Forced Suppression of let-7a-5p in Mouse Blastocysts Improves Implantation Rate.

Embryo implantation requires appropriate communication between the blastocyst and endometrium. Recurrent implantation failure is an essential component of assisted reproductive technology. Also, miRNA-mediated gene expression impacts the implantation process, and the downregulation of some miRs, such as mmu-let-7a, improves this process. In the present study, we evaluated the effect of let-7a forced suppression on the mouse implantation rate. In total, 100 adult female mice and 10 adult male mice were included (Strain CD-1). We analysed the expression of let-7a and its potential mRNAs targets (Igf1, Il1a, Itgb3 and Tgfb1) in control, sham and antagomir-treated blastocysts using quantitative reverse transcription PCR (qRT-PCR). The control and treated blastocysts were transferred to the 20 pseudopregnant mice so that the effect of let-7a suppression on the rate of implantation could be determined. The expression level of let-7a in the treatment group was significantly downregulated (P=0.001) In contrast, no significant expression changes were observed for let-7a or mRNAs targets when the sham and control groups were compared (P>0.05). In comparison to the controls, the antagomir-treated group exhibited significantly upregulated expression levels of Igf1 (0.0167), Itgb3 (0.045) and Tgfb1 (0.0115). Additionally, the implantation rate was significantly higher in the treatment group (78%) than the control group (61%) (P=0.0098). We found that forced suppression of mmu-let-7a-5p through successful transfection of Anti-miR leads to upregulation of downstream genes, Igf1, Itgb3 and Tgfb1, which directly involved in the trophoblast-endometrium attachment and improve the implantation rate.

The effects of Finasteride on the expression of Dazl, Tsga10, Sycp3, Prm2 genes during spermatogenesis in testes of NMRI mice.

OBJECTIVE: In this study, the impact of Finasteride was assessed on the expression of four biomarkers of the spermatogenesis process, namely Dazl, Tsga10, Sycp3, and Prm2 using the Real-Time PCR technique. MATERIALS AND METHODS: The experimental protocol was carried out on male NMRI mice for 35 days in which three animal groups received three different doses of Finasteride (1, 5, and 20 mg/body weight). RESULTS: The results showed that the expression levels of both Dazl and Prm2 genes were significantly decreased only at a dose of 20 mg/body weight, but at doses of 5 and 20 mg/body weight, the expression levels of Sycp3 and Tsga10 genes were significantly reduced. CONCLUSIONS: It seems that Finasteride, at a dose of 5 mg/body weight or higher, may have adverse effects on male spermatogenesis and fertility.

The effect of maternal sleep deprivation on differentiation of mesenchymal stem cells in the presence of neonates brain cerebrospinal fluid of Wistar rats.

OBJECTIVE: Cerebrospinal fluid (CSF) contains proliferation, differentiation and maturation signals that are essential factors for brain development. Due to presence of such factors this fluid has proliferative and differentiation potential. A previous study showed that maternal sleep deprivation (MSD) decrease the number of newborn neurons in development of hippocampus. Also, it impairs hippocampus-dependent spatial learning and memory in the young offspring rat. MSD can change CSF factors. In the present study, the effect of MSD-CSF on differentiation of mesenchymal stem cells to neural cells was examined, and expression of Nestin, Neun, and NeurD1 that are neurogenic markers was investigated. MATERIAL AND METHODS: In this study, bone marrow mesenchymal stem cells were aspirated from the femur and tibia of young male Wistar rats. Then cell suspension was cultured in DMEM medium supplemented with 10 % FBS and 1 % antibiotics. Pregnant rats were subjected to sleep deprivation for 6 h by gentle handling during 4th, 7th, and 18th day of pregnancy. CSF was collected from cisterna magna of neonate rats. CSF was added to culture media with a 5 % ratio (v/v). Then cell viability was determined with MTT assay. Total cellular RNA was extracted, cDNA was synthesized and NeuN, Nestin, NeuroD1 and IL-6 genes were analyzed by Real-time PCR. RESULTS: Real time-PCR analysis showed that expression of Neun and NeurD1gene decreased compared with culture in normal CSF (N-CSF), and also showed that expression of Nestin did not decrease, inflammatory gene (IL-6) showed high expression compared to culture with N-CSF. CONCLUSION: Based on our results, MSD-CSF could inhibit neurogenesis process in mesenchymal stem cells and also, this result suggests that MSD can suppress neural differentiation and decrease of neurogenesis gene expression. Overall these findings suggest that insomnia and sleep loss may active inflammatory responses in the brain and change CSF-markers (Fig. 3, Ref. 34).

