Expression of paralogs of cytochrome P 450 21a1 pseudogene (Cyp21a1-ps) and endogenous retrovirus SC1 (SC1) in the rat ovary during the ovulatory process.

This study assesses the relatively high incidence of the expression of paralogs of several pseudogenes within the cascade of expression of functional genes in the rat ovary in response to an ovulation-stimulating dose of gonadotropin. Immature Wistar rats were primed with 10 IU equine chorionic gonadotropin subcutaneously, and 48 h later the 12-h ovulatory process was initiated by 10 IU hCG subcutaneously. Ovarian RNA was extracted at 0, 2, 4, 8, 12, and 24 h after injecting the animals with hCG. The RNA extracts were used for RT-PCR differential display to detect gene expression in the ovarian tissue. Sequence analyses of differentially expressed cDNAs revealed that approximately 27% (i.e. 22/82 clones) of the transcripts were fragments of paralogs of known pseudogenes. Out of the 22 clones reported here, 12 have high sequence similarity to the cytochrome P450 pseudogene Cyp21a1-ps, and 5 have high sequence similarity to both the Cyp21a1-ps and the aldo-keto reductase gene Akr1c6. The remaining five clones were paralogs of the endogenous retrovirus SC1 that has heavily infested the rat genome. Northern analyses reveal that peak expression of all the 22 paralogs occurs at 4-8 h into the ovulatory process. In situ hybridization shows that expression of these pseudogenes is primarily in the granulosa layer of ovulatory follicles. In summary, the results reveal that ovarian expression of Cyp21a1-ps- and SC1-like pseudogenes occurs concurrently with the ovulatory process.

Comprehensive analysis of ovarian gene expression during ovulation using differential display.

Mammalian ovulation is a normal biological process that is initiated when a gonadotropic hormone stimulates G protein-coupled receptors in the plasma membrane of cells in ovarian follicles. This article outlines differential display (DD) protocols and associated methods that have been used to discover more than 30 genes that are expressed in the rat ovary during the ovulatory process. Details are provided regarding the methods for total RNA extraction, reverse transcription (RT), DD-polymerase chain reaction (PCR), polyacrylamide gel electrophoresis (PAGE), Northern analysis of the differentially expressed cDNA fragments, cloning of the cDNA fragments, sequencing of the cDNA, and in situ hybridization of the cDNA fragments with sections of ovarian tissue. These methods provide clear evidence of the temporal and spatial patterns of expression of ovulation-specific genes in the ovary. Most of the genes that have been discovered to date have been associated previously with cascades of gene expression in acute inflammatory reactions. Therefore, the data support the working hypothesis that the ovary becomes inflamed at the time of ovulation, and this acute condition softens local connective tissues and causes ovarian follicles to rupture and release fertile eggs.

Temporal and spatial patterns of ovarian gene transcription following an ovulatory dose of gonadotropin in the rat.

In recent years, there have been a number of efforts to identify genes that are expressed in mature ovarian follicles in response to an ovulatory dose of LH or its homologue hCG. This review keys on 20 ovulation-specific genes that we have identified by the molecular procedure known as differential display. The objective is to use this sampling of genes to illustrate the diversity in the temporal and spatial patterns of expression of genes in the ovary following the stimulus of this gonadal target tissue by a single glycoprotein hormone. The specific genes that are surveyed include 5-aminolevulinate synthase; early growth response protein-1; gamma-glutamylcysteine synthetase; cyclooxygenase-2; epiregulin; pituitary adenylate cyclase-activating polypeptide; tumor necrosis factor-stimulated gene-6; regulator of G-protein signaling protein-2; adrenodoxin; steroidogenic acute regulatory protein; 3alpha-hydroxysteroid dehydrogenase; CD63, a disintegrin and metalloproteinase with thrombospondin motifs; tissue inhibitor of metalloproteinase-1; carbonyl reductase, a G-protein-coupled receptor; pancreatitis-associated protein-III; glutathione S-transferase; and metallothionein-1. The ovulatory expression of these different genes is predominantly within the granulosa layer of mature follicles. However, there were also instances of expression in the thecal and stromal tissue of the ovary, as well as in vascular endothelial cells and in luteal tissue. The overwhelming impression is that the molecular events of ovulation are far more complex, and therefore more highly ordered, than originally imagined.

Ovulation: new dimensions and new regulators of the inflammatory-like response.

Ovulation is a complex process that is initiated by the lutenizing hormone surge and is controlled by the temporal and spatial expression of specific genes. This review focuses on recent endocrine, biochemical, and genetic information that has been derived largely from the identification of new genes that are expressed in the ovary, and from knowledge gained by the targeted deletion of genes that appear to impact the ovulation process. Two main areas are described in most detail. First, because mutant mouse models indicate that appropriate formation of the cumulus matrix is essential for successful ovulation, genes expressed in the cumulus cells and those that control cumulus expansion are discussed. Second, because mice null for the progesterone receptor fail to ovulate and are ideal models for dissecting the critical events downstream of progesterone receptor, genes expressed in mural granulosa cells that regulate the expression of novel proteases are described.