Growth effects of protein hormones on cultured rabbit ovarian surface epithelial cells.

Throughout life, the ovarian surface epithelium (OSE) undergoes morphogenetic changes that may be hormonally regulated. To investigate this possibility, a population of cells morphologically identical to native OSE cells was isolated from estrous rabbits with collagenase, unit gravity sedimentation, and trypsin-EDTA. Cells were incubated with various concentrations of protein hormones in serum-rich medium or in a chemically defined medium containing fibronectin. Tritiated thymidine was added 24 h before interruption of cultures. Growth-promoting effects of tested hormones were more pronounced and consistent in serum-free cultures. Under these conditions, human chorionic gonadotropin (10,000 mIU/ml) caused a 2.8-fold increase in cell number and a 3.4-fold stimulation of thymidine incorporation. Luteinizing hormone (NIAMDD-oLH-24, 1.0 micrograms/ml) and follicle-stimulating hormone (NIADDK-oFSH-16, 1.0 micrograms/ml) produced, respectively, a 1.7-fold and 1.5-fold increase in cell proliferation, and over 1.4-fold and 1.3-fold stimulations of thymidine uptake. When used together, no growth stimulation by these gonadotropins was seen. Slight but significant increases in cell number (1.4-fold) and in radiolabel incorporation (1.3-fold) were observed with prolactin (NIADDK-oPrl-16, 10 ng/ml). These data indicate that some protein hormones promote the growth of OSE cells. This property may be important in regulating these cells during normal and pathologic states.

Growth characteristics of rabbit ovarian mesothelial (surface epithelial) cells.

This study analyzes the morphology and growth characteristics of rabbit ovarian mesothelial (surface epithelial) cells explanted in vitro. Best cell growth and expression of differentiation were obtained using a polystyrene substrate, 100% air, and a nutrient composed of 85% medium 199 and 15% fetal bovine serum. Surface epithelia gave rise to cell outgrowths within 36 h of explantation and formed confluent epithelial monolayers after 1 week. Cells exhibited a marked proliferative activity as indicated by: (a) a labeling index of 80% at day 4, and of 40% at days 10 and 38; (b) a cell doubling time of 30-36 h during the logarithmic phase of growth; and (c) an estimated 24 cell population doublings during 6 weeks. Confluent cultures contained cells complexed by desmosomes and tight junctions and displaying numerous microvilli, intracellular lumina, and focal cilia. Cells organized often into nests and gland-like spaces and developed occasional hemicysts. High-density cultures developed also a multilayered configuration, villous processes, epithelial invaginations or crypts, and focal cytological features suggestive of steroidogenesis. This study shows that rabbit ovarian surface epithelial cells can be maintained in vitro with a high degree of differentiation. This model is being used to investigate the pathobiology of ovarian surface epithelium.

Ultrastructural immunocytochemical localization of lysozyme in the mucociliary epithelium of the rabbit endocervix in different hormonal states.

The distribution of lysozyme in the endocervix of estrous, pseudopregnant, and ovariectomized rabbits was studied using two different immunocytochemical techniques--the unlabeled antibody enzyme method of Sternberger et al. (1970) and the peroxidase-labeled antibody method of Taylor and Burns (1974). With both procedures, a fine immunostaining precipitate was seen over the entire area of basal mucous granules, while immunodeposits were coarser and mostly located in the outer zone of central and apical granules. A nonspecific staining was noted when tissues were reacted with peroxidase-antiperoxidase complex alone. This troublesome artifact was abolished by preincubating tissues with human IgA. This step did not affect the specific immunostaining for lysozyme yet nonspecific staining was absent from specificity and method controls carried out for both immunocytochemical procedures. The presence of high levels of lysozyme in the endocervical epithelium of estrous rabbits was also confirmed in enzymatically isolated endocervical epithelia using the lysoplate method of Osserman and Lawlor (1966). Mucous granules and immunostainable intracellular lysozyme were abundant during estrus, decreased during early pseudopregnancy, and were absent after long-term ovariectomy. However, they were restored by the administration of estradiol (5 micrograms/12 hours/10 days) to ovariectomized animals. These data indicate a common hormonal regulation and secretory mechanism for endocervical mucous glycoproteins and lysozyme.

Isolation and ultrastructure of rabbit ovarian mesothelium (surface epithelium).

This study evaluated the efficacy of various dissociating procedures for isolating mesothelial or surface epithelial cells from rabbit ovaries. The procedure which provided the highest and most homogenous cell yield involved the following sequence: (a) incubation of whole ovaries for 60 min in Clostridium histolyticum collagenase, type I (300 U/ml); (b) vortexing of ovaries at 30 rpm for 60 s in medium 199; (c) removal of partially detached surface epithelium by gentle scraping with a microdissecting blade; and (d) unit gravity enrichment of isolated epithelial organoids (surface layers and villous processes). As shown by light and ultrastructural microscopy, this procedure cleaved the surface epithelium from over two-thirds of the underlying basement membrane and tunica albuginea. As estimated by further dispersal of epithelial organoids with trypsin-EDTA, 0.8 to 1.5 X 10(6) surface cells were routinely obtained from a single ovary. This isolation procedure is simple and provides an experimental tool for investigating in vitro the regulation and developmental behavior of ovarian surface epithelium.

Morphology of rabbit endocervical mucosa after mating.

The distribution and morphology of rabbit endocervical mucous cells before and after mating were studied histochemically with Alcian blue, pH 2.5, and periodic acid-Schiff (AB-PAS) and by scanning and transmission electron microscopy. Before mating, the lower and upper segments of the endocervical canal contained approximately 75% and 93% of cells engorged with AB-PAS positive granules. Two hours after mating, the percentage of histochemically positive cells decreased to less than 46% and 85%, respectively. Before insemination, mucous cells were prominent among ciliated cells and contained numerous secretory granules some of which appeared to be undergoing release through exocytosis. Granule-engorged apices of mucous cells were rarely seen after insemination. Instead, cells displayed a markedly reduced complement of mucous granules and their apical surface contained numerous exocytotic pits. In many areas, a blanket of mucous material covered mucous and ciliated cells. As for the observed variation in mucous cell distribution, these changes were predominant in the endocervical mucosa of the lower cervix. We have previously shown that both native and isolated mucous cells respond to serum with rapid and massive granule release. Degranulation of endocervical mucous cells in response to semen may play an important role in regulating sperm access to the upper reproductive tract under physiologic and pathologic conditions.