Study of the effect of hypothyroidism on the apoptotic index in rat ovarian follicles, using the TUNEL technique

Among the endocrine diseases, hypothyroidism is the commonest after diabetes. Thyroid hormones [T3, T4] are essential for genital organs function. Apoptosis process in ovarian cells plays a significant role of development in ovarian follicles. The aim of the present study was determine the apoptotic index induced by hypothyroidism in rat ovarian follicles. In the present study, twenty female mature wistar rats were used with age of 2.5 months and weight of 200-250 [g]. Rats were divided into test and control groups. In test group chemical hypothyroidism induced by propylthiouracil [PTU; 500 mg/L] in drinking water. The control group only received normal drinking water. After three weeks the rats were killed and their ovaries removed and were fixed for tissue preparation. TUNEL technique were used for determine of apoptosis. Cells count done by stereological method. Data were analyzed by t-test and one-way ANOVA followed by Tukey test. Significance was accepted at P<0.05. The findings showed that the apoptotic index had a significant decrease in late antral and graffian follicles [P=0.000] and no significant decrease in preantral and early follicles [P>0.05] in hypothyroid group. All granulose cells were TUNEL-positive in primary follicles but no cell was seen in primordial follicles in groups. The results of the present study showed that the hypothyroidism may be vigorous decreased of apoptotic index in antral and graffian follicles. Hypothyroidism increased the number of luteal bodies and decreased the number of graffian follicles in ovarian tissue

Tamoxifen Mimics the Effects of Endogenous Ovarian Hormones on Repeated Seizures Induced by Pentylenetetrazole in Rats

In the present study, the effects of tamoxifen on pentylenetetrazole (PTZ)-induced repeated seizures and hippocampal neuronal damage in ovariectomized rats were investigated. Thirty seven virgin female Wistar rats were divided to: (1) control, (2) sham-PTZ, (3) sham-PTZ-tamoxifen (sham-PTZ-T), (4) Ovariectomized -PTZ (OVX-PTZ) and (5) OVX-PTZ-tamoxifen (OVX-PTZ-T) groups. The animals of groups 3 and 5 were injected by tamoxifen (10 mg/kg) on 7 consecutive days. After 7 days of tamoxifen injection, they also were then injected by tamoxifen 30 min prior each PTZ injection. PTZ (40 mg/kg) was injected on 6 consecutive days and the animal behaviors were observed for 60 min. The histological methods were then used to determine dark neurons in hippocampus. A significant decrease in the seizure score was seen in OVX-PTZ group compared to Sham-PTZ. The animals of OVX-PTZ-T group had a significant higher seizure score compared to OVX-PTZ group. The dark neurons in DG of OVX group were lower than sham group (p<0.01). The numbers of dark neurons in CA1 area of OVX-PTZ-T group was higher than OVX-PTZ group (p<0.05) compared to control, the numbers of dark neurons in CA3 area showed a significant increase in Sham-PTZ and OVX-PTZ group (p<0.05 and p<0.01 respectively). Dark neurons in OVX-PTZ-T group were higher than OVX-PTZ group (p<0.05). It is concluded that pretreatment of the ovariectomized rats by tamoxifen increased PTZ-induced seizure score and dark neurons. It might be suggested that tamoxifen has agonistic effects for estrogen receptors to change the seizure severity.

Regulatory changes of n-acetylgalactosamine terminal sugar in early mouse embryonic paraxial mesenchyme

