Human ovarian theca-derived multipotent stem cells have the potential to differentiate into Oocyte-Like cells in vitro

Objective: In this study, we have examined human theca stem cells [hTSCs] in vitro differentiation capacity into human oocyte like cells [hOLCs]

Materials and Methods: In this interventional experiment study, hTSCs were isolated from the theca layer of small antral follicles [3-5 mm in size]. Isolated hTSCs were expanded and cultured in differentiation medium, containing 5% human follicular fluid, for 50 days. Gene expressions of PRDM1, PRDM14, VASA, DAZL, OCT4, ZP1, 2, 3 GDF9, SCP3 and DMC1 were evaluated by quantitative reverse transcription polymerase chain reaction [qRT-PCR] on days 0, 18, and 25 after monoculture as well as one week after co-culture with human granulosa cells [hGCs]. In addition, GDF9, OCT4, DAZL, VASA, and ZP3 proteins were immune-localized in oocyte-like structures

Results: After 16-18 days, the color of the medium became acidic. After 25 days, the cells started to differentiate into round-shaped cells [20-25 ìm diameter]. One week after co-culturing with hGCs, the size of the round cells increased 60 to70 ìm and convert to hOLCs. However, these growing cells expressed some primordial germ cell [PGC]- and germ cell genes [PRDM1, PRDM14, VASA, DAZL, and OCT4] as well as oocyte specific genes [ZP1, 2, 3 and GDF9], and meiotic-specific markers [SCP3 and DMC1]. In addition, GDF9, OCT4, DAZL, VASA, and ZP3 proteins were present in hOLCs

Conclusion: To sum up, hTSCs have the ability to differentiate into hOLCs. This introduced model paved the way for further in vitro studies of the exact mechanisms behind germ cell formation and differentiation. However, the functionality of hOLCs needs further investigation

Optimization of the electroporation conditions for transfection of human factor IX into the goat fetal fibroblasts

Electroporation is the most common method used for the transfection of DNA. Although this method has been attributed for various cell using different buffer compositions, the effects of DNA concentration on the transfection efficiency has not yet been studied. Here the correlation between the efficiency of electroporation reaction and increments of DNA concentration has been investigated. Following this investigation, a study was set out to produce a transgenic goat which is capable of secreting recombinant human coagulation factor IX in its milk. In this experimental study, a linearized recombinant vector [pBC1] entailing human coagulation factor IX [5.7 kb] cDNA was introduced into goat fetal fibroblast cells [three sub passages] via electroporation in four separate experiments [while other variables were kept constant], beginning with 10 micro g DNA per pulse in the first experiment and increments of 10 micro g/pulse for the next three reactions. Thereafter, polymerase chain reaction [PCR]-positive cell culture plates were diluted by several factors for further analysis of the transfected wells. Subsequently, positive cells were isolated for fluorescence in situ hybridization. Logistic regression model was used for data analyzing. Significance was defined as p< 0.05. The results showed no significant difference among three first concentrations except for group 1 [10 micro g/pulse] and group 3 [30 micro g/pulse], but there was a significant difference between these groups and the fourth group [p<0.05], as this group [40 micro g/pulse] statistically showed the highest rate of transfection. As the fluorescence in situ hybridization [FISH] results indicated the transgene was integrated in a single position in PCR positive cells. Increasing amount of using the vector 40micro g/pulse efficiently increased the number of transfected cells. Besides of voltage and buffer strength which had been previously shown to play a critical role in electroporation efficiency, our results showed an increase in DNA concentration could affect an exponential surge in the electroporation efficiency

Effect of Beta Mercaptaethanol with and without BSO [DL-Buthionine Sulfoximine] on Resumption of Meisosis, in vitro maturation and Embryo development of immature mouse oocytes

The purpose of this study was to evaluated the effect of beta-mercaptoethanol on resumption of meiosis, in vitro maturation of immature mouse oocytes and resulting embryo development with and without BSO [DL-Buthionine sulfoximine] Material and Germinal vesicle [GV] were recovered from 6-8 weeks old NMRI ovaries and cultured in maturation medium in MEMalpha supplemented with 7.5IU/ml hCG, 100mIU/ml rhFSH, 5% FCS [control group] and adding 100 micro m beta-mercaptoethanol [group 1] or with 5mM BSO + 100 micro m beta-mercaptoethanol [group 2] for 24h. The matured oocytes then were fertilized and cultured for 5 days. Fertilization and development were accomplished in T6 medium.The percentage of GV oocytes reaching to metaphase I [or undergo GVBD] were 78.5%, 85%, 86% in control group, group 1 and group 2 respectively, that no significant difference was detected between groups. The proportion of oocytes that progressed to the metaphase II [MII] stage was minimum within 5mM BSO group [group 2] and maximum within beta-mercaptoetanol group [group 1] with significant difference comparing with control and each other [P<=0.05]. The percentage of embryos reaching to morula stage within beta-mercaptoetanol group was significantly higher than the control group [5% and 12.2% respectively]. None of oocytes treated with BSO could pass the 8 cell stage. beta-mercaptoetanol enhances IVM and improves embryo development. While adding BSO into the maturation medium even with beta-mercaptoetanol decreases maturation and declines the embryo development

Effect of DEHP[Di-2ethyl hexyl-phthalate] on resumption of meiosis and in -virto maturation of mouse oocytes and development of resulting embryos

DEHP [di[2-ethylhexyl] phthalate]] is widely used in plastic industry and some reproductive toxicity has been shown with it. So, this study was designed to evaluate DEHP effects on resumption of meiosis and in vitro maturation of mouse oocytes as well as development of embryos resulted from them. Mice of 4-6 weeks old were administered daily doses of 50, 100, 200 microl of 2.56 micro M DEHP solution for 12 days. Immature mouse oocytes were recovered from all experimental groups and matured in MEM-alpha medium containing 5% FCS with and without 7.5 IU hCG and 100 mIU rFSH. IVF was performed T6 medium. Resumption of meiosis and in vitro maturation were significantly lower in all experimental groups in culture media without hormones compared to controls. Fertilization and embryo development were also significantly decreased in both culture media [with and without hormones]. This study showed the adverse effects of DEHP on in vitro maturation and embryo development in a dose dependent manner