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Ultrastructural and morphalogical changes of mouse ovarian tissues following direct cover vitrification with different cryoprotectants.

Introduction

Ovarian tissue cryopreservation is a successful technique that enables the storage of the large numbers of follicles and preserves the structural integrity of somatic and reproductive cells. Therefore, many attempts have been made to improve cryopreservation conditions by using simple and efficient vitrification procedures (1-3). Vitrification of ovarian tissue is a useful method for preservation of female mouse germ cells and live pups following transplantation of vitrifiedwarmed ovarian tissue (4-6).

However, it has been shown that vitrification is often associated with ultrastructural damage of follicular cells and oocytes (7-11).

Vitrification protocols depended on several factors such as appropriate cryopreservation techniques, type and concentration of cryoprotectant, number of equilibration, warming steps and cryopreservation devices (12-14). Toxicity of cryoprotectant is the hot spot of cryobiology studies; therefore, the main challenge is to find the optimal concentration of cryoprotectant solution.

Cryoprotectant toxicity is probably reduced by lowering the temperature of exposure. Therefore, the recent advances in the cryopreservation of ovarian tissue were designed based on the development of ultra-rapid vitrification procedures, including several modifications, such as direct cover vitrification (DCV). Recently developed DCV method is suitable for freezing ovarian tissue with a high follicular viability (15, 16). In DCV as an ultrarapid cooling technique, vitrification (cryoprotectant) solutions have direct contact with the liquid nitrogen (N2).

Chen et al. showed that DCV could facilitate the maximum cooling rate of vitrification and prevent ice crystal injury (16). The isolated follicles from cryopreserved ovarian tissues by DCV can develop in in vitro culture (17), however ovarian tissue cryopreservation by supercool of EG and DMSO affect maturation and development of follicles (18). The data on the success of this method for ovarian tissue cryopreservation remain limited and conflicting and therefore further investigation is required.

However, it was previously reported that EG is an efficient and optimal cryoprotectant for cryopreservation of mouse ovarian tissue with a little change in the structure of ovarian follicles because of low toxicity and rapid permeation from cell membrane (19, 20). However, optimal cryoprotectant is one of the important factors in ovarian tissue vitrification, but still many of them are toxic to cells.

It has been revealed that combination of EG+DMSO with sucrose could improve follicular preservation (21, 22), whereas others have reported no added benefit on combination of cryoprotectants (EG+DMSO) (23). In contrast, Nagano et al. showed that exposure of secondary follicles to EG (2 M) causes morphological alterations in follicles (24).

Although the cryodamage may be decreased following ovarian tissue vitrification, the optimal cryoprotectant with low toxicity is important for ovarian tissue vitrification.

Despite many reports on improvement of vitrification procedure, the quality is still lower in vitrified than non-vitrified cells and tissues. There are no reports about the characteristics of follicular structure after DCV.

The aim of this study was modified direct cover vitrification of mouse ovarian tissue using different concentrations of EG and/or DMSO and its mixture. Also, histological examination, viability and ultrastructural analyses were evaluated.

Methods

Chemicals: All chemicals and media used in the study were purchased from Sigma-Aldrich Co. (Hamburg-Germany) unless otherwise indicated.

Animals and Ovarian Tissue Preparation: Five to six week-old Balb/c female mice (n=85) were housed in light and temperature controlled condition (12 hr light/12 hr dark, 22-24[degrees]C and 55% humidity). Food and water were freely available at all times. All the experiments using animals were in accordance with the International Animal Care and ethical Committee of Tabriz University of Medical Sciences.

Animal were sacrificed by cervical dislocation and their ovaries were dissected free of fat and mesentery. One ovary of each mouse was considered for vitrification procedure and the other as the intact control.

The equilibration and vitrification solution: The collected ovaries were randomly allocated to three groups: control or non-vitrified (n=8), toxicity test (n=18) and DCV (n=59). The ovaries were vitrified based on the Chen method (16). The cryoprotectant and warming solutions were prepared in Dulbecco's phosphate- buffered saline (DPBS). In this experiment, six concentrations of cryoprotectants in the equilibration and vitrification solutions were used: 5% EG in DPBS with 0.5 M sucrose and 20% FBS (group DCV1), 10% EG in DPBS with 0.5 M sucrose and 20% FBS (group DCV2), 5% DMSO in DPBS with 0.5 M sucrose and 20% FBS (group DCV3), 10% DMSO in DPBS with 0.5 M sucrose and 20% FBS (group DCV4), 5% EG+5% DMSO in DPBS with 0.5 M sucrose and 20% FBS (group DCV5), 10% EG+10% DMSO in DPBS with 0.5 M sucrose and 20% FBS (group DCV6). In DCV7 group, ovaries were equilibrated in DCV5 and DCV6 solution respectively, and then vitrified in DCV6 solution.