Evaluation of the repair of diaphyseal fracture of femoral bone using bone marrow mesenchymal stem cells in nicotine-bearing rat.

OBJECTIVE: Following bone fractures during accidents, some patients suffer from poor repair of bone fractures and subsequent aesthetic and psychological problems. One of the treatments is based on transplantation of stem cells (seeded on scaffolds) to the lesion site. Bone marrow stromal cells (BMSCs) are multivalent stem cells which are able to reproduce and differentiate into osteogenic cells. The objective of this study was to evaluate the treatment of bone fractures by means of transplantation of the latter cells in rats. METHODS: In this study, the therapeutic effect of mesenchymal stem cells from bone marrow adipocytes was evaluated in bone fractures. BMSCs were isolated from rat femur. Two sources of differentiated and non-differentiated osteocyte cells were provided and mixed with collagen in order to be transferred to animals divided in three main groups of model: nicotine, non-nicotine and control groups. After four weeks, the repair of the fracture that had been inflicted by a 2­mm drill into the diaphyseal region of the femoral bone was investigated by radiographic tests and histopathologic staining. RESULTS: The radiographic results as well as those of histopathologic staining showed that osteogenesis was more intensive in the non-nicotine group than in the nicotine group with differentiated and non-differentiated osteocyte cells. CONCLUSION: The transplantation of differentiated BMSCs to a bone lesion affected the repair of bone fractures while the nicotine agent played an important role in delaying the bone regeneration (Tab. 1, Fig. 8, Ref. 31).

Stemness of spermatogonial stem cells encapsulated in alginate hydrogel during cryopreservation.

This study investigated the effect of spermatogonial stem cell encapsulated in alginate hydrogel during cryopreservation, as cells were protected against damage during cryopreservation within the hydrogel. Spermatogonial stem cells were isolated from the testes of Balb/c mice pups (6 days old), purified in laminin-coated dishes and CD90.1 microbeads, encapsulated in alginate hydrogel and then cryopreserved. After thawing, cell viability and Spermatogonial stem cell (SSC) colony diameter were evaluated. After RNA was isolated and cDNA was synthesised, the expression of stemness genes was considered using RT real-time PCR. Finally, spermatogonial stem cells labelled with BrdU were transplanted to busulfan azoospermic mouse models. Lin28a and Sall4 genes were significantly upregulated after cryopreservation in alginate hydrogel. However, cell viability was significantly decreased. The diameter of colonies consisting of spermatogonial stem cells freeze-thawed in alginate microbeads showed no significant difference with fresh spermatogonial stem cells and the control group. The injection of freeze-thawed spermatogonial stem cells encapsulated in alginate hydrogel resulted in spermatogenesis recovery. Alginate mimics the extracellular matrices (ECM) for spermatogonial stem cells; therefore, it can support stemness potential during the cell cryopreservation process and restart spermatogenesis after transplantation.

Sperm ubiquitination and DNA fragmentation in men with occupational exposure and varicocele.

