The Effects of Ovarian Encapsulation on Morphology and Expression of Apoptosis-Related Genes in Vitrified Mouse Ovary

Shirazi Tehrani, Atefeh; Mazoochi, Tahereh; Akhavan Taheri, Maryam; Aghadavood, Esmat; Salehnia, Mojdeh

doi:10.18502/jri.v22i1.4992

The Effects of Ovarian Encapsulation on Morphology and Expression of Apoptosis-Related Genes in Vitrified Mouse Ovary

Authors

Atefeh Shirazi Tehrani ¹

Tahereh Mazoochi ¹

Maryam Akhavan Taheri ²

Esmat Aghadavood ³

Mojdeh Salehnia ⁴

¹ Gametogenesis Research Center, Kashan University of Medical Sciences, Kashan, Iran

² Anatomical Sciences Research Center, Kashan University of Medical Sciences, Kashan, Iran

³ Department of Biochemistry, Faculty of Medicine, Kashan University of Medical Sciences, Kashan, Iran

⁴ Department of Anatomy, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran

10.18502/jri.v22i1.4992

Abstract

Background: The purpose of this study was to determine the effects of alginate hydrogel as a capsule to protect the ovary against possible detrimental effects of vitrification and warming on morphology and expression of apoptosis-related genes in the mouse ovary. Methods: In this experimental study, the ovaries from twenty-five female 8-week-old mice were divided into five groups of non-vitrified ovaries, vitrified ovaries, ovaries that were encapsulated with concentrations of 0.5, 0.75 and 1% of alginate hydrogel. The morphological study was performed using hematoxylin and eosin staining. Expression levels of apoptosis-associated genes were quantified in each group by real-time RT-PCR. The one-way ANOVA and post hoc test were used to analyze the data and values of p<0.05 were considered statistically significant. Results: The results of follicle count showed that the mean of total follicles in all groups was not significantly different. The average number of atretic follicles in vitrified and experimental groups significantly increased in comparison with the non-vitrified group (p=0.001). The results of the evaluation of apoptosis-related genes showed that the ratio of Bax/Bcl-2 in experimental groups 1 and 2 was significantly higher than the vitrified group and experimental group 3 (p=0.000). The expression level of caspase 3 gene was not significantly different among all groups. Conclusion: Ovarian encapsulation with used concentrations of alginate hydrogel failed to improve the morphology and molecular aspects of follicles and it was not able to better preserve the intact follicles of vitrified ovaries. However, morphological and molecular findings appear to improve with increasing alginate hydrogel concentration.

Keywords

Alginate Hydrogel

Mouse

Ovary

Vitrification

Dittrich R, Maltaris T, Hoffmann I, Oppelt PG, Beckmann MW, Mueller A. Fertility preservation in cancer patients. Minerva Ginecol. 2010;62(1):63-80.

Fathi R, Rezazadeh Valojerdi M, Salehnia M, Ebrahimi B, SalmanYazdi R. Ovarian tissue transplantation: advantages, disadvantages and upcoming challenges (A review article). J Mazandaran Univ Med Sci. 2014;24(113):253-65.

Donnez J. Introduction: fertility preservation, from cancer to benign disease to social reasons: the challenge of the present decade. Fertil Steril. 2013;99(6):1467-8.

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

Zhou XH, Wu YJ, Shi J, Xia YX, Zheng SS. Cryopreservation of human ovarian tissue: comparison of novel direct cover vitrification and conventional vitrification. Cryobiology. 2010;60(2):101-5.

Choi J, Lee B, Lee E, Yoon BK, Bae D, Choi D. Cryopreservation of ovarian tissues temporarily suppresses the proliferation of granulosa cells in mouse preantral follicles. Cryobiology. 2008;56(1):36-42.

Gandolfi F, Paffoni A, Brambilla EP, Bonetti S, Brevini TA, Ragni G. Efficiency of equilibrium cooling and vitrification procedures for the cryopreservation of ovarian tissue: comparative analysis between human and animal models. Fertil Steril. 2006;85 Suppl 1:1150-6.

Keros V, Xella S, Hultenby K, Pettersson K, Sheikhi M, Volpe A, et al. Vitrification versus controlled-rate freezing in cryopreservation of human ovarian tissue. Hum Reprod. 2009;24(7):1670-83.

