Calcium-Free Vitrification Reduces Cryoprotectant-Induced Zona

REPRODUCTIONRESEARCH

Calcium-free vitriﬁcation reduces cryoprotectant-induced zona pellucida hardening and increases fertilization rates in mouse oocytes

Mark G Larman, Courtney B Sheehan and David K Gardner Colorado Center for Reproductive Medicine, 799 East Hampden Avenue, Suite 520, Englewood, Colorado 80113, USA Correspondence should be addressed to D K Gardner; Email: [email protected]

Abstract Despite the success of embryo cyropreservation, routine oocyte freezing has proved elusive with only around 200 children born since the first reported birth in 1986. The reason for the poor efficiency is unclear, but evidence of zona pellucida hardening following oocyte freezing indicates that current protocols affect oocyte physiology. Here we report that two cryoprotectants commonly used in vitrification procedures, dimethyl sulfoxide (DMSO) and ethylene glycol, cause a large transient increase in intracellular calcium concentration in mouse metaphase II (MII) oocytes comparable to the initial increase triggered at fertilization. Removal of extracellular calcium from the medium failed to affect the response exacted by DMSO challenge, but significantly reduced the ethylene glycol-induced calcium increase. These results suggest that the source of the DMSO-induced calcium increase is solely from the internal calcium pool, as opposed to ethylene glycol that causes an influx of calcium across the plasma membrane from the external medium. By carrying out vitrification in calcium-free media, it was found that zona hardening is significantly reduced and subsequent fertilization and development to the two-cell stage significantly increased. Furthermore, such calcium-free treatment appears not to affect the embryo adversely, as shown by development rates to the blastocyst stage and cell number/allocation. Since zona hardening is one of the early activation events normally triggered by the sperm-induced calcium increases observed at fertilization, it is possible that other processes are negatively affected by the calcium rise caused by cryoprotectants used during oocyte freezing, which might explain the current poor efficiency of this technique. Reproduction (2006) 131 53–61

Introduction Currently, there are two techniques employed for cryopreservation - slow-freezing and vitrification. The principal The ability to routinely cryopreserve human oocytes will difference between these two procedures, when using the have a significant impact on assisted reproductive technology (ART). Changes in government guidelines in certain same freezing straws, is the rate of cooling (approximately . 8 countries now restrict the number of oocytes that can be 1vs 1000 C/min respectively). For both protocols, first inseminated. This means there is now an increased need the cells must be treated with a combination of cell-per- for gamete cryopreservation. Furthermore, not only will meating and non-permeating agents to minimize oocyte cryopreservation circumvent the ethical and legal ice formation that can severely damage the cell. Pre- problems associated with embryo freezing, but it will also viously, high concentrations of cryoprotectant were benefit numerous patient groups including women at risk required for vitrification, but novel containers and sub- of ovarian function loss through premature menopause, microliter volumes result in super-fast cooling and sub- surgical treatments or radio/chemotherapy (for review see sequent warming rates (.20 000 8C/min; Steponkus et al. Falcone et al. 2004). An increasing number of women are 1990, Mukaida et al. 2001) so that concentrations closer deciding to delay starting a family and although currently to those used in slow-freezing can now be used (Mukaida controversial, one could envisage cryostorage of oocytes et al. 2002, Kasai & Mukaida 2004). from younger women to alleviate the problems associated Embryo freezing has been implemented very success- with maternal ageing and the sharp drop in egg quality fully in ART and now allows supernumerary embryos gen- that occurs between 30 and 40 years of age. erated through in vitro fertilization (IVF) to be routinely