Assessment of sperm ubiquitination and DNA fragmentation as sperm functional markers are proposed to complement routine semen analysis. This study focuses on the evaluation of these markers in infertile men with varicocele or exposed to occupational background. The results were compared with normozoospermic men. Semen parameters in both groups were lower than those in the control group. Ubiquitination median, as a marker for functionality of the ubiquitin-proteasome system, was also lower in both groups. The ubiquitination median showed a significant positive correlation with motility in both groups, while it showed only a negative correlation with sperm morphology in the varicocele group. DNA fragmentation showed a significant correlation with semen parameters, in total varicocele and also total exposure groups. In conclusion, significant difference of sperm ubiquitination between normal and study groups further validates that sperm ubiquitination as a potential molecular marker for sperm evaluation in addition to routine semen analysis in clinical laboratories.

Cloning and expression of human vascular endothelial growth factor gene and inhibition of its expression by antisense in prokaryotic system.

BACKGROUND AND THE PURPOSE OF THE STUDY: Angiogenesis is an important process in physiology and disease pathogenesis and is controlled in a healthy body by a number of stimulatory and inhibitory factors. The aim of this study was to determine the effect of antisense transcript on the sense transcript of the endothelial growth factor (EGF) gene in bacterial system as an approach for the gene regulation in tumors. METHODS: The hepatoma cell line (HepG2) was stimulated by PMA. VEGF mRNA was used for RT-PCR. VEGF cDNA was synthesised and cloned into T-vector pTZ57R, then sense fragment of VEGF subcloned into pACYC Duet-1 expression vector and antisense VEGF subcloned into pCDNA3 expression vector. Recombinant plasmids were transforemed into BL21 bacterial cells. Expression of recombinant plasmid was analysed by western blot technique. RESULTS: The recombinant pCDNA3-VEGF (pYZantiVEGF) was successfully expressed in BL21 cells. Western blot analysis showed that the expression of VEGF decreased significantly in the cells transfected with VEGF antisense RNA compared with the pACYCDUET-1-VEGF (pYZsenseVEGF) transfected and control. MAJOR CONCLUSIONS: The expression of VEGF in BL21 cells was strong. In vitro, antisense of VEGF inhibited VEGF expression significantly in BL21 cells.

Neurodegenerative, with expression ATF-2 by p38 in cortical neurons.

DNA damage, as an important initiator of neuronal cell death, has been implicated in numerous neurodegenerative conditions. We previously delineated several pathways that control embryonic cortical neuronal cell death evoked by the DNA-damaging agent, camptothecin. The topisomerase-1 inhibitor, camptothecin, has been shown to induce cortical neuronal cell death in a reproducible and synchronistic manner. Primary embryonic neuronal cell culture cortical neurons were prepared. In the study, the survival % of neurons in camptothecin P38 group, after 6 hours (85%), 24 hours (64%) and 48 hours (50%), compared to camptothecin ATF-2 and P38 group after 4 hours (97 and 95%), have been significantly lower, and the expression % of neurons in camptothecin P38 group , after 6 hours (20%), 24 hours (40%) and 48 hours (55%), compared to camptothecin ATF-2 and P38 group after 4 hours (5 and 3%) have been significant lower (p<0.05). The expression % of neurons in camptothecin P38 group, after 24 hours (40%), compared to camptothecin ATF-2 group after 24hours (30%), have been significant lower (p<0.05). This study revealed that camptothecin induces P38 expression and P38 in embryonic cortical neurons to determine the importance of the P38 pathway in neuronal death following DNA damage, and P38 is induce phosphorylation of ATF-2 in embryonic cortical neurons following DNA damage.

Comparative study between intact and non-intact intramuscular auto-grafted mouse ovaries.

Anticancer treatments often lead to ovarian failure and infertility. Cryopreservation and subsequent transplantation of the ovaries is one of the solutions that has been adopted as a means of preserving fertility, but primitive ischaemia in the grafted ovary that damages the oocyte pool is considered to be a possible problem. In order to improve blood supply and follicle preservation, two incisions were made in the ovaries before an intramuscular auto-grafting procedure and these non-intact ovaries were compared with the intact ovaries that were also auto-grafted intramuscularly. Follicle numbers and apoptosis were examined in intact and non-intact groups after 1, 2 and 3 weeks post-grafting. The results were compared with the control ovaries, which were not incised and grafted. Although follicle survival in both grafted groups was lower than in the controls (P

Model-based drug development.

