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Human oocyte cryopreservation and the fate of cortical granules
Human oocyte cryopreservation and the fate of cortical granules Yehudith Ghetler, Ph.D.,a,b,c Ehud Skutelsky, Ph.D.,b Isaac Ben Nun, M.D.,c Liah Ben Dor, M.Sc.,a,b Dina Amihai, M.Sc.,b and Ruth Shalgi, Ph.D.a a
Department of Cell and Developmental Biology and b Department of Pathology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; and c IVF Unit, Meir Hospital (affiliated with Tel Aviv University), Kfar Saba, Israel
Objective: To examine the effect of the commonly used oocyte cryopreservation protocol on the cortical granules (CGs) of human immature germinal vesicle (GV) and mature metaphase II (MII) oocytes. Design: Laboratory study. Setting: IVF unit. Intervention(s): Unfertilized, intracytoplasmic sperm injected (ICSI) oocytes, and immature oocytes were cryopreserved using a slow freezing–rapid thawing program with 1,2-propanediol (PROH) as a cryoprotectant. Main Outcome Measure(s): Cortical granule exocytosis (CGE) was assessed by either confocal microscopy or transmission electron microscopy (TEM). Result(s): The survival rates of frozen-thawed oocytes (mature and immature) were significantly lower compared with zygotes. Both mature and immature oocytes exhibited increased fluorescence after cryopreservation, indicating the occurrence of CGE. Mere exposure of oocytes to cryoprotectants induced CGE of 70% the value of control zygotes. The TEM revealed a drastic reduction in the amount of CGs at the cortex of frozen-thawed GV and MII oocytes, as well as appearance of vesicles in the ooplasm. Conclusion(s): The commonly used PROH freezing protocol for human oocytes resulted in extensive CGE. This finding explains why ICSI is needed to achieve fertilization of frozen-thawed human oocytes. (Fertil Steril威 2006; 86:210 – 6. ©2006 by American Society for Reproductive Medicine.) Key Words: Cryopreservation, cortical granules, exocytosis, mature human oocyte, immature human oocyte
Cryopreservation of human embryos at different developmental stages is successfully applied in IVF treatment programs. However, the cryopreservation of human oocytes still generally yields unsatisfactory results and is therefore considered experimental. The ability to cryopreserve human oocytes is of great importance because it avoids serious ethical and legal issues arising from embryo freezing, such as disposal, donation, or dispute over ownership. Furthermore, cryopreservation of human oocytes could lay the ground for egg donation programs and for establishing oocyte banks, offering the opportunity to preserve the fertility potential of women who face chemotherapy or radiation therapy. Successful cryopreservation of mammalian oocytes had already been reported by several investigators (1, 2). The technologies for cryopreservation of human oocytes used in IVF clinics are derived from traditional protocols of embryo cryopreservation and, as such, render results that are inconsistent and suboptimal. Cryopreservation of oocytes results in low survival rate, hardening of the zona pellucida (ZP), and various types of injuries, including damage to the meiotic spindle and microfilaments (3–7). Attempts have been Received June 8, 2005; revised and accepted December 5, 2005. Reprint requests: Ruth Shalgi, Ph.D., Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel Aviv University, Ramat Aviv, Tel Aviv 69978, Israel (FAX: 972-3-6406149; E-mail: shalgir@ post.tau.ac.il).
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made to freeze immature oocytes at the germinal vesicle (GV) stage, where the DNA is still enclosed within the nucleus and protected by a membrane and may be less sensitive to stressful environmental conditions (8). Immature oocytes, excised from unstimulated follicles or from ovarian tissue, could, in part, survive and mature, but their developmental capability is still very low (9, 10). The common protocol used for oocyte cryopreservation is slow freezing–rapid thawing in 1,2-propanediol (PROH) (11–13). Other protocols such as slow freezing–rapid thawing in dimethyl sulphoxyde (DMSO) (14, 15) and an ultrarapid method (16, 17) have also been introduced. Some research groups have used variations of vitrification (18, 19), including vitrification at specially enhanced cooling rates using open pooled straws (20), electron microscope grids (21), and minimum drop size in supercooled liquid nitrogen (22). Human oocytes that survived cryopreservation had, reportedly, a low fertilization rate (23), indicating that the normal process of the sperm penetration is impaired. Therefore, cryopreserved human oocytes are fertilized by intracytoplasmic sperm injection (ICSI) in an effort to improve their fertilization rate (5, 13). The cortical granules (CGs) are Golgi-derived membranebound spherical or slightly ovoid organelles formed during the early stages of oocyte growth and maturation and are
Fertility and Sterility姞 Vol. 86, No. 1, July 2006 Copyright ©2006 American Society for Reproductive Medicine, Published by Elsevier Inc.
