Slow cooling of human oocytes: ultrastructural injuries and apoptotic status

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1 Slow cooling of human oocytes: ultrastructural injuries and apoptotic status Roberto Gualtieri, Ph.D., a Mirella Iaccarino, B.S., b Valentina Mollo, B.S., a Marina Prisco, Ph.D., c Stefania Iaccarino, M.D., b and Riccardo Talevi, Ph.D. a a Department of Structural and Functional Biology, University of Naples Federico II, Monte S Angelo, Naples; b Mediterranean Center of Assisted Reproduction, Mediterranean Clinic, Naples; and c Department of Biological Sciences, University of Naples Federico II, Monte S Angelo, Naples, Italy Objective: To identify the damages caused by slow cooling human metaphase II (MII) oocytes comparing the ultrastructure, inner mitochondrial membrane potential (DJm), and apoptotic status of fresh and cryopreserved oocytes. Design: Experimental study. Setting: University biology research unit and private IVF unit. Patient(s): Fresh and cryopreserved supernumerary MII oocytes donated from women undergoing IVF cycles. Main Outcome Measure(s): Ultrastructure was assessed by transmission electron microscopy (TEM), mitochondrial function by means of the fluorescent DJm reporter JC-1, and apoptotic status through fluorescent labeling with the pan-caspase inhibitor fluorescein isothiocyanate conjugate (FITC)-VAD FMK, and terminal deoxynucleotidyl transferase-mediated dutp nick-end labeling. Result(s): Compared to fresh oocytes, frozen/thawed (F/T) oocytes showed reduced cortical granule densities (F/T /10 mm vs. fresh /10 mm), swelling of smooth endoplasmic reticulum (F/T mm 2 vs. fresh mm 2 ), decreased electron density of the mitochondrial matrix and damage to the mitochondrial membranes, low DJm of pericortical mitochondria, but no signs of apoptosis. Conclusion(s): Slow cooling is associated with cortical granule exocytosis, swelling of smooth endoplasmic reticulum vesicles, and mitochondrial damage, but does not induce early or late apoptotic events. The observed injuries might be responsible for the reduced developmental competence of cryopreserved oocytes. (Fertil Steril Ò 2009;91: Ó2009 by American Society for Reproductive Medicine.) Key Words: Assisted reproduction, cryopreservation, human oocyte, ultrastructure, mitochondria, apoptosis, cryodamage Interest in the cryopreservation of unfertilized human and animal oocytes spans two decades and has been reinforced by recent legislations that banned embryo freezing in several countries (e.g., Italy, Law no. 40, February 19, 2004). Oocyte cryopreservation offers several advantages: [1] it avoids the ethical and legal problems encountered in embryo cryopreservation, [2] it preserves female fertility in cases of pathologies or therapies that damage or destroy ovarian reserves, and [3] it would introduce cryobanking for oocyte donation. Although the first two births from cryopreserved oocytes were reported about 20 years ago (1, 2), such results could not be reproduced until 1997 (3) after replacement of dimethyl sulfoxide (DMSO) with propane-1,2-diol (PrOH) (4). The increase in sucrose concentration from M in a slow-cooling protocol based on 1.5 M PrOH (5) greatly Received November 7, 2007; revised January 11, 2008; accepted January 22, 2008; published online March 25, R.G. has nothing to disclose. M.I. has nothing to disclose. V.M. has nothing to disclose. M.P. has nothing to disclose. S.I. has nothing to disclose. R.T. has nothing to disclose. Supported by the Italian Ministry of University and Research, PRIN 2005 grant _004 to Roberto Gualtieri Influence of Cryopreservation on Qualitative Parameters of In Vivo Matured Human Oocytes. Reprint requests: Roberto Gualtieri, Ph.D., Dipartimento di Biologia Strutturale e Funzionale, Universita di Napoli Federico II, Complesso Universitario di Monte S Angelo, Via Cinthia, Napoli, Italy (FAX: ; roberto.gualtieri@unina.it). improved survival, fertilization, and embryo development of frozen-thawed (F/T) oocytes. Currently this method is adopted by several IVF units worldwide with conflicting results (6 12; reviewed in Ref. 13), which may depend on oocyte quality (i.e., the ability to withstand the stress of cryopreservation). The metaphase II (MII) oocyte represents the final product of molecular and cellular processes that confer the ability to normally fertilize, develop, and produce a healthy offspring. Cellular and molecular alterations associated with osmotic forces produced during oocyte dehydration rehydration cycles or by cryoprotectants may affect the molecular architecture of the meiotic spindle and chromosomes, as well as the distribution and activity of other cytoplasmic components. As a consequence, the competence for fertilization and pre- and postimplantation embryo development may be reduced or completely lost. The first reports on the influence of cryoprotectants and decreased temperature on the MII spindle depolymerization in the mouse (14, 15) raised considerable concerns on human oocyte cryopreservation. However, later reports on human MII oocytes, frozen slowly with PrOH and sucrose, showed that surviving oocytes had normal spindles and karyotypes (16, 17), and that microtubule repolymerization occurs during post-thawing culture reconstituting normal spindles in the majority of oocytes (18, 19). Therefore, little is known about the nature of structures and processes affected by cryopreservation that are /09/$36.00 Fertility and Sterility â Vol. 91, No. 4, April doi: /j.fertnstert Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc.

