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Reproductionresearch 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 cryo- preservation - 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 technol- ogy (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 02:36:58PM 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
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