Cell and Tissue Journal

Abstract Aim: The lipid compositions of mammalian plasma membrane of spermatozoa are highly sensitive to oxidative stress due to the presence of high amounts of unsaturated fatty acids. If the production of active oxygen compounds is more than the antioxidant capacity of sperm to neutralize its effects, the sperm will undergo oxidative damage, which is characterized by the reduction of qualitative and viability parameters of spermatozoa. In recent years, research has been done on the antioxidant properties of some plants and their protective role for animal spermatozoa. So, the purpose of this research was to investigate the in vitro effect of Syzygium aromaticum extract on the spermatozoa qualitative parameters and preservation of sperm viability under the semen storage in liquid condition.
Material and Methods: Semen collection was done from 8 Arabi rams weekly for 6 weeks and their semen was immediately mixed and after dilution, divided into 5 parts. The treatments included the addition of different levels of zero (control), 25, 50, 75 and 100 µg/ml of clove bud extract to the semen diluent. At zero, 24, 48, 72 and 96 hours after storage the semen samples at 5°C, the quality parameters of the spermatozoa included the motility, viability, plasma membrane integrity and morphological defect rates were evaluated.
Results: At zero time, spermatozoa quality parameters were not affected by the treatments (P<0.05). In 24 hours, the percentage of motility, viability and plasma membrane integrity of spermatozoa in 100 µg/ml of Syzygium aromaticum extract decreased (P<0.05), and lower levels of extract were ineffective on the mentioned parameters of spermatozoa compared to the control. But, after the storage of samples up to 96 hours, 25 and 50 µg/ml of extract significantly improved the percentage of motility, viability and plasma membrane integrity of spermatozoa compared to the control (P<0.05). There was no significant difference in the percentage of spermatozoa morphological abnormalities among the treatments (P<0.05). 
Conclusion: In general, adding 25 µg/ml of Syzygium aromaticum extract to semen diluent of Arabian ram improved the motility, viability and plasma membrane integrity of spermatozoa as well as maintain their viability during the storage of semen under 5˚C. 

Abstract Aim: The sperm membrane is rich in unsaturated fatty acids, which is very sensitive to the damage caused by ROS as a result of lipid peroxidation. Using suitable antioxidants in semen extenders can reduce the level of ROS. Rutin is a naturally occurring antioxidant polyphenolic flavonoid with powerful radical scavenging and lipid peroxidation inhibition properties. However, no studies have investigated the potential impact of rutin supplementation in cooling preservation of ram epididymal sperm. The current research was aimed to compare different concentrations of rutin antioxidant in the cooling process.
Material and methods: In this experimental study, testes of mature rams were collected from a local slaughterhouse and transferred to the laboratory within 1 hours in saline (0.9% NaCl) solution. In the laboratory, the epididymis was separated from the testis and cleaned from any connective tissues and blood vessels. For sperm recovery, the cauda epididymis was trimmed with a scalpel and epididymal sperm was obtained by cutting into small pieces in 5 ml of tris base extender pre-warmed at 37°C and incubated for 10 min to allow sperm swim out. The diluted sperm samples were divided into five equal experimental groups including: 1) tris base extender (Control), 2) extender containing 0.5 mM rutin, 3) extender containing 0.75 mM rutin, 4) extender containing 1 mM rutin, and 5) extender containing 1.25 mM rutin. The sperm samples were cooled gradually from 37°C to 4°C in 2 hours and held at 4°C. Sperm motility, viability, membrane integrity and lipid peroxidation were evaluated after extension (0 h) and at 24 and 48 hours of storage. The motility, viability, membrane integrity and morphology of sperm samples were evaluated immediately after extension (0 h) and at 24 and 48 h of storage at 4°C. The sperm motion kinematics were objectively evaluated by using the computer-assisted sperm analysis (CASA) system. The eosin-nigrosin staining technique was applied to evaluate viability. The hypo-osmotic swelling test (HOST) was performed to assess membrane integrity. Sperm morphology was determined by staining sperm smears with Hancock’s solution. Lipid peroxidation was evaluated by measuring malondialdehyde (MDA) concentration.
Results: The data obtained from this experiment show that after 24 and 48 hours of cooling, the addition of rutin antioxidant at a concentration of 0.75 and 1 mM causes a significant increase in total motility, progressive motility and straight path velocity. The results of the present study show that experimental treatments at 0, 24 and 48 hours after cooling have no effect on VAP, VCL and STR. The results of this experiment show that after 24 and 48 hours of cooling, the addition of rutin antioxidants at concentrations of 0.75 and 1 mM significantly increases the viability and integrity of the membrane. Also, the results show that the experimental treatments reduce the amount of MDA. The results show that the applied treatments have no effect on sperm morphology.
Conclusion: The results show that using rutin antioxidant in concentrations of 0.75 and 1 mM improves the quality of ram sperm.