q 2006 Society for Reproduction and Fertility DOI: 10.1530/rep.1.00878 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org Downloaded from Bioscientifica.com at 10/02/2021 10:38:26PM via free access 54 M G Larman and others frozen and stored (Liebermann 2003, Veeck 2003). requires the presence of the oocyte (Vincent et al. 1990). Blastocysts thawed and subsequently transferred yield an DMSO reduces the number of cortical granules (Vincent acceptable pregnancy rate, which demonstrates that cryo- et al. 1991), suggesting that the DMSO-induced zona preserved human blastocysts do not have a dramatic loss hardening is caused by cortical granule exocytosis. Since of viability (Gardner et al. 2003, Leibo 2004, Veeck et al. DMSO has been shown to cause a transient intracellular 2004). However, the oocyte has proved to be far more calcium rise in various cell lines (Morley & Whitfield troublesome (Coticchio et al. 2004). Chen (1986) reported 1993) and fusion of cortical granules to the oocyte plasma the first pregnancy following IVF of a cryopreserved membrane is calcium-dependent (Kline & Kline 1992, human oocyte. Despite this, to date only around 200 chil- Tahara et al. 1996), it is tempting to suggest that DMSO dren have resulted from oocyte cryopreservation tech- triggers cortical granule release by increasing intracellular niques (Leibo 2004). Together with the fact that the calcium. DMSO appears not to be the only cryoprotectant number of births per number of oocytes frozen is typically that increases calcium since high concentrations of ethyl- no greater than 3%, it is clear that oocyte cryopreservation ene glycol (EG, 10–40%) have been shown recently to is an inefficient process at present. increase intracellular calcium in mouse oocytes (Takaha- The stark difference between the success of oocyte and shi et al. 2004). embryo cryopreservation has yet to be strictly explained, Therefore, we have investigated whether exposure to but differences in cell size and membrane permeability cryoprotectants causes a calcium increase that leads to have been proposed (Leibo 2004, Stachecki & Cohen zona hardening. The data presented here show that the 2004). The oocyte is unique in that the maternal DNA is two cryoprotectants used in vitrification protocols, DMSO held suspended in the cytoplasm on the meiotic spindle and EG, cause large transient increases in intracellular and not within the protective confines of the nuclear calcium in mouse oocytes. The increase in calcium is suf- membrane, as it is for the majority of the time during sub- ficient to cause zona hardening, which significantly sequent preimplantation development. Damage to the reduces fertilization. By making the vitrification medium DNA and/or microtubules could explain the limited suc- nominally calcium-free we have determined that zona cess of oocyte cryopreservation. However, a further con- hardening is significantly reduced, which allows IVF with- sideration is that the oocyte is arrested in a state primed out the use of ICSI or laser-assisted zona removal. Further- for activation and changes in its environment can cause more, calcium-free vitrification does not seem to affect parthenogenetic activation. embryo developmental rates and cell number/allocation. A current potential problem with oocyte freezing is the induction of a primary activation event, zona hardening, Materials and Methods which significantly inhibits IVF (Matson et al. 1997) and may affect subsequent implantation. Zona hardening is Unless otherwise stated all Materials were obtained from brought about by fusion of cortical granules to the plasma Sigma Chemical Co. (St Louis, MO, USA). This work membrane and the release of their contents into the zona received prior approval from the Institutional Animal Care pellucida layers (for review see Sun 2003). The membrane and Users Committee. Sperm was collected from 12- to fusion event is calcium-dependent and is normally trig- 20-week-old F1 (C57BL/6 £ CBA) male mice and oocytes gered by the increase in intracellular calcium initiated by were collected from 4- to 5-week-old F1 (C57BL/6 £ sperm–egg fusion (Kline & Kline 1992). Upon fusion, the CBA/Ca) female mice (Jackson Laboratory, Bar Harbor, sperm triggers a series of calcium oscillations. The initial ME, USA). Female mice were administered 5 iu pregnant calcium increase lasts for around 5 min and causes the cor- mare’s serum gonadotropin and 48–52 h later 5 iu human tical granules to fuse with the plasma membrane and chorionic gonadotropin (hCG) were administered. release their contents into the zona pellucida layers (Tahara Oocytes were collected 12.5–13.5 h post-hCG and et al. 1996). Proteolytic enzymes target sperm binding pro- denuded by incubation in G-MOPS (Lane & Gardner teins to prevent further sperm from fertilizing the egg. Zona 2004) (supplemented with 5 mg/ml human serum albumin hardening can be overcome by intracytoplasmic sperm (HSA)) with 0.5 mg/ml hyaluronidase. Oocytes were injection (ICSI) (Porcu et al. 2000), but the fact that the washed three times and held in droplets of G-MOPS oocyte has undergone the cortical granule reaction indi- under Ovoil (Vitrolife, Gothenburg, Sweden) at 37 8C. cates that the oocyte has been artificially activated prior to Intracellular calcium changes were monitored by load- actual fertilization. This could, therefore, explain the poor ing the oocytes with 10 mmol l21 Indo-1 AM (Molecular efficiency of oocyte freezing, for even though a sperm is Probes, Eugene, OR, USA) for 30 min. The loading media injected via ICSI, the oocyte has already begun its acti- included 250 mmol l21 sulfinpyrazone to reduce dye com- vation events such as cortical granule exocytosis and poss- partmentalization and extrusion (Lawrence et al. 1997). ible down-regulation of cell cycle control proteins. Single oocytes were held in G-MOPS with or without cal- It has previously been reported that the cryoprotectant cium (with a holding pipette) in a heated chamber on a dimethyl sulfoxide (DMSO) causes zona hardening and a Nikon TE300 microscope equipped with an SFX-2 micro- reduced rate of fertilization (Vincent et al. 1991). fluorimeter (Solamere Technology Group, Salt Lake City, The effect of DMSO is not directly on the zona, but UT, USA). Oocytes were incubated in calcium-free