The development of vertebrae is a complex phenomenon that is correlated with distinct morphological and biochemical alterations in the paraxial mesenchyme and glycoconjugates. The purpose of this study is to investigate the glycosylation pattern in paraxial mesenchyme-forming vertebrae by using the lectin histochemical technique. In this descriptive-analytic study, B4G fixed paraffin sections of 9 to 15 day Balb/c mouse embryos were processed for histochemical studies using seven different HRP-labelled lectins: Glycin max [SBA], Maclura pomifera [MPA], Wistaria floribunda [WFA], Vicia villosa [VVA] which all of them are specific for N-acetylgalactosamine [GalNAc], Ulex europius [UEA1, binds to alpha-L-fucose], wheat germ agglutinin [WGA, binds to sialic acid], and Griffonia simplicifolia [GSA1-B4, binds to galactose terminal sugars]. The sections were observed separately by three examiners who were blinded to the lectins. Grading was done according to the intensity of the tested lectins' reactions with the specimen, from negative [-] to severe [+++]. Data was analysed with SPSS software [version 11.5] and the non-parametric Kruskal Wallis test; p<0.05 was considered significant. Our findings showed that among the tested lectins, only GalNAc residue sensitive lectins showed regulated changes in paraxial mesenchyme. Reactions of WFA and MPA lectins with paraxial mesenchyme were severe on GD9. Reactions of WFA continued to GD15 constantly, while MPA reactions continued strongly to GD12, significantly decreased thereafter [p<0.001], and then disappeared. VVA and SBA bindings initiated weakly on GD10 and continued to GD12 without changing. These reactions increased significantly [p<0.001] thereafter, became severe to GD14, and later disappeared. The other tested lectins did not reveal regulated changes. According to these findings it can be concluded that only the GalNAc terminal sugar showed temporally regulated changes during the early embryonic development of vertebrae in mice. Therefore it most likely plays a key role [s] in the development of vertebrae, especially in the conversion of mesenchymal cells into chondroblasts. The other tested terminal sugars may have no role in this phenomenon

Lectin histochemical study of vasculogenesis during rat pituitary morphogenesis

The aim of this study was to investigate glycoconjugates distribution patterns as well as their changes during the course of pituitary portal vasculogenesis and angiogenesis. Formalin fixed paraffin sections of 10 to 20 days of Sprague Dawly rat fetuses were processed for histochemical studies using four different horseradish peroxidase [HRP] conjugated lectins. Orange peel fungus [OFA], Vicica villosa [VVA], Glycine max [SBA] and Wistaria floribunda [WFA] specific for alpha-L-Fucose, D-Gal, alpha, beta-D-GalNAc and D-GalNAc terminal sugars of glycoconjugates respectively. Our finding indicated that adenohypophysal cells reacted with OFA on gestational day 10 E[10] and increased progressively to E[14]. Staining intensity did not change from days 14 to17, then after increased following days to E[20] significantly [P< 0.05]. A few cells around Rathke's pouch reacted with VVA on E[13], increased to E[14] and decreased significantly afterward [P< 0.05]. Reaction of some cells around Rathke's pouch reacted with SBA on E[14]. This visible reaction was the same as E[18] and decreased later [P< 0.05]. Many cells around Rathke's pouch reacted with WFA on E[13] and increased on E[14] and E[15] and decreased thereafter [P<0.05]. Reactions of OFA and other tested lectins with endothelial cells around Rathke's pouch and developing pars distalis were different. These results suggest that embryonic origin of hypophiseal pituitary portal [HPP] system endothelial cells are not the same and our finding also indicated that glycoconjugates with terminal sugars alpha-L-Fucose, D-Gal, alpha, beta-D-GalNAc may play critical role[s] in cell interactions and tissue differentiations such as vasculogensis and angiogenesis as well as other developmental precursors in formation of the pituitary gland

Morphometrical study of polysialylated neural cell adhesion molecule positive cells in rat pups hippocampus following induction of seizure during pregnancy

The polysialylated neural cell adhesion molecule [PSA-NCAM] is expressed in developing brain. Fetal brain damage is caused by different conditions such as seizure and hypoxia. The present study was designed to investigate the effect of maternal seizures on the number of PSA-NCAM positive cells in pup's hippocampus. Female Wistar rats were divided into four groups: [a] kindled rats which received PTZ [40 mg/kg, i.p.] during pregnancy from embryonic day 14-19 [E14-E19] every 48 h, [b] kindled rats which did not receive PTZ during pregnancy, [c] non-kindle, pregnant rats which received PTZ injection [40 mg/kg, i.p.] during pregnancy from E14 to E19 every 48 h, and [d] non-kindle, pregnant rats which received injection with an equal volume of normal saline as sham controls. At postnatal day 14 [PD[14]], rat pups were perfused, and their brain were fixed, embedded and coronal sections stained by immunohistochemistry method. The number of PSA-NCAM positive cells per unit area in the pup's hippocampus was counted. The number of PSA-NCAM positive cells in the CA1, CA3, and DG fields of pup's hippocampus, which was obtained from mothers who experienced PTZ injection during pregnancy, was decreased approximately 2.6 [P = 0.001], 2 [P = 0.001], and 2.1 [P = 0.001] times compared with non-PTZ treated maternal groups, respectively. Our study showed that maternal seizures reduced the number of neurons and also PSA-NCAM positive cells per unit area in the offspring hippocampus that it may cause impairment in hippocampal functions