Abstract Aim: The main purpose of the recent study was to evaluate gill chloride cells in Cyprinus carpio during adaptation to different environmental salinities over a short period.
Material and Methods: For this research, Cyprinus carpio by an average weight of 41.4±1 g and length of 16.9±1 cm in four groups with three replications was used. The first group as control was located in fresh water and the next three groups respectively were kept in 4 ppt, 8 ppt, and 12 ppt salinity with the exact condition. On 0, 6, 12, 24, 48, 72, and 96 h the second-gill arch with a maximum thickness of 0.5 cm from the left side was prepared and placed in formalin’s buffer 10%, glutaraldehyde 2.5% solution, and pH 7.4. Then the standard method of preparing tissue sections was performed and paraffin sections with a thickness of 4-6 microns were prepared and stained with hematoxylin and eosin methods and examined with a light microscope. Also, changes in the structure and number of mitochondrial-rich cells, distribution, and dispersion of Na+/K+-ATPase enzyme were studied by scanning electron microscope and immunohistochemical technique.
Results: Results showed obvious modifications in the number and distribution of chloride cells in two filament and lamellar positions at different sampling times. The highest number of chloride cells in the filament and lamella, 26.28±1.33; 19.11±1.27 was reported in 12ppt and the lowest number belonged to the control group. The largest size of chloride cells in the filament and lamella, 22.51±1.56; 12.72±1.32 was reported in 12ppt and the lowest size belonged to the control group.
Conclusion: The findings of this study showed that C. carpio gill chloride cells in the face of different salinities in a short period can change the number and size and adapt to the new condition.

Abstract Aim: About 80% of deaths caused by diabetes are related to cardiovascular diseases. Diabetes and morphine are the main risk factors for cardiovascular diseases that lead to endothelial dysfunction and defective angiogenesis. Today, many angiogenic and angiostatic factors have been identified that play an important role in cardiovascular health, and the most important of them are VEGF (angiogenic factor) and endostatin (angiostatic factor). In this study, the effect of eight weeks of endurance training on vascular endothelial growth factor  (VEGF) and endostatin  (ES) protein levels in the heart tissue of diabetic male Wistar rats with morphine withdrawal syndrome was investigated.
Material and methods: In this experimental study, 32 male Wistar rats were randomly divided into 4 groups of 8 including diabetes control  (D), morphine diabetes  (DM), diabetes + endurance training  (D.ET) and morphine diabetes+ endurance training  (D.M.ET). Then induction of diabetes and addiction to morphine was done. 72 hours after the injection, in order to ensure diabetes, the samples whose blood sugar level was more than 250 mg/dL were considered as diabetic. In order to ensure the dependence of the rats on morphine, at the end of the 21st day, by intraperitoneal injection of naloxone to the samples at the rate of 2 mg/kg of body weight, the signs of withdrawal from addiction such as: jumping, climbing, scratching, teeth grinding , redness around the eyes, diarrhea, tremors, eyelid drooping, erection and standing on two legs for 30 minutes were evaluated. The training groups performed an 8-week endurance training program. At the end of the study, all rats were sacrificed and their heart tissue was removed. VEGF and ES protein levels were measured by ELISA method. Data were analyzed using one-way ANOVA test at a significance level of p≤0.05.
Results: The results showed that in diabetic and morphine rats, the level of VEGF and ES is significantly lower and higher than other groups, respectively  (P<0.05). Also, endurance training is associated with improvement of VEGF and ES values in diabetes + endurance training and morphine diabetes+ endurance training groups  (P<0.05). On the other hand, the level of VEGF and ES in group D with D.M.ET showed no significant difference (P>0.05).
Conclusion: In the present study, it was shown that eight weeks of endurance training caused a significant increase in VEGF in the heart tissue of diabetic rats with morphine withdrawal syndrome. Also, the level of ES protein as one of the most important internal inhibitory factors of angiogenesis decreased significantly. Our findings show that the intervention of endurance training is likely to be effective and useful in regulating the levels of angiogenic factors in the heart tissue of diabetic and diabetic rats in withdrawal syndrome and as a low-cost non-pharmacological solution in the treatment of cardiovascular diseases caused by addiction and diabetes are recommended to specialists in these fields.

Abstract Aim: In this review article, we intend to investigate and review the factors that lead to embryonic developmental arrest and explore different strategies to address this phenomenon. We aim to pave the way for further research in this field and enhance the efficiency of the IVF method for infertility treatment.
 
Introduction: To ensure proper embryonic development, embryos need to progress through their developmental stages unimpeded. Any disruptions in these stages including oocyte factors, sperm factors, and embryonic factors can result in infertility due to pre-implantation developmental arrest. Therefore, it can be very helpful to understand the effective factors behind this phenomenon.
 
