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The effect of liposomes on thermotropic membrane phase transitions of bovine spermatozoa and oocytes: implications for reducing chilling sensitivity
Cryobiology 45 (2002) 143–152 www.academicpress.com
The effect of liposomes on thermotropic membrane phase transitions of bovine spermatozoa and oocytes: implications for reducing chilling sensitivityq Y. Zeron,a M. Tomczak,b J. Crowe,b and A. Arava,* a
Institute of Animal Science, Agricultural Research Organization (ARO), The Volcani Center, Bet Dagan, 50250, Israel b Section of Molecular and Cellular Biology, University of California, Davis, CA 95616, USA Received 2 January 2002; accepted 2 October 2002
Abstract We have examined the effects of combinations between egg-phosphatidylcholine (EPC) or dipalmitoylphosphatidylcholine (DPPC) liposomes with either bovine spermatozoa or oocytes on cellular chilling sensitivity, lipid phase transition temperature (Tm ), and the ability of the oocytes to develop to the blastocyst stage. Spermatozoa and oocytes were exposed to EPC and DPPC liposomes at various temperatures (spermatozoa: 4, 12, 16, and 25 °C; oocytes: 4, 16, and 32 °C). The membrane integrity of the spermatozoa-control group decreased significantly following exposure to 16 or 12 °C, compared to ambient temperature (25 °C). In contrast, the EPC-sperm group had a greater resistance to chilling at each temperature and showed a decline in membrane integrity only at the lowest temperatures investigated. However, the DPPC-sperm group was injured significantly at all temperatures tested. Similar to the sperm, oocytes from the control group that were exposed to 16 °C were injured more severely than oocytes that were electrofused with EPC or DPPC liposomes. The membrane integrity of the oocytes at 16 °C that were electrofused with either EPC or DPPC liposomes was approximately the same as the control group held at 32 °C (normalized to 100%), compared to 46% in the control group at 16 °C (P < 0:01). The transition temperatures of the sperm and oocyte membranes revealed different Tm for the different liposome treatments. All groups had a significantly higher cleavage rate, as well as increased blastocyst formation when oocytes were exposed to temperatures above or below their Tm . We suggest that the Tm of spermatozoa or oocytes can be changed by spontaneous association or electrofusion of liposomes with cellular membranes and, consequently, the chilling sensitivity can be altered. The resulting possibility is that embryo development after cryopreservation could be improved with such a method. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Liposomes; Chilling sensitivity; Lipid phase transition; Membrane integrity; Oocytes; Sperm
q This work was funded in part by a grant from the US–Israel Binational Agricultural Research and Development Fund (BARD-2528-96). Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel. No. 401/01. * Corresponding author. Fax: +972-8-947-5075. E-mail address: [email protected] (A. Arav).
Chilling injury to mammalian gametes was first described in the 1930s as the irreversible damage that occurs upon cooling to low, but nonfreezing, temperatures (referred in [43]). Several studies suggested that injury at sub-physiological
0011-2240/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 1 1 - 2 2 4 0 ( 0 2 ) 0 0 1 2 3 - 2
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temperatures, termed chilling injury, is the major limiting factor for successful cryopreservation of gametes [2,24,33]. Other studies suggested that membranes are the primary site for structural and functional chilling injury in sperm [10,11] and oocytes [2]. Additionally, membrane chilling injury is dependent on the biochemical, and biophysical properties of membranes [39,43]. Phospholipid (PL) fatty acids (FA) are a major structural component of biological membranes and affect both the biophysical properties and functions of the cell [31,39]. Previous studies showed that palmitic (C16:0) and oleic (C18:1) acids were the most abundant fatty acids in the phospholipid fraction of oocytes from cattle [16,46], pigs [15] and sheep [7], and may function as energy reserves [40] or as precursors for the elongation of long chain fatty acids [23]. These studies also showed that polyunsaturated fatty acid (PUFA) was found in low concentrations in oocyte membranes. In contrast to oocytes, the spermatozoa of most mammals contain high concentrations of PUFA [26,29], which could partially account for the difference in fluidity between spermatozoa and oocyte membranes. Recently we showed that oocyte membrane fluidity is affected by temperature alterations between seasons, as well as by changes in FA composition [46]. This is consistent with other studies suggesting an association between FA composition and membrane fluidity [37,39]. The thermotropic lipid phase transition (Tm ) from the fluid liquid crystalline phase to the more rigid gel phase [8] is associated with chilling injury in sperm cells [3,11]. The temperature at which Tm occurs is greatly affected by the FA composition of the membrane [3], and chilling injury is maximized in the temperature range surrounding Tm [45]. Membranes that have lipids with multiple unsaturations or short carbon chains are more fluid at low temperatures [32,44] and, therefore, will be more chilling resistant. Furthermore, Arav et al. [3] suggested that the unsaturated FA content of PUFA was different between gamete membranes in different species and that an increase in the unsaturated FA content in PUFA had a major influence on this difference in Tm . There are several ways to change the chilling sensitivity and Tm in mammalian gametes. Various reports showed that enrichment of diet with PUFA can alter the biophysical properties or reproductive performance of sperm [4] and oocytes [38,47]. Other studies showed that depletion of intracellular lipids changed the chilling suscepti-
bility of oocytes in vitro [25,27] but they did not clarify how it affected the lipid membrane composition. Spermatozoa can spontaneously interact with liposomes as described in several studies [9,12,13,28,34]. Previous work suggested that phospholipids adsorbed on to the sperm cells, which were then cooled to 4 °C, increased the chilling resistance of the sperm cells from bovine [28] and ram [34]. Using 14 C-labeled egg yolk, Foulkes [13] reported that the radioactive label remained associated with bovine sperm lipids. It is well documented that liposomes prepared with different acyl chains have different Tm [6]. Fusing liposomes that have different Tm to gametes may influence their biophysical properties and decrease their chilling sensitivity. In the present study, we examined the effects of altering the lipid composition of spermatozoa and cumulus-oocyte complexes (COCs) on chilling injury, Tm , and the ability of these gametes to maintain developmental competence. Spermatozoa were coincubated with liposomes under conditions that result in spontaneous fusion. COCs were coincubated with liposomes and subjected to electrofusion.
