Cell Journal (Yakhteh)

7-14

Authors: Fatemeh Ghasemian, Mohammad Hadi Bahadori, Seyedeh Zahra Hosseini Kolkooh, Maryam Esmaeili

Objective: It is necessary to evaluate fertility effective agents to predict assisted reproduction outcomes. This study was designed to examine sperm vacuole characteristics, and its association with sperm chromatin status and protamine-1(PRM1) to protamine-2 (PRM2) ratio, to predict assisted pregnancy outcomes. Materials and Methods: In this experimental study, ninety eight semen samples from infertile men were classified based on Vanderzwalmen’s criteria as follows: grade I: no vacuoles; grade II: ≤2 small vacuoles; grade III: ≥1 large vacuole and grade IV: large vacuole with other abnormalities. The location, frequency and size of vacuoles were assessed using high magnification, a deep learning algorithm, and scanning electron microscopy (SEM). The chromatin integrity, condensation, viability and acrosome integrity, and protamination status were evaluated for vacuolated samples by toluidine blue (TB) staining, aniline blue, triple staining, and CMA3 staining, respectively. Also, Protamine-1 and protamine-2 genes expression was analysed by reverse transcription-quantitative polymerase chain reaction (PCR). The assisted reproduction outcomes were also followed for each cycle. Results: The results show a significant correlation between the vacuole size (III and IV) and abnormal sperm chromatin condensation (P=0.03 and P=0.02, respectively), and also, protamine-deficient (P=0.04 and P=0.03, respectively). The percentage of reacting acrosomes was significantly higher in the grades III and IV spermatozoa in comparison with normal group. The vacuolated spermatozoa with grade IV showed a high protamine mRNA ratio (PRM-2 was underexpressed, P=0.01). In the IVF cycles, we observed a negative association between sperm head vacuole and fertilization rate (P=0.01). This negative association was also significantly observed in pregnancy and live birth rate in the groups with grade III and IV (P=0.04 and P=0.03, respectively). Conclusion: The results of our study highlight the importance sperm parameters such as sperm head vacuole characteristics, particularly those parameters with the potency of reflecting protamine-deficiency and in vitro fertilization/ intracytoplasmic sperm injection (IVF/ICSI) outcomes predicting.

22-27

Authors: Fatemah Sadat Mostafavi, Abbas Bakhteyari, Parvaneh Nikpour, Nahid Eskandari, Roshanak Aboutorabi

Objective: Given the prevalence of fertility problems in couples and the defect in embryo implantation as well as the low success rate of assisted reproductive techniques, it is necessary to investigate the causes of this phenomenon. Type 2 diabetes mellitus (T2DM) is a metabolic disease with multiple effects on various organs as well as the endometrium. In this study, the effects of endometrial cell culture on the expression of α3 and β1 integrin genes and protein in type 2 diabetic rats were investigated. Materials and Methods: In this experimental study, 35 female rats were divided into five groups: control, sham, diabetic, Pioglitazone-treated and Metformin-treated groups. First, rats were maintained in diabetic condition for 4 weeks. Then, treatment was performed for the next four weeks. Four weeks after induction of diabetes, rats were sacrificed at the time of embryo implantation. The uterus was removed. Endometrial cells were isolated and cultured for 7 days. Immunocytochemistry staining was used to confirm endometrial cells. Expression of α3 and β1 integrin genes was determined by real-time polymerase chain reaction (PCR) technique and the α3β1 protein content measured using Western blot both before and after endometrial cell culture. Result: The expression level of α3 integrin gene in the Pioglitazone-treated group compared with metformin-treated group was significantly decreased (P<0.001). The same result was observed in β1 integrin gene expression (P=0.004). Also, the α3β1 protein level increased in all diabetic groups, but its reduction was significantly greater in pioglitazonetreated group (P=0.004). Conclusion: T2DM altered the expression of α3 and β1 integrin genes and related proteins, which endometrial cell culture regulated this disorder. According to these results, may be the endometrial cell culture can reduce the adverse effects of diabetes on α3 and β1 integrin expression at the level of gene and protein, in endometrial cells.

36-43

Authors: Mohamad Pezeshki-Modaress, Mohadeseh Akbarzadeh, Dariush Ebrahimibagha, Mojgan Zandi, Tayyeb Ghadimi, Amin Sadeghi, Sarah Rajabi

Objective: Poly(ε-caprolactone) (PCL) has considerable mechanical and biological properties that make it a good candidate for tissue engineering applications. PCL alongside proteins and polysaccharides, like gelatin (GEL) and chondroitin sulphate (CS), can be used to fabricate composite scaffolds that provide mechanical and biological requirements for skin tissue engineering scaffolds. The aim of this study was fabricating novel composite nanofibrous scaffold containing various ratios of GEL/CS and PCL using co-electrospinning process. Materials and Methods: In this experimental study, PCL mixed with a GEL/CS blend has limitations in miscibility and the lack of a common solvent. Here, we electrospun PCL and GEL/CS coincide separately on the same drum by using different nozzles to create composite nanofibrous scaffolds with different ratios (2:1, 1:1 and 1:2) of GEL to CSPCL, and we mixed them at the micro/nanoscale. Morphology, porosity, phosphate-buffered saline (PBS) absorption, chemical structure, mechanical property and in vitro bioactivity of the prepared composite scaffolds were analysed. Results: Scanning electron microscopy (SEM) images showed beadless nanofibres at all ratios of GEL to CS-PCL. The composite scaffolds (2:1, 1:1 and 1:2) had increased porosity compared to the PCL nanofibrous scaffolds, in addition to a significant increase in PBS absorption. The mechanical properties of the composite scaffolds were investigated under different conditions. The results demonstrated that all of the composite specimens had better strength when compared with the GEL/CS nanofibres. The increase in PCL ratio led to an increase in tensile strength of the nanofibres. Human dermal fibroblasts (HDF) were cultured on the fabricated composite scaffolds and evaluated by 3-(4,5-dimethylthiazol- 2-yl)-5-(3 carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) analysis and SEM. The results showed the bioactivity of these nanofibres and the potential for these scaffolds to be used for skin tissue engineering applications. Conclusion: The fabricated co-electrospun composite scaffolds had higher porosity and PBS absorption in comparison with the PCL nanofibrous scaffolds, in addition to significant improvements in mechanical properties under wet and dry conditions compared to the GEL/CS scaffold.

51-54

Authors: Da Som Jeong, Yu Cheon Kim, Ji Hoon Oh, Myoung Hee Kim

General control non-derepressible 5 (Gcn5) is a member of histone acetyltransferase (HAT) that plays key roles during embryogenesis as well as in the development of various human cancers. Gcn5, an epigenetic regulator of Hoxc11, has been reported to be negatively regulated by Akt1 in the mouse embryonic fibroblasts (MEFs). However, the exact mechanism by which Akt1 regulates Gcn5 is not well understood. Using protein stability chase assay, we observed that Gcn5 is negatively regulated by Akt1 at the post-translational level in MEFs. The stability of Gcn5 protein is determined by the competitive binding with the protein partner that interacts with Gcn5. The interaction of Gcn5 and Cul4a-Ddb1 complex predominates and promotes ubiquitination of Gcn5 in the wild-type MEFs. On the other hand, in the Akt1-null MEFs, the interaction of Gcn5 and And-1 inhibits binding of Gcn5 and Cul4a-Dbd1 E3 ubiquitin ligase complex, thereby increasing the stability of the Gcn5 protein. Taken together, our study indicates that Akt1 negatively controls Gcn5 via the proteasomal degradation pathway, suggesting a potential mechanism that regulates the expression of Hox genes.