Lipid peroxidation and its role in the expression of NLRP1a and NLRP3 genes in testicular tissue of male rats: a model of spinal cord injury

Background: The majority of male patients with spinal cord injury [SCI] suffer from infertility. Nucleotide-binding oligomerization domain-like receptors NOD-like receptors [NLRs] are a kind of receptors that corporate in the inflammasome complex. Recent studies have introduced the inflammasome as the responsible agent for secreting cytokines in semen. Reactive oxygen species [ROS] is one of the elements that trigger inflammasome activation. Genital infections in SCI can lead to ROS generation. We investigated the relation between lipid peroxidation and inflammasome complex activity in testicular tissue of SCI rats

Methods: Adult male rats [n=20], weighting 200- 250 g, were included and divided into four groups: three experimental groups, including SCI1, SCI3, and SCI7, i.e. the rats were subjected to SCI procedure and sacrificed after one, three, and seven days, respectively and a control group. We performed a moderate, midline spinal contusion injury at thoracic level 10. The animals were anesthetized, and testes were collected for measurement of gene expression by real-time PCR. Caudal parts of epididymis were collected for malondialdehyde [MDA] measurement

Results: No NLRP1a mRNA overexpression was seen in the testes of control and SCI groups. After seven days from SCI surgery, NLRP3 mRNA expression was significantly increased in SCI7 animals [p

Conclusion: NLRP3 overexpression occurs due to the increased ROS production in testis tissue of SCI rats

Flaxseed can reduce hypoxia-induced damages in rat testes

Background: Hypoxia causes detrimental effects on the structure and function of tissues through increased production of reactive oxygen species that are generated during the re-oxygenation phase of intermittent and continuous hypobaric hypoxia. This study was carried out to evaluate the effects of flaxseed [Fx] in reducing the incidence of hypoxia in rat testes

Materials and Methods: In this experimental study, 24 adult Wistar rats were randomly divided into four groups: i. Control group [Co] that received normal levels of oxygen and food, ii. Sham group [Sh] that were placed in hypoxia chamber but received normal oxygen and food, iii. Hypoxia induction group [Hx] that were placed in hypoxia chamber and treated with normal food, iv. Hypoxia induction group [Hx+Fx] that were placed in hypoxia chamber and treated with 10% flaxseed food. Both the Hx and Hx+Fx groups were kept in a hypoxic chamber for 30 days; during this period rats were exposed to reduced pressure [oxygen 8% and nitrogen 92%] for 4 hours/day. Then, all animal were sacrificed and their testes were removed. Malondialdehyde [MDA] and total antioxidant capacity [TAC] levels were evaluated in the testis tissue. Tubular damages were examined using histological studies. Blood samples and sperm were collected to assess IL-18 level and measure sperms parameters, respectively. All data were analyzed using SPPSS-22 software. One way-ANOVA or Kruskal-Wallis tests were performed for statistical analysis

Results: A significant difference was recorded in the testicular mass/body weight ratio in Hx and Hx+Fx groups in comparison to the control [P=0.003 and 0.027, respectively] and Sh [P=0.001 and 0.009, respectively] groups. The sperm count and motility in Hx+Fx group were significantly different from those of the Hx group [P=0.0001 and 0.028, respectively]. Also sperm viability [P=0.0001] and abnormality [P=0.0001] in Hx+Fx group were significantly different from Hx group

Conclusion: This study therefore suggests that the oral administration of flaxseed can be useful for prevention from the detrimental effects of hypoxia on rat testes structure and sperm parameters

Methylation status of H19/IGF2 differentially methylated region in in vitro human blastocysts donated by healthy couples

Backgrund: Imprinted genes are a unique subset of few genes, which have been differentially methylated region [DMR] in a parental origin-dependent manner during gametogenesis, and these genes are highly protected during pre-implantation epigenetic reprogramming. Several studies have shown that the particular vulnerability of imprinting genes during suboptimal pre- and peri-conception micro-environments often is occurred by assisted reproduction techniques [ART]. This study investigated the methylation status of H19/IGF2 DMR at high-quality expanding/expanded human blastocysts donated by healthy individuals to evaluate the risks linked to ART