Materials and methods Chemicals and lipids Unless stated otherwise, all chemicals used were obtained from Sigma Chemical (St. Louis, MO, USA). Egg-phosphatidylcholine (EPC), which is composed of the following fatty acids: 16:0 (34%), 16:1 (1.7%), 18:0 (11%), 18:1 (32%), 18:2 (18%), and 20:4 (3.3%), and dipalmitoylphosphatidylcholine (DPPC, 16:0) in chloroform were purchased from Avanti Polar Lipids (Alabaster, Alabama, USA). Oocytes Oocytes collection and in vitro maturation Ovaries were obtained at a local abattoir from slaughtered Holstein cows and placed in an insulated vessel containing physiological saline (0.9% NaCl, w/v) with 1 106 IU/ml of penicillin and 1 106 IU/ml of streptomycin at 32–36 °C. The ovaries were washed with 0.9% NaCl (w/v) and their centers were cut as previously described [46]. Cumulus-oocyte complexes were aspirated from the follicles using an 18 g needle on a 10 ml syringe and expelled into 65 mm petri dish (Corning Glass Works, NY, USA) containing Hepes-TALP
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(H-TALP). Oocytes with three or more layers of cumulus cells surrounding a homogeneous cytoplasm were used for electofusion treatments or maturation studies. For maturation studies, 40–60 COCs per well were placed in a 4-well culture multidish (Nunc, Denmark) in 500 ll of maturation medium consisting of 25 mM Hepes TCM199, supplemented with 10% (v/v) heat-inactivated fetal calf serum (Bio-Lab, Jerusalem, Israel), 0.2 mM Na pyruvate, 5 lg/L gentamicin, 10 lg/ml oLH (NIADDK-NIH-26, AFP5551B, Bethesda, MD), 1 lg/ml oFSH (NIADDK-NIH-20, AFP 7028D, Bethesda, MD) and 1 lg/ml estradiol [17]. COCs were then incubated for 24 h at 39 °C in a humidified atmosphere of 5% CO2 in air. Activation and development of oocytes After maturation, oocytes were examined for their ability to develop to the blastocyst stage after activation, as previously described [20]. Briefly, COCs were denuded from cumulus cells in the maturation wells and placed for 5 min in the ionomycin medium: 10 ml of TCM-199 supplemented with 25 mM Hepes, 10% (v/v) heat-inactivated FCS, 0.2 mM sodium pyruvate, 5 lg gentamicin, and 5 lM of ionomycin. Oocytes were transferred to 6-dimethylaminopurine (6-DMAP) medium (10 ml of TCM 199 supplemented with 2 mM of 6-DMAP) for 4.5 h. The oocytes were then washed three times in a cleavage medium (Sydney IVF Cleavage Medium, Cook, Australia) and transferred in groups of 10 into 50 ll cleavage drops under mineral oil. Cleaved embryos were counted on day 4 (activation day ¼ 0) and transferred to 50 ll drops of blastocysts medium (Sydney IVF Blastocyst Medium, Cook, Australia) under mineral oil. Blastocysts were counted after 8–10 days. Sperm Semen from Israeli Holstein Friesian bulls (five-years-old) was collected once a week between January and March at the Sion Artificial Insemination Center and transported (undiluted semen) with liposomes (see below) to the Lab within 30 min at 25 °C. Chilling and viability staining of oocytes and sperm Cumulus-oocyte complexes were aspirated in 10 ll of H-TALP (without BSA) into a mouthoperated micropipette and expelled into 1 ml of H-TALP precooled to 4 or 16 °C for 15 min as previously described [45]. Subsequently, COCs
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were placed immediately in TCM-199, supplemented with 10% heat inactivated FCS, 0.2 mM Na pyruvate, 5 lg/L gentamicin, and incubated for 1 h at 39 °C in a humidified atmosphere of 5% CO2 . Following incubation, COCs were denuded and exposed to 5-carboxyfluorescein diacetate (cFDA) (Molecular Probes, Leiden, The Netherlands) as previously described [45]. After being washed with H-TALP, the oocytes were placed on a slide under a coverslip and cFDA fluorescence was evaluated using a fluorescence microscope connected to a CCD video camera (Applitec LIS700, Israel) and imaging data acquisition software (NIH-1.55, MD, USA). The levels of fluorescence intensity were determined using the imaging data processing software on a PowerPC 7100/80 computer. Mean intensity of the pixels from each treatment were normalized to controls. Sperm (10 ll sperm in a glass tube) were incubated in 11 lg/ml of cFDA for 20 min at room temperature, followed by immersion into a precooled 1 L water bath (4 °C, 12 °C or 16 °C), or incubated at 25 °C (control) for 30 min, after which all samples were washed with H-TALP. The number of live (fluorescent) sperm were then determined using a fluorescence microscope connected to a CCD video camera. Liposome preparation and introducing them with bovine COCs and sperm EPC or DPPC (20 mg each) were dried under nitrogen gas, and subsequently hydrated to 100 mg/ml in 200 ll of H-TALP supplemented with 0.5% PVP (w/v) at 25 °C. The suspension was vortexed for 10 min and then loaded into a glass syringe in a hand held extruder (Avestin, Ottawa, Canada; [21]). The lipid suspension was extruded through 0.1 lm pore filters and then placed into a 3 ml glass tube. After addition of 0.04% (v/v) phytohemaglutinin to the liposomes, the tubes were purged with nitrogen gas and the liposomes (0.1 lm diameter) were stored at ambient temperature in the dark. COCs (50/group) were incubated for 5 min in 100 ll of unilamellar liposome solution at 25 °C, followed by transfer into 200 ll of non-electrolyte fusion solution (0.3 M mannitol, 0.05 mM Caþþ , and 0.1 mM Mgþþ supplemented with 10 ll liposome solution) in the electrofusion chamber (CF150 chamber, Biochemical Laboratory Service, Budapest, Hungary). Electrofusion of 10 COCs/ group was done in 3 pulses of 1.5 kV (DC), each lasting 80 ls. Subsequently, the electrofused COCs were divided into two groups: half were chilled to
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16 °C as described by Zeron et al. [45], and half were exposed to 32 °C for 15 min. All COCs were then washed 3 times in maturation medium and examined for their membrane integrity or development as described in Materials and methods. Spermatozoa (3 ml pooled from three different bulls in concentration of 1:1 106 cells/ml) were incubated with EPC or DPPC liposomes (100 mg/ ml) for 30 min at room temperature, followed by exposure to low temperatures as described above. Measurements of membrane phase transitions The membrane phase transition of oocytes and sperm were determined with a Bruker-Equinox 55 Fourier transform infrared (FTIR) spectrometer connected to a Bruker FTIR microscope A590 (Ettlingen, D-76275, Germany), as previously described [46]. The sperm were concentrated by centrifugation (12,000g, 10 min). Oocytes or pelleted sperm were placed between two sapphire windows. Sample temperature was regulated by a microprocessor feedback system, which measures sample temperature with a thermocouple placed directly on the surface of the windows. The temperature was adjusted to the desired level, within 0.1 °C, and allowed to equilibrate for 2 min before the sample was scanned. The temperature was controlled with two thermoelectric coolers placed on the microscopic stage using directional solidification (IMT, Israel). The FTIR spectra were baseline subtracted to monitor the frequency of the symmetric CH2 stretching vibration of the lipid fatty acyl chains. The center of the lipid phase transition curve from liquid crystalline to gel phase is designated Tm , and is determined by statistical analysis as described by Crowe et al. [8].
(Fig. 1). After exposure to 16 or 12 °C for 30 min, the percentage of sperm cells with intact membranes decreased by 54 and 57%, respectively, compared with the control sperm cells that were held at 25 °C. In contrast, the sperm cells that interacted with EPC liposomes had a greater resistance to chilling at each temperature. The EPCsperm cells did show a slight decline in membrane integrity at 16 °C (P < 0:1) and a significant decline at 12 and 4 °C compared to the controls incubated at ambient temperature. At 25 °C there was already significant injury to the DPPC-sperm cells compared to the control and EPC groups, and their membrane integrity decreased as the incubation temperature decreased (Fig. 1). These differences led us to evaluate the biophysical characteristics of the sperm cell membranes. The transition temperatures of the sperm cell membranes from the liquid crystalline to the gel phase revealed a different Tm for the different liposome treatments (Fig. 2). The control group had a Tm around 13 °C, in contrast to higher Tm of the sperm cells incubated with DPPC, which was centered on 23 °C. However, the wave number of the sperm cells incubated with EPC decreased only slightly below 2852 cm1 at the lowest temperatures investigated, which indicates that the sperm membrane was still in the liquid crystalline phase and that the transition temperature was lower than 4 °C.
