Altered methyltransferase gene expression, mitochondrial copy number and 4977-bp common deletion in subfertile men with variable sperm parameters.

Semen parameters have been found to predict reproductive success poorly and are the most prevalent diagnostic tool for male infertility. There are few conflicting reports regarding the correlation of DNMT genes expression, mitochondrial DNA copy number (mtDNAcn) and deletion (mtDNAdel) with different sperm parameters. To investigate DNMT mRNA level, mtDNAcn and deletion in infertile men, with different sperm parameters, compared with fertile men, semen samples from 30 men with unknown male infertility and normal sperm parameters (experimental group I), 30 infertile patients with at least two abnormal sperm parameters (experimental group II) and 30 fertile normozoospermic men (control group) were collected. After semen analysis, total RNA and DNA were extracted. The isolated DNA was used for assessing the respective mtDNAcn and the presence of common 4977 bp deletion in mtDNA by applying real-time quantitative PCR and multiplex PCR, respectively. Synthesized cDNA from total RNAs was used to quantify DNMT1, DNMT3A and DNMT3B transcripts in study groups by using real-time quantitative reverse-transcription PCR. Significantly higher proportions of mtDNAcn were found in experimental group II. DNMT1 was significantly downregulated in both experimental groups and 4977 bp deletion was not detected. Progressive motility and normal morphology were significantly and negatively correlated with mtDNAcn. A significant positive correlation was detected between sperm parameters and DNMT1 mRNA levels. In conclusion, infertile men with different sperm parameter qualities showed elevated mtDNA content. Abnormal sperm parameters associated with DNMT1 gene expression indicate the possibility of changes in some epigenetic aspects of spermatogenesis in subfertile men with different sperm parameters.

Effect of titanium dioxide nanoparticles on the stereological parameters of the dentate gyrus and the morphology of granular hippocampal neurons in mice / Efecto de las nanopartículas de dióxido de titanio sobre los parámetros estereológicos del giro dentado y morfología de las neuronas granulares del hipocampo en ratones

SUMMARY: This study aims to investigate the Effects of Titanium dioxide nanoparticles (TiO2 NPs) on the stereological parameters in the dentate gyrus and the morphology of granular hippocampal neurons in adult mice. Adult male mice (n=20, weight average: 45 g) were randomly divided into four groups including: group receiving saline (controls), low-dose (LD) 2.5 mg/kg TiO TiO2 NPs, medium-dose (MD) 5 mg/kg TiO2 NPs and high-dose (HD) 10 mg/kg TiO2 NPs, daily using gavage for 35 days. To estimate the volume of the hippocampus, dentate gyrus, and sub-layers of dentate gyrus the Cavalieri principle was used. The physical dissector was used to determine the numerical density of dentate gyrus granular cells. For analyzing the morphology of dentate gyrus granular cells the qualitative Golgi staining was used. Our data showed that the total volume of the hippocampus, dentate gyrus and its sublayers including molecular, granular and polymorph in TiO2 treated mice decreased significantly compared to the control group. Moreover, the total number and numerical density of dentate gyrus granular sub layer cells showed a significant reduction in all three experimental groups compared to the control group. The granular cells of the dentate gyrus had shorter dendritic length and decreased dendritic branches in the TiO2-treated in comparison with the control mice. These data can justify the disorders related to memory, learning and hippocampus neurons damages due to using of TiO2 NPs.

RESUMEN: En este estudio se analizaron los efectos de las nanopartículas de dióxido de titanio (TiO2 NP) sobre los parámetros estereológicos en el giro dentado y la morfología de las neuronas granulares del hipocampo en ratones adultos. Se dividieron aleatoriamente ratones machos adultos (n = 20, promedio de peso: 45 g) en cuatro grupos: grupo que recibió solución salina (controles), dosis baja (LD) 2,5 mg/kg NP de TiO2, dosis media (MD) 5 mg/kg de NP de TiO2 y dosis altas (HD) de 10 mg/kg de NP de TiO2, por vía utilizando sonda durante 35 días. Para estimar el volumen del hipocampo, el giro dentado y las subcapas del giro dentado se utilizó el principio de Cavalieri. Se utilizó el disector físico para determinar la densidad numérica de las células granulares del giro dentado. Para analizar la morfología de las células granulares del giro dentado se usó la tinción cualitativa de Golgi. Nuestros datos mostraron que el volumen total del hipocampo, el giro dentado y sus subcapas, incluyendo la molecular, granular y polimorfos, en ratones tratados con TiO2, disminuyó significativamente en comparación con el grupo de control. Además, el número total y la densidad numérica de las células de la subcapa granular del giro dentado mostró una reducción significativa en los tres grupos experimentales en comparación con el grupo control. Las células granulares del giro dentado tenían una longitud dendrítica menor y ramas dendríticas disminuidas en los ratones tratados con TiO2 en comparación con los ratones del grupo control. Estos datos pueden justificar los trastornos relacionados con la memoria, el aprendizaje y los daños en las neuronas del hipocampo debido al uso de NP de TiO2.

