**1. Introduction**

204 Current Frontiers in Cryopreservation

Salle, B., J. Lornage, B. Demirci, F. Vaudoyer, M. T. Poirel, M. Franck, R. C. Rudigoz&J. F.

Salle, B., J. Lornage, M. Franck, L. Isoard, R. C. Rudigoz&J. F. Guerin. (1998). Freezing,

Santos, R. R., C. Amorim, S. Cecconi, M. Fassbender, M. Imhof, J. Lornage, M. Paris, V.

Soede, N. M., P. Langendijk&B. Kemp. (2011). Reproductive cycles in pigs. *Anim Reprod Sci*,

Stevens, V. C. (1997). Some reproductive studies in the baboon. *Hum Reprod Update*, 3, 533-

Weinbauer, G. F., M. Niehoff, M. Niehaus, S. Srivastav, A. Fuchs, E. Van Esch&J. M. Cline.

Yang, M. Y.&J. E. Fortune. (2006). Testosterone stimulates the primary to secondary follicle

Yeoman, R. R., D. P. Wolf&D. M. Lee. (2005). Coculture of monkey ovarian tissue increases

Zhang, J. M., Y. Sheng, Y. Z. Cao, H. Y. Wang&Z. J. Chen. (2011). Cryopreservation of whole

Zhang, J. M., Y. Sheng, Y. Z. Cao, H. Y. Wang&Z. J. Chen. (2011). Effects of cooling rates and

*Pathol*, 36, 7S-23S, 1533-1601 (Electronic) 0192-6233 (Linking)

*Genet*, 28, 445-452, 1573-7330 (Electronic) 1058-0468 (Linking)

*Genet*, 28, 627-633, 1573-7330 (Electronic) 1058-0468 (Linking)

(2008). Physiology and Endocrinology of the Ovarian Cycle in Macaques. *Toxicol* 

transition in bovine follicles in vitro. *Biol Reprod*, 75, 924-932, 0006-3363 (Print) 0006-

survival after vitrification and slow-rate freezing. *Fertil Steril*, 83 Suppl 1, 1248-1254,

ovaries with vascular pedicles: vitrification or conventional freezing? *J Assist Reprod* 

ice-seeding temperatures on the cryopreservation of whole ovaries. *J Assist Reprod* 

gamete morphology. *Hum Reprod*, 17, 612-619, 0268-1161 (Print)

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Guerin. (1999). Restoration of ovarian steroid secretion and histologic assessment after freezing, thawing, and autograft of a hemi-ovary in sheep. *Fertil Steril*, 72, 366-

thawing, and autograft of ovarian fragments in sheep: preliminary experiments and histologic assessment. *Fertil Steril*, 70, 124-128, 0015-0282 (Print) 0015-0282

Schoenfeldt&B. Martinez-Madrid. (2010). Cryopreservation of ovarian tissue: an emerging technology for female germline preservation of endangered species and breeds. *Anim Reprod Sci*, 122, 151-163, 1873-2232 (Electronic) 0378-4320 (Linking) Schnorr, J., S. Oehninger, J. Toner, J. Hsiu, S. Lanzendorf, R. Williams&G. Hodgen. (2002).

Functional studies of subcutaneous ovarian transplants in non-human primates: steroidogenesis, endometrial development, ovulation, menstrual patterns and

540, 1355-4786 (Print) 1355-4786 (Linking) Ting, A. Y., R. R. Yeoman, M. S. Lawson&M. B. Zelinski. (2011). In vitro development of secondary follicles from cryopreserved rhesus macaque ovarian tissue after slow-rate freeze or vitrification. *Hum Reprod*, 26, 2461-2472, 1460-2350 (Electronic) 0268-1161 (Linking) Vandeberg, J. L. (2004). Need for primate models in biomedical research. *Gynecol Obstet Invest*, 57, 6-8, 0378-7346 (Print) 0378-7346 (Linking) Wallin, A., M. Ghahremani, P. Dahm-Kahler&M. Brannstrom. (2009). Viability and function of the cryopreserved whole ovary: in vitro studies in the sheep. *Hum Reprod*, 24, 1684-1694, 1460-2350 Cryopreservation has been attempted for most of the developmental stages of both male and female reproductive cells, ranging from the immature gametes residing in ovarian or testicular tissues through the mature oocytes and spermatozoa.