Statistics Means were calculated and differences between treatments examined by t tests using the General Linear Model procedure of JMP [35]. Significance was P < 0:05 unless otherwise stated.
Results The effect of different liposomes on sperm chilling sensitivity and lipid phase transitions Sperm and oocytes showed different responses to chilling. The membrane integrity (measured by cFDA fluorescence intensity) of the control group of sperm cells declined substantially following exposure to hypothermic temperature for 30 min
Fig. 1. The effect of different temperatures on sperm cell membrane integrity after treatment with different liposomes (egg-phosphatidylcholine, EPC; dipalmitoylphosphatidylcholine, DPPC) for 30 min. Liposomes (100 mg/ ml) were added to the sperm cells immediately after ejaculation. Membrane integrity was determined using cFDA (11 lg/ml). Values presented were normalized to the control at 25 °C from pixel values of cFDA fluorescence. Each column represents data pooled from three different bulls (five-years-old) in 4 replicates. Bars represent SE and values with a common letter do not differ significantly between treatments at P < 0:05.
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Fig. 2. The effect on sperm membrane lipid phase transitions after exposure to different liposomes. The sperm cells were collected from bulls (five-years-old), and egg-phosphatidylcholine (EPC) or dipalmitoylphosphatidylcholine (DPPC) were added (100 mg/ml) immediately after ejaculation. The phase transition of sperm cell membranes was measured by Fourier transform infrared (FTIR) microscopy. The center of the lipid phase transition curve, from liquid crystalline to gel phase is designated Tm . Tm were calculated from the frequencytemperature plots according to Crowe et al. [8].
The effect of different liposomes on oocyte chilling sensitivity and lipid phase transition The effects of short term chilling on the membrane integrity of oocytes following electrofusion of different liposomes with the oocytes were determined (Fig. 3). Preliminary experiments of introducing liposomes composed of various lipids to the COCs without electrofusion revealed the same results as the control group (data not shown).
Fig. 3. The effect on oocyte membrane integrity after electofusion with different liposomes. Membrane integrity of oocytes at the germinal vesicle stage following exposure to egg-phosphatidylcholine (EPC) or dipalmitoylphosphatidylcholine, (DPPC) liposomes and incubated at 32 °C (white column) or 16 °C (black column) were determined using cFDA. Values presented were normalized to the control at 32 °C from pixel values of cFDA fluorescence. Each column represents data pooled from 110 15 oocytes in the different treatments (5 replicates). Bars represent SE and values with a common letter do not differ significantly at P < 0:05.
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Oocytes from the electrofusion control group that were exposed to 16 °C for 15 min were injured more severely than oocytes that were electrofused with EPC or DPPC liposomes. All results were normalized to the control group at 32 °C to make a direct comparisons between treatments, therefore some of the percentages are greater than 100%. The membrane integrity of the oocytes at 16 °C that were electrofused with either EPC or DPPC liposomes was 107 and 92%, respectively, compared to 46% membrane integrity in the control group at 16 °C (P < 0:01; normalized values). Furthermore, under chilling conditions the oocytes electrofused with either EPC or DPPC liposomes had similar membrane integrity as the control oocytes that were incubated at 32 °C (Fig. 3). However, the DPPC group revealed a significant decrease in membrane integrity when incubated at 32 °C. These results indicate that the sensitivity of the oocytes to chilling was different after the different liposome treatments. The electrofusion process by itself did not improve the chilling resistance of oocytes, as the oocytes electrofused without liposomes showed no increased chilling tolerance (electrofusion control, Fig. 3). Therefore, we examined the Tm of the oocyte membranes after electrofusion with liposomes and, subsequently, their ability to develop to the blastocyst stage. The effect of liposomes on bovine oocyte lipid phase transitions and on chilling sensitivity is shown in Fig. 4. The control oocytes had a different phase transition pattern from the liquid crystalline to the gel phase than the electrofused oocyte–liposome groups. The Tm of the control oocytes (control group, Fig. 4A) was 16 °C, compared to a Tm of less than 4 °C in the electrofused EPC-oocytes (EPC group, Fig. 4B), and at 25 °C in the electrofused DPPC-oocytes (DPPC group, Fig. 4C). The wave number excursion between groups was also different: all groups had an initial wave number in the liquid crystalline phase between 2854 and 2853 cm1 , but only the control and DPPC groups had endpoint wave numbers characteristic of the gel phase (between 2851.2 and 2850.8 cm1 , Figs. 4A and C). The sensitivity of oocytes to chilling was measured by their ability to cleave and develop to the blastocyst stage after in vitro maturation and chemical activation at 39 °C. All groups had a significantly higher cleavage rate, as well as increased blastocyst formation when oocytes were exposed to temperatures above or below their Tm , but not after incubation at temperatures in the
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had significantly higher rates of cleavage and blastocyst formation after exposure to 16 °C (39 and 9%, respectively), compared to the control group. By contrast, after exposure to 32 °C the oocytes electrofused with DPPC liposomes had a significantly lower rate of cleavage and development to the blastocyst stage (1 and 0.2%, respectively) compared to the control group. Again, exposure of this group to 16 °C resulted in a significantly higher rate of cleavage and blastocyst formation (40 and 10%, respectively).
Discussion
Fig. 4. The effect of electrofusion on the lipid phase transition and embryo development of bovine oocytes subjected to chilling temperatures. Germinal vesicle oocytes (control, A) and oocytes electrofused with eggphosphatidylcholine, (EPC, B) or dipalmitoylphosphatidylcholine, (DPPC, C) (n ¼ 300, 275 and 285, respectively) were exposed to temperatures of 4, 16, and 32 °C (control) or 16 and 32 °C (EPC and DPPC groups) for 15 min. Chilling tolerance was determined by their ability to cleave after chemical activation (2–4 cell stage, white columns) and developed to the blastocyst stage (black columns). Bars represent SE and values with a common letter (small letters between cleavage stage; capital letters between blastocysts stage) do not differ significantly at P < 0:05. The phase properties of cytoplasmatic membranes of oocytes (n ¼ 13, each group) were measured by Fourier transform infrared (FTIR) microscopy. The center of the lipid phase transition curve, from liquid crystalline to gel phase is designated Tm . Tm were calculated from the frequency-temperature plots according to Crowe et al. [8].
