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Department of Animal Science, Cornell University, Ithaca, New York 14853
Address all correspondence and requests for reprints to: Dr. Susan M. Quirk, Department of Animal Science, 258 Morrison Hall, Cornell University, Ithaca, New York 14853. E-mail: SMQ1{at}cornell.edu
The Fas antigen is a cell surface receptor that, when engaged by Fas
ligand or specific agonistic antibodies, triggers apoptosis. The effect
of an agonistic monoclonal antibody to mouse Fas antigen (Fas mAb,
clone J02) on the viability of cells from dispersed mouse corpora lutea
(CL cultures) was tested. Cultures were prepared by enzymatic digestion
of CL from day 4–7 pseudopregnant mice. Cultures were pretreated with
0, 1, 10, 100, or 1000 U/ml murine interferon-
(IFN) at 72 h of
culture. IFN has been shown to increase Fas antigen expression in a
number of cell types. At 96 h (time zero), cultures were treated
with Fas mAb or IgG. By 4 h after Fas mAb treatment, discrete
homogeneous patches of cells within the cultures showed characteristic
signs of apoptosis, including blebbing of cell membranes, detachment,
and disappearance from the culture. CL cultures contain luteal,
stromal, and endothelial cells; fibroblasts; and surface epithelial
cells (OSE). Cells dying in response to Fas mAb were identified as OSE.
Affected cells had the cobblestone appearance and distinct nuclei
typical of epithelial cells. Unlike luteal cells, OSE did not stain
with the lipophilic dye, Nile red. The cells did not stain with
acetylated low density lipoprotein conjugated to the fluorescent marker
octadecyl indocarbocyanine, a marker for endothelial cells and
monocytes. Cells in patches stained positively for cytokeratin, a
marker for epithelial cells. Fas-mediated cytotoxicity was quantified
by counting the number of cells present in discrete patches of OSE 0
and 8 h after Fas mAb treatment. Fas mAb treatment had no effect
in cultures pretreated with 0 or 1 U/ml IFN, but induced significant
death of OSE in cultures pretreated with 10, 100, and 1000 U/ml IFN
(37 ± 11%, 54 ± 18%, and 60 ± 11%, respectively).
There was no apparent effect of Fas mAb on other cell types within the
CL cultures. To confirm that cells dying in response to Fas mAb were
OSE, experiments were also performed on enriched cultures of OSE
prepared by enzymatic digestion of the outer surface of the ovary. In
enriched OSE cultures pretreated with 200 U/ml IFN, there was 44%
killing in response to Fas mAb, whereas in cells not pretreated with
IFN, there was no effect. In situ fluorescent end
labeling of DNA in CL cultures indicated that treatment with IFN and
Fas mAb induced DNA fragmentation in OSE typical of apoptosis.
Immunocytochemistry of CL cultures indicated that Fas antigen was
expressed in OSE pretreated with IFN. Quantitative reverse
transcriptase-PCR showed that IFN pretreatment increased Fas antigen
messenger RNA levels 2.3-fold in enriched cultures of OSE. In summary,
OSE in CL cultures and enriched cultures of OSE undergo apoptosis in
response to Fas mAb when pretreated with IFN. In vivo,
OSE undergo programmed cell death before ovulation and rapidly
proliferate to repair the surface of the ovulatory follicle after
ovulation. Most ovarian cancers are derived from the OSE. The results
have implications for both normal ovarian function and oncogenesis in
the ovary.
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