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present in the cortex of mature unfertilized oocytes of most animal species (24). Both density and distribution of CGs change when they translocate to the cortical region of the oocyte during the process of oocyte maturation (25, 26). The CG’s content is released into the perivitelline space (PVS) immediately after oocyte fertilization or activation and is believed to alter the ZP glycoproteins, establishing the block to polyspermy, and preventing the penetration of additional spermatozoa (27). Due to the major role of CGs in inducing the block to polyspermy, their fate in frozen-thawed oocytes is of great importance, but research results have been inconsistent, ranging from a reduction in the number of CGs following cryopreservation (3, 28) to an apparently normal density and distribution of CGs in frozen-thawed oocytes (29 –31). In view of this inconsistency, we aimed to examine the affect of the commonly used oocyte PROH cryopreservation protocol on the CGs of human immature GV and mature metaphase II (MII) oocytes by using confocal microscopy as well as transmission electron microscopy (TEM). MATERIALS AND METHODS Oocytes included in this study were donated by consenting patients undergoing IVF-ICSI procedures and represent material that is otherwise discarded. The study was approved by the local review board. The ovulation induction (by a “long” protocol of GnRH analog and hMG) and the ICSI procedure used were as described previously (32, 33). Oocytes that failed to be fertilized by ICSI (unfertilized [UF]) and immature oocytes not suitable for ICSI were used. The UF oocytes were reassessed 24 h after ICSI to rule out late fertilization occurrence and only then were they frozen. The immature oocytes were handled according to their developmental state: GV oocytes were frozen on the retrieval day, whereas oocytes at the first metaphase (MI) stage were further cultured for 24 h in the routinely used culture media (Cook Culture System; Cook, Brisbane, Australia). Only oocytes that were able to mature in vitro to the MII stage were cryopreserved. The UF as well as in vitro–matured MII oocytes served as a model of mature oocytes throughout this research. Abnormally fertilized (one pronucleus [1PN] or three pronuclei [3PN]) oocytes served as positive control for occurrence of cortical granule exocytosis (CGE). Study Groups Several experimental groups were included in the research. Group 1: Untreated mature oocytes do not undergo CGE (34) served as a negative control for assessing CG status (n ⫽ 13). Group 2: Mature oocytes that were subjected to the freezing-thawing solutions, according to the protocol, but not to the actual freezing procedure, served as a control for the effect of the solutions on the CGs’ fate, (n ⫽ 16). Group 3 consisted of mature oocytes that were frozen ⱖ1 week before thawing (n ⫽ 23). Group 4 consisted of GV oocytes Fertility and Sterility姞
that were frozen ⱖ1 week before thawing (n ⫽ 10). Group 5: Abnormally fertilized oocytes (1PN or 3PN) after ICSI served as a positive control for CGE occurrence (n ⫽ 11); in a previous study we demonstrated that these abnormally fertilized ICSI oocytes exhibit an intense CGE (34). All oocytes were assessed for CGE by confocal microscopy or TEM. Cryopreservation Protocol The slow freezing–rapid thawing method was used throughout the study. The freezing/thawing solutions (Cook) and protocols were the routine protocols used in our IVF program for zygote and early embryo cryopreservation. In brief, a three-step cryopreservation system was used with HEPESbuffered salt solution (HBSS) containing PROH and sucrose as cryoprotectants: step 1: incubation in prewarmed (37°C) HBSS for 10 minutes at room temperature (RT); step 2: incubation in HBSS containing 1.5 mol/L PROH for 10 minutes at RT; step 3: transfer into HBSS containing 1.5 mol/L PROH and 0.1 mol/L sucrose, loading the samples into straws at RT, and transfering them into an automated Kryo 10 biologic vertical freezer (Kryo 10 series III, Planer, Middlesex, UK). Cooling steps: starting at 20°C, cooling at ⫺2°C/min down to ⫺7°C, soaking for 10 minutes, manual seeding, holding 10 minutes, cooling at ⫺0.3°C/min down to ⫺30°C, cooling at ⫺50°C/min down to ⫺150°C and plunging into liquid nitrogen (⫺196°C). Samples were stored in liquid nitrogen for ⱖ1 week. For thawing, straws were removed from liquid nitrogen and kept 30 seconds at RT before being immersed in a 30°C water bath until all the ice melted. The cryoprotectant was removed with four consecutive steps of 5 minutes each incubation at RT: step 1: HBSS containing 1.0 mol/L PROH and 0.2 mol/L sucrose; step 2: HBSS containing 0.5 mol/L PROH and 0.2 mol/L sucrose; step 3: HBSS containing 0.2 mol/L sucrose; step 4: only HBSS followed by additional 5 minutes at 37°C. Finally, the oocytes were incubated in culture medium (Cook) at 37°C with 5% CO2 in humidified air for 1 hour and then assessed morphologically. Only oocytes that survived the whole process of freezing-thawing were further examined. Assessment of CGE The assessment of cortical reaction was performed by labeling the oocytes with the lectin Lens culinaris agglutinin (LCA), which binds specifically to CG content and exudate (35). The studied oocytes were briefly exposed to 0.25% pronase (Sigma, St. Louis, MO) for ZP removal, followed by three rinses in HTF-HEPES medium (Irvine, Santa Ana, CA) supplemented with 10% synthetic serum substitute (Irvine). The ZP-free oocytes were fixed with 3% paraformaldehyde in DPBS for 30 minutes at RT and washed three times in DPBS supplemented with 1% bovine serum albumin (Fraction V; Sigma). The fixated oocytes were stained for 30 minutes with 211
uranyl acetate followed by lead citrate. The sections were examined using a JOEL-100CX TEM at 80 kV (37).
TABLE 1 Survival rate of frozen thawed oocytes and zygotes.
Study group
Number of oocytes/ zygotes
Survival rate* (mean % ⴞ SEM)
Zygotes Mature ocytes GV oocytes
173 109 92
74.7 ⫾ 4.2a 40.8 ⫾ 4.9b 31.4 ⫾ 7.1b
Note: Survival of thawed oocytes and zygotes was morphologically determined after 1 h of culture. a,b Different superscripts within column denote significant differences (P⬍.001). Ghetler. Cortical granules in cryopreserved human oocytes. Fertil Steril 2006.