Method: Methylation levels of H19/IGF2 DMR were analyzed by bisulfite conversion and sequencing at 18 CpG sites [CpGs] located in this region

Result: The overall percentage of methylated CpGs and the proportion of hyper-methylated clones of H19/IGF2 DMR in analyzed blastocysts were 37.85 +/- 4.87% and 43.75 +/- 5.1%, respectively. For validation of our technique, the corresponding methylation levels of peripheral human lymphocytes were defined [49.52 +/- 1.86% and 50%, respectively]

Conclusion: Considering the absence of in vivoproduced human embryos, it is not possible to conclude that the methylation found in H19/IGF2 DMR is actually normal or abnormal. Regarding the possible risks associated with ART, the procedures should be optimized in order to at least reduce some of the epigenetic risks

Role of somatic testicular cells during mouse spermatogenesis in three-dimensional collagen gel culture system

Spermatogonial stem cells [SSCs] are the only cell type that can restore fertility to an infertile recipient following transplantation. Much effort has been made to develop a protocol for differentiating isolated SSCs in vitro. Recently, three-dimensional [3D] culture system has been introduced as an appropriate microenvironment for clonal expansion and differentiation of SSCs. This system provides structural support and multiple options for several manipulation such as addition of different cells. Somatic cells have a critical role in stimulating spermatogenesis. They provide complex cell to cell interaction, transport proteins and produce enzymes and regulatory factors. This study aimed to optimize the culture condition by adding somatic testicular cells to the collagen gel culture system in order to induce spermatogenesis progression. In this experimental study, the disassociation of SSCs was performed by using a two-step enzymatic digestion of type I collagenase, hyaluronidase and DNase. Somatic testicular cells including Sertoli cells and peritubular cells were obtained after the second digestion. SSCs were isolated by Magnetic Activated Cell Sorting [MACS] using GDNF family receptor alpha-1 [Gfr Alpha -1] antibody. Two experimental designs were investigated. 1. Gfr Alpha -1 positive SSCs were cultured in a collagen solution. 2. Somatic testicular cells were added to the Gfr Alpha -1 positive SSCs in a collagen solution. Spermatogenesis progression was determined after three weeks by staining of synaptonemal complex protein 3 [SCP3]-positive cells. Semi-quantitative Reverse Transcription PCR was undertaken for SCP3 as a meiotic marker and, Crem and Thyroid transcription factor-1 [TTF1] as post meiotic markers. For statistical analysis student t test was performed Testicular supporter cells increased the expression of meiotic and post meiotic markers and had a positive effect on extensive colony formation. Collagen gel culture system supported by somatic testicular cells provides a microenvironment that mimics seminiferous epithelium and induces spermatogenesis in vitro

[Neurotrophic effect of tacrolimus on pyramidal cells of CA1 region of hippocampus following transient global ischemia/reperfusion in male wistar rat]

Cerebral ischemia is known as a major problem in the world. Reperfusion following the ischemia ultimately leads to programmed cell death or apoptosis. Specific regions of the brain and certain types of neurons are more susceptible to cerebral ischemia, such as pyramidal neurons of CA1 region of hippocampus. Recently, using of immunophilin ligands has been considered to be a potential and appropriate strategy for neuroprotective and neurogenitor activity. Up to now, the right time of injection for providing the suitable effect on pyramidal cells of CA1 has not been assessed precisely. In this study, the neurotrophic effects of tacrolimus on CA1 cells were studied on 40 male Wistar rats in 8 experimental groups. Ischemia model was induced by ligation of bilateral common carotid arteries. For detecting the most appropriate time for 6mg/kg, Injection was done via single and double doses with intervals of 6, 24, 48 and hours. The repeated doses of 6mg/kg with interval of 48 hours are the suitable dose and time of injection. It seems that tacrolimus can be an appropriate strategy as a neurotrophic drug for treating brain ischemia

