopically [[14], [15], [16], [17]], by electronic particle size measurements [18,19], or via monitoring quenching properties of entrapped fluorophores [20]. Different formalisms are available to fit cell volume response data to derive the membrane permeability to water (Lp) and to specific solutes/CPAs (Ps); including models that consider solute-solvent interactions or assume water and solute transport as independent processes [21,22]. Moreover, simplified models are available for determining permeability coefficients of artificial phospholipid vesicles and planar membranes [23,24].
In case of tissues, both the extracellular matrix and cellular membranes hinder free diffusion of CPAs. Times needed to load tissues with enough amounts of CPAs increase with increasing tissue thickness, molecular weight of the CPA, and medium viscosity [25,26]. CPA and water diffusion or permeation in tissues can be studied using a variety of methods including osmometer measurements combined with tissue weight measurements [27,28], magnetic resonance imaging [29], computer tomography [30,31], and Raman and infrared spectroscopic approaches [32,33]. Formalisms based on Fick's law of diffusion can be used to derive CPA diffusion coefficients from experimentally determined plots of the tissue CPA content versus time [27,28,34]. In addition, more complex models exist that also consider biomechanical forces (i.e., shrinking and swelling) taking place [25,35]. Similar as is the case with single cells, tissue dehydration precedes CPA diffusion when tissues are exposed to CPA solutions [34].
The aim of this study was to investigate permeation/diffusion kinetics of CPAs and water both in equine oocytes and ovarian tissue. Porcine material was investigated for comparison. CPAs that were studied included glycerol (GLY), propylene glycol (PG), dimethyl sulfoxide (DMSO) and ethylene glycol (EG). For oocytes, in addition to membrane permeability to various CPAs, water permeability in hypo- and hypertonic media was compared. For tissues, effects of different CPA concentrations were tested, and tissue dehydration and CPA diffusion were also studied during step-wise loading protocols. Parameters describing cell membrane permeability towards water and solutes were inferred from video microscopic imaging of oocyte volume responses during perfusion with different solutions. CPA and water transport processes in ovarian tissue were studied using osmometer and tissue weight measurements. In the current manuscript is shown that CPA diffusion in ovarian tissue fundamentally differs from that in oocytes where CPAs only encounter the plasma membrane as a selective barrier.
Section snippets
Isolation and preparation of equine oocytes and ovarian cortex tissue pieces
Genital tracts were collected from mares at a local slaughterhouse and transported to the lab within 2–4 h, in an isolated container. Mares (19 in total) had ages ranging from 4.5–18 years (14.3 ± 3.9), and material from at least 3 different animals was included in each experiment. At arrival, ovaries were dissected, rinsed with saline and further handling was done at room temperature (22 ± 2 °C).
Follicles were aspirated singly using a 12-G needle connected to a 25 mL syringe. In addition, an
Equine oocytes volume behavior in anisotonic media and membrane permeability to water
Oocytes were held, using a micro capillary, and volume responses were recorded during perfusion with anisotonic solution. Oocytes shrink and swell when exposed to hyper and hypotonic media, respectively, due to water moving out and into the cell (Fig. 1A–B). Equilibrium is reached within 5 min, and the final volume is dependent on the medium osmolality. The original cell volume, Vo in isotonic medium of 300 mOsm kg−1, was 6.84 ± 1.38 × 105 μm3. The osmotically inactive volume, Vb, was derived
Discussion
Membrane hydraulic permeability primarily depends on the cell specific membrane composition and the temperature [4]. Membrane permeability to water, however, is not a static value, but depends on the medium composition and the level of osmotic stress that is used to study the rate of water transport across the membrane. Also, the direction of the water flow across the membrane may affect the observed membrane permeability to water. Rectification, which refers to a difference in the rate at
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This work was supported by the German Research Foundation/Deutsche Forschungsgemeinschaft (grant numbers WO1735/6-2, SI1462/4-2). Heinke Eylers is acknowledged for technical assistance, and Juezhu Fan for her role in data analysis. Fleischhandel Kappei in Seesen, Fleischerei Dohrmann in Bremen and Leine-Fleisch in Laatzen are acknowledged for providing equine and porcine ovaries from slaughterhouse material.
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