Primordial germ cells can be differentiated by retinoic acid and progesterone induction from embryonic stem cells.

This study aimed to examine the expression of the genes associated with different development stages of primordial germ cells (PGCs) in differentiating mouse embryonic stem cells (mESCs). The cells were cultured in three groups of control, 10-8 M of all-trans retinoic acid and the combination of 10-7 M of Progesterone and retinoic acid for 7, 12, 17, and 22 days. Immunofluorescent and Quantitative RT-PCR were used to evaluate the effect of progesterone on the differentiation of mESCs into primordial germ cells. RA-treated cells exhibited increased expression of Fragilis, Stella, Dazl, Stra8, Sycp3, and Gdf9 genes and decreased expression of Oct4, Mvh genes compared to the non-treated controls. Furthermore, RA in combination with progesterone (RA?P) led to increased expression of Oct4, Fragilis, Stella, Dazl, Sycp3, Gdf9 and decreased expression of Mvh, and Stra8 genes compared to the RA-treated scenario. Immunofluorescence detection of Stella and Mvh showed that the expression levels of the cells treated with RA+P are much higher than those of the other groups. Our project showed that under the influence of the induced factors, mESCs can spontaneously differentiate into germ cells. Also, the combination of RA+P can enhance and accelerate the differentiation of mESCs into germ cells.

Anticancer Effect of Enterocin A-Colicin E1 Fusion Peptide on the Gastric Cancer Cell.

Cancer is one of the most causes of death all over the world, although improvements in its treatment and recognition. Due to the limitations of common anticancer methods, including surgery, chemotherapy, and radiotherapy, attention has been drawn to other anti-cancer compounds, especially natural peptides such as bacteriocins. In this study, we used a combination of two bacteriocins, colicin E1 and enterocin A, against AGS gastric cancer cell lines. In order to evaluate anticancer properties of fusion peptide, we applied MTT assay, real-time PCR, and flow cytometry tests. This is the first report to show the cell growth inhibitory activity of the enterocin A in combination with colicin E1 against AGS human cancer cells. The results of this study showed that this fusion peptide at a concentration of 60.4 µg/mL and 24 h was able to kill half of the tested cancer cells, and treatment of the cells with this concentration increased the expression of bax and caspase 3 genes and reduced the expression of bacl-2 in 24 h. Flow cytometry analysis of annexin V-FITC/propidium iodide results also showed that our peptide was able to induce apoptosis in treated cells compared with control. Taken together, enterocin A-colicin E1 (ent A-col E1) can be considered as a good candidate for anticancer therapies.

l-carnitine reduces the adverse effects of ROS and up-regulates the expression of implantation related genes in in vitro developed mouse embryos.

In vitro developed embryos are inevitably exposed to various reactive oxygen species (ROS) which may decrease the embryo's competence in assisted reproductive technology (ART) procedures. Optimization of embryo culture media using antioxidant agents could help to improve embryo quality and could overcome failures in current ART. The aim of this study was to evaluate the effects of l-carnitine (LC), an enhancer of mitochondrial activity and free radical scavenger, in culture media on early embryo competence and expression of ErbB1 and ErbB4 implantation related genes. Two-cell mouse embryos were cultured in the following four conditions: 1. LC group in media containing LC; 2.H 2O2 group exposed to H2O2 for 30 min and then transferred into a simple media; 3.H2O2+LC group exposed to H2O2 for 30 min and then transferred into a simple media containing LC; 4.the control group kept throughout in simple media. All groups were allowed to develop until the blastocyst stage. ErbB1 and ErbB4 expression were evaluated by Real-time PCR and immunocytochemistry. The expression of Sirt3 gene was also evaluated. Intracellular ROS levels were examined by DCFH-DA fluorescence intensity. In order to assess the morphological quality of the embryos, ICM and OCM number blastocyst cells were evaluated by using Hoechst and propidium iodide (PI) staining. ErbB1, ErbB4, ROS levels and cell number were compared across all in vitro groups. Our data reveal that LC significantly increases ErbB1 and ErbB4 gene and protein expression with intracellular ROS levels and Sirt3 gene expression significantly decreased after LC treatment. It is worth noting that an elevated cell number was observed in the LC-treated group compared with the other groups suggesting increased viability and/or proliferation. Our findings suggest that the use of LC could be helpful to improve preimplantation embryo culture media through its effects in decreasing ROS levels and the increase of implantation-related genes.