5 g/mL LCA-biotin (Vector, Burlingame, CA), washed, and stained with 2 g/mL Texas red–streptavidin (Vector) for 30 minutes, thus labeling the membrane of exocytosed granules and the exudate. For high-quality assessment of the CGE, a confocal laser scanning microscope (CLSM) (LSM 410; Ziess, Oberkochen, Germany) equipped with a 25 mW krypton-argon laser was used. The oocytes were scanned every 5m and the composite image of micrographs taken at all focal planes of each oocyte was displayed. The fluorescence intensity of each individual oocyte was quantified, with the aid of the LSM software, and presented as the ratio between the fluorescent area and the total area of the oocyte, which was then related to the fluorescence intensity of the positive control (group 5) which was set arbitrarily as 1. Assessment of Meiotic Status Oocytes were incubated for 10 minutes in the presence of 2 g/mL DNA-specific fluorochrome (Hoechst 33342, Sigma) and then washed three times in DPBS. Analysis of each oocyte was performed at the time of CGE assessment, using the CLSM. Electron Microscopy (EM) The ultrastructure of CGs of oocytes from the various experimental groups was examined using TEM. At least three oocytes from each group were sectioned and analyzed. The oocytes were fixed for 1 hour in Karnovsky fixative (36), washed twice in 0.2 mol/L cacodylate buffer, pH 7.4, postfixed in 1% osmium tetroxide, washed, and gradually dehydrated through ascending grades of ethanol and propylene oxide. Each oocyte was individually embedded in Araldite (Polysciences, Warrington, PA) and sectioned (0.075 ⫾ 0.015 nm) on an LKB III Ultratome. The ultrathin sections were mounted on grids and stained with 212
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Statistical Analysis The significance of differences between experimental groups was determined by one-way analysis of variance after arcsin square root of proportion transformation (for survival study) and ln transformation (for the fluorescence study) to achieve normal distribution, combined with Tukey’s method for multiple comparisons. RESULTS Survival rate of the frozen-thawed oocytes was compared to the survival rate of zygotes (2 PN) cryopreserved in our IVF program (Table 1). The overall survival rate of oocytes was low and inconsistent (0%–92%). The survival rate of zygotes in our IVF unit during the same period of time was 74.7% ⫾ 4.2% (mean ⫾ SEM), similar to other reports (38) and significantly (P⬍.001) higher than the survival rate of either immature (31.4% ⫾ 7.1%) or mature oocytes (40.8% ⫾ 4.9%). There was no significant difference between the survival rate of mature and immature oocytes. We used the CLSM for assessing the CGE occurrence as an indicator of the fate of CGs in frozen-thawed oocytes. The treated oocytes (groups 2, 3, 4) and the negative control
FIGURE 1 Detection of cortical granule exocytosis (CGE) by labeling with biotinylated lectin Lens culinaris agglutinin and Texas red–strepavidin. Composite confocal microscopy images (A–D) and light microscopy images (a– d) of untreated mature oocyte (A, a), untreated 3PN zygote (B, b), frozenthawed (slow freezing–rapid thawing in 1,2 propanediol) mature oocyte (C, c), and frozenthawed (slow freezing–rapid thawing in 1,2 propanediol) germinal vesicle stage oocyte (D, d), illustrating the degree of CGE at the various treatments. Bar ⫽ 35 m.
Ghetler. Cortical granules in cryopreserved human oocytes. Fertil Steril 2006.
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TABLE 2 Fluorescence intensity of frozen-thawed oocytes. Study group
Number of oocytes
Fluorescence intensity (mean ⴞ SEM)
1 2 3 4 5
14 16 23 10 11
0.23 ⫾ 0.09a 0.73 ⫾ 0.21b 1.51 ⫾ 0.33b,c 2.51 ⫾ 1.08c 1
Note: Relative fluorescence intensity expressed as ratio of the abnormally ICSI-fertilized oocytes (group 5). Group 1: untreated mature oocytes. Group 2: mature oocytes following exposure to freezingthawing solutions. Group 3: frozen-thawed mature oocytes. Group 4: frozen-thawed germinal vesicle oocytes. Group 5: abnormally fertilized oocytes, fluorescence intensity arbitrarily defined as 1. a,b,c Different superscripts within column denote significant differences P⬍.001). Ghetler. Cortical granules in cryopreserved human oocytes. Fertil Steril 2006.
procedure (Figs. 2, bottom, and 3, bottom). Only very few dark CGs were observed within frozen-thawed oocytes. Mature frozen-thawed oocytes had only a few scattered CGs deeper in the cytoplasm, whereas GV frozen-thawed oocytes had a few CGs at the cortex and aggregates of light granules throughout the cytoplasm. All the frozen-thawed oocytes formed membrane-coated electron-transparent vesicles, which in some cases were aggregated with CGs (Figs. 2, bottom, and 3, bottom). No such vesicles could be observed within the oocytes before cryopreservation.
FIGURE 2 The effect of cryopreservation on mature oocytes. (Top) An untreated oocyte: Abundant dark electron-dense granules (white arrow) are present in the cortex, and light (black arrow) granules are present mainly deeper in the cytoplasm. (Bottom) A frozen-thawed oocyte: Very few granules are present at the cortex. Light (black arrow) and a few dark (white arrow) granules are in the inner region. A large number of membrane-coated transparent vesicles (V) are observed, scattered throughout the ooplasm. ZP ⫽ zona pellucida. Bar ⫽ 2 m.