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G-MOPS for 30 min before cryoprotectant exposure. Sol- Results utions without calcium were termed ‘nominally-free’ since calcium was simply omitted from the stock solution. To Current vitrification protocols typically employ both ensure rapid exposure that would best mimic the vitrifica- DMSO and EG. To investigate the effect of these two tion procedure, oocytes were held in a small droplet cryoprotectants on intracellular calcium concentration, (approx. 20 ml) of G-MOPS before 980 ml of the cryoprotec- mouse oocytes were loaded with the ratiometric fluorescent indicator, Indo-1 and the cryoprotectant solution tant solution (made up in G-MOPS with 12 mg/ml HSA) 21 was added to the heated chamber (37 8C). rapidly applied. Both 1.5 mol l DMSO and EG caused Vitrification and warming were carried out at 37 8Cina transient increases in intracellular calcium that base solution of G-MOPS supplemented with 12 mg/ml increased the fluorescence ratio 2–3 times and lasted HSA. Vitrification was carried out using a two-step method for approximately 5 min (Fig. 1a,b). The ratio increase and cryoloop, as previously described (Lane & Gardner caused by both cryoprotectants was similar to the initial 2001). Briefly, oocytes were placed in the initial cryopro- increase caused by sperm entry, although slightly longer tectant solution (8% DMSO, 8% EG v/v; equates to in duration (Fig. 1c). It appears that the kinetics of 1.13 mol l21 DMSO, 1.43 mol l21 EG) for 1 min. Oocytes calcium release are different as the mean base-to-peak ^ were then moved to the second solution (16% DMSO, ratio for DMSO (3.20 0.16) was elevated compared ^ 16% EG v/v, 0.65 mol l21 sucrose; equates to 2.25 mol l21 with EG (2.66 0.18; P ¼ 0.06). DMSO, 2.86 mol l21 EG) for less than 30 s before being In an attempt to reduce the amount of calcium being pipetted onto a cryoloop, which had been preloaded with released, oocytes were incubated in calcium-free media this solution. The cryoloop was then plunged into a cryo- prior to application of the cryoprotectants (again in vial filled with liquid nitrogen to facilitate vitrification. For calcium-free media). The effect of calcium-free treatment warming, oocytes were moved through 1 ml serial dilutions was different for DMSO compared with EG (Fig. 1d,e). of sucrose (0.25 mol l21 for 1 min; 0.125 mol l21 for 2 min; The DMSO-induced increase in calcium was unaffected 0 mol l21 for 5 min) to reduce osmotic stress (Lane & Gard- by the absence of extracellular calcium. The mean base- ner 2001). Oocytes were allowed to recover for 1 h. At this to-peak ratio increase was not significantly different for stage oocytes were either fertilized or the degree of zona DMSO challenge in the presence or absence of calcium ^ ^ hardening was determined. (3.20 0.16 and 3.11 0.15 respectively; P ¼ 0.68; To load the calcium chelator 1,2-bis(o-aminophen- Fig. 2a). In contrast, the EG response was significantly oxy)ethane-N,N,N0,N0-tetraacetic acid (BAPTA-AM), decreased by removal of calcium (2.66 ^ 0.18 and oocytes were incubated with 10 mmol l21 BAPTA-AM 1.58 ^ 0.1 respectively; P ¼ 0.0001; Fig. 2b). (Molecular Probes) for 30 min. For zona hardening assess- It has previously been reported that removing calcium ment, oocytes were transferred to a 1% w/v solution of from the cryoprotectant medium reduces the cytotoxicity chymotrypsin (in G-MOPS) on a heated stage (37 8C) and of high levels of EG (Takahashi et al. 2004). Therefore, the integrity of the zona pellucida was monitored over reducing the increase in intracellular calcium in response time, the end point being when the zona was no longer to cryoprotectant exposure offers an advantage in improv- clearly visible (Matson et al. 1997). For fertilization, ing cell survival. Since an increase in calcium normally oocytes were transferred to G-FERT (Vitrolife). Twenty triggers cortical granule release, which leads to zona hard- oocytes were placed in 20 ml droplets of G-FERT. One ening, it was determined whether removing extracellular microliter sperm suspension that had been allowed to calcium also reduced zona hardening following cryopro- capacitate for 2 h in 1 ml G-FERT was added to each 20 ml tectant exposure during vitrification. Oocytes were either droplet. When required, laser-assisted fertilization was vitrified in the presence or absence of extracellular carried out using a Fertilase 670 nm wavelength laser sys- calcium. Zona hardening was then assessed by monitoring tem (MTM Medical Technologies, Montreux, Switzerland) the time taken for zona dissolution in 1% chymotrypsin to create a 5–10 mm hole in the zona. Oocytes were then (Matson et al. 1997). Figure 3a shows that vitrified oocytes maintained at 37 8Cin6%CO2,5%O2 and 89% N2. in the presence of calcium have a zona pellucida that Twenty four hours after the sperm were added, oocytes takes 6 times longer to dissolve than non-vitrified control were scored for development to the two-cell stage. Those oocytes. However, removing extracellular calcium signifi- embryos that had cleaved were transferred to G1 medium cantly reduces (by 40%) the zona hardening induced (Gardner & Lane 2004) and cultured for a further 24 h during vitrification. before being transferred to G2 medium (Gardner & Lane To demonstrate that it is indeed a rise in intracellular 2004) for an additional 48 h. On day 4 and day 5, calcium that triggers zona hardening during the vitrifica- embryos were scored for developmental stages. Cell num- tion procedure and not a direct physical effect, oocytes ber and allocation to the trophectoderm and inner cell were loaded with the calcium chelator, BAPTA-AM, mass were assessed using differential cell staining (Hardy which has been used to block calcium increases at fertili- et al. 1989). Statistical analysis was performed using zation and mitosis entry in mouse embryos (Kono et al. either Students unpaired t-test or ANOVA followed by 1996, Tombes et al. 1992). By directly preventing an Bonferonni comparison. increase in intracellular calcium with BAPTA, zona www.reproduction-online.org Reproduction (2006) 131 53–61

