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

Abstract

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.

Keywords

• Ovaryum
• kriyoprezervasyon
• vitrifikasyon
• mTOR
• p-mTOR

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

Abstract

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.

Keywords

• Ovary
• cryopreservation
• vitrification
• mTOR
• p-mTOR

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