(group 1) did not show activation, as indicated by Hoechst staining. However, our results demonstrate that both mature and immature frozen-thawed oocytes underwent CGE (Figs. 1C and D, respectively) compared with untreated oocytes (Fig. 1A) or fertilized oocytes (Fig. 1B). The calculated CGE intensity is presented in Table 2. Untreated mature oocytes (group 1, negative control) exhibited very low fluorescence relative to the positive control group of abnormally fertilized oocytes (group 5). The mere exposure to cryopreservation solutions (group 2) resulted in a strong CGE: about 70% of the positive control value and significantly different from the untreated mature oocytes (P⬍.001). The CGE of oocytes, both mature (group 3) and immature (group 4), passing through the complete freezingthawing procedure was significantly stronger than that of untreated oocytes. The GV frozen-thawed oocytes exhibited a higher (but not statistically different) degree of CGE relative to the frozen-thawed mature oocytes. We examined ultrathin sections of mature and immature untreated oocytes by TEM and compared them to sections of oocytes that survived the freezing-thawing procedure. Mature untreated oocytes exhibited an abundant number of CGs consisting of dark (electron-dense) and light subpopulations (Fig. 2, top). The dark granules were restricted only to the cortical region, whereas light granules were scattered all through the cytoplasm as well. The GV untreated oocytes contained few granules at the cortex (mostly dark) and many clusters of light granules throughout the cytoplasm (Fig. 3, top). A change in the CG distribution pattern was observed in both mature and immature oocytes after freezing-thawing Fertility and Sterility姞
Ghetler. Cortical granules in cryopreserved human oocytes. Fertil Steril 2006.
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FIGURE 3 The effect of cryopreservation on immature (geminal vesicle stage) oocytes. (Top) An untreated oocyte: Dark electron-dense granules (white arrow) are present in the cortex. Numerous aggregated light (black arrow) granules appear throughout the cytoplasm. (Bottom) A frozenthawed oocyte: Very few dark (white arrow) granules are present at the cortex. Light (black arrow) aggregated granules are in the inner cytoplasm. A large number of membrane-coated transparent vesicles (V) were observed scattered throughout the ooplasm. ZP ⫽ zona pellucida. Bar ⫽ 2 m.
The initial step of the cryopreservation procedure, the dehydration step, which included exposure to cryoprotectants at RT, had already caused the release of content from a significant number of CGs. Similar effect on CGs of mouse and human oocytes was demonstrated following exposure to PROH or DMSO (28). Further exocytosis was demonstrated after the freezing-thawing process, as indicated by the fluorescence of CG exudate in mature as well as immature cryopreserved oocytes. Although not statistically significant, GV oocytes appear more susceptible than MII oocytes to the harch procedure of freezing-thawing regarding their survival rate and degree of CGE. The difference might be attributed to possible differences in the composition of membranes of GV and MII oocytes. Cortical granule exocytosis might occur because of changes in the properties and permeability of the oocyte’s membrane after chilling injury (40), toxicity of the cryoprotectant solutions (41), or osmotic shock induced by osmotic pressure changes that involve an Na⫹/H⫹ exchange–mediated signal transduction pathway (42). Mattioli et al. (43) demonstrated a clear biphasic Ca⫹2 rise in immature pig oocytes during cooling from 30 to 14°C which was related to DNA fragmentation. Ben-Yosef et al. (44) demonstrated cooling-induced rat egg activation, as manifested by intracellular calcium concentration (Ca⫹2i) transients and second polar body extrusion. Such a rise in Ca⫹2i may trigger CGE as well. The overall fluorescence of frozen-thawed oocytes (mature and immature) was higher than that of ICSI-fertilized (1PN and 3PN) positive control oocytes. It is possible that owing to changes in membrane properties and/or permeability that occur during cryopreservation the content of granules located deeper within the oocyte is also released into the PVS, whereas only cortically located granules are released during fertilization, or that there is leakage of lectin (LCA)– binding molecules, which contributes to the massive fluorescent labeling.
Ghetler. Cortical granules in cryopreserved human oocytes. Fertil Steril 2006.
DISCUSSION Oocyte cryopreservation is a feasible procedure yielding birth of healthy babies, although clinical efficiency is still low (39). In the present research we tried to elucidate one of the pitfalls of fertilizing frozen-thawed oocytes, i.e., the premature occurrence of CGE. We demonstrated that the most commonly used procedure of cryopreserving human oocytes, the PROH slow freezing-rapid thawing procedure, has a detrimental effect on the CGs. Our findings explain the low fertilization rate of human frozen-thawed oocytes and justify the need for ICSI for this population of oocytes. 214
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We demonstrated, by TEM analysis, changes in the distribution pattern of the CGs as well as cytoplasmic structural changes following cryopreservation. The cryopreservation procedure resulted in the loss of CGs from the cortical area and in the appearance of vesicles within the cytoplasm of both immature and mature cryopreserved oocytes, which might indicate structural damage occurring from the freezing-thawing process. Similar findings were reported after exposure of bovine oocytes to vitrification solutions (45). Further research is needed for determining the origin and meaning of the newly formed vesicles. Standard embryo cryopreservation technologies appear to have limitations when applied to oocytes. Cryosurvival is low and inconsistent, and oocytes that manage to survive this procedure exhibit structural and functional damage. An improved survival rate for human oocytes was obtained by raising the seeding temperature to ⫺4.5°C, as close as possible to the melting point of the solution (46), indicat-
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ing that the commonly practiced seeding temperature of ⫺7°C is not optimal. Because the initial step of cryopreservation (i.e., dehydration by exposure to cryoprotectants) had already caused an extensive release of CGs, effort should be directed toward modifying that step to minimize the damage and optimize the results. A new freezing protocol should be tailored for the cryopreservation of human oocytes to avoid oocyte damage and improve oocyte post-thaw survival rate. Acknowledgements: The authors thank Ruth Kaplan-Kraicer, M.Sc., Sackler Faculty of Medicine, Tel Aviv University, and Tal Rom, M.Sc., and Ayelet Itzkovitz, M.Sc., IVF Unit, Sapir Medical Center, Kfar Saba, for their technical assistance and Leonid Mittelman, M.D., Sackler Faculty of Medicine, for his assistance in confocal microscopy. This work is in partial fulfillment of the requirements for the Ph.D. degree of Y. Ghetler, Sackler Faculty of Medicine, Tel Aviv University.