Lee D, Ouhibi N, Battaglia D. Cryopreservation of ovarian tissue: banking reproductive potential for the future. Curr womens Health Rep. 2001;1(2):152-6.

Mazoochi T, Salehnia M, Valojerdi MR, Mowla SJ. Morphologic, ultrastructural, and biochemical identification of apoptosis in vitrified-warmed mouse ovarian tissue. Fertil Steril. 2008;90(4 Suppl):1480-6.

Mazoochi T, Khamechian T, Ehteram M, Kashani HH. The effect of melatonin on expression of p53 and ovarian preantral follicle development isolated from vitrified ovary. Comp Clin Path. 2018;27(1):83-8.

Posillico S, Kader A, Falcone T, Agarwal A. Ovarian tissue vitrification: Modalities, challenges and potentials. Curr Womens Health Rev. 2010;6(4):352-66.

Huang H, Choi JK, Rao W, Zhao S, Agarwal P, Zhao G, et al. Alginate hydrogel microencapsulation inhibits devitrification and enables large‐volume low‐CPA cell vitrification. Adv Funct Mater. 2015;25(44):6839-50.

Abdi S, Salehnia M, Hosseinkhani S. Comparison of Survival and Developmental rates of Mouse Ovarian Follicles after Two- and Three-Dimensional Cultures. Pathobiol Res. 2013;16(2):51-63.

Brito IR, Lima IM, Xu M, Shea LD, Woodruff TK, Figueiredo JR. Three-dimensional systems for in vitro follicular culture: overview of alginate-based matrices. Reprod Fertil Dev. 2014;26(7):915-30.

Shikanov A, Zhang Z, Xu M, Smith RM, Rajan A, Woodruff TK, et al. Fibrin encapsulation and vascular endothelial growth factor delivery promotes ovarian graft survival in mice. Tissue Eng Part A. 2011;17(23-24):3095-104.

Akhavan Taheri M, Rezazadeh Valojerdi M, Ebrahimi B. Intramuscular autotransplantation of vitrified rat ovary encapsulated with hyaluronic acid hydrogel. Biopreserv Biobank. 2016;14(2):114-21.

Gao JM, Yan J, Li R, Li M, Yan LY, Wang TR, et al. Improvement in the quality of heterotopic allotransplanted mouse ovarian tissues with basic fibroblast growth factor and fibrin hydrogel. Hum Reprod. 2013;28(10):2784-93.

Caligioni CS. Assessing reproductive status/stages in mice. Curr Protoc Neurosci. 2009;48(1):A-4I.

Mofarahe ZS, Novin MG, Jafarabadi M, Salehnia M, Noroozian M, Ghorbanmehr N. Effect of human ovarian tissue vitrification/warming on the expression of genes related to folliculogenesis. Iran Biomed J. 2015;19(4):220-5.

Ramezani M, Salehnia M, Jafarabadi M. Short term culture of vitrified human ovarian cortical tissue to assess the cryopreservation outcome: Molecular and morphological analysis. J Reprod Infertil. 2017;18(1):162-71.

Xu M, West E, Shea LD, Woodruff TK. Identification of a stage-specific permissive in vitro culture environment for follicle growth and oocyte development. Biol Reprod. 2006;75(6):916-23.

Hornick JE, Duncan FE, Shea LD, Woodruff TK. Isolated primate primordial follicles require a rigid physical environment to survive and grow in vitro. Hum Reprod. 2012;27(6):1801-10.

Sadeghnia S, Akhondi MM, Hossein G, Mobini S, Hosseini L, Naderi MM, et al. Development of sheep primordial follicles encapsulated in alginate or in ovarian tissue in fresh and vitrified samples. Cryobiology. 2016;72(2):100-5.

Camboni A, Van Langendonckt A, Donnez J, Vanacker J, Dolmans MM, Amorim CA. Alginate beads as a tool to handle, cryopreserve and culture isolated human primordial/primary follicles. Cryobiology. 2013;67(1):64-9.

O'Brien MJ, Pendola JK, Eppig JJ. A revised protocol for in vitro development of mouse oocytes from primordial follicles dramatically improves their developmental competence. Biol Reprod. 2003;68(5):1682-6.