Investigation of the Distribution and Expression Level of Pro and Anti-Apoptotic Bax and Bcl-2 in Ovarian Follicles at Different Developmental Stages

Yıl 2021, , 424 - 428, 29.12.2021

Uğur Şeker , Funda Eşki , Serdal Kurt

https://doi.org/10.35440/hutfd.995512

Cited By: 1

Öz

Background: In this study we aimed to investigate the distribution and expression level of pro and anti-apoptotic proteins, Bax and Bcl-2, in different developmental stages of ovarian follicles and any relations between these proteins and follicle atresia.
Materials and Methods: For that purpose, bilateral 16 ovaries of adult 8 mice were received and the tissues were fixed in 10% neutral buffered formalin. Routine tissue processing protocol was performed and the samples were embedded into paraffin blocks. Five µm thick sections were received and the tissue sections were stained with Bax and Bcl-2 immunohistochemistry. The ovarian follicles were classified as primordial, primary, secondary and antral. Distribution and expression levels of Bax and Bcl-2 were evaluated among and within the developmental stages. The expression levels of Bax and Bcl-2 were also compared with atretic follicle ratio.
Results: Immunopositivity of Bax and Bcl-2 were observed in ovarian stromal cells, granulosa, oocytes, and lutheal cells in a varying range. Despite of some immunpositivity, most of the primordial and primary follicle granulosa cells and oocytes were negative for these apoptosis regulator proteins. The intensity of immunopositivity increased at the farther developmental process in follicles. In addition, the immunoexpression level significantly increased just with the beginning of the secondary follicular stage and the expression levels were the most intense in antral follicles. Furthermore, some of the antral follicles were intense Bax positive which were observed with atretic follicle morphology.
Conclusions: Bax and Bcl-2 are crucial regulators of ovarian follicle development. Although Bcl-2 contributes on development, correlation analyses indicated that Bax decides stronger than Bcl-2 on the atresia or development fate of follicle.

Anahtar Kelimeler

Bax, Bcl-2, Ovary, Follicle, Atresia

Farklı Gelişim Dönemlerindeki Ovaryum Foliküllerinde Pro ve Anti-Apoptotik Bax ve Bcl-2'nin Dağılım ve Ekspresyon Düzeyinin İncelenmesi

Öz

Amaç: Bu çalışmada, farklı gelişimsel evredeki ovaryum foliküllerinde Bax ve Bcl-2 ekspresyon düzeylerini ve bu proteinlerle folikül atrezisi arasındaki ilişkiyi araştırmayı amaçladı.
Materyal ve Metod: Bu amaçla 8 fareye ait bilateral toplam 16 ovaryum toplandı ve dokular 10%’luk tamponlanmış formalin içerisinde fikse edildi. Dokulara rutin doku takibi protokolü uygulandı ve örnekler erimiş parafine gömülerek bloklandı. Parafin bloklara gömülü doku örneklerinden alınan 5 µm kalınlığındaki kesitler Bax ve Bcl-2 immunohistokimya boyandı. Ovaryum folikülleri primordiyal, primer, seconder ve antral şeklinde sınıflandırılıd. Gelişim aşamalarının içinde ve aşamalar arasındaki Bax ve Bcl-2 dağılımı ve ekspresyon düzeyi değerlendirildi. Ekspresyon düzeyleri aynı zamanda folikül atrezisi ile kıyaslandı.
Bulgular: Ovaryum stroma hücrelerinde, granuloza, oosit ve luteal hücrelerde Bax ve Bcl-2 immunopozitivitesi değişen oranlarda izlendi. Yer yer gözlenen immunopozitiviteye karşın, primordiyal ve primer folikül granuloza hücreleri ve oositleri büyük bir oranda apoptozis düzenleyici proteinler yönünden negatifti. İmmunopozitivite yoğunluğu ileri gelişim dönemlerinde artış eğilimindeydi. Ayrıca, immunoekspresyon düzeyi sekonder foliküler aşamadan sonra önemli bir artış gösteriyordu ve antral foliküllerde en yüksek düzeyde ekspresyon yoğunluğu izlendi. Dahası bazı antral foliküllerdeki yoğun Bax pozitifliğine atretik folikül morfolojisi eşlik etmekteydi.
Sonuç: Bax ve Bcl-2 ovaryum folikül gelişiminin önemli düzenleyicileridir. Her ne kadar Bcl-2 gelişim sürecinde etkili olsa da, korelasyon analizleri Bax’ın folikül atrezi veya gelişim kararını vermede Bcl-2’den daha güçlü olduğunu göstermiştir.

