In vivo levels of mRNA and the specificity of the extrauterine environment on matrix metalloproteinase (MMP)-3, MMP-2, and tissue inhibitor of matrix metalloproteinase (TIMP)-1 were evaluated in eutopic and ectopic endometrial tissue during the establishment of endometriosis in a rat model. Uteri and endometriotic implants were collected and frozen at 36 h, 2 wk, and 4 wk postsurgery to study in vivo mRNA levels. Intact uteri, uterine tissues implanted in the peritoneum or under the skin, and peritoneal adipose implants were collected at 2 wk, halved, and either frozen or cultured. Gene-specific reverse transcriptase-polymerase chain reaction was performed to detect and quantify MMP-2, MMP-3, and TIMP-1 mRNA levels. The peritoneal endometriotic implants progressed from avascularized implants, to vascularized red lesions, to well-established encapsulated cysts. In vivo, MMP-3 mRNA was detectable at all times in ectopic tissues but not in eutopic uterine tissues, whereas MMP-2 and TIMP-1 were ubiquitously expressed at all times in both tissues. In vitro, only MMP-3 mRNA levels were elevated in endometrial tissues collected from the intact uterine and from under the skin, at levels similar to in vivo endometriotic implant MMP-3. In conclusion, ectopic endometrial MMP-3 may participate in the process of invasion and tissue remodeling that is hypothesized to occur in the pathogenesis of endometriosis.

Uterine stromal cells undergo mitosis and differentiate into the decidua just prior to the expected time of implantation in humans and rodents. We have utilized a culture system that will be suitable for study of the molecular mechanisms regulating stromal cell proliferation. Stromal cells were isolated from the uteri of ovariectomized rats and were cultured in chemically defined medium. Cultured cells express the mesenchymal markers vimentin and desmin. They do not express the epithelial marker cytokeratin. Serum-starved stromal cells were stimulated to proliferate in a time frame consistent with the cell cycle through addition of a panel of growth factors (basic fibroblast growth factor [bFGF], epidermal growth factor, platelet-derived growth factor, transforming growth factor alpha, insulin-like growth factor I) and hormones to the culture medium. None of the growth factors tested significantly stimulated proliferation in the absence of progesterone. Furthermore, progesterone was the only steroid of those tested that stimulated mitosis in the presence of growth factors. Stromal cell proliferation in response to progesterone and bFGF was dose dependent and saturable. Addition of the progesterone receptor antagonist mifepristone (RU486) and an inhibitor of tyrosine kinase receptor activation (suramin) abolished stromal cell mitosis. Progesterone receptors and fibroblast growth factor receptor 1 (FGFR1) were identified by immunoblot analysis in proliferating stromal cells. Taken together, these results show that cultured stromal cells maintain progesterone-dependent cell cycle control that is mediated via progesterone receptors. Moreover, the data indicate that bFGF control of stromal cell proliferation is modulated via a specific isoform of FGFR1 containing the three-loop immunoglobulin-like domain.

Endometriosis protein-I (ENDO-I) mRNA expression and protein localization were evaluated using in-situ hybridization and immunohistochemistry in endometriotic lesions and eutopic endometrium from women with endometriosis, and in eutopic endometrium from women without endometriosis (controls). When present, ENDO-I mRNA and protein were observed in the functionalis zone of endometrial stroma and the stroma of endometriotic lesions. Expression and localization differences were scored and statistically analysed. During the secretory stage, ENDO-I mRNA expression by endometriotic lesions and eutopic endometrium from women with disease was significantly greater than ENDO-I mRNA expression by proliferative stage eutopic endometrium from women with disease or eutopic endometrium from controls, regardless of cycle stage (P < 0.001). More ENDO-I protein was localized in endometriotic lesions and eutopic endometrium from women with disease than in eutopic endometrium from controls, regardless of cycle stage (P < 0.001). Differential expression and localization of ENDO-I may help develop minimally invasive diagnostic strategies for endometriosis. Further, as ENDO-I shares nucleotide sequence and amino acid sequence with hepatic haptoglobin-which in certain disease states is immunosuppressive and angiogenic-differences in ENDO-I expression and localization in the peritoneal cavity may contribute to the pathogenesis of endometriosis and/or facilitate development of unprecedented diagnostic or therapeutic approaches for management of this enigmatic disease.

