Follicular development and the expression of BAX and vascular endothelial growth factor in transplanted ovaries in uni- and bilateral ovariectomized mice: An experimental study


Background: Several conflicting results have been reported on the survival and function of transplanted ovaries.

Objective: Evaluation of the follicular development and the expression of vascular endothelial growth factor (VEGF) and Bcl-2-associated X protein (BAX) in ovaries transplanted into uni- and bilaterally ovariectomized mice.

Materials and Methods: In this experimental study, 40 female NMRI mice (21-days-old, 12-15 gr) were ovariectomized uni- and bilaterally (n = 20/ group), while the 8-wk-old mice were considered as intact control group (n = 6). 5 weeks after transplantation at the proestrus stage, the morphology of recovered transplanted ovaries and the proportion of follicles were studied at different developmental stages. The apoptosis cell death by pro-apoptotic protein BAX and the expression of VEGF were evaluated using immunohistochemistry.

Results: In the bilaterally ovariectomized mice, among the 455 counted normal follicles, a lower rate of primordial and primary follicles and a higher rate of preantral and antral follicles were observed (p = 0.002). However, the percentages of preantral and antral follicles, and the corpus luteum were significantly lower in the intact control group (among the 508 counted normal follicles in this group) compared to other transplanted groups (p = 0.002). The number of BAX-positive cells in all groups was not significantly different. The VEGF expression was prominent in vessels of the corpus luteum, and also in the theca layer of large follicles of studied groups.

Conclusion: Early discharge of ovarian reserve was prominent in the bilaterally ovariectomized group but the incidence of apoptotic cells and VEGF expression as angiogenic factor did not differ in both ovariectomized mice. Thus, unilaterally ovariectomy has less side effects on the ovarian reserve compared to bilateral ovariectomy.

Key words: Autotransplantation, BAX protein, Vascular endothelial growth factor, Ovariectomy, Mice.

[1] Soleimani R, Heytens E, Oktay K. Enhancement of neoangiogenesis and follicle survival by sphingosine-1- phosphate in human ovarian tissue xenotransplants. PLoS One 2011; 6: e19475.

[2] Gellert SE, Pors SE, Kristensen SG, Bay-Bjørn AM, Ernst E, Andersen CY. Transplantation of frozen-thawed ovarian tissue: An update on worldwide activity published in peer-reviewed papers and on the Danish cohort. J Assist Reprod Genet 2018; 35: 561–570.

[3] Kim S, Lee Y, Lee S, Kim T. Ovarian tissue cryopreservation and transplantation in patients with cancer. Obstet Gynecol Sci 2018; 61: 431–442.

[4] Marin L, Bedoschi G, Kawahara T, Oktay KH. History, evolution and current state of ovarian tissue autotransplantation with cryopreserved tissue: A successful translational research journey from 1999 to 2020. Reprod Sci 2020; 27: 955–962.

[5] Xie S, Zhang X, Chen W, Xie C, Chen W, Cheng P, et al. Developmental status: Impact of short-term ischemia on follicular survival of whole ovarian transplantation in a rabbit model. PloS One 2015; 10: e0135049.

[6] Da Costa MM, Gonçalves LP, Lemos MS, Marangon ARM, Lucci CM. Investigation on revascularization time and initial damage after transplantation of fresh and cryopreserved ovarian tissue in domestic cats. Cell Tissue Bank 2020; 21: 303–312.

[7] Gavish Z, Spector I, Peer G, Schlatt S, Wistuba J, Roness H, et al. Follicle activation is a significant and immediate cause of follicle loss after ovarian tissue transplantation. J Assist Reprod Genet 2018; 35: 61–69.

[8] Soleimani R, heytens E, Van den Broecke R, Rottiers I, Dhont M, Cuvelier CA, et al. Xenotransplantation of cryopreserved human ovarian tissue into murine back muscle. Hum Reprod 2010; 25: 1458–1470.

[9] Cho IA, Lee YJ, Lee HJ, Choi IY, Shin JK, Lee SA, et al. Angiopoietin-1 and-2 and vascular endothelial growth factor expression in ovarian grafts after cryopreservation using two methods. Clin Exp Reprod Med 2018; 45: 143– 148.

[10] Labied S, Delforge Y, Munaut C, Blacher S, Colige A, Delcombel R, et al. Isoform 111 of vascular endothelial growth factor (VEGF111) improves angiogenesis of ovarian tissue xenotransplantation. Transplantation 2013; 95: 426–433.

[11] Zand-Vakili M, Golkar-Narenji A, Mozdziak PE, Eimani H. An in vitro study on oocyte and follicles of transplanted ovaries treated with vascular endothelial growth factor. J Turk Ger Gynecol Assoc 2017; 18: 167–173.

