Effectiveness of slow freezing and vitrification for long-term preservation of mouse ovarian tissue

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Theriogenology 75 (2011) 1045–1051 www.theriojournal.com

Effectiveness of slow freezing and vitrification for long-term preservation of mouse ovarian tissue G.A. Kima, H.Y. Kima, J.W. Kimb, G. Leeb, E. Leec, J.Y. Ahna, J.H. Parka, J.M. Lima,d,* a Department of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Korea Dental Research Institute, School of Dentistry, Seoul National University, Seoul 110-749, Korea c School of Veterinary Medicine, Kangwon National University, Chunchon 200-701, Korea d Laboratory of Stem Cell and Bioevaluation, WCU Biomodulation Program, Seoul National University, Seoul 151-742, Korea b

Received 1 March 2010; received in revised form 2 November 2010; accepted 2 November 2010

Abstract This study was conducted to evaluate the interaction between cryo-damage and ART outcome after cryopreservation of mouse ovarian tissues with different methods. Either a vitrification or a slow freezing was employed for the cryopreservation of B6CBAF1 mouse ovaries and follicle growth and the preimplantation development of intrafollicular oocytes following parthenogenesis or IVF were monitored. Both cryopreservation protocols caused significant damage to follicle components, including vacuole formation and mitochondrial deformities. Regardless of the cryopreservation protocols employed, a sharp (P ⬍ 0.0001) decrease in follicle viability and post-thaw growth was detected. When IVF program was employed, significant (P ⬍ 0.05) decrease in cleavage and blastocyst formation was notable in both modes of cryopreservation. However, such retardation was not found when oocytes were parthenogenetically activated. In the IVF oocytes, slow freezing led to better development than vitrification. In conclusion, a close relationship between cryopreservation and ART methods should be considered for the selection of cryopreservation program. © 2011 Elsevier Inc. All rights reserved. Keywords: Ovarian follicle; Slow freezing; Vitrification; IVF; Parthenogenesis; Preimplantation development

1. Introduction Previously [1], we demonstrated that the ovarian tissue cryopreservation programs employed in our assisted reproductive technology (ART) system induced vacuole formation and mitochondrial deformities in ovarian follicles and intrafollicular oocytes. These further suggested that cryopreservation procedures influence developmental potential of preantral follicles de-

* Corresponding author. Tel.: ⫹822-880-4806; fax: ⫹822-8742555. E-mail address: [email protected] 0093-691X/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2010.11.012

rived from cryopreserved ovaries in terms of maturation and preimplantation development. From different viewpoint, the oocytes used in various ART programs vary in terms of their susceptibility to cryoinjury, and that each microorganelle responsible for a specific biological event has a different susceptibility to damage from freezing. Cryodamage to follicles may retard follicle growth and oocyte maturation, while deformation of the zona pellucida may block the penetration of a mature oocyte by sperm. It is highly possible that mitochondrial damage following cryopreservation inhibits various cellular events during preimplantation development.

