NCBI Summary:
Synaptic vesicle membrane docking and fusion is mediated by SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) located on the vesicle membrane (v-SNAREs) and the target membrane (t-SNAREs). The assembled v-SNARE/t-SNARE complex consists of a bundle of four helices, one of which is supplied by v-SNARE and the other three by t-SNARE. For t-SNAREs on the plasma membrane, the protein syntaxin supplies one helix and the protein encoded by this gene contributes the other two. Therefore, this gene product is a presynaptic plasma membrane protein involved in the regulation of neurotransmitter release. Two alternative transcript variants encoding different protein isoforms have been described for this gene.
Development and Application of a Rat Ovarian Gene Expression Database (rOGED) Jo M, et al 2004 . The pituitary gonadotropins play a key role in follicular development and ovulation through the induction of specific genes. To identify these genes, we have constructed a genome-wide rat ovarian gene expression database (rOGED). The database was constructed from total RNA isolated from intact ovaries, granulosa cells, or residual ovarian tissues collected from immature PMSG/hCG-treated rats at 0 (no PMSG), 12, and 48 h post-PMSG, as well as 6 and 12 h post-hCG. The total RNA was used for DNA microarray analysis using Affymetrix Rat Expression Arrays 230A and B. The microarray data were compiled and used for display of individual gene expression profiles through specially developed software. The final rOGED provides immediate analysis of temporal gene expression profiles for over 28,000 genes in intact ovaries, granulosa cells, and residual ovarian tissue during follicular growth and the preovulatory period. The accuracy of the rOGED was validated against the gene profiles for over 20 known genes. The utility of the rOGED was demonstrated by identifying six genes which have not been described in the rat periovulatory ovary. The mRNA expression patterns and cellular localization for each of these six genes, estrogen sulfotransferase, synaptosomal-associated protein 25 kDa, runt related transcription factor, calgranulin B, alpha1-macroglobulin, and MAP kinase phosphotase-3, were confirmed by Northern blot analyses and in situ hybridization, respectively. The current findings demonstrate that the rOGED can be used as an instant reference for ovarian gene expression profiles, as well as a reliable resource for identifying important yet, to date, unknown ovarian genes.
Gene whose expression is detected by cDNA array hybridization: transcription factors, cell signaling and extracellular communication Rozenn Dalbi?Tran and Pascal Mermilloda
Synaptosomal associated protein 25 (Snap25) gene expression is hormonally regulated during ovulation and is involved in cytokine/chemokine exocytosis from granulosa cells. Shimada M et al. During ovulation, granulosa cells and cumulus cells synthesize and secrete a wide variety of factors including members of the Interleukin (IL) cytokine family via the process of exocytosis. Exocytosis is controlled by the SNARE complex consisting of proteins residing in the vesicle membrane and the plasma membrane. One of the SNARE proteins, SNAP25, is expressed abundantly in neuronal cells and is also induced transiently in the rat ovary in response to LH. Therefore, we sought to determine the molecular mechanisms controlling ovarian expression of the Snap25 gene, and the role of SNAP25 in exocytosis of secreted factors, such as ILs from cumulus cells and granulosa cells. In preovulatory (PO) follicles of eCG-primed mice, expression of Snap25 mRNA was neglible but was induced markedly 8hr post-hCG stimulation. In Pgr null mice Snap25 mRNA and protein levels were significantly lower at 8 hr post-hCG compared to wild type mice. To analyze the molecular mechanisms by which PGR regulates this gene, a 1517 bp murine Snap25 promoter-luciferase reporter construct was generated and transfected into granulosa cell cultures. Three SP1/SP3 sites but not consensus AP1 or CRE sites were essential for basal and Forskolin/PMA-induced promoter activity in granulosa cells. The induction was significantly suppressed by PGR antagonist, RU486. Treatment of cumulus oocyte complexes (COCs) or granulosa ceslls with FSH/Amphiregulin, LH or Forskolin/PMA induced elevated expression of Snap25 mRNA and increased the secretion of 8 cytokine and chemokine factors. Transfection of granulosa cells with Snap25 siRNA significantly reduced the levels of both SNAP25 protein and the secretion of cytokines. From these results, we conclude that progesterone-PGR-mediated SNAP25 expression in COCs and granulosa cells regulates cytokine and chemokine secretion via an exocytosis system.
