John K. Critser

Fundamental Cryobiology of Mammalian Spermatozoa

Publisher Summary This chapter discusses the fundamental cryobiology of mammalian spermatozoa. The low motility of cryopreserved mammalian spermatozoa and the often lower conception rates may be due to the fact that procedures for cryopreservation of many mammalian cell types, including sperm, have evolved empirically. The primary assay of sperm function is the use of insemination and measurement of pregnancy initiation. The approaches to measuring sperm plasma membrane integrity include supervital staining and hyposmotic swelling. The other general approach to evaluating plasma membrane integrity is to assay the maintenance of membrane semipermeability by testing the cells ability to change its volume when exposed to anisomotic conditions. Survival of cells subjected to cryopreservation depends not only on the presence of a permeating cryoprotective agent (CPA) but also on the concentration of the CPA. Viability of mammalian sperm is very sensitive to osmotic stress and the associated cell volume excursion. The optimization of CPA addition and removal procedures is also elaborated.

Cryopreservation of human spermatozoa. IV. The effects of cooling rate and warming rate on the maintenance of motility, plasma membrane integrity, and mitochondrial function

OBJECTIVEnTo test the hypotheses that there is a two-factor aspect of cellular damage during cryopreservation that occurs in human sperm (osmotic effects versus intracellular ice formation) and that there is a cooling rate by warming rate interaction related to this damage.nnnDESIGNnEjaculates from healthy men were cooled at 0.1, 1.0, 10, 175, or 800 degrees C/min to -80 degrees C in a solution of 0.85 M glycerol and plunged into liquid nitrogen. Samples were warmed at 400 degrees C/min (experiment 1) or either 1 degrees C or 400 degrees C/min (experiment 2). After warming, sperm were assessed for survival using motility as the endpoint in experiment 1 and motility, plasma membrane integrity, and mitochondrial function in experiment 2.nnnRESULTSnIn experiment 1, over the various cooling rates with a standard 400 degrees C/min warming rate, a plot of motility versus cooling rate produced a classical inverted U-shaped curve (n = 6) with maximum motility at the 10 degrees C/min cooling rate. In experiment 2, over the various cooling rates, both 1 and 400 degrees C/min warming rates produced similar but shifted plots of motility, plasma membrane integrity, and mitochondrial function versus cooling rate, which also produced inverted U-shaped patterns (n = 11). Maximal survival for each of the three endpoints occurred at 10 degrees C/min cooling rate for the rapidly warmed sperm and at 1 degree C/min for the slowly warmed sperm.nnnCONCLUSIONSnThese data support the hypotheses that a two-factor hypothesis of cryodamage applies to human spermatozoa and that an interaction exists between cooling rate and warming rate. These data also suggest that motility, plasma membrane integrity, and mitochondrial function are not differently affected by cooling and warming during cryopreservation.

Reproductive tissue banking: Scientific principles

Utility of Viable Tissues Ex Vivo: Banking of Reproductive Cells and Tissues. Tissue Maturation in Vivo and in Vitro: Gamete and Embryo Ontogeny. Metabolic Support at Normothermia. Pharmacological Intervention in Vitro. Hypothermia and Mammalian Gametes. Fundamental Cryobiology of Mammalian Spermatozoa. The Cryobiology of Mammalian Oocyte. Cryopreservation of Multicellular Embryos and Reproductive Tissues. Genome Resource Banking: Impact on Biotic Conservation and Society. Implications of Tissue Banking for Human Reproductive Medicine. Subject Index.

Effect of developmental stage on bovine oocyte plasma membrane water and cryoprotectant permeability characteristics

Knowledge of bovine oocyte plasma membrane permeability characteristics at different developmental stages in the presence of cryoprotective agents (CPAs) is limited. The objective of this study was to determine the oolema hydraulic conductivity (Lp), cryoprotectant permeability (PCPA), and reflection coefficient (σ) for immature (germinal vesicle stage, GV) and in vitro–matured (metaphase II, MII) bovine oocytes. Two commonly used cryoprotective agents, dimethyl sulfoxide (DMSO) and ethylene glycol (EG), were studied. Osmometric studies were performed using a micromanipulator connected to an inverted microscope at 22 ± 2°C. Each oocyte was immobilized via a holding pipette, and osmotically induced volume changes over time (dv/dt) were recorded. The Lp values for GV and MII oocytes in DMSO (LpDMSO) were 0.70 ± 0.06 and 1.14 ± 0.07 μm/min/atm (mean ± SEM) and in EG (LpEG) were 0.50 ± 0.06 and 0.83 ± 0.07 μm/min/atm, respectively. Estimates of PDMSO for GV and MII oocytes were 0.36 ± 0.03 and 0.48 ± 0.03 μm/sec, and PEG values for GV and MII oocytes were 0.22 ± 0.03, 0.37 ± 0.03 μm/sec, respectively. The σ values for GV and MII oocytes in DMSO (σDMSO) were 0.86 ± 0.03 and 0.90 ± 0.04 and in EG (σEG) were 0.94 ± 0.03 and 0.76 ± 0.04, respectively. These data demonstrate that bovine oolema permeability coefficients to water and cryoprotectants change after in vitro maturation. Furthermore, the bovine oocyte PDMSO is higher than the PEG. These results may provide a biophysical basis for developing criteria for choosing optimal CPAs and for minimizing damage during addition and removal of the CPAs. Additionally, these data support the hypothesis that different procedures may be required for optimal cryopreservation of different oocyte developmental stages. Mol. Reprod. Dev. 49:408–415, 1998.