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To confirm this hypothesis, we evaluated the relationship between ovarian freezing methods and ART programs. We subsequently investigated isolated follicle growth after ovary cryopreservation and intrafollicular oocyte maturation. The cleavage and blastocyst formation from intact or cryopreserved oocytes were monitored following different activation program (parthenogenesis or IVF). Cryopreservation by slow freezing–thawing or vitrification–warming, which are part of our standard ART program, was employed for ovarian tissue cryopreservation [1]. 2. Materials and methods 2.1. Animals Female F1 hybrid (B6CBAF1) mice were produced by the mating of female C57BL/6 and male CBA/Ca mice under controlled conditions as follows: lighting (14L:10D), temperature (20 to 22 °C), and humidity (40 to 60%). Prepubertal 2-wk-old F1 female mice were used for retrieval of the ovaries in this study. All procedures for animal management, breeding, and surgery followed the standard operating protocols of Seoul National University. Appropriate management of the experimental samples and quality control of the laboratory facility and equipment were also made. The protocols employed in this study were approved by our institutional animal care and use committee (approval number: SNU-070423-4). 2.2. Slow-freezing The ovaries retrieved from donor mice were sliced into 0.7 to 0.8 ⫻ 0.2 to 0.3 ⫻ 0.1 mm in size and were subsequently cryopreserved using a modified method of Gosden et al (1994) [2]. Briefly, they were suspended in L-15 medium (Gibco BRL, Grand Island, NY, USA) containing 10% (v:v) fetal bovine serum (FBS; HyClone Laboratories, Logan, UT, USA) and 1.5 M DMSO on ice then placed individually into the middle of a 0.5 mL plastic straw (FHK, Tokyo, Japan) using a small volume of medium. After being sealed with straw powder (FHK, Tokyo, Japan), the straws were placed in a programmable freezer pre-cooled to 4 °C for 30 min. The slowfreezing procedure was as follows: cooled at 1 °C/ min to ⫺7 °C and held for 5 min, seeded manually, held at ⫺7 °C for 10 min, and cooled to ⫺30 °C at 0.3 °C/min. The straws were then exposed to liquid nitrogen vapor for 5 min and stored in liquid nitrogen for up to 3 mo. To thaw, the straws were placed on

a warm plate at 37 °C for 30 sec and the ovaries were removed. The cryoprotectant was removed by serial dilutions of the cryoprotectants with one or more washes in medium, which could avoid rapid osmotic processes in the cryopreserved tissue. After removing cryoprotectant, the thawed ovaries were washed repeatedly in L-15 medium containing 10% (v:v) FBS at 37 °C before experimental treatment. 2.3. Vitrification Whole ovarian tissues were also cryopreserved using the vitrification method described by Choi et al (2007) [3]. Briefly, they were equilibrated in a stepwise manner in L-15 medium containing 20% (v:v) FBS, 7.5% (v:v) EG, and 7.5% (v:v) DMSO for 15 min at 37 °C incubation, then exposed to a vitrification solution containing 15% (v:v) EG, 15% (v:v) DMSO, and 0.5 M sucrose for 5 min. Following equilibration, each of the ovaries was loaded onto an electron microscopic (EM) grid (IGC 400; Pelco International, Redding, CA, USA) and excess vitrification solution was removed using sterilized filter paper. The ovarian tissue on the grids were immediately plunged into liquid nitrogen for 30 sec. Vitrified ovaries attached the EM grids were then placed in cryovials filled with liquid nitrogen and stored for up to 3 mo. To thaw, the EM grids in the cryovials were placed in L-15 medium containing 20% (v:v) FBS and 1.0 M sucrose for 10 min at 37 °C incubation. The ovaries detached from the grids were further incubated for 10 min in L-15 medium containing 10% (v:v) FBS before use. 2.4. Evaluation of ultrastructure For ultrastructural observation with TEM, postthawed ovaries were fixed in modified Karnovsky’s solution at 4 °C overnight and treated osmium tetroxide at 4 °C for 2 h [4]. Post-fixed specimens were dehydrated gradually in ethanol solutions of different concentration and en-bloc staining with aqueous uranyl acetate was subsequently conducted at 4 °C overnight. After embedding into resin, ultrathin sectioning into 60 nm in thickness was performed with an ultramicrotome. The sections were placed on copper grids, and stained with uranyl acetate and Reynolds’ lead citrate. The sectioned surfaces were observed with a TEM (LIBRA 120; Carl Zeiss, Oberkochen, Germany). 2.5. In vitro-culture of follicles Female F1 mice were euthanized by cervical dislocation and their ovaries were immediately removed aseptically. The ovaries were placed in 2 mL of pre-warmed

