Farede prematüre over yetmezliğinde mTERT telomeraz katalitik alt ünitesinin rolünün belirlenmesi

Memelilerde prenatal hayatta oluşan oosit rezervi, postnatal hayatta azalarak tükenmektedir. Foliküler atrezi olarak adlandırılan bu mekanizma ile ovariyal yaşlanma meydana gelmektedir. Primordiyal folikül havuzu tekrardan yenilenemediği için bu tükeniş sonucunda infertilite ya da Prematüre Over Yetmezliği (POY) ile karşı karşıya kalınmaktadır. Polisiklik aromatik hidrokarbon 7, 12-dimetilbenz (7,12-dimethylbenz-[a]anthracene (DMBA)), ovaryum da dahil olmak üzere pekçok tümörü tetikleyen ve günlük hayatta da sıklıkla maaruz kaldığımız bir çevresel karsinojendir. Telomeraz, hücrenin bölünmesi esnasında kromozomların kararlı yapılarının korunmasını sağlar ve fare telomeraz ters transkriptaz (mTERT), telomeraz kompleksinin gerekli bir bileşenidir. c-Abl protein tirozin kinaz, DNA çift sarmalında kırıkların oluşması ile aktive olan ve telomer kontrolü esnasında bu kırıkların tamir edilmesine katılan bir proteindir. Telomer kısalması, hücre bölünmesini zamanla durduran bir işlemdir. Bu nedenle çalışmamızda DMBA indüklü ovotoksisite sonucu oluşan POY’de c-Abl ve mTERT’in önemli bir role sahip olabileceğini düşünmekteyiz. Buradan yola çıkarak günlük yaşamda sürekli olarak maaruz kalınan DMBA’nın ovaryumdaki oosit rezervi, oosit kalitesi ve granuloza hücreleri üzerindeki etkisini c-Abl (Abelson Tirozin Kinaz) ve mTERT (telomeraz katalitik altünitesi) belirteçleri ile açıklamayı hedefledik. Çalışmamızda postnatal (PND) 28 günlük BalbC türü farelere 7 gün boyunca susam yağı içerisinde çözülen 1mg/kg DMBA uygulaması yapıldı. İzole edilen ovaryum dokularında Hematoksilen-Eozin boyama ile morfolojik değerlendirme ve folikül sayımı yapıldı. İmmünofloresan yöntemi ile c-Abl ve mTERT lokalizasyonları gösterildi. Ayrıca ELISA, western blot ve qRT-PCR yöntemleri kullanılarak fare ovaryumunda DMBA tedavisinin telomeraz aktivitesi üzerine olan etkisi gösterildi. Ayrıca, Transmisyon Elektron Mikroskobi yöntemi ile oosit-granuloza hücresi arasındaki ilişki ultrastrüktürel düzeyde incelendi. Çalışmamızda DMBA indüklü ovotoksisite sonucu oluşan POY’de c-Abl ve mTERT, ilk defa gösterilmiştir. Bu proje ile DMBA uygulanmış fare ovaryumunda c-Abl ve mTERT ekspresyonlarındaki değişimin gösterilmesiyle, projemizin sonuçlarının POY’de erken over yaşlanması ve kadın infertilitesine ilişkin sinyal mekanizmalarına ışık tutacağını düşünmekteyiz.(TÜBİTAK-215S867)

mTOR Controls Ovarian Follicle Growth by Regulating Granulosa Cell Proliferation

We have shown that inhibition of mTOR in granulosa cells and ovarian follicles results in compromised granulosa proliferation and reduced follicle growth. Further analysis here using spontaneously immortalized rat granulosa cells has revealed that mTOR pathway activity is enhanced during M-phase of the cell cycle. mTOR specific phosphorylation of p70S6 kinase and 4E-BP, and expression of Raptor are all enhanced during M-phase. The predominant effect of mTOR inhibition by the specific inhibitor Rapamycin (RAP) was a dose-responsive arrest in the G1 cell cycle stage. The fraction of granulosa cells that continued to divide in the presence of RAP exhibited a dose-dependent increase in aberrant mitotic figures known as anaphase bridges. Strikingly, estradiol consistently decreased the incidence of aberrant mitotic figures. In mice treated with RAP, the mitotic index was reduced compared to controls, and a similar increase in aberrant mitotic events was noted. RAP injected during a superovulation regime resulted in a dose-dependent reduction in the numbers of eggs ovulated. Implications for the real-time regulation of follicle growth and dominance, including the consequences of increased numbers of aneuploid granulosa cells, are discussed

The p38 MAPK signalling pathway is required for glucose metabolism, lineage specification and embryo survival during mouse preimplantation development