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(d) (a) 4 4 DMSO DMSO 3 3 -Ca2+ +Ca2+ 2 2 Ratio Ratio 1 1

0 0 0 5 10 15 0 51015 Time (mins) Time (mins) (b) (e) 4 4 EG EG 3 3 -Ca2+ +Ca2+ 2 2 Ratio Ratio 1 1

0 0 0 5 10 15 0510 15 Time (mins) Time (mins) (c) 4

2 Ratio

0 0 5 10 15 Time (mins)

Figure 1 The effect of DMSO and EG on intracellular calcium in metaphase II (MII) mouse oocytes. Mouse oocytes loaded with Indo-1 were held in a heated chamber (37 8C) and changes in calcium were monitored with a PMT (photomultiplier tube)-based system. Oocytes were treated with 1.5 mol l21 DMSO (a, d) or EG (b, e) in the presence or absence of calcium (n ¼ greater than 12 for each treatment, with at least 6 replicates). (c) The initial increase in calcium triggered by sperm at fertilization. hardening was further reduced to levels not significantly that physically alters the zona pellucida (Wood et al. different from non-vitrified oocytes (Fig. 3a). 1992). To examine this, oocytes were treated with the A previous report has suggested that it is part of the same vitrifcation regime (i.e. vitrification and warming freeze/thaw process and not cryoprotectant exposure solutions) without exposure to liquid nitrogen and

(a) (b) 4 4

3 3

2 2 ** Mean base to peak ratio increase

Mean base to peak ratio increase 1 1 +Ca2+ -Ca2+ +Ca2+ -Ca2+

Figure 2 Mean base-to-peak ratio increase following DMSO and EG exposure in the presence and absence of extracellular calcium. The increase in calcium was measured by the fold-increase in ratio from base to peak (n ¼ greater than 12 for each treatment, with at least 6 replicates). (a) The difference between DMSO challenge in the presence or absence of calcium was insigniﬁcant (P ¼ 0.68), whereas (b) EG showed a signiﬁcant reduction in calcium release in the absence of extracellular calcium. **P ¼ 0.0001.