Vitrification and warming: Ovarian tissue was equilibrated in the DCV1-6 solution at room temperature for 8 min, and then placed in vitrification solution at room temperature for 2 min. In DCV7 group, ovarian tissue was equilibrated in DCV5 (4 min) and then DCV6 (4 min) solutions at room temperature and placed in the vitrification solution of DCV6 for 2 min.

The cryotubes were partially filled with a minimum volume of the vitrification solution. The liquid nitrogen was directly applied onto the vitrification solution (DCV), then the cap of the cryotubes was closed and they were placed into liquid nitrogen and stored for one week.

For warming, vitrified ovaries were warmed in room temperature for 10 s and then placed in 25 [degrees]C water bath for 10 s. The content of each cryotube was expelled into 1 ml of descending concentrations of sucrose (1, 0.5 and 0.25 M) and DPBS at room temperature for 10 min.

The warmed ovaries were equilibrated for 30 min in a-MEM medium supplemented with 20% fetal bovine serum (FBS) before preparing them for viability evaluation. For toxicity test, the ovaries were exposed to the cryoprotectant solution and passed through all stages of vitrification and warming procedure except plunging in liquid nitrogen.

Ovarian follicular viability: Fresh and vitrified-warmed ovaries were placed in 100 [micro]l microdroplets of medium as described earlier. Ovarian follicles in different stages were mechanically isolated from the ovaries using 29-gauge needles under a stereomicroscope (SZ-STS, Olympus, Tokyo, Japan) and were transferred to new microdroplets (20 [micro]l) of medium and covered with mineral oil.

For each group, only follicles containing layers of membrane-enclosed granulosa cells with a centrally located oocyte were examined. It was stained using 0.4% trypan blue and examined under an inverted microscope. The follicles were classified as viable or non-viable; in non-viable follicles, oocyte or the surrounding granulosa cells had blue coloration and viable ones were not stained and the oocyte and the surrounding granulosa cells were clear (Figure 1) (7).

Histological evaluation: The follicle morphology was examined by histological staining and by using light microscope (magnification x400) after vitrification and warming. Vitrified, non-vitrified and toxicity tested ovaries were embedded in paraffin wax and serially sectioned at 5 [micro]m-thickness. Then every 10th section of each ovary was mounted on glass slides, and stained with hematoxylin and eosin.

For this study, primordial follicles were defined as those containing single layer of flattened granulosa cells surrounding the oocyte (30-50 [micro]m), primary follicles as those with single layer of cuboidal granulosa cells (50-90 [micro]m), and secondary follicles as those with two or more layers of cuboidal granulosa cells and no antrum (100-150 [micro]m). For each group, only follicles with a visible nucleus in the oocyte were considered for counting to avoid duplicate counts of a follicle.

The follicles were histologically classified as normal, when they contained an intact oocyte and complete layer of granulosa cells (Figure 2A). The follicle was regarded as influenced if it had an intact oocyte with partial detachment of the oocyte from surrounding granulose cells (Figure 2B). The follicle was regarded as degenerated if it contained a cytoplasmic microvacuolation, pyknotic nucleus, shrunken ooplasm, and/or disrupted granulosa cells (Figure 2C).

Electron microscopy: All chemicals were obtained from TAAB Laboratories Ltd. (Berkshire, UK). Vitrified and non-vitrified ovaries were randomly collected (n=3 from each groups) after equilibration in medium for 30 min and fixed in 2.5% glutaraldehyde in PBS (pH=7.4) for 2 hr, and post fixed with 1% osmium tetroxide in the same buffer for 2 hr. Following dehydration in an ascending series of ethanol, specimens were placed in propylene oxide and embedded in Epon 812. The ovarian tissue was cut into 0.5 [micro]m sections (semithin sections), stained with toluidine blue and observed in light microscopy. Ultrathin sections (6080 nm) were contrasted with uranyl acetate and lead citrate for evaluation by electron microscopy (Zeiss-Gottingen-Germany).

The granulosa cells and oocyte were studied to find the integrity of the cytoplasmic membrane, the mitochondrial cristae, nuclear membrane, density of cytoplasm, cytoplasmic organelles, the numbers and size of vesicles in the cytoplasm and attachment of the cytoplasm to granulosa cells.

Statistical analysis: Quantitative variables were expressed as mean[+ or -]SD. Categorical variables were presented as values or percentages. One-way analysis of variance (ANOVA) and Tukey's test were used to compare the proportion of normal morphology or variable number of follicles for nonvitrified and vitrified ovaries. Statistical analysis was done with SPSS software 16.0. A p<0.05 was considered statistically significant.

