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Dondurulmuş ve çözülmüş ovaryum dokusunda mTOR ve p-mTOR ekspresyonunun immünohistokimyasal olarak gösterilmesi

Yıl 2026, Cilt: 19 Sayı: 1, 27 - 35, 16.01.2026
https://doi.org/10.31362/patd.1709595
https://izlik.org/JA75PX55UH

Öz

Amaç: Ovarian kriyoprezervasyon, doğurganlığı korumak için tercih edilen toksik etkileri en aza indiren oldukça faydalı bir yöntemdir. Mammalian target of rapamycin (mTOR) yolağı hücre büyümesi, çoğalması, otofaji, besin sinyalizasyonu ve hayatta kalması için kritik öneme sahiptir. Amacımız immünohistokimyasal yöntemle overin dondurulmasından önce ve sonra mTOR ve Fosforile (p-mTOR) ekspresyonlarını araştırmaktır.
Gereç ve yöntem: Over örnekleri iki gruba ayrıldı — kontrol grubu (C; n=6) ve vitrifikasyon–çözdürme grubu (V/T; n=6). C grubu tamponlu formalin içinde 48 saat fikse edildi. V/T grubu -196ºC'de sıvı nitrojen içinde donduruldu. Bir hafta sonra dokular çözüldü. Her iki grupta da parafin takibi yapıldı. Ovaryumların Hematoksilen ve Eozin ile boyanmasını takiben folikül sayımı yapıldı. mTOR ve p-mTOR ekspresyonu için immünohistokimya incelemesi yapıldı.
Bulgular: Folikül sayıları gruplar arasında istatistiksel olarak farklı değildi. C ve V/T grubu overlerdeki foliküllerde güçlü mTOR ekspresyonu gözlendi. Kontrol grubunda oositlerde ve granüloza hücrelerinde pozitif p-mTOR ekspresyonu gözlenirken, V/T grubunda ekspresyon negatifti.
Sonuç: Kriyoprezervasyon yöntemleri over dokusunda mTOR ekspresyonunu değiştirmemiştir. Bununla birlikte, dondurulmuş ovaryum dokularında oosit kalitesi ile ilişkili p-mTOR aktivitesinin olmaması, kriyoprezervasyonun oosit kalitesini etkilediğini düşündürmüştür.

Etik Beyan

Pamukkale Üniversitesi Deney Hayvanları Etik Kurulu (Onay numarası: 2016/24).

Destekleyen Kurum

Pamukkale Üniversitesi Bilimsel Araştırma Projeleri Koordinasyon Birimi (2017SABE007)

Proje Numarası

Pamukkale University Scientific Research Projects Coordination Unit (grant number 2017SABE007)

