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Viability of oocytes and granulosa cells from cryopreserved ovine ovarian primordial, primary and secondary follicles
Small Ruminant Research 99 (2011) 203–207
Contents lists available at ScienceDirect
Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres
Short communication
Viability of oocytes and granulosa cells from cryopreserved ovine ovarian primordial, primary and secondary follicles R.R. Santos a,b,∗ , R. Van den Hurk a , A.P.R. Rodrigues c , J.R. Figueiredo c a b c
Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands Laboratory of Biology and Medicine of Wild Mammals from Amazonia, Federal University of Pará, Belém, Pará, Brazil Laboratory of Manipulation of Oocytes and Ovarian Preantral Follicles, Faculty of Veterinary, State of Ceará University, Fortaleza, CE, Brazil
a r t i c l e
i n f o
Article history: Received 8 October 2010 Received in revised form 23 February 2011 Accepted 3 March 2011 Available online 14 July 2011 Keywords: Sheep Ovarian tissue Cryopreservation Viability markers
a b s t r a c t The study aimed to evaluate the effect of the addition of sucrose to the freezing and washing medium on the morphology and viability of ovine primordial, primary and secondary follicles and their enclosed oocytes and granulosa cells. Ovine primordial, primary and secondary ovarian follicles were cryopreserved in the absence or presence of 0.5 M sucrose, with or without 1.0 M ethylene glycol (EG) or 1.0 M dimethyl sulphoxide (DMSO). After thawing, all follicles were washed in minimum essential medium (MEM), with or without 0.3 M sucrose. Inclusion of sucrose in the freezing media generally improved the postthaw morphology of the preantral follicles. The addition of sucrose to the washing media did not affect the percentage of normal follicles cryopreserved in the sucrose-containing media. However, its addition to the washing medium of ovarian tissue cryopreserved in the presence of EG, did increase the percentage of normal follicles. Although all the cryopreservation treatments lowered the percentage viability of the different isolated preantral follicle classes and their oocytes, the addition of sucrose to the cryoprotectants EG and DMSO appeared beneficial for the viability of all preantral follicles. The percentages recorded were lowest for the secondary follicles. In the presence of sucrose, cryopreservation of oocytes and granulosa cells from primordial and primary follicles was better than that from the cellular compartments of secondary follicles. It can thus be said that oocytes and granulosa cells of primordial and primary ovine follicles are well-cryopreserved in the presence of EG or DMSO, supplemented with sucrose, followed by a thawing-washing procedure in a sucrose-free medium. © 2011 Published by Elsevier B.V.
1. Introduction For the cryopreservation of ovarian tissue, different freezing techniques and several successful cryoprotectants have been described. The most commonly used cryoprotectants to preserve ovine ovarian tissue have been reported to
∗ Corresponding author at: Utrecht University, Faculty of Veterinary Medicine, Department of Equine Sciences, Veterinary Pharmaceuticals, Pharmacology and Toxicology Division, Yalelaan 114, 3584 CM, Utrecht, The Netherlands. Tel.: +31 30 253 1078. E-mail addresses: [email protected], [email protected] (R.R. Santos). 0921-4488/$ – see front matter © 2011 Published by Elsevier B.V. doi:10.1016/j.smallrumres.2011.03.006
be ethylene glycol (EG) and dimethyl sulphoxide (DMSO), supplemented or not with sucrose (Amorim et al., 2003). However, there is a lack of research regarding ovarian tissue freezing in the presence of an extra-cellular cryoprotectant, like sucrose (Santos et al., 2006a; Marsella et al., 2008; Fabbri et al., 2010). Although ovarian tissue cryopreservation is often performed aimed at preserving the primordial follicles (Ksiazkiewicz, 2006; Santos et al., 2010), more advanced stages of preantral follicles, i.e. primary and secondary follicles, may also survive the cryopreservation process (Santos et al., 2008; Barrett et al., 2010). However, compared to the more advanced follicular stages, primordial follicles are
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more effectively cryopreserved due to their low metabolic rate, smaller oocytes, absence of a zona pellucida, and a lower quantity of intracytoplasmic lipids. Additionally, the lower number of granulosa cells may also play a role in this respect (Shaw et al., 2000). In most cryopreservation studies, histological analyses are commonly used to evaluate the quality of ovarian follicles. Such an evaluation facilitates the identification of signs of advanced atresia, like, e.g. nuclear pycnosis, detachment of the granulosa cells from the oocyte and abnormalities of the basement membrane. Morphologically intact follicles or their cellular components may, however, still be non-viable (Van den Hurk et al., 1998). Various fluorescent probes are currently available to discriminate between viable (presence of mitochondrial or esterase activity) and non-viable (loss of membrane integrity) cells, respectively. Such markers have been previously used to evaluate the quality of bovine (Schotanus et al., 1997; Van den Hurk et al., 1998), caprine (Santos et al., 2006a), canine (Lopes et al., 2009), murine (Cortvrindt and Smitz, 2001) and human (Martinez-Madrid et al., 2004) preantral ovarian follicles. Thus far, however, little attention has been paid to the viability of oocytes and the granulosa cells per preantral follicle. The present study thus aimed to examine (i) the effects of cryopreservation using EG and/or DMSO, supplemented or not with sucrose on the morphology of ovine preantral follicles; (ii) the effects of sucrose addition to the washing (thawing) medium on the morphology of the preantral follicles; and (iii) the effects of freezing–thawing on the viability of ovine primordial, primary and secondary follicles and their enclosed oocytes and granulosa cells. 2. Materials and methods
2.2.2. Experiment II: Viability of oocytes and granulosa cells from primordial, primary and secondary follicles After dissection of the ovaries from 5 sheep, 5 fragments of 1 mm3 were obtained from each ovarian pair. One fragment (control) was immediately allocated to follicular isolation, according to a procedure as described by Amorim et al. (2000). Follicles presenting a normal morphology under light microscopy were submitted to a viability test, using a marker for live (calcein-AM) or dead (ethidium homodimer-1) cells. The remaining 4 fragments were subjected to the freezing–thawing procedure using the different cryoprotectants: (i) 1.0 M DMSO, (ii) 1.0 M EG, (iii) 1.0 M DMSO + 0.5 M sucrose or (iv) 1.0 M EG + 0.5 M sucrose. After thawing, and subsequent removal of the cryoprotectant, preantral follicles were isolated and those considered morphologically normal were evaluated for viability using an epifluorescence microscope (BH2-RFCA microscope, Olympus, Tokyo, Japan), equipped with a digital camera (Coolpix900, Nikon Instruments Europe, Badhoevedorp, The Netherlands).