2 responsible for the low competence of F/T oocytes for fertilization and embryogenesis. In the present study, the influence of slow freezing/rapid thawing with 1.5 M PrOH and 0.3 M sucrose was studied on MII oocyte ultrastructure and mitochondrial function, by means of the inner mitochondrial membrane potential (DJm) reporter JC-1, and apoptotic status through terminal deoxynucleotidyl transferase-mediated dutp nick-end labeling (TUNEL) and fluorescent labeling with the pan-caspase inhibitor fluorescein isothiocyanate conjugate (FITC)-VAD FMK. MATERIALS AND METHODS Source of Oocytes The present study was approved by the Ministry of University and Research of the Italian Government (PRIN 2005 grant _004). In this study, patients undergoing intracytoplasmic sperm injection (ICSI) at the Centro Mediterraneo di Fecondazione Assistita, Naples, Italy, between March 2004 and April 2006, from whom were retrieved more than three good quality oocytes, were offered the opportunity to cryopreserve supernumerary eggs for clinical or research use. Supernumerary fresh MII oocytes included in this study were collected from patients (mean age SD: years) whose infertility was due to a male factor or to a tubal factor and donated for research under written consent. All patients were down-regulated with GnRH analogue (Enantone Die; Takeda, Catania, Italy) starting on day 21 of the previous cycle and continuing until the day of hcg administration. Ovarian stimulation was performed by a step-down protocol with a decreasing dose of gonadotropins (Gonal-F 75 IU; Serono, Rome, Italy or Puregon IU; Organon, Oss, The Netherlands and Menogon; Ferring Spa, Milan, Italy) until the day before hcg injection. Follicle development was monitored by ultrasound and E 2 measurements. Ovulation was induced by 10,000 IU of hcg (Gonasi HP 5000 IU; Amsa Srl, Rome, Italy) when at least two follicles of R18 mm in diameter were observed, with E 2 concentrations corresponding to the number of follicles. Transvaginal ultrasound-guided oocyte retrieval was performed 35 hours after hcg administration, under general anesthesia. Cumulus removal was performed by a brief exposure to hyaluronidase (20 IU/mL; Sigma Aldrich Srl, Milan, Italy) and mechanical manipulation with fine-bore glass pipettes. Denuded oocytes were evaluated with a Nikon TE 2000 inverted microscope (Nikon, Kanagawa, Japan) under Hoffmann modulation contrast, and only oocytes at the MII stage showing a homogeneous cytoplasm and no dysmorphisms (20) were either directly processed for the analysis or slow frozen within 2 3 hours after collection. Oocyte culture was performed in 20-mL drops of Quinn s Advantage Protein Plus Cleavage Medium (Sage BioPharma, Bedminster, NJ) under mineral oil at 37 C, 5% CO 2. Oocytes from the same patients were randomly allocated to the fresh or F/T experimental groups. Cryopreservation Slow freezing/fast thawing was performed according to the original protocol based on 1.5 M PrOH and 0.3 M sucrose described by Fabbri et al. (5). All solutions were prepared using Dulbecco s phosphate-buffered saline (PBS) (GIBCO, Life Technologies Ltd., Paisley, United Kingdom), PrOH (Fluka Chemika; Sigma Aldrich SrL), and a serum protein supplement (SPS; MediCult, Florence, Italy). Procedures were performed at room temperature unless otherwise stated. The MII oocytes were equilibrated in 1.5 M PrOH, 20% SPS in Dulbecco s PBS for 10 minutes, transferred in 1.5 M PrOH, 0.3 M sucrose, 20% SPS in Dulbecco s PBS for 5 minutes and loaded into plastic straws (Paillettes Crystal 133 mm; Cryo Bio System, Paris, France). A maximum of four oocytes were loaded into each straw, and then placed into the CryoMed Freezer 7457 (Thermo Forma, Marietta, OH). The temperature was gradually decreased from 20 to 8 C at a rate of 2 C/min. Manual seeding was induced during the 10-minute holding ramp at 8 C. The temperature was decreased to 30 C at a rate of 0.3 C/min and then to 150 C at a rate of 50 C/min. The straws were plunged into liquid nitrogen and stored until thawing. Straws were thawed in air for 30 seconds and then into a 30 C water bath for 40 seconds. Oocytes were treated in 1.0 M PrOH, 0.3 M sucrose, 20% SPS in Dulbecco s PBS for 5 minutes, equilibrated in 0.5 M PrOH, 0.3 M sucrose, 20% SPS in Dulbecco s PBS for 5 minutes, and then in 0.3 M sucrose, 20% SPS in Dulbecco s PBS for 10 minutes. Finally, they were placed in 20% SPS in Dulbecco s PBS for 10 minutes at room temperature and 10 minutes at 37 C. The thawed oocytes were cultured for 3 hours in 20-mL drops of IVF medium (MediCult) under mineral oil at 37 C, 5% CO 2 in air and all surviving oocytes were processed for