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.

Association of serum content of 25-hydroxy vitamin D with semen quality in normozoospermic and oligoasthenoteratozoospermic men

Background: vitamin D has multifaceted function in human reproductive physiology. It has been revealed that vitamin D is involved in spermatogenesis, and semen quality can be linked to vitamin D status in men

Objective: evaluating the correlation of 25-hydroxy vitamin D [25-OHD] levels in serum with basic and advanced semen parameters and essential determinants of spermatozoa function

Materials and Methods: participants were categorized, based on semen parameters, into normozoospermic [NS] and oligoasthenoteratozoospermic [OAT] men. Serum level of 25-OHD was measured. Apoptotic status of spermatozoa, mitochondrial membrane potential and reactive oxygen species content of semen were assessed

Results: difference of 25-OHD concentration in serum of NS men versus OAT ones did not meet significance threshold. DNA fragmentation, reactive oxygen species content of semen and mitochondrial membrane potential state revealed significant difference between NS and OAT subjects. There were no significant differences in basic and functional semen parameters when men were stratified based on serum 25-OHD level. Taking both 25-OHD and semen categories [NS and OAT] into consideration did not indicate any significant difference in studied parameters. Total motility of spermatozoa was positively correlated with serum concentration of 25-OHD in all studied subjects. In addition, normal morphology of spermatozoa in NS men revealed a positive and significant correlation with levels of 25-OHD in serum

Conclusion: vitamin D may affect motility and morphology of spermatozoa. Lower content of serum vitamin D may affect fertility of men and should be considered in examination of men with abnormal spermogram

Isolation and enrichment of mouse female germ line stem cells

The existence of female germ-line stem cells [FGSCs] has been the subject of a wide range of recent studies. Successful isolation and culture of FGSCs could facilitate studies on regenerative medicine and infertility treatments in the near future. Our aim in the present study was evaluation of the most commonly used techniques in enrichment of FGSCs and in establishment of the best procedure. In this experimental study, after digesting neonate ovary from C57Bl/6 mice, we performed 2 different isolation experiments: magnetic activated cell sorting [MACS] and pre-plating. MACS was applied using two different antibodies against mouse vasa homolog [MVH] and stage-specific embryonic antigen-1 [SSEA1] markers. After the cells were passaged and proliferated in vitro, colony-forming cells were characterized using reverse transcription-polymerase chain reaction [RT-PCR] [for analysis of expression of Oct4, Nanog, C-kit, Fragilis, Mvh, Dazl, Scp3 and Zp3], alkaline phosphatase [AP] activity test and immunocytochemistry. Data showed that colonies can be seen more frequently in pre-plating technique than that in MACS. Using the SSEA1 antibody with MACS, 1.98 +/- 0.49% [Mean +/- SDV] positive cells were yield as compared to the total cells sorted. The colonies formed after pre-plating expressed pluripotency and germ stem cell markers [Oct4, Nanog, C-kit, Fragilis, Mvh and Dazl] whereas did not express Zp3 and Scp3 at the mRNA level. Immunocytochemistry in these colonies further confirmed the presence of OCT4 and MVH proteins, and AP activity measured by AP-kit showed positive reaction. We established a simple and an efficient pre-plating technique to culture and to enrich FGSCs from neonatal mouse ovaries

Differentiation of human umbilical cord matrix-derived mesenchymal stem cells into germ-like cells