WOS: 000365991800016PubMed ID: 26025760Preimplantation embryo development is an important and unique period and is strictly controlled. This period includes a series of critical events that are regulated by multiple signal-transduction pathways, all of which are crucial in the establishment of a viable pregnancy. The p38 mitogen-activated protein kinase ( MAPK) signalling pathway is one of these pathways, and inhibition of its activity during preimplantation development has a deleterious effect. The molecular mechanisms underlying the deleterious effects of p38 MAPK suppression in early embryo development remain unknown. To investigate of the effect of p38 MAPK inhibition on late preimplantation stages in detail, we cultured 2-cell stage embryos in the presence of SB203580 for 48 h and analysed the 8-cell, morula, and blastocyst stages. We determined that prolonged inhibition of the p38 MAPK altered the expression levels of Glut1 and Glut4, decreased glucose uptake during the 8-cell to blastocyst transition, changed the expression levels of transcripts which will be important to lineage commitment, including Oct4/Pou5f1, Nanog, Sox2, and Gata6, and increased cell death in 8-16 cell stage embryos onwards. Strikingly, while the expression levels of Nanog, Gata6 and Oct4/Pou5f1 mRNAs were significantly decreased, Sox2 mRNA was increased in SB203580-treated blastocysts. Taken together, our results provide important insight into the biological processes controlled by the p38 MAPK pathway and its critical role during preimplantation development.Akdeniz UniversityTürkiye Bilimsel ve Teknolojik Araştırma Kurumu (TUBITAK

Protective effect of chemically modified SOD on lipid peroxidation and antioxidant status in diabetic rats

WOS: 000347577900012PubMed ID: 25124383Reactive oxygen species mediated oxidative stress play an important role on the injury of tissue damage and increased attention has been focused on the role of free radicals in diabetes mellitus (DM). In the present study firstly superoxide dismutase (SOD) enzyme was chemically modified with two different polymer and physicochemical properties of these conjugates clearly analyzed. Then, the stability of carboxymethylcellulose-SOD (CMC-SOD) and poly methyl vinyl ether-co-maleic anhydride-SOD (PMVE/MA-SOD) conjugates was investigated against temperature and externally added H2O2. Moreover, we investigated the effect of chemically modified SOD enzyme on lipid peroxidation and antioxidant status in streptozotocin (STZ)-induced diabetic rats. PMVE/MA-SOD conjugate treatment significantly reduced MDA level compared with the control groups, native and CMC-SOD conjugate treated groups in brain, kidney and liver tissue. GSH and SOD enzyme activity in diabetic groups was significantly increased by treatment of CMC-SOD and PMVE/MA-SOD conjugates. The protective effects on degenerative changes in diabetic rats were also further confirmed by histopathological examination. This study provides the preventative activity of SOD-polymer conjugates against complication of oxidative stress in experimentally induced diabetic rats. These results suggest that chemically modified SOD is effective on the oxidative stress-associated disease and offer a therapeutic advantage in clinical use. (C) 2014 Elsevier B.V. All rights reserved.Research Foundation of Yildiz Technical University [2010-07-04-GEP01]This work was supported by Research Foundation of Yildiz Technical University under project number 2010-07-04-GEP01. We wish to thank Animal Laboratory of Bezmialem Vakif University staff for animal care and technical assistance

Expression of CCM2 and CCM3 during mouse gonadogenesis

WOS: 000363272800010PubMed ID: 26386873Three cerebral cavernous malformation (CCM) proteins, CCM1, CCM2, and CCM3, regulate cell-cell adhesion, cell shape and polarity, and most likely cell adhesion to extracellular matrix. Recently, CCM2 and CCM3 are known to be expressed in control and varicocele-induced rat testes, but little is known about these proteins during gonadogenesis. This led us to study the CCM proteins during the mouse gonadogenesis. Neonatal (PND 0), postnatal, and adult mice testes and ovaries were obtained from mice. CCM2 and CCM3 expression were analyzed during mouse testicular and ovarian development by immunohistochemistry and quantitative real-time PCR. The results showed that in both sexes, Ccm2 and Ccm3 mRNA and protein were first detectable after gonadogenesis when the gonads were well differentiated and remained present until the adult stage. In the testis, CCM2 and CCM3 expression were restricted to the nuclei of Sertoli cells, suggesting a conserved role in testicular differentiation. In the ovary, the CCM2 and CCM3 proteins were localized in the cytoplasm of oocytes, suggesting an unexpected role during oogenesis. Quantitative real-time PCR (qRT-PCR) results showed that expression of Ccm2 and Ccm3 genes could play a role in the regulation of mouse gonadogenesis translational activation upon testicular and ovarian development. The localization of CCM2 and CCM3 proteins show their different functions for CCM2 and CCM3 which may have important roles in testicular and ovarian differentiation. In conclusion, CCM2 and CCM3 may be involved in establishing the differential expression pattern in developing mouse testis and ovary

Effect of c-Abl gene silencing by siRNA on proliferation of mouse granulosa cells

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Superovulation alters embryonic poly(A)-binding protein (Epab) and poly(A)-binding protein, cytoplasmic 1 (Pabpc1) gene expression in mouse oocytes and early embryos