analysis. Electron Microscopy Procedures were carried out at room temperature unless otherwise stated. The MII fresh (n ¼ 16) and surviving F/T oocytes (n ¼ 25) were fixed within 2 3 hours after collection or thawing in 2.5% glutharaldehyde (SIC, Rome, Italy) in 0.1 M sodium cacodylate at ph 7.3, washed three times for 10 minutes in the same buffer, postfixed in 1% osmium tetroxide (SIC) in 0.1 M sodium cacodylate at ph 7.3 on ice, washed three times for 10 minutes in the same buffer, treated in 0.1% tannic acid in cacodylate for 10 minutes and dehydrated in ascending series of ethanol (Carlo Erba Reagenti, Milan, Italy) on ice. Oocytes were treated twice for 5 minutes with propylene oxide (FLUKA, Milan, Italy), infiltrated in 1:1 propylene oxide/epon 812 (Agar Scientific, Stansted, United Kingdom) overnight, and individually embedded in fresh resin. Thick (0.5 1 mm) and thin sections (60 80 nm) were cut with a diamond knife (Diatome, Biel, Switzerland) at a Reichert-Jung Ultracut E ultramicrotome and collected on glass slides or 200-mesh thin bar copper grids (SIC). Thick sections were stained with 0.1% toluidine blue in sodium borax, examined by light microscopy (LM) (Zeiss Axioskop, Jena, Germany) and photographed using a Nikon DS-cooled camera head DS-5Mc connected to a Nikon DS 1024 Gualtieri et al. Cryodamage in slow frozen human oocytes Vol. 91, No. 4, April 2009

3 camera control unit DS-L1. Thin sections were stained with saturated uranyl acetate in methanol and Reynold s lead citrate and observed and photographed with a Philips (Eindhoven, The Netherlands) EM 208 S electron microscope (EM) at 80 KV. The morphometry of the cortical granule (CG) numerical density was carried out on images acquired at a magnification of 7,100 of whole surface profile on two to three equatorial sections for each fresh and F/T oocyte. The CG density was expressed as CG number per 10 mm of oocyte surface profile. The same image series were analyzed to determine the mean area of smooth endoplasmic reticulum (SER) vesicles in the pericortical cytoplasm of fresh and F/ T oocytes. Fluorescence Analysis of Inner Mitochondrial Membrane Potential Fresh (n ¼ 17) and F/T (n ¼ 24) oocytes were incubated with the inner mitochondrial membrane potential probe JC-1 (5,5 0,6,6 0 - tetrachloro-1,1 0,3,3 0 -tetraethylbenzimidazolylcarbocyanine iodide; Invitrogen, Milan, Italy) at 2 mm in IVF medium (MediCult) for 30 minutes at 37 C, 5% CO 2 in air (21, 22). Negative controls (n ¼ 3) were prepared by treatment with the mitochondrial membrane potential disrupter carbonyl cyanide 3-chlorophenylhydrazone at 50 mm for 5 minutes at 37 C, 5% CO 2 in air followed by exposure to JC-1 as described previously. Images were acquired using a Nikon DS-cooled camera head DS-5Mc connected to a Nikon DS camera control unit DS-L1. Assessment of Activated Caspase FITC-VAD-FMK (Promega, Milan, Italy), a cell-permeant fluorochrome derivative of the caspase inhibitor Val-Ala- DL-Asp-fluoromethylketone, was used to detect activated caspases in oocytes by epifluorescent microscopy. Fresh (n ¼ 5) and F/T (n ¼ 17) oocytes were incubated in 1 ml of IVF medium (MediCult) containing 5 mm of FITC-VAD-FMK for 1 hour at 37 C, 5% CO 2 in air, washed twice for 10 minutes in the same medium and observed at a Nikon TE 2000 fluorescence microscope. Images were acquired using a Nikon DS-cooled camera head DS-5Mc connected to a Nikon DS camera control unit DS-L1. TUNEL Assay The DNA fragmentation was determined using the TUNEL procedure (In Situ Cell Death Detection Kit, Fluorescein; Roche Diagnostics, Milan, Italy). Free 3 OH ends of DNA were labeled with fluorescein isothiocyanate-conjugated dutp (FITC-dUTP) by means of the enzyme terminal deoxynucleotidyl transferase (TdT). Fresh (n ¼ 8) and F/T (n ¼ 15) oocytes were fixed in 4% freshly prepared paraformaldehyde in PBS for 1 hour at room temperature, washed three times for 10 minutes in PBS 3 mg/ml polyvinylpyrrolidone (PBS-PVP), permeabilized in 0.1% (vol/vol) Triton X-100 containing 0.1% (wt/vol) sodium citrate in PBS for 30 minutes at 4 C and finally washed three times for 10 minutes in PBS-PVP. Samples were then incubated in 50-mL drops of TUNEL reaction mixture (containing FITC-dUTP and TdT) for 1 hour at 37 C in the dark. Oocytes were then washed in PBS-PVP, transferred to 50-mL drops of 1 mg/ ml Hoechst in PBS-PVP for 30 minutes at room temperature, washed three times in PBS-PVP, and mounted on glass slides. In each procedure negative (n ¼ 2) and positive (n ¼ 2) fresh oocyte controls were prepared by omission of TdT in the reaction mixture or by pretreatment with 1 mg/ ml DNase I (Roche Diagnostics) for 