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Leibovitz L-15 medium (Gibco-BRL) supplemented with 10% (v:v) heat-inactivated FBS and 1% (v:v) penicillin streptomycin solution at 37 °C and then cryopreserved by either vitrification or slow freezing. To enhance the penetration of the cryoprotectants such as DMSO, ethylene glycol (EG), and sucrose, the retrieved ovaries were punctured with a 30 ga needle. All medium substrates were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA) unless otherwise stated. Follicle collection and subsequent in vitro manipulation were achieved by standard protocols [5,6]. Preantral follicles were mechanically isolated from frozen–thawed ovaries using a 30 ga needle. The follicles were then washed three times in 10 ␮L droplets of L-15 medium. Among these, early secondary follicles 100 to 125 ␮m in diameter [5,6] were placed individually in a 10 ␮L culture droplet overlaid with washed mineral oil in 60 ⫻ 15-mm culture dishes (SPL, Gyeonggi, Korea) for in vitro culture. The follicles were cultured for 9 days in ␣-MEMglutamax medium (with ribonucleoside and deoxyribonucleoside; Gibco-BRL, Grand Island, NY, USA) supplemented with 5% (v:v) FBS, 5 ␮g/mL insulin, 5 ␮g/mL transferrin, 5 ng/mL selenium, 100 mIU/mL recombinant human FSH (Organon, Oss, The Netherlands), and 1% (v:v) antibiotic solution. On day 1 of culture, an additional 10 ␮L of fresh medium was added to each droplet; half of the medium was changed every other day. To enable the maturation of the intrafollicular oocytes in the follicles, 2.5 IU/mL hCG (Pregnyl™; Organon, Oss, The Netherlands) and 5 ng/mL EGF were added to the culture medium 16 to 18 h before the end of the culture period (day 9 of culture). Oocyte maturation (to metaphase II) was determined by extrusion of the first polar body and by expansion of the cumulus cells in the cumulus-oocyte complexes (COCs). To identify extrusion of the first polar body, the oocytes were freed from the surrounding cumulus cells with 200 IU/mL hyaluronidase [5,6]. Viability of follicles was determined by morphological parameters such as the integrity of follicular cells and intrafollicular oocytes. Any type of damages such as cytoplasm disruption and cell shrinkage was considered to monitor cell viability. Developmental retardation was also monitored for evaluating cell viability. On the other hand, follicular growth during in vitro culture was classified into three stages; follicular, diffuse and pseudoantral stages [5–7]. Briefly, the follicles remain intact without expansion

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of follicular cells were classified as the follicular stage of the follicles, while the diffuse stage represents the expansion of follicular cell layers by proliferating of granulosa cells and the beginning of the

Fig. 1. Ultrastructure of preantral follicles derived from ovaries cryopreserved by different methods. An ultrastructural comparison was made among preantral follicles derived from intact, non-frozen ovaries (A,B), slowly frozen–thawed ovaries (C,D), and vitrified– warmed ovaries (E,F). Methods of cryopreservation previously identified as optimal were employed. The preantral follicles derived from slow freezing (C,D) or vitrification (E,F) possessed numerous vacuoles (V) in follicular components. Intact (white arrow) and damaged/ deformed mitochondria (black arrow) were observed in the ooplasm (C–F), which was greatly reduced in the non-frozen follicles (A,B). CH, Chromosome; D, Debris; GC, Granulosa cells; Lip, Lipid; MV, Microvillus; N, Nucleus; NE, Nuclear envelope; O, oocyte; SER, Smooth endoplasmic reticulum; TC, Theca cells; ZP, Zona pellucida.

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theca cell attachment to the bottom of culture dish. Diffused follicle having antrum-like cavities in follicular cell layer and a morphologically normal oocyte was classified as the pseudoantral stage. 2.6. Activation of oocytes Oocytes matured by the follicle culture for 9 days were either activated parthenogenetically or inseminated in vitro. For the parthenogenesis, the oocytes were cultured for 4 h in Ca2⫹-free potassium simplex optimized medium (KSOM) supplemented with 10 mM SrCl2 and 5 ␮g/mL cytochalasin B [5,6]. The Cytochalasin B is used to trigger the progression of meiotic maturation from MII and to retain the polar body in the cytoplasm for the diploidization of the activated oocytes. For IVF, the oocytes were inseminated with epididymal semen in KSOM for 4 to 6 h.