Development of a novel microperfusion chamber for determination of cell membrane transport properties

A novel microperfusion chamber was developed to measure kinetic cell volume changes under various extracellular conditions and to quantitatively determine cell membrane transport properties. This device eliminates modeling ambiguities and limitations inherent in the use of the microdiffusion chamber and the micropipette perfusion technique, both of which have been previously validated and are closely related optical technologies using light microscopy and image analysis. The resultant simplicity should prove to be especially valuable for study of the coupled transport of water and permeating solutes through cell membranes. Using the microperfusion chamber, water and dimethylsulfoxide (DMSO) permeability coefficients of mouse oocytes as well as the water permeability coefficient of golden hamster pancreatic islet cells were determined. In these experiments, the individual cells were held in the chamber and perfused at 22 degrees C with hyperosmotic media, with or without DMSO (1.5 M). The cell volume change was videotaped and quantified by image analysis. Based on the experimental data and irreversible thermodynamics theory for the coupled mass transfer across the cell membrane, the water permeability coefficient of the oocytes was determined to be 0.47 micron. min-1. atm-1 in the absence of DMSO and 0.65 microns. min-1. atm-1 in the presence of DMSO. The DMSO permeability coefficient of the oocyte membrane and associated membrane reflection coefficient to DMSO were determined to be 0.23 and 0.85 micron/s, respectively. These values are consistent with those determined using the micropipette perfusion and microdiffusion chamber techniques. The water permeability coefficient of the golden hamster pancreatic islet cells was determined to be 0.27 microns. min-1. atm-1, which agrees well with a value previously determined using an electronic sizing (Coulter counter) technique. The use of the microperfusion chamber has the following major advantages: 1) This method allows the extracellular condition(s) to be readily changed by perfusing a single cell or group of cells with a prepared medium (cells can be reperfused with a different medium to study the response of the same cell to different osmotic conditions). 2) The short mixing time of cells and perfusion medium allows for accurate control of the extracellular osmolality and ensures accuracy of the corresponding mathematical formulation (modeling). 3) This technique has wide applicability in studying the cell osmotic response and in determining cell membrane transport properties.

The Cryobiology of Mammalian Oocytes

Publisher Summary This chapter describes the cryobiology of mammalian oocytes. Oocytes present with a relatively complex subcellular structure within which many of the subcellular components are particularly temperature and osmotically sensitive. It has been reported that cumulus cells and tranzonal processes have a metabolic role and also play an important part in the development of immature oocytes. There are subtle, but potentially critical, subcellular differences which exist among oocytes from different species. It is found that fundamental changes in the structure and function of the oocyte occur as they develop from the germinal vesicle (GV) stage to the MII stage. It is apparent that effective cryopreservation of bovine oocytes will certainly enhance the utilization of oocytes from animals with high genetic value. It is found that the lipid phase transition of the membrane lipids for GV stage oocytes is 13°C–20°C no phase transition was observed in MII stage oocytes. Comparison of the cytoskeletal systems of murine and human oocytes has indicated significant differences in the organization in their respective responses to cooling and exposure to cryoprotectant compounds. It is suggested that if oocyte cryopreservation is to become a safe and efficacious clinical application in the rapidly growing assisted reproductive technology field, an in-depth understanding of the biophysical factors which lead to success or failure must be developed.