10 minutes at room temperature. Samples were observed with a Nikon TE 2000 fluorescence microscope. Images were acquired using a Nikon DS-cooled camera head DS-5Mc connected to a Nikon DS camera control unit DS-L1. Statistical Analysis The data are presented as mean SD. Overall analysis was performed by the estimate model of analysis of variance (ANOVA) (23) followed by the Tukey s honestly significant difference test for pairwise comparisons when overall significance was detected. Percentage data were compared by c 2 or Fisher s exact test. Statistical significance was defined as P<.05. RESULTS Ultrastructure and Morphometry of Fresh and F/T Oocytes Among 107 MII oocytes cryopreserved and thawed for analysis, 81 survived after 2 3 hours of post-thawing culture (survival rate, 75.7%). The transmission electron microscopy (TEM) analysis was performed on 16 fresh and 25 F/T oocytes. Figure 1a,b shows the typical ultrastructure of two representative fresh oocytes at low magnification. These are characterized by long and thin microvilli projecting into a narrow subzonal space, numerous and highly electron-dense CGs arranged in single or multiple rows, SER vesicles, spherical clusters of SER tubular cisternae (Fig. 1a, inset), and numerous mitochondria uniformly scattered within the pericortical cytoplasm. Mitochondria are characterized by a few cristae rarely crossing an electron-dense matrix and are often in close apposition to SER vesicles characterized by a granular content of medium electron density (Fig. 2a,b). A certain degree of ultrastructural heterogeneity was observed among different fresh oocytes with regard to the number of CGs and the distribution of mitochondria and SER vesicles in the pericortical cytoplasm. In fact, the apparent numerical density of CGs was variable in different oocytes (Fig. 1a,b) and within single oocytes, whereas mitochondria and SER vesicles were observed grouped in clumps (Fig. 1b) rather than homogeneously scattered in the pericortical cytoplasm (Fig. 1a) in 56% of the oocytes. Such heterogeneity did not correspond to a different morphological grading under Hoffmann interferential contrast in living oocytes before fixation. Morphometrical measures of CG density were performed on the collection of images of whole surface profiles at a magnification of 7,100 on two to three equatorial sections for each oocyte. The CG densities were markedly variable with a minimum and maximum value per oocyte of 5 and 18 CGs per 10 mm of linear surface profile and a mean SD of / 10 mm. One or more vacuoles mm in diameter and with interrupted limiting membrane and a translucent content were Fertility and Sterility â 1025

4 FIGURE 1 Transmission electron micrographs of fresh (a,b) and frozen/thawed (c e) human metaphase II oocytes. The pericortical cytoplasm of fresh oocytes (a,b) contains a variable number of cortical granules (CG) beneath the plasma membrane and numerous mitochondria (M) sometimes grouped in discrete sites (b). In frozen/thawed oocytes (c e), the pericortical cytoplasm is often devoid of organelles (c,d) and shows an apparently reduced CG numerical density and mitochondria grouped with smooth endoplasmic reticulum vesicles (SER) in the deeper cytoplasmic regions. Some frozen/thawed oocytes show enlarged SER vesicles (d, arrows) and figures of CG exocytosis (e, asterisks). Clusters of SER tubular cisternae in fresh (a inset) and frozen/thawed (d inset) oocytes have a similar ultrastructure. ZP ¼ zona pellucida. Bar, 3 mm (a d), 1 mm (a,d insets; e) Gualtieri et al. Cryodamage in slow frozen human oocytes Vol. 91, No. 4, April 2009

5 FIGURE 2 Transmission electron micrographs of fresh (a,b) and frozen/thawed (c,d) human metaphase II oocytes. Mitochondria (M) and smooth endoplasmic reticulum (SER) vesicles are scattered in the pericortical cytoplasm of fresh oocytes (a,b). In frozen/thawed oocytes SER vesicles are often found in association with mitochondria and appear swollen (c,d). In several frozen/thawed oocytes, mitochondria appear degenerated (d, asterisks) and SER vesicles markedly swollen. Bar, 1 mm (a d). detected in 12.5% of the fresh oocytes. In F/T oocytes (Fig. 1c,d), mitochondria and SER vesicles were more often observed grouped in clumps (F/T 76% vs. fresh 56%, P ¼.18) and a cytoplasmic pericortical layer 5 10 mm thick beneath the oolemma appeared cleared of such elements in 52% of the oocytes (F/T 52% vs. fresh 0, P<.001). The apparent CG density was markedly variable among different oocytes and generally reduced compared to fresh oocytes (Fig. 1c,d). Fusion of the CG membrane with the oolemma or association with exocytozed CG contents with the