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Department of Anatomy and Human Biology (A.M.D., S.H., B.S., S.P.), University of Western Australia, Nedlands, Perth, Western Australia 6907, Australia; and Vincent Center for Reproductive Biology, Department of Obstetrics and Gynecology (K.I.T., J.L.T.), Massachusetts General Hospital/Harvard Medical School, Boston, Massachusetts 02114
Address all correspondence and requests for reprints to: Dr. A. M. Dharmarajan, Department of Anatomy and Human Biology, University of Western Australia, Nedlands, Western Australia 6907, Australia. E-mail: dharma{at}anhb.uwa.edu.au
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Introduction |
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The possible involvement of apoptosis, or physiological cell death, in ovarian function was first demonstrated by early morphological studies that described the occurrence of chromatolysis in granulosa cells of rabbit ovarian follicles during atresia (7). Biochemical evidence for the existence of a calcium- and magnesium-dependent endonuclease activity in rat ovarian granulosa and luteal cell nuclei capable of generating the mono- and oligonucleosomes that comprise the apoptotic DNA ladder was subsequently reported (8). Since then, a number of experimental paradigms have been employed to substantiate a role for apoptosis in various events of cell turnover in the ovary (9), including germ cell attrition (10, 11, 12), granulosa and thecal-interstitial cell death during follicular atresia (12, 13), ovarian surface epithelial death during ovulation (15, 16), and luteolysis (17, 18, 19, 20, 21).
Recent studies using functional and regressing CL collected from pseudopregnant rabbit ovaries have provided evidence that internucleosomal DNA cleavage, a hallmark of apoptosis, is present in luteal cells of regressing, but not functional, CL (19). These findings confirmed earlier reports of a role for apoptosis in luteolysis in the rat (17) and cow (18), and have since been supported by numerous studies in various species, including humans (21). To complement the in vivo analyses of luteal cell death described above, an in vitro system employing individual CL collected from pseudopregnant rabbits was developed and used to demonstrate a time-dependent onset of apoptosis in this tissue in serum-free organ culture (19). Furthermore, using this model it was reported that hCG blocks apoptosis in the rabbit CL cultured in vitro, suggesting that this pregnancy-associated gonadotropin in humans may be important for luteal maintenance via its survival actions in the CL (19). However, the mechanisms by which this hormone, and other as yet unidentified endocrine factors, regulate apoptosis in the CL remain to be fully identified.
Several investigations have shown that the final series of events that trigger physiological cell death, regardless of species or cell type, may involve changes in the expression of a conserved subset of genes (22, 23). For example, the Bcl-2 protooncogene product, when elevated in many cell types, delays the normal course of cell death induced by trophic factor deprivation or harmful stimuli (24, 25). Immunolocalization of Bcl-2 within cells has revealed its association with mitochondrial membranes (26, 27), implying that the actions of Bcl-2 may be linked at least in part to the reduction-oxidation (redox) status of the cell (28). In this regard, it has been reported that Bcl-2 reduces accumulation of reactive oxygen species (ROS) in transfected cells and can protect a variety of cells from apoptosis induced by oxidative stressors (29, 30). Conversely, the Bcl-2-related protein, Bax (31), causes increased mitochondrial production of ROS and apoptosis when overexpressed in cultured cells (32). Aside from Bcl-2, other factors that play a role in protecting cells from oxidative stress include superoxide dismutases (SOD), which are responsible for the conversion of superoxide radical anion to peroxide intermediates, and glutathione peroxidase and catalase, which convert peroxides to water (33).
In agreement with these findings, it has been shown in vivo that gonadotropin-mediated suppression of apoptosis in granulosa cells of the immature rat ovary is associated with increased expression of secreted and mitochondrial (manganese-containing, Mn-SOD) isoforms of SOD (34) and reduced expression of bax (35). The involvement of Bax in precipitating follicular atresia has been further supported by immunolocalization studies in the human ovary (36) as well as by analysis of mice harboring a targeted disruption in the bax gene (37, 38). Additionally, apoptosis induced in granulosa cells of rat ovarian follicles by in vitro culture without hormonal support is prevented by treatment with SOD and by a number of other antioxidant factors, including catalase, ascorbic acid, and N-acetyl-L-cysteine (34). A parallel series of studies with CL obtained from bovine ovaries has shown that apoptosis during structural lueolysis in vivo is associated with reduced expression of oxidative stress response genes (20) and elevated bax levels (39).