However, each variant of the reproductive cells has introduced their own problems, and it has been realized that many aspects of the particular physiology of the cells will dictate how they respond to cryopreservation. Both male and female gametes have acquired highly specialized structural components (essential to fertilization and development) that may respond to the freezing process in ways different from that of basic cell structures.

#### **1.1 Cryopreservation of spermatozoa**

Semen is one of the most practical means of storing germplasm due to its abundant availability and ease of application (Holt and Pickard, 1999; FAO, 2007). Stored spermatozoa could be introduced back into existing populations either immediately or decades or centuries afterwards. In this way, cryopreservation of spermatozoa associated with artificial insemination (AI) or *in vitro* fertilization (IVF) facilitates the management of domestic animals herds, especially cow dairy herds where it is now used since 60 years. Cryopreservation better allows the use of semen from genetically superior males of threatened livestock breeds and has the potential to protect existing diversity and maintain heterozygosity while minimizing the movement of living animals and the transmission of venereal diseases (Johnston and Lacy, 1995; Andrabi and Maxwell, 2007).

Spermatozoon is a very small cell containing low amounts of cytoplasm and consequently low quantity of water. Furthermore sperm nuclear material is compacted and protected

Gérard Louis2, Pierre Guérin1 and Samuel Buff1

<sup>\*</sup> Thierry Joly1, Loris Commin1, Pierre Bruyère1, Anne Baudot2,

*<sup>1</sup>Université de Lyon, VetAgro Sup – Veterinary Campus of Lyon, UPSP ICE* 

*<sup>&#</sup>x27;Interactions between Cells and their Environment', Team Cryobio, France* 

*<sup>2</sup>Université Paris Descartes, Sorbonne Paris Cité, UFR Biomedical, France*

New Approaches of Ovarian Tissue Cryopreservation from Domestic Animal Species 207

Sperm obtained after stem cell transplantation were shown to be able to fertilize mouse oocytes. Fertile offspring were obtained through artificial reproductive technologies following the establishment of complete spermatogenesis by grafting testis tissue from newborn mice, pigs, or goats into mouse host (Honaramooz et al., 2002; Schlatt et al., 2002). Different freezing protocols have been developed in several species but without a clearly identified procedure (Travers et al., 2011). Testicular tissue from prepubertal boys facing gonadotoxic treatment could be banked for several years for spermatogonial stem cell transplantation. Pregnancies have been achieved with ICSI using immature spermatids and secondary spermatocytes extracted from testicular tissue in men with spermatogenic arrest

Oocyte cryopreservation offers many advantages. It permits to preserve endangered species and low effective breeds, and to preserve fertility of high genetic value females. In human it permits to overcome some fertility problems. Oocyte banks would also enlarge the gene pool, facilitate several assisted reproductive procedures, salvage female genetics after unexpected death, and avoid controversy surrounding the preservation of embryos (Ledda et al., 2001; Checura and Seidel, 2007). Like semen, oocyte cryopreservation is beneficial for international exchange of germplasm, as it avoids injury and sanitary risks involved in live animal transportation (Pereira and Marques, 2008). But oocyte cryopreservation gives much

This is due essentially to the much important size and complexity of oocytes. For example, nuclear material is much more exposed in prophase I or metaphase II oocyte than in compacted chromatin of sperm cells. Oocytes collected from slaughterhouse derived ovaries are at the germinal vesicle (GV) stage in which the genetic material is contained within the nucleus. Since this stage has no spindle present, GVs are assumed to be less prone to chromosomal and microtubular damage during cryopreservation. However, oocytes can also be cryopreserved at the metaphase II stage of maturation. During the metaphase II stage, the cumulus cells surrounding the oocyte are expanded, microfilaments of actin are involved in cell shape and movements, and microtubules form the spindle apparatus (Massip, 2003). Moreover, cryopreservation of oocytes necessitates the success of the following steps: *in vitro* maturation, *in vitro* fertilization and embryo culture. Progress in female gametes cryopreservation has gone hand in hand with that for *in vitro* maturation and embryo culture. In livestock animals, oocytes collected by *in vivo* pickup or at slaughter can be frozen for extended periods of time for subsequent IVF to produce embryos. However, in some species such as *canidae* the collection of oocytes is difficult and *in vitro* maturation (IVM), *in vitro* fertilization (IVF) and embryo culture are not yet under control (Luvoni et al., 2006). For these reasons precise cooling and thawing rates and the use of a programmable cell freezer are necessary for oocyte (and also embryo) cryoconservation and