oocyte surface was detected in some F/T oocytes (Fig. 1e). Results of morphometrical analysis showed minimum and maximum values per oocyte of 0.1 and 7/10 mm, respectively, and a mean SD of /10 mm. Mean CG densities of the fresh and F/T groups were significantly different (P<.001). Careful comparison of ultrastructural features of fresh and F/T oocytes revealed other conspicuous differences in SER vesicles and associated mitochondria within the pericortical cytoplasm. The SER vesicles in the majority of F/T oocytes (Figs. 1d and 2c,d) appeared swollen compared to vesicles in fresh oocytes (Figs. 1a,b and 2a,b). Clusters of SER tubular cisternae scattered in the pericortical cytoplasm had a similar area and distribution in fresh and cryopreserved samples (Fig. 1, insets). Morphometrical analysis performed on the fresh and F/T groups to determine the mean area of SER vesicles demonstrated markedly variable SER vesicle areas. In the fresh group, minimum and maximum values of SER vesicle area were and mm 2 per oocyte, respectively, with a mean SD of mm 2. In the F/T group, the lowest and highest SER vesicle area were and mm 2 per oocyte, respectively, with a mean SD of mm 2. Differences of SER vesicle area between the fresh and F/T groups were highly significant (P<.001). One or more vacuoles resembling those found in fresh oocytes were retrieved in 64% of the F/T oocytes. As stated, in fresh samples, mitochondria had the typical morphological features described in human oocytes (24, 25). They occur as spherical/oval elements, approximately Fertility and Sterility â 1027

6 TABLE 1 Ultrastructure, mitochondrial function, and apoptotic status in fresh and frozen/thawed (F/T) human oocytes. Fresh F/T P value CG (No./10 mm surface profile) a <.001 Clearing of pericortical cytoplasm (%) b 0 52 <.001 Mean SER vesicle area (mm 2 ) a <.001 Vacuoles (%) b <.01 Degenerated mitochondria (%) b 0 71 <.001 High polarized mitochondria (%) b <.01 Activated caspases (%) b 0 0 NS DNA fragmentation (%) b 0 0 NS Note: NS ¼ not significant. a Values are mean SD. b Percentage of oocytes. 0.5 mm in diameter, and typically contain a few short cristae rarely crossing a highly electron dense matrix (Fig. 2a,b). In sharp contrast, a high percentage of F/T oocytes (72%) showed a variable and, in some cases, very high fraction of mitochondria with a decreased electron density of the matrix or with ruptures of the outer and inner membranes visible on the cut plane (Fig. 2d). In addition, the fraction of F/Toocytes showing mitochondrial damage was also characterized by SER swelling (Fig. 2d), higher than that observed in the rest of F/T oocytes (Fig. 2c). Mean SER vesicle area in F/T oocytes with degenerated mitochondria ( mm 2 ) was more than twice the value detected in F/T oocytes with intact mitochondria ( mm 2 )(P<.05). No significant differences in CG densities and percentages of vacuolated oocytes were detected between the F/T subgroups with intact and degenerated mitochondria. Ultrastructural data are summarized in Tables 1 and 2. Mitochondrial Inner Membrane Potential in Fresh and F/T Oocytes To evaluate whether the mitochondrial structural damage detected through TEM analysis in the pericortical cytoplasm of F/T oocytes was correlated with an altered mitochondrial function, live fresh (n ¼ 17) and F/T (n ¼ 24) oocytes were labeled with the inner membrane potential reporter JC-1. This probe is known to differentially label low- and highpolarized mitochondria. In fact, at DJm <100 mv, JC-1 remains as a monomer and emits green fluorescence in the FITC channel, whereas at DJm >140 mv it forms J-aggregates and emits red fluorescence in the rhodamine isothiocyanate channel (26). As reported by Van Blerkom et al. (21), fresh oocytes in the present study also had particulate red fluorescence in the pericortical region (i.e., high polarized mitochondria) and green fluorescence in the deeper cytoplasm. In particular, 77% of fresh oocytes had a variable number of high polarized mitochondria in the pericortical cytoplasm and sometimes in the first polar body (Fig. 3a d). In sharp contrast, only 25% of the F/T oocytes had high polarized pericortical mitochondria. In the remainder of the F/T oocytes, particulate red fluorescence was absent or retained only in the first polar body (Fig. 3e h). In addition, in 37.5% of F/T oocytes the pericortical cytoplasm did not fluoresce at all (Fig. 3g,h), suggesting a total collapse of DJm or a displacement toward deeper cytoplasmic areas as observed with TEM. Control fresh oocytes (n ¼ 3) treated with the mitochondrial membrane potential disrupter carbonyl cyanide 3- chlorophenylhydrazone were negative both in the red and green channels. Data are summarized in Table 1. TABLE 2 Ultrastructure in frozen/thawed (F/T) human oocytes with normal and degenerated mitochondria. F/T normal mitochondria F/T degenerated mitochondria P value CG (No./10 mm surface profile) a NS Mean SER vesicle area (mm 2 ) a <.05 Vacuoles (%) b NS Note: NS ¼ not significant. a Values are mean SD. b Percentage of oocytes Gualtieri et al. Cryodamage in slow frozen human oocytes Vol. 91, No. 4, April 2009

