Özlem Özden Akkaya

The formation of primordial follicles determines the pool size of follicles in the ovary, and is crucial for female reproductivity. Oocyte nest breakdown, and the formation of primordial follicles, largely depend upon the communication between oocytes and the surrounding pregranulosa cells. The neurogenic locus notch homolog protein (Notch) signaling pathway is the key player for this cell-to-cell communication, and is responsible for primordial folliculogenesis. However, different endocrine disruptors, including bisphenol A (BPA; a plasticizer and a constituent of reusable plastic containers) may affect the Notch signaling pathway, and might induce ovary dysfunction via Notch signaling. Consequently, we investigated the possible influence of BPA treatment on the proportional distribution of the follicular stages, follicle numbers, levels of apoptosis, and on Notch2 and Jagged2 expressions in the ovary. BPA was administered at doses of either 50 µg/kg/day or 50 mg/kg/day, at different time intervals, during neonatal and fetal periods in vivo. After collecting the ovaries from the various experimental groups, follicles were counted, and frequency of apoptosis was determined by TUNEL assay. In addition, Notch2 and Jagged2 expressions were assessed by immunohistochemical staining and qPCR. In summary, BPA treatment affected the follicle numbers and apoptosis level, and Notch2 and Jagged2 expressions varied with follicular stage. It was also observed that these parameters were dose and time dependent with respect to BPA exposure.

Objective: Leptin, a 16 kDa hormone encoded by the obese (Ob) gene, is known for its role in regulating food intake, body composition, and energy expenditure. Leptin receptor expression has been demonstrated in several tissues, including the small intestine. Weight gain may occur in humans after menopause or in animals following ovariectomy. Estrogen affects leptin and leptin receptor expressions. In this study, we aimed to contribute to the etiology of obesity by investigating the effects of E2 on leptin receptors in the small intestines of ovariectomized rats as a model of postmenopausal conditions. Materials and Methods: Bilateral ovariectomy was performed on 6-month-old Sprague-Dawley female rats. Ovariectomized rats (Ovx) were injected with 0.2 ml of sesame oil/rat/day or E2 (25 µg/rat/day) and euthanized at the 18th, 90th, or 162nd hours. Duodenum, jejunum, and ileum samples were fixed and embedded in paraffin using standard methods. The expression of leptin receptors were detected in the small intestine through immunohistochemistry. Results: Leptin receptor expression was found in the villi and crypt epithelium of the small intestine and in Brunner’s gland of the duodenum. E2 administration increased the leptin receptor expressions on the epithelium of villi and crypt in the duodenum and jejunum at the 90th hour (p<0.05); ileum at the 18th hour (p<0.05); and also on the epithelium of villi in the duodenum at the 162nd hour (p<0.05). Conclusion: Our results indicate that E2 may upregulate the expression of leptin receptors in the small intestine, where glucose and other nutrients are absorbed after food intake and digestion, depending on the timing.

Synthetic pyrethroid insecticides have been widely used for years to prevent harmful effects of insects and control disease vectors. In this study, the effects of resveratrol against the potential toxicity of permethrin, an effective pyrethroid derivative, on the fetus were investigated. Accordingly, Wistar female rats were divided into four groups as Control, Sham, Permethrin, and Permethrin + Resveratrol. Lung, liver, kidney and small intestine of developing fetuses were evaluated histopathologically. Also, Bone Morphogenetic Protein-4 (BMP-4) in bone tissue development and Fibroblast Growth Factor-1 (FGF-1) expressions in lung were examined immunohistochemically. All structures in the Control and Sham groups were normal. Permethrin caused epithelial damage, regression in bronchial and primitive alveolar development in the lung; congestion, edema and sinusoidal dilatation around the central vein in the liver; tubular epithelial degeneration, regression in glomeruli and tubule formation in the kidney; epithelial degeneration and irregularity in the villus structure in the small intestine. Immunohistochemical results indicated that permethrin administration decreased BMP-4 levels in bone tissue and FGF-1 levels in lung. Resveratrol application was found to greatly alleviate histopathological and immunohistopathological variability in all tissues. Oral consumption of permethrin by pregnant rats caused growth retardation and tissue damage in many different tissues in offspring. Intake of resveratrol during pregnancy showed protective effects against fetotoxicity caused by permethrin.