Results

The ovarian follicles were mechanically isolated from fresh and vitrified ovaries including 638 primordial follicles (approximately 80 follicles in each group), 721 primary follicles (approximately 90 follicles in each group) and 640 secondary follicles (approximately 80 follicles in each group) were examined for their viability (Table 1). There were no significant differences in viability of primordial and primary follicles in all verified concentrations (p>0.05).

Although there were no significant differences in viability of secondary follicles of control and combination of cryoprotectants (DCV5-7 groups), in combination of cryoprotectant (EG+DMSO), a higher percentage of viable secondary follicles was observed than single cryoprotectant after cryopreservation (p<0.05).

Follicle morphology: The normality of various developmental stages of follicles in the non-vitrified and vitrified groups are presented in table 2. The percentages of normal follicles in toxicity tested groups were the same as those of vitrification tested groups and no significant differences could be found (p>0.05) (Table 3).

The number of normal primordial follicles in DCV1 and DCV3 were significantly lower than other groups (p<0.05). Primordial follicles with an oocyte surrounded by one layer of flattened or flattened cuboidal granulosa cells (Figure 3.) and granulosa cells had a high nuclear/cytoplasmic ratio and laid on a continuous basement membrane.

In total, 119 to 138 primary follicles were examined for morphological analysis in fresh and DCV groups.

The percentage of normal primary follicles observed in ovarian tissue vitrified in combination of cryoprotectants (DCV5-7) was not significant compared with control, while the percentage of normal primary follicles in single cryoprotectant groups (DCV 1-4) was lower than control group (p<0.05).

In primary follicles, the oocyte was surrounded by one layer of cuboidal granulosa cells (Figure 3A). The morphology of primary follicles was well preserved in the combination of cryoprotectants groups and the oocytes cytoplasm was clear and normal. Also, the granulosa cells were intact and firmly attached to the related basement membranes (Figures 3 G-H).

In total, 1164 secondary follicles were histologically examined in non-vitrified and vitrified groups. A very low rate of degeneration was observed in non-vitrified ovarian tissue and DCV7 groups (1.90% vs. 4.97%). The ovarian tissue exposed to DCV7 solution had significantly higher normal follicles in comparison to those exposed to DCV1-6 solutions.

Secondary follicles consisted of an oocyte surrounded by two or more layers of granulosa cells though the number of layers was very irregular (Figures 2A-C).

In this study, an attempt was made to evaluate morphologically normal follicles that they were either round or oval oocytes by presenting a well-delimited nucleus with uncondensed chromatin, surrounded by healthy granulosa cells juxtaposed to oocyte (Figure 3A). The morphology of follicles was well preserved in the DCV7 and the cytoplasm of the secondary follicles and oocytes was normal and also the granulose layers and the ca interna and externa were intact and firmly attached to the related basement membranes. The stromal cells were normal with distinct boundaries and prominent nucleus (Figure 3H).

Two types of degeneration could be distinguished. The most predominant degeneration type was characterized by the disruption of intercellular contacts among innermost granulosa cells and the oocyte.

This type of degeneration and cryoinjury was observed most often in secondary follicles in vitrified ovarian tissue in DCV1-6 groups (Figures 3B-G). However, this kind of degeneration was also observed in low level in the control and DCV7 group (Figure 3H).

The other type of degeneration was characterized by shrinkage and pyknosis of the oocyte and granulosa cells, detachment of the follicle from the surrounding stroma, cytoplasmic retraction and numerous cytoplasmic vacuoles. This kind of degeneration was observed with great frequency in secondary follicles of ovarian tissue vitrified in DCV1-6 groups (Figures 3B-G).

In all cases, there was no difference between the percentages of normal primordial, primary and secondary follicles of toxicity tests and vitrification groups (p>0.05).

Therefore, ovarian tissue frozen in combination of cryoprotectants had significantly higher percentages of normal follicles than single cryoprotectants.

Ultrastructural ovarian tissue: The ultrastructure of the ovarian tissue was well preserved in DCV7 concentration and it was very similar to the nonvitrified group. In addition, there was no evidence of subcellular alterations in ovaries vitrified in DCV7 solution. All ovarian tissues vitrified in single cryoprotectant showed a poor ultrastructure. Indeed, compared to oocyte, follicular granulosa cells showed a good ultrastructure.

Primordial follicle ultrastructure: In primordial follicles, oocytes were surrounded by 4-8 flattened granulosa cells and it had shown a homogenous cytoplasm with a large number of vesicle and centrally located nucleus.