7 FIGURE 3 Fluorescent micrographs of fresh (a d) and frozen/thawed (e h) human metaphase II oocytes labeled with the inner mitochondrial membrane potential probe JC-1. Images of the oocytes in a,c,e,g have been acquired in the green channel, whereas b,d,f,h show images of the same oocytes acquired in the red channel. Red particulate fluorescence indicate high polarized mitochondria that in most fresh oocytes are localized in the pericortical cytoplasm and sometimes in the first polar body (PB) (a d). Green fluorescence indicate low polarized mitochondria localized in the deeper cytoplasm. In most frozen/thawed oocytes, high polarized mitochondria are absent or retained only in the polar body (e h). In some frozen/thawed oocytes (g,h) the pericortical cytoplasm is negative and the fluorescence appears restricted to the deeper cytoplasm. Bar, 50 mm. Fertility and Sterility â 1029

8 FIGURE 3 Continued Assessment of Activated Caspases and DNA Fragmentation in Fresh and F/T Oocytes The cell-permeant fluorochrome derivative of the pancaspase inhibitor Val-Ala-DL-Asp-fluoromethylketone, FITC-VAD-FMK, was used to detect activated caspases in fresh (n ¼ 5) and F/T (n ¼ 17) oocytes. All oocytes analyzed were negative (Fig. 4). Previously reported punctate autofluorescence associated with lipid refractile bodies (22) was sometimes detected in the oocyte cytoplasm (Fig. 4; see also Fig. 5a,c). The possibility that cryopreservation causes DNA fragmentation was evaluated in F/T (n ¼ 15) oocytes by means of the TUNEL reaction in situ. Fresh oocytes (n ¼ 4) were identically processed for comparison. Both fresh (Fig. 5a,b) and F/T (Fig. 5c,d) oocytes were TUNEL negative. Residual cumulus cells of both fresh and F/T oocytes were sometimes TUNEL positive (Fig. 5c). Control fresh oocytes (n ¼ 2) prepared by omission of TdT in the reaction mixture were negative, whereas positive controls pretreated with DNase I (n ¼ 2) had markedly positive chromosomes both in the oocyte and polar body (Fig. 5e,f). Also in these samples punctate autofluorescence was sometimes detected in the oocyte cytoplasm (Fig. 5a,c). Data are summarized in Table 1. DISCUSSION The possibility to successfully cryopreserve human oocytes represents an important aim in assisted reproduction technologies (ART). However, because the efficiency of oocyte cryopreservation methods is still unsatisfactory, as they reduce the embryo competence to undergo correct development, the assessment of cryodamaged processes and organelles is fundamental in the evaluation and refinement of current and future cryopreservation protocols. The main results in the present study demonstrated that slow cooling with 1.5 M PrOH and 0.3 M sucrose induces a premature CG exocytosis, affects the pericortical distribution and morphofunctional integrity of mitochondria and SER vesicles, causes a moderate microvacuolization, but does not induce early or late apoptotic events. Morphometrical TEM analysis demonstrated greatly reduced CG numerical densities in F/T oocytes. In fact, the mean CG density in the F/T group accounted for only a third of that observed in the fresh group. The statistically significant numerical reduction of CGs in F/T oocytes and the retrieval of clear aspects of exocytosis in some fields demonstrate that cryopreservation with 1.5 M PrOH and 0.3 M sucrose triggers a massive CG exocytosis. Previous data on the possibility that cryopreservation stimulates a premature CG exocytosis in animals and humans are conflicting. Such discrepancies may depend on the type of cryoprotectant and the method used to detect CGs. In fact, DMSO and cooling of mouse oocytes have been reported to induce zona pellucida (ZP) hardening and numerical decrease of CGs (27).On the other hand, slow cooling of human oocytes has classically been afforded using PrOH and sucrose as cryoprotectants. Although exposure of human oocytes to DMSO or PrOH at room temperature has been reported to trigger CG exocytosis (28), several investigators did not detect a numerical reduction of CGs assessed through fluorescent labeling with Lens culinaris agglutinin in human oocytes slow cooled with PrOH and sucrose (8, 16, 17, 22). Lens culinaris agglutinin staining has been applied both to permeabilized and unpermeabilized oocytes to reveal CGs in the cortex (8, 16, 17, 22) or their exocytozed contents associated with the oocyte surface (29). This latter method may avoid possible ambiguity in the localization of CG contents in permeabilized samples. In fact, Lens culinaris agglutinin staining of unpermeabilized samples revealed a surface labeling due to CG exocytosis that 1030 Gualtieri et al. Cryodamage in slow frozen human oocytes Vol. 91, No. 4, April 2009