[12] Shultz LD, Goodwin N, Ishikawa F, Hosur V, Lyons BL, Greiner DL. Subcapsular transplantation of tissue in the kidney. Cold Spring Harb Protoc 2014; 2014: 737–740.

[13] Gao J, Huang Y, Li M, Zhao H, Zhao Y, Li R, et al. Effect of local basic fibroblast growth factor and vascular endothelial growth factor on subcutaneously allotransplanted ovarian tissue in ovariectomized mice. PLoS One 2015; 10: e0134035.

[14] Bagis H, Akkoç T, Tasş A, Aktoprakligil D. Cryogenic effect of antifreeze protein on transgenic mouse ovaries and the production of live offspring by orthotopic transplantation of cryopreserved mouse ovaries. Mol Reprod Dev 2008; 75: 608–613.

[15] Morales-Ledesma L, Ramírez DA, Vieyra E, Trujillo A, Chavira R, Cárdenas M, et al. Effects of acute unilateral ovariectomy to pre-pubertal rats on steroid hormones secretion and compensatory ovarian responses. Reprod Biol Endocrinol 2011; 9: 41.

[16] Mason JB, Parkinson KC, Habermehl TL. Orthotopic ovarian transplantation procedures to investigate the life and health-span influence of ovarian senescence in female mice. J Vis Exp 2018; 132: 56638.

[17] Albamonte MI, Albamonte MS, Bou-Khair RM, Zuccardi L, Vitullo AD. The ovarian germinal reserve and apoptosis-related proteins in the infant and adolescent human ovary. J Ovarian Res 2019; 12: 22.

[18] Slot KA, Voorendt M, De Boer-Brouwer M, Van Vugt HH, Teerds KJ. Estrous cycle dependent changes in expression and distribution of Fas, Fas ligand, Bcl-2, Bax, and pro- and active caspase-3 in the rat ovary. J Endocrinol 2006; 188: 179–192.

[19] Jafarabadi M, Abdollahi M, Salehnia M. Assessment of vitrification outcome by xenotransplantation of ovarian cortex pieces in γ-irradiated mice: Morphological and molecular analyses of apoptosis. J Assist Reprod Genet 2015; 32: 195–205.

[20] Tavana S, Azarnia M, Rezazadeh Valojerdi M, Shahverdi A. Hyaluronic acid-based hydrogel scaffold without angiogenic growth factors enhances ovarian tissue function after autotransplantation in rats. Biomed Mater 2016; 11: 055006.

[21] Tamadon A, Raayat Jahromi A, Rahmanifar F, Ayaseh M, Koohi-Hosseinabadi O, Moghiminasr R. Histomorphometric evaluation of superovulation effect on follicular development after autologous ovarian transplantation in mice. Vet Med Int 2015; 2015: 236436.

[22] Liu J, Van der Elst J, Van den Broecke R, Dhont M. Early massive follicle loss and apoptosis in heterotopically grafted newborn mouse ovaries. Hum Reprod 2002; 17: 605–611.

[23] Amorim CA, Dolmans MM, David A, Jaeger J, Vanacker J, Camboni A, et al. Vitrification and xenografting of human ovarian tissue. Fertil Steril 2012; 98: 1291–1298.

[24] Khalili MA, Dehghan M, Nazari S, Agha-Rahimi A. Assessment of ovarian tissues autografted to various body sites followed by IVM in mouse. Iran J Reprod Med 2014; 12: 199–204.

[25] Byers SL, Wiles MV, Dunn SL, Taft RA. Mouse estrous cycle identification tool and images. PLoS One 2012; 7: e35538.

[26] Albamonte MI, Albamonte MS, Stella I, Zuccardi L, Vitullo AD. The infant and pubertal human ovary: Balbiani’s body-associated VASA expression, immunohistochemical detection of apoptosis-related BCL2 and BAX proteins, and DNA fragmentation. Hum Reprod 2013; 28: 698–706.

[27] Bhattacharya K. Ovulation and rate of implantation following unilateral ovariectomy in mice. J Hum Reprod Sci 2013; 6: 45–48.

[28] Ma WZ, Zheng XM, Hei CC, Zhao CJ, Xie SS, Chang Q, et al. Optimal FSH usage in revascularization of allotransplanted ovarian tissue in mice. J Ovarian Res 2017; 10: 5.

[29] Heidegger H, Jeschke U. Human chorionic gonadotropin (hCG)-an endocrine, regulator of gestation and cancer. Int J Mol Sci 2018; 19: 1502.