2.3. Freeze and thawing procedure Ovarian samples were individually placed in straws and equilibrated in a programmable freezer (Planner Kryo 10 Series II, Cryotech Benelux, Schagen, The Netherlands) for 20 min, at 20 ◦ C in 1.5 ml minimum essential medium (MEM) (Sigma Chemicals, Poole, Dorset, UK), supplemented with 0.5 M sucrose (Sigma), 1.0 M DMSO (Merck, Darmstadt, Germany) or 1.0 M EG (Sigma), alone or in combination. The cooling rate used was 2 ◦ C/min from 20 ◦ C to −7 ◦ C, whereby ice-out growth induction (seeding) was manually performed – by touching the straws with a forceps pre-cooled in liquid nitrogen. After seeding, the straws were maintained at this temperature (−7 ◦ C) for 10 min, then cooled at a rate of 0.3 ◦ C/min to −30 ◦ C, and finally at 0.15 ◦ C/min to −33 ◦ C, after which the straws were plunged into the liquid nitrogen (−196 ◦ C) and stored for 1 week. When required, the straws were thawed in air for 1 min at room temperature (∼25 ◦ C) and then immersed in a water bath at 37 ◦ C until the cryopreservation medium had completely melted. The cryoprotectant was then removed from the tissue at room temperature using a three-step equilibration (5 min each) method in MEM, supplemented or not with 0.3 M sucrose. When sucrose was used, an additional wash was performed, using MEM.
2.1. Source and preparation of ovarian tissue
2.4. Assessment of oocyte and granulosa cell viability
This study was subdivided into 2 experiments. For each experiment, female reproductive organs (ovaries) from adult mixed breed ewes were obtained at a local slaughterhouse (Montfoort, The Netherlands). The material was transported to the laboratory immediately after slaughter (∼1 h) in thermo flasks at 30 ◦ C. At the laboratory, the ovaries were trimmed from adhering tissue, washed in 70% alcohol and subsequently washed twice in a phosphate buffer solution (PBS).
Preantral follicles were mechanically isolated from ovarian tissue by applying a mechanical procedure for the isolation of ovine preantral follicles as described by Amorim et al. (2000). Briefly, the ovarian cortex was cut into small fragments, using a tissue chopper (Meyvis, Gouda, The Netherlands). The ovarian fragments were then placed in PBS supplemented with 0.1% (v/v) penicillin/streptomycin (Gibco, Paisley, UK) at room temperature (25 ◦ C), and then pipetted 40 times using a Pasteur pipette. The suspension was successively filtered through 500 and 100 m nylon mesh filters. Preantral follicles smaller than 100 m were collected using a dissecting stereomicroscope (SZ-STS, Olympus, Tokyo, Japan) and transferred to a HEPES buffered M199 + 1% BSA (holding medium) solution. Isolated follicles were incubated in the holding medium for 10 min at 37 ◦ C, in a mixture of 4 M of calcein AM, 2 M ethidium homodimer1 (Molecular Probes Europe B.V., Leiden, The Netherlands) and 10 M Hoechst 33342 (Sigma) to detect esterase enzyme activity, membrane integrity and to enable the counting of the nuclei, respectively (Schotanus et al., 1997). After labeling, stained follicles were washed three times in the holding medium, then mounted on a glass microscope slide in a 5 l of antifade medium (Vectashield, Vector Lab., Burlingame, CA), to prevent photobleaching, and finally examined using an epifluorescent microscope (BH2-RFCA, Olympus, Tokyo, Japan), equipped with a digital camera (Coolpix900, Nikon Instruments Europe B.V., Badhoevedorp, The Netherlands). The emitted fluorescent signals of Hoechst, calcein and ethidium homodimer were recorded at 350, 488 and 568 nm, respectively. Oocytes and granulosa cells were classified as viable, if the ooplasm was stained positively with calcein, and the chromatin was not labeled with ethidium homodimer. The percentage viable granulosa cells was calculated in relation to the total number of Hoechst positive nuclei. Follicles were considered viable when viable oocytes were surrounded by >90% viable granulosa cells (Santos et al., 2006a).
2.2. Experimental design 2.2.1. Experiment I: Morphological analyses of cryopreserved preantral follicles After dissection of the ovaries from 10 sheep, 15 fragments of 1 mm3 were collected from each ovarian pair. One (control) sample was fixed for routine histological analysis, while the remaining 14 fragments were subjected to a freezing–thawing procedure as previously described by Santos et al. (2006a), in the presence of 0.5 M sucrose, 1.0 DMSO or 1.0 EG alone or a mixture of these compounds (1:1) – and subsequently fixed for histological analyses. Preantral follicles were classified as follicles without an antrum and with an oocyte, surrounded by one layer of flattened (primordial follicle) or cuboidal granulosa cells (primary follicle), or with an oocyte surrounded by two or more layers of cuboidal granulosa cells (secondary follicle). Ovarian follicular quality was thus evaluated based on the morphological integrity of the oocyte, granulosa cells and the basement membrane. These preantral follicles were thus classified as morphologically normal or degenerated according to Santos et al. (2006b), i.e. (i) normal, when follicles contained an intact oocyte and intact granulosa cells; (ii) degenerative, when containing a pyknotic oocyte nucleus, shrunken ooplasm and disorganized granulosa cells, e.g. enlargement in volume and detachment from the basement membrane.