In mixed cryoprotectants, the mitochondria were predominantly round with normal cristae and continuous membranes without electron-dense granules. The rough endoplasm reticulum cisternae and organelle was very well developed and the cellular membranes of oocyte and granulosa cells were in close connection.

In single cryoprotectants, elongated mitochondria were observed in some cases and their cristae disappeared.

Primary follicle ultrastructure: A single layer of 820 granulosa cells surrounded the oocyte of primary follicles. In the primary follicles, ooplasm remained full of vesicles and the large amount of organelle was very similar to the one observed in the primordial follicles, but elongated mitochondria were observed in some cases and gap junctions were observed between oocyte and granulosa cell membranes in primary follicles of ovarian tissue vitrified in single cryoprotectants (Figures 4A, B).

Secondary follicles ultrastructure: The oocytes were surrounded by two or three layers of cuboidal granulosa cells in secondary follicles.

The integrity of cell organelles in the secondary follicles in controls (Figure 5A) and DCV7 group (Figure 5F) was well preserved. Round mitochondria with continuous membranes and normal cristae were still more abundant, and elongated mitochondria were rarely observed (Figure 6C). Patches of ZP material were observed in all structures and were usually associated with erect microvilli (Figure 6D).

Our ultrastructural studies in single cryoprotectants groups showed that after warming of ovaries, noticeable changes occurred in the organelles of oocytes and follicular cells (Figures 5B, C and Figure 6A). In addition, extremely vacuolated oocytes with nuclear shrinkage and elongated mitochondria with a few cristae were observed as well (Figure 6A). In some cases, granulose cells had disappeared and were swollen. Moreover, they exhibited irregularly-shaped nuclei, irregular distribution of cytoplasmic organelles and vacuolation (Figures 5D, 5F and Figure 6B).

Discussion

Our data showed that direct cover vitrification of ovarian tissue using combination of cryoprotectants (stepwise concentration of EG with DMSO) had no harmful effect on the morphology and ultrastructure of ovarian tissue, where the cell structure is complex.

Many studies consider vitrification to be the best optimal cryopreservation technique for whole organ with heterogeneous and complex structure (19-25). Thus, ovarian tissue cryopreservation involves a compromise between effects on the oocyte, follicular cells and stroma. This makes it difficult to select an appropriate cryoprotectant concentration and cooling rate for ovarian tissue.

An important factor in the selection of a vitrification solution will be its toxicity and permeability into cells. EG and DMSO are the most commonly used permeable cryoprotectants in vitrification; EG has highest permeability due to lowest molecular weight compared with DMSO (62.07 g/mol vs. 78.13 g/mol) (26).

In this study, EG+DMSO was better than EG or DMSO for ovarian tissue vitrification; when ovaries equilibrated and vitrified in combination of cryoprotectants have enough penetration into cells and avoid the intracellular ice formation, then they could improve the efficiency of cryoprotectants.

A disadvantage of the vitrification procedure is toxicity of the cryoprotectant agents, which can cause cell damage. To overcome cryoprotectant toxicity, in this study, the cooling rate was decreased by direct plunging of ovarian tissue to nitrogen and exposing ovarian tissue to combination of cryoprotectants (5% to 10%). This could probably be due to sufficient permeation of the cryoprotectants into the ovarian tissue and reduction of osmotic stress; therefore, maybe it improves the success of cryopreserving follicles in different stages.

In our study, when single cryoprotectants were applied, the morphologically normal follicles reduced from 94% to 34%, which was consistent with previous reports in which mouse ovarian tissues were vitrified either in DMSO alone (3) or in a mixture of DMSO, acetamide, propylene glycol (PG) and polyethylene glycol (15, 18, 25, 27) or when ovarian tissues were vitrified in DMSO, acetamide and PG (28-30).

In this study, the importance of the presence of both non-permeating (sucrose) and permeating (EG and DMSO) cryoprotectants in a vitrification solution was shown for successful follicular preservation.

Non-permeating cryoprotectants such as sugars could reduce ice crystal formation during cryopreservation, stabilize lipid membrane and facilitate the dehydration and rehydration of cells prior to cooling (31). The previous studies approved these results. i.e., the protective effect of vitrification on the ultrastructure of follicles obtained after vitrifying/warming with EG and sucrose (21, 32).

In agreement with Choi et al., more resistance of primordial follicle to cryodamage were observed in vitrified ovarian tissues compared to other stages. Primordial follicles have small size, low number of granulose cells around the small oocyte, and there is the absence of the zona pellucida and peripheral cortical granules in them (33). In addition, the primordial follicles have more potential to repair sublethal damage to organelles during their growth phase (34). In our study, more than 90% of primordial follicles survived in all vitrification groups. Moreover, the primordial follicles were free of ultrastructural sign of damages.