Mesenchymal Stem Cells [MSCs] are multipotent cells that can be collected from different sources. Under specific conditions, MSCs can be differentiated to tissue specific cells in vitro. Human Umbilical Cord Mesenchymal Stem Cells [hUCMSCs] can easily be harvested and cultured in in vitro conditions. Production of germ cells from mesenchymal stem cells is a very interesting and promising area in the field of reproductive medicine. In the present study, the possible trans-differentiation of hUCMSCs into Primordial like Germ Cell [PGC] was performed in vitro under specific condition. Human umbilical cord mesenchymal stem cells were cultured and expanded in DMEM medium containing 10% FBS. The cultured cells were studied for differentiation ability to adipocytes and osteocytes. Furthermore, MSCs related markers were identified by flow cytometry method. For PGC differentiation, hUCMS cells were cultured in differentiation medium containing Bone Morphogenetic Protein 4 [BMP4] and it was followed by retinoic acid [RA]. Real time PCR and immunocytochemistry analysis were performed to evaluate the expression of PGC specific genes and proteins, respectively. Our results showed that hUCMSCs cultured in the presence of BMP4 and RA are able to trans differentiate in to PGC like cells in vitro. Real time PCR and immunocytochemistry results showed that differentiated cells expressed PGC specific markers after 14 days of culture. Based on these results, it was concluded that hUCMSC may be considered as a promising alternative cell source in reproductive medicine. More studies including laboratory and also animal models are needed to evaluate the functionality of differentiated PGCs before introducing them to clinical applications

Comparison of differentiation potential of male mouse adipose tissue and bone marrow derived-mesenchymal stem cells into germ cells

Recent publications about differentiation of stem cells to germ cells have motivated researchers to make new approaches to infertility. In vitro production of germ cells improves understanding differentiation process of male and female germ cells. Due to the problem of using embryonic stem cells [ESC], it's necessary the mentioned cells be replaced with some adult multi-potent stem cells in laboratories. The aim of this study was to obtain germ cells from appropriate source beyond ESC and compare differential potentials of adipocytes derived stem cells [ADMSCs] with bone marrow derived stem cells [BMMSCs]. To find multi-potential entity, after providing purified ADMSCs and BMMSCs, differentiation to osteoblast and adipocyte was confirmed by using appropriate culture medium. To confirm mesenchymal lineage production superficial markers [expression of CD90 and CD44 and non-expression of CD45 and CD31] were investigated by flowcytometry. Then the cells were differentiated to germ cells in inductive medium containing retinoic acid for 7days. To evaluate germ cells characteristic markers [Dazl [Deleted in azoospermia-like], Mvh [Mouse vasa homolog gene], Stra8 [Stimulated by retinoic acid] and Scp3 [Synaptonemal complex protein 3]] flowcytometry, imunoflorescence and real time PCR were used. Both types of cells were able to differentiate into osteoblast and adipocyte cells and presentation of stem cell superficial markers [CD90, CD44] and absence of endothelial and blood cell markers [CD31, CD45] were confirmative The flowcytometry, imunoflorescence and real time PCR results showed remarkable expression of germ cells characteristic markers [Mvh, Dazl, Stra8, and Scp3]. It was found that although ADMSCs were attained easier and also cultured and differentiated rapidly, germ cell markers were expressed in BMMSCs significantly more than ADMSCs

Functional concentrations of BMP4 on differentiation of mouse embryonic stem cells to primordial germ cells

Bone morphogenetic protein 4 [BMP4] has a significant role in primordial germ cells [PGCs] differentiation from mouse embryonic stem cell [mESC]. The aim of this study is to determine the best concentration of BMP4 at a time of two days on differentiation PGCs from mESC. To differentiate PGCs, embryoid bodies [EBs] from mESCs were cultured in concentrations of 0, 5 and 10 ng/ml BMP4 for two days. Germ cell markers Oct4 [Pou5f1], Stella [Dppa3] and Mvh [Ddx4] were analyzed by flow cytometry, immunocytochemistry and reverse transcriptase polymerase chain reaction [RT-PCR]. Flow cytometry data demonstrated most Mvh-positive cells were observed only in the treated groups. Immunocytochemistry of EBs in the treated groups identified cells positive for Mvh. PCR results showed expression of Oct4 in the control group and treated groups. Stella and Mvh were expressed only in the treated groups. Low concentrations of BMP4 during two days had an optimal effect on differentiation of PGCs from mESC

Repair of spinal cord injury by co-transplantation of embryonic stem cell-derived motor neuron and olfactory ensheathing cell