Evaluation of melatonin for prevention of radiation myelopathy in irradiated cervical spinal cord

Radiation myelopathy [RM] is known as a serious complication of head and neck radiation therapy. Furthermore, the radioprotective roles of melatonin have been investigated on different tissues. The aim of this study was to assess the radio protective effects of melatonin on biochemical, histopathological and clinical manifestations of RM in the rat cervical spinal cord. Four groups of rats were investigated as follows: The control group was treated with vehicle. The second group [melatonin only] was intraperitoneally injected with 100 mg/kg melatonin. The third group's [radiation] cervical spinal cord area was irradiated with 22 Gy cobalt-60 gamma-rays. The fourth group [melatonin plus irradiation] received 100 mg/kg melatonin intraperitoneally, and after 30 minutes their spinal cord area was irradiated with 22 Gy gamma radiation. Five animals from each group were randomly selected. 72 hours, 8 and 22 weeks after irradiation for analysis of malondialdehyde [MDA] and glutathione [GSH] levels, and underwent histopathological studies. The MDA levels in the irradiation group were significantly higher than in the control group [p < 0.001]. Furthermore, the GSH levels in this group were significantly lower than that of those in the control group [p < 0.001]. Administration of melatonin markedly reduced MDA [p < 0.001] and increased GSH [p < 0.05] levels in this group. Demyelination and clinical signs of myelopathy were decreased in the melatonin plus irradiation group in comparison to the irradiated group. Our study confirms the radioprotective effects of melatonin at early stages of biochemical, as well as late histological and clinical changes in the spinal cord

Home based training: main strategy of community based rehabilitation in Iran

Study of effectiveness of "home based training" in community based rehabilitation program on disabled people, who were trained and evaluated at the end of the course, under supervision of 21 pilot health and medical networks. In a cross-sectional study, 614 disabled people who had participated in "home based training" were selected with stratified random sampling method. They were evaluated according to function progress level variables by community based rehabilitation program experts. Age groups, sex, disability groups, employment state and teacher's relation variables were studied from their files and recording data. Statistical analysis was performed with Chi-square test. There was a relationship between age group and disability group with functional progress level [P value =0.014 and P value <0.001]. Low age groups, visual disabled group, epileptic patients and individuals with learning disability had the best results. High age groups, mixed age disability group and individuals with verbal and hearing problems had the least favorable results. There was a relationship between teacher's relation with progress or no progress state [P value = 0.038]. Individuals that were teachers had the best results and individuals with teachers other than first or second relation or health worker had the least favorable results. Home based training in community based rehabilitation program is an effective method for improving the functions of disabled people in some selected groups

Induction of liver metallothionein contribute to the developmental toxicity of valproic acid in rat

There is an increased risk of neural tube defects and axial skeletal malformations among infants born by mothers who had received valproic acid. The aim of the present study is, if administration of valproic acid can induce maternal hepatic Metallothionein [MT] synthesis and so secondary decrease of plasma Zn. In the present experimental study, mated rats were divided into four groups of 12 animals each [control, valproic acid [VPA], valproic acid + zinc [VPA+ Zn] and Zinc [Zn] groups. The VPA group received 300 mg/kg valproic acid; daily. The control group received an equal volume of 0.9% NaCI. The VPA+ Zn group received 300 mg/kg VPA as well as 30 mg/kg zinc sulfate, and the Zn group received 30 mg/kg zinc sulfate, daily. These drugs were administered intraperitoneally from day 6 through day 15 of gestation. Dams were killed on GD 16 or 20. Blood was drawn to determine plasma zinc; furthermore, maternal liver Zn and MT were also determined. The zinc concentration in the plasma of rats treated with valproic acid was significantly lower than those of the other groups on GD 16 [p=0.004], but liver Zn [p=0.016] and MT [p=0.004] were significantly higher than those of the control group. On GD 20 the incidence of skeletal malformations and neural tube defects tended to be higher in VPA group than VPA+ Zn treated group and no anomalies were seen in the control group. The results from the present experiment support hypothesis that one of biochemical lesions causing the teratogenicity of VPA is a drug -induced maternal plasma zinc deficiency secondary to Metallothionein induction in liver

role of L-arginine in control of apoptosis in preimplantation mouse embryos cultured in high glucose media