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3.2. Experiment II: Viability of cryopreserved preantral follicles
Fig. 1. Mean percentage (±SE) of histological normal preantral follicles in control ovarian fragments and in fragments frozen/thawed in the presence (FT+) or absence (FT−) of sucrose and washed in the presence (W+) or absence (W−) of sucrose. [* Value differs from control (P < 0.05); a–c, Values are significantly different within the same cryoprotectant (P < 0.05); A and B, Values are significantly different between cryoprotectants under same freezing–thawing/washing conditions]. 2.5. Statistical analysis In experiment I, preantral follicle quality after cryopreservation was assessed morphologically using histology, and the mean percentage of normal primordial, primary and secondary follicles between frozenthawed and control fragments compared using an one-way ANOVA and Tukey’s test. The viability of the oocytes and granulosa cells (for experiment II) within each follicular class, was analyzed using the chi-square test. The number of total nuclei per follicle was compared using an oneway ANOVA. In all cases, the statistical tests were performed using Stat View for Windows, and differences were considered to be significant, when P < 0.05.
A total of 580 preantral follicles were mechanically isolated from cryopreserved ovarian fragments shortly after thawing, and then examined for viability (evaluating oocytes and granulosa cells), using a fluorescent labeling technique. All cryopreservation treatments significantly reduced the percentage of viable primordial, primary and secondary follicles, compared to the controls. In all three isolated preantral follicle classes, post-thaw viability was significantly better maintained after freezing of the ovarian tissue in sucrose and DMSO or sucrose and EG, than in EG or DMSO alone (Fig. 2A and C). After cryopreservation of the ovarian tissue in the presence of a DMSO or an EG solution supplemented with sucrose, followed by thawing of the ovarian tissue and isolation of primordial follicles, the percentage of viable granulosa cells in these early-stage follicles were maintained at the control levels – but they were significantly
3. Results 3.1. Experiment I: Histology of cryopreserved preantral follicles At least 150 follicles were evaluated per treatment (30 ± 5 follicles per fragment). Fig. 1 summarizes the results obtained following histological analyses of the preantral follicles in non-frozen (control) and cryopreserved ovarian tissue. Normal histomorphology was recorded in 81.4 ± 2.6% of the non-frozen or control follicles. All the applied freezing–thawing treatments significantly reduced the percentage of histological normal preantral follicles, compared to the control ovarian tissue. Inclusion of sucrose in the freezing medium improved the post-thaw morphology of the follicles in all the frozen-thawed groups (Fig. 1). The percentages of morphological normal follicles cryopreserved in sucrose-containing media and washed in sucrose-free media were not significantly different from those cryopreserved and washed in sucrose-containing media. The same trend was observed for follicles that were cryopreserved in sucrose-free media, except when EG was used as a cryoprotectant. This latter method of follicle cryoprotection resulted in an increased percentage of normal follicles when a sucrose-containing washing medium was used. Freezing in a mixture of EG and DMSO in the presence or absence of sucrose and washing in the presence or absence of sucrose was not beneficial for the morphology of follicles, compared to the corresponding treatments in which EG or DMSO was used. Therefore, further test mixtures of EG and DMSO for viability evaluation of isolated follicles and their cellular components were not performed.
Fig. 2. Mean percentage (±SE) of viable preantral follicles (black bars), oocytes (grey bars), and granulosa cells (white bars). A = primordial follicles; B = primary follicles; C = secondary follicles. [* Value differs from control within each group: viable preantral follicles, viable oocytes and viable granulosa cells (P < 0.05); a–c, Values are significantly different between preantral follicles, oocytes and granulosa cells within each group (P < 0.05)].
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reduced (P < 0.05), when such tissue was cryopreserved in EG or DMSO alone (Fig. 2A). Compared to the percentage of viable oocytes from the control primordial follicles, all cryopreservation treatments resulted in significantly lower (P < 0.05) viability rates of such oocytes (Fig. 2A). Among these latter values, the viability of primordial follicle oocytes was lowest, when the ovarian tissue was frozen in the presence of DMSO or EG alone (Fig. 2A). Concerning the granulosa cells from the primary follicles, cryopreservation in the presence of DMSO plus sucrose, EG plus sucrose or EG alone, but not in DMSO alone, resulted in viable rates or similar to that of the control follicles (Fig. 2B). Although the percentages of viable oocytes from the primary follicles were significantly reduced (P < 0.05) after cryopreservation in all tested solutions, the addition of sucrose to DMSO or EG resulted in significantly higher percentages of viable oocytes – compared to those from follicles frozen in DMSO or EG alone (P < 0.05; Fig. 2B). The percentage of viable granulosa cells from the secondary follicles was only similar to those of the control secondary follicles, when the cryopreservation was performed in a medium containing EG, with or without sucrose (Fig. 2C). All cryopreservation treatments resulted in a reduction (P < 0.05) of viable oocytes from the secondary follicles (Fig. 2C). 4. Discussion The present trial describes the viability of ovine ovarian preantral follicles after freezing in the presence of DMSO or EG, supplemented or not with sucrose, followed by the cryoprotectant removal in the presence, or absence of sucrose. The applied freezing–thawing treatments lowered the percentage of ovarian tissue enclosed in the histological normal preantral follicles, and those of post-thawing isolated viable primordial, primary and secondary follicles – compared to the controls. These findings are contradictory to those of Tsuribe et al. (2009), who claimed the maintenance of ovine follicular morphology after cryopreservation of the ovine ovarian tissue to be only in EG. Differently to these researchers, however, cryopreservation was performed in serum-free solutions, as serum supplementation to cryoprotectant solutions may also induce the risk of infection (Hreinsson et al., 2003). This can hinder normal development of frozen-thawed follicular follicles in future in vitro culture, or transplantation experiments. The histological evaluations of ovine ovarian cortical tissue and the viability analyses of the different preantral follicle classes showed that these follicles, like caprine ovarian tissue enclosed preantral follicles (Santos et al., 2006a), can best be frozen in a medium containing EG or DMSO – to which sucrose is added. Adding sucrose to the washing solution was effective only when the ovarian tissue was cryopreserved in EG alone. Recently, Faustino et al. (2010) have shown efficient cryopreservation of caprine and ovine preantral follicles using an EG cryoprotectant solution, followed by the washing in a sucrose-containing medium.
