Enhanced development of mouse single blastomeres into blastocysts via the simultaneous inhibition of TGF‐β and ERK pathways in microdroplet culture

Optimization of an in vitro culture that supports blastocyst (BL) development from single blastomeres (SBs) is essential to generate additional embryos for farm animals and humans and unravel the mechanisms that underlie totipotency. In this study, we have examined BL development from SBs that were derived from 2‐cell and 4‐cell mouse embryos in different media. Moreover, BLs were assessed for inner cell mass (ICM) by staining with Oct4. We found that BL development was improved in a lower volume of medium (1 µL) compared with a higher volume (5 µL). Furthermore, the supplementation of medium with the inhibitors of ERK1/2 and TGFβ (R2i) signaling pathways in 1 µL droplets of T6 medium improved BL development. The co‐culture of SBs with intact embryos in the presence of R2i showed more BL development and ICM to trophectoderm cell number ratio in comparison with SB culture and SB group culture. We also observed reduced total cell number, ICM, and trophectoderm cell numbers in all of the SB culture conditions versus intact embryo development. These findings might facilitate the successful generation of additional embryos for biomedical applications and elucidate the mechanisms that underlie totipotency.     

Nitric Oxide and Protein Disulfide Isomerase Explain the Complexities of Unfolded Protein Response Following Intra-hippocampal Aβ Injection

Several pathways involved in regulation of intracellular protein integrity are known as the protein quality control (PQC) system. Molecular chaperones as the main players are engaged in various aspects of PQC system. According to the importance of these proteins in cell survival, in the present study, we traced endoplasmic reticulum-specific markers and chaperone-mediated autophagy (CMA)-associated factors as two main arms of PQC system in intra-hippocampal amyloid beta (Aβ)-injected rats during 10 days running. Data analysis from Western blot indicated that exposure to Aβ activates immunoglobulin heavy-chain-binding protein (Bip) which is the upstream regulator of unfolded protein responses (UPR). Activation of UPR system eventually led to induction of pro-apoptotic factors like CHOP, calpain, and caspase-12. Moreover, our data revealed that protein disulfide isomerase activity dramatically decreased after Aβ injection, which could be attributed to the increased levels of nitric oxide. Besides, Aβ injection induced levels of 2 members of heat shock proteins (Hsp) 70 and 90. Elevated levels of Hsps family members are accompanied by increased levels of lysosome-associated membrane protein type-2A (Lamp-2A) that are involved in CMA. Despite the reduction in CHOP, calpain, caspase-12, and Lamp-2A protein levels, the levels of molecular chaperones Bip, Hsps70, and 90 increased 10 days after Aβ injection in comparison to the control group. Based on our results, 10 days after Aβ injection, despite the activation of protective chaperones, markers associated with neurotoxicity were still elevated.     

Up-regulation of Arl4a gene expression by broccoli aqueous extract is associated with improved spermatogenesis in mouse testes

Introduction: Broccoli (Brassica oleracea) is well known for its properties as an anticancer, antioxidant, and scavenger of free radicals. However, its benefits in enhancing spermatogenesis have not been well established.Objective: To study broccoli aqueous extract effects on sperm factors and the expression of genes Catsper1, Catsper2, Arl4a, Sox5, and Sox9 in sperm factors in mice.Material and methods: Male mice were divided randomly into six groups: (1) Control; (2) cadmium (3 mg/kg of mouse body weight); (3) orally treated with 200 µl broccoli aqueous extract (1 g ml^-1); (4) orally treated with 400 µl of broccoli aqueous extract; (5) orally treated with 200 broccoli aqueous extract plus cadmium, and (6) orally treated with 400 µl of broccoli aqueous extract plus cadmium. We analyzed the sperms factors and Catsper1, Catsper2, Arl4a, Sox5, and Sox9 gene expression.Results: An obvious improvement in sperm count and a slight enhancement in sperm motility were observed in mice treated with broccoli extract alone or with cadmium. Sperm viability was reduced by broccoli extract except for the 200 µl dose with cadmium, which significantly increased it. Interestingly, Arl4a gene expression increased in the 400 µl broccoli- treated group. Likewise, the Arl4a mRNA level in mice treated with cadmium and 200 µl of broccoli extract was higher than in the cadmium-treated mice. Furthermore, broccoli extract enhanced the mRNA level of Catsper2 and Sox5 genes in mice treated with 200 µl and 400 µl broccoli extract plus cadmium compared with the group treated solely with cadmium.Conclusion: The higher sperm count in broccoli-treated mice opens the way for the development of pharmaceutical products for infertile men.     