The activated oocytes were subsequently cultured in 5 ␮L droplets of modified Chatot, Ziomek, and Bavister (CZB) medium. The first cleavage division and blastocyst formation were monitored at 48 and 120 h, respectively. 2.7. Statistical analysis A prospective, randomized trial was conducted for statistical analysis and all experiments were replicated at least three times. A generalized linear model (PROCGLM) in the Statistical Analysis System (SAS Institute, Cary, NC, USA) program was used to evaluate follicle growth and oocyte development. When ANOVA detected a significant effect, a least-squares analysis employing DUNCAN’s pairwise comparison test was conducted. Differences between the treatments were considered significant at P values less than 0.05.

Fig. 2. Morphological integrity (A) and in vitro-development (B) of follicles isolated from slow-frozen or vitrified ovaries. (A) A sharp decrease in the number of intact follicles was detected in the slowly frozen and vitrified groups on day 3 of culture. (B) No difference was seen in the number of follicles developing to the diffuse stage, but more intact or vitrified–thawed follicles developed to the pseudoantral stage than slowly frozen follicles. A significant difference in the number of follicles developing to the pseudoantral stage was detected between the intact and both cryopreserved groups.
: P ⬍ 0.0001, ⌿: P ⬍ 0.05.

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Table 1 Maturation and preimplantation development of parthenogenetically activated or IVF intrafollicular oocytes derived from cryopreserved ovariesa. Activation

Methods of Cryopreservation

Cultured

Perthenogenesis

None Slow freezing Vitrification None Slow freezing Vitrification

73 73 67 80 130 59

IVF

No (%)b of oocytes Matured

Developed to 2-cell embryos

Developed to blastocysts

39 (53)c 20 (27)d 21 (31)d — — —

21 (29) 13 (18) 17 (25) 38 (48)c 21 (16)d 2 (3)e

8 (11) 12 (16) 8 (12) 10 (13)c 5 (4)d 1 (2)d

None, no freezing-intact follicle; Model effects of treatment, which were indicated as P value, were 0.0021 in maturation, 0.2871 in cleavage and 0.5852 in blastocyst within the parthenogenesis group, while 0.0001 in cleavage and 0.0014 in blastocyst in the IVF group. a Either slow freezing or vitrification of whole ovaries was employed for follicle cryopreservation (referred to Gosden et al and Choi et al, respectively). b Percentage of the number of follicles cultured. c– e Different superscripts within a column are significantly different, P ⬍ 0.05.

3. Results 3.1. Ultrastructure of cryopreseved follicles The ultrastructure of preantral follicles derived from either slowly frozen–thawed or vitrified–warmed ovaries was monitored. As shown in Figure 1, numerous vacuoles were detected in the oocytes with follicular cells regardless of the cryopreservation protocol. The intact follicles had no vacuoles, while granular or granular cytoplasmic vacuoles were noted near the endoplasmic reticulum of follicles derived from cryopreserved ovaries. Severe mitochondrial deformities, which were not detected in the intact follicles, were also noted in the same follicles from cryopreserved ovaries. 3.2. Development of cryopreserved follicles As shown in Figure 2, a significant decrease in viability was detected in the follicles derived from cryopreserved ovaries, while no difference in viability was seen between the two freezing protocols. This decrease continued throughout the culture period from day 3 (94 to 100% vs 60 to 82%, respectively; P ⬍ 0.0001). In the follicles, a significant delay in development to the pseudoantral stage was detected on day 7 in the slow-freezing compared to the intact and vitrification groups (22% vs 38 to 48%, respectively; P ⬍ 0.05); however, no significant difference in the number of follicles developing to the diffuse stage was observed. On day 9, development to the pseudoantral stage was the highest in the intact group compared to the cryopreservation groups (94% vs 46 to 47%, respectively; P ⬍ 0.0001). More of the follicles that had