phase transition range. In the control group, the rate of cleavage and development to the blastocyst stage was higher after exposure to 32 °C (68 and 18%, respectively), compared to incubation at 16 °C (20 and 1%, respectively; Fig. 4A). However, the oocytes electrofused with EPC liposomes
During cryopreservation cells are exposed to low temperatures and therefore are vulnerable to chilling injury [18,43]. Chilling damage has been studied extensively in sperm [24,42]. One possible cause of chilling injury is the membrane damage that is associated with the thermotropic lipid phase transition. De Leeuw et al. [10] examined the ultrastructural changes in different sperm plasma membranes and showed that at reduced temperatures the intramembranous components aggregated and redistributed in the head and principal piece of the sperm. This led to phenomenon known as lateral phase separation. Drobnis et al. [11] emphasized this hypothesis with results showing that spermatozoa chilling injury is associated with the membrane lipid phase transition and suggested lateral phase separation to be the main cause of damage to the membrane. Phospholipid fatty acid composition influences the phase transition profile of the sperm membranes. In the present study we presented evidence that spontaneous association between spermatozoa and liposomes occurs. Mixing two types of lipids with two different Tm Õs results in a single Tm intermediate between the two. Contrary, if the two populations are not mixed, two Tm Õs are observed at the original values for the two lipids. We clearly showed that one cooperative phase transition that occurred for both DPPC and EPC treated sperm cells. Several studies have examined the lipid and fatty acid composition in spermatozoa membranes from different species [3,14,29]. Similar to a previous report [3], we found that the lipid phase transition of the spermatozoa occurs over a broad temperature range, which suggests the presence of a variety of fatty acids in the membrane as well as a high content of long chain fatty acids. The present study has shown that sperm cells were extremely sensitive to chilling, but were less
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chilling sensitive after the addition of EPC liposomes. The EPC liposomes decreased the lipid phase transition of the spermatozoa to lower than 4 °C, from 13 °C in the control sperm cells. Exposure of the EPC-sperm cells to higher temperatures than lipid phase transition temperature range, followed by chilling to 16 °C did not cause damage to the membranes. Presumably, damage was avoided because the EPC-sperm had not passed through their Tm , so the injury that would result normally was avoided. In artificial insemination centers, the sperm freezing protocol starts by slowly chilling the sperm cells, in the presence of egg-yolk, to temperatures between 4 and 5 °C (< 1 °C/min) and holding them at that temperature. This protocol requires that association of the liposomes to spermatozoa would have to occur under these conditions (probably spontaneously) to be effective for cryoprotection. Evidence for the spontaneous association between exogenous lipids and the spermatozoa plasma membrane have been shown by radioactive labeling [13]. However, this is a complex process, dependent on temperature and cooling rate [9], and more work will have to be done to optimize the conditions for increased sperm survival after cryopreservation. Egg phosphatidylcholine liposomes also might stabilize the sperm as they spontaneously associate or interact with the membrane [12]. Thus, the current results showing that EPC-sperm have greater survival than the controls supports the previous findings. In contrast, the DPPC-sperm group showed greater membrane sensitivity at all temperatures investigated. This is probably due to the exposure of this group to temperatures at which the membrane was in the center of the phase transition range (Tm ¼ 25–30 °C). Additionally, chilling this group to lower temperatures did not reverse the damage that occurred at 25 °C. The chilling sensitivity of oocytes is well documented [2,22,30,45] and, consequently, their successful cryopreservation is limited. Studies conducted on the chilling sensitivity of mature oocytes (MII stage) found that the main damage occurred due to meiotic spindle disorganization followed by microtubule depolymerization [1,36]. Cryopreservation of immature oocytes could be a solution for this problem, as oocytes do not contain polymerized microtubules at this stage. However, immature oocytes at the germinal vesicle stage (GV) are extremely sensitive to chilling temperatures, with the damage occurring at the cytoplasmic membrane [2]. Recently, evidence
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describing the influence of phospholipid fatty acid composition on the biophysical properties of immature oocyte membranes was reported [3,46]. These studies showed that the ratio of unsaturated to saturated fatty acids and the position of the double-bond in the carbon chain, and, thus, the transition temperature of the membranes, affected their chilling sensitivity. The present study has shown that chilling sensitivity changed after electrofusion of the oocytes with liposomes prepared from egg phosphatidylcholine. We exposed the oocytes, surrounded by their cumulus cells, to liposomes (electrofusing and chilling procedures) as it was shown that cumulus cells are essential for maturation [5]. We used very small size of liposomes (0.1 ll) to facilitate penetration of the liposomes through the zona pellucida, as it was previously described that zona pellucida pores are 187 ll in diameter [41]. Therefore, the changes observed clearly were due to an alteration in the cellular membrane fluidity, caused by electrofusion with the liposomes. We observed greater than 10% survival of blastocyst following chilling of EPC or DPPC-oocytes compared with the unchilled control oocytes. Electrofusing EPC liposomes with GV oocytes changed the composition of unsaturated fatty acids present in the membrane and, therefore, changed the physical properties of the oocytes. We assume the oocytes membranes were saturated with exogenous lipids following electrofusion due to the large ratio of exogenous lipid to endogenous membrane lipid. Our present study shows evidence of changes in oocyte membrane fluidity following electrofusion with liposomes to change their lipid composition. Our direct evidence that the liposomes were fused to the oocytes is the significant changes in both the Tm of the lipid phase transition and the chilling sensitivity. In addition, the lipid phase transition of control COCs (approximately 16 °C) and of EPC or DPPC alone (Tm below 0 and 40 °C, respectively [19]), are very different from the lipid phase transitions we found after electrofusion with these liposomes. We showed that the oocyte Tm was altered after electrofusion with EPC and DPPC liposomes. The resulting Tm of the EPC group was lower, and of the DPPC group higher, than the control oocytes. Recently, we have shown that bovine oocytes are sensitive to chilling around the temperature range where the Tm occurs (Tm centered at 16 °C) and less sensitive at temperatures above or below this range [45]. Exposing spermatozoa or oocytes to the lipid
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phase transition temperature damaged their membrane integrity and physiological functions. In the present study we showed that oocyte membrane integrity was maintained after exposure to 16 °C, which was lower (DPPC group) or higher (EPC group) than the control-group Tm . However, it is not clear why membrane integrity was lower when oocytes from the DPPC-group exposed to 32 °C compared to 16 °C. One explanation may be that the DPPC-electrofused oocytes are damaged during incubation at temperatures near the lipid phase transition temperature, at which the oocytes were incubated (39 °C). The alteration of Tm affected the developmental ability of the embryos after low temperature exposure. The EPC and DPPC groups had significantly higher cleavage rates and blastocyst formation than the control group after exposure to temperature of 16 °C. This finding confirms results from other studies that associated Tm and chilling sensitivity with fatty acids composition [3,11]. However, changing the Tm to higher temperature with DPPC liposomes decreased the rate of embryo development. Oocytes and sperm have different Tm profiles, due to differences in fatty acid and lipid composition in fatty acid and lipid composition [3,16,23,29,46]. These studies showed that sperm have a greater diversity of fatty acids than oocytes. Particularly, sperm have a high percentage of lipids with long polyunsaturated fatty acids, while oocytes almost exclusively have lipids with fully saturated fatty acids. We also showed that the oocyte Tm profile changed after liposome treatments, and this is due probably to the resulting alteration of the fatty acid composition in the membrane. We suggest that the lipid phase transition of spermatozoa or oocytes can be changed by the addition of liposomes and consequently, the chilling sensitivity could be altered. These finding may improve cryopreservation of gametes as well as embryonic development after cryopreservation.
Acknowledgments We would like to express our gratitude to Professor David Sklan for fruitful comments on the manuscript. The author thanks Ana Ocheretny for technical assistance. Thanks are expressed to Sion A.I. Center for donating the bovine semen used in this study and NHPP for generous provision of FSH and LH.
