Serap Cllaker MICILI, Seda VATANSEVER, Kemal ÖZBİLGİN

The Role of proteins in apoptosis of somatic and germ cells in the mouse Ovary

Bu çalışmada gelişen foliküllerde hücre ölüm mekanizmasını belirlemek için büyüyen fare foliküllerinde Bcl-2, p53 ve Bax antikorlarının eskprese edilip edilmediğini araştırmayı amaçladık. Ovaryum dokuları ve oositler 30 adet Mus Musculus tip fareden elde edildi. Ovaryum doku kesitleri ve oositler immunhistokimyasal olarak anti-Bcl-2, anti-Bax ve anti-p53 antikorları ve DNA fragmentasyonunu belirlemek için terminal deoxynucleotidyl transferase (TdT) (TUNEL) yöntemi ile incelendi. Ovaryum kesitlerinin incelenmesinde sekonder follikülerinin antrumunda ve Graaf follikülerinin granuloza hücrelerinde TUNEL pozitif hücreler gözlendi. Bcl-2 immunreaktivitesi primer ve sekonder folliküllerinde orta şiddetteydi. p53 immunboyanması Graaf follikülerinin granuloza hücrelerinde orta şiddetteyken, Bax immunreaktivitesi sekonder foliküllerde orta, Graff follikülerinde ise şiddetli olarak gözlendi. Zona pellusidadan ayrılan granuloza hücreleri ise TUNEL pozitif boyanma gösterdi. Fertilize olmamış oositlerin değerlendirilmesinde, zona pellusidadan ayrılan granuloza hücrelerinde TUNEL pozitif hücreler gözlendi. Zonaya yapışık hücreler TUNEL negatif özellikteydi. Bcl-2 immunreaktivitesi oosit ve granuloza hücrelerinin her ikisinde de zona pellusidaya yapışık granuloza hücrelerinde gözlenirken, p53 immunboyanması sadece zona pelusidadan ayrılan granuloza hücrelerinde gözlendi. Sonuç olarak, granuloza hücrelerinde apoptozisin düzenlenmesinde zona pellusidadan ayrılan granuloza hücrelerinin apoptotik kaskada uğrayabileceği ve bunun Bax ekspresyonu ile kontrol edilebileceği düşünülmüştür. Bu nedenle, granuloza hücre ölümünün Graaf folikülünde intrinsic pathway, ovulasyondan sonra ise extrinsic pathway ile düzenlendiğini düşünmekteyiz. Bu sonuçlara ek olarak daha ayrıntılı çalışmalara ihtiyaç olduğunu düşünmekteyiz.

Anahtar Kelimeler:

antikor, programlanmış hücre ölümü, hayvan modelleri, eşey hücreleri, tanecikli hücreler, morfogenez, oosit, yumurtalık follikülleri, yumurtalık gelişimi, yumurtalıklar, Zona pellucida

Fare Ovaryumunda somatik ve germ hücrelerinin apoptozisinde proteinlerin rolü

In this study, to determine the mechanisms of cell death in developing follicles, we investigated whether expression of Bcl-2, p53 and Bax play a role throughout the growth of follicles in the mice. Ovarian tissues and oocytes were obtained from 30 Mus Musculus type mouse. The immunostaining of ovarian tissue sections and oocytes was performed using anti-Bcl-2, anti-Bax and anti-p53 antibodies and terminal deoxynucleotidyl transfrase (TdT) assay (TUNEL) were also used for detection of internucleosomal DNA fragmentation. In ovarian tissue section evaluation, granulosa cells in antrum of secondary and Graaf follicles were positive with TUNEL staining. Immunoreactivity of Bcl-2 was moderate in primary and secondary follicles of granulosa cells. While immunostaining of p53 was moderate in granulosa cells of Graaf follicles, Bax immunoreactivity was moderate and strong in secondary and Graaf follicles, respectively.When granulosa cells were break off from zona pellucida, there were TUNEL positive cells.In unfertilised oocytes evaluation, TUNEL positive cells were observed in the granulosa cells which were free from zona pellucida. When the granulosa cells were attached with zona pellucida, they were TUNEL negative. While immunoreactivity of Bcl-2 was detected in both oocytes and granulosa cells which were attached with zona pellucida, immunostaining of p53 were only detected in granulosa cells which break off from zona pellucida. In conclusion, regulation of apoptosis in granulosa cells may be controlled by Bax expression and when the granulosa cells were not attached with zona pellucida, they may go into the apoptotic cascade. Therefore; we suggest that, the death of granulosa cells may control signals from intrinsic pathways in the Graaf follicule or from extrinsic pathways after ovulation. However, we consider of further studies to be necessary.

Keywords:

antibodies, apoptosis, animal models, germ cells, granulosa cells, morphogenesis, oocytes, ovarian follicles, ovarian development, ovaries, zona pellucida,

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