Acute and chronic effects of gold nanoparticles on sperm parameters and chromatin structure in Mice.

BACKGROUND: The particles in the range of 1-100 nm are called nanoparticles. Gold nanoparticle is one of the most important metal nanoparticles with wide usage. OBJECTIVE: This study investigated the effects of gold nanoparticles on sperm parameters and chromatin structure in mice. MATERIALS AND METHODS: In this experimental study, 72 male bulb-c mice were divided into 9 groups including: 4 Sham groups (Sc 1-4), 4 experimental groups (Au 1-4), and 1 control group (C). Experimental groups received 40 and 200 µg/kg/day soluble gold (Au) nano-particles for 7 and 35 days, by intra peritoneal injection, respectively. Sham groups were treated with 1.2 mM sodium citrate solution with 40 and 200 µg/kg/day doses for same days and control group did not receive any materials. Motility and Morphology of spermatozoa were analyzed. Chromatin quality was also evaluated using AB (Aniline blue), TB (Toluidine blue) and CMA3 (Chromomycin A3) staining methods. RESULTS: The sperm analysis results showed that motility and morphology of sperm in experimental groups (especially in groups that have been treated for 35 days with nano-particles) had significant decrease in comparison with control group. TB, AB and CMA3 results showed a significant increase in abnormal spermatozoa from all Au-treated groups. CONCLUSION: Gold nano-particles firstly can reduce the sperm parameters such as motility and normal morphology and secondly affect sperm chromatin remodeling and cause the increase instability of chromatin and also increase the rate of sperm DNA damage. These deleterious effects were more obvious in maximum dose and chronic phase.

Beneficial effect of Boswellia serrata gum resin on spatial learning and the dendritic tree of dentate gyrus granule cells in aged rats.

OBJECTIVE: The hippocampal formation, particularly the dentate gyrus (DG), shows age-related morphological changes that could cause memory decline. It is indicated that Boswellia resins attenuates memory deficits and the major component of Boswellia serrata (Bs) gum resin, beta boswellic acid increased neurite outgrowth and branching in hippocampal neurons. This study was designed to investigate the effect of Boswellia treatment on spatial learning performance and the morphology of dentate granule cells in aged rats. MATERIALS AND METHODS: Sixteen male Wistar rats (24 months old) were divided into experimental and control groups. Experimental group was intragastrically administered with the aqueous extract of Bs (100 mg/kg/d for 8 weeks) and control group received a similar volume of water. Spatial learning performance of rats was tested using Morris water maze task. At the end of experiment, the brain was removed and the right hippocampus was serially sectioned for morphometric analysis. The Cavalieri principle was employed to estimate the volume of the DG. A quantitative Golgi study was used to analyze the dendritic trees of dentate granule cells. RESULTS: Chronic treatment with Bs improved spatial learning capability during the three acquisition days. Comparisons also revealed that Bs-treated aged rat had greater DG with increased dendritic complexity in the dentate granule cells than control rats. Hippocampal granule cells of Bs-treated aged rats had more dendritic segments, larger arbors, more numerical branching density and more dendritic spines in comparison to control animals. CONCLUSION: This study provided a neuro-anatomical basis for memory improvement due to chronic treatment with Bs.

Effects of Ginkgo biloba extract on the structure of Cornu Ammonis in aged rat: A morphometric study.

