The mesenchymal stem cells (MSC) has been investigated as a source of stem cell therapy to replace and treat damaged cells. Human endometrial epithelial and stromal cells was isolated from hysterectomy tissue and the direct evidence of stem/progenitor cells in the human endometrium was identified. Endometrium derived stem cells (EnMSCs) are known to have a high proliferative ability, genetic stability, lack of tumorigenicity and low immnunogenicity during long-term cultivation. Here, we aimed to identify MSC in canine endometrium and characterize its potential to differentiate into decidua cells. EnMSCs were isolated from thrown-away spayed uterus of adult canine depending on their estrus cycle, and identified by flow cytometry, immunocytochemistry and flow cytometry with MSC specific markers. We then characterized the ability of EnMSCs by the doubling-time analysis, colony-forming units and MSC differentiation assays. Isolated EnMSCs expressed stem cell specific genes (Sox2, Oct4, Nanog, MCAM, Endoglin, Susd2 and IGTB) and MSC surface markers (CD90, CD44 and CD117). EnMSCs are also differentiated into adipogenic, osteogenic and chondrogenic cells morphologically under modified conditions with the expression of lineage specific genetic markers. EnMSCs showed higher proliferation ability than canine amniotic fluid derived MSCs which were used as a positive control. EnMSCs were cultured at low density (10, 20, cells/cm2) and initiated to form small colonies of loosely-arranged cells and gradually formed large colonies of densely-packed cells which underwent self-renewal with high proliferative potential which is similar to the clonogenicity feature of human endometrium-derived stem cells. EnMSCs were then induced to differentiate into decidua cells with 0.5 mM dbcAMP. After 14 days, EnMSCs changed their morphology into the elongated and rounded shape. The induced decidual cells expressed PRL and IGFBP1 which are typically expressed in decidua cells. In conclusion, we successfully isolated and characterized MSC in the canine endometrium which differentiated into decidua cells. These results showed that endometrium may be a promising source of stem cells, and furthermore raise the possibility of canine EnMSCs as a novel hypothetical decidualisation model of infertility associated with decidualisation insufficiency and implantation failure.

Somatic cell nuclear transfer (SCNT) has been considered for preserving genetically valuable or endangered animals. Sapsaree is a Natianal Monument in Korea to maintian a pure pedigree. The aim of this study was to produce azoospermia Sapsaree using SCNT and identify normal reproductive ability of cloned azoospermia Sapsaree. Ear skin biopsy was performed on a thirteen-year-old azoospermia Sapsaree and ear skin fibroblasts were isolated for SCNT as donor cells. The fibroblasts were injected into enucleated in vivo matured oocytes, the couplets were electrical fused by two pulse of direct current (55 V for 15 μs) using titanium and platinum fusion needle and activated by calcium ionophore. Cloned embryos were surgically transferred into oviducts of natuarally estrus cycle synchronized recipient dogs. The fusion rate of platinum needle was 70%, which was higher than those of titanium needle (64.1%). Developmental rate to the 8 cells and 10 cell stages was higher in platinum needle group (24% and 16%, respectively) than those of platinum needle group (14.8% and 3.1%, respectively). Total 35 SCNT embryos were transferred into oviducts of 3 recipient dogs and one recipient finally delivered a puppy by caesarean section. As results, this study demonstrated that platinum fusion needle could be successfully make the reconstructed embryos and improve the efficiency of canine SCNT. Cloning azoospermia Sapsaree may contribute to conserve genetically valuable and unique pedigree. And further study should be confirm whether cloned live dog is azoospermia.

Prolonged communication between oocytes and the surrounding somatic cells is one of the unique reproductive physiology in canine. Paracrine Kit ligand (KITL) signaling is a well-known communication between granulosa cells and the oocyte. KITL is a cytokine growth factor secreted by granulosa cells that signals via the c-kit receptor expressed by oocytes. Paracrine factors, including growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), exert their effects by binding with the kinase receptors expressed on the granulosa cells. However, the regulations of GDF9 and BMP15 in the canine KITL expression are currently poorly understood. Therefore, we investigated the effects of GDF9 and BMP15 on the expression of KITL in canine ovarian granulosa cells in vitro. In Annexin V assay recombinant GDF9 and BMP15 did not induce apoptosis in the cultured ovarian granulosa cells. When treated, FSH significantly increased KITL expression, and hCG suppressed its expression. When both FSH and hCG were treated, the expression of KITL was affected by GDF9 and BMP15 in dose and time dependent manner in the luteal granulosa cells. GDF9 (10 ng/mL) significantly decreased KITL expression after12 h. BMP15 (10 ng/mL) significantly also decreased KITL expression after 24 h. Western blot and immunochemistry results indicate that GDF9 activated Smad2/3. After blocking ALK 4/5/7 receptors by SB, GDF9 failed to activate Smad2/3, also BMP15 did not activate Smad1/5/8 after blocking ALK 2/3/6 receptors by DM. So GDF9 exerts its effects via using ALK 4/5/7 receptors to activate SMAD2/3 signaling, and BMP15 binds ALK 2/3/6 receptors to activate SMAD1/5/8 signaling. The expression of KITL was not changed by SB or DM treatment. However, the effect of GDF9 and BMP15, which decreased the expression of KITL, was suppressed by SB or DM treatment. In conclusion, this study provides the first evidence that recombinant GDF9 and BMP15 decrease KITL expression in canine ovarian granulosa cells.

The cancer and Parkinson's disease associated protein DJ-1 is multifunctional protein that involves in diverse cellular process. DJ-1 protein has a cellular protective role and promoted cell survival under an oxidative stress. However, the cellular protective mechanism of DJ-1 is not fully understand, and we needs to be further study their functions in novel organisms. In the present study, we investigated the protective role of DJ-1 against induced oxidative stress in canine cell line. On the basis of these experiments, canine DJ-1 overexpressing and null cell lines were established. The stable overexpression and down regulation of DJ-1 efficiency confirmed by the western blot analysis. Subsequently, the DJ-1 gene transfected cell lines and control cells were subjected to induced the oxidative stress, and then cell viability, cell proliferation assay, cellular apoptosis detection analysis (Annexin V and TUNEL assay), intracellular ROS and mitochondrial activity were measured appropriately. The results showed that DJ-1 overexpressed cells were up-regulated cell viability under oxidative stress conditions induced by the rotenone and hydrogen peroxide (H2O2), whereas loss of DJ-1 cells were down-regulated the cell survival activity. Additionally, overexpression of DJ-1 cells increased cell resistance to oxidative stress and inhibited the elevation of cell death and cellular ROS induced apoptosis. Moreover, DJ-1 overexpressed cells was increased mitochondrial functions by using confocal microscopy with MitoTracker staining. On the contrary to this, DJ-1 null cells show defective cellular protection and mitochondria activity against oxidative stress conditions. Our data indicate that canine DJ-1 protein attenuates cellular apoptosis and ROS generation, enhances the cellular survival activity and promote mitochondrial function under the oxidative stress, likewise other mammalian cells. Importantly, DJ-1 overexpression may be an important part of a protective strategy as a sensor for oxidative stress.