The low productivity and escalating costs of drug development have been well documented over the past several years. Less than 10% of new compounds that enter clinical trials ultimately make it to the market, and many more fail in the preclinical stages of development. These challenges in the "critical path" of drug development are discussed in a 2004 publication by the US Food and Drug Administration. The document emphasizes new tools and various opportunities to improve drug development. One of the opportunities recommended is the application of "model-based drug development (MBDD)." This paper discusses what constitutes the key elements of MBDD and how these elements should fit together to inform drug development strategy and decision-making.

Effect of a new phenanthroline derivative on testicular tissue and serum testosterone concentration and its contraceptive affects on adult Balb/C mouse strain.

In this research, the effect of 2, 6-diaminopyridinum as a new phenanthroline derivative was studied on the hypophysis-gonad axis, testicular tissue and sperm production in male Balb/C mice. Fifty adult male Balb/C mice were divided in five groups. First group was considered as untreated control. Saline was injected to second group and the remaining three groups received intraperitoneal injection of 15, 20 and 25 mg kg(-1) of 2, 6-diaminopyridinum every other day for 20 days. The LD50 was determined to be 35 mg kg(-1) body weight. The testicular tissues were studied morphologically and the serum concentration of FSH, LH and testosterone were measured. The results showed that 25 mg kg(-1) diaminopyridinum decreased the number of germ cells significantly and serum testosterone level with no change on FSH and LH levels. This study indicates that 25 mg kg(-1) of phenanthroline may directly affect testicular tissue causing a lower testosterone level and spermatogenesis in mice.

Organ culture studies on the development of mouse embryo limb buds under EMF influence.

PURPOSE: To investigate the effects of an electromagnetic field (EMF) on limb bud development in vitro, an organ culture system was applied. MATERIALS AND METHODS: Three test groups of amputated mouse limb buds included the experimental (E) group which received EMF (50 Hz/13.1 mT, for 2 h), a sham (Sh) group exposed to no EMF treatment and the control (C) group. The limb buds of E and Sh groups (n = 20 per group) were amputated from mouse embryos on day 11.5 of development and cultured in minimum essential medium Eagle (MEM Eagle), supplemented with 15% human embryo cord serum, for 2 days, while those of group C (n = 20) were removed on day 13.5 of development. All samples were fixed in Bouin's fluid, embedded in paraffin, serially sectioned (5 microm thick) and stained with Hematoxylin and Eosin. Limb bud measurements were performed using a scaled graticule. RESULTS: Morphological and histological examinations showed significant changes in the experimental limb bud group as compared with the sham and control groups. The growth rate in both fore and hindlimb buds in proximal-distal (P-D) and anterior-posterior (A-P) axes were significantly increased. Chondrocyte counts and mitotic figures of mesenchymal and red blood cells were significantly increased as compared with those of sham and control groups. There was also a significant reduction of mesenchymal cell counts, while no significant difference was observed in the degenerated cell counts among the three groups. CONCLUSIONS: These findings suggest that EMF, under the conditions applied, has progressive effects on the limb bud development and that both proliferation and differentiation can be stimulated in vitro.

Pharmacokinetics of eltanolone in male and female patients following intravenous bolus injection.

