Evaluation of prooxidant-antioxidant balance in in vitro fertilization-conceived mice / 대한생식의학회지

OBJECTIVE: Concerns about the safety of assisted reproductive technology (ART) have been raised, as some studies have shown elevated incidence rates of childhood cancer, asthma, allergies, and other diseases in ART-conceived babies. Findings regarding the health of ART-conceived babies are controversial. The present study was conducted to evaluate the prooxidant-antioxidant balance (PAB) in in vitro fertilization (IVF)-conceived mice in comparison to naturally conceived offspring. METHODS: Mice (6–8 weeks) were divided into two groups (IVF-conceived and naturally conceived) matched by sex, age, weight, and litter size. A 1-mL blood sample was taken and the sera were separated. The oxidant-antioxidant balance was evaluated using a fast and reliable PAB assay. The results were expressed as mean±standard deviation. RESULTS: The mean PAB values (HK units) in the IVF-conceived and naturally conceived groups were 59.70±22.30 and 54.70±18.22, respectively (p=0.82). CONCLUSION: Since free radicals contribute to several pathological conditions and antioxidants play an important protective role against oxidative stress, evaluating the oxidant-antioxidant balance is very important. Although the results of this study showed that the quality of the defense mechanism against free radicals was not significantly different between the IVF-conceived and naturally conceived mice, other parameters of metabolic dysfunction need to be measured.

effects of sericin on cryopreserved sperm cells and subsequent embryo development in mice

Background: Sericin, because of its ability to remove free radicals and its antioxidant properties, has been used to successfully cryopreserve various mammalian cell types. However, the effects of sericin on cryopreservation of mouse sperm has not been reported

Objective: The current study intended to determine the protective role of different concentrations of sericin [0, 0.25, 0.5, and 0.75%] on mouse spermatozoa during cryopreservation, in addition to its effect on in vitro fertilization and subsequent embryo development

Materials and Methods: Mouse sperm from epididymides were frozen in cryoprotective agent with 18% raffinose, 3% skim milk, and different concentrations of sericin [0, 0.25, 0.5, 0.75%]. Thawed sperm were used for in vitro fertilization. The obtained embryos were cultured in Ksom medium for 6 days. The post-thawed motility, viability, fertilizing ability, and subsequent development to the 2-cell embryo and blastocyst stages were evaluated

Results: Our findings show that frozen-thawed sperm cells with 5% sericin indicate significantly [p

Conclusion: Supplementation by 0.5% sericin in cryoprotective agent improved frozen-thawed mouse epididymal sperm cell quality and resulted in increased embryo development

Combined effect of retinoic acid and basic fibroblast growth factor on maturation of mouse oocyte and subsequent fertilization and development

Background: Many autocrine and paracrine elements that are produced within follicular niche have been the focus of much in vitro maturation [IVM] research. The present study was carried out to compare retinoic acid [RA] and basic fibroblast growth factor [bFGF] efficacy on IVM of mouse oocytes, and their further dual consumption to reach an optimal protocol

Materials and Methods: In this experimental study, germinal vesicle [GV] oocytes obtained from two-months-old NMRI mice were randomly divided into control, sham and three experimental groups. The basic culture medium was alpha-MEM supplemented with 10% fetal bovine serum [FBS], 50 mg/l streptomycin, 60 mg/l penicillin and 10 ng/ ml epidermal growth factors. Each of the experimental groups received one of the following treatments: RA [2 microM], bFGF [20 ng/ml] or combination of RA and bFGF with the indicated concentrations. After 24 hours, capacitated spermatozoa were added to in vitro matured oocytes. Five hours later, the oocytes were cultured in fresh droplets of M2 medium for 24 hours and assessed for cleavage to the two-cells stage

Results: As compared with the control group, the rate of maturation was significantly increased in the RA [P<0.001] and bFGF+RA [P<0.02] groups with 58 +/- 10 and 57 +/- 3.46, respectively. The rate of maturation was significant in the RA [P<0.02] and bFGF+RA [P<0.03] groups, in comparison with the bFGF group. The bFGF+RA group had higher rate [83 +/- 1.52] of two-cells development, than control [33 +/- 1, P<0.001]

Conclusion: Our findings showed beneficial effects of 2 micro M RA and 20 ng/ml bFGF combination on mouse oocyte IVM

Embryonic stem cell conditioned medium supports in vitro maturation of mouse oocytes

Background: This study aimed to investigate the maturation and fertilization rates of immature mouse oocytes using Embryonic Stem Cell Conditioned Medium [ESCM]