WOS: 000368127000013PubMed ID: 25034140Embryonic poly(A)-binding protein (EPAB) and poly(A)-binding protein, cytoplasmic 1 (PABPC1) play critical roles in translational regulation of stored maternal mRNAs required for proper oocyte maturation and early embryo development in mammals. Superovulation is a commonly used technique to obtain a great number of oocytes in the same developmental stages in assisted reproductive technology (ART) and in clinical or experimental animal studies. Previous studies have convincingly indicated that superovulation alone can cause impaired oocyte maturation, delayed embryo development, decreased implantation rate and increased postimplantation loss. Although how superovulation results in these disturbances has not been clearly addressed yet, putative changes in genes related to oocyte and early embryo development seem to be potential risk factors. Thus, the aim of the present study was to determine the effect of superovulation on Epab and Pabpc1 gene expression. To this end, low- (5IU) and high-dose (10IU) pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotrophin (hCG) were administered to female mice to induce superovulation, with naturally cycling female mice serving as controls. Epab and Pabpc1 gene expression in germinal vesicle (GV) stage oocytes, MII oocytes and 1- and 2-cell embryos collected from each group were quantified using quantitative reverse transcription-polymerase chain reaction. Superovulation with low or high doses of gonadotropins significantly altered Epab and Pabpc1 mRNA levels in GV oocytes, MII oocytes and 1- and 2-cell embryos compared with their respective controls (P<0.05). These changes most likely lead to variations in expression of EPAB- and PABPC1-regulated genes, which may adversely influence the quality of oocytes and early embryos retrieved using superovulation.Akdeniz University [2008.01.0103.006]This study was supported by a grant from the Akdeniz University Research Fund (2008.01.0103.006). The authors thank Donald Desmond for helpful comments on and corrections to this article

Superovulation alters embryonic poly(A)-binding protein (Epab) and poly(A)-binding protein, cytoplasmic 1 (Pabpc1) gene expression in mouse oocytes and early embryos

İstanbul Bilim Üniversitesi, Tıp Fakültesi.Embryonic poly(A)-binding protein (EPAB) and poly(A)-binding protein, cytoplasmic 1 (PABPC1) play critical roles in translational regulation of stored maternal mRNAs required for proper oocyte maturation and early embryo development in mammals. Superovulation is a commonly used technique to obtain a great number of oocytes in the same developmental stages in assisted reproductive technology (ART) and in clinical or experimental animal studies. Previous studies have convincingly indicated that superovulation alone can cause impaired oocyte maturation, delayed embryo development, decreased implantation rate and increased postimplantation loss. Although how superovulation results in these disturbances has not been clearly addressed yet, putative changes in genes related to oocyte and early embryo development seem to be potential risk factors. Thus, the aim of the present study was to determine the effect of superovulation on Epab and Pabpc1 gene expression. To this end, low- (5 IU) and high-dose (10 IU) pregnant mare’s serum gonadotropin (PMSG) and human chorionic gonadotrophin (hCG) were administered to female mice to induce superovulation, with naturally cycling female mice serving as controls. Epab and Pabpc1 gene expression in germinal vesicle (GV) stage oocytes, MII oocytes and 1- and 2-cell embryos collected from each group were quantified using quantitative reverse transcription–polymerase chain reaction. Superovulation with low or high doses of gonadotropins significantly altered Epab and Pabpc1 mRNA levels in GV oocytes, MII oocytes and 1- and 2-cell embryos compared with their respective controls (P < 0.05). These changes most likely lead to variations in expression of EPAB- and PABPC1-regulated genes, which may adversely influence the quality of oocytes and early embryos retrieved using superovulation

Impaired follicular development and ovulation in PCOS mouse model can rescued by rapamycin treatment

34th Annual Meeting of the European-Society-of-Human-Reproduction-and-Embryology (ESHRE) -- JUN 01-04, 2018 -- Barcelona, SPAINWOS: 000438519902203Polycystic ovary syndrome (PCOS) is a common and complex endocrine disorder affecting 5-10% of women in reproductive age that is characterized by hyperandrogenism, oligo- or anovulation and infertility. However the pathophysiology of PCOS still remains unknown. The mammalian target of rapamycin (mTOR) is a central component that regulates various processes including cell growth, proliferation, metabolism, and angiogenesis. mTOR signaling cascade has recently been examined in ovarian follicles where it regulates granulosa cell proliferation and differentiation. mTOR functions as two complexes, mTOR complex 1 and 2. Therefore, we hypothesized that mTORC1 and/or 2 may have important role in proliferation of theca and granulosa cells in PCOS. In the present study, we sought to determine the mTOR signaling pathway in PCOS mouse ovary. We designed 3 groups: Control (C, no treatment), PCOS (P, The injection of DHEA (6 mg/100 g BW in 0.1 ml of sesame oil) (s.c) for 20 consecutive days), Vehicle (V, daily (s.c) sesame oil alone injection). Our results showed that mTORC1 and mTORC2-mediated signaling may play a role in PCOS mouse ovary. These findings provide evidence that mTORC1 and mTORC2 may have responsibility in increased ovarian follicular cell proliferation and growth in PCOS. Consequently, these results suggest that the mTOR signaling pathways (mTORC1 and mTORC 2) may create new clinical strategies to optimize developmental competence of PCOS should target correction of the entire follicle growth, oocyte development process and anovulatory infertility in PCOSEuropean Society of Human Reproduction and Embryolog