BACKGROUND: The pharmacokinetics of the steroid anesthetic eltanolone have been studied in male volunteers. However, steroids may exhibit gender-related differences in pharmacokinetics and surgery may alter drug disposition. METHODS: Male (n = 12) and female (n = 9) ASA 1-2 patients (age 26-45 yrs) undergoing discectomy with microsurgical technique were included. Anesthesia was induced with eltanolone 0.75 mg/kg and maintained with nitrous oxide, fentanyl and atracurium. Venous blood was sampled for up to 12 h and analyzed for eltanolone and its major metabolites. RESULTS: Induction was smooth and anesthesia uneventful, except that five cases developed a mild transient erythema. Loss of verbal contact occurred within 20-60 s. Pharmacokinetics in one person deviated significantly from the rest of the subjects. No difference between groups with respect to the primary outcome variable noncompartmental clearance (Cl, 1/min) 1.7 vs 1.6, was found. However, the volume of distribution at steady state (Vss, 1/kg) was larger in women (3.1) compared to men (1.3). The pharmacokinetics followed a three-compartment model. The half-lives (min) of the alpha, beta and gamma phases (men vs women, medians) were 1.5 vs 2.2, 42 vs 40 and 222 vs 360, respectively. Area under the curve (AUC, min microgram/l) was 39,810 vs 34,905. Context-sensitive modelling indicated that it may take 10 min more for women than men to recover from an eltanolone infusion of 2 h duration. CONCLUSION: The gender-related differences in the pharmacokinetics of eltanolone were small, and of little clinical significance for induction of anesthesia with eltanolone.

Pharmacodynamic interaction of eltanolone and alfentanil during lower abdominal surgery in female patients.

We have studied the influence of eltanolone on intraoperative alfentanil requirements in 18 female patients undergoing lower abdominal surgery receiving target-controlled infusions of eltanolone and alfentanil. While target concentrations of eltanolone were maintained constant, target concentrations of alfentanil changed in response to the presence or absence of responses. With serum eltanolone concentrations increasing from 500 to 2000 ng ml-1, the EC50 of alfentanil for suppression of responses to surgical stimulation decreased from 233 to 9 ng ml-1. The findings suggest that the interaction between eltanolone and alfentanil is synergistic.

Pharmacokinetics of eltanolone following bolus injection and constant rate infusion.

Disposition of intravenous anaesthetic eltanolone was studied when administered as a bolus injection (B) of 0.75 mg/kg and constant rate intravenous infusion at 2 mg/kg/hr (12) and 3.5 mg/ kg/hr (13.5) for 2 hr in healthy male volunteers. Venous blood samples were collected for 12 hr and 20 hr following bolus injection and intravenous infusion, respectively. Serum eltanolone concentrations were determined by a specific gas chromatographic mass spectrometric assay. Using a nonlinear regression analysis, the individual data sets were best fitted by a three-compartment mamillary model with central elimination. Derived pharmacokinetic parameters expressed as median and 95% confidence intervals indicated an initial fast distribution with a half-life of 1.80 (0.23-5.47) min (B), 1.44 (0.97-2.06) min (12) and 1.44 (0.95-2.39) min (13.5), an intermediate phase with a half-life of 35.4 (28.7-45.2) min (B), 39.6 (31.0-47.9) min (12) and 35.4 (33.3-44.9) min (13.5) and a moderately short terminal phase with a half-life of 3.8 (2.7-5.9) hr (B), 5.0 (4.2-6.1) hr (12) and 4.6.(4.0-4.8) hr (13.5). The serum clearance after bolus injection was 1.37 (1.23-1.67) L/hr/kg and after infusion was 1.36 (1.25-1.52) L/hr/kg (12) and 1.17 (1.11-1.31) L/hr/kg (13.5). The pharmacokinetics of eltanolone appear to be linear over the dosage range studied. Pharmacokinetic parameters obtained after bolus injection were very much similar to the parameters obtained after infusion with the exception of t1/2 beta which was longer after the infusion (significant) and the volume of central compartment which was lower after infusion (not significant). Context sensitive times were estimated for a 30%, 50% and 80% drop in the concentration of eltanolone after different infusion times. A 30% drop in concentration is estimated to take about 2 to 3 min. A 50% drop in concentration is estimated to take about 8 min when duration of infusion is 3 hr and reaches a value of about 10 min by a duration of infusion of 10 hr. A 80% drop in concentration is estimated to take about 55 min following an infusion of 1 hr and it reaches a value of 70-80 min following an infusion of 10 hr.