Bone marrow derived stem cells (BMSC) are the most utilized cell type in the field of bone regeneration. Although BMSC are both safe and efficacious, the search for alternative sources for stem cells continues. We investigated bovine BMSC and adipose tissue derived mesenchymal stem cells (ATSC) using immunofluorescence and PCR. We further compared the osteogenic differentiation potentials of both sources of stem cells. We assessed alkaline phosphatase (ALP) enzyme levels and calcium deposition in differentiating cells at days 7, 14 and 21 to compare the osteogenic differentiation capability of both cell types. We found that ATSC expressed significantly higher ALP levels compared to BMSC throughout osteogenic differentiation. Calcium deposition was greater in ATSC than BMSC at days 7 and 14. By the end of day 21, BMSC produced greater calcium deposition. We found that ATSC undergo osteogenic differentiation more rapidly than BMSC, but BMSC provide greater mineralization over longer periods.

The molecular mechanisms involved in the progression of chondrogenic differentiation require considerable studies, despite significant advancement in recent years. Chondrocytes are known for their paracrine effect yet there is little understanding about the impact of the paracrine factors released by chondrocytes in chondrogenic differentiation of mesenchymal stem cells. In this study, it was aimed to investigate the role of Notch signaling pathway during chondrogenesis of rat amniotic fluid stem cells (RAFSCs) in 3D culture in alginate beads using various induction media including the conditioned media from chondrocytes culture as well, for a period of 21 days. Our results indicated that Notch1 and VEGF genes were expressed during chondrogenic differentiation of RAFSCs along with SOX9 and COL2A1 upregulation in all different groups. However, SOX9 was significantly upregulated in group containing both conditioned media and standard chondrogenic induction medium (CM + IM group). The results suggest that conditioned medium can initiate chondrogenic differentiation, but it does not sufficiently contain all the factors potent for strong upregulation of SOX9. However, conditioned medium maintains the Notch1 gene expression at a sufficient level during the chondrogenesis stage. We propose that studies in this area are needed to understand the exact mechanisms involved in progression of differentiation under the influence of conditioned media.

Mesenchymal stem cells are widely used in the treatment of many diseases, including osteoarthritis, due to their ability to differentiate into cartilage and self-renewal features. The high chondrogenic differentiation potential of synovial fluid-derived mesenchymal stem cells (SFMSCs) increases the importance of these cells in osteoarthritis treatments. Bone Morphogenic Protein (BMP)-9 and Transforming Growth Factor Beta (TGFß)-3 growth factors added to chondrogenic differentiation medium increase chondrogenic differentiation and they also possess hypertrophic effects on chondrocytes. In this study, it was aimed to demonstrate the possible hypertrophic effects of BMP-9 and TGFß-3 added to the chondrogenic basal medium during in vitro induced chondrogenesis in coculture of synovial fluid-derived mesenchymal stem cells at Passage(P)5 and chondrocytes at P1. Six experimental groups were formed. Cell hypertrophy was demonstrated by examining Matrix Metalloproteinase (MMP)-13 and Runt Related Transcription Factor (RUNX)-2 gene expressions, which are hypertrophic markers, in stem cells where chondrogenesis were induced in Transwell coculture. Although the addition of BMP-9 and TGFß-3 to the chondrogenic medium increased hypertrophic gene expressions in experimental groups compared to control, they were not statistically significant. The addition of BMP-9 and TGFß-3, separately or together, during the chondrogenic differentiation of stem cells does not cause significantly cartilage hypertrophy.