To current knowledge, this study is the first in which the viability of the follicles, oocytes and granulosa cells has been studied per preantral follicle class. The findings regarding follicular viability correspond with those recorded for both the oocyte and granulosa cells from primordial and primary follicles, respectively. However not from those of the secondary follicles, whose viability was not significantly altered by the different cryopreservation treatments. Thus, contrary to primordial and primary follicles, the addition of sucrose did not result in more viable oocytes and granulosa cells in secondary follicles. Possibly, in ovarian tissue enclosed with multi-layered follicles, increased physical barriers hampered the penetration of the cryoprotectants. A recent study on vitrification of isolated bovine secondary follicles has shown that in a cryoprotectant solution containing EG, DMSO and fetal calf serum, the presence of sucrose was essential for the maintenance of the morphology and their developmental potential to grow to the antral stages – after xenografting in SCID mice (Bao et al., 2010). The different results obtained with bovine and ovine secondary follicles may be due to differences in applied cryopreservation procedures, i.e. slow freezing vs. vitrification and/or the freezing of ovarian tissue enclosed preantral follicles vs. freezing of isolated secondary follicles. In conclusion, the normal morphology of ovine preantral follicles and the viability of the various preantral follicle classes are best maintained when ovarian cortical tissue is frozen in a medium with EG or DMSO, supplemented with sucrose – and thereafter washed in a medium without sucrose. After the freezing–thawing, the viability of isolated ovine secondary follicles is markedly reduced, compared to that of primordial and primary follicles. The oocytes from these secondary follicles were particularly affected. Acknowledgement R.R. Santos’ cryopreservation research is supported by the project 483439/2009-6 from CNPq, Brazil. References Amorim, C.A., Rodrigues, A.P., Lucci, C.M., Figueiredo, J.R., Gonc¸alves, P.B., 2000. Effect of sectioning on the number of isolated ovine preantral follicles. Small Rumin. Res. 37, 269–277. Amorim, C.A., Gonc¸alves, P.B., Figueiredo, J.R., 2003. Cryopreservation of oocytes from pre-antral follicles. Hum. Reprod. Update 9, 119–129. Bao, R.M., Yamasaka, E., Moniruzzaman, M., Hamawaki, A., Yoshikawa, M., Miyano, T., 2010. Development of vitrified bovine secondary and primordial follicles in xenografts. Theriogenology 74, 817–827. Barrett, S.L., Shea, L.D., Woodruff, T.K., 2010. Noninvasive index of cryorecovery and growth potential for human follicles in vitro. Biol. Reprod. 82, 1180–1189. Cortvrindt, R., Smitz, J., 2001. In vitro follicle growth: achievements in mammalian species. Reprod. Domest. Anim. 36, 3–9. Fabbri, R., Pasquinelli, G., Keane, D., Magnani, V., Paradisi, R., Venturoli, S., 2010. Optimization of protocols for human ovarian tissue cryopreservation with sucrose, 1,2-propanediol and human serum. Reprod. Biomed. Online 21, 819–828. Faustino, L.R., Santos, R.R., Silva, C.M., Pinto, L.C., Celestino, J.J., Campello, C.C., Figueiredo, J.R., 2010. Goat and sheep ovarian tissue cryopreservation: effects on the morphology and development of primordial follicles and density of stromal cell. Anim. Reprod. Sci. 122, 90–97. Hreinsson, J., Zhang, P., Swahn, M.L., Hultenpy, K., Hovatta, O., 2003. Cryopreservation of follicles in human ovarian cortical tissue. Comparison
R.R. Santos et al. / Small Ruminant Research 99 (2011) 203–207 of serum and human serum albumin in the cryoprotectant solutions. Hum. Reprod. 18, 2420–2428. Ksiazkiewicz, L.K., 2006. Recent achievements in in vitro culture and preservation of ovarian follicles in mammals. Reprod. Biol. 6, 3–16. Lopes, C.A.P., Santos, R.R., Celestino, J.J., Melo, M.A.P., Chaves, R.N., Campello, C.C., Silva, J.R.V., Báo, S.N., Jewgenow, K., Figueiredo, J.R., 2009. Short-term preservation of canine preantral follicles: effect of temperature, medium and time. Anim. Reprod. Sci. 115, 201–214. Marsella, T., Sena, P., Xella, S., La Marca, A., Giuli, S., De Pol, A., Volpe, A., Marzona, L., 2008. Human ovarian tissue cryopreservation: effect of sucrose concentration on morphological features after thawing. Reprod. Biomed. Online 16, 257–267. Martinez-Madrid, B., Dolmans, M.M., Van Langendonckt, A., Defrère, S., Donnez, J., 2004. Freeze-thawing intact human ovary with its vascular pedicle with a passive cooling device. Fertil. Steril. 82, 1390–1394. Santos, R.R., Tharasanit, T., Figueiredo, J.R., Van Haeften, T., Van den Hurk, R., 2006a. Preservation of caprine preantral follicle viability after cryopreservation in sucrose and ethylene glycol. Cell. Tissue Res. 325, 523–531. Santos, R.R., Rodrigues, A.P., Costa, S.H., Silva, J.R.V., Matos, M.H., Lucci, C.M., Báo, S.N., Van den Hurk, R., Figueiredo, J.R., 2006b. Histological and ultrastructural analysis of cryopreserved sheep preantral follicles. Anim. Reprod. Sci. 91, 249–263.