The follicular changes after vitrifying-warming suggested the sensitivity of the secondary follicles to cryoprotectant solutions relative to that of the primordial and primary follicles. This can be explained by the increased complexity of the growing follicle and gap junctional communication, which makes them more sensitive to cryodamage (35). This hypothesis was in agreement with results of Luz et al that demonstrated the secondary follicle has lower viability and normal morphology than other follicular development stages (36).

Our ultrastructural studies showed that after vitrifying-warming of ovaries in DCV7 group, no noticeable changes occurred in the organelles of oocytes and follicular cells and the integrity of cell organelles was well preserved. This method may be an efficient alternative for cryopreservation of mammalian ovarian tissue and can be used to improve fertility among cancer patients.

The mitochondrial organization and morphology and cytoskeletal network is a necessary key factor for cryopreservation assessment (37, 38). The primary function of mitochondria is to generate ATP, necessary for cell survival and cell function. The movement of mitochondria within different areas of the cell is mediated by cytoskeletal network of microtubules (41). Our results suggested that the mitochondrial damages during ovarian tissue vitrification might be involved in the reduced viability of ovarian follicles. Moreover, mitochondria played an important role in [Ca.sup.2+] signaling that mediated oocyte activation and development, and also apoptosis (39). As was mentioned before, the ultrastructural alternation in mitochondria in mixed cryoprotectants group, especially DCV7 group was lower than the single cryoprotectants groups. These results suggested that the follicles from combination of cryoprotectants group have developmen tal potential in comparison to single cryoprotectants groups (Figures 5A-D).

Conclusion

In conclusion, following exposure of ovarian tissue to sucrose, DMSO, EG in alone or a mixture of these cryoprotectants by using DCV, a combination of EG and DMSO and sucrose appeared to be the best cryoprotectant for the preservation of follicular morphology and viability.

Acknowledgement

We would like to thank Ayda Azami for technical assistance in electron microscopy. This work was supported by a grant from Tabriz University of Medical Sciences.

Conflict of Interest

The authors declare no conflict of interest.

References

(1.) Kuleshova LL, Lopata A. Vitrification can be more favorable than slow cooling. Fertil Steril. 2002;78 (3):449-54.

(2.) Courbiere B, Odagescu V, Baudot A, Massardier J, Mazoyer C, Salle B, et al. Cryopreservation of the ovary by vitrification as an alternative to slowcooling protocols. Fertil Steril. 2006;86(4 Suppl): 1243-51.

(3.) Li YB, Zhou CQ, Yang GF, Wang Q, Dong Y. Modified vitrification method for cryopreservation of human ovarian tissues. Chin Med J. 2007; 120 (2):110-4.

(4.) Hani T, Tachibe T, Shingai S, Kamada N, Ueda O, Jishage K. Fertility of mice receiving vitrified adult mouse ovaries. Reproduction. 2006;131(4):681-7.

(5.) Hasegawa A, Mochida N, Ogasawara T, Koyama K. Pup birth from mouse oocytes in preantral follicles derived from vitrified and warmed ovaries followed by in vitro growth, in vitro maturation, and in vitro fertilization. Fertil Steril. 2006;86(4 Suppl):1182-92.

(6.) Newton H, Illingworth P. In-vitro growth of murine pre-antral follicles after isolation from cryopreserved ovarian tissue. Hum Reprod. 2001;16(3):423-9.

(7.) Xu M, Banc A, Woodruff TK, Shea LD. Secondary follicle growth and oocyte maturation by culture in alginate hydrogel following cryopreservation of the ovary or individual follicles. Biotechnol Bioeng. 2009;103(2):378-86.

(8.) Abdollahi M, Salehnia M, Salehpour S, Ghorbanmehr N. Human ovarian tissue vitrification/warming has minor effect on the expression of apoptosisrelated genes. Iran Biomed J. 2013;17(4):179-86.

(9.) Salehnia M, Abbasian Moghadam E, Rezazadeh Velojerdi M. Ultrastructure of follicles after vitrification of mouse ovarian tissue. Fertil Steril. 2002; 78(3):644-5.

(10.) Choi WJ, Yeo HJ, Shin JK, Lee SA, Lee JH, Paik WY. Effect of vitrification method on survivability, follicular growth and ovulation of preantral follicles in mice. J Obstet Gynaecol Res. 2007;33(2): 128-33.

