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Background: Sperm cryopreservation reduces sperm quality. Kisspeptin (KP) has beneficial effects on sperm functions. This study compares the effect of KP and Glutathione (GSH) on mitigating the detrimental effects of the freeze-thaw cycle on sperm. Methods: An experimental study was conducted in Birjand (Iran) during 2018-2020. Thirty normal swim-up semen samples were treated with Ham’s F10 medium (negative control), 1 mM GSH (positive control), or KP (10 µM) for 30 min before freezing. The motility, acrosome reaction, capacitation, and DNA quality of the frozen-thawed sperms were assessed according to the WHO guidelines. Statistical analysis was performed using paired t test, one-way analysis of variance, and least significant difference.Results: Pre-incubation with KP significantly increased the percentage of sperm motility (34.00±6.7, P=0.003) compared to the control (20.44±7.4) and GSH-treated (31.25±12.2) aliquots. The frequency of non-capacitated spermatozoa was significantly higher in the KP-treated group (98.73%) than in the control (96.46%) and GSH-treated (96.49%) aliquots (P

Background: In mammalian ovaries, loss of over two-thirds of germ cells happens due to cell death. Nonetheless, the exact mechanism of cell death has yet to be determined. The present basic practical study was designed to detect 3 types of programmed cell death, namely apoptosis, autophagy, and necrosis, in murine embryonic gonadal ridges and neonatal ovaries.Methods: Twenty gonadal ridges and ovaries from female mouse embryos 13.5 days post coitum and newborn mice 1 day postnatal were collected. The TUNEL assay was performed to evaluate apoptosis. The interplay of autophagy was evaluated by immunohistochemistry for beclin-1. Necrotic cell death was analyzed by propidium iodide (PI) staining. The count and percentage of the labeled oocytes in the gonadal ridges and ovaries were evaluated and compared using the independent t test and one-way ANOVA. A P value less than 0.05 was considered statistically significant.Results: We detected TUNEL-positive reaction in the embryonic germ cells and in the small and large oocytes of the neonatal ovaries. The germ cells and small oocytes reacted to beclin-1. PI absorption was detected in the embryonic germ cells and the large oocytes of the neonatal ovaries, but not in the small oocytes. The percentage of the TUNEL-positive and PI-labeled oocytes in the gonadal ridges was significantly higher than that in the neonatal ovaries (P

Background: Platelet-rich plasma (PRP) and bioceramics such as hydroxyapatite (HA) and zirconium oxide (ZrO2) are used to reconstruct mandibular defects. We sought to determine the synergistic effects of HA/ZrO2 and PRP and compare their osteogenic activity. Methods: ZrO2 scaffolds were constructed by the slurry method and were then coated with HA and impregnated by PRP/heparan sulfate (HS). Bilateral mandibular defects were created in 26 male rabbits. In 20 rabbits, the left defects were treated with HA/ZrO2/PRP (Group 1) and the corresponding right defects were filled with HA/ZrO2 (Group 2). The 6 remaining models were treated with PRP gels at both sides (Group 3). The osteoconductivity of HA/ZrO2/PRP was compared with that of HA/ZrO2 or PRP by radiological and histological methods after the follow-up period, at weeks 6, 8, and 12. The statistical analyses were performed by ANOVA and LSD using SPSS, version 16.0, for Windows (P

Background: Human Wharton’s jelly mesenchymal stem cells (HWJMSCs) express liver-specific markers such as albumin, alpha-fetoprotein, cytokeratin-19, cytokeratin-18, and glucose-6-phosphatase. Therefore, they can be considered as a good source for cell replacement therapy for liver diseases. This study aimed to evaluate the effects of various culture systems on the hepatocyte-specific gene expression pattern of naïve HWJMSCs. Methods: HWJMSCs were characterized as MSCs by detecting the surface CD markers and capability to differentiate toward osteoblast and adipocyte. HWJMSCs were cultured in 2D collagen films and 3D collagen scaffolds for 21 days and were compared to control cultures. Real time RT-PCR was used to evaluate the expression of liver-specific genes. Results: The HWJMSCs which were grown on non-coated culture plates expressed cytokeratin-18 and -19, alpha-fetoprotein, albumin, glucose-6-phosphatase, and claudin. The expression of the hepatic nuclear factor 4 (HNF4) was very low. The cells showed a significant increase in caludin expression when they cultured in 3D collagen scaffolds compared to the conventional monolayer culture and 2D collagen scaffold. Conclusion: Various culture systems did not influence on hepatocyte specific marker expression by HWJMSCs, except for claudin. The expression of claudin showed that 3D collagen scaffold provided the extracellular matrix for induction of the cells to interconnect with each other.