Oocytes are extremely sensitive to chilling, and the technique is not as established as in semen or embryos, due to the fact that oocytes typically have a low permeability to cryoprotectants (Woods et al., 2004). The major differences between oocytes and embryos are the plasma membrane, presence of cortical granules, and spindle formation at

(Fishel et al., 1997; Vanderzwalmen et al., 1997; Sofikitis et al., 1998).

**1.3 Cryopreservation of oocytes** 

lower results when compared with spermatozoa.

very few studies have been conducted in animals.

against physical injuries. For these reasons, cryopreservation of spermatozoa gives excellent results in term of viability and fertility, and is today widely used in human and animal assisted reproduction.

Semen from most mammalian and a few avian species has been successfully frozen in the past several years (FAO, 2007). Indeed, much better results have been obtained with sperm cryopreservation than with oocytes (and embryos) in term of viability. For these reasons, sperm cryobanking is used since the middle of the last century in domestic animal species and lately in human. Numerous storage facilities such as the French National Cryobank were also created for the preservation of valuable or endangered species. However, protocols currently used to conserve semen are still suboptimal and cannot be easily applied across species (Woods et al., 2004). First-service conception rates vary drastically between different breeding programs, but on average conception rates are fairly high in cattle, pigs, goats, and sheep. In wild species cryopreservation of gametes is currently used to preserve endangered species or breeds, and to overcome some fertility troubles. Breed reconstruction solely from semen is possible through a series of back-cross generations; however, the entire genetics of the original breed will not be recovered (Boettcher et al., 2005).

In human, gametes cryopreservation has also been developed to overcome fertility troubles (eg. genital duct problems, sexual dysfunction …). Sperm cryopreservation that has produced live birth has been available for over 50 years (Sherman and Bunge, 1953). Anonymous donor sperm banking has been a fundamental concept of reproductive medicine for several decades, and artificial insemination and donor sperm cryobanking are widely used in reproductive medicine centers (Critser, 1998). The availability of frozen donor sperm has become a mainstay for the treatment of serious male infertility worldwide. More recently, gametes cryopreservation has been used to preserve fertility of men (or women) submitted to gonadotoxic treatments and elective sperm cryopreservation programs have been provided from cancer patients all over the world. Using vitrification of sperm obtained from testicular biopsy, epididymal fluid, or a semen sample after electroejaculation could create new hope for infertile men (Edge et al., 2006).

### **1.2 Cryopreservation of testicular tissue**

Cryopreservation of testicular tissue has been studied since about ten years in animals (Jezek et al., 2001; Jahnukainen et al., 2007; Milazzo et al., 2008; Zeng et al., 2009; Abrishami et al., 2010; Milazzo et al., 2010; Curaba et al., 2011) and human (Bahadur and Ralph, 1999; Bahadur et al., 2000; Guerin, 2005; Revel and Mejia, 2010). It is the only available solution for pre-pubertal boys who must receive a gonadotoxic treatment (eg. cancer therapy;(Keros et al., 2005; Keros et al., 2007; Wyns et al., 2007; Wyns et al., 2008; Wyns et al., 2010).

In contrast to the situation in the ovary, it is well established that spermatogonial stem cells exist in the testis and are responsible for maintaining spermatogenesis from puberty for the lifetime of the male. Human testicular cells might be harvested, cryopreserved before a gonadotoxic treatment and re-introduced into the testis upon its completion. Possibilities include transplantation back into the inactive testes (ipsigenic germ cell transplantation), maturation *in vivo* in another host (xenogenic germ cell transplantation), or *in vitro* spermatogenesis. Mature sperm could then be used for fertilization by ICSI.