Amniotic membrane derived stem cells (AMSCs) are reported to have a comparatively higher potency than multipotent stem cells. These cells are shown to have low immunogenicity and no teratogenicity. Among various conventional methods of isolation using enzymes, explant culture method is believed to be an easy and cost-effective way to harvest stem cells. The purpose of this study was to isolate AMSCs from amniotic membrane of rats and to characterize them for multilineage differentiation, including generation of neurospheres to use them later in in-vivo experiments. Amniotic membranes were collected from Wistar rats on 17th day of pregnancy. After processing of the tissues, AMSCs were isolated by the explant culture method and continued to grow until 10th passage. The doubling time was estimated and the cells were analyzed for growth curve parameters at passages 5 and 9. The osteogenic and adipogenic differentiation studies were carried out from the same cells after 3rd passage. Neurospheres generation from AMSCs was performed using neurogenic induction media. The cells were further assessed for their mesenchymal, haemopoietic, and neurogenic marker expressions by immunofluorescence staining and PCR analysis The study suggests that AMSCs isolated through explant culture are reliable stem cells which could generate neurospheres under proper induction conditions and could be a potential candidate to be used on in-vivo neural degeneration models.

Bisphenol A (BPA) is the object of great concern because of its widespread use, shown by the several millions of tons currently produced throughout the world each year. BPA is an endocrine disrupting chemical and was classified as a probable human carcinogen. Fetal and perinatal exposure to environmentally relevant doses of BPA induces multigenerational impairments of male fertility. Notch signaling, functions to regulate cell-fate by modulating differentiation, proliferation, and survival of cells. Notch signaling system play a significant role in male germ cell differentiation and survival. In this study, it was aimed to explore if the protein expression of Notch I receptor is altered following BPA treatments during embriyonal (E) days 18 to 21, postnatal (P) days 0 to 3, and P4 to P7 time intervals in the rat testes in vivo. As a result, it was determined that the application of BPA did not cause a change in the expression of Notch 1 in the rat testis. Endokrin bozucu kimyasallar arasında Bisphenol A (BPA) her yıl dünyada bir kaç milyon ton üretilmesi ve yaygın olarak kullanılmasıyla en çok dikkat çeken bir ajandır. BPA endokrin bozucu bir ajandır ve insanlar için karsinojen olarak sınıflandırılmaktadır. Fötal ve perinatal dönemde çevredeki dozlarla uyumlu olarak BPA’ya maruz kalmak nesiller boyunca erkek infertilitesinin bozulmasını indüklemektedir. Notch sinyal fonksiyonları hücrelerin farklılaşmasını, çoğalmasını ve canlı kalmasını düzenleyerek hücre kaderini belirler. Notch sinyal sistemi erkek germ hücrelerinin farklılaşması ve yaşamasında önemli bir rol oynamaktadır. Bu çalışmada in vivo embriyonal (E) 18-21, postnatal (P) 0-3 ve P4-7. günlerde BPA uygulamasını takiben rat testislerinde Notch I reseptör protein ekspresyonunun değişip değişmediğini araştırmak amaçlandı. Sonuç olarak BPA uygulamasının testiste Notch 1 ekspresyonunu değiştirmediğini belirledik.

Easy harvesting and handy application of stem cells therapy want convenient and alternative source. Equine amniotic fluid derived mesenchymal stem cells make hope for scientists as of no ethical concern and of higher proliferation and differentiation potency. Equine AFSCs were isolated, cultured and propagated for characterization of potency of growth and multilineage differentiation study. Successful harvesting and optimum differentiation of equine AFSCs into osteogenic, adipogenic and neurospheres through this study could make a ground for their clinical application for various purposes. Further extensive and comprehensive molecular studies are important to conclude the precious procedures for clinical approach of amniotic fluid derived stem cells.

Mesenchymal stem cells (MSCs) have become a potent source for cell therapy due their inherent properties of self-renewal and ability to undergo unlimited divisions in the field of regenerative medicine. In this study, equine adipose tissue stem cells (EASCs) have been evaluated on the basis of their morphology, proliferation potential and multilineage differentiation capabilities for their future usage in cell therapy. The cells were further differentiated into osteogenic and adipogenic cell lineages as well as into adipose derived neurospheres to ensure the stemness of propagated cells. In our study, we further successfully cultured the neurosphere derived cells which were cultured from neurospheres after enzymatic dissociation. The study exhibits that EASCs offer a suitable choice for cellular regenerative therapy in equines. Nonetheless, there is a still need to molecular characterization of EASCs in order to establish a standard practice in equine medicine.