Background: Activation of bone morphogenetic protein 4 (BMP4) signaling pathway in embryonic stem (ES) cells plays an important role in controlling cell proliferation, differentiation, and apoptosis. Adverse effects of BMP4 occur in a time dependent manner; however, little is known about the effect of different time exposure of this growth factor on cell number in culture media. In this study, we investigated the role of two different exposure times to BMP4 in cell viability, embryoid body (EB), size, and cavitation of ES cells. Methods: Embryonic stem cells (R1 and B1 lines) were released from the feeder cell layers and were cultured using EBs protocol by using the hanging drop method and monolayer culture system. The cells were cultured for 5 days with 100 ng/mL BMP4 from the beginning (++BMP4) or after 48 h (+BMP4) of culture and their cell number were counted by trypan blue staining. The data were analyzed using non-parametric two-tailed Mann-Whitney test. P

Background: Wharton’s jelly is an unlimited source of stem cells that can be used in cell therapy and tissue engineering without any ethical concern. It has been revealed the cell-free extract could be effective to induce cell differentiation. The objective of this study was to induce Wharton’s jelly-derived mesenchymal stem cells (MSCs) into hepatocyte-like cells by premeabilization of the cells in the presence of HepG2 cell line extract. Methods: MSCs were isolated from the umbilical cord, CD marker profile and their differentiation potential into adipogenic and osteogenic lineages were determined. The cells were then, permeabilized by streptolysin O in the presence of HepG cell extract. The treated cells were cultured for 17 days. The cell phenotype was evaluated and the hepatocyte specific markers were detected by immunofluorescence and immunocytochemistry. The Periodic Acid Schiff (PAS) reaction and the cellular uptake of indocyanine green were performed to evaluate the functional behavior of the differentiated cells. Results: The phenotype of extract-treated MSCs changed into a round or polygonal cells with few short processes and they could express high level of albumin, cytokeratin 18 and 19. The MSCs also could store glycogen and uptake and release indocyanine green. Conclusion: We demonstrated for the first time that Wharton’s jelly-derived MSCs could differentiate into hepatocyte-like cells by premeabilization of them in the presence of HepG2 cell extract. This study suggests a feasible method to differentiate MSCs into functional hepatocyte-like cells.

Background: The regenerative capacity of the mammalian heart is quite limited. Recent reports have focused on reprogramming mesenchymal stem cells into cardiomyocytes. We investigated whether fibroblasts could transdifferentiate into myocardium. Methods: Mouse embryonic fibroblasts were treated with Trichostatin A (TSA) and 5-Aza-2-Deoxycytidine (5-aza-dC). The treated cells were permeabilized with streptolysin O and exposed to the mouse cardiomyocyte extract and cultured for 1, 10, and 21 days. Cardiomyocyte markers were detected by immunohistochemistry. Alkaline phosphatase activity and OCT4 were also detected in cells treated by chromatin-modifying agents. Results: The cells exposed to a combination of 5-aza-dC and TSA and permeabilized in the presence of the cardiomyocyte extract showed morphological changes. The cells were unable to express cardiomyocyte markers after 24 h. Immunocytochemical assays showed a notable degree of myosin heavy chain and α-actinin expressions after 10 days. The expression of the natriuretic factor and troponin T occurred after 21 days in these cells. The cells exposed to chromatin-modifying agents also expressed cardiomyocyte markers; however, the proportion of reprogrammed cells was clearly smaller than that in the cultures exposed to 5-aza-dC , TSA, and extract. Conclusion: It seems that the fibroblasts were able to eliminate the previous epigenetic markers and form new ones according to the factors existing in the extract. Since no beating was observed, at least up to 21 days, the cells may need an appropriate extracellular matrix for their function.

Background: The glycoconjugate content of sperms indicates their physiological and fertility properties. Lectin reactivity is indicative of intact, capacitated, and acrosome-reacted sperms. In the epididymis, sperms experience maturation, glycoconjugate modification, and simultaneously, higher L-carnitine (LC) concentrations. The aim of this project was to evaluate the effects of LC and Pentoxifylline (PF) on the integrity, capacitation, and acrosomal reaction of sperms by studying their lectin reactivity.Methods: Mouse testicular sperm samples were divided into three parts. Each sample was added Ham’s F10 (control) or media containing 1.76 mM LC or PF. At 30 and 90 minutes after incubation, sperm motility was assessed. Peanut agglutinin (PNA), wheat germ agglutinin (WGA), and Concanavalin A (Con A) were used to detect non-acrosome-reacted, non-capacitated, and acrosome-reacted sperms, respectively and the frequency was evaluated by flow cytometry. Statistical analysis was performed using the ANOVA. Results: Sperm motility increased after 30 and 90 minutes of incubation in the LC- and PF-treated cultures (P=0.001). LC administration created a significant increase in the percentage of the non-acrosome-reacted sperms compared to the control sperms after 30 and 90 minutes (P=0.02 and P=0.03, respectively). The frequency of the non-capacitated sperms in the LC-treated group increased compared to the control sperms after 30 minutes significantly (P=0.01). Conclusion: Although the administration of LC and PF enhanced sperm motility, LC also impacted glycoconjugates on the sperm surface. Glycoconjugates are involved in the interaction between the sperm and the zona pellucida and subsequently fertilization, thereby probably influencing the male fertility state.