undergone cryopreservation ceased their development at the diffuse stage compared to the intact follicles (13 to 19%). 3.3. Preimplantation development after cryopreservation by different protocols Comparison was made within the same activation protocol (parthenogenesis and IVF). A significant (P ⬍ 0.0021) model effect was detected only for the comparison of oocyte maturation in the parthenogenesis group, which showed a significant decrease after cryopreservation (53% vs 27 to 31%; Table 1). However, no difference was found in cleavage (18 to 29%; P ⫽ 0.2871) or blastocyst formation among the treatments (11 to 16%; P ⫽ 0.5852). In the IVF group, all comparisons yielded significant decreases in cleavage (3% vs 16% vs 48%, respectively; P ⬍ 0.0001) and blastocyst formation (2 to 4% vs 13%, respectively; P ⬍ 0.0014) in the cryopreservation groups compared to the intact (no cryopreservation) group. No difference in developing oocyte morphology was observed among the treatments (Fig. 3). 4. Discussion The results of this study clearly demonstrate a close relationship between cryopreservation methods and ART outcomes. In cryopreserved tissues, vacuole formation and mitochondrial damages in the components of ovarian follicles are commonly occurred in both the vitrification and the slow freezing employed. However, the effectiveness of each protocol is determined by the ART conducted after cryopreservation, and protocol-

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Fig. 3. Post-thaw development of preantral follicles after being either frozen slowly (A,C,E,G) or vitrified (B,D,F,H) as punctured ovaries. (A,B) Preantral follicles isolated immediately after thawing or warming; (C,D) mature oocytes retrieved at the end of the culture period (day 10). The first polar body was observed in both groups. (E,F) Embryo development 48 h after parthenogenesis: cleaving embryos were observed in both groups. (G,H) Embryo development 120 h after culture: blastocyst formation was visible in both groups. Scale bar ⫽ 100 ␮m.

specific damage following cryopreservation affected each activation program. In this study, freezing exerted no effect on cleavage and blastocyst formation in the parthenogenesis group, whereas an effect was noted in the IVF group. The follicles derived from slowly frozen ovarian tissue showed delayed growth in vitro compared to intact follicles. In contrast, better development after IVF was observed in the slow-freezing case than in the vitrified case, while no difference in preimplantation development of parthenogenetic embryos was detected between the intact and cryopreservation groups. In the ultrastructural study, vacuoles in zona pellucida and

mitochondrial deformities were detached after cryopreservation. Cohen et al [8] suggested that integrity of zona pellucida could be employed as an indicator of ART outcome after cryopreservation. It has been known that mitochondria significantly contribute to maintaining physiological normality and cellular activity during embryogenesis [9]. In this study, we did not examine in detail how mitochondrial damage influenced specific cellular events during folliculogenesis and oocyte development. From different viewpoint, however, ovarian follicles and intrafollicular oocytes can be tolerable for certain degree of damage, while the latent disorder incurred by the tissue banking should be addressed before clinical application. Slow freezing has been employed as a standard protocol for the cryopreservation of oocytes and various somatic tissues, while vitrification is an alternative method that simplifies the cryopreservation process. Several studies have been performed with both methods using oocytes from humans [10] and mice [11] and ovarian tissue from humans [12–15] and mice [1,3]. Cryopreserved ovarian tissue has been successfully engrafted leading to the production of live offspring in mice [16 –22] and in humans [See Demeestere et al for a review; 23]. Similar to previous reports, we found that the oocytes retrieved from preantral follicles of cryopreserved ovaries were able to develop to the blastocyst stage more prominently, although the efficiency was lower compared with that in intact oocytes. In conclusion, cryo-damage induced by slow freezing or vitrification procedure influences freezing susceptibility of oocytes provided for different ART programs. Although similar damages may be incurred by a vitrification or by a slow freezing, the optimal freezing procedure is determined by the ART program being employed. However, both modes of cryopreservation, slow freezing and vitrification, can be employed for ovarian tissue banking.

Acknowledgments This research was supported by a grant (Code 20080401034072) from BioGreen 21 Program, Rural Development Administration, Republic of Korea. This study was also supported by educational grants of Brain Korea 21 and WCU (World Class University) programs (R31-10056) through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology.

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