Sperm obtained after stem cell transplantation were shown to be able to fertilize mouse oocytes. Fertile offspring were obtained through artificial reproductive technologies following the establishment of complete spermatogenesis by grafting testis tissue from newborn mice, pigs, or goats into mouse host (Honaramooz et al., 2002; Schlatt et al., 2002). Different freezing protocols have been developed in several species but without a clearly identified procedure (Travers et al., 2011). Testicular tissue from prepubertal boys facing gonadotoxic treatment could be banked for several years for spermatogonial stem cell transplantation. Pregnancies have been achieved with ICSI using immature spermatids and secondary spermatocytes extracted from testicular tissue in men with spermatogenic arrest (Fishel et al., 1997; Vanderzwalmen et al., 1997; Sofikitis et al., 1998).

#### **1.3 Cryopreservation of oocytes**

206 Current Frontiers in Cryopreservation

against physical injuries. For these reasons, cryopreservation of spermatozoa gives excellent results in term of viability and fertility, and is today widely used in human and animal

Semen from most mammalian and a few avian species has been successfully frozen in the past several years (FAO, 2007). Indeed, much better results have been obtained with sperm cryopreservation than with oocytes (and embryos) in term of viability. For these reasons, sperm cryobanking is used since the middle of the last century in domestic animal species and lately in human. Numerous storage facilities such as the French National Cryobank were also created for the preservation of valuable or endangered species. However, protocols currently used to conserve semen are still suboptimal and cannot be easily applied across species (Woods et al., 2004). First-service conception rates vary drastically between different breeding programs, but on average conception rates are fairly high in cattle, pigs, goats, and sheep. In wild species cryopreservation of gametes is currently used to preserve endangered species or breeds, and to overcome some fertility troubles. Breed reconstruction solely from semen is possible through a series of back-cross generations; however, the entire

In human, gametes cryopreservation has also been developed to overcome fertility troubles (eg. genital duct problems, sexual dysfunction …). Sperm cryopreservation that has produced live birth has been available for over 50 years (Sherman and Bunge, 1953). Anonymous donor sperm banking has been a fundamental concept of reproductive medicine for several decades, and artificial insemination and donor sperm cryobanking are widely used in reproductive medicine centers (Critser, 1998). The availability of frozen donor sperm has become a mainstay for the treatment of serious male infertility worldwide. More recently, gametes cryopreservation has been used to preserve fertility of men (or women) submitted to gonadotoxic treatments and elective sperm cryopreservation programs have been provided from cancer patients all over the world. Using vitrification of sperm obtained from testicular biopsy, epididymal fluid, or a semen sample after

Cryopreservation of testicular tissue has been studied since about ten years in animals (Jezek et al., 2001; Jahnukainen et al., 2007; Milazzo et al., 2008; Zeng et al., 2009; Abrishami et al., 2010; Milazzo et al., 2010; Curaba et al., 2011) and human (Bahadur and Ralph, 1999; Bahadur et al., 2000; Guerin, 2005; Revel and Mejia, 2010). It is the only available solution for pre-pubertal boys who must receive a gonadotoxic treatment (eg. cancer therapy;(Keros et

In contrast to the situation in the ovary, it is well established that spermatogonial stem cells exist in the testis and are responsible for maintaining spermatogenesis from puberty for the lifetime of the male. Human testicular cells might be harvested, cryopreserved before a gonadotoxic treatment and re-introduced into the testis upon its completion. Possibilities include transplantation back into the inactive testes (ipsigenic germ cell transplantation), maturation *in vivo* in another host (xenogenic germ cell transplantation), or *in vitro*

genetics of the original breed will not be recovered (Boettcher et al., 2005).

electroejaculation could create new hope for infertile men (Edge et al., 2006).

al., 2005; Keros et al., 2007; Wyns et al., 2007; Wyns et al., 2008; Wyns et al., 2010).

spermatogenesis. Mature sperm could then be used for fertilization by ICSI.

**1.2 Cryopreservation of testicular tissue** 

assisted reproduction.

Oocyte cryopreservation offers many advantages. It permits to preserve endangered species and low effective breeds, and to preserve fertility of high genetic value females. In human it permits to overcome some fertility problems. Oocyte banks would also enlarge the gene pool, facilitate several assisted reproductive procedures, salvage female genetics after unexpected death, and avoid controversy surrounding the preservation of embryos (Ledda et al., 2001; Checura and Seidel, 2007). Like semen, oocyte cryopreservation is beneficial for international exchange of germplasm, as it avoids injury and sanitary risks involved in live animal transportation (Pereira and Marques, 2008). But oocyte cryopreservation gives much lower results when compared with spermatozoa.