Methods: Germinal Vesicle [GV] stage oocytes were observed in 120 NMRI mice, aged 4-6 weeks. GV oocytes with or without cumulus cells were subjected to IVM in either ESCM, Embryonic Stem Cell Growth Medium [ESGM], or alpha-minimum essential medium [alpha-MEM]. After recording the Metaphase II [MII] oocyte maturation rate, the oocytes were fertilized in vitro. The fertilization success rate was recorded after 24 hr. The embryos were maintained in potassium Simplex Optimization Medium [KSOM] for 96 hr and allowed to grow until the blastocyst stage. After recording developmental competence, they were transferred into the uteri of pseudopregnant mice and their birth rates were recorded

Results: No significant difference existed between the maturation rates in alpha-MEM [68.18%] and ESCM [64.67%; p>0.05], whereas this rate was significantly higher for both alpha-MEM and ESCM compared to ESGM [32.22%; p<0.05]. A significant difference in IVF success rate existed for oocytes grown in alpha-MEM [69.44%], ESCM [61.53%], and ESGM [0%]. A significantly higher developmental competence was observed at the blastocyst stage for oocytes grown in alpha-MEM [51.2%] compared to ESCM [35%; p<0.05]. 17 days after embryo transfer into the uteri of pseudopregnant mice, there was a nonsignficant [p>0.05], similar birth rate between alpha-MEM and ESCM [47 vs. 40%]

Conclusion: ESCM is an effective medium for preantral follicle growth, oocyte maturation, and subsequent embryo development

The expressions of stem cell markers: Oct4, Nanog, Sox2, nucleostemin, Bmi, Zfx, Tcl1, Tbx3, Dppa4, and Esrrb in bladder, colon, and prostate cancer, and certain cancer cell lines / 대한해부학회지

Uncontrolled self-renewal plays a direct function in the progression of different types of carcinomas. The same molecular pathway that manages self-renewal in normal stem cells also seems to manage cancer stem cells. Here, we examine the expressions of self-renewal regulatory factors Oct4, Nanog, Sox2, nucleostemin, Zfx, Esrrb, Tcl1, Tbx3, and Dppa4 in tissue samples of colon, prostate, and bladder carcinomas as well as cancer cell lines HT-29, Caco-2, HT-1376, LNCaP, and HepG2. We used reverse transcriptase polymerase chain reaction to examine expressions of the above mentioned regulatory factors in cancer cell lines HT-29, Caco-2, HT-1376, LNCaP, and HepG2 and in 20 tumor tissue samples. Total RNA was isolated by the ISOGEN method. RNA integrity was checked by agarose gel electrophoresis and spectrophotometry. Expressions of Oct4 and nucleostemin at the protein level were determined by immunocytochemistry. A significant relationship was found between tumor grade and self-renewal gene expression. Expressions of stem cell specific marker genes were detected in all examined cancer cell lines, in 40% to 100% of bladder cancer samples, and in 60% to 100% of colon and prostate cancer samples. Oct4 expressed in 100% of tumor tissue samples. Our data show that stem cell markers Oct4, Nanog, Sox2, nucleostemin, Bmi, Zfx, Esrrb, Tcl1, Tbx3, and Dppa4 significantly express in cancer cell lines and cancer tissues. Hence, these markers might be useful as potential tumor markers in the diagnosis and/or prognosis of tumors.

Production of cloned mice by nuclear transfer of cumulus cells

Over the past several years, mammals have been successfully cloned by either the splitting of an early stage embryo or nuclear transfer of adult somatic cells [NT] into oocytes. Although it has been 15 years since the generation of the first cloned mammals from somatic cells by NT, the success rate for producing live offspring by this technique is low regardless of the cell type and animal species used. However, these techniques have the potential to be important tools for future research in basic biology. In the present study, we described our experiences in producing successfully cloned mouse using NT method and piezo-actuated micromanipulator. B6D2F1 mice, 8-12 weeks old, were superovulated with injections of 5 IU of pregnant mare serum gonadotropin and 5 IU of human chorionic gonadotropin administered 48 hr apart. Enucleation and donor nuclei cumulus cell injection were performed with a piezo-actuated micromanipulator after which activation and trichostatin A treatment were used for reconstructed oocytes. Two-cell stage cloned embryos that developed in the mWM medium were transferred into the oviducts of pseudopregnant NMRI mice. Of 367 oocytes collected, 131 [69%] developed into 2-cell stage embryos. Of these, 5 [1%] live pups were successfully delivered. We used NMRI foster mother to raise the pups by lactation. One adult cloned mouse was mated, after which she delivered and raised normal offspring. For mouse cloning, the present study also successfully tested the capability of somatic cell nuclear transfer SCNT using a piezo unit