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Santos, R.R., Van Haeften, T., Roelen, B.A., Knijn, H.M., Colenbrander, B., Gadella, B.M., Van den Hurk, R., 2008. Osmotic tolerance and freezability of isolated caprine early-staged follicles. Cell Tissue Res. 333, 323–331. Santos, R.R., Amorim, C., Cecconi, S., Fassbender, M., Imhof, M., Lornage, J., Paris, M., Schoenfeldt, V., Martinez-Madrid, B., 2010. Cryopreservation of ovarian tissue: an emerging technology for female germline preservation of endangered species and breeds. Anim. Reprod Sci. 122, 151–163. Schotanus, K., Hage, W.J., Vanderstchele, H., Van den Hurk, R., 1997. Effects of conditioned media from murine granulosa cell lines on the growth of isolated bovine preantral follicles. Theriogenology 48, 471–483. Shaw, J.M., Oranratnachai, A., Trounson, A.O., 2000. Fundamental cryobiology of mammalian oocytes and ovarian tissue. Theriogenology 53, 59–72. Tsuribe, P.M., Gobbo, C.A., Landim-Alvarenga, F.C., 2009. Viability of primordial follicles derived from cryopreserved ovine ovarian cortex tissue. Fertil. Steril. 91, 1976–1983. Van den Hurk, R., Spek, E.R., Hage, W.J., Fair, T., Ralph, J.H., Schotanus, K., 1998. Ultrastructure and viability of isolated bovine preantral follicles. Hum. Reprod. Up. 4, 833–841.
Contents lists available at ScienceDirect
Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres
Short communication
Viability of oocytes and granulosa cells from cryopreserved ovine ovarian primordial, primary and secondary follicles R.R. Santos a,b,∗ , R. Van den Hurk a , A.P.R. Rodrigues c , J.R. Figueiredo c a b c
Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands Laboratory of Biology and Medicine of Wild Mammals from Amazonia, Federal University of Pará, Belém, Pará, Brazil Laboratory of Manipulation of Oocytes and Ovarian Preantral Follicles, Faculty of Veterinary, State of Ceará University, Fortaleza, CE, Brazil
a r t i c l e
i n f o
Article history: Received 8 October 2010 Received in revised form 23 February 2011 Accepted 3 March 2011 Available online 14 July 2011 Keywords: Sheep Ovarian tissue Cryopreservation Viability markers
a b s t r a c t The study aimed to evaluate the effect of the addition of sucrose to the freezing and washing medium on the morphology and viability of ovine primordial, primary and secondary follicles and their enclosed oocytes and granulosa cells. Ovine primordial, primary and secondary ovarian follicles were cryopreserved in the absence or presence of 0.5 M sucrose, with or without 1.0 M ethylene glycol (EG) or 1.0 M dimethyl sulphoxide (DMSO). After thawing, all follicles were washed in minimum essential medium (MEM), with or without 0.3 M sucrose. Inclusion of sucrose in the freezing media generally improved the postthaw morphology of the preantral follicles. The addition of sucrose to the washing media did not affect the percentage of normal follicles cryopreserved in the sucrose-containing media. However, its addition to the washing medium of ovarian tissue cryopreserved in the presence of EG, did increase the percentage of normal follicles. Although all the cryopreservation treatments lowered the percentage viability of the different isolated preantral follicle classes and their oocytes, the addition of sucrose to the cryoprotectants EG and DMSO appeared beneficial for the viability of all preantral follicles. The percentages recorded were lowest for the secondary follicles. In the presence of sucrose, cryopreservation of oocytes and granulosa cells from primordial and primary follicles was better than that from the cellular compartments of secondary follicles. It can thus be said that oocytes and granulosa cells of primordial and primary ovine follicles are well-cryopreserved in the presence of EG or DMSO, supplemented with sucrose, followed by a thawing-washing procedure in a sucrose-free medium. © 2011 Published by Elsevier B.V.