(11.) Boonkusol D, Faisaikarm T, Dinnyes A, Kitiyanant Y. Effects of vitrification procedures on subsequent development and ultrastructure of in vitromatured swamp buffalo (Bubalus bubalis) oocytes. Reprod Fertil Dev. 2007;19(2):383-91.

(12.) Vajta G, Holm P, Kuwayama M, Booth PJ, Jacobsen H, Greve T, et al. Open Pulled Straw (OPS) vitrification: a new way to reduce cryoinjuries of bovine ova and embryos. Mol Reprod Dev. 1998; 51(1):53-8.

(13.) Wani NA, Maurya SN, Misra AK, Saxena VB, Lakhchaura BD. Effect of cryoprotectants and their concentration on in vitro development of vitrifiedwarmed immature oocytes in buffalo (Bubalus bubalis). Theriogenology. 2004;61(5):831-42.

(14.) Schiewe MC, Rall WF, Stuart LD, Wildt DE. Analysis of cryoprotectant, cooling rate and in situ dilution using conventional freezing or vitrification for cryopreserving sheep embryos. Theriogenology. 1991;36(2):279-93.

(15.) Isachenko E, Isachenko V, Rahimi G, Nawroth F. Cryopreservation of human ovarian tissue by direct plunging into liquid nitrogen. Eur J Obstet Gynecol Reprod Biol. 2003;108(2):186-93.

(16.) Chen SU, Chien CL, Wu MY, Chen TH, Lai SM, Lin CW, et al. Novel direct cover vitrification for cryopreservation of ovarian tissues increases follicle viability and pregnancy capability in mice. Hum Reprod. 2006;21(11):2794-800.

(17.) Huang LS, Tan SQ, Qu QH. [Morphology and celluar proliferation of follicles from cryopreserved human ovarian tissues]. Sichuan Da Xue Xue Bao Yi Xue Ban. 2010;41(2):261-4. Chinese.

(18.) Tan X, Song E, Liu X, Liu G, Cheng H, Wan F. Successful vitrification of mouse ovaries using less-concentrated cryoprotectants with Supercool X-1000 supplementation. In Vitro Cell Dev Biol Anim. 2012;48(2):69-74.

(19.) Abedelahi A, Salehnia M, Abdolamir A, Hajizadeh E. Comparison of ultrastructure and morphology of mouse ovarian follicles after conventional and direct cover vitrification using different concentrations of ethylene glycol. Iran J Reprod Med. 2009; 7(2):45-52.

(20.) Abedelahi A, Salehnia M, Allameh AA, Davoodi D. Sodium selenite improves the in vitro follicular development by reducing the reactive oxygen species level and increasing the total antioxidant capacity and glutathione peroxide activity. Hum Reprod. 2010;25(4):977-85.

(21.) Fathi R, Valojerdi MR, Salehnia M. Effects of different cryoprotectant combinations on primordial follicle survivability and apoptosis incidence after vitrification of whole rat ovary. Cryo Letters. 2013; 34(3):228-38.

(22.) Sheikhi M, Hultenby K, Niklasson B, Lundqvist M, Hovatta O. Preservation of human ovarian follicles within tissue frozen by vitrification in a xeno-free closed system using only ethylene glycol as a permeating cryoprotectant. Fertil Steril. 2013; 100(1):170-7.

(23.) Faheem MS, Carvalhais I, Baron E, Moreira da Silva F. Effect of bovine ovarian tissue vitrification on the structural preservation of antral follicles. Reprod Domest Anim. 2013;48(5):774-80.

(24.) Nagano M, Atabay EP, Atabay EC, Hishinuma M, Katagiri S, Takahashi Y. Effects of isolation method and pre-treatment with ethylene glycol or raffinose before vitrification on in vitro viability of mouse preantral follicles. Biomed Res. 2007;28(3): 153-60.

(25.) Isachenko V, Isachenko E, Rahimi G, Krivokharchenko A, Alabart JL, Nawroth F. Cryopreservation of human ovarian tissue by direct plunging into liquid nitrogen: negative effect of disaccharides in vitrification solution. Cryo Letters. 2002;23(5): 333-44.

(26.) Chi HJ, Koo JJ, Kim MY, Joo JY, Chang SS, Chung KS. Cryopreservation of human embryos using ethylene glycol in controlled slow freezing. Hum Reprod. 2002;17(8):2146-51.

(27.) Rodrigues AP, Amorim CA, Costa SH, Matos MH, Santos RR, Lucci CM, et al. Cryopreservation of caprine ovarian tissue using glycerol and ethylene glycol. Theriogenology. 2004;61(6):1009-24.