Retinoic acid and/or progesterone differentiate mouse induced pluripotent stem cells into male germ cells in vitro.

Numerous reagents were employed for differentiating induced pluripotent stem cells (iPSCs) into male germ cells; however, the induction procedure was ineffective. The aim of this study was to improve the in vitro differentiation of mice iPSCs (miPSCs) into male germ cells with retinoic acid (RA) and progesterone (P). miPSCs were differentiated to embryoid bodies (EBs) in suspension with RA with or without progesterone for 0, 4, and 7 days. Then, the expression of certain genes at different stages of male germ cell development including Ddx4 (pre meiosis), Stra8 (meiosis), AKAP3 (post meiosis), and Mvh protein was examined in RNA and/or protein levels by real-time polymerase chain reaction or flow cytometry, respectively. The Stra8 gene expression increased in the RA groups on all days. But, expression of this gene declined in RA + P groups. In addition, an increased expression of Ddx4 gene was observed on day 0 in the P group. Also, a significant upregulation was observed in the expression of AKAP3 gene in the RA + P group on days 0 and 4. However, gene expression decreased in P and RA groups on day 7. The expression of Mvh protein significantly increased in the RA group on day 7. The Mvh expression was also enhanced in the P group on day 4, but it decreased on day 7, while this protein upregulated on day 0 and 7 in the RA + P group. The miPSCs have the capacity for in vitro differentiation into male germ cells by RA and/or progesterone. However, the effects of these inducers depend on the type of combination and an effective time.

Differential Expression of HSP90ß in MDA-MB-231 and MCF-7 Cell Lines after Treatment with Doxorubicin.

BACKGROUND: Breast cancer is a complex, heterogeneous disease and one of the most common malignancies in women worldwide. The efficacy of chemotherapy as an important breast cancer treatment option has been severely limited because of the inherent or acquired resistance of cancer cells. The molecular chaperone heat shock protein 90 (HSP90) upregulated in response to cellular stress is required for functions such as conformational maturation, activation and stability in more than 200 client proteins, mostly of the signaling type. In this study, the expression of HSP90 isoforms including HSP90α and HSP90ß in breast cancer cell lines before and after treatment with doxorubicin (DOX) was assessed. MATERIAL AND METHODS: The cell cytotoxicity of DOX in MDA-MB-231 and MCF-7 cell lines was determined using the MTT assay. Immunofluorescence and western blotting techniques were used to determine the expression of HSP90ß in the cell lines before and after DOX treatment. Immunofluorescence was also conducted to ascertain the expression of HSP90α. RESULTS: The MTT assay results showed that the MDA-MB- 231 cells (IC50=14.521 µM) were more sensitive than the MCF-7 cells (IC50=16.3315 µM) to DOX. The immunofluorescence results indicated that the expression of HSP90α in both cell lines decreased after exposure to DOX. The western blot and immunofluorescence analyses showed that HSP90ß expression decreased in the MCF-7 cells but increased in the MDA-MB- 231 cells after DOX treatment. Conclusion: The obtained results suggested that HSP90α and HSP90ß expression levels were reduced in the MCF-7 cells after exposure to DOX. In the MDA-MB-231 cells, HSP90α expression was reduced while HSP90ß was found to be overexpressed following DOX treatment.

The role of microRNAs in embryonic stem cell and induced pluripotent stem cell differentiation in male germ cells.

New perspectives have been opened by advances in stem cell research for reproductive and regenerative medicine. Several different cell types can be differentiated from stem cells (SCs) under suitable in vitro and in vivo conditions. The differentiation of SCs into male germ cells has been reported by many groups. Due to their unlimited pluripotency and self-renewal, embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) can be used as valuable tools for drug delivery, disease modeling, developmental studies, and cell-based therapies in regenerative medicine. The unique features of SCs are controlled by a dynamic interplay between extrinsic signaling pathways, and regulations at epigenetic, transcriptional and posttranscriptional levels. In recent years, significant progress has been made toward better understanding of the functions and expression of specific microRNAs (miRNAs) in the maintenance of SC pluripotency. miRNAs are short noncoding molecules, which play a functional role in the regulation of gene expression. In addition, the important regulatory role of miRNAs in differentiation and dedifferentiation has been recently demonstrated. A balance between differentiation and pluripotency is maintained by miRNAs in the embryo and stem cells. This review summarizes the recent findings about the role of miRNAs in the regulation of self-renewal and pluripotency of iPSCs and ESCs, as well as their impact on cellular reprogramming and stem cell differentiation into male germ cells.