Gene dosage analysis of proximal spinal muscular atrophy carriers using real-time PCR

Autosomal recessive spinal muscular atrophy is a disease resulting from homozygous absence of SMN1 gene in approximately 94% of SMA patients. To identify patients who retained a single SMN1 copy, SMN1 dosage analysis was performed by quantitative Real-time PCR using SYBR green dye. SMN1 dosage analysis results were utilized to identify carriers before offering prenatal diagnosis. Carrier testing was performed for 150 individuals. Copy number of the SMN1 gene was determined by the comparative threshold cycle [Ct] method and human serum albumin gene was used as a reference. Analysis of 150 DNA samples with quantitative PCR determined the number of SMN1 gene copies. Of these, 50 [33.33%] cases had one SMN1 gene copy, 87 [58%] had two copies and 13 [8.66%] did not have any copies of SMN1. The homozygous SMN1 deletion ratio was 0.00 and deletion of one copy of SMN1 gene ratio ranged from 0.3 to 0.58. This report demonstrates modification of risk estimation for the diagnosis and detection of SMA carriers by accurate determination of SMN1 copy number. SMN1 copy number analysis is an important parameter for identification of couples at risk of having children affected with SMA. It also reduces unwarranted prenatal diagnosis for SMA. Furthermore, the dosage analysis might be useful for the counseling of clinically suspected SMA patients with negative diagnostic SMA tests

[ effect of ethanol injection on kidney histological structure in mice]

Destructive effects of ethanol consumption have been confirmed on several organs of the body. Nevertheless, classic research which has been done on kidney in this field had less attention. The aim of this study was to evaluate of ethanol effects on light microscopic structure of Mice kidneys. In this experimental study, Balb/c strain mice, equally 40 males and females, weighing 30-35 grams were divided in two equal experimental and control groups randomly. The Mice in control and expermented groups further divided in two sub groups. One sub group received 1mg/gr body weight daily of ethanol for one month, intraperitoneally and other sub group received 1mg/gr body weight of ethanol for two months, intraperitoneally. Control groups divided in two sub groups and Mice received daily salin with same volume as experimental groups. Then, the Mice have been anesthetized and sacrificed and then, subsequently kidneys were removed. Routine histological methods and Hematoxylin and Eosin staining were done. Slides were observed by light microscope. In experimental group, Mice which received ethanol for one month, interstitial bleeding and inflammatory cells infiltration in cortical zone of kidney was observed. In Mice which received ethanol for two months, we observed massive and concentrated lymphoblastic infiltration, especially on deep medulla around the blood vessels with extending to calyxes and pelvis of the kidney. No histological changes were observed in control groups. This study showed that the intraperitoneal injection of ethanol cause progressive destruction effects on cortex and medulla of the kidney. These effects depend on duration of ethanol consumption

Distribution of specific glycoconjugates in early mouse embryonic notochord and paraxial mesenchyme

It is well known that glycoconjugate components of the cell surface and extracellular matrix, play an essential role[s] in many developmental phenomena such as cell differentiation, migration, and cellular interactions. The purpose of this study was to investigate distribution of this macromolecules during differentiation of the notochord and paraxial mesoderm. Formalin fixed paraffin sections of 9 to 16 days of BALB/c mouse embryos were processed for histochemical studies using four different horseradish peroxidase conjugated lectins including wheat germ agglutinin [WGA], Griffonia simplicifolia [GSA1-B4], Arachis hypogaea [PNA] and Lotus tetragonolobus [LTA], specific for sialic acid, galactose, N-acetylgalactosamine [GalNAc] and fucose terminal sugar, respectively. Our results showed that in primordial of the developing vertebrae PNA sensitive glycoconjugate appeared on gestational day 13 and increased to gestational day 15 significantly [P<0.05] and disappeared later. GSA1-B4 revealed no reaction and LTA and WGA reactions were observed only in notochord. Among the lectins that were used in this study, only PNA showed continuous and strong reaction just during gestational day 13 to 15. This finding suggests that PNA temporally regulated changes occurred during early vertebral development in mouse embryos and probably their specific terminal sugar plays a key role[s] during prechondrogenic vertebrae