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[43] P.F. Watson, G.J. Morris, Cold shock injury in animal cells, Symp. Soc. Exp. Biol. 41 (1987) 311– 340. [44] I.G. White, Lipids and calcium uptake of sperm in relation to cold shock and preservation: a review, Reprod. Fertil. Dev. 5 (1993) 639–658. [45] Y. Zeron, M. Pearl, A. Borochov, A. Arav, Kinetic and temporal factors influence chilling injury to germinal vesicle and mature bovine oocytes, Cryobiology 38 (1999) 35–42.
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The effect of liposomes on thermotropic membrane phase transitions of bovine spermatozoa and oocytes: implications for reducing chilling sensitivityq Y. Zeron,a M. Tomczak,b J. Crowe,b and A. Arava,* a
Institute of Animal Science, Agricultural Research Organization (ARO), The Volcani Center, Bet Dagan, 50250, Israel b Section of Molecular and Cellular Biology, University of California, Davis, CA 95616, USA Received 2 January 2002; accepted 2 October 2002
Abstract We have examined the effects of combinations between egg-phosphatidylcholine (EPC) or dipalmitoylphosphatidylcholine (DPPC) liposomes with either bovine spermatozoa or oocytes on cellular chilling sensitivity, lipid phase transition temperature (Tm ), and the ability of the oocytes to develop to the blastocyst stage. Spermatozoa and oocytes were exposed to EPC and DPPC liposomes at various temperatures (spermatozoa: 4, 12, 16, and 25 °C; oocytes: 4, 16, and 32 °C). The membrane integrity of the spermatozoa-control group decreased significantly following exposure to 16 or 12 °C, compared to ambient temperature (25 °C). In contrast, the EPC-sperm group had a greater resistance to chilling at each temperature and showed a decline in membrane integrity only at the lowest temperatures investigated. However, the DPPC-sperm group was injured significantly at all temperatures tested. Similar to the sperm, oocytes from the control group that were exposed to 16 °C were injured more severely than oocytes that were electrofused with EPC or DPPC liposomes. The membrane integrity of the oocytes at 16 °C that were electrofused with either EPC or DPPC liposomes was approximately the same as the control group held at 32 °C (normalized to 100%), compared to 46% in the control group at 16 °C (P < 0:01). The transition temperatures of the sperm and oocyte membranes revealed different Tm for the different liposome treatments. All groups had a significantly higher cleavage rate, as well as increased blastocyst formation when oocytes were exposed to temperatures above or below their Tm . We suggest that the Tm of spermatozoa or oocytes can be changed by spontaneous association or electrofusion of liposomes with cellular membranes and, consequently, the chilling sensitivity can be altered. The resulting possibility is that embryo development after cryopreservation could be improved with such a method. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Liposomes; Chilling sensitivity; Lipid phase transition; Membrane integrity; Oocytes; Sperm
q This work was funded in part by a grant from the US–Israel Binational Agricultural Research and Development Fund (BARD-2528-96). Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel. No. 401/01. * Corresponding author. Fax: +972-8-947-5075. E-mail address: [email protected] (A. Arav).
Chilling injury to mammalian gametes was first described in the 1930s as the irreversible damage that occurs upon cooling to low, but nonfreezing, temperatures (referred in [43]). Several studies suggested that injury at sub-physiological
0011-2240/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 1 1 - 2 2 4 0 ( 0 2 ) 0 0 1 2 3 - 2
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temperatures, termed chilling injury, is the major limiting factor for successful cryopreservation of gametes [2,24,33]. Other studies suggested that membranes are the primary site for structural and functional chilling injury in sperm [10,11] and oocytes [2]. Additionally, membrane chilling injury is dependent on the biochemical, and biophysical properties of membranes [39,43]. Phospholipid (PL) fatty acids (FA) are a major structural component of biological membranes and affect both the biophysical properties and functions of the cell [31,39]. Previous studies showed that palmitic (C16:0) and oleic (C18:1) acids were the most abundant fatty acids in the phospholipid fraction of oocytes from cattle [16,46], pigs [15] and sheep [7], and may function as energy reserves [40] or as precursors for the elongation of long chain fatty acids [23]. These studies also showed that polyunsaturated fatty acid (PUFA) was found in low concentrations in oocyte membranes. In contrast to oocytes, the spermatozoa of most mammals contain high concentrations of PUFA [26,29], which could partially account for the difference in fluidity between spermatozoa and oocyte membranes. Recently we showed that oocyte membrane fluidity is affected by temperature alterations between seasons, as well as by changes in FA composition [46]. This is consistent with other studies suggesting an association between FA composition and membrane fluidity [37,39]. The thermotropic lipid phase transition (Tm ) from the fluid liquid crystalline phase to the more rigid gel phase [8] is associated with chilling injury in sperm cells [3,11]. The temperature at which Tm occurs is greatly affected by the FA composition of the membrane [3], and chilling injury is maximized in the temperature range surrounding Tm [45]. Membranes that have lipids with multiple unsaturations or short carbon chains are more fluid at low temperatures [32,44] and, therefore, will be more chilling resistant. Furthermore, Arav et al. [3] suggested that the unsaturated FA content of PUFA was different between gamete membranes in different species and that an increase in the unsaturated FA content in PUFA had a major influence on this difference in Tm . There are several ways to change the chilling sensitivity and Tm in mammalian gametes. Various reports showed that enrichment of diet with PUFA can alter the biophysical properties or reproductive performance of sperm [4] and oocytes [38,47]. Other studies showed that depletion of intracellular lipids changed the chilling suscepti-
bility of oocytes in vitro [25,27] but they did not clarify how it affected the lipid membrane composition. Spermatozoa can spontaneously interact with liposomes as described in several studies [9,12,13,28,34]. Previous work suggested that phospholipids adsorbed on to the sperm cells, which were then cooled to 4 °C, increased the chilling resistance of the sperm cells from bovine [28] and ram [34]. Using 14 C-labeled egg yolk, Foulkes [13] reported that the radioactive label remained associated with bovine sperm lipids. It is well documented that liposomes prepared with different acyl chains have different Tm [6]. Fusing liposomes that have different Tm to gametes may influence their biophysical properties and decrease their chilling sensitivity. In the present study, we examined the effects of altering the lipid composition of spermatozoa and cumulus-oocyte complexes (COCs) on chilling injury, Tm , and the ability of these gametes to maintain developmental competence. Spermatozoa were coincubated with liposomes under conditions that result in spontaneous fusion. COCs were coincubated with liposomes and subjected to electrofusion.