This is due essentially to the much important size and complexity of oocytes. For example, nuclear material is much more exposed in prophase I or metaphase II oocyte than in compacted chromatin of sperm cells. Oocytes collected from slaughterhouse derived ovaries are at the germinal vesicle (GV) stage in which the genetic material is contained within the nucleus. Since this stage has no spindle present, GVs are assumed to be less prone to chromosomal and microtubular damage during cryopreservation. However, oocytes can also be cryopreserved at the metaphase II stage of maturation. During the metaphase II stage, the cumulus cells surrounding the oocyte are expanded, microfilaments of actin are involved in cell shape and movements, and microtubules form the spindle apparatus (Massip, 2003). Moreover, cryopreservation of oocytes necessitates the success of the following steps: *in vitro* maturation, *in vitro* fertilization and embryo culture. Progress in female gametes cryopreservation has gone hand in hand with that for *in vitro* maturation and embryo culture. In livestock animals, oocytes collected by *in vivo* pickup or at slaughter can be frozen for extended periods of time for subsequent IVF to produce embryos. However, in some species such as *canidae* the collection of oocytes is difficult and *in vitro* maturation (IVM), *in vitro* fertilization (IVF) and embryo culture are not yet under control (Luvoni et al., 2006). For these reasons precise cooling and thawing rates and the use of a programmable cell freezer are necessary for oocyte (and also embryo) cryoconservation and very few studies have been conducted in animals.

Oocytes are extremely sensitive to chilling, and the technique is not as established as in semen or embryos, due to the fact that oocytes typically have a low permeability to cryoprotectants (Woods et al., 2004). The major differences between oocytes and embryos are the plasma membrane, presence of cortical granules, and spindle formation at

New Approaches of Ovarian Tissue Cryopreservation from Domestic Animal Species 209

been frozen and stored at -79°C (Parrott, 1960). Vitrification of ovarian tissue was also investigated. Nevertheless, Isachenko et al suggested that in human, low freezing protocols

This technique has also been developed in rabbit (Neto et al., 2007a), mouse (Candy et al., 1997), rat (Aubard et al., 1998), ewe (Gosden et al., 1994; Demirci et al., 2001), cow (Paynter et al., 1999). We have obtained newborn rabbits after autografting of cryopreserved ovarian cortex (Neto et al., 2007b). Also, our team developed this technique in cat (Neto et al., 2006)

Several techniques have been applied to ovarian cortex cryopreservation: slow freezing, vitrification. Simultaneously to ovarian tissue cryopreservation, numerous researches have been conducted about ovarian tissue grafting: orthotopic, heterotopic, auto-, allo- and

**2. Development of optimized methods for the cryopreservation of the ovarian** 

The most common cryopreservation method is the slow freezing procedure, consisting of an initial slow, controlled-rate cooling to subzero temperatures followed by rapid cooling as the sample is plunged into liquid nitrogen for storage (−196°C). At such a low temperature, biological activity is effectively stopped, and the cells functional status may be preserved for centuries. However, several physical stresses damage the cells at these low temperatures. Intracellular ice formation is one the largest contributors to cell death; therefore, freezing protocols use a combination of dehydration, freezing point depression, supercooling, and

Currently used ovarian cortex cryopreservation protocols have been direct, or slight modifications of the methods developed for isolated oocytes and embryos. There were primarily developed by trial and error adjustments of cooling and warming rates, and choice of CPA and CPA concentrations. However, because there are a large number of protocol variables potentially affecting cell viability, an exhaustive experimental search for the optimal combination of these parameters has long been considered to be prohibitively

**2.1 Chemical and physical parameters affecting equilibration and freezing processes** 

The result of a cryopreservation process is influenced by several chemophysical parameters affecting directly or not the functions and the integrity of the ovarian cells along the freezing process, from the equilibration to the thawing. Among these parameters, the method of equilibration, the freezing rate, the composition of the freezing solution and notably the nature of the permeating CPAs and the non-permeating CPAs, the concentration of each CPA, the use of serum, or the rate of thawing may be investigated to know the relative

In general, we can expect coupled flows of water and CPAs when CPAs are added, during freezing, thawing and when CPAs are removed from the cells, resulting in a series of

were more promising than vitrification protocols (Isachenko et al., 2009).

and dog (Commin et al., 2011).

hetero-grafting (Pullium et al., 2008).