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Department of Cell and Developmental Biology and b Department of Pathology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; and c IVF Unit, Meir Hospital (affiliated with Tel Aviv University), Kfar Saba, Israel
Objective: To examine the effect of the commonly used oocyte cryopreservation protocol on the cortical granules (CGs) of human immature germinal vesicle (GV) and mature metaphase II (MII) oocytes. Design: Laboratory study. Setting: IVF unit. Intervention(s): Unfertilized, intracytoplasmic sperm injected (ICSI) oocytes, and immature oocytes were cryopreserved using a slow freezing–rapid thawing program with 1,2-propanediol (PROH) as a cryoprotectant. Main Outcome Measure(s): Cortical granule exocytosis (CGE) was assessed by either confocal microscopy or transmission electron microscopy (TEM). Result(s): The survival rates of frozen-thawed oocytes (mature and immature) were significantly lower compared with zygotes. Both mature and immature oocytes exhibited increased fluorescence after cryopreservation, indicating the occurrence of CGE. Mere exposure of oocytes to cryoprotectants induced CGE of 70% the value of control zygotes. The TEM revealed a drastic reduction in the amount of CGs at the cortex of frozen-thawed GV and MII oocytes, as well as appearance of vesicles in the ooplasm. Conclusion(s): The commonly used PROH freezing protocol for human oocytes resulted in extensive CGE. This finding explains why ICSI is needed to achieve fertilization of frozen-thawed human oocytes. (Fertil Steril威 2006; 86:210 – 6. ©2006 by American Society for Reproductive Medicine.) Key Words: Cryopreservation, cortical granules, exocytosis, mature human oocyte, immature human oocyte
Cryopreservation of human embryos at different developmental stages is successfully applied in IVF treatment programs. However, the cryopreservation of human oocytes still generally yields unsatisfactory results and is therefore considered experimental. The ability to cryopreserve human oocytes is of great importance because it avoids serious ethical and legal issues arising from embryo freezing, such as disposal, donation, or dispute over ownership. Furthermore, cryopreservation of human oocytes could lay the ground for egg donation programs and for establishing oocyte banks, offering the opportunity to preserve the fertility potential of women who face chemotherapy or radiation therapy. Successful cryopreservation of mammalian oocytes had already been reported by several investigators (1, 2). The technologies for cryopreservation of human oocytes used in IVF clinics are derived from traditional protocols of embryo cryopreservation and, as such, render results that are inconsistent and suboptimal. Cryopreservation of oocytes results in low survival rate, hardening of the zona pellucida (ZP), and various types of injuries, including damage to the meiotic spindle and microfilaments (3–7). Attempts have been Received June 8, 2005; revised and accepted December 5, 2005. Reprint requests: Ruth Shalgi, Ph.D., Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel Aviv University, Ramat Aviv, Tel Aviv 69978, Israel (FAX: 972-3-6406149; E-mail: shalgir@ post.tau.ac.il).
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made to freeze immature oocytes at the germinal vesicle (GV) stage, where the DNA is still enclosed within the nucleus and protected by a membrane and may be less sensitive to stressful environmental conditions (8). Immature oocytes, excised from unstimulated follicles or from ovarian tissue, could, in part, survive and mature, but their developmental capability is still very low (9, 10). The common protocol used for oocyte cryopreservation is slow freezing–rapid thawing in 1,2-propanediol (PROH) (11–13). Other protocols such as slow freezing–rapid thawing in dimethyl sulphoxyde (DMSO) (14, 15) and an ultrarapid method (16, 17) have also been introduced. Some research groups have used variations of vitrification (18, 19), including vitrification at specially enhanced cooling rates using open pooled straws (20), electron microscope grids (21), and minimum drop size in supercooled liquid nitrogen (22). Human oocytes that survived cryopreservation had, reportedly, a low fertilization rate (23), indicating that the normal process of the sperm penetration is impaired. Therefore, cryopreserved human oocytes are fertilized by intracytoplasmic sperm injection (ICSI) in an effort to improve their fertilization rate (5, 13). The cortical granules (CGs) are Golgi-derived membranebound spherical or slightly ovoid organelles formed during the early stages of oocyte growth and maturation and are