Topic: Among couples attempting to conceive, infertility persists despite their best efforts. Couples are typically considered infertile if they have tried to conceive for twelve months without success and without using protection; but if the woman is over 37 years old, this twelve months will be reduced to six months. Assisted reproductive techniques (ART) encompass various methods that are aimed at aiding these couples in overcoming infertility. This definition includes any manipulation of embryos and eggs for infertility treatment. Assisted reproductive techniques include in vitro maturation (IVM), in vitro fertilization (IVF), and intracytoplasmic sperm injection (ICSI). Among these, IVF/ICSI is particularly effective in treating couples infertility. However, one common challenge in this process is embryonic developmental arrest during the pre-implantation stages, a primary cause of infertility in treatment cycles. Approximately 40 to 50 percent of IVF cycles do not progress to the blastocyst stage and arrest. This arrest characterized by a lack of cell division for at least 24 hours. Most embryonic arrests occur on the second and third day after fertilization, during the two to eight-cell stages. These arrested embryos retain their developmental potential, and their gene expression program related to their arrested stage remains unaffected. Arrested embryos can be classified into three categories: those with impaired activation of the embryonic genome (EGA), those with low levels of glycolysis, and those with variable levels of oxidative phosphorylation. Solutions to overcome embryonic developmental arrest include maternal spindle transfer (MST)/nuclear transfer (NT), optimizing culture media, employing antioxidants, and synthesizing complementary RNA (cRNA)/small interfering RNA (siRNA).
 
Conclusion: Pre-implantation arrest can result from various factors, either of embryonic, maternal, and paternal origin. Among embryonic factors, disturbances in fields like gene expression, mitochondrial activity, methylation patterns, chromosomal abnormalities, small non-coding RNAs, and embryonic metabolic status have the most significant impact on embryonic arrest induction. Additionally, parental factors, like genetic factors, and infertility etiology can lead to embryonic developmental arrest. External factors, such as laboratory conditions, ART methods, and the role of the physician, also play a role in embryonic developmental arrest. While treatment studies for overcoming embryonic arrest are very limited, they are generally based on animal models, which is why it is necessary to conduct more studies and enter the human phase. A comprehensive understanding of the causes of embryonic arrest and solutions to address them can enhance infertility treatment technologies and improve the effectiveness of the IVF method for infertility treatment.

Abstract Aim: Spermatogonial stem cells (SSCs) are the basis of male spermatogenesis and fertility. SSCs are distinguished by their ability to self-renew and differentiate into spermatozoa throughout the male reproductive life and pass genetic information to the next generation. Immunohistochemical analysis showed PLZF-positive cells in the basement membrane of the seminiferous tubule. It seems that the PLZF germ cell marker is specifically expressed in the spermatogonial cells of the testis. In mice with genetic deletion of the VASA gene, males show reproductive deficiency with reduced sperm production. In this study, we looked at the expression of PLZF and VASA in spermatogenic tubules and germ cells in vivo and in vitro. Material and method: Isolation of spermatogonial stem cells, Cultivation of testis on STO feeder layer, Immunohistochemistry (IHC), Immunocytochemistry (ICC) and Fluidigm reverse transcriptase-polymerase chain reaction (RT-PCR), Network analysis of protein-protein interactions (PPI), Pathway enrichment analysis and gene analysis (GO), were used to analyze the expression of PLZF and VASA in mice testis tissue. Results: In this experimental study, whereas undifferentiated spermatogonial cells sharply express PLZF, other types of germ cells located in the seminefrous tubul were negative for this marker. In other hand, the germ cells near the basal membrane of seminefrous tubul showed expression of VASA wheras the undifferentiated germ cells located on the basal membrane were negative. The ICC analysis indicated higher expression of PLZF in the isolated undifferetiated cells in compare to the differentiated germ cells. Fluidigm real-time RT-PCR result demonstrated a significant expression (P<0.05) of VASA in the spermatogonial stem cells compared to differentiated cells and also showed expression of PLZF in undifferentiated spermatogonia. In the present experiment, after the production of SSCs under the stimulation of growth factors FGF, EGF, and GDNF, immunocytochemical staining showed a clear expression of PLZF and VASA in SSCs compared to in-vivo conditions, and VASA is less expressed in these cells. The data obtained from IHC analysis showed that VASA is expressed in the center of testicular cords. The data set related to protein-protein interaction members was used in this research due to the lack of information about PLZF expression in different stages of spermatogenesis. The study of gene expression showed that several biological and functional pathways are involved in the expression of PLZF in different stages of spermatogenesis. Conclusion: These results clearly proved the role of PLZF as a specific marker for spermatogonial stem cells, and can be beneficial for advance reserach about in-vitro differentiation of SSCs to functional sperms.