The failure of regeneration after spinal cord injury [SCI] has been attributed to axonal demyelination and neuronal death. Cellular replacement and white matter regeneration are both necessary for SCI repair. In this study, we evaluated the co-transplantation of olfactory ensheathing cells [OEC] and embryonic stem [ES] cell-derived motor neurons [ESMN] on contused SCI. OEC cultured from olfactory nerve rootlets and olfactory bulbs. ESMN was generated by exposing mouse ES cells to retinoic acid and sonic hedgehog. Thirty female rats were used to prepare SCI models in five groups. Control and medium-injected groups was subjected to induce lesion without cell transplantation. OEC or ESMN or both were transplanted into the site of the lesion in other groups. The purity of OEC culture was 95%. Motor neuron progenitor markers [Olig2, Nkx6.1 and Pax6] and motor neuron markers [Is11, Is12 and Hb9] were expressed. Histological analysis showed that significantly more [P<0.001] spinal tissue was spared in OEC, ESMN and OEC+ ESMN groups but the OEC+ ESMN group had a significantly greater percentage of spared tissue and myelination than other groups [P< 0.05]. The numbers of ESMN in co-transplanted group were significantly higher than ESMN group [P<0.05]. A significant [P<0.05] recovery of hindlimb function was observed in rats in the transplanted groups. We found that the co-transplantation of ESMN and OEC into an injured spinal cord has a synergistic effect, promoting neural regeneration, ESMN survival and partial functional recovery

in vitro fertilization rate of mouse ova in the absence or presence of recombinant human leukemia inhibitory factor

To examine the effect of human recombinant leukemia inhibitory factor in different doses on rate of fertilization of mouse ova. Prospective study. Department of Anatomy, laboratory of cell culture. Animals: Female NMRI mice 6 to 8 weeks old. Mice were killed at 12-14 hours after hCG or 36-38 hours after hMG injection. Mature oocytes were obtained and divided randomly into 5 groups. Oocytes in group A [n=157] were cultured as the control group in TYH medium. Oocytes in groups B, C, D, E [n=137, 154, 166 and 159, respectively] were cultured in the same medium supplemented with recombinant human leukemia inhibitory factor in four different concentrations [5, 7.5, 10, 20ng/ml, respectively] for 1 hour. After that time 100000 spermatozoa were added to every drop and after 24-26 hours two cell embryos were recorded. Fertilization was assessed by recording the number of 2-cell embryos and analysed by X[2] tests. Two cell embryos. No significant difference was detected in the rate of two cell embryos in the studied experimental groups as compared with the rate of two cell embryos in control group [Group A]. This study showed that, different concentrations of recombinant human of leukemia inhibitory factor in standard medium does not enhance in vitro fertilization rate of mouse oocytes

Time course of degradation and deadenylation of maternal c-mos and cyclin A2 mRNA during early development of one-cell embryo in mouse

Early in the development of many animals, before transcription begins, any change in the pattern of protein synthesis is attributed to a change in the translational activity or stability of mRNA in the egg and early embryo. As a result, translational control is critical for a variety of developmental decisions, including oocyte maturation and initiation of preimplantation development. In this study, using real-time RT-PCR method, we defined the time course of degradation and deadenylation of an oocyte specific gene [c-mos] more precisely and a gene that is re-synthesized after ZGA [cyclin A2]. Our data indicate that oocyte-specific transcript, c-mos, degrades rapidly while cyclin A2 mRNA does not and the deadenylation of c-mos mRNA precedes the process of degradation. Our findings suggest that time-dependent elimination of different maternal mRNA is a way for regulation of translation in early development of mouse embryos

effect of nitric oxide on mouse Pre-implantation embryo development and apoptosis

Clinical studies have shown that in pathological conditions such as endometriosis and reproductive tract infection [male and female] there is an activation status of macrophages that produce large quantities of nitric oxide [NO] in addition to other effector molecules. Large amounts of NO may have embryotoxic roles and produce infertility. This study was designed to evaluate the effect of different concentrations of NO on mouse pre-implantation embryo development in vitro. Mouse embryos [2-cell stage] were cultured in media containing different concentrations of sodium nitroprusside [SNP], an NO donor, or L-arginine methyl ester [L-NAME], an NO syntase [NOS] inhibitor. At the end of culture, cell apoptosis was studied by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling [TUNEL] technique. The results showed that development of preimplantation embryos were inhibited by high concentration of SNP [1 and 10