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(a) not only to the zona hardening, but also to a speciﬁc 800 a change in a sperm binding protein. The zona pellucida glycoprotein (ZP2) becomes converted to the fertilized 600 form, which does not support sperm binding. This a * has resulted in the use of ICSI or partial zona removal 400 following freezing to permit sperm access to the plasma membrane and fertilization (Carroll et al. 1990, Kazem 200 * et al. 1995, Porcu et al. 2000). Since zona hardening is signiﬁcantly reduced in the absence of extracellular

Time for zona dissolution (s) 0 calcium, we wanted to determine if this increased the fer- Control +Ca2+ -Ca2+ BAPTA tilization rate and subsequent development to the two-cell stage. Oocytes were vitrified in the presence or absence of extracellular calcium and then fertilized in vitro. Develop- Vitrification (b) ment to the two-cell stage was assessed at 24 h. Oocytes 100 vitrified in the presence of calcium had very poor fertilization rates and poor development to the two-cell stage (less 80 * than 10%; Fig. 3b). Removing the extracellular calcium significantly increased the number of oocytes reaching the 60 b two-cell stage to a rate that approached the non-vitrified * 40 control (Fig. 3b). To show that it was zona hardening that reduced the level of fertilization and development, 20 a oocytes that had been vitrified in the presence of calcium

had a 5–10 mm hole made in the zona with a laser. When 0 Oocytes reaching 2-cell stage (%) Control +Ca2+ -Ca2+ Laser sperm access to the oocyte was permitted with this laser- assistance procedure, fertilization and two-cell development were comparable to non-vitrified oocytes (Fig. 3b). Vitrification Removing extracellular calcium appears to significantly reduce zona hardening and therefore dramatically Figure 3 The effect of removing extracellular calcium from the vitrifi- improve fertilization rates. To ensure that the removal of cation medium on zona hardening and subsequent fertilization. Oocytes were collected from F1 hybrid (C57BL/6 £ CBA) female calcium during the vitrification procedure does not com- mice at 12.5 h post-hCG. (a) Oocytes were divided into 3 treatment promise embryo development, fertilized oocytes were groups and a non-vitrified control. Following vitrification in the pre- subsequently cultured and monitored for developmental sence or absence of calcium or treatment with BAPTA-AM, the rate to the blastocyst stage and cell number/allocation oocytes were incubated in 1% chymotrypsin to assess the level of through differential staining. Oocytes that had been ferti- zona hardening. The time taken for the zona to completely disap- pear was recorded for each treatment (n ¼ greater than 60 eggs for lized and developed to two-cell embryos were cultured in each treatment, with at least 3 replicates). Calcium-free vitrification sequential media for a total of 96 h. Embryos were scored and BAPTA-AM treatment significantly reduced zona hardening, on day 4 and day 5 for embryo development. The vitrifica- when compared to vitrification with calcium (*; P ¼ 0.001). Zona tion protocol we used has previously been reported not to hardening following vitrification with or without calcium was stat- affect embryo development or cell number/allocation istically different from control (a; P ¼ 0.001). (b) Following vitrification (.95% survival rate) oocytes were fertilized 15 h post-hCG in (Lane & Gardner 2001). It was found that carrying out G-FERT. Laser-assisted fertilization (Laser) was included as an calcium-free vitrification did not affect embryo develop- additional treatment group. The ability of the sperm to penetrate ment (Fig. 4a). On day four 39.1% of control (non-vitri- the zona and fertilize the oocyte was assessed by monitoring fied) embryos had reached the blastocyst stage, which was the number of two-cell stage embryos 24 h after addition of sperm comparable to the number of embryos following calcium- (n ¼ greater than 250 eggs for each treatment). Calcium-free vitrification and laser-assistance significantly increased fertilization and free vitrification (37.2%). On day five the percentage of development to the two-cell stage, when compared to vitrification embryos from the control and calcium-free vitrification with calcium (*; P ¼ 0.001). Development to the two-cell stage, treatments that had reached the hatching blastocyst stage following vitrification with or without calcium was statistically was also similar (94.2 and 92.2% respectively). different from control (a; P ¼ 0.001, b; P ¼ 0.01). Using differential staining (Hardy et al. 1989) cell num- subsequent vitrification. The time for zona dissolution ber and allocation between the trophectoderm (TE) and following this treatment was 780.2 ^ 26.42 s, indicating inner cell mass (ICM) were determined for non-vitrified that the effect on the zona is caused by the cryoprotec- control embryos and those that were vitrified in calcium- tant solution and not by the freeze/thaw process. free media. Figure 4b shows that as with the regular vitrifi- The outcome of zona hardening following vitrification cation protocol the calcium-free treatment had no detri- is severely reduced fertilization rates with IVF. This is due mental effect on TE or ICM numbers. www.reproduction-online.org Reproduction (2006) 131 53–61