1. Introduction For the cryopreservation of ovarian tissue, different freezing techniques and several successful cryoprotectants have been described. The most commonly used cryoprotectants to preserve ovine ovarian tissue have been reported to
∗ Corresponding author at: Utrecht University, Faculty of Veterinary Medicine, Department of Equine Sciences, Veterinary Pharmaceuticals, Pharmacology and Toxicology Division, Yalelaan 114, 3584 CM, Utrecht, The Netherlands. Tel.: +31 30 253 1078. E-mail addresses: [email protected], [email protected] (R.R. Santos). 0921-4488/$ – see front matter © 2011 Published by Elsevier B.V. doi:10.1016/j.smallrumres.2011.03.006
be ethylene glycol (EG) and dimethyl sulphoxide (DMSO), supplemented or not with sucrose (Amorim et al., 2003). However, there is a lack of research regarding ovarian tissue freezing in the presence of an extra-cellular cryoprotectant, like sucrose (Santos et al., 2006a; Marsella et al., 2008; Fabbri et al., 2010). Although ovarian tissue cryopreservation is often performed aimed at preserving the primordial follicles (Ksiazkiewicz, 2006; Santos et al., 2010), more advanced stages of preantral follicles, i.e. primary and secondary follicles, may also survive the cryopreservation process (Santos et al., 2008; Barrett et al., 2010). However, compared to the more advanced follicular stages, primordial follicles are
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more effectively cryopreserved due to their low metabolic rate, smaller oocytes, absence of a zona pellucida, and a lower quantity of intracytoplasmic lipids. Additionally, the lower number of granulosa cells may also play a role in this respect (Shaw et al., 2000). In most cryopreservation studies, histological analyses are commonly used to evaluate the quality of ovarian follicles. Such an evaluation facilitates the identification of signs of advanced atresia, like, e.g. nuclear pycnosis, detachment of the granulosa cells from the oocyte and abnormalities of the basement membrane. Morphologically intact follicles or their cellular components may, however, still be non-viable (Van den Hurk et al., 1998). Various fluorescent probes are currently available to discriminate between viable (presence of mitochondrial or esterase activity) and non-viable (loss of membrane integrity) cells, respectively. Such markers have been previously used to evaluate the quality of bovine (Schotanus et al., 1997; Van den Hurk et al., 1998), caprine (Santos et al., 2006a), canine (Lopes et al., 2009), murine (Cortvrindt and Smitz, 2001) and human (Martinez-Madrid et al., 2004) preantral ovarian follicles. Thus far, however, little attention has been paid to the viability of oocytes and the granulosa cells per preantral follicle. The present study thus aimed to examine (i) the effects of cryopreservation using EG and/or DMSO, supplemented or not with sucrose on the morphology of ovine preantral follicles; (ii) the effects of sucrose addition to the washing (thawing) medium on the morphology of the preantral follicles; and (iii) the effects of freezing–thawing on the viability of ovine primordial, primary and secondary follicles and their enclosed oocytes and granulosa cells. 2. Materials and methods
2.2.2. Experiment II: Viability of oocytes and granulosa cells from primordial, primary and secondary follicles After dissection of the ovaries from 5 sheep, 5 fragments of 1 mm3 were obtained from each ovarian pair. One fragment (control) was immediately allocated to follicular isolation, according to a procedure as described by Amorim et al. (2000). Follicles presenting a normal morphology under light microscopy were submitted to a viability test, using a marker for live (calcein-AM) or dead (ethidium homodimer-1) cells. The remaining 4 fragments were subjected to the freezing–thawing procedure using the different cryoprotectants: (i) 1.0 M DMSO, (ii) 1.0 M EG, (iii) 1.0 M DMSO + 0.5 M sucrose or (iv) 1.0 M EG + 0.5 M sucrose. After thawing, and subsequent removal of the cryoprotectant, preantral follicles were isolated and those considered morphologically normal were evaluated for viability using an epifluorescence microscope (BH2-RFCA microscope, Olympus, Tokyo, Japan), equipped with a digital camera (Coolpix900, Nikon Instruments Europe, Badhoevedorp, The Netherlands).
2.3. Freeze and thawing procedure Ovarian samples were individually placed in straws and equilibrated in a programmable freezer (Planner Kryo 10 Series II, Cryotech Benelux, Schagen, The Netherlands) for 20 min, at 20 ◦ C in 1.5 ml minimum essential medium (MEM) (Sigma Chemicals, Poole, Dorset, UK), supplemented with 0.5 M sucrose (Sigma), 1.0 M DMSO (Merck, Darmstadt, Germany) or 1.0 M EG (Sigma), alone or in combination. The cooling rate used was 2 ◦ C/min from 20 ◦ C to −7 ◦ C, whereby ice-out growth induction (seeding) was manually performed – by touching the straws with a forceps pre-cooled in liquid nitrogen. After seeding, the straws were maintained at this temperature (−7 ◦ C) for 10 min, then cooled at a rate of 0.3 ◦ C/min to −30 ◦ C, and finally at 0.15 ◦ C/min to −33 ◦ C, after which the straws were plunged into the liquid nitrogen (−196 ◦ C) and stored for 1 week. When required, the straws were thawed in air for 1 min at room temperature (∼25 ◦ C) and then immersed in a water bath at 37 ◦ C until the cryopreservation medium had completely melted. The cryoprotectant was then removed from the tissue at room temperature using a three-step equilibration (5 min each) method in MEM, supplemented or not with 0.3 M sucrose. When sucrose was used, an additional wash was performed, using MEM.
2.1. Source and preparation of ovarian tissue
2.4. Assessment of oocyte and granulosa cell viability
This study was subdivided into 2 experiments. For each experiment, female reproductive organs (ovaries) from adult mixed breed ewes were obtained at a local slaughterhouse (Montfoort, The Netherlands). The material was transported to the laboratory immediately after slaughter (∼1 h) in thermo flasks at 30 ◦ C. At the laboratory, the ovaries were trimmed from adhering tissue, washed in 70% alcohol and subsequently washed twice in a phosphate buffer solution (PBS).
Preantral follicles were mechanically isolated from ovarian tissue by applying a mechanical procedure for the isolation of ovine preantral follicles as described by Amorim et al. (2000). Briefly, the ovarian cortex was cut into small fragments, using a tissue chopper (Meyvis, Gouda, The Netherlands). The ovarian fragments were then placed in PBS supplemented with 0.1% (v/v) penicillin/streptomycin (Gibco, Paisley, UK) at room temperature (25 ◦ C), and then pipetted 40 times using a Pasteur pipette. The suspension was successively filtered through 500 and 100 m nylon mesh filters. Preantral follicles smaller than 100 m were collected using a dissecting stereomicroscope (SZ-STS, Olympus, Tokyo, Japan) and transferred to a HEPES buffered M199 + 1% BSA (holding medium) solution. Isolated follicles were incubated in the holding medium for 10 min at 37 ◦ C, in a mixture of 4 M of calcein AM, 2 M ethidium homodimer1 (Molecular Probes Europe B.V., Leiden, The Netherlands) and 10 M Hoechst 33342 (Sigma) to detect esterase enzyme activity, membrane integrity and to enable the counting of the nuclei, respectively (Schotanus et al., 1997). After labeling, stained follicles were washed three times in the holding medium, then mounted on a glass microscope slide in a 5 l of antifade medium (Vectashield, Vector Lab., Burlingame, CA), to prevent photobleaching, and finally examined using an epifluorescent microscope (BH2-RFCA, Olympus, Tokyo, Japan), equipped with a digital camera (Coolpix900, Nikon Instruments Europe B.V., Badhoevedorp, The Netherlands). The emitted fluorescent signals of Hoechst, calcein and ethidium homodimer were recorded at 350, 488 and 568 nm, respectively. Oocytes and granulosa cells were classified as viable, if the ooplasm was stained positively with calcein, and the chromatin was not labeled with ethidium homodimer. The percentage viable granulosa cells was calculated in relation to the total number of Hoechst positive nuclei. Follicles were considered viable when viable oocytes were surrounded by >90% viable granulosa cells (Santos et al., 2006a).