**tissue in domestic mammalian species** 

expensive in terms of time and resources.

**of ovarian tissue in mammalian species** 

influence of each of them and the induced cell injuries.

intracellular vitrification in an attempt to avoid cell damage.

metaphase II stage of meiosis (Chen et al., 2006; Salvetti et al., 2010). To date, there has been no consistent oocyte cryopreservation method established in any species, although, there has been significant progress and offspring have been born from frozen-thawed oocytes in cattle, sheep, and horses (Otoi et al., 1996; Maclellan et al., 2002; Woods et al., 2004). During the process of cryopreservation, oocytes suffer considerable morphological and functional damage, although, the extent of cryoinjuries depends on the species and the origin (*in vivo* or *in vitro* produced). The mechanism for cryoinjuries is yet to be fully understood, and until more insight is gained, improvement of oocyte cryopreservation will be difficult. Also, it is noticeable that immature oocyte present in primordial follicles seems more resistant to cryopreservation when compared to mature oocyte. Consequently, cryopreservation of ovarian cortex may constitute an interesting alternative to isolated mature oocyte cryopreservation.

#### **1.4 Cryopreservation of ovarian tissue**

At birth, the ovaries contain the lifetime complement of primary oocytes which are arrested in the prophase stage of meiosis 1 and are surrounded by a single-layered epithelium to form the primordial follicles. Ovarian cortex presents several advantages when compared with isolated oocytes: *(i)* it contains the important pool of growing follicles; *(ii)* it does not necessitate the *in vitro* maturation /*in vitro* fertilization /embryo culture steps if it is associated with grafting; *(iii)* no previous ovarian stimulation is necessary. Consequently cryopreservation of ovarian cortex is an alternative to cryopreservation of isolated oocytes or embryos. It could be used as an emergency preservation and as infertility therapy method for valuable animals. Ovarian cortex cryopreservation has been developed in human in order to preserve fertility in young women submitted to gonadotoxic therapy (Stahler et al., 1976; Gook et al., 2004). In human newborns were obtained after orthotopic autograft of frozen-thawed ovarian cortices (Donnez et al., 2004).

It is obvious that, to achieve successful cryopreservation of ovarian tissue, it is essential to maintain the functional status of the whole mixture of different cell types: oocytes, granulosa cells, epithelial cells, fibroblasts… This represents a major difficulty, because the optimum kinetic of cooling is different for each cell type. Oocytes are large cells, with a low surface to volume ratio, surrounded by zona pellucida. Immediately adjacent to the oocyte are corona radiata cells that have long cytoplasmic extensions which penetrate the zona pellucida, ending in oocyte membrane. These processes and gap junctions are important in the metabolic cooperation between the oocyte and surrounding layers of granulosa cells, which form the cumulus-oocyte complex during the growth phase. Consequently, at the opposite to cryopreservation of isolated cells, a cryopreservation protocol for a tissue represents a compromise between the requirements of the different constitutive cells.

The early work on ovarian tissue cryopreservation was performed in animal studies: rabbit (Smith, 1952) and rat (Parkes and Smith, 1953; Deanesly, 1954). The earliest positive results were obtained when glycerol (15%) plus serum were used as cryoprotective agents (CPAs) for cryopreservation of rabbit granulosa cells, via a slow cooling protocol (Smith, 1952). An equilibration period was necessary to achieve CPA penetration into the tissue. For this reason small samples were recommended. A rapid rewarming by plunging the samples into a water bath at 40°C was the most effective procedure (Parkes and Smith, 1953; 1954). Normal offspring were obtained from mice with orthotopic ovarian grafts of tissue that had