Kaynakça

  • Donnez J, Dolmans MM. Ovarian cortex transplantation: 60 reported live births brings the success and worldwide expansion of the technique towards routine clinical practice. J Assist Reprod Genet. 2015;32(8):1167-1170. doi:10.1007/s10815-015-0544-9
  • Liu W, Zhang J, Wang L. et al. The protective effects of rapamycin pretreatment on ovarian damage during ovarian tissue cryopreservation and transplantation. Biochem Biophys Res Commun. 2021;534:780-786. doi:10.1016/j.bbrc.2020.10.110
  • Donnez J, Dolmans MM, Pellicer A, et al. Fertility preservation for age-related fertility decline. Lancet. 2015;385(9967):506-507. doi:10.1016/S0140-6736(15)60199-4
  • Suzuki N, Yoshioka N, Takae S, et al. Successful fertility preservation following ovarian tissue vitrification in patients with primary ovarian insufficiency. Hum Reprod. 2015;30(3):608-615. doi:10.1093/humrep/deu353
  • Alessi DR, Pearce LR, García Martínez JM. New insights into mTOR signaling: mTORC2 and beyond. Sci Signal. 2009;2(67):pe27. doi:10.1126/scisignal.267pe27
  • Dunlop EA, Tee AR. Mammalian target of rapamycin complex 1: signalling inputs, substrates and feedback mechanisms. Cell Signal. 2009;21(6):827-835. doi:10.1016/j.cellsig.2009.01.012
  • Foster KG, Fingar DC. Mammalian target of rapamycin (mTOR): conducting the cellular signaling symphony. J Biol Chem. 2010;285(19):14071-14077. doi:10.1074/jbc.R109.094003
  • Juhasz G, Neufeld TP. Experimental control and characterization of autophagy in Drosophila. Methods Mol Biol. 2008;445:125-133. doi:10.1007/978-1-59745-157-4_8.
  • 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(4):e19475. doi:10.1371/journal.pone.0019475
  • Saxton RA, Sabatini DM. mTOR Signaling in Growth, Metabolism, and Disease. Cell. 2017;168(6):960-976. doi:10.1016/j.cell.2017.02.004 Erratum in Cell. 2017;169(2):361-371. doi:10.1016/j.cell.2017.03.035.
  • Tayefi Nasrabadi H, Gavami M, Akbarzadeh A, Beheshti R, Mohammadnejad D, Abedelahi A. Preservation of mouse ovarian tissue follicle morphology and ultra-structure after vitrifying in biotechnological protocols. J Ovarian Res. 2015;8:7. doi:10.1186/s13048-015-0137-3
  • Caldwell AS, Middleton LJ, Jimenez M, et al. Characterization of reproductive, metabolic, and endocrine features of polycystic ovary syndrome in female hyperandrogenic mouse models. Endocrinology. 2014;155(8):3146-3159. doi:10.1210/en.2014-1196
  • Çil N, Oğuz EO, Mete E, Çetinkaya A, Mete GA. Effects of umbilical cord blood stem cells on healing factors for diabetic foot injuries. Biotech Histochem. 2017;92(1):15-28. doi:10.1080/10520295.2016.1243728
  • Celik S, Ozkavukcu S, Celik Ozenci C. Altered expression of activator proteins that control follicle reserve after ovarian tissue cryopreservation/transplantation and primordial follicle loss prevention by rapamycin. J Assist Reprod Genet. 2020;37(9):2119-2136. doi:10.1007/s10815-020-01875-7
  • Bindels J, Squatrito M, Bernet L, Nisolle M, Henry L, Munaut C. The mTOR Inhibitor Rapamycin Counteracts Follicle Activation Induced by Ovarian Cryopreservation in Murine Transplantation Models. Medicina (Kaunas). 2023;59(8):1474. doi:10.3390/medicina59081474
  • Wang J, Zhang Y, Wu C, et al. Effects of AavLEA1 Protein on Mouse Ovarian Tissue Cryopreservation by Vitrification. Biopreserv Biobank. 2022;20(2):168-175. doi:10.1089/bio.2021.0048
  • Damous LL, Shiroma ME, Carvalho AETS, Soares Jr JM, Krieger JE, Baracat EC. Gene expression profile in experimental frozen-thawed ovarian grafts treated with scaffold-base delivery of adipose tissue-derived stem cells. Clinics (Sao Paulo). 2022;77:100066. doi:10.1016/j.clinsp.2022.100066
  • Wu YT, Tan HL, Huang Q, Ong CN, Shen HM. Activation of the PI3K-Akt-mTOR signaling pathway promotes necrotic cell death via suppression of autophagy. Autophagy. 2009;5(6):824-834. doi:10.4161/auto.9099
  • Zha X, Hu Z, He S, Wang F, Shen H, Zhang H. TSC1/TSC2 inactivation inhibits AKT through mTORC1-dependent up-regulation of STAT3-PTEN cascade. Cancer Lett. 2011;313(2):211-217. doi:10.1016/j.canlet.2011.09.006
  • Hunzicker Dunn ME, Lopez Biladeau B, Law NC, Fiedler SE, Carr DW, Maizels ET. PKA and GAB2 play central roles in the FSH signaling pathway to PI3K and AKT in ovarian granulosa cells. Proc Natl Acad Sci USA. 2012;109(44):E2979-2988. doi:10.1073/pnas.1205661109
  • Guo J, Zhang T, Guo Y, et al. Oocyte stage-specific effects of MTOR determine granulosa cell fate and oocyte quality in mice. Proc Natl Acad Sci USA. 2018;115(23):E5326-E5333. doi:10.1073/pnas.1800352115
  • Carvalho AA, Faustino LR, Silva CM, et al. Catalase addition to vitrification solutions maintains goat ovarian preantral follicles stability. Res Vet Sci. 2014;97(1):140-147. doi:10.1016/j.rvsc.2014.05.006
  • Lee JR, Youm HW, Kim SK, Jee BC, Suh CS, Kim SH. Effect of necrostatin on mouse ovarian cryopreservation and transplantation. Eur J Obstet Gynecol Reprod Biol. 2014;178:16-20. doi:10.1016/j.ejogrb.2014.04.040
  • Lee J, Lee JR, Youm HW, Suh CS, Kim SH. Effect of preoperative simvastatin treatment on transplantation of cryopreserved-warmed mouse ovarian tissue quality. Theriogenology. 2015;83(2):285-293. doi:10.1016/j.theriogenology.2014.09.027
  • Shu WH, Yang SH, Wei M, et al. Effects of sericin on oxidative stress and PI3K/AKT/mTOR signal pathway in cryopreserved mice ovarian tissue. Cryobiology. 2023;111:16-25. doi:10.1016/j.cryobiol.2023.03.003
  • Pham NA, Schwock J, Iakovlev V, Pond G, Hedley DW, Tsao MS. Immunohistochemical analysis of changes in signaling pathway activation downstream of growth factor receptors in pancreatic duct cell carcinogenesis. BMC Cancer. 2008;8:43. doi:10.1186/1471-2407-8-43
  • Chen JS, Wang Q, Fu XH, et al. Involvement of PI3K/PTEN/AKT/mTOR pathway in invasion and metastasis in hepatocellular carcinoma: Association with MMP-9. Hepatol Res. 2009;39(2):177-186. doi:10.1111/j.1872-034X.2008.00449.x
  • Johnson SM, Gulhati P, Rampy BA, et al. Novel expression patterns of PI3K/Akt/mTOR signaling pathway components in colorectal cancer. J Am Coll Surg. 2010;210(5):767-776, 776-778. doi:10.1016/j.jamcollsurg.2009.12.008
  • Kogasaka Y, Hoshino Y, Hiradate Y, Tanemura K, Sato E. Distribution and association of mTOR with its cofactors, raptor and rictor, in cumulus cells and oocytes during meiotic maturation in mice. Mol Reprod Dev. 2013;80(4):334-348. doi:10.1002/mrd.22166