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present in the cortex of mature unfertilized oocytes of most animal species (24). Both density and distribution of CGs change when they translocate to the cortical region of the oocyte during the process of oocyte maturation (25, 26). The CG’s content is released into the perivitelline space (PVS) immediately after oocyte fertilization or activation and is believed to alter the ZP glycoproteins, establishing the block to polyspermy, and preventing the penetration of additional spermatozoa (27). Due to the major role of CGs in inducing the block to polyspermy, their fate in frozen-thawed oocytes is of great importance, but research results have been inconsistent, ranging from a reduction in the number of CGs following cryopreservation (3, 28) to an apparently normal density and distribution of CGs in frozen-thawed oocytes (29 –31). In view of this inconsistency, we aimed to examine the affect of the commonly used oocyte PROH cryopreservation protocol on the CGs of human immature GV and mature metaphase II (MII) oocytes by using confocal microscopy as well as transmission electron microscopy (TEM). MATERIALS AND METHODS Oocytes included in this study were donated by consenting patients undergoing IVF-ICSI procedures and represent material that is otherwise discarded. The study was approved by the local review board. The ovulation induction (by a “long” protocol of GnRH analog and hMG) and the ICSI procedure used were as described previously (32, 33). Oocytes that failed to be fertilized by ICSI (unfertilized [UF]) and immature oocytes not suitable for ICSI were used. The UF oocytes were reassessed 24 h after ICSI to rule out late fertilization occurrence and only then were they frozen. The immature oocytes were handled according to their developmental state: GV oocytes were frozen on the retrieval day, whereas oocytes at the first metaphase (MI) stage were further cultured for 24 h in the routinely used culture media (Cook Culture System; Cook, Brisbane, Australia). Only oocytes that were able to mature in vitro to the MII stage were cryopreserved. The UF as well as in vitro–matured MII oocytes served as a model of mature oocytes throughout this research. Abnormally fertilized (one pronucleus [1PN] or three pronuclei [3PN]) oocytes served as positive control for occurrence of cortical granule exocytosis (CGE). Study Groups Several experimental groups were included in the research. Group 1: Untreated mature oocytes do not undergo CGE (34) served as a negative control for assessing CG status (n ⫽ 13). Group 2: Mature oocytes that were subjected to the freezing-thawing solutions, according to the protocol, but not to the actual freezing procedure, served as a control for the effect of the solutions on the CGs’ fate, (n ⫽ 16). Group 3 consisted of mature oocytes that were frozen ⱖ1 week before thawing (n ⫽ 23). Group 4 consisted of GV oocytes Fertility and Sterility姞
that were frozen ⱖ1 week before thawing (n ⫽ 10). Group 5: Abnormally fertilized oocytes (1PN or 3PN) after ICSI served as a positive control for CGE occurrence (n ⫽ 11); in a previous study we demonstrated that these abnormally fertilized ICSI oocytes exhibit an intense CGE (34). All oocytes were assessed for CGE by confocal microscopy or TEM. Cryopreservation Protocol The slow freezing–rapid thawing method was used throughout the study. The freezing/thawing solutions (Cook) and protocols were the routine protocols used in our IVF program for zygote and early embryo cryopreservation. In brief, a three-step cryopreservation system was used with HEPESbuffered salt solution (HBSS) containing PROH and sucrose as cryoprotectants: step 1: incubation in prewarmed (37°C) HBSS for 10 minutes at room temperature (RT); step 2: incubation in HBSS containing 1.5 mol/L PROH for 10 minutes at RT; step 3: transfer into HBSS containing 1.5 mol/L PROH and 0.1 mol/L sucrose, loading the samples into straws at RT, and transfering them into an automated Kryo 10 biologic vertical freezer (Kryo 10 series III, Planer, Middlesex, UK). Cooling steps: starting at 20°C, cooling at ⫺2°C/min down to ⫺7°C, soaking for 10 minutes, manual seeding, holding 10 minutes, cooling at ⫺0.3°C/min down to ⫺30°C, cooling at ⫺50°C/min down to ⫺150°C and plunging into liquid nitrogen (⫺196°C). Samples were stored in liquid nitrogen for ⱖ1 week. For thawing, straws were removed from liquid nitrogen and kept 30 seconds at RT before being immersed in a 30°C water bath until all the ice melted. The cryoprotectant was removed with four consecutive steps of 5 minutes each incubation at RT: step 1: HBSS containing 1.0 mol/L PROH and 0.2 mol/L sucrose; step 2: HBSS containing 0.5 mol/L PROH and 0.2 mol/L sucrose; step 3: HBSS containing 0.2 mol/L sucrose; step 4: only HBSS followed by additional 5 minutes at 37°C. Finally, the oocytes were incubated in culture medium (Cook) at 37°C with 5% CO2 in humidified air for 1 hour and then assessed morphologically. Only oocytes that survived the whole process of freezing-thawing were further examined. Assessment of CGE The assessment of cortical reaction was performed by labeling the oocytes with the lectin Lens culinaris agglutinin (LCA), which binds specifically to CG content and exudate (35). The studied oocytes were briefly exposed to 0.25% pronase (Sigma, St. Louis, MO) for ZP removal, followed by three rinses in HTF-HEPES medium (Irvine, Santa Ana, CA) supplemented with 10% synthetic serum substitute (Irvine). The ZP-free oocytes were fixed with 3% paraformaldehyde in DPBS for 30 minutes at RT and washed three times in DPBS supplemented with 1% bovine serum albumin (Fraction V; Sigma). The fixated oocytes were stained for 30 minutes with 211
uranyl acetate followed by lead citrate. The sections were examined using a JOEL-100CX TEM at 80 kV (37).
TABLE 1 Survival rate of frozen thawed oocytes and zygotes.
Study group
Number of oocytes/ zygotes
Survival rate* (mean % ⴞ SEM)
Zygotes Mature ocytes GV oocytes
173 109 92
74.7 ⫾ 4.2a 40.8 ⫾ 4.9b 31.4 ⫾ 7.1b
Note: Survival of thawed oocytes and zygotes was morphologically determined after 1 h of culture. a,b Different superscripts within column denote significant differences (P⬍.001). Ghetler. Cortical granules in cryopreserved human oocytes. Fertil Steril 2006.
