Brain tissue cysts in infected mice with RH-strain of Toxoplasma gondii and evaluation of BAG1 and SAG1 genes expression

Toxoplasma gondii is an obligate intracellular parasite that infects humans at high prevalence rates. The virulent RH strain of T. gondii is generally considered to have lost its cyst forming capacity. This study performed to obtain tissue cysts in mice infected with tachyzoites of RH strain treated with sulfadiazine [SDZ]. It provides the opportunity to analyze the conversion of tachyzoite to bradyzoite stage of the RH strain, followed by stage-specific gene-expression analyzing. Two groups of Swiss-Webster and BALB/c mice were infected subcutaneously with 10[4] tachyzoites of T. gondiiy RH strain and given SDZ [300 mg/l] with NaHCO3 [5 g[-1]] in drinking water from day 1 to day 14 post infection [p.i]. The infected mice were sacrificed on day 50 post infection. Their brains were removed and the numbers of tissue cysts were microscopically counted. Total RNA was extracted from brains and cDNA synthesis was carried out. Finally, RT-PCR [Reverse transcription PCR] was used to detect the expression of bradyzoite [BAG[1]] and tachyzoite [SAG[1]] specific genes during tachyzoite / bradyzoite stage conversion. Sixty five percent of all infected mice were survived. Cysts were detectable in mice brain [45%] on day 50 p.i. Also RT-PCR of the brain samples was positive for SAG1 and BAG1. It seems that conversion of tachyzoites to bradyzoites in brain of mice undergoing SDZ was not completed until 50 days after inoculation

Production and evaluation of Toxoplasma gondii recombinant surface antigen 1 [SAG1] for serodiagnosis of acute and chronic toxoplasma infection in human sera

The assays currently available for the detection of specific anti-Toxoplasma antibodies may vary in their abilities to detect serum immunoglobulins, due to the Lack of a purified standardized antigen. The aim of this study was evaluation the recombinant Toxoplasma gondii SAG1 antigen for the serodiagnosis of acute and chronic toxoplasmosis. This study describes an ELISA using recombinant SAG1 for detection of IgM and IgG antibodies against Toxoplasma gondii in human sera. Genomic DNA of T. gondii [RH Strain] was isolated and PCR reaction was performed. Recovered DNA was cloned into PTZ57R cloning vector. The recombinant plasmid was detected by restriction analysis. The SAG1 gene was subcloned in the pET- 28a expression vector. Protein production was then induced with 1 mM isopropyl-D - thiogalactopyranoside [IPTG]. A total of 204 sera were tested using a commercial IgG and IgM ELISA kit [Trinity, USA] as gold standard prior to testing them with the recombinant antigen. Tested sera were divided into the following groups:[a] The 74 T. gondii IgG positive [b] 70 T.gondii IgM positive [c] 60 sera who had no serological evidence of toxoplasmosis as negative sera.To determine the specificity of the test, we used other parasitic diseases including echinococusis [N=5], malaria [N=14], leishmaniasis [N=7],fasciolasis [N=4], sterengyloidiasis [N=1]. Sensitivity and specificity of the generated recombinant IgG ELISA in comparison with commercial ELISA [Com ELISA] were 93% and 95%, and the sensitivity and specificity of the generated recombinant IgM ELISA were 87% and 95% respectively. The results acquired here show that this antigen is useful for diagnostic purposes and could be replaced by lysed, whole cell antigens for diagnosis of chronic toxoplasmosis

Deletion and testicular expression of DAZ [deleted in azoospermia] gene in patients with non-obstructive azoospermia

Deletions of the DAZ [deleted in azoospermia] genes within the human Y chromosome's AZFc region are the most common cause of spermatogenesis failure. These deletions are usually assessed by analyses of genomic DNA extracted from peripheral leukocytes. DAZ genes are expressed in male germ cells. In this prospective study, we investigated DAZ expression and deletion in 102 consecutive infertile men presenting with non-obstructive azoospermia in Avesina Research Institute, Tehran, Iran during 2005-6. In this prospective study, we extracted genomic DNA from peripheral blood leukocytes for detection of DAZ deletions and testicular biopsies for histopathological assessment and analyses of DAZ expression level by reverse transcription polymerase chain reaction. DAZ levels were normalized to expression of the housekeeping Phosphoglucomutase 1 gene. In four out of 102 patients [3.9%], we found DAZ deletion. DAZ expression was observed in 60 [61.2%] of 98 other patients. Expression was not detected in patient with Sertoli cell-only syndrome, but observed in 37 of 40 [92.5%] patients with maturation arrest and 20 of 26 [76.9%] with hypospermatogenesis. The absence of DAZ expression could result in quantitative reduction of germ cells and might be observed despite of normal genomic DNA constitution. We recommend to check DAZ testicular expression and genomic DNA deletion, in non-obstructive azoospermia. This is more recommended to avoid transmission of genetic abnormalities which might lead to infertility in male offspring, when assisted reproductive techniques [ART] are performed

Expression of synaptonemal complex protein 3 [SYCP3] m RNAin the testis: a molecular marker for spermatogenesis in azoospermic men

Men with unexplained infertility and azoospermia are often observed in the context of genetic defects. The expression of a wide variety of genes is developmentally regulated during human meiosis. Synaptonemal Protein 3 [SYCP3] gene, located on chromosome 12, encodes a DNA-binding protein as the structural component of the synaptonemal complex,which mediates the synopsis or homologous pairing of chromosomes during meiosis. Absence of SYCP3 in mice may lead to male infertility as well as female sub-fertility. SYCP3 expression analysis could be a tool for the prediction of human spermatogenesis progression, especially in infertile men. SYCP3 mRNA expression in testicular samples of 110 patients with non-obstructive azoospermia were studied in Avesina Infertility Clinic in Tehran, Iran during 2005 and early 2006. Semi-quantitative nested reverse transcriptase-PCR was employed in order to find the strength of gene expression. Using histopathological scoring for all samples, the expression level of SYCP3 during spermatogenesis was also evaluated. Testicular SYCP3 mRNA expression was observed in 67 patients [60.9%]. The expression level correlated with the degree of spermatogenic failure [p<0.0001]. While this gene had been expressed in patients with hypo-spermatogenesis and maturation arrest, a lack of expression was seen in those with spermatogonial arrest, Sertoli cell-only syndrome and testicular atrophy. These data indicate that SYCP3 is expressed in the human testis and it is restricted to germ cells. Our findings, in association with those obtained in experimental animals, show that lack of SYCP3 expression may have negative effects on spermatogenesis and male fertility. SYCP3 gene expression may help detect specific spermatogenesis stages in conjunction with histopathological findings