Materials and methods Chemicals and lipids Unless stated otherwise, all chemicals used were obtained from Sigma Chemical (St. Louis, MO, USA). Egg-phosphatidylcholine (EPC), which is composed of the following fatty acids: 16:0 (34%), 16:1 (1.7%), 18:0 (11%), 18:1 (32%), 18:2 (18%), and 20:4 (3.3%), and dipalmitoylphosphatidylcholine (DPPC, 16:0) in chloroform were purchased from Avanti Polar Lipids (Alabaster, Alabama, USA). Oocytes Oocytes collection and in vitro maturation Ovaries were obtained at a local abattoir from slaughtered Holstein cows and placed in an insulated vessel containing physiological saline (0.9% NaCl, w/v) with 1 106 IU/ml of penicillin and 1 106 IU/ml of streptomycin at 32–36 °C. The ovaries were washed with 0.9% NaCl (w/v) and their centers were cut as previously described [46]. Cumulus-oocyte complexes were aspirated from the follicles using an 18 g needle on a 10 ml syringe and expelled into 65 mm petri dish (Corning Glass Works, NY, USA) containing Hepes-TALP
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(H-TALP). Oocytes with three or more layers of cumulus cells surrounding a homogeneous cytoplasm were used for electofusion treatments or maturation studies. For maturation studies, 40–60 COCs per well were placed in a 4-well culture multidish (Nunc, Denmark) in 500 ll of maturation medium consisting of 25 mM Hepes TCM199, supplemented with 10% (v/v) heat-inactivated fetal calf serum (Bio-Lab, Jerusalem, Israel), 0.2 mM Na pyruvate, 5 lg/L gentamicin, 10 lg/ml oLH (NIADDK-NIH-26, AFP5551B, Bethesda, MD), 1 lg/ml oFSH (NIADDK-NIH-20, AFP 7028D, Bethesda, MD) and 1 lg/ml estradiol [17]. COCs were then incubated for 24 h at 39 °C in a humidified atmosphere of 5% CO2 in air. Activation and development of oocytes After maturation, oocytes were examined for their ability to develop to the blastocyst stage after activation, as previously described [20]. Briefly, COCs were denuded from cumulus cells in the maturation wells and placed for 5 min in the ionomycin medium: 10 ml of TCM-199 supplemented with 25 mM Hepes, 10% (v/v) heat-inactivated FCS, 0.2 mM sodium pyruvate, 5 lg gentamicin, and 5 lM of ionomycin. Oocytes were transferred to 6-dimethylaminopurine (6-DMAP) medium (10 ml of TCM 199 supplemented with 2 mM of 6-DMAP) for 4.5 h. The oocytes were then washed three times in a cleavage medium (Sydney IVF Cleavage Medium, Cook, Australia) and transferred in groups of 10 into 50 ll cleavage drops under mineral oil. Cleaved embryos were counted on day 4 (activation day ¼ 0) and transferred to 50 ll drops of blastocysts medium (Sydney IVF Blastocyst Medium, Cook, Australia) under mineral oil. Blastocysts were counted after 8–10 days. Sperm Semen from Israeli Holstein Friesian bulls (five-years-old) was collected once a week between January and March at the Sion Artificial Insemination Center and transported (undiluted semen) with liposomes (see below) to the Lab within 30 min at 25 °C. Chilling and viability staining of oocytes and sperm Cumulus-oocyte complexes were aspirated in 10 ll of H-TALP (without BSA) into a mouthoperated micropipette and expelled into 1 ml of H-TALP precooled to 4 or 16 °C for 15 min as previously described [45]. Subsequently, COCs
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were placed immediately in TCM-199, supplemented with 10% heat inactivated FCS, 0.2 mM Na pyruvate, 5 lg/L gentamicin, and incubated for 1 h at 39 °C in a humidified atmosphere of 5% CO2 . Following incubation, COCs were denuded and exposed to 5-carboxyfluorescein diacetate (cFDA) (Molecular Probes, Leiden, The Netherlands) as previously described [45]. After being washed with H-TALP, the oocytes were placed on a slide under a coverslip and cFDA fluorescence was evaluated using a fluorescence microscope connected to a CCD video camera (Applitec LIS700, Israel) and imaging data acquisition software (NIH-1.55, MD, USA). The levels of fluorescence intensity were determined using the imaging data processing software on a PowerPC 7100/80 computer. Mean intensity of the pixels from each treatment were normalized to controls. Sperm (10 ll sperm in a glass tube) were incubated in 11 lg/ml of cFDA for 20 min at room temperature, followed by immersion into a precooled 1 L water bath (4 °C, 12 °C or 16 °C), or incubated at 25 °C (control) for 30 min, after which all samples were washed with H-TALP. The number of live (fluorescent) sperm were then determined using a fluorescence microscope connected to a CCD video camera. Liposome preparation and introducing them with bovine COCs and sperm EPC or DPPC (20 mg each) were dried under nitrogen gas, and subsequently hydrated to 100 mg/ml in 200 ll of H-TALP supplemented with 0.5% PVP (w/v) at 25 °C. The suspension was vortexed for 10 min and then loaded into a glass syringe in a hand held extruder (Avestin, Ottawa, Canada; [21]). The lipid suspension was extruded through 0.1 lm pore filters and then placed into a 3 ml glass tube. After addition of 0.04% (v/v) phytohemaglutinin to the liposomes, the tubes were purged with nitrogen gas and the liposomes (0.1 lm diameter) were stored at ambient temperature in the dark. COCs (50/group) were incubated for 5 min in 100 ll of unilamellar liposome solution at 25 °C, followed by transfer into 200 ll of non-electrolyte fusion solution (0.3 M mannitol, 0.05 mM Caþþ , and 0.1 mM Mgþþ supplemented with 10 ll liposome solution) in the electrofusion chamber (CF150 chamber, Biochemical Laboratory Service, Budapest, Hungary). Electrofusion of 10 COCs/ group was done in 3 pulses of 1.5 kV (DC), each lasting 80 ls. Subsequently, the electrofused COCs were divided into two groups: half were chilled to
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16 °C as described by Zeron et al. [45], and half were exposed to 32 °C for 15 min. All COCs were then washed 3 times in maturation medium and examined for their membrane integrity or development as described in Materials and methods. Spermatozoa (3 ml pooled from three different bulls in concentration of 1:1 106 cells/ml) were incubated with EPC or DPPC liposomes (100 mg/ ml) for 30 min at room temperature, followed by exposure to low temperatures as described above. Measurements of membrane phase transitions The membrane phase transition of oocytes and sperm were determined with a Bruker-Equinox 55 Fourier transform infrared (FTIR) spectrometer connected to a Bruker FTIR microscope A590 (Ettlingen, D-76275, Germany), as previously described [46]. The sperm were concentrated by centrifugation (12,000g, 10 min). Oocytes or pelleted sperm were placed between two sapphire windows. Sample temperature was regulated by a microprocessor feedback system, which measures sample temperature with a thermocouple placed directly on the surface of the windows. The temperature was adjusted to the desired level, within 0.1 °C, and allowed to equilibrate for 2 min before the sample was scanned. The temperature was controlled with two thermoelectric coolers placed on the microscopic stage using directional solidification (IMT, Israel). The FTIR spectra were baseline subtracted to monitor the frequency of the symmetric CH2 stretching vibration of the lipid fatty acyl chains. The center of the lipid phase transition curve from liquid crystalline to gel phase is designated Tm , and is determined by statistical analysis as described by Crowe et al. [8].
(Fig. 1). After exposure to 16 or 12 °C for 30 min, the percentage of sperm cells with intact membranes decreased by 54 and 57%, respectively, compared with the control sperm cells that were held at 25 °C. In contrast, the sperm cells that interacted with EPC liposomes had a greater resistance to chilling at each temperature. The EPCsperm cells did show a slight decline in membrane integrity at 16 °C (P < 0:1) and a significant decline at 12 and 4 °C compared to the controls incubated at ambient temperature. At 25 °C there was already significant injury to the DPPC-sperm cells compared to the control and EPC groups, and their membrane integrity decreased as the incubation temperature decreased (Fig. 1). These differences led us to evaluate the biophysical characteristics of the sperm cell membranes. The transition temperatures of the sperm cell membranes from the liquid crystalline to the gel phase revealed a different Tm for the different liposome treatments (Fig. 2). The control group had a Tm around 13 °C, in contrast to higher Tm of the sperm cells incubated with DPPC, which was centered on 23 °C. However, the wave number of the sperm cells incubated with EPC decreased only slightly below 2852 cm1 at the lowest temperatures investigated, which indicates that the sperm membrane was still in the liquid crystalline phase and that the transition temperature was lower than 4 °C.