Maternal hyperglycemia causes delay in early stages of embryonic growth and development, higher incidence of congenital malformations and spontaneous miscarriage compared with those of non-diabetic conditions. High glucosis tratogenicity seems to be related to reduction of Nitric Oxide production [NO] in hyperglycemic condition. In order to test this hypothesis, 2-cell stage embryos of normal mice were cultured with high concentration of glucose [30mM] and different concentrations of L-arginine [5,10,20 mM] or L-NAME, an NO syntase [NOS] inhibitor. In the end of culture, blastocysts were stained by by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling [TUNEL] technique and apoptotic cells were detected by using a Fluorescence microscope. Finally the amount of nitrite in the cultured media was assayed by Griess method. The results indicated that high glucose reduces Nitric Oxide production by preimplantation embryos and increases apoptosis of embryonic cells, but 5-20mM of L-arginine significantly increases Nitric Oxide production and decreases apoptosis. On the contrary L-NAME significantly inhibits the development of pre-implantation embryos. In conclusion, this study indicated that reduced nitric oxide production in high glucosis condition is a main factor for embryonic damage, and supplementation of high glucose media with L-arginine has an important role in prevention of high glucosis embryotoxicity

Human sperm aster formation and chromatin configuration in rabbit oocytes following intracytoplasmic sperm injection using a Piezo-Micromanipulator

In human fertilization, the sperm centrosome nucleates a radial array of microtubules called the sperm aster. The sperm aster is responsible for apposition of male and female pronuclei, and later gives rise to the first meiotic spindle. The objective of this study was to determine microtubule assembly and chromatin configuration in rabbit oocytes following intracytoplasmic injection with human sperm by piezo-driven pipette. Oocytes were collected from superovulated dose 14-15 h after hCG injection and were fertilized by injection of a single human sperm into the ooplasm of each oocyte without additional activation treatment. Four hours post heterologous intracytoplamic sperm injection [ICSI], rabbit eggs were fixed and microtubule organization and chromatin configuration were examined by immunofluorescence microscopy. In unfertilized oocytes, microtubules were present only in the metaphase-arrested second meiotic spindle. Following human sperm injection, an aster of microtubules formed adjacent to the sperm head, around mid-piece, and sperm aster was enlarged and assembled around male and female pronuclei. During pronuclear centration, male and female pronuclei were surrounded by a microtubule array without nucleation sites. With fertile human sperm, the sperm aster formation rate was 54.6%. From our data we concluded that human spermatozoa can be injected successfully into rabbit oocytes, resulting in a reasonable survival rate, and that rabbit oocytes provide a reliable tools for assessing human sperm centrosomal function using the Piezo-ICSI system

effect of nitric oxide on mouse Pre-implantation embryo development and apoptosis

Clinical studies have shown that in pathological conditions such as endometriosis and reproductive tract infection [male and female] there is an activation status of macrophages that produce large quantities of nitric oxide [NO] in addition to other effector molecules. Large amounts of NO may have embryotoxic roles and produce infertility. This study was designed to evaluate the effect of different concentrations of NO on mouse pre-implantation embryo development in vitro. Mouse embryos [2-cell stage] were cultured in media containing different concentrations of sodium nitroprusside [SNP], an NO donor, or L-arginine methyl ester [L-NAME], an NO syntase [NOS] inhibitor. At the end of culture, cell apoptosis was studied by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling [TUNEL] technique. The results showed that development of preimplantation embryos were inhibited by high concentration of SNP [1 and 10