Application of induced pluripotent stem cell and embryonic stem cell technology to the study of male infertility.

Stem cells (SCs) are classes of undifferentiated biological cells existing only at the embryonic, fetal, and adult stages that can divide to produce specialized cell types during fetal development and remain in our bodies throughout life. The progression of regenerative and reproductive medicine owes the advancement of respective in vitro and in vivo biological science on the stem cell nature under appropriate conditions. The SCs are promising therapeutic tools to treat currently of infertility because of wide sources and high potency to differentiate. Nevertheless, no effective remedies are available to deal with severe infertility due to congenital or gonadotoxic stem cell deficiency in prepubertal childhood. Some recent solutions have been developed to address the severe fertility problems, including in vitro formation of germ cells from stem cells, induction of pluripotency from somatic cells, and production of patient-specific pluripotent stem cells. There is a possibility of fertility restoration using the in vitro formation of germ cells from somatic cells. Accordingly, the present review aimed at studying the literature published on the medical application of stem cells in reproductive concerns.

Protective effect of Allium cepa (onion) seeds (AC) extract on histopathology of testis in STZ-induced male rats / Efecto protector del extracto de semillas de Allium cepa (cebolla) (AC) en la histopatología del testículo en ratas macho inducidas por STZ

SUMMARY: The aim of the present study was to evaluate the effect of the extract of Allium cepa (Onion) seeds (AC) on morphometric and histology of testis and biochemical parameters in STZ-induced male rats. Forty adult male Wistar rats (2 month old) were allocated into four groups of control, diabetic control, diabetic treated with 200 or 400 mg/kg/day of onion seed extract. Diabetes mellitus was induced using 60 mg/kg body weight of Streptozotocin as a single intraperitoneal injection. The extract was administered by stomach gavage for 28 days. The morphometric and histological structure of the testis, biochemical factors like glucose and testosterone levels were assessed. All analyses were done at the end of the four week study period. Data were compared by using Kruskal Wallis Test, Dunnett T3 and the degree of significance was set at P < 0.05 and P < 0.01. In diabetic+200 rats, the numbers of primary spermatocytes were significantly increased. In diabetic+400 rats, seminiferous tubular diameter was significantly increased and the level of testosterone hormone and testis weight was decreased significantly. In diabetic+200 and 400 rats, the numbers of spermatid, FBS and lumen diameter were significantly increased and the numbers of spermatozoa cells, body weight and volume density (VD) % lumen were decreased. Also, the numbers of spermatid in control diabetic rats was decreased. Our finding indicated that onion seed extract might be useful as a supplementary protective agent against adverse effects of diabetes on reproductive system in diabetic men.

RESUMEN: El objetivo del presente estudio fue evaluar el efecto del extracto de semillas de Allium cepa (cebolla) sobre la morfometría e histología de testículos y parámetros bioquímicos en ratas macho inducidas por estreptozotocina (STZ). Se asignaron cuarenta ratas macho Wistar adultas (2 meses de edad) en cuatro grupos: control diabético y diabético tratados con 200 o 400 mg / kg / día de extracto de semilla de cebolla. Se indujo diabetes mellitus utilizando 60 mg/kg de peso corporal de estreptozotocina por inyección única intraperitoneal. El extracto se administró por sonda gástrica durante 28 días. Se evaluaron la estructura morfométrica e histológica de los testículos, factores bioquímicos como la glucosa y los niveles de testosterona. Todos los análisis se realizaron al final del período de estudio de cuatro semanas. Los datos se compararon mediante el uso de Kruskal Wallis Test, Dunnett T3 y el grado de significación se estableció en P <0,05 y P <0,01. En el grupo diabético + 200, el número de espermatocitos primarios aumentó significativamente. En el grupo diabético + 400, el diámetro tubular seminífero aumentó significativamente en cambio el nivel de testosterona y el peso del testículo disminuyeron significativamente. En el grupo diabéticos + 200 y 400, los números de espermátidas, FBS y diámetro de luz se incrementaron significativamente y el número de espermatozoides, peso corporal y densidad de volumen (VD)% de lumen disminuyeron. Además, disminuyó el número de espermátidas en ratas diabéticas control. Nuestro estudio indicó que el extracto de semilla de cebolla podría ser útil como un agente protector adicional contra los efectos adversos de la diabetes en el sistema reproductivo en hombres diabéticos.