Comparative capability of menstrual blood versus bone marrow derived stem cells in neural differentiation

In order to characterize the potency of menstrual blood stem cells (MenSCs) for future cell therapy of neurological disorders instead of bone marrow stem cells (BMSCs) as a well-known and conventional source of adult stem cells, we examined the in vitro differentiation potential of these stem cells into neural-like cells. The differentiation potential of MenSCs to neural cells in comparison with BMSCs was assessed under two step neural differentiation including conversion to neurosphere-like cells and final differentiation. The expression levels of Nestin, Microtubule-associated protein 2, gamma-aminobutyric acid type B receptor subunit 1 and 2, and Tubulin, beta 3 class III mRNA and/or protein were up-regulated during development of MenSCs into neurosphere-like cells (NSCs) and neural-like cells. The up-regulation level of these markers in differentiated neural-like cells from MenSCs was comparable with differentiated cells from BMSCs. Moreover, both differentiated MenSCs and BMSCs expressed high levels of potassium, calcium and sodium channel genes developing functional channels with electrophysiological recording. For the first time, we demonstrated that MenSCs are a unique cell population with differentiation ability into neural-like cells comparable to BMSCs. In addition, we have introduced an approach to generate NSCs from MenSCs and BMSCs and their further differentiation into neural-like cells in vitro. Our results hold a promise to future stem cell therapy of neurological disorders using NSCs derived from menstrual blood, an accessible source in every woman.     

Castration attenuates myelin repair following lysolecithin induced demyelination in rat optic chiasm: an evaluation using visual evoked potential, marker genes expression and myelin staining

Multiple sclerosis (MS) is a demyelinating disease that affects the central nervous system. MS is the most common neurological disorder in young adults with a greater incidence among females. Male gonadal hormones have a protective effect on neural system development and myelin maturation. In this study, we investigate the effect of castration on lysolecithin-induced demyelination and remyelination processes using visual evoked potentials, in addition to measuring the expressions of Olig2, MBP, Nogo-A and GFAP mRNAs as oligodendrocyte or astrocyte markers; and histological assessments by myelin-specific staining. We observed more expanded demyelination with delayed repair process in castrated rats. Expression levels of the aforementioned marker genes confirmed histological and electrophysiological observations. Our results showed a pivotal role for endogenous male hormones in the context of demyelinating insults. It may also account for the different prognosis of MS between male and female genders and provide new insights for therapeutic treatments.     

Nogo Receptor Inhibition Enhances Functional Recovery following Lysolecithin-Induced Demyelination in Mouse Optic Chiasm

Background

Inhibitory factors have been implicated in the failure of remyelination in demyelinating diseases. Myelin associated inhibitors act through a common receptor called Nogo receptor (NgR) that plays critical inhibitory roles in CNS plasticity. Here we investigated the effects of abrogating NgR inhibition in a non-immune model of focal demyelination in adult mouse optic chiasm.

Methodology/Principal Findings

A focal area of demyelination was induced in adult mouse optic chiasm by microinjection of lysolecithin. To knock down NgR levels, siRNAs against NgR were intracerebroventricularly administered via a permanent cannula over 14 days, Functional changes were monitored by electrophysiological recording of latency of visual evoked potentials (VEPs). Histological analysis was carried out 3, 7 and 14 days post demyelination lesion. To assess the effect of NgR inhibition on precursor cell repopulation, BrdU was administered to the animals prior to the demyelination induction. Inhibition of NgR significantly restored VEPs responses following optic chiasm demyelination. These findings were confirmed histologically by myelin specific staining. siNgR application resulted in a smaller lesion size compared to control. NgR inhibition significantly increased the numbers of BrdU+/Olig2+ progenitor cells in the lesioned area and in the neurogenic zone of the third ventricle. These progenitor cells (Olig2+ or GFAP+) migrated away from this area as a function of time.