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(a) down-regulation (Lorca et al. 1991, Kline & Kline 1992, 100 Nixon et al. 2002, Marangos & Carroll 2004) which allows Morula completion of meiosis. Precocious initiation of cortical Blastocyst 80 granule exocytosis has already been demonstrated during Hatching cryopreservation (Carroll et al. 1990). Together with the 60 observation that the cryoprotectant agent DMSO causes 40 zona hardening through cortical granule release, which is a calcium-dependent event, the aim of this study was to 20 investigate if vitriﬁcation affects intracellular calcium. 21

Stage of development (%) Previous studies have reported that 1.5 mol l 0 1,2-propanediol increased intracellular calcium in aged 2+ 2+ Control -Ca Control -Ca mouse oocytes (Litkouhi et al. 1999) and high levels of EG Day 4 Day 5 caused sustained calcium increases (Takahashi et al. 2004). (b) However, to our knowledge no study has investigated the 100 effect of cryoprotectants (DMSO and EG) at levels commonly used for vitrification on young oocytes (14–17 h 80 post-hCG). By loading mouse oocytes with a fluorescent 60 calcium indicator and exposing them to DMSO and EG, we have found that both of these agents cause a dramatic 40 transient increase in intracellular calcium. The amplitude Cell number of the calcium increase caused by the cryoprotectants is 20 comparable to the initial increase triggered at fertilization, but lasts approximately 50% longer in duration (Saunders 0 et al. 2002). DMSO appeared to induce a larger and more TE ICM Total TE ICM Total sustained increase in calcium compared with EG, which 2+ Control -Ca may be attributable to the higher permeability of the oocyte Figure 4 The effect of calcium-free vitrification on embryo develop- to DMSO (Paynter 2005). The exact mechanism for the ment. (a) Two-cell embryos derived from either non-vitrified (control) cryoprotectant-induced calcium increase is unknown. The or calcium-free vitrified oocytes were cultured for 5 days. Embryo lipophilic properties of DMSO and EG would be expected development was scored on day 4 and day 5 for morula, blastocyst to have a non-specific effect on the plasma membrane and ¼ and hatching blastocyst formation (n greater than 140 embryos for other internal membranes such as the endoplasmic reticu- each treatment). (b) Following the day 5 score, cell numbers compos- ing the trophectoderm (TE) and inner cell mass (ICM) were deter- lum, which would lead to either calcium influx and/or mined using differential cell staining (n ¼ greater than 60 for each calcium release from internal stores. However, the osmotic treatment). No statistical differences were observed. contraction induced by cryoprotectants might also contrib- ute to the increase in intracellular calcium. Discussion Removing external calcium failed to affect the intracellular calcium increase caused by DMSO, which suggests that Despite the high success rate of embryo cryopreservation internal stores, such as the endoplasmic reticulum, are the and subsequent clinical pregnancies, efficient human source of the rise. This is in agreement with observations oocyte cryopreservation remains elusive. Pronucleate with several different cell lines (Morley & Whitfield 1993). stage embryos have been frozen using numerous tech- In contrast, observations presented here show that with EG, niques and different mammalian species, which suggests removal of external calcium significantly reduces the intra- that the surface area to volume ratio, per se, does not cellular calcium increase, which suggests the majority of account for the problematic nature of oocyte freezing the EG-induced calcium increase is via influx across the (Coticchio et al. 2004). The permeability of the oocyte to plasma membrane from the medium. cryoprotectants and their ability to replace intracellular Earlier studies using a fluorescent lectin binding assay to water is another well documented consideration (Pfaff assess cortical granule exudate reported no premature et al. 1998, Pedro et al. 2005), but rarely is the cell cycle release following freezing of mouse or human oocytes stage contemplated. The oocyte is primed for activation (Wood et al. 1992, Gook et al. 1993). However, as dis- and completion of meiosis, which is triggered by the cussed by Wood and colleagues (1992) it is possible that sperm. The sperm delivers the signal to resume develop- the assay may not be sensitive enough to detect a lower ment by introducing a sperm-specific protein upon threshold of release or the population of cortical granules sperm–egg fusion (Saunders et al. 2002). The protein released may not contain the appropriate binding substrate. (phospholipase C zeta) triggers calcium oscillations that Since the amount of calcium released is comparable to that last several hours and control a number of activation at fertilization, it would be capable of causing cortical events such as cortical granule exocytosis (block to poly- granule exocytosis, which would explain zona hardening. spermy) and maturation promoting factor (MPF) This conclusion is also in agreement with the findings of

