ABSTRACT
Bone morphogenetic protein (BMP15) is a member of the transforming growth factor ß (TGF-ß) superfamily with a key role in regulating follicle development in mammals and birds. However, potential ovarian roles of BMPs remain unexplored in teleosts. In this study, the full-length sequences of bmp15 were obtained using rapid-amplification of cDNA ends (RACE). The full-length cDNA sequence of bmp15 is 2217 bp which contained 214 bp 5'-UTR and 845 bp 3'-UTR. The open reading frame (ORF) sequence of bmp15 is 1158 bp, encoding a predicted protein of 385 amino acid residues. BMP15 has a specific RXXR protease cleavage site of TGF-ß superfamily (is RIRR) and six conserved cysteine residues. Using real-time quantitative PCR revealed that bmp15 mRNA was largely expressed in the ovary and testis and mostly in oocytes within the follicle, slightly expressed in muscle, liver and pituitary. BMP15 is mainly present at stage I follicles by real-time quantitative PCR and immunohistochemistry. Phylogenetic analysis showed that gibel carp bmp15 was similar to bmp15 of zebrafish and other fish species. Treatment with human chorionic gonadotropin (hCG) in isolated follicles of gibel carp in vitro showed altered bmp15 mRNA expression: when treated with 10 ng/mL hCG for 10h, the expression level of bmp15 was significantly increased. However, with proceeding cultivation, the expression level of BMP15 mRNA decreased. The results of this study indicate that bmp15 may play a key role during development of follicles in gibel carp, especially in early stage follicles.
Subject(s)
Bone Morphogenetic Protein 15/chemistry , Bone Morphogenetic Protein 15/metabolism , Carps/metabolism , Chorionic Gonadotropin/pharmacology , Ovarian Follicle/drug effects , Ovarian Follicle/metabolism , Amino Acid Sequence , Animals , Base Sequence , Bone Morphogenetic Protein 15/genetics , Carps/genetics , Female , Gene Expression Regulation, Developmental/drug effects , Gene Expression Regulation, Developmental/genetics , Humans , Molecular Sequence Data , Sequence AlignmentABSTRACT
Evodiamine (1), a biologically active alkaloid isolated from Evodia rutaecarpa (known in Chinese as Wu-Chu-Yu), has antioxidant, anti-inflammatory, and anticancer activities. It has recently been demonstrated that the cytotoxic activities of 1 might be due to its ability to inhibit cell growth and induce apoptosis. In this study, we investigated the effects of 1 on growth and apoptosis in COLO-205 cells by MTT assay, fluorescence microscopy, transmission electron microscopy, DNA fragmentation assay, flow cytometry, immunohistochemical analysis, Western blotting, and caspase-3 activity assay. Our data revealed that 1 could significantly inhibit COLO-205 cell proliferation in a dose-dependent manner, and 1-treated COLO-205 cells displayed typical morphological apoptotic characteristics and formation of DNA ladders in agarose gel electrophoresis. The COLO-205 cell cycle was arrested in G(2)/M phase by 1. Meanwhile, 1 increased the expression of Bax and p53, decreased the expression of Bcl-2, lowered the mitochondrial transmembrane potential, and induced the activation of caspase-3. These activities may contribute to the anticarcinogenic action of 1.
Subject(s)
Anticarcinogenic Agents/toxicity , Apoptosis , Carcinoma/metabolism , Colorectal Neoplasms/metabolism , Plant Extracts/toxicity , Quinazolines/toxicity , Anticarcinogenic Agents/chemistry , Anticarcinogenic Agents/isolation & purification , Carcinoma/pathology , Caspase 3/metabolism , Cell Cycle , Cell Proliferation , Colorectal Neoplasms/pathology , Evodia/chemistry , G2 Phase , Humans , Membrane Potentials , Mitochondria/drug effects , Plant Extracts/chemistry , Plant Extracts/isolation & purification , Proto-Oncogene Proteins c-bcl-2/metabolism , Quinazolines/chemistry , Quinazolines/isolation & purification , Tumor Cells, Cultured , Tumor Suppressor Protein p53/metabolism , bcl-2-Associated X Protein/metabolismABSTRACT
Follicular growth, development and ovulation are highly ordered processes that involve the expression of many genes under precise temporal and spatial regulation. However, information on stage-specific gene expression during the antral follicle phase in sheep is not well understood. In the present study, suppressive subtractive hybridization (SSH) was performed to screen genes that were differentially expressed in the granulosa cells between large follicles (LF, >5mm) and small follicles (SF, 3-5mm), and subtractive cDNA library was constructed. Furthermore, with dot-blot analysis, a total of 90 clones randomly selected from the library were proven to be differentially expressed in the granulosa cells. Among these, 38 exhibited high homology to known genes, 14 sequences were corresponding to novel expressed sequence tags (ESTs). Four ESTs, LAPTM4A, SERPINE2, GSTA1, and INHBA, were further examined the reproducibility of the SSH data by the real-time quantitative PCR. Results confirmed an increase expression of respective mRNA in granulosa cells of large follicles compared with that of small follicles. It is concluded that we have identified several genes (known or unknown) that may effect follicular growth, dominance or ovulation in ewes.
Subject(s)
Granulosa Cells/physiology , Animals , DNA Primers , DNA, Complementary/genetics , Expressed Sequence Tags , Female , Follicular Phase , Gene Expression Regulation , Glutathione Transferase/genetics , Inhibin-beta Subunits/genetics , Isoenzymes/genetics , Membrane Proteins/genetics , Nucleic Acid Hybridization , Polymerase Chain Reaction , RNA/genetics , RNA/isolation & purification , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Serpins/genetics , SheepABSTRACT
We investigated the mRNA expression patterns of receptor genes for bone morphogenetic proteins-15 (BMP15) and growth differentiation factor-9 (GDF-9) in granulosa cells of sheep treated with FSH. The effects of FSH and estradiol (E2) on the regulation of BMPRII, BMPRIB and ALK-5 in ovine granulosa cells were also examined. Ovaries were collected on day 16 of the estrous cycle and granulose cells were harvested from follicles of two sizes (3-5 and >5mm in diameter). For in vitro studies, granulosa cells were obtained from follicles of 3-5mm in diameter and cultured in serum-free McCoy's 5A medium supplemented with different doses of FSH (0, 1, 5, 10ng/ml) or a combination of 5ng/ml FSH with 1ng/ml E2. Expression of BMPRII, BMPRIB and ALK-5 mRNA was estimated by quantitative real-time PCR. Our results demonstrated that BMPRII, BMPRIB and ALK-5 expression was significantly higher in the granulosa cells of large follicles than of small follicles. Treatment of granulose cells with FSH (1-10ng/ml) alone down-regulated the expression of BMPRIB (P<0.05). BMPRII and ALK-5 mRNA expression was not significantly different at an FSH concentration of 5ng/ml compared to control. A further increase in FSH (10ng/ml) down-regulated the expression of BMPRII and ALK-5 (P<0.05). The combination of FSH (5ng/ml) and E2 (1ng/ml) up-regulated the expression of BMPRII, BMPRIB and ALK-5 in granulose cells (P<0.05). Therefore, the present study establishes the expression levels of the receptor genes of BMP15 and GDF-9 and suggests that the expression of BMPRII, BMPRIB and ALK-5 may be regulated by FSH and E2 in ovine granulosa cells.