5 g/mL LCA-biotin (Vector, Burlingame, CA), washed, and stained with 2 g/mL Texas red–streptavidin (Vector) for 30 minutes, thus labeling the membrane of exocytosed granules and the exudate. For high-quality assessment of the CGE, a confocal laser scanning microscope (CLSM) (LSM 410; Ziess, Oberkochen, Germany) equipped with a 25 mW krypton-argon laser was used. The oocytes were scanned every 5m and the composite image of micrographs taken at all focal planes of each oocyte was displayed. The fluorescence intensity of each individual oocyte was quantified, with the aid of the LSM software, and presented as the ratio between the fluorescent area and the total area of the oocyte, which was then related to the fluorescence intensity of the positive control (group 5) which was set arbitrarily as 1. Assessment of Meiotic Status Oocytes were incubated for 10 minutes in the presence of 2 g/mL DNA-specific fluorochrome (Hoechst 33342, Sigma) and then washed three times in DPBS. Analysis of each oocyte was performed at the time of CGE assessment, using the CLSM. Electron Microscopy (EM) The ultrastructure of CGs of oocytes from the various experimental groups was examined using TEM. At least three oocytes from each group were sectioned and analyzed. The oocytes were fixed for 1 hour in Karnovsky fixative (36), washed twice in 0.2 mol/L cacodylate buffer, pH 7.4, postfixed in 1% osmium tetroxide, washed, and gradually dehydrated through ascending grades of ethanol and propylene oxide. Each oocyte was individually embedded in Araldite (Polysciences, Warrington, PA) and sectioned (0.075 ⫾ 0.015 nm) on an LKB III Ultratome. The ultrathin sections were mounted on grids and stained with 212
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Statistical Analysis The significance of differences between experimental groups was determined by one-way analysis of variance after arcsin square root of proportion transformation (for survival study) and ln transformation (for the fluorescence study) to achieve normal distribution, combined with Tukey’s method for multiple comparisons. RESULTS Survival rate of the frozen-thawed oocytes was compared to the survival rate of zygotes (2 PN) cryopreserved in our IVF program (Table 1). The overall survival rate of oocytes was low and inconsistent (0%–92%). The survival rate of zygotes in our IVF unit during the same period of time was 74.7% ⫾ 4.2% (mean ⫾ SEM), similar to other reports (38) and significantly (P⬍.001) higher than the survival rate of either immature (31.4% ⫾ 7.1%) or mature oocytes (40.8% ⫾ 4.9%). There was no significant difference between the survival rate of mature and immature oocytes. We used the CLSM for assessing the CGE occurrence as an indicator of the fate of CGs in frozen-thawed oocytes. The treated oocytes (groups 2, 3, 4) and the negative control
FIGURE 1 Detection of cortical granule exocytosis (CGE) by labeling with biotinylated lectin Lens culinaris agglutinin and Texas red–strepavidin. Composite confocal microscopy images (A–D) and light microscopy images (a– d) of untreated mature oocyte (A, a), untreated 3PN zygote (B, b), frozenthawed (slow freezing–rapid thawing in 1,2 propanediol) mature oocyte (C, c), and frozenthawed (slow freezing–rapid thawing in 1,2 propanediol) germinal vesicle stage oocyte (D, d), illustrating the degree of CGE at the various treatments. Bar ⫽ 35 m.
Ghetler. Cortical granules in cryopreserved human oocytes. Fertil Steril 2006.
Cortical granules in cryopreserved human oocytes
Vol. 86, No. 1, July 2006
TABLE 2 Fluorescence intensity of frozen-thawed oocytes. Study group
Number of oocytes
Fluorescence intensity (mean ⴞ SEM)
1 2 3 4 5
14 16 23 10 11
0.23 ⫾ 0.09a 0.73 ⫾ 0.21b 1.51 ⫾ 0.33b,c 2.51 ⫾ 1.08c 1
Note: Relative fluorescence intensity expressed as ratio of the abnormally ICSI-fertilized oocytes (group 5). Group 1: untreated mature oocytes. Group 2: mature oocytes following exposure to freezingthawing solutions. Group 3: frozen-thawed mature oocytes. Group 4: frozen-thawed germinal vesicle oocytes. Group 5: abnormally fertilized oocytes, fluorescence intensity arbitrarily defined as 1. a,b,c Different superscripts within column denote significant differences P⬍.001). Ghetler. Cortical granules in cryopreserved human oocytes. Fertil Steril 2006.
procedure (Figs. 2, bottom, and 3, bottom). Only very few dark CGs were observed within frozen-thawed oocytes. Mature frozen-thawed oocytes had only a few scattered CGs deeper in the cytoplasm, whereas GV frozen-thawed oocytes had a few CGs at the cortex and aggregates of light granules throughout the cytoplasm. All the frozen-thawed oocytes formed membrane-coated electron-transparent vesicles, which in some cases were aggregated with CGs (Figs. 2, bottom, and 3, bottom). No such vesicles could be observed within the oocytes before cryopreservation.
FIGURE 2 The effect of cryopreservation on mature oocytes. (Top) An untreated oocyte: Abundant dark electron-dense granules (white arrow) are present in the cortex, and light (black arrow) granules are present mainly deeper in the cytoplasm. (Bottom) A frozen-thawed oocyte: Very few granules are present at the cortex. Light (black arrow) and a few dark (white arrow) granules are in the inner region. A large number of membrane-coated transparent vesicles (V) are observed, scattered throughout the ooplasm. ZP ⫽ zona pellucida. Bar ⫽ 2 m.