OBJECTIVES: Growing evidence indicates that extract of Ginkgo biloba (EGb) attenuates hippocampal-dependent memory deficit in aged individuals; however, very little is known about the effect of EGb on the structure of hippocampus. Therefore we examined the EGb-induced morphological changes of the Cornu Ammonis (CA) region in aged rats. MATERIALS AND METHODS: Sixteen aged male Wistar rats, 24 months old, were randomly divided into experimental and control groups. Experimental group was orally administered EGb (100 mg/kg/d for 8 weeks), and the control group received a similar volume of water. The volume estimation of CA hippocampal field was done by Cavalieri principle and a quantitative Golgi study was also used for analysis of dendritic arborizations of CA3 and CA1 pyramidal cells. RESULTS: Results revealed that EGb-treated aged rats had greater volumes than control animals in the layers of pyramidal and radiatum lacunosum moleculare in both CA3 and CA1 subfields. The neurons of CA3 and CA1 in experimental rats had more dendritic segments and larger total dendritic length compared to the control. The results also showed that the aged rats treated by EGb had more numerical branching density in the apical dendrites of CA3 and CA1 pyramidal cells. CONCLUSION: The results of the present study show that long-term administration of EGb could produce morphometrical changes in hippocampal pyramidal cells in aged rats. Results also provide a neuroanatomical basis for memory improvement due to chronic treatment with EGb.

Effect of Boswellia serrata gum resin on the morphology of hippocampal CA1 pyramidal cells in aged rat.

Experimental evidence indicates that administration of Boswellia resin, known as olibanum or Frankincense, increases memory power. It is reported that beta boswellic acid, the major component of Boswellia serrata gum resin, could enhance neurite outgrowth and branching in hippocampal neurons. We therefore studied whether Boswellia treatment produces morphological changes in the superior region of cornu ammonis (CA1) in aged rats. Sixteen male Wistar rats, 24 months of age, were randomly divided in experimental and control groups. The experimental group was orally administered Boswellia serrata gum resin (100 mg/kg per day for 8 weeks) and the control group received a similar volume of water. The Cavalieri principle was employed to estimate the volumes of CA1 hippocampal field, and a quantitative Golgi study was used to analysis of dendritic arborizations of CA1 pyramidal cells. Comparisons revealed that Boswellia-treated aged rats had greater volumes than control animals in stratum pyramidale and stratum radiatum lacunosum-moleculare. The neurons of CA1 in experimental rats had more dendritic segments (40.25 ± 4.20) than controls (30.9 ± 4.55), P = 0.001. The total dendritic length of CA1 neurons was approximately 20 % larger in the experimental group compared to control. Results also indicated that the aged rats treated with Boswellia resin had more numerical branching density in the apical dendrites of CA1 pyramidal neurons. The results of the present study show that long-term administration of Boswellia resin can attenuate age-related dendritic regression in CA1 pyramidal cells in rat hippocampus.

Stereological estimation of granule cell number and purkinje cell volume in the cerebellum of noise-exposed young rat.

In spite of the existing reports on behavioural and biochemical changes related to the cerebellum due to noise stress, not much is known about the effect of noise stress on the neuronal changes in the cerebellum. The present study aims at investigating the effects from one week noise exposure on granule cell number and Purkinje cell volume within the neonate rat cerebellum. 15-day-old male Wistar rats were randomly divided into noise exposed (NE) and control groups (n=8 in each group). NE rats exposed to loud noise (100 dB/30 min/3 times per day) during the third postnatal week. One cerebellar half was selected at random for estimating the volume of the cerebellar layers and neuronal quantifications and the other was used for estimating individual somal volume of Purkinje cells. Cavalieri's principle, physical disector and nucleator methods were employed respectively for unbiased estimation of the volumes of the cerebellar layers, the numerical density of neurons and the individual volume of Purkinje cells. Results of this study show that noise stress significantly decreases the volume of granule layer together with decreased numerical density and total number of granule cells in the cerebellum. Furthermore, a decrease in somal volume of Purkinje cells was found in NE rats. These results, for the first time, demonstrate an effect of noise stress on the granule cell number and individual volume of Purkinje cells in rat cerebellum.

Stereological analysis of cornu ammonis in prenatally stressed rats: a heuristic neurodevelopmental model of schizophrenia.