Expression regulated by
FSH, LH
Comment
To study a presumed regulation of SNAP-25, Grosse et al 2000 used
a rat GC line (GFSHR-17), which expresses FSH receptors, and luteinizing
human GC, which express LH receptors. FSH
elevated SNAP-25 mRNA and protein levels about fivefold within 24 h in
GFSHR-17 cells. In contrast, SNAP-25 protein and
mRNA-levels were not altered by LH/hCG in
luteinized human GC.
Ovarian localization
Oocyte, Granulosa, Theca, Luteal cells
Comment
Grosse et al 2000 reported synaptosome-Associated Protein of 25 Kilodaltons in
Oocytes and Steroid-Producing Cells of Rat and Human
Ovary.
The synaptosome-associated protein of 25 kDa (SNAP-25) is crucially
involved in exocytosis in neurons. The authors found
SNAP-25 to be expressed in nonneuronal cells of the rat
and human ovary, namely in all oocytes and in steroidogenic cells,
including granulosa cells (GC) of large antral follicles
and luteal cells. Both isoforms, SNAP-25a and b, were found in the ovary.
Oocytes obtained by laser capture
microdissection were shown to express SNAP-25b, whereas SNAP-25a was found
in rat GC and human luteinized GC.
Immunohistochemical observations of strong SNAP-25 staining in GC of large
growing antral follicles compared with absent
or weak staining in small follicles suggested a role in folliculogenesis.
Follicle stages
Antral, Preovulatory
Comment
[P1-243] Identification and Characterization of Five Genes Induced by hCG Administration during the Preovulatory Period in the Rat Ovary.
(Endocrine society 2004)
Misung Jo, Mary C Gieske, Sarah E Wheeler, Charles E Payne, Chemyong Ko, Thomas E Curry, Jr. Ob and Gyn, Univ of Kentucky, Lexington, KY; Clin Scis, Univ of Kentucky, Lexington, KY
The LH surge initiates the ovulatory process by triggering a series of biochemical and molecular events that induce the spatiotemporal expression of specific genes. In the present study, we utilized a newly developed rat ovarian gene expression database (rOGED) to identify unknown genes involved in the ovulatory process. Five genes that showed elevated levels of mRNA expression after hCG treatment in rOGED were selected. These genes included mitogen-activated protein kinase phosphotase-3 (MKP-3), runt related transcription factor (Runx 1), calgranulin B, 1-macroglobulin, and synaptosome-associated protein 25 kDa (Snap 25). To confirm and characterize the spatiotemporal expression pattern of these genes during the periovulatory period, immature rats were injected with PMSG; 48 h later hCG was administered. Ovaries were collected at 0 (48 h post-eCG), 6, 12, and 24 h post-hCG. Northern blots revealed dramatic, yet transient increases in levels of mRNA for Runx 1, calgranulin B, MKP-3, and 1-macroglobulin at 6 h post-hCG, which are consistent with the profiles seen in rOGED. Two transcripts of the Snap 25 gene were detected, which were expressed differentially after hCG injection. In situ hybridization showed a distinct localization pattern of expression of these genes. High expression of both Runx 1 and MKP-3 was localized to the granulosa cell layer of preovulatory follicles at 6 and 12 h post-hCG, but the expression was also detected in the theca layer at 6 h post-hCG. MKP-3 can inactivate ERKs while Runx 1 is involved in the cell differentiation/ proliferation process. Snap 25 mRNA was localized to granulosa cells of preovulatory follicles and the theca layer at 12 h post-hCG. Snap 25 is a key component of SNARE proteins required for intracellular membrane fusion events during exocytosis. 1-Macroglobulin can inhibit a broad spectrum of proteases and was localized only to the theca layer at 6 h post-hCG. Calgranulin B mRNA was localized to cells scattered in the interstitial and stroma layer of ovaries obtained at 6 h and 12 h post-hCG, but not to follicular cells. Calgranulin B is highly expressed in neutrophils and monocytes that are found in a variety of inflammatory conditions. The fact that the ovulatory hCG stimulus induces dramatic, yet transient increases in levels of mRNA for these genes in a cell-type specific manner suggests that the expression of each of these genes may be important for successful ovulation.