Immunohistochemical demonstration of mTOR and p-mTOR expression in frozen and thawed ovarian tissue

Yıl 2026, Cilt: 19 Sayı: 1, 27 - 35, 16.01.2026
https://doi.org/10.31362/patd.1709595
https://izlik.org/JA75PX55UH

Öz

Purpose: Ovarian cryopreservation is a highly effective method that minimizes toxic effects and is prefered for preserving fertility. The mammalian target of rapamycin (mTOR) pathway is critically important for cell growth, proliferation, autophagy, nutrient signaling, and survival. Our aim was to investigate the expression of mTOR and Phosphorylated (p-mTOR) in the ovary before and after freezing using an immunohistochemical method.
Material and methods: Ovarian samples were allocated into two groups: control (C; n=6) and vitrification–thawing (V/T; n=6). Control samples were fixed in buffered formalin for 48 hours. The vitrification–thawing (V/T) group was frozen in liquid nitrogen at -196ºC and stored for one week before thawing. All samples were processed for paraffin embedding and sectioning. Ovaries were stained with Hematoxylin and Eosin, and follicle counting was performed. Immunohistochemistry was carried out to evaluate mTOR and p-mTOR expression.
Results: Follicle numbers did not differ significanlty between the groups. Strong mTOR expression was observed in follicles of both C and V/T group ovaries. Positive p-mTOR expression was detected in oocytes and granulosa cells in the control group, whereas the expression was negative in the V/T group.
Conclusion: Cryopreservation methods did not induce to alter mTOR expression in ovarian tissue. However, the absence of p-mTOR activity associated with oocyte quality in frozen ovarian tissues suggested that cryopreservation effected the quality of oocyte.

Etik Beyan

Pamukkale University Experiments Ethics Committee (Approval number: 2016/24).

Destekleyen Kurum

Pamukkale University Scientific Research Projects Coordination Unit (grant number 2017SABE007)

Proje Numarası

Pamukkale University Scientific Research Projects Coordination Unit (grant number 2017SABE007)