2.2. Experimental design 2.2.1. Experiment I: Morphological analyses of cryopreserved preantral follicles After dissection of the ovaries from 10 sheep, 15 fragments of 1 mm3 were collected from each ovarian pair. One (control) sample was fixed for routine histological analysis, while the remaining 14 fragments were subjected to a freezing–thawing procedure as previously described by Santos et al. (2006a), in the presence of 0.5 M sucrose, 1.0 DMSO or 1.0 EG alone or a mixture of these compounds (1:1) – and subsequently fixed for histological analyses. Preantral follicles were classified as follicles without an antrum and with an oocyte, surrounded by one layer of flattened (primordial follicle) or cuboidal granulosa cells (primary follicle), or with an oocyte surrounded by two or more layers of cuboidal granulosa cells (secondary follicle). Ovarian follicular quality was thus evaluated based on the morphological integrity of the oocyte, granulosa cells and the basement membrane. These preantral follicles were thus classified as morphologically normal or degenerated according to Santos et al. (2006b), i.e. (i) normal, when follicles contained an intact oocyte and intact granulosa cells; (ii) degenerative, when containing a pyknotic oocyte nucleus, shrunken ooplasm and disorganized granulosa cells, e.g. enlargement in volume and detachment from the basement membrane.
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3.2. Experiment II: Viability of cryopreserved preantral follicles
Fig. 1. Mean percentage (±SE) of histological normal preantral follicles in control ovarian fragments and in fragments frozen/thawed in the presence (FT+) or absence (FT−) of sucrose and washed in the presence (W+) or absence (W−) of sucrose. [* Value differs from control (P < 0.05); a–c, Values are significantly different within the same cryoprotectant (P < 0.05); A and B, Values are significantly different between cryoprotectants under same freezing–thawing/washing conditions]. 2.5. Statistical analysis In experiment I, preantral follicle quality after cryopreservation was assessed morphologically using histology, and the mean percentage of normal primordial, primary and secondary follicles between frozenthawed and control fragments compared using an one-way ANOVA and Tukey’s test. The viability of the oocytes and granulosa cells (for experiment II) within each follicular class, was analyzed using the chi-square test. The number of total nuclei per follicle was compared using an oneway ANOVA. In all cases, the statistical tests were performed using Stat View for Windows, and differences were considered to be significant, when P < 0.05.
A total of 580 preantral follicles were mechanically isolated from cryopreserved ovarian fragments shortly after thawing, and then examined for viability (evaluating oocytes and granulosa cells), using a fluorescent labeling technique. All cryopreservation treatments significantly reduced the percentage of viable primordial, primary and secondary follicles, compared to the controls. In all three isolated preantral follicle classes, post-thaw viability was significantly better maintained after freezing of the ovarian tissue in sucrose and DMSO or sucrose and EG, than in EG or DMSO alone (Fig. 2A and C). After cryopreservation of the ovarian tissue in the presence of a DMSO or an EG solution supplemented with sucrose, followed by thawing of the ovarian tissue and isolation of primordial follicles, the percentage of viable granulosa cells in these early-stage follicles were maintained at the control levels – but they were significantly
3. Results 3.1. Experiment I: Histology of cryopreserved preantral follicles At least 150 follicles were evaluated per treatment (30 ± 5 follicles per fragment). Fig. 1 summarizes the results obtained following histological analyses of the preantral follicles in non-frozen (control) and cryopreserved ovarian tissue. Normal histomorphology was recorded in 81.4 ± 2.6% of the non-frozen or control follicles. All the applied freezing–thawing treatments significantly reduced the percentage of histological normal preantral follicles, compared to the control ovarian tissue. Inclusion of sucrose in the freezing medium improved the post-thaw morphology of the follicles in all the frozen-thawed groups (Fig. 1). The percentages of morphological normal follicles cryopreserved in sucrose-containing media and washed in sucrose-free media were not significantly different from those cryopreserved and washed in sucrose-containing media. The same trend was observed for follicles that were cryopreserved in sucrose-free media, except when EG was used as a cryoprotectant. This latter method of follicle cryoprotection resulted in an increased percentage of normal follicles when a sucrose-containing washing medium was used. Freezing in a mixture of EG and DMSO in the presence or absence of sucrose and washing in the presence or absence of sucrose was not beneficial for the morphology of follicles, compared to the corresponding treatments in which EG or DMSO was used. Therefore, further test mixtures of EG and DMSO for viability evaluation of isolated follicles and their cellular components were not performed.
Fig. 2. Mean percentage (±SE) of viable preantral follicles (black bars), oocytes (grey bars), and granulosa cells (white bars). A = primordial follicles; B = primary follicles; C = secondary follicles. [* Value differs from control within each group: viable preantral follicles, viable oocytes and viable granulosa cells (P < 0.05); a–c, Values are significantly different between preantral follicles, oocytes and granulosa cells within each group (P < 0.05)].