OBJECTIVE(S): The hippocampus has been implicated in pathophysiology of schizophrenia. Prenatal stress is a contributing risk factor for a wide variety of neuropsychiatric diseases including schizophrenia. This study examined long-term effects of prenatal restraint stress on the stereological parameters in the Cornu Ammonis (CA) of adult male rats as an animal model of schizophrenia. MATERIALS AND METHODS: Wistar pregnant dams in experimental group were stressed in a cylindrical Plexiglas restrainer daily for 1 hr during last week of gestation. Controls remained in the animal room and were exposed only to normal animal room conditions. At 2 months of age, the volume of the pyramidal cell layer of the CA, the numerical density and the somal volume of the respective neurons were assessed in the male offspring generated from stressed and control pregnancies. Cavalieri's principle, physical disector and nucleator were applied for stereological analyses. RESULTS: This study showed that prenatal stress significantly decreased the volume of CA3 pyramidal cell layer and the individual somal volume of CA3 pyramidal neurons. However, there were no markedly differences in the numerical density, total number of CA3 pyramidal neurons and stereological parameters in CA1 of prenatally stressed and control animals. CONCLUSION: These data indicate that prenatal stress exposure induced neuronal changes in the CA3 subfield of hippocampus which are similar to what is observed in schizophrenia.

The effect of prenatal exposure to restraint stress on hippocampal granule neurons of adult rat offspring.

OBJECTIVES: It is well known that prenatal stresses (PS) induce a variety of neurobiological and behavioral alterations, some of them involving the hippocampal formation. This study aimed to determine whether restraint stress influences the neuronal volume and number of granule cells in the hippocampus of adult rat offspring. MATERIALS AND METHODS: TEN WISTAR PREGNANT RATS WERE RANDOMLY DIVIDED: stressed and control groups. Pregnant dams in the stressed group were placed in a Plexiglas restraint tube for 1 hr daily from days 15-21 of gestation. Neuroendocrinological consequences of prenatal stress exposure were evaluated in the male offspring on postnatal day 60. The total numbers and the individual volume of granule cells in the hippocampus were also estimated with the optical fractionator and the rotator methods, respectively. RESULTS: Prenatally stressed rats exhibited prolonged elevation in plasma glucocorticoid levels following acute exposure to restraint stress. Data also indicated that there is a decrease in neuronal volume of hippocampal granule cells in prenatally stressed compared with their controls (625±64.1 µm(3) vs. 741±80.6 µm(3)). There was no significant difference in the total number of granule cells between prenatally stressed and control animals. CONCLUSION: The present study indicated that exposure of pregnant female during last week of pregnancy leads to a decline in neuronal size in hippocampus of adult male rats without neuronal loss. The present results may provide a basis for the understanding of the reported disturbances in behavior and learning of PS offspring.

Prenatal stress induces learning deficits and is associated with a decrease in granules and CA3 cell dendritic tree size in rat hippocampus.

Exposure to gestational stress impairs hippocampal-dependent learning in offspring. In spite of the known decisive role of hippocampal dendritic architecture in learning and memory, there has been no study to date that examines the effect of prenatal stress on the morphology of the hippocampal neurons. Therefore we performed a quantitative morphological analysis of the dendritic architecture of Golgi-impregnated hippocampal neurons in prenatally stressed rats. Subjects included male rat offspring (2 months old) for which the mothers had been restrained for 1 h/day during the last week of gestation. Spatial learning performance levels using Morris water maze and changes in the morphology of hippocampal dendritic trees were studied. Results indicated that the study group had lower spatial learning capabilities along with decreased length and number of dendritic segments, branching of granules and cornu ammonis (CA)3 pyramidal cells. There was no change in the dendritic morphology of CA1 pyramidal cells. These results suggest that prenatal stress in rat results in spatial learning deficits and profound alterations in the neurites of the hippocampal cells of male offspring.

Design-based estimation of neuronal number and individual neuronal volume in the rat hippocampus.

Tools recently developed in stereology were employed for unbiased estimation of the neuronal number and volume in three major subdivisions of rat hippocampus (dentate granular, CA1 and CA3 pyramidal layers). The optical fractionator is used extensively in quantitative studies of the hippocampus; however, the classical optical fractionator design may be affected by tissue deformation in the z-axis of the section. In this study, we applied an improved optical fractionator design to estimate total number of neurons on 100 microm thick vibratome sections that had been deformed, in the z-dimension, during histological processing. For estimating cell number, it is necessary to randomize only the location of section planes. But, in the local stereological methods, like cell volume estimation, the orientation of sections must also be randomized. We present a detailed application of a method for making vertical sections from the hippocampus. The volume of hippocampal neurons was estimated using the rotator principle on 40 microm thick plastic vertical uniform random sections and corrected for tissue shrinkage. Application of the proposed new design should result in more accurate estimates of neuron number and neuronal volumes in tissue sections affected by homogenous non-uniform shrinkage.