(28.) Santos RR, Rodrigues AP, Costa SH, Silva JR, Matos MH, Lucci CM, et al. Histological and ultrastructural analysis of cryopreserved sheep preantral follicles. Anim Reprod Sci. 2006;91(3-4):249-63.

(29.) Celestino JJ, dos Santos RR, Lopes CA, Martins FS, Matos MH, Melo MA, et al. Preservation of bovine preantral follicle viability and ultrastructure after cooling and freezing of ovarian tissue. Anim Reprod Sci. 2008;108(3-4):309-18.

(30.) Yamada C, Caetano HV, Simoes R, Nicacio AC, Feitosa WB, Assumpcao ME, et al. Immature bovine oocyte cryopreservation: comparison of different associations with ethylene glycol, glycerol and dimethylsulfoxide. Anim Reprod Sci. 2007;99 (3-4):384-8.

(31.) Begin I, Bhatia B, Baldassarre H, Dinnyes A, Keefer CL. Cryopreservation of goat oocytes and in vivo derived 2- to 4-cell embryos using the cryoloop (CLV) and solid-surface vitrification (SSV) methods. Theriogenology. 2003;59(8):1839-50.

(32.) Abedelahi A, Salehnia M, Allameh AA. The effects of different concentrations of sodium selenite on the in vitro maturation of preantral follicles in serum-free and serum supplemented media. J Assist Reprod Genet. 2008;25(9-10):483-8.

(33.) Choi J, Lee JY, Lee E, Yoon BK, Bae D, Choi D. Cryopreservation of the mouse ovary inhibits the onset of primordial follicle development. Cryobiology. 2007;54(1):55-62.

(34.) Kim SS, Yang HW, Kang HG, Lee HH, Lee HC, Ko DS, et al. Quantitative assessment of ischemic tissue damage in ovarian cortical tissue with or without antioxidant (ascorbic acid) treatment. Fertil Steril. 2004;82(3):679-85.

(35.) Cecconi S, Capacchietti G, Russo V, Berardinelli P, Mattioli M, Barboni B. In vitro growth of preantral follicles isolated from cryopreserved ovine ovarian tissue. Biol Reprod. 2004;70(1):12-7.

(36.) Luz VB, Santos RR, Pinto LC, Soares AA, Celestino JJ, Mafezoli J, et al. Dimethyl sulfoxide perfusion in caprine ovarian tissue and its relationship with follicular viability after cryopreservation. Fertil Steril. 2009;91(4 Suppl):1513-5.

(37.) Rho GJ, Kim S, Yoo JG, Balasubramanian S, Lee HJ, Choe SY. Microtubulin configuration and mitochondrial distribution after ultra-rapid cooling of bovine oocytes. Mol Reprod Dev. 2002;63(4):46470.

(38.) Van Blerkom J. Microtubule mediation of cytoplasmic and nuclear maturation during the early stages of resumed meiosis in cultured mouse oocytes. Proc Natl Acad Sci U S A. 1991;88(11): 5031-5.

(39.) Liu L, Hammar K, Smith PJ, Inoue S, Keefe DL. Mitochondrial modulation of calcium signaling at the initiation of development. Cell Calcium. 2001; 30(6):423-33.

Maryam Ghavami [1], Daryoush Mohammadnejad [2], Rahim Beheshti [3], Jafar Solmani-rad [1], Ali Abedelahi [1] *

[1]- Department of Anatomical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran

[2]- Drug Applied Research Center, Tabriz University of Medical Science, Tabriz, Iran

[3]- Department of Veterinary, Shabestar Branch, Islamic Azad University, Shabestar, Iran

* Corresponding Author: Ali Abedelahi, Department of Anatomical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran

E-mail: abedelahia@yahoo.com

Received: Oct. 20, 2014

Accepted: Nov. 23, 2014

Table 1. Number and percentage of intact (Int) and degenerated
(Deg) follicles of various stages isolated from vitrified and
non-vitrified ovarian tissue after Trypan blue staining

Group     Total number   Primordial follicles (%)
          of follicles
                         Total    Int      Deg

Control       247         84     82(98)   2(2)
DCV1          248         77     70(89)   7(11)
DCV2          276         86     80(92)   6(8)
DCV3          268         88     80(90)   8(10)
DCV4          238         74     67(92)   7(8)
DCV5          224         80     75(94)   5(6)
DCV6          237         74     70(94)   4(6)
DCV7          261         75     72(96)   3(4)

Group       Primary follicles (%)      Secondary follicles (%)