Concentration-effect relationships of eltanolone given as a bolus dose or constant rate intravenous infusion to healthy male volunteers.

BACKGROUND: The primary purpose of this study was to evaluate concentration-effect relationships of the new steroid anesthetic eltanolone during recovery from a bolus dose and constant rate intravenous infusion in healthy male volunteers. METHODS: Ten subjects received a bolus dose of 0.75 mg/kg eltanolone over 20 s. A 2-h constant rate intravenous infusion of eltanolone was given to five subjects at a rate of 2 mg.kg-1.h-1 and to another five subjects at a rate of 3.5 mg.kg-1.h-1. Recovery performance was assessed as the time required to reach different end-points and by means of three different psychomotor tests. RESULTS: A low interindividual variability was found in the serum concentration of eltanolone at the pharmacodynamic end-points during recovery. The Cp50 value for "eye opening" was 382 micrograms/L (95% confidence interval, 285-489) after a bolus dose corresponding to a median time of 16 min (range 8-25). After eltanolone infusion, the Cp50 value for "eye opening" was 507 micrograms/L (95% confidence interval, 425-605) and the corresponding median time was 21 min (range 8-25) in the low-dose group and 49 min (range 31-66) in the high-dose group. The Cp50 values at the same effect end-points in the bolus group were less than those in the infusion groups, probably because of insufficient equilibration time between serum and the effect compartment. CONCLUSIONS: Recovery characteristics of eltanolone were predictable because of a relatively low interindividual variability in serum concentrations but with a slow blood:effect compartment equilibration.

Elimination of non-reactive and weakly reactive human alpha 1-acid glycoprotein after induction of the acute phase response in rats.

The disposition of concanavalin A (Con A)-non-reactive and Con A-reactive human alpha 1-acid glycoprotein (AAG) was studied in normal male rats and in acute phase response-activated male rats. Activation of the acute phase response was made using a subcutaneous administration of ethynyloestradiol in sesame oil. This technique increased the endogenous AAG concentration 7-fold. In control rats the two forms of human AAG showed identical kinetics with an average clearance of 5.4 mL h-1 kg-1, terminal half-life of 13.5 h and a volume of distribution (steady state) of 91 mL kg-1. In the acute phase response-activated animals, both the clearance and volume of distribution were larger: the average clearance of the Con A-non-reactive AAG was 10.2 mL h-1 kg-1, the volume of distribution (steady state) 152 mL kg-1 and the terminal half-life 11.7 h. The Con A-reactive AAG had a clearance of 14.7 mL h-1 kg-1, a volume of distribution (steady state) of 262 mL kg-1 and a half-life of 15.8 h. The results indicate that not only does activation of the acute phase response alter the kinetics of AAG but that the change is different for the different types of AAG.

Spermatogenesis and sperm dimorphism in land slug Arion after L. (Pulmonata, Mollusca).

The fine structure of spermatogonia, spermatocytes, spermatids and spermatozoa has been examined in the hermaphrodite gland of Arion ater in detail. Intercellular bridges link spermatogonia, primary and secondary spermatocytes and spermatids. The primary spermatocytes appear in groups, connected to a common cytoplasmic mass, the Cytophore. Two types of spermatids and sperm are recognized (eupyrene and apyrene). The apyrene spermatids differ from the eupyrene type, particularly in the later spermatid stage, by residual cytoplasm with many vesiculose bodies around the nucleus, which is electron-dense. The eupyrene sperm have an acrosome and DNA-histone fibers in the nucleus. Organelle function of the spermatogenic cells at different stages of differentiation, and also the occurrence of sperm dimorphism have been discussed.