Statistics Means were calculated and differences between treatments examined by t tests using the General Linear Model procedure of JMP [35]. Significance was P < 0:05 unless otherwise stated.
Results The effect of different liposomes on sperm chilling sensitivity and lipid phase transitions Sperm and oocytes showed different responses to chilling. The membrane integrity (measured by cFDA fluorescence intensity) of the control group of sperm cells declined substantially following exposure to hypothermic temperature for 30 min
Fig. 1. The effect of different temperatures on sperm cell membrane integrity after treatment with different liposomes (egg-phosphatidylcholine, EPC; dipalmitoylphosphatidylcholine, DPPC) for 30 min. Liposomes (100 mg/ ml) were added to the sperm cells immediately after ejaculation. Membrane integrity was determined using cFDA (11 lg/ml). Values presented were normalized to the control at 25 °C from pixel values of cFDA fluorescence. Each column represents data pooled from three different bulls (five-years-old) in 4 replicates. Bars represent SE and values with a common letter do not differ significantly between treatments at P < 0:05.
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Fig. 2. The effect on sperm membrane lipid phase transitions after exposure to different liposomes. The sperm cells were collected from bulls (five-years-old), and egg-phosphatidylcholine (EPC) or dipalmitoylphosphatidylcholine (DPPC) were added (100 mg/ml) immediately after ejaculation. The phase transition of sperm cell membranes was measured by Fourier transform infrared (FTIR) microscopy. The center of the lipid phase transition curve, from liquid crystalline to gel phase is designated Tm . Tm were calculated from the frequencytemperature plots according to Crowe et al. [8].
The effect of different liposomes on oocyte chilling sensitivity and lipid phase transition The effects of short term chilling on the membrane integrity of oocytes following electrofusion of different liposomes with the oocytes were determined (Fig. 3). Preliminary experiments of introducing liposomes composed of various lipids to the COCs without electrofusion revealed the same results as the control group (data not shown).
Fig. 3. The effect on oocyte membrane integrity after electofusion with different liposomes. Membrane integrity of oocytes at the germinal vesicle stage following exposure to egg-phosphatidylcholine (EPC) or dipalmitoylphosphatidylcholine, (DPPC) liposomes and incubated at 32 °C (white column) or 16 °C (black column) were determined using cFDA. Values presented were normalized to the control at 32 °C from pixel values of cFDA fluorescence. Each column represents data pooled from 110 15 oocytes in the different treatments (5 replicates). Bars represent SE and values with a common letter do not differ significantly at P < 0:05.
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Oocytes from the electrofusion control group that were exposed to 16 °C for 15 min were injured more severely than oocytes that were electrofused with EPC or DPPC liposomes. All results were normalized to the control group at 32 °C to make a direct comparisons between treatments, therefore some of the percentages are greater than 100%. The membrane integrity of the oocytes at 16 °C that were electrofused with either EPC or DPPC liposomes was 107 and 92%, respectively, compared to 46% membrane integrity in the control group at 16 °C (P < 0:01; normalized values). Furthermore, under chilling conditions the oocytes electrofused with either EPC or DPPC liposomes had similar membrane integrity as the control oocytes that were incubated at 32 °C (Fig. 3). However, the DPPC group revealed a significant decrease in membrane integrity when incubated at 32 °C. These results indicate that the sensitivity of the oocytes to chilling was different after the different liposome treatments. The electrofusion process by itself did not improve the chilling resistance of oocytes, as the oocytes electrofused without liposomes showed no increased chilling tolerance (electrofusion control, Fig. 3). Therefore, we examined the Tm of the oocyte membranes after electrofusion with liposomes and, subsequently, their ability to develop to the blastocyst stage. The effect of liposomes on bovine oocyte lipid phase transitions and on chilling sensitivity is shown in Fig. 4. The control oocytes had a different phase transition pattern from the liquid crystalline to the gel phase than the electrofused oocyte–liposome groups. The Tm of the control oocytes (control group, Fig. 4A) was 16 °C, compared to a Tm of less than 4 °C in the electrofused EPC-oocytes (EPC group, Fig. 4B), and at 25 °C in the electrofused DPPC-oocytes (DPPC group, Fig. 4C). The wave number excursion between groups was also different: all groups had an initial wave number in the liquid crystalline phase between 2854 and 2853 cm1 , but only the control and DPPC groups had endpoint wave numbers characteristic of the gel phase (between 2851.2 and 2850.8 cm1 , Figs. 4A and C). The sensitivity of oocytes to chilling was measured by their ability to cleave and develop to the blastocyst stage after in vitro maturation and chemical activation at 39 °C. All groups had a significantly higher cleavage rate, as well as increased blastocyst formation when oocytes were exposed to temperatures above or below their Tm , but not after incubation at temperatures in the
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had significantly higher rates of cleavage and blastocyst formation after exposure to 16 °C (39 and 9%, respectively), compared to the control group. By contrast, after exposure to 32 °C the oocytes electrofused with DPPC liposomes had a significantly lower rate of cleavage and development to the blastocyst stage (1 and 0.2%, respectively) compared to the control group. Again, exposure of this group to 16 °C resulted in a significantly higher rate of cleavage and blastocyst formation (40 and 10%, respectively).
Discussion
Fig. 4. The effect of electrofusion on the lipid phase transition and embryo development of bovine oocytes subjected to chilling temperatures. Germinal vesicle oocytes (control, A) and oocytes electrofused with eggphosphatidylcholine, (EPC, B) or dipalmitoylphosphatidylcholine, (DPPC, C) (n ¼ 300, 275 and 285, respectively) were exposed to temperatures of 4, 16, and 32 °C (control) or 16 and 32 °C (EPC and DPPC groups) for 15 min. Chilling tolerance was determined by their ability to cleave after chemical activation (2–4 cell stage, white columns) and developed to the blastocyst stage (black columns). Bars represent SE and values with a common letter (small letters between cleavage stage; capital letters between blastocysts stage) do not differ significantly at P < 0:05. The phase properties of cytoplasmatic membranes of oocytes (n ¼ 13, each group) were measured by Fourier transform infrared (FTIR) microscopy. The center of the lipid phase transition curve, from liquid crystalline to gel phase is designated Tm . Tm were calculated from the frequency-temperature plots according to Crowe et al. [8].