          Total    Int     Deg    Total    Int         Deg

Control    82     80(97)   2(3)    81     78(97)       3(3)
DCV1       91     83(91)   8(9)    80     68(85)   12(15) (a,b)
DCV2       97     89(92)   8(8)    93     81(86)    12(14) (a)
DCV3       95     87(91)   8(9)    85     73(85)   12(15) (a,b)
DCV4       91     83(92)   8(8)    73     61(84)   12(16) (a,b)
DCV5       88     83(95)   5(5)    56     50(88)      6(12)
DCV6       87     83(95)   4(5)    76     69(90)      7(10)
DCV7       90     87(97)   3(3)    96     92(96)       4(4)

a: p < 0.05 versus non-vitrified control group; b: p < 0.05
versus vitrified tissue in DCV7 group DCV= Direct Cover
Vitrification

Table 2. Number and percentage of intact (Int), influenced (Inf)
and degenerated (Deg) follicles of various stages evaluated in
vitrified and non-vitrified ovarian tissue after Hematoxylin-
eosin staining

Group       Total                  Primordial follicles (%)
          number of
          follicles
                      Total        Int         Inf      Deg     Total

Control      532       255       253(99)      1(0.5)   1(0.5)    133
DCV1         574       280    265(95) (a,b)    8(3)     7(2)     124
DCV2         541       235       227(96)       4(2)     4(2)     152
DCV3         494       262    245(94) (a,b)    9(3)     8(3)     119
DCV4         542       255       246(96)       6(2)     3(2)     126
DCV5         519       302       290(96)       9(3)     3(1)     101
DCV6         632       354       343(97)       8(2)     3(1)     138
DCV7         758       470       460(98)       7(1)     3(1)     122

Group               Primary follicles (%)

               Int         Inf    Deg    Total

Control      129(97)      2(2)    2(1)    144
DCV1      108(86) (a,b)   8(7)    8(7)    170
DCV2      134(88) (a,b)   10(7)   8(5     154
DCV3      104(87) (a,b)   10(9)   5(4)    113
DCV4      190(86) (a,b)   10(8)   7(6)    161
DCV5         92(91)       4(4)    5(5)    116
DCV6         128(93)      6(4)    4(3)    140
DCV7         115(94)      4(4)    3(2)    166

Group          Secondary follicles (%)

              Int         Inf      Deg

Control     136(94)       5(4)     3(2)
DCV1      53(36) (a,b)   81(44)   36(20)
DCV2      60(39) (a,b)   43(28)   51(33)
DCV3      27(23) (a,b)   53(48)   33(29)
DCV4      53(38) (a,b)   80(46)   28(16)
DCV5      49(42) (a,b)   48(42)   19(16)
DCV6      62(45) (a,b)   46(33)   32(22)
DCV7        144(86)      14(9)     8(5)

(a): p < 0.05 versus non-vitrified control group; (b): p < 0.05
versus vitrified tissue in DCV7 group DCV= Direct Cover
Vitrification

Table 3. The percentage of intact (Int), influenced (Inf) and
degenerated (Deg) follicles at different developmental stages
evaluated in toxicity tested ovaries

Group   Total number        Primordial follicles (%)
        of follicles
                       Total     Int     Inf    Deg

DCV1        357         200    192(96)   6(3)   2(1)
DCV2        356         190    182(96)   4(2)   4(2)
DCV3        385         205    194(95)   6(3)   4(2)
DCV4        355         186    178(96)   5(3)   3(1)
DCV5        364         173    166(96)   5(3)   2(1)
DCV6        375         220    213(97)   4(2)   1(1)
DCV7        385         219    210(98)   7(3)   2(1)

Group          Primary follicles (%)        Preantral follicles (%)

        Total    Int     Inf    Deg    Total    Int     Inf    Deg

DCV1     88     82(94)   3(3)   3(3)    69     65(95)   3(4)   1(2)
DCV2     93     86(93)   4(4)   3(3)    73     70(96)   2(3)   1(1)
DCV3     100    95(95)   4(4)   1(1)    80     75(94)   2(2)   3(4)
DCV4     78     73(94)   4(5)   1(1)    91     86(95)   1(1)   4(4)
DCV5     103    98(95)   3(3)   2(2)    88     84(96)   2(2)   2(2)
DCV6     90     87(97)   2(2)   1(1)    65     63(97)   1(2)   1(1)
DCV7     95     91(96)   4(4)   0(0)    75     72(97)   2(2)   1(1)

DCV= Direct Cover Vitrification
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Title Annotation:Original Article
Author:Ghavami, Maryam; Mohammadnejad, Daryoush; Beheshti, Rahim; Solmani-rad, Jafar; Abedelahi, Ali
Publication:Journal of Reproduction and Infertility
Article Type:Report
Date:Jul 1, 2015
Words:5552
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