Evaluating the expression of self-renewal genes in human endothelial progenitor cells

Endothelial progenitor cells [EPCs] have a potential application for cell therapy, however, their biological nature is not well-understood. EPCs also possess some stemness features, such as their clonogenicity and differentiation capacity. The main aim of this study was to evaluate the expression of certain transcription factors regulating self-renewal property of stem cells. In this experimental study, peripheral blood mononuclear cells were isolated from fresh human blood of several volunteers and were cultured in fibronectin- coated plates. EPCs were identified based on their morphology and growth characteristic. Then, the expression of some markers implicated in self-renewal capacity was assessed in the isolated cells using reverse transcription-polymerase chain reaction [RT-PCR] and immunocytochemistry. Expression of the cell surface markers, CD31 and CD34, was determined by RT-PCR and immunocytochemistry. Furthermore, these cells had the ability for Di-AC-LDL incorporation as well as attachment to lectin I. EPCs did not express the main stem cell markers, like OCT4-A, Nanog, and Sox2; nevertheless, they expressed the weaker pluripotent markers, including OCT4B and OCT4-B1 spliced variants, such as Nucleostemin and ZFX. Furthermore, the expression of Nucleostemin and ZFX genes revealed a decreasing pattern from days 4[th] to 11[th]. The main regulators of stem cell self-renewal genes, including OCT4-A, Nanog, and Sox2 are not expressed in EPCs. Forced expression of these genes can elevate the stemness property and clinical application of EPCs

[Evaluating the expression of Oct-4, NANOG, Sox2 and nucleostemin in colon cancer cell lines [Caco-2 and HT-29]]

Evaluating the expression of Oct-4, NANOG, Sox2 and Nucleostemin in colon cancer cell lines [Caco-2 and HT-29]. Caco-2 and HT-29 human colon cancer cell lines were cultured in Dulbecco's modified eagles medium [DMEM] and Roswell Park Memorial Institute medium [RPMI] respectively, containing 10% fetal bovin serum [FBS] with 1% peniciline and streptomycinen in 37°, 5% CO2 incubator. Total RNA was isolated using the ISOGEN method. RNA integrity was checked with the use of agarose gel electrophoresis and spectrophotometry. Reverse transcriptase polymerase chain reaction [RT-PCR] was used to examin the samples. The expression of Oct-4 and Nucleostemin at the protein level was further determined using immunocytochemistry. RT-PCR analysis of Caco2 and HT-29 colon cancer cell lines showed expression of Oct-4, NANOG, Sox2 and Nucleostemin genes. Also immunocytochemical analysis confirmed the cytoplasmic and nuclear expression of the Oct-4 protein and Nucleostemin proteins. Collectively, our data confirmed the expression of Oct-4, NANOG, Sox2 and Nucleostemin in colon cancer cells and suggested that their expression can be used as potential tumor markers in diagnosis and /or prognosis of colon tumors. These results confirm the potential value of the cancer stem-cell theory in cancer therapy

Murine Adherent CD34 [+] cell population expanded by single cell cloning

While human endothelial progenitor cells [EPCs] have been a subject of somehow extensive investigation, EPCs from adult mouse hematopoietic system were poorly studied. Present investigation is focused on FVB mouse endothelial progenitor cells in terms of their isolation, purification, and expansion. Material and Mononuclear cells collected from murine peripheral blood were cultured in fibronectin coated plate for two weeks, at which point, the adherent cell population were lifted and analyzed in terms of some surface markers. Using FACS Vantage equipped with one-cell deposition unit, single CD34 positive cells were plated per well already containing medium optimized for single cell growth. Several clones were then emerged, expanded, and examined in terms of some surface markers. Furthermore, the cells were investigated regarding ability to uptake DiI-ac-LDL and form capillary network on matrigel surfaces Adherent population of mononuclear cells from mouse peripheral blood was appeared morphologically heterogeneous. About 5% of the adherent cells were CD34 positive. Having optimized their culture condition, several CD34 positive clones were expanded. The cells comprising the clones were DiI-ac-LDL+ and formed capillary-like tube when being seeded on matrigel surfaces.The primary culture of the mononuclear cells from murine peripheral blood contains a very limited number of cells positive for endothelial lineage markers. These cells [adherent CD34 positive] could be expanded by single cell cloning technique