metaphase II stage of meiosis (Chen et al., 2006; Salvetti et al., 2010). To date, there has been no consistent oocyte cryopreservation method established in any species, although, there has been significant progress and offspring have been born from frozen-thawed oocytes in cattle, sheep, and horses (Otoi et al., 1996; Maclellan et al., 2002; Woods et al., 2004). During the process of cryopreservation, oocytes suffer considerable morphological and functional damage, although, the extent of cryoinjuries depends on the species and the origin (*in vivo* or *in vitro* produced). The mechanism for cryoinjuries is yet to be fully understood, and until more insight is gained, improvement of oocyte cryopreservation will be difficult. Also, it is noticeable that immature oocyte present in primordial follicles seems more resistant to cryopreservation when compared to mature oocyte. Consequently, cryopreservation of ovarian cortex may constitute an interesting alternative to isolated mature oocyte

At birth, the ovaries contain the lifetime complement of primary oocytes which are arrested in the prophase stage of meiosis 1 and are surrounded by a single-layered epithelium to form the primordial follicles. Ovarian cortex presents several advantages when compared with isolated oocytes: *(i)* it contains the important pool of growing follicles; *(ii)* it does not necessitate the *in vitro* maturation /*in vitro* fertilization /embryo culture steps if it is associated with grafting; *(iii)* no previous ovarian stimulation is necessary. Consequently cryopreservation of ovarian cortex is an alternative to cryopreservation of isolated oocytes or embryos. It could be used as an emergency preservation and as infertility therapy method for valuable animals. Ovarian cortex cryopreservation has been developed in human in order to preserve fertility in young women submitted to gonadotoxic therapy (Stahler et al., 1976; Gook et al., 2004). In human newborns were obtained after orthotopic autograft of

It is obvious that, to achieve successful cryopreservation of ovarian tissue, it is essential to maintain the functional status of the whole mixture of different cell types: oocytes, granulosa cells, epithelial cells, fibroblasts… This represents a major difficulty, because the optimum kinetic of cooling is different for each cell type. Oocytes are large cells, with a low surface to volume ratio, surrounded by zona pellucida. Immediately adjacent to the oocyte are corona radiata cells that have long cytoplasmic extensions which penetrate the zona pellucida, ending in oocyte membrane. These processes and gap junctions are important in the metabolic cooperation between the oocyte and surrounding layers of granulosa cells, which form the cumulus-oocyte complex during the growth phase. Consequently, at the opposite to cryopreservation of isolated cells, a cryopreservation protocol for a tissue

represents a compromise between the requirements of the different constitutive cells.

The early work on ovarian tissue cryopreservation was performed in animal studies: rabbit (Smith, 1952) and rat (Parkes and Smith, 1953; Deanesly, 1954). The earliest positive results were obtained when glycerol (15%) plus serum were used as cryoprotective agents (CPAs) for cryopreservation of rabbit granulosa cells, via a slow cooling protocol (Smith, 1952). An equilibration period was necessary to achieve CPA penetration into the tissue. For this reason small samples were recommended. A rapid rewarming by plunging the samples into a water bath at 40°C was the most effective procedure (Parkes and Smith, 1953; 1954). Normal offspring were obtained from mice with orthotopic ovarian grafts of tissue that had

cryopreservation.

**1.4 Cryopreservation of ovarian tissue** 

frozen-thawed ovarian cortices (Donnez et al., 2004).

been frozen and stored at -79°C (Parrott, 1960). Vitrification of ovarian tissue was also investigated. Nevertheless, Isachenko et al suggested that in human, low freezing protocols were more promising than vitrification protocols (Isachenko et al., 2009).

This technique has also been developed in rabbit (Neto et al., 2007a), mouse (Candy et al., 1997), rat (Aubard et al., 1998), ewe (Gosden et al., 1994; Demirci et al., 2001), cow (Paynter et al., 1999). We have obtained newborn rabbits after autografting of cryopreserved ovarian cortex (Neto et al., 2007b). Also, our team developed this technique in cat (Neto et al., 2006) and dog (Commin et al., 2011).

Several techniques have been applied to ovarian cortex cryopreservation: slow freezing, vitrification. Simultaneously to ovarian tissue cryopreservation, numerous researches have been conducted about ovarian tissue grafting: orthotopic, heterotopic, auto-, allo- and hetero-grafting (Pullium et al., 2008).