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reduced (P < 0.05), when such tissue was cryopreserved in EG or DMSO alone (Fig. 2A). Compared to the percentage of viable oocytes from the control primordial follicles, all cryopreservation treatments resulted in significantly lower (P < 0.05) viability rates of such oocytes (Fig. 2A). Among these latter values, the viability of primordial follicle oocytes was lowest, when the ovarian tissue was frozen in the presence of DMSO or EG alone (Fig. 2A). Concerning the granulosa cells from the primary follicles, cryopreservation in the presence of DMSO plus sucrose, EG plus sucrose or EG alone, but not in DMSO alone, resulted in viable rates or similar to that of the control follicles (Fig. 2B). Although the percentages of viable oocytes from the primary follicles were significantly reduced (P < 0.05) after cryopreservation in all tested solutions, the addition of sucrose to DMSO or EG resulted in significantly higher percentages of viable oocytes – compared to those from follicles frozen in DMSO or EG alone (P < 0.05; Fig. 2B). The percentage of viable granulosa cells from the secondary follicles was only similar to those of the control secondary follicles, when the cryopreservation was performed in a medium containing EG, with or without sucrose (Fig. 2C). All cryopreservation treatments resulted in a reduction (P < 0.05) of viable oocytes from the secondary follicles (Fig. 2C). 4. Discussion The present trial describes the viability of ovine ovarian preantral follicles after freezing in the presence of DMSO or EG, supplemented or not with sucrose, followed by the cryoprotectant removal in the presence, or absence of sucrose. The applied freezing–thawing treatments lowered the percentage of ovarian tissue enclosed in the histological normal preantral follicles, and those of post-thawing isolated viable primordial, primary and secondary follicles – compared to the controls. These findings are contradictory to those of Tsuribe et al. (2009), who claimed the maintenance of ovine follicular morphology after cryopreservation of the ovine ovarian tissue to be only in EG. Differently to these researchers, however, cryopreservation was performed in serum-free solutions, as serum supplementation to cryoprotectant solutions may also induce the risk of infection (Hreinsson et al., 2003). This can hinder normal development of frozen-thawed follicular follicles in future in vitro culture, or transplantation experiments. The histological evaluations of ovine ovarian cortical tissue and the viability analyses of the different preantral follicle classes showed that these follicles, like caprine ovarian tissue enclosed preantral follicles (Santos et al., 2006a), can best be frozen in a medium containing EG or DMSO – to which sucrose is added. Adding sucrose to the washing solution was effective only when the ovarian tissue was cryopreserved in EG alone. Recently, Faustino et al. (2010) have shown efficient cryopreservation of caprine and ovine preantral follicles using an EG cryoprotectant solution, followed by the washing in a sucrose-containing medium.
To current knowledge, this study is the first in which the viability of the follicles, oocytes and granulosa cells has been studied per preantral follicle class. The findings regarding follicular viability correspond with those recorded for both the oocyte and granulosa cells from primordial and primary follicles, respectively. However not from those of the secondary follicles, whose viability was not significantly altered by the different cryopreservation treatments. Thus, contrary to primordial and primary follicles, the addition of sucrose did not result in more viable oocytes and granulosa cells in secondary follicles. Possibly, in ovarian tissue enclosed with multi-layered follicles, increased physical barriers hampered the penetration of the cryoprotectants. A recent study on vitrification of isolated bovine secondary follicles has shown that in a cryoprotectant solution containing EG, DMSO and fetal calf serum, the presence of sucrose was essential for the maintenance of the morphology and their developmental potential to grow to the antral stages – after xenografting in SCID mice (Bao et al., 2010). The different results obtained with bovine and ovine secondary follicles may be due to differences in applied cryopreservation procedures, i.e. slow freezing vs. vitrification and/or the freezing of ovarian tissue enclosed preantral follicles vs. freezing of isolated secondary follicles. In conclusion, the normal morphology of ovine preantral follicles and the viability of the various preantral follicle classes are best maintained when ovarian cortical tissue is frozen in a medium with EG or DMSO, supplemented with sucrose – and thereafter washed in a medium without sucrose. After the freezing–thawing, the viability of isolated ovine secondary follicles is markedly reduced, compared to that of primordial and primary follicles. The oocytes from these secondary follicles were particularly affected. Acknowledgement R.R. Santos’ cryopreservation research is supported by the project 483439/2009-6 from CNPq, Brazil. References Amorim, C.A., Rodrigues, A.P., Lucci, C.M., Figueiredo, J.R., Gonc¸alves, P.B., 2000. Effect of sectioning on the number of isolated ovine preantral follicles. Small Rumin. Res. 37, 269–277. Amorim, C.A., Gonc¸alves, P.B., Figueiredo, J.R., 2003. Cryopreservation of oocytes from pre-antral follicles. Hum. Reprod. Update 9, 119–129. Bao, R.M., Yamasaka, E., Moniruzzaman, M., Hamawaki, A., Yoshikawa, M., Miyano, T., 2010. Development of vitrified bovine secondary and primordial follicles in xenografts. Theriogenology 74, 817–827. Barrett, S.L., Shea, L.D., Woodruff, T.K., 2010. Noninvasive index of cryorecovery and growth potential for human follicles in vitro. Biol. Reprod. 82, 1180–1189. Cortvrindt, R., Smitz, J., 2001. In vitro follicle growth: achievements in mammalian species. Reprod. Domest. Anim. 36, 3–9. Fabbri, R., Pasquinelli, G., Keane, D., Magnani, V., Paradisi, R., Venturoli, S., 2010. Optimization of protocols for human ovarian tissue cryopreservation with sucrose, 1,2-propanediol and human serum. Reprod. Biomed. Online 21, 819–828. Faustino, L.R., Santos, R.R., Silva, C.M., Pinto, L.C., Celestino, J.J., Campello, C.C., Figueiredo, J.R., 2010. Goat and sheep ovarian tissue cryopreservation: effects on the morphology and development of primordial follicles and density of stromal cell. Anim. Reprod. Sci. 122, 90–97. Hreinsson, J., Zhang, P., Swahn, M.L., Hultenpy, K., Hovatta, O., 2003. Cryopreservation of follicles in human ovarian cortical tissue. Comparison
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