phase transition range. In the control group, the rate of cleavage and development to the blastocyst stage was higher after exposure to 32 °C (68 and 18%, respectively), compared to incubation at 16 °C (20 and 1%, respectively; Fig. 4A). However, the oocytes electrofused with EPC liposomes
During cryopreservation cells are exposed to low temperatures and therefore are vulnerable to chilling injury [18,43]. Chilling damage has been studied extensively in sperm [24,42]. One possible cause of chilling injury is the membrane damage that is associated with the thermotropic lipid phase transition. De Leeuw et al. [10] examined the ultrastructural changes in different sperm plasma membranes and showed that at reduced temperatures the intramembranous components aggregated and redistributed in the head and principal piece of the sperm. This led to phenomenon known as lateral phase separation. Drobnis et al. [11] emphasized this hypothesis with results showing that spermatozoa chilling injury is associated with the membrane lipid phase transition and suggested lateral phase separation to be the main cause of damage to the membrane. Phospholipid fatty acid composition influences the phase transition profile of the sperm membranes. In the present study we presented evidence that spontaneous association between spermatozoa and liposomes occurs. Mixing two types of lipids with two different Tm Õs results in a single Tm intermediate between the two. Contrary, if the two populations are not mixed, two Tm Õs are observed at the original values for the two lipids. We clearly showed that one cooperative phase transition that occurred for both DPPC and EPC treated sperm cells. Several studies have examined the lipid and fatty acid composition in spermatozoa membranes from different species [3,14,29]. Similar to a previous report [3], we found that the lipid phase transition of the spermatozoa occurs over a broad temperature range, which suggests the presence of a variety of fatty acids in the membrane as well as a high content of long chain fatty acids. The present study has shown that sperm cells were extremely sensitive to chilling, but were less
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chilling sensitive after the addition of EPC liposomes. The EPC liposomes decreased the lipid phase transition of the spermatozoa to lower than 4 °C, from 13 °C in the control sperm cells. Exposure of the EPC-sperm cells to higher temperatures than lipid phase transition temperature range, followed by chilling to 16 °C did not cause damage to the membranes. Presumably, damage was avoided because the EPC-sperm had not passed through their Tm , so the injury that would result normally was avoided. In artificial insemination centers, the sperm freezing protocol starts by slowly chilling the sperm cells, in the presence of egg-yolk, to temperatures between 4 and 5 °C (< 1 °C/min) and holding them at that temperature. This protocol requires that association of the liposomes to spermatozoa would have to occur under these conditions (probably spontaneously) to be effective for cryoprotection. Evidence for the spontaneous association between exogenous lipids and the spermatozoa plasma membrane have been shown by radioactive labeling [13]. However, this is a complex process, dependent on temperature and cooling rate [9], and more work will have to be done to optimize the conditions for increased sperm survival after cryopreservation. Egg phosphatidylcholine liposomes also might stabilize the sperm as they spontaneously associate or interact with the membrane [12]. Thus, the current results showing that EPC-sperm have greater survival than the controls supports the previous findings. In contrast, the DPPC-sperm group showed greater membrane sensitivity at all temperatures investigated. This is probably due to the exposure of this group to temperatures at which the membrane was in the center of the phase transition range (Tm ¼ 25–30 °C). Additionally, chilling this group to lower temperatures did not reverse the damage that occurred at 25 °C. The chilling sensitivity of oocytes is well documented [2,22,30,45] and, consequently, their successful cryopreservation is limited. Studies conducted on the chilling sensitivity of mature oocytes (MII stage) found that the main damage occurred due to meiotic spindle disorganization followed by microtubule depolymerization [1,36]. Cryopreservation of immature oocytes could be a solution for this problem, as oocytes do not contain polymerized microtubules at this stage. However, immature oocytes at the germinal vesicle stage (GV) are extremely sensitive to chilling temperatures, with the damage occurring at the cytoplasmic membrane [2]. Recently, evidence
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describing the influence of phospholipid fatty acid composition on the biophysical properties of immature oocyte membranes was reported [3,46]. These studies showed that the ratio of unsaturated to saturated fatty acids and the position of the double-bond in the carbon chain, and, thus, the transition temperature of the membranes, affected their chilling sensitivity. The present study has shown that chilling sensitivity changed after electrofusion of the oocytes with liposomes prepared from egg phosphatidylcholine. We exposed the oocytes, surrounded by their cumulus cells, to liposomes (electrofusing and chilling procedures) as it was shown that cumulus cells are essential for maturation [5]. We used very small size of liposomes (0.1 ll) to facilitate penetration of the liposomes through the zona pellucida, as it was previously described that zona pellucida pores are 187 ll in diameter [41]. Therefore, the changes observed clearly were due to an alteration in the cellular membrane fluidity, caused by electrofusion with the liposomes. We observed greater than 10% survival of blastocyst following chilling of EPC or DPPC-oocytes compared with the unchilled control oocytes. Electrofusing EPC liposomes with GV oocytes changed the composition of unsaturated fatty acids present in the membrane and, therefore, changed the physical properties of the oocytes. We assume the oocytes membranes were saturated with exogenous lipids following electrofusion due to the large ratio of exogenous lipid to endogenous membrane lipid. Our present study shows evidence of changes in oocyte membrane fluidity following electrofusion with liposomes to change their lipid composition. Our direct evidence that the liposomes were fused to the oocytes is the significant changes in both the Tm of the lipid phase transition and the chilling sensitivity. In addition, the lipid phase transition of control COCs (approximately 16 °C) and of EPC or DPPC alone (Tm below 0 and 40 °C, respectively [19]), are very different from the lipid phase transitions we found after electrofusion with these liposomes. We showed that the oocyte Tm was altered after electrofusion with EPC and DPPC liposomes. The resulting Tm of the EPC group was lower, and of the DPPC group higher, than the control oocytes. Recently, we have shown that bovine oocytes are sensitive to chilling around the temperature range where the Tm occurs (Tm centered at 16 °C) and less sensitive at temperatures above or below this range [45]. Exposing spermatozoa or oocytes to the lipid
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phase transition temperature damaged their membrane integrity and physiological functions. In the present study we showed that oocyte membrane integrity was maintained after exposure to 16 °C, which was lower (DPPC group) or higher (EPC group) than the control-group Tm . However, it is not clear why membrane integrity was lower when oocytes from the DPPC-group exposed to 32 °C compared to 16 °C. One explanation may be that the DPPC-electrofused oocytes are damaged during incubation at temperatures near the lipid phase transition temperature, at which the oocytes were incubated (39 °C). The alteration of Tm affected the developmental ability of the embryos after low temperature exposure. The EPC and DPPC groups had significantly higher cleavage rates and blastocyst formation than the control group after exposure to temperature of 16 °C. This finding confirms results from other studies that associated Tm and chilling sensitivity with fatty acids composition [3,11]. However, changing the Tm to higher temperature with DPPC liposomes decreased the rate of embryo development. Oocytes and sperm have different Tm profiles, due to differences in fatty acid and lipid composition in fatty acid and lipid composition [3,16,23,29,46]. These studies showed that sperm have a greater diversity of fatty acids than oocytes. Particularly, sperm have a high percentage of lipids with long polyunsaturated fatty acids, while oocytes almost exclusively have lipids with fully saturated fatty acids. We also showed that the oocyte Tm profile changed after liposome treatments, and this is due probably to the resulting alteration of the fatty acid composition in the membrane. We suggest that the lipid phase transition of spermatozoa or oocytes can be changed by the addition of liposomes and consequently, the chilling sensitivity could be altered. These finding may improve cryopreservation of gametes as well as embryonic development after cryopreservation.
Acknowledgments We would like to express our gratitude to Professor David Sklan for fruitful comments on the manuscript. The author thanks Ana Ocheretny for technical assistance. Thanks are expressed to Sion A.I. Center for donating the bovine semen used in this study and NHPP for generous provision of FSH and LH.
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