Effect of sesame-supplemented diet on prostate and seminal vesicle histology of adult rat / Efecto de la dieta suplementada con sésamo en la histología de la próstata y vesícula seminal de ratas adultas

Infertility is a great concern among the people of reproductive age. The use of natural products obtained from traditional herbs is appealing. Studies show that antioxidants are important in improving male infertility. Thirty adult male rats were randomly divided into two regimen and control groups. The regimen group received diet containing 30 % sesame seed, while the control group received standard diet for 12 weeks. Histology of prostate and seminal vesicle were evaluated and serum levels of FSH, LH and testosterone concentrations were assessed as well. The results showed that, the diameter of peripheral epithelium and the volume density of the prostate epithelium increased but the volume density of the prostate stroma was decreased significantly in the regimen group compared to the control group. Central epithelium diameter and the volume density of the prostate lumen did not change significantly between two groups. In seminal vesicle, volume density of fibromascular and lumen decreased significantly in regimen group compared to control group. FSH and Testosterone level did not change while LH concentration increased significantly in the regimen group compared to the control group (P <0.03). This study shows that the sesame seed might improve male rat reproductive systems by histopathological changes in prostate and seminal vesicle.

La infertilidad es una gran preocupación para las personas en edad reproductiva y el uso de productos naturales obtenidos a partir de hierbas tradicionales es interesante. Los estudios demuestran que los antioxidantes son importantes en la mejora de la infertilidad masculina. Treinta ratas macho, adultas, se dividieron aleatoriamente en dos grupos, experimental y control. El grupo experimental recibió dieta con un 30 % de semillas de sésamo, mientras que el grupo control recibió dieta estándar durante 12 semanas. Fueron evaluadas la histología de próstata y vesícula seminal, así como los niveles séricos de las concentraciones de FSH, LH y testosterona. Los resultados mostraron que el diámetro del epitelio periférico y la densidad de volumen del epitelio de la próstata aumentaron, pero la densidad de volumen del estroma de próstata se redujo significativamente en el grupo experimental en comparación con el grupo control. El diámetro del epitelio central y la densidad de volumen del lumen de la próstata no presentaron cambios significativos entre los dos grupos. En la vesícula seminal, la densidad de volumen fibromuscular y el lumen se redujeron significativamente en el grupo de régimen en comparación con el grupo control. FSH y el nivel de testosterona no cambiaron, mientras que la concentración de LH aumentó significativamente en el grupo de régimen en comparación con el grupo control (P <0,03). Este estudio indica que la semilla de sésamo podría mejorar los sistemas reproductivos de ratas macho a partir de cambios histopatológicos en la próstata y las vesículas seminales.

Protective effect of combined pumpkin seed and ginger extracts on sperm characteristics, biochemical parameters and epididymal histology in adult male rats treated with cyclophosphamide.

Reproductive toxicity is one of the side effects of cyclophosphamide (CP) in cancer treatment. Pumpkin seeds and Zingiber officinale are natural sources of antioxidants. We investigated the possible protective effect of combined pumpkin seed and Zingiber officinale extracts on sperm characteristics, epididymal histology and biochemical parameters of CP-treated rats. Male adult Wistar rats were divided randomly into six groups. Group 1, as a control, received an isotonic saline solution injection intraperitoneally (IP). Group 2 were injected IP with a single dose of CP (100 mg/kg) once. Groups 3 and 4 received CP plus 300 and 600 mg/kg combined pumpkin seed and Zingiber officinale extract (50:50). Groups 5 and 6 received only 300 and 600 mg/kg combined pumpkin seed and Zingiber officinale extract. Six weeks after treatment, sperm characteristics, histopathological changes and biochemical parameters were assessed. In CP-treated rats, motile spermatozoa were decreased, and abnormal or dead spermatozoa increased significantly (P < 0.001) but administration of the mixed extract improved sperm parameters. Epididymal epithelium and fibromascular thickness were also improved in extract-treated rats compared to control or CP groups. Biochemical analysis showed that the administration of combined extracts could increase the total antioxidant capacity (TAC) level significantly in groups 3, 4, 5 and 6. Interestingly, the mixed extract could decrease most of the side effects of CP such as vacuolization and separation of epididymal tissue. Our findings indicated that the combined extracts might be used as a protective agent against CP-induced reproductive toxicity.