Cryobiology of Rat Embryos II: A Theoretical Model for the Development of Interrupted Slow Freezing Procedures

Abstract Current mammalian embryo cryopreservation protocols typically employ an interrupted slow freezing (ISF) procedure. In general, ISF consists of initial slow cooling, which raises the extracellular solute concentration, and results in cell dehydration. Permeating cryoprotective agents (CPAs), such as dimethyl sulfoxide (DMSO), are typically included in the medium to protect the cells against high solute concentrations. As this ISF procedure continues, slow cooling is terminated at an intermediate temperature (Tp), followed by plunging into liquid nitrogen (LN2). If the slow cooling step allowed a critical concentration ([CPA]c) of CPA to be reached within the cell, the CPA will interact with the remaining intracellular water during rapid cooling, resulting in the majority of the intracellular solution becoming vitrified and preventing damaging intracellular ice formation (IIF). This study presents a theoretical model to develop efficient ISF procedures, on the basis of previously developed data for the rat zygote. The model was used to select values of initial CPA concentrations and slow cooling rates (from initial estimated ranges of 0 to 4 molal DMSO and 0 to 2.5°C/min cooling rates) that would allow the intracellular solute concentration to exceed the critical concentration. The optimal combination was then determined from this range based on minimizing the duration of slow cooling.

Human spermatozoa glycerol permeability and activation energy determined by electron paramagnetic resonance

The permeability of human spermatozoa to glycerol and its activation energy were determined using electron paramagnetic resonance (EPR) techniques. EPR was used to monitor the aqueous cell volume change vs. time during the glycerol permeation process using the aqueous spin label 15N-tempone and the membrane impermeable broadening agent potassium trioxalatochromiate (chromium oxalate). The permeation process was completed in tens of seconds, requiring the use of a stopped-flow methodology. The glycerol permeability coefficient (Pg) was determined by fitting a simple theoretical model to the experimental data. The permeabilities of human spermatozoa in 1 molar and 2 molar glycerol at 20 degrees C are (10.3 +/- 0.3).10(-4) cm/min (mean +/- S.D.) and (6.0 +/- 1.4).10(-4) cm/min, respectively. The permeabilities of human spermatozoa in 2 molar glycerol at 30, 20, 10, and 0 degrees C are (8.3 +/- 1.3).10(-4) cm/min, (6.0 +/- 1.4).10(-4) cm/min, (2.1 +/- 0.4).10(-4) cm/min, and (1.1 +/- 0.3).10(-4) cm/min, respectively. The activation energy (Ea) for glycerol permeation between 30 degrees C and 0 degrees C was found to be 11.6 kcal/mol.

Cryobiology of Rat Embryos I: Determination of Zygote Membrane Permeability Coefficients for Water and Cryoprotectants, Their Activation Energies, and the Development of Improved Cryopreservation Methods

Abstract New rat models are being developed at an exponential rate, making improved methods to cryopreserve rat embryos extremely important. However, cryopreservation of rat embryos has proven to be difficult and expensive. In this study, a series of experiments was performed to characterize the fundamental cryobiology of rat fertilized 1-cell embryos (zygotes) and to investigate the effects of different cryoprotective agents (CPAs) and two different plunging temperatures (Tp) on post-thaw survival of embryos from three genetic backgrounds. In the initial experiments, information on the fundamental cryobiology of rat zygotes was determined, including 1) the hydraulic conductivity in the presence of CPAs (Lp), 2) the cryoprotectant permeability (PCPA), 3) the reflection coefficient (σ), and 4) the activation energies for these parameters. PCPA values were determined for the CPAs, ethylene glycol (EG), dimethyl sulfoxide (DMSO), and propylene glycol (PG). Using this information, a cryopreservation method was developed and the cryosurvival and fetal development of Sprague-Dawley zygotes cryopreserved in either EG, DMSO, or PG and plunged at either −30 or −80°C, were assessed. The highest fetal developmental rates were obtained using a Tp of −30°C and EG (61.2% ± 2.4%), which was not different (P > 0.05) from nonfrozen control zygotes (54.6% ± 3.0%).

Tissue Maturation in Vivo and in Vitro: Gamete and Early Embryo Ontogeny

Publisher Summary This chapter discusses various aspects of tissue maturation in vivo and in vitro. The primary oocyte stage of gamete differentiation in most female mammals is reached during fetal life after proliferation of primordial germ cells and oogonia with subsequent entry into meiosis and transformation of the gamete into the primary oocyte. Primary oocytes from tertiary follicles are most frequently recovered postmortem, surgically, with the aid of a laparoscope or most recently through ultrasound-guided follicular aspiration. Each in vitro protocol has its own rate of success and the outcome of the total process will be a multiplicative factor comprising all the steps. A system for producing bovine embryos in vitro has actually worked at some level of efficiency for a number of nondomestic ungulates. The buffering system in the medium selected for maturation will dictate the CO2 concentration required for culture. Macromolecular supplements used in tissue culture perform two basic roles. One of the roles is to provide a fixed source of nitrogen and the other is to reduce stickiness of the cell surfaces to make handling and manipulation easier. The follicular and ovarian factors affecting oocyte competency are also elaborated.