Ovarian mesothelial and extramesothelial cells in interactive culture.

The ovarian mesothelium (OM) represents the tissue of origin of ovarian epithelial cancer. To gain insight into the regulation of this tissue, OM organoids and submesothelial ovarian stromal cells (SC) were isolated from New Zealand White rabbits by a stepwise tissue dispersal technique, while granulosa cells (GC) were aspirated from mature follicles (14 +/- 4 groups/animal). OM and SC dispersal were sequentially accomplished by: a) 1-h incubation in collagenase type I (300 U/ml), gentle scraping of the ovarian surface, and 1 g sedimentation of OM organoids (equivalent to 0.93 +/- 0.40 x 10(6) cells/animal) on 5% bovine serum albumin (BSA); b) 2-h incubation in pronase-collagenase (0.5%-300 U/ml) under periodical resuspension and gentle scraping of SC (1.40 +/- 0.25 x 10(6)/animal) from OM-denuded ovaries. After a week-long in vitro expansion, OM cells (OMC) were cultured alone and with SC or GC within monocameral vessels or bicameral transfilter vessels in serumless, fibronectinrich (4 micrograms/ml) HL-1 medium. After 7 d of contact cell-cell interaction, cytokeratin-positive OMC became surrounded by fibroblastoid, vimentin-positive SC or by cytokeratin and vimentin-weakly positive GC. Filter-bound OMC humorally interacting with underlying SC or GC displayed a biphasic, epithelioid and spindle, morphology with universal cytokeratin expression. Bromo-2'-deoxyuridine (BrdU) immunoperoxidase revealed mean cell proliferation indices of 14.88% for OMC cultured alone, 11.21% and 19.39% for OMC cultured with GC or SC in monocameral dishes, and 15.25% or 22.47% for OMC cultured in bicameral vessels over GC or SC, respectively. This model provides an experimental tool for investigating the unexplored role of stromal-mesothelial interaction in OM pathobiology.

Cell proliferation and apoptosis during development and aging of the rabbit corpus luteum.

Corpora lutea (CL) are endocrine ovarian structures that regulate fundamental reproductive events in mammals. The functional lifespan of these structures is finite as CL regress and cease secreting progesterone after species-dependent intervals during nonfertile postovulatory cycles or pregnancy. The signals that regulate CL aging are poorly understood. This study investigates cell growth and programmed cell death or apoptosis in corpora lutea of New Zealand White rabbits. To study cell growth, CL were obtained at various postovulatory days (POD) from animals injected with the deoxyribonucleic acid (DNA) precursor analog bromodeoxyuridine (BUdR). The BUdR-labeled cells were identified by avidin-biotin-complex immunocytochemistry, and the mean proliferation index area computed by image analysis. Apoptotic cells were scored and further identified by in situ demonstration of DNA fragmentation. Proliferation in parenchymal, stromal, and endothelial CL cells was significantly elevated at POD 3, 5, 18, and 21 and highest at POD 3 (P < 0.001). The number of apoptotic cells was elevated (P < 0.001) at POD 18 and 21, while 1 percent or less of CL cells were apoptotic at POD 3, 5, and 12. Apoptosis was accompanied by shrinkage or vacuolization of CL cells and increased mean number (P < 0.001) of heterophilic leucocytes at POD 18. These data demonstrate that cell growth is more intense during early luteal development and that cell deletion via apoptosis plays an important role in CL regression. The role of paracrine signals such as microphagic cytokines in CL aging remains to be elucidated.

Influence of a corpus luteum tissue extract on rabbit ovarian mesothelial cells.

This study investigates rabbit ovarian mesothelial (OM) cells exposed in vitro to a crude corpus luteum extract (CLE; 60 micrograms/ml). The growth of OM cells was evaluated by measuring the change in cell number (mean % +/- standard error of mean, SEM), the number of cell population doublings (CPD +/- SEM), and the cell population doubling time in hours (CPDT +/- SEM) after 7.5 days of culture in a serum-poor medium. Quantitative estimates of surface morphology changes were obtained by analyzing the total number (mean no. +/- SEM), density (mean no./100 microns 2 +/- SEM), and length-to-diameter ratio (mean L/D +/- SEM) of microvilli. OM cells in control medium formed loosely cohesive monolayers, and grew 152.53 +/- 11.01% with a CPD of 0.59 +/- 0.08 and a CPDT of 117.29 +/- 6.43 hours. The exposed surface area of these cells was over 8,000 microns 2 and was covered in its epinuclear region by long and slender microvilli with a L/D of 6.01 +/- 0.29. The total number of microvilli in each control cell was 1977.52 +/- 120.49 with a density of 0.58 +/- 0.03/100 microns 2 in the epinuclear region and of 0.05 +/- 0.003/150 microns 2 in the remaining surface area (5,161.62 +/- 354.43 microns 2). In contrast, CLE-rich cells cultures grew 329.57 +/- 16.65%, with a CPD of 1.71 +/- 0.07 and a CPDT of 53.43 +/- 2.93 hours.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and characterization of rabbit peritoneal mesothelial cells.

Four enriched mesothelial cell populations distinguished by different size and density were obtained when a unit gravity sedimentation procedure was applied to pronase-dispersed rabbit peritoneal mesothelial cells. Cell integrity was confirmed by trypan blue, ultrastructural and biosynthetic analyses, as well as by explantation in tissue culture. The enriched mesothelial cells displayed the immunocytochemical, ultrastructural and growth characteristics of native mesothelial cells. This isolation and separation procedure should provide a valuable experimental tool to study the pathobiology of mesothelial cells.