Kaynakça

  • Donnez J, Dolmans MM. Ovarian cortex transplantation: 60 reported live births brings the success and worldwide expansion of the technique towards routine clinical practice. J Assist Reprod Genet. 2015;32(8):1167-1170. doi:10.1007/s10815-015-0544-9
  • Liu W, Zhang J, Wang L. et al. The protective effects of rapamycin pretreatment on ovarian damage during ovarian tissue cryopreservation and transplantation. Biochem Biophys Res Commun. 2021;534:780-786. doi:10.1016/j.bbrc.2020.10.110
  • Donnez J, Dolmans MM, Pellicer A, et al. Fertility preservation for age-related fertility decline. Lancet. 2015;385(9967):506-507. doi:10.1016/S0140-6736(15)60199-4
  • Suzuki N, Yoshioka N, Takae S, et al. Successful fertility preservation following ovarian tissue vitrification in patients with primary ovarian insufficiency. Hum Reprod. 2015;30(3):608-615. doi:10.1093/humrep/deu353
  • Alessi DR, Pearce LR, García Martínez JM. New insights into mTOR signaling: mTORC2 and beyond. Sci Signal. 2009;2(67):pe27. doi:10.1126/scisignal.267pe27
  • Dunlop EA, Tee AR. Mammalian target of rapamycin complex 1: signalling inputs, substrates and feedback mechanisms. Cell Signal. 2009;21(6):827-835. doi:10.1016/j.cellsig.2009.01.012
  • Foster KG, Fingar DC. Mammalian target of rapamycin (mTOR): conducting the cellular signaling symphony. J Biol Chem. 2010;285(19):14071-14077. doi:10.1074/jbc.R109.094003
  • Juhasz G, Neufeld TP. Experimental control and characterization of autophagy in Drosophila. Methods Mol Biol. 2008;445:125-133. doi:10.1007/978-1-59745-157-4_8.
  • 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(4):e19475. doi:10.1371/journal.pone.0019475
  • Saxton RA, Sabatini DM. mTOR Signaling in Growth, Metabolism, and Disease. Cell. 2017;168(6):960-976. doi:10.1016/j.cell.2017.02.004 Erratum in Cell. 2017;169(2):361-371. doi:10.1016/j.cell.2017.03.035.
  • Tayefi Nasrabadi H, Gavami M, Akbarzadeh A, Beheshti R, Mohammadnejad D, Abedelahi A. Preservation of mouse ovarian tissue follicle morphology and ultra-structure after vitrifying in biotechnological protocols. J Ovarian Res. 2015;8:7. doi:10.1186/s13048-015-0137-3
  • Caldwell AS, Middleton LJ, Jimenez M, et al. Characterization of reproductive, metabolic, and endocrine features of polycystic ovary syndrome in female hyperandrogenic mouse models. Endocrinology. 2014;155(8):3146-3159. doi:10.1210/en.2014-1196
  • Çil N, Oğuz EO, Mete E, Çetinkaya A, Mete GA. Effects of umbilical cord blood stem cells on healing factors for diabetic foot injuries. Biotech Histochem. 2017;92(1):15-28. doi:10.1080/10520295.2016.1243728
  • Celik S, Ozkavukcu S, Celik Ozenci C. Altered expression of activator proteins that control follicle reserve after ovarian tissue cryopreservation/transplantation and primordial follicle loss prevention by rapamycin. J Assist Reprod Genet. 2020;37(9):2119-2136. doi:10.1007/s10815-020-01875-7
  • Bindels J, Squatrito M, Bernet L, Nisolle M, Henry L, Munaut C. The mTOR Inhibitor Rapamycin Counteracts Follicle Activation Induced by Ovarian Cryopreservation in Murine Transplantation Models. Medicina (Kaunas). 2023;59(8):1474. doi:10.3390/medicina59081474
  • Wang J, Zhang Y, Wu C, et al. Effects of AavLEA1 Protein on Mouse Ovarian Tissue Cryopreservation by Vitrification. Biopreserv Biobank. 2022;20(2):168-175. doi:10.1089/bio.2021.0048
  • Damous LL, Shiroma ME, Carvalho AETS, Soares Jr JM, Krieger JE, Baracat EC. Gene expression profile in experimental frozen-thawed ovarian grafts treated with scaffold-base delivery of adipose tissue-derived stem cells. Clinics (Sao Paulo). 2022;77:100066. doi:10.1016/j.clinsp.2022.100066