9 FIGURE 4 Bright field (a,c) and fluorescent micrographs (b,d) of representative fresh (a,b) and frozen/thawed (c,d) human metaphase II oocytes labeled with FITC-VAD-FMK for detection of activated caspases. Both fresh and frozen/ thawed oocytes were negative. Asterisks indicate autofluorescent lipid inclusions commonly found in human oocytes. Bar, 50 mm. was confirmed by TEM analysis in oocytes slow cooled with 1.5 M PrOH and 0.2 M sucrose (29). Recently, Nottola et al. (30) observed a CG numerical reduction or a decreased CG electron density both with 0.1 and 0.3 M sucrose in about 40% of F/T oocytes. Morphometrical assessment of CG numerical density on whole surface profiles in several equatorial sections for each oocyte, performed in the present study, confirms that the slow cooling protocol with 1.5 M PrOH and 0.3 M sucrose leads to a marked CG numerical reduction and provides quantitative data on the extent of CG exocytosis. Because CG exocytosis is physiologically triggered at fertilization by intracellular calcium ([Ca 2þ ] i ) oscillations, an eventual reduction of CG densities in F/T oocytes might be the consequence of a premature [Ca 2þ ] i increase. Recently, Larman et al. (31) demonstrated that exposure to 1.5 M PrOH causes a protracted [Ca 2þ ] i elevation and ZP hardening in mouse oocytes, probably due to a passive Ca 2þ transport from the external medium as the cryoprotectant permeates the plasma membrane. Therefore, exposure to PrOH in calcium-containing media is likely to induce a premature [Ca 2þ ] i increase sufficient to trigger CG exocytosis also in human oocytes. As described in the Results section, cryopreservation affects the pericortical distribution of mitochondria and SER vesicles. In fact, a pericortical layer 5 10 mm thick, devoid of such organelles, was retrieved in about 50% of the F/T oocytes analyzed. Because it is known that these organelles are associated with the cytoskeleton, cryopreservation might cause their displacement, directly affecting the associated cytoskeletal elements or merely through physical forces that break their links with the cytoskeleton. The TEM analysis revealed that slow cooling also induces a moderate microvacuolization and a swelling of SER vesicles. The ultrastructure and size of the vacuoles, as well as the incidence of vacuolization, are similar to the data shown by Nottola et al. (30). The area of the SER vesicles in the F/T group had a mean value twice as high as that observed in the fresh group. Such an effect was not observed on clusters of SER tubular cisternae Fertility and Sterility â 1031

10 FIGURE 5 Fluorescent micrographs of fresh (a,b,e,f) and frozen/thawed (c,d) human metaphase II oocytes labeled with the TUNEL reaction (a,c,e) and counterstained with Hoechst (b,d,f) to localize DNA. Fresh (a,b) and frozen/ thawed (c,d) oocytes were TUNEL negative. Fresh control oocytes treated with DNase I (e,f) show markedly positive chromosomes (CH), both in the oocyte and in the first polar body (PB). Residual cumulus cells (CC) (a d) were sometimes TUNEL positive (c). Asterisks in a and c indicate autofluorescent lipid inclusions commonly found in human oocytes. Accidental displacement of the first polar body from its original position in b,e,f was caused by sample permeabilization and flattening. Bar, 50 mm Gualtieri et al. Cryodamage in slow frozen human oocytes Vol. 91, No. 4, April 2009

11 that had a similar area and distribution both in fresh and cryopreserved samples. In addition, the data clearly showed a direct relationship between mitochondrial damage and swelling of SER vesicles. To our knowledge this is the first report showing that oocyte cryopreservation may induce a swelling of SER elements and that such damage is markedly higher if mitochondria are also injured. In mammalian oocytes, SER is the main calcium store involved in the development of [Ca 2þ ] i transients initiated by inositol trisphosphate at fertilization (32, 33). The marked swelling of SER in F/T oocytes raises concerns about its correct functionality at fertilization or with ICSI. Present data demonstrated for the