Alternation of apoptotic and implanting genes expression of mouse embryos after re-vitrification

Background: nowadays, oocytes and embryos vitrification has become a routine technique. Based on clinical judgment, re-vitrification maybe required. But little is known about re-vitrification impact on genes expression

Objective: the impact of re-vitrification on apoptotic and implanting genes, Bax, Bcl-2 and ErbB4, at compaction stage embryos were evaluated in this study

Materials and Methods: in this experimental study, 8 cell embryos [n=240] were collected from female mature mice, 60-62 hr post HCG injection. The embryos were divided randomly to 3 groups included: fresh [n=80], vitrified at 8 cell stage [n=80], vitrified at 8 cell stage thawed and re-vitrified at compaction stage [n=80]. Embryos were vitrified by using cryolock, [open system] described by Kuwayama. Q-PCR was used to examine the expression of Bax, Bcl2 ErbB4 genes in derived blastocysts

Results: our result showed that expanded blastocyst rate was similar between vitrified and re-vitrified groups, while re-vitrified embryos showed significant decrease in expanded blastocyst rate comparing with fresh embryos [p=0.03]. In addition, significant difference was observed on apoptotic gene expression when comparing re-vitrified and fresh embryos [p=0.004], however expression of Bax and Bcl-2 [apoptotic] genes didn't demonstrate a significant difference between re-vitrified and vitrified groups. The expression rate of ErbB4, an implantation gene was decreased in re-vitrified embryos comparing with fresh embryos [p=0.003], but it was similar between re-vitrified and vitrified embryos

Conclusion: re-vitrification can alter the expression of Bax, Bcl-2 and ErbB4 genes and developmental rate of mouse embryos in compaction stage

Autologous transplantation of adult mice spermatogonial stem cells into gamma irradiated testes

We evaluated structural and functional changes of fresh and frozen-thawed adult mouse spermatogonial stem cells following auto-transplantation into gamma-irra-diated testes. In this experimental research, the right testes from adult mice [n=25] were collected, then Sertoli and spermatogonial cells were isolated using two-step enzymatic digestion, lectin immobilization and differential plating. Three weeks after cultivation, the Bromodeoxyuridine [BrdU]-labeled spermatogonial cells were transplanted, via rete testis, into the other testis of the same mouse, which had been irradiated with 14Gy. The mice were transplanted with: fresh cells [control 1], fresh cells co-cultured with Sertoli cells [control 2], the frozen-thawed cells [experimental 1] and frozen-thawed cells co-cultured with Sertoli cells [experimental 2]. The morphological changes between different transplanted testes groups were compared in 8 weeks after transplantation. The statistical significance between mean values was determined by Kruskal Wallis and one-way analysis of variance in efficiency of transplantation. The statistical analysis revealed significant increases in the mean percentage of testis weight and normal seminiferous tubules following spermatogonial stem cells transplantation in the recipient's testes. The normal seminiferous tubules percentage in the co-culture system with fresh cells and frozen-thawed groups were more than those in non-transplanted and fresh cell transplanted groups [p

Localization of herpes simplex virus type 1 DNA in latently infected BALB/c mice neurons using in situ polymerase chain reaction

Herpes simplex virus type-1 [HSV-1] establishes a lifelong latent infection in neurons following primary infection. The existence of latent HSV-1 DNA in the trigeminal ganglia of infected BALB/c mice was examined using a direct in situ PCR technique, based on Digoxigenin-11-dUTP detection system with anti-digoxigenin-peroxidase and 3,3f-diaminobenzidine [DAB] substrate. Eight-week-old male BALB/c mice were inoculated via the eye by 10[4] plaque forming unit of wild type Iranian isolates of HSV-1. After establishment of latency, trigeminal ganglia were removed and examined using in situ PCR to detect HSV-1 genome. Finally, the results of in situ PCR were verified by a two-round PCR method, using amplification cocktail of in situ reaction, as a template for a conventional gel base PCR. The results suggest that a direct in situ PCR method using a peroxidase and DAB detection system is a useful means for detection of latent HSV-1 DNA in the latently infected ganglia