An antiserum to the beta 2 subunit of the rat gamma-aminobutyric acid (GABAA) receptor was prepared by immunizing a rabbit with a fusion protein expressed in bacteria. The fusion protein had the large, intracellular loop expanding between the putative M3 and M4 transmembrane domains of the beta 2 subunit fused to staphylococcal protein A (SPA). The antiserum immunoprecipitated both the solubilized and the affinity-purified GABAA receptors. The anti-beta 2 antibodies were affinity purified on immobilized beta 2 intracellular loop peptide. The antibodies recognized a 55-57 kDa peptide in immunoblots of either crude membranes from rat cerebral cortex or affinity-purified GABAA receptors from bovine cerebral cortex. Immunocytochemistry with the affinity-purified antibody has revealed for the first time the localization of the beta 2 subunit in the rat brain. A comparative study of the regional and cellular immunoreactivities of the affinity-purified anti-beta 2 antibody and the monoclonal antibody 62-3G1 (which recognizes both beta 2 and beta 3 subunits) is presented. The procedure described for generating and preparing specific anti-beta 2 subunit antibodies that are valuable for immunocytochemistry could be extended to other GABAA receptor subunits.

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YGR150C gene product (Ygr150cp) is one of the three mitochondrially located Saccharomyces cerevisiae proteins with pentatricopeptide repeat (PPR) motifs. Ygr150cp is essential for mitochondrial functionality but its molecular targets are still unknown. This study was undertaken to define the role of Ygr150cp in mitochondria biogenesis. Repression of Ygr150cp expression in complemented mutants prevented their use of glycerol or lactate, but allowed limited growth on ethanol-containing medium. RNA hybridization studies showed that Δygr150c meiotic segregants produced COB and COX1 transcripts but failed to process them into the mature forms. Detailed RT-PCR assays revealed that Δygr150c specifically failed to remove the fourth intron of both COB and COX1 pre-mRNAs while all other group I introns were excised. Expression of Ygr150cp mutants without any of the PPR motifs did not complement the growth phenotype. Accordingly, we designate YGR150C as CCM1 (COB and COX1 mRNA Maturation). This report provides the first evidence of PPR protein involvement in the specific removal of Group I introns in mitochondria of Saccharomyces cerevisiae.

To explore the identity and possible function of endometriosis protein-I (ENDO-I), which is an acidic glycoprotein synthesized and secreted by endometriotic lesions, partial amino acid sequence and cDNA sequence were determined. Partially purified, de novo-synthesized rat endometriosis glycoproteins were separated by two-dimensional SDS-PAGE, transferred to polyvinyl difluoride membranes, and stained with Coomassie blue. Protein corresponding to the size and pI of ENDO-I was cut from the membranes and analyzed by automated Edman degradation. ENDO-I amino acid sequence analysis identified 15 residues that shared significant homology with the beta-chain of rat, mouse, and human haptoglobin (Hp) and human Hp-related protein. Western blot analyses using anti-Hp antibody demonstrated cross-reactivity with de novo-synthesized ENDO-I protein in endometriosis culture media. For nucleotide sequence analysis, poly A-enriched mRNA was isolated from rat endometriotic tissues. A gene-specific oligonucleotide primer was designed and used for 3' rapid amplification of cDNA ends (RACE). Automated sequencing of RACE cDNA fragments identified 859 base pairs, of which 858 were identical to rat Hp. Reverse transcription-polymerase chain reaction was used to demonstrate that ENDO-I transcripts are differentially expressed by endometriosis but not by uterine tissues. In the human, distinct subtypes of Hp as well as proteins sharing epitopes with Hp have been used to diagnose a variety of diseases; therefore, Hp-like ENDO-I may prove to be a nonsurgical diagnostic tool to assess endometriosis. Hepatic Hp, induced by acute-phase stimuli, modulates macrophage function and angiogenic activity. If ENDO-I possesses similar activities, it may be involved with anomalies of the immune system or the etiology and pathophysiology of endometriosis.

A unique glycoprotein, called endometriosis protein-I (ENDO-I), has previously been shown to be synthesized and secreted by rat and human endometriotic explants. Furthermore, the N-terminal amino acid sequence analysis showed that ENDO-I shares homology with haptoglobin. The present study was designed to determine this sequence of ENDO-I cDNA from human peritoneal endometriotic lesions and to determine the relative expression of the ENDO-I gene in several human tissues. Poly A-enriched RNA was isolated and reverse-transcribed. To determine the sequence of ENDO-I cDNA, a polymerase chain reaction was performed on cDNA from human endometriotic lesions and a gene-specific primer based on the human haptoglobin sequence. A similar procedure was followed to assess the relative expression of the ENDO-I gene in various tissues. Glyceraldehyde-3-phosphate-dehydrogenase was used as an internal control. ENDO-I gene expression was quantified by densitometry. Sequence analysis of ENDO-I cDNA identified 873 nucleotides that displayed 99.4% homology with the human haptoglobin beta-chain. Relative expression of ENDO-I mRNA by peritoneal endometriotic lesions was 19-fold greater than peritoneum, 28-fold greater than endometrioma and 37-fold greater than eutopic endometrium (P<0.01). Haptoglobin-like ENDO-I may be associated with localized angiogenesis and altered immune response involved with the aetiology/pathophysiology of endometriosis.