first time that a high percentage of F/T oocytes had mitochondria with a markedly decreased matrix electron density or with ruptures of the outer and inner membranes. Mitochondria represent the most abundant organelles in mammalian oocytes and their dysfunction or abnormalities may be a critical determinant of human embryo developmental competence. Therefore, live fresh and F/T oocytes were labeled with the inner membrane potential reporter JC-1, which allows the detection of mitochondrial function. As reported by Van Blerkom et al. (21), the fresh oocytes in the present study also had particulate red fluorescence in the pericortical region (i.e., high polarized mitochondria). In particular, the majority of fresh oocytes were positive and had a variable number of high polarized pericortical mitochondria, whereas, as previously reported by Jones et al. (22), F/T oocytes had a markedly decreased mitochondrial function. High polarized mitochondria have been reported to comprise a very small fraction of the total complement in human oocytes, specifically located in the pericortical cytoplasm (34, 35). In several studies, differences of DJm among individual oocytes have been associated with developmental competence heterogeneity (21, 22, 35 37). Taken together, these data demonstrate that mitochondrial functionality is a specific target of cryodamage that should be monitored during the development of cryopreservation protocols. On the other hand, mitochondrial damage can impair redox and Ca 2þ homeostasis, and release proapoptotic factors (38). To our knowledge, the possibility that cryopreservation induces an apoptotic process has been studied only in animals. In fact, temperature decrease during oocyte maturation (39), or oocyte cryopreservation (40) in cows, and cooling of mature oocytes (41) in pigs, triggers apoptosis and DNA fragmentation in surviving oocytes. Although, in the present study, assessment of DNA fragmentation and activation of caspases in F/T oocytes did not show any sign of apoptosis, this does not rule out that inheritance of a reduced complement of functional mitochondria may compromise developmental processes. Data collected in this study demonstrate that the main organelles cryoinjured in slow cooled human oocytes are the pericortical mitochondria and associated SER vesicles. Several reports indicate that these organelles play a key role in the regulation of [Ca 2þ ] i transients at fertilization. In the mouse, sperm-triggered [Ca 2þ ] i transients directly stimulate mitochondrial activity (42). Experimental collapse of DJm demonstrated that mitochondrial activity is absolutely required to maintain basal [Ca 2þ ] i in the unfertilized oocyte and to recover the basal level once [Ca 2þ ] i oscillations begin at fertilization (42). Therefore, we can hypothesize that cryodamage of SER and mitochondria could modify the normal pattern of [Ca 2þ ] i oscillations at fertilization or ICSI. This is supported by the reported decreased ability of F/T oocyte to elevate Ca 2þ in response to the Ca 2þ ionophore A23187 (22). Recently, several articles dealt with the roles of Ca 2þ signaling at oocyte activation and its long-term effects on embryo development. In the mouse, Ca 2þ signaling controls CG exocytosis, cell cycle resumption, and recruitment of maternal messenger RNAs (mrna), and each of these events is initiated and completed by a different number of Ca 2þ transients (43). In the rabbit, manipulation of frequency and amplitude of Ca 2þ pulses during parthenogenetic oocyte activation reduces the implantation rate and affects postimplantation development (44). Interestingly, under- and overexpression of Ca 2þ signals in mouse oocytes fertilized in vivo has no effects on the rate and quality of blastocyst development, but reduces the ability of blastocysts to implant or develop postimplantation, and significantly affects blastocyst global gene transcription (45). Because embryos generated from human oocytes cryopreserved with the slow-cooling 0.3 M sucrose protocol have a reduced implantation potential (9 11) and high rates of first trimester abortions (10, 11), the data reported in the present study raise concerns on a possible causal link between clinical outcomes and misfunctioning of Ca 2þ signaling in F/T oocytes. Such concerns are strengthened by the demonstration that slow cooling with 1.5 M PrOH causes a protracted Ca 2þ increase and proteome alterations in mouse oocytes. However, in oocytes exposed to PrOH in Ca 2þ -free media, a Ca 2þ increase is suppressed and proteome more closely resemble that of fresh controls (31). 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