Anahtar Kelimeler

Bax, Bcl-2, Ovaryum, Folikül, Atrezi

Kaynakça

• Treesh S,Khair N. Effect of thyroid disorders on the adult female albino rats (Histological and Histochemical Study). J Cytol Histol. 2014;5(4):1.
• Carlsson IB, Scott JE, Visser J, Ritvos O, Themmen A,Hovatta O. Anti-Müllerian hormone inhibits initiation of growth of human primordial ovarian follicles in vitro. Hum Reprod. 2006;21(9):2223-2227.
• Hsueh AJ, Kawamura K, Cheng Y,Fauser BC. Intraovarian control of early folliculogenesis. Endocr Rev. 2015;36(1):1-24.
• Kumar TR, Wang Y, Lu N,Matzuk MM. Follicle stimulating hormone is required for ovarian follicle maturation but not male fertility. Nat Genet. 1997;15(2):201-204.
• Orisaka M, Miyazaki Y, Shirafuji A, Tamamura C, Tsuyoshi H, Tsang BK, et al. The role of pituitary gonadotropins and intraovarian regulators in follicle development: A mini‐review. Reprod Med Biol. 2021;20(2):169-175.
• Rajkovic A, Pangas SA,Matzuk MM. Follicular development: mouse, sheep, and human models. Knobil and Neill’s physiology of reproduction. 2006;1:383-424.
• Yu YS, Sui HS, Han ZB, Wei L, Luo MJ,Tan JH. Apoptosis in granulosa cells during follicular atresia: relationship with steroids and insulin-like growth factors. Cell Res. 2004;14(4):341-346.
• D’Arcy MS. Cell death: a review of the major forms of apoptosis, necrosis and autophagy. Cell Biol Int. 2019;43(6):582-592.
• Xia X, Wang X, Cheng Z, Qin W, Lei L, Jiang J, et al. The role of pyroptosis in cancer: pro-cancer or pro-“host”? Cell Death Dis. 2019;10(9):1-13.
• Brunelle JK,Letai A. Control of mitochondrial apoptosis by the Bcl-2 family. Journal of cell science. 2009;122(4):437-441.
• Sonigo C, Jankowski S, Yoo O, Trassard O, Bousquet N, Grynberg M, et al. High-throughput ovarian follicle counting by an innovative deep learning approach. Sci Rep. 2018;8(1):1-9.
• Costa L, Moreia-Pinto B, Felgueira E, Ribeiro A, Rebelo I,Fonseca B. The major endocannabinoid anandamide (AEA) induces apoptosis of human granulosa cells. Prostaglandins, Leukotrienes and Essential Fatty Acids. 2021:102311.
• Monniaux D, Cadoret V, Clément F, Tran RD, Elis S, Fabre S, et al. Folliculogenesis, In: Huhtaniemi I, Martini L, eds. Encyclopedia of Endocrine Diseases. Netherlands: Elsevier, 2018:1-22.
• Vaskivuo TE, Anttonen M, Herva R, Billig Hk, Dorland M, te Velde ER, et al. Survival of human ovarian follicles from fetal to adult life: apoptosis, apoptosis-related proteins, and transcription factor GATA-4. J Clin Endocrinol Metab. 2001;86(7):3421-3429.
• Rolaki A, Drakakis P, Millingos S, Loutradis D,Makrigiannakis A. Novel trends in follicular development, atresia and corpus luteum regression: a role for apoptosis. Reprod Biomed Online. 2005;11(1):93-103.
• Hakuno N, Koji T, Yano T, Kobayashi N, Tsutsumi O, Taketani Y, et al. Fas/APO-1/CD95 system as a mediator of granulosa cell apoptosis in ovarian follicle atresia. Endocrinology. 1996;137(5):1938-1948.
• Sakamaki K, Yoshida H, Nishimura Y, Nishikawa SI, Manabe N,Yonehara S. Involvement of Fas antigen in ovarian follicular atresia and luteolysis. Mol Reprod Dev. 1997;47(1):11-18.
• Roughton SA, Lareu RR, Bittles AH,Dharmarajan AM. Fas and Fas ligand messenger ribonucleic acid and protein expression in the rat corpus luteum during apoptosis-mediated luteolysis. Biol Reprod. 1999;60(4):797-804.
• Gürsoy E, Ergin K, Başaloğlu H, Koca Y,Seyrek K. Expression and localisation of Bcl-2 and Bax proteins in developing rat ovary. Res Vet Sci. 2008;84(1):56-61.