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Vincent and colleagues (1990), who demonstrated that the However, in human IVF a more defined system is required oocyte must be present for zona hardening to occur follow- and human serum albumin has been shown not to afford ing exposure to DMSO. The data presented here show that the same protection as BSA (George & Johnson 1993). cryoprotectant exposure alone causes zona hardening. Lowering the temperature during cryoprotectant exposure Since introduction of the calcium chelating agent, BAPTA, prevents zona hardening, most likely through the decrease prevents zona hardening, it appears that the cryoprotec- in temperature-sensitive enzymatic activity and cellular tants used increase intracellular calcium to levels sufficient function, but by carrying out the procedure at 37 8C it alle- to induce cortical granule release and subsequent zona viates potential cooling injury to the cytoskeleton (Picker- hardening. Removing calcium during vitrification signifi- ing & Johnson 1987, Pickering et al. 1990). cantly reduces the degree of zona hardening, which dra- The data presented here demonstrate that the cryopro- matically improves fertilization and development to the tectants, EG and DMSO, increase intracellular calcium, two-cell stage. which leads to the induction of zona hardening. Sub- Although zona hardening and its negative effect on ferti- sequent removal of calcium from the vitrification medium lization rates can be circumvented through ICSI or partial facilitated IVF and development to the two-cell stage at a zona removal, cortical granule release is but one of the rate approaching that of control (non-vitrified) oocytes. initial activation events. A large increase in calcium might The calcium-free treatment did not affect embryo develop- also be detrimental for further embryo development by ment rates or cell number/allocation in the blastocyst, affecting cell cycle proteins such as MPF whose down- which suggests that the removal of calcium during cryo- regulation is normally triggered by the calcium preservation is not detrimental. In this present study we oscillations at fertilization (Lorca et al. 1991, Marangos & wished to examine the effect of calcium-free vitrification Carroll 2004). Calcium is a ubiquitous signaling agent that on a common protocol that used EG and DMSO. How- is involved in a number of biological processes, so strict ever, the fact that the response to DMSO challenge was homeostatic regulation of release is critical (for review see unaffected by removing extracellular calcium suggests that Berridge et al. 1998). A sustained level of micromolar DMSO should either be omitted from the cryoprotectant calcium is sufficient to cause the inappropriate activation solutions or reduced to a concentration that does not dra- of calcium-dependent enzymes such as proteases and matically increase intracellular calcium. The kind of ana- phospholipases, which can have severe cellular conse- lysis presented here provides the opportunity for further quences (Orrenius et al. 2003). Furthermore, oocytes acti- experiments, which include using the measurement of vated with high frequency calcium oscillations can calcium during cryoprotectant challenge to empirically undergo apoptosis (Gordo et al. 2000, 2002). A large determine the optimum concentration and duration of increase in intracellular calcium caused by cryoprotectants exposure for vitrification protocols that minimize the may therefore decrease viability and explain the low effi- increase in oocyte intracellular calcium. By carrying out ciency of oocyte cryopreservation. Further support for this such studies it is anticipated that not only will zona hard- idea is provided by removing extracellular calcium and ening be significantly reduced, but also that normal cell introducing BAPTA to reduce the intracellular calcium rise function will be facilitated allowing IVF and thereby during cryoprotectant exposure. Takahashi and colleagues increasing the efficiency of oocyte cryopreservation. (2004) demonstrated that reducing the calcium increase in mouse oocytes with such an approach significantly reduced the calcium rise caused by 40% EG (for 3 min), Acknowledgements which, in turn, increased the number of intact oocytes from The authors wish to thank Laura Reed for her assistance and 0% to 100%. Vitrolife for their support of these studies. The authors declare The results presented here demonstrate that both DMSO that there is no conflict of interest that would prejudice the and EG cause a transient increase in intracellular calcium. impartiality of this scientific work. This increase in calcium is responsible for zona hardening and explains the requirement for zona removal or ICSI to References permit fertilization. Zona hardening, caused by exposure Berridge MJ, Bootman MD & Lipp P 1998 Calcium - a life and death to the cryoprotectant, can be overcome by inclusion of signal. Nature 395 645–648. bovine serum albumin (BSA) in the medium (George et al. Carroll J, Depypere H & Matthews CD 1990 Freeze–thaw-induced 1992). This, together with the employment of lower tem- changes of the zona pellucida explains decreased rates of fertiliza- peratures during cryoprotectant exposure, would account tion in frozen–thawed mouse oocytes. Journal of Reproduction and Fertility 89 253–259. for the apparent lack of zona hardening and high fertiliza- Chen C 1986 Pregnancy after human oocyte cryopreservation. Lancet tion rates reported in some previous studies (Carroll et al. 1 884–886. 1990, Hotamisligil et al. 1996, dela Pena et al. 2001). It is Coticchio G, Bonu MA, Borini A & Flamigni C 2004 Oocyte cryopre- thought that the active component in BSA is fetuin servation: a biological perspective. European Journal of Obstetrics, Gynecology and Reproductive Biology 115 (Supp 1) S2–S7. (George & Johnson 1993) which presumably acts as either Falcone T, Attaran M, Bedaiwy MA & Goldberg JM 2004 Ovarian a competitive substrate for proteolytic enzymes released function preservation in the cancer patient. Fertility and Sterility 81 from the cortical granules or as a direct inhibitor. 243–257. www.reproduction-online.org Reproduction (2006) 131 53–61

