Search Results

Atrial natriuretic peptide (ANP) as well as its receptors is found in mammalian ovary and follicular cells and its function in oocyte meiotic maturation has also been reported in Xenopus, hamster and rat. But the results are controversial and the physiological mechanism of ANP on oocyte maturation is not clear, especially the relationship between gonadotrophin and ANP as well as the signal transduction, and these need further study. The present study conducted experiments to examine these questions by using drug treatment and Western blot analysis and focused on pig oocyte meiotic maturation and cumulus expansion in vitro. The results revealed that ANP could inhibited FSH-induced pig oocyte maturation and cumulus expansion and prevent the full phosphorylation of mitogen-activated protein kinase in both oocytes and cumulus cells, and that these inhibitory effects could be mimicked by 8-Br-cyclic guanosine 5′-monophosphate (8-Br-cGMP), but blocked by a protein kinase G (PKG) inhibitor KT5823. Zaprinast, a cGMP-specific phosphodiesterase inhibitor, could enhance the inhibitory effect of ANP on oocyte maturation. A specific analogue of ANP, C-ANP-(4–23), which binds to the natriuretic peptide receptor-C (NPRC), had no effect in either FSH-induced or spontaneous oocyte maturation. Treatment with forskolin, a stimulator of adenylate cyclase, had a biphasic effect; 44 h treatment induced cumulus expansion but inhibited oocyte maturation while 2 h treatment induced maturation of cumulus-enclosed oocytes (CEOs). Both ANP and C-ANP-(4–23) could inhibit the effect of forskolin on CEO maturation, and these inhibitory effects of ANP/C-ANP-(4–23) could be blocked by preincubation with pertussis toxin (PT), consistent with mediation by a Gi protein(s) in the cumulus cells. All these results suggest that ANP is a multifunctional regulator of FSH and forskolin on pig CEO maturation by two signalling mechanisms: one is via a cGMP/PKG pathway, the other is via NPRC receptors in cumulus cells and the activation of the PT-sensitive Gi protein(s).

Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders; it is characterized by polycystic ovaries, hyperandrogenism and chronic anovulation. To obtain a global view of those genes that might be involved in the development of this complex clinical disorder, we used recently developed cDNA microarray technology to compare differential gene expressions between normal human ovary and ovaries from PCOS patients. A total of 9216 clones randomly selected from a commercial human ovary cDNA library were screened. Among them, 290 clones showed differential expressions, including 119 known genes and 100 known or unknown expressed sequence tags (ESTs). Among 119 known genes, 88 were upregulated and 31 downregulated in the PCOS ovary, as compared with normal human ovary. These differentially expressed genes are involved in various biologic functions, such as cell division/apoptosis, regulation of gene expression and metabolism, reflecting the complexity of clinical manifestations of PCOS. The molecular characteristics established from our study will further our understanding of the pathogenesis of PCOS and help us to identify new targets for further studies and for the development of new therapeutic interventions.

To clarify tissue-specificity of pancreatic β cells, comparison of mRNA expression in various conditions of the tissue of multiple organisms is important. Although the developed methodologies for mRNA monitoring such as microarray, rely on the growth of dbEST (database of expressed sequence tag), a large number of unknown genes in the genome, especially in the rat, have not been shown to be expressed. In this study, we have established the first database of ESTs from rat pancreatic islet and RINm5F cells. Two cDNA libraries were constructed using mRNAs from rat pancreatic islet and RINm5F cells to cover a wider spectrum of expressed genes. Over 40 000 clones were randomly selected from the two libraries and partially sequenced. The sequences obtained were subjected to BLAST database analyses. This large-scale sequencing generated 40 710 3′-ESTs. Clustering analysis and homology search of nucleotide and peptide databases using both 3′- and 5′-ESTs revealed 10 406 non-redundant transcripts representing 4078 known genes or homologs and 6328 unknown genes. To confirm actual expression, the unknown sequences were further subjected to dbEST search, resulting in the identification of 5432 significant matches to those from other sources. Interestingly, of the remaining sequences showing no match, 779 were found to be encoded by exon–intron organization in the corresponding genomic sequences, suggesting that these are newly found as actually expressed in this study. Since many genes are up- or down-regulated in differing conditions, applications of the expression profile should facilitate identification of the genes involved in cell-specific functions in normal and disease states.

In order to elucidate the roles of 17β-HSDs in fish gonadal steroidogenesis, three types of 17β-HSDs (17β-HSD1, 17β-HSD8 and putative 17β-HSD12) were cloned and characterized from the Nile tilapia, Oreochromis niloticus. The cloned cDNAs of 17β-HSD type 1, 8 and 12 were 1504, 1006 and 1930 bp long, with open reading frames encoding proteins of 289, 256 and 314 aminoacids, respectively. Tissue distribution pattern analyzed by RT-PCR and Northern blot showed that 17β-HSD1 was dominantly expressed in the ovary, while the putative 17β-HSD12, one of the two duplicates found in fish, is a male specific enzyme and expressed exclusively in testis (detected by RT-PCR only). On the other hand, 17β-HSD8 was expressed in the brain, gill, heart, liver, intestine, gonad, kidney and muscle of both male and female. Enzymatic assays of the three types of 17β-HSDs were performed using recombinant proteins expressed in E. coli or HEK 293 cells. Tilapia 17β-HSD1 expressed in E. coli had the preference for NADP(H) as cofactor and could catalyze the inter-conversion between estrone and estradiol efficiently as well as the inter-conversion between androstenedione and testosterone, but less efficiently. Tilapia 17β-HSD8 recombinant protein expressed in HEK 293 cells could catalyze the conversion of testosterone to androstenedione, as well as the inter-conversion between estrone and estradiol. However, the putative 17β-HSD12 expressed in E. coli or in HEK 293 cells showed no conversion to any of the four substrates tested in this study. Based on enzyme characterization and tissue distribution, it is plausible to attribute crucial roles to 17β-HSDs in the gonadal steroidogenesis of teleosts.

In order to understand the tIssue specificity of the endocrine pancreas, it is important to clarify the expression profile of mRNAs in various states of the tIssue. A total of approximately 9000 non-redundant expressed genes from human pancreatic islets and insulinoma have so far been determined as expressed sequence tags (ESTs) and deposited in public databases. In the present study towards the identification of a complete set of genes expressed in human pancreatic islets, we have determined 3'-ESTs of 21267 clones randomly selected from a cDNA library of human pancreatic islet tumors. Clustering analysis generated 6157 non-redundant sequences comprising 2323 groups and 3834 singletons. Nucleotide and peptide database searches show that 3103 of them represent known human sequences or homologs of genes identified in other species and 58 are new members of structurally related families. The sequences were classified on the basis of the putative protein functions encoded, and were assigned to the respective chromosome by database analysis. The sequences were also compared with the EST databases (dbEST and EPConDB) including ESTs from normal pancreatic islet, insulinoma, and fetal pancreas. Since 3384 genes were newly found to be expressed in human pancreatic islets and 587 of them were unique to the islets, this study has considerably expanded the catalog of genes expressed in the endocrine pancreas. The larger collection of pancreatic islet-related ESTs should provide a better genome source for molecular studies of differentiation, tIssue-specific functions, and tumorigenesis of the endocrine pancreas as well as for genetic studies of diabetes mellitus.