(group 1) did not show activation, as indicated by Hoechst staining. However, our results demonstrate that both mature and immature frozen-thawed oocytes underwent CGE (Figs. 1C and D, respectively) compared with untreated oocytes (Fig. 1A) or fertilized oocytes (Fig. 1B). The calculated CGE intensity is presented in Table 2. Untreated mature oocytes (group 1, negative control) exhibited very low fluorescence relative to the positive control group of abnormally fertilized oocytes (group 5). The mere exposure to cryopreservation solutions (group 2) resulted in a strong CGE: about 70% of the positive control value and significantly different from the untreated mature oocytes (P⬍.001). The CGE of oocytes, both mature (group 3) and immature (group 4), passing through the complete freezingthawing procedure was significantly stronger than that of untreated oocytes. The GV frozen-thawed oocytes exhibited a higher (but not statistically different) degree of CGE relative to the frozen-thawed mature oocytes. We examined ultrathin sections of mature and immature untreated oocytes by TEM and compared them to sections of oocytes that survived the freezing-thawing procedure. Mature untreated oocytes exhibited an abundant number of CGs consisting of dark (electron-dense) and light subpopulations (Fig. 2, top). The dark granules were restricted only to the cortical region, whereas light granules were scattered all through the cytoplasm as well. The GV untreated oocytes contained few granules at the cortex (mostly dark) and many clusters of light granules throughout the cytoplasm (Fig. 3, top). A change in the CG distribution pattern was observed in both mature and immature oocytes after freezing-thawing Fertility and Sterility姞
Ghetler. Cortical granules in cryopreserved human oocytes. Fertil Steril 2006.
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FIGURE 3 The effect of cryopreservation on immature (geminal vesicle stage) oocytes. (Top) An untreated oocyte: Dark electron-dense granules (white arrow) are present in the cortex. Numerous aggregated light (black arrow) granules appear throughout the cytoplasm. (Bottom) A frozenthawed oocyte: Very few dark (white arrow) granules are present at the cortex. Light (black arrow) aggregated granules are in the inner cytoplasm. A large number of membrane-coated transparent vesicles (V) were observed scattered throughout the ooplasm. ZP ⫽ zona pellucida. Bar ⫽ 2 m.
The initial step of the cryopreservation procedure, the dehydration step, which included exposure to cryoprotectants at RT, had already caused the release of content from a significant number of CGs. Similar effect on CGs of mouse and human oocytes was demonstrated following exposure to PROH or DMSO (28). Further exocytosis was demonstrated after the freezing-thawing process, as indicated by the fluorescence of CG exudate in mature as well as immature cryopreserved oocytes. Although not statistically significant, GV oocytes appear more susceptible than MII oocytes to the harch procedure of freezing-thawing regarding their survival rate and degree of CGE. The difference might be attributed to possible differences in the composition of membranes of GV and MII oocytes. Cortical granule exocytosis might occur because of changes in the properties and permeability of the oocyte’s membrane after chilling injury (40), toxicity of the cryoprotectant solutions (41), or osmotic shock induced by osmotic pressure changes that involve an Na⫹/H⫹ exchange–mediated signal transduction pathway (42). Mattioli et al. (43) demonstrated a clear biphasic Ca⫹2 rise in immature pig oocytes during cooling from 30 to 14°C which was related to DNA fragmentation. Ben-Yosef et al. (44) demonstrated cooling-induced rat egg activation, as manifested by intracellular calcium concentration (Ca⫹2i) transients and second polar body extrusion. Such a rise in Ca⫹2i may trigger CGE as well. The overall fluorescence of frozen-thawed oocytes (mature and immature) was higher than that of ICSI-fertilized (1PN and 3PN) positive control oocytes. It is possible that owing to changes in membrane properties and/or permeability that occur during cryopreservation the content of granules located deeper within the oocyte is also released into the PVS, whereas only cortically located granules are released during fertilization, or that there is leakage of lectin (LCA)– binding molecules, which contributes to the massive fluorescent labeling.
Ghetler. Cortical granules in cryopreserved human oocytes. Fertil Steril 2006.
DISCUSSION Oocyte cryopreservation is a feasible procedure yielding birth of healthy babies, although clinical efficiency is still low (39). In the present research we tried to elucidate one of the pitfalls of fertilizing frozen-thawed oocytes, i.e., the premature occurrence of CGE. We demonstrated that the most commonly used procedure of cryopreserving human oocytes, the PROH slow freezing-rapid thawing procedure, has a detrimental effect on the CGs. Our findings explain the low fertilization rate of human frozen-thawed oocytes and justify the need for ICSI for this population of oocytes. 214
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We demonstrated, by TEM analysis, changes in the distribution pattern of the CGs as well as cytoplasmic structural changes following cryopreservation. The cryopreservation procedure resulted in the loss of CGs from the cortical area and in the appearance of vesicles within the cytoplasm of both immature and mature cryopreserved oocytes, which might indicate structural damage occurring from the freezing-thawing process. Similar findings were reported after exposure of bovine oocytes to vitrification solutions (45). Further research is needed for determining the origin and meaning of the newly formed vesicles. Standard embryo cryopreservation technologies appear to have limitations when applied to oocytes. Cryosurvival is low and inconsistent, and oocytes that manage to survive this procedure exhibit structural and functional damage. An improved survival rate for human oocytes was obtained by raising the seeding temperature to ⫺4.5°C, as close as possible to the melting point of the solution (46), indicat-
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ing that the commonly practiced seeding temperature of ⫺7°C is not optimal. Because the initial step of cryopreservation (i.e., dehydration by exposure to cryoprotectants) had already caused an extensive release of CGs, effort should be directed toward modifying that step to minimize the damage and optimize the results. A new freezing protocol should be tailored for the cryopreservation of human oocytes to avoid oocyte damage and improve oocyte post-thaw survival rate. Acknowledgements: The authors thank Ruth Kaplan-Kraicer, M.Sc., Sackler Faculty of Medicine, Tel Aviv University, and Tal Rom, M.Sc., and Ayelet Itzkovitz, M.Sc., IVF Unit, Sapir Medical Center, Kfar Saba, for their technical assistance and Leonid Mittelman, M.D., Sackler Faculty of Medicine, for his assistance in confocal microscopy. This work is in partial fulfillment of the requirements for the Ph.D. degree of Y. Ghetler, Sackler Faculty of Medicine, Tel Aviv University.
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