Conclusions/Significance

Our results show that inhibition of NgR facilitate myelin repair in the demyelinated chiasm, with enhanced recruitment of proliferating cells to the lesion site. Thus, antagonizing NgR function could have therapeutic potential for demyelinating disorders such as Multiple Sclerosis.     

Revision of the foraminiferal biozonation scheme in Upper Cretaceous carbonates of the Dezful Embayment,Zagros, Iran: Integrated palaeontological,sedimentological and geochemical investigation

Cretaceous carbonate successions of the Bangestan Group, such as the Sarvak and Ilam formations, are among the most prolific hydrocarbon reserves of the Middle East. However, relatively little is known about their detailed palaeontology and biostratigraphy. Moreover, due to lithological similarity of these carbonate formations recognition of their boundaries in subsurface studies is problematic. To investigate these units, biostratigraphic analyses were carried out on nearly 1100 m of cores, including core plug samples and thin sections prepared from five giant and supergiant oilfields in the northern and southern Dezful Embayment, SW Iran. Accordingly, 59 species of foraminifera (assigned to 43 genera) as well as 11 species of non-foraminifera (10 genera) were recognized. As a result, three biozones were identified, which in stratigraphic order are: Nezzazata-Alveolinids Assemblage Zone; Moncharmontia apenninica-Nezzazatinella-Dicyclina Assemblage Zone; and Rotalia skourensis-algae Assemblage Zone. These are compared with the Wynd's (1965) biozonation scheme, previously introduced in the Zagros area, and a revised scheme is presented. Accordingly, a Cenomanian–Turonian age and a Coniacian–Campanian age are envisaged for the Upper Sarvak and Ilam formations, respectively. In our new biostratigraphic scheme, the Sarvak–Ilam formations boundary is considered to be located above the Moncharmontia apenninica-Nezzazatinella-Dicyclina Assemblage Zone (equivalent of Valvulammina-Dicyclina Assemblage Zone of Wynd, 1965), that is Turonian in age. This zone is bounded by two palaeoexposure surfaces, which correspond approximately to the C–T boundary transitional interval and a post-Turonian, which can be possibly assigned to the Coniacian. Significant sedimentological features of these disconformities include bauxitic–lateritic horizons, karstified profiles and solution-collapsed breccias. Geochemical signatures of these meteorically altered surfaces are also considered to calibrate biofacies and biozones. Finally, we compared our new biozonation scheme with other studies in neighboring areas of SW Iran and the Middle East.     

bFGF-mediated pluripotency maintenance in human induced pluripotent stem cells is associated with NRAS-MAPK signaling

Background

Human pluripotent stem cells (PSCs) open new windows for basic research and regenerative medicine due to their remarkable properties, i.e. their ability to self-renew indefinitely and being pluripotent. There are different, conflicting data related to the role of basic fibroblast growth factor (bFGF) in intracellular signal transduction and the regulation of pluripotency of PSCs. Here, we investigated the effect of bFGF and its downstream pathways in pluripotent vs. differentiated human induced (hi) PSCs.

Methods

bFGF downstream signaling pathways were investigated in long-term culture of hiPSCs from pluripotent to differentiated state (withdrawing bFGF) using immunoblotting, immunocytochemistry and qPCR. Subcellular distribution of signaling components were investigated by simple fractionation and immunoblotting upon bFGF stimulation. Finally, RAS activity and RAS isoforms were studied using RAS assays both after short- and long-term culture in response to bFGF stimulation.

Results

Our results revealed that hiPSCs were differentiated into the ectoderm lineage upon withdrawing bFGF as an essential pluripotency mediator. Pluripotency markers OCT4, SOX2 and NANOG were downregulated, following a drastic decrease in MAPK pathway activity levels. Notably, a remarkable increase in phosphorylation levels of p38 and JAK/STAT3 was observed in differentiated hiPSCs, while the PI3K/AKT and JNK pathways remained active during differentiation. Our data further indicate that among the RAS paralogs, NRAS predominantly activates the MAPK pathway in hiPSCs.

Conclusion

Collectively, the MAPK pathway appears to be the prime signaling pathway downstream of bFGF for maintaining pluripotency in hiPSCs and among the MAPK pathways, the activity of NRAS-RAF-MEK-ERK is decreased during differentiation, whereas p38 is activated and JNK remains constant.