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fertilization in living mouse eggs. American Journal of Physiology Vincent C, Pickering SJ & Johnson MH 1990 The hardening effect of 270 C1354–C1361. dimethyl sulphoxide on the mouse zona pellucida requires the Takahashi T, Igarashi H, Doshida M, Takahashi K, Nakahara K, presence of an oocyte and is associated with a reduction in the Tezuka N & Kurachi H 2004 Lowering intracellular and extracellu- number of cortical granules present. Journal of Reproduction and lar calcium contents prevents cytotoxic effects of ethylene glycol- Fertility 89 253–259. based vitriﬁcation solution in unfertilized mouse oocytes. Molecu- Vincent C, Turner K, Pickering SJ & Johnson MH 1991 Zona lar Reproduction and Development 68 250–258. pellucida modiﬁcations in the mouse in the absence of oocyte ac- Tombes RM, Simerly C, Borisy GG & Schatten G 1992 Meiosis, egg activation. Molecular Reproduction and Development 28 394–404. tivation and nuclear envelope breakdown are differentially reliant on Wood MJ, Whittingham DG & Lee SH 1992 Fertilization failure of Ca2þ, whereas germinal vesicle breakdown is Ca2þ-independent in frozen mouse oocytes is not due to premature cortical granule the mouse oocyte. Journal of Cell Biology 117 799–811. release. Biology of Reproduction 46 1187–1195. Veeck LL 2003 Does the developmental stage at freezing impact on clinical results post-thaw? Reproductive Biomedicine Online 79 407–410. Veeck LL, Bodine R, Clarke RN, Berrios R, Libraro J, Moschini RM, Received 8 July 2005 Zaninovic N & Rosenwaks Z 2004 High pregnancy rates can be First decision 25 August 2005 achieved after freezing and thawing human blastocysts. Fertility Revised manuscript received 24 September 2005 and Sterility 82 1418–1427. Accepted 3 October 2005

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