To further clarify and extend our understanding of the role of antioxidant pathways in regulating luteal cell survival, in the present report we used the in vitro CL organ culture system to determine if antioxidant factors suppress apoptosis in the rabbit CL, to investigate if antioxidant enzymes directly modulate expression of bcl-2-related genes in the rabbit CL, and to evaluate if hCG promotes rabbit luteal cell survival by altering the expression of Mn-SOD and/or the bcl-2-related genes bax and bcl-x.
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Materials and Methods |
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Animals
Sexually mature New Zealand White rabbits (Animal Services,
University of Western Australia), with an average weight of 3.5 kg,
were used for all experiments. Rabbits were caged individually for a
minimum period of 4 days under controlled light and temperature for
acclimation and were given rabbit pellets (Glen Forrest Stockfeeders,
Glen Forrest, Australia) and water ad libitum.
Pseudopregnancy was induced by administration of 100 IU hCG via the
marginal ear vein, and the day of hCG injection was defined as day 0 of
pseudopregnancy. The selection of days during pseudopregnancy on which
tissues were collected was based upon earlier studies demonstrating
that progesterone secretion by the pseudopregnant rabbit CL increases
from day 1 to a maximum on day 11 and then declines to baseline (day 1)
levels by day 18. Pseudopregnancy lasts for 21 days in this model, as
defined by structural regression of the CL at this time (2, 41, 42).
All procedures involving animals were conducted after approval by the
animal ethics committee of the University of Western Australia.
Analysis of bcl-x, bax, and Mn-SOD expression in the CL during
pseudopregnancy in vivo
On days 1, 11, and 18 after hCG administration, rabbits were
anesthetized with sodium pentobarbital (32 mg/kg) and subjected to
laparotomy. Both ovaries from each animal were removed, and the CL were
quickly dissected, cleaned of adherent stromal tissue, snap-frozen in
liquid nitrogen, and stored at -70 C until RNA extraction and analysis
of bax, bcl-x, and Mn-SOD messenger RNA (mRNA)
levels by Northern blot (see below).
In vitro organ culture of isolated CL: apoptosis studies
To assess the effects of inhibitors of oxidative stress on
apoptosis in the CL, a previously described organ culture system was
used (19, 40). Individual intact CL were collected from ovaries of day
11 pseudopregnant rabbits using nonenzymatic dissection. Isolated CL
were fully cleaned of adherent tissue and snap-frozen immediately (0 h)
or placed in sterile 20-ml glass scintillation vials containing 2 ml
Eagle’s MEM supplemented with 2 mM
L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin
sulfate, and 0.1% fatty acid-free BSA (fraction V; Sigma Chemical Co.). Incubations were carried out at 37 C for 2 h
without or with increasing doses of SOD (1.5–150 U/ml), AA (1–100
mM), NALC (25 or 50 mM), or catalase (10–1000
U/ml). CL were then collected from incubations, snap-frozen, and
subsequently analyzed for the occurrence and extent of internucleosomal
DNA breakdown associated with apoptosis (see below).
Extraction and analysis of DNA for internucleosomal cleavage
Genomic DNA was prepared from individual CL as originally
described (43) and modified (44). After extraction, purification, and
quantitation of DNA, 500 ng of DNA from each sample were labeled on
3'-ends with [32P]dideoxy-ATP (3000 Ci/mmol; Amersham
Australia, Sydney, Australia) using the terminal transferase reaction,
as described previously (45). Radiolabeled DNA samples were resolved by
electrophoresis through 2% agarose gels at 50 V (6.5 V/cm) for 3–3.5
h. The gels were dried for 2 h without heat in a slab gel drier,
sealed in plastic wrap, and exposed to Kodak X-Omat films
(Eastman Kodak Co., Rochester, NY) at -70 C for
autoradiographic analysis. Low mol wt DNA fractions (<15 kb) were then
excised from gels, mixed with 3 ml scintillation fluid, and counted in
a ß-counter to provide a quantitative estimate of the degree of
internucleosomal DNA cleavage among samples (44).
In vitro organ culture of isolated CL: mRNA analysis
CL isolated from day 11 pseudopregnant rabbit ovaries were
incubated as described above in the absence or presence of hCG (1
µg/ml) or SOD (100 U/ml; determined in this study to be the maximally
effective dose for the inhibition of apoptosis in incubated CL; see
Results). After incubation, CL were collected, snap-frozen,
and analyzed for changes in levels of bax, bcl-x,
and Mn-SOD mRNA levels by Northern blot (see below).
Preparation of complementary RNA (cRNA) and cDNA probes
Antisense RNA probes