  • Wu YT, Tan HL, Huang Q, Ong CN, Shen HM. Activation of the PI3K-Akt-mTOR signaling pathway promotes necrotic cell death via suppression of autophagy. Autophagy. 2009;5(6):824-834. doi:10.4161/auto.9099
  • Zha X, Hu Z, He S, Wang F, Shen H, Zhang H. TSC1/TSC2 inactivation inhibits AKT through mTORC1-dependent up-regulation of STAT3-PTEN cascade. Cancer Lett. 2011;313(2):211-217. doi:10.1016/j.canlet.2011.09.006
  • Hunzicker Dunn ME, Lopez Biladeau B, Law NC, Fiedler SE, Carr DW, Maizels ET. PKA and GAB2 play central roles in the FSH signaling pathway to PI3K and AKT in ovarian granulosa cells. Proc Natl Acad Sci USA. 2012;109(44):E2979-2988. doi:10.1073/pnas.1205661109
  • Guo J, Zhang T, Guo Y, et al. Oocyte stage-specific effects of MTOR determine granulosa cell fate and oocyte quality in mice. Proc Natl Acad Sci USA. 2018;115(23):E5326-E5333. doi:10.1073/pnas.1800352115
  • Carvalho AA, Faustino LR, Silva CM, et al. Catalase addition to vitrification solutions maintains goat ovarian preantral follicles stability. Res Vet Sci. 2014;97(1):140-147. doi:10.1016/j.rvsc.2014.05.006
  • Lee JR, Youm HW, Kim SK, Jee BC, Suh CS, Kim SH. Effect of necrostatin on mouse ovarian cryopreservation and transplantation. Eur J Obstet Gynecol Reprod Biol. 2014;178:16-20. doi:10.1016/j.ejogrb.2014.04.040
  • Lee J, Lee JR, Youm HW, Suh CS, Kim SH. Effect of preoperative simvastatin treatment on transplantation of cryopreserved-warmed mouse ovarian tissue quality. Theriogenology. 2015;83(2):285-293. doi:10.1016/j.theriogenology.2014.09.027
  • Shu WH, Yang SH, Wei M, et al. Effects of sericin on oxidative stress and PI3K/AKT/mTOR signal pathway in cryopreserved mice ovarian tissue. Cryobiology. 2023;111:16-25. doi:10.1016/j.cryobiol.2023.03.003
  • Pham NA, Schwock J, Iakovlev V, Pond G, Hedley DW, Tsao MS. Immunohistochemical analysis of changes in signaling pathway activation downstream of growth factor receptors in pancreatic duct cell carcinogenesis. BMC Cancer. 2008;8:43. doi:10.1186/1471-2407-8-43
  • Chen JS, Wang Q, Fu XH, et al. Involvement of PI3K/PTEN/AKT/mTOR pathway in invasion and metastasis in hepatocellular carcinoma: Association with MMP-9. Hepatol Res. 2009;39(2):177-186. doi:10.1111/j.1872-034X.2008.00449.x
  • Johnson SM, Gulhati P, Rampy BA, et al. Novel expression patterns of PI3K/Akt/mTOR signaling pathway components in colorectal cancer. J Am Coll Surg. 2010;210(5):767-776, 776-778. doi:10.1016/j.jamcollsurg.2009.12.008
  • Kogasaka Y, Hoshino Y, Hiradate Y, Tanemura K, Sato E. Distribution and association of mTOR with its cofactors, raptor and rictor, in cumulus cells and oocytes during meiotic maturation in mice. Mol Reprod Dev. 2013;80(4):334-348. doi:10.1002/mrd.22166
Toplam 29 adet kaynakça vardır.

Ayrıntılar

Birincil Dil İngilizce
Konular Üreme Tıbbı (Diğer)
Bölüm Araştırma Makalesi
Yazarlar

Umray Dede 0000-0001-9831-2456

Murat Serkant Ünal 0000-0003-1992-7909

Nazlı Çil 0000-0002-2164-8688

Gülçin Abban Mete 0000-0001-6794-3685

Proje Numarası Pamukkale University Scientific Research Projects Coordination Unit (grant number 2017SABE007)
Gönderilme Tarihi 30 Mayıs 2025
Kabul Tarihi 1 Temmuz 2025
Erken Görünüm Tarihi 1 Ekim 2025
Yayımlanma Tarihi 16 Ocak 2026
DOI https://doi.org/10.31362/patd.1709595
IZ https://izlik.org/JA75PX55UH
Yayımlandığı Sayı Yıl 2026 Cilt: 19 Sayı: 1

Kaynak Göster

AMA 1.Dede U, Ünal MS, Çil N, Abban Mete G. Immunohistochemical demonstration of mTOR and p-mTOR expression in frozen and thawed ovarian tissue. Pam Tıp Derg. 2026;19(1):27-35. doi:10.31362/patd.1709595
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