Mahmoud Hashemitabar; Elham Heidari; Mahmoud Orazizadeh; Susan Sabbagh; Mahsa Afrough; Maryam Dastoorpoor; Ata A. Ghadiri
Volume 20, Issue 12 , December 2018, , Pages 1-10
Abstract
Background: Asthenozoospermia (astheno) is a common male infertility disorder associated with low sperm motility. The pro- gressive movement of sperm is an important factor in the fertilization rate, and it requires a high level of adenosine triphosphate (ATP). Objectives: This experimental study aimed ...
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Background: Asthenozoospermia (astheno) is a common male infertility disorder associated with low sperm motility. The pro- gressive movement of sperm is an important factor in the fertilization rate, and it requires a high level of adenosine triphosphate (ATP). Objectives: This experimental study aimed to identify the role of cytochrome c oxidase 6B2 (COX6B2) as an important functional subunit of Cytochrome-c Oxidase in sperm motility. Methods: According to the World Health Organization (WHO) criteria, Semen samples were collected from 14 asthenozoospermia and 16 normospermia individuals that were referring to the Infertility Research and Treatment Center of Khuzestan, Iran in October2016- May 2017. The sperm from two groups was isolated via the Percoll density gradient centrifugation to prepare healthy, motile sperm for COX6B2 immunofluorescent staining and real-time polymerase chain reaction (PCR). In addition, apoptosis assessment was carried out simultaneously to compare apoptosis and the COX6B2 expression level. To analyze the data, descriptive statistics including frequency and mean and analytical statistics including Fisher’s exact test and two independent samples were used. Results: COX6B2 was detected in midpiece of the sperm by immunofluorescence assays. In addition, the percentage of COX6B2 positive sperm in the astheno samples was almost half that of the normal group (49.0 ± 15.8 to 28.7 ± 14.1, P = 0.641). Real-time PCR definitely reconfirmed the immunofluorescent staining result. A decrease in apoptosis was shown in as the no samples compared with the normal group (19.1 ± 0.4 to 9 ± 0.2, P = 0.04). Conclusions: The expression of COX6B2 in the sperm midpiece represents the OXPHOS pathway and functionality of mitochondria in sperm. This study introduced COX6B2 staining as a potential functional test for the recognition of competent mitochondria in sperm and it could be assigned as a biomarker in male-factor patients.
Behrouz Taheri; Mohammad-Hossein Modarressi; Mahmoud Hashemitabar; Mohammad Miryounesi; Elahe Motevaseli
Volume 20, Issue 8 , August 2018, , Pages 1-6
Abstract
Background: Leukemia Inhibitory Factor (LIF) is largely used in stem cell researches for the maintenance of Embryonic Stem Cells (ESCs) in a pluripotent state. However, the relatively high costs of LIF is a potential limit of such researches.Objectives: The aim of this study was prokaryotic expression ...
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Background: Leukemia Inhibitory Factor (LIF) is largely used in stem cell researches for the maintenance of Embryonic Stem Cells (ESCs) in a pluripotent state. However, the relatively high costs of LIF is a potential limit of such researches.Objectives: The aim of this study was prokaryotic expression and purification of the recombinant hexahistidine-tagged human LIF (His6-hLIF) fusion protein and assessment of its ability to maintain a pluripotent or undifferentiated state of ESCs. Methods: Encoding the DNA sequence of mature hLIF was codon optimized for expression in Escherichia coli and chemically syn- thesized and cloned in the expression vector pET- 28a (+). Immobilized Metal Affinity Chromatography (IMAC) was performed to purify the recombinant His6-hLIF. Then, His6-hLIF was tested for its ability to maintain mESC by comparison with commercial LIF as a control. Results: The yield for the recombinant His6-hLIF was assessed to be approximately 1.7 mg from one liter of culture. There were no statistically significant differences in expression of two pluripotency gene markers, oct-4 and Nanog, between mESCs treated withHis6-hLIF and those with commercial hLIF (P = 0.09 and P = 0.13, respectively). Besides, morphological characteristics (round cellular morphology) were similar between them. Conclusions: Collectively, the findings showed that the ability of the recombinant His6-hLIF protein in maintaining pluripotent state of ESCs was comparable to commercial hLIF, providing evidence that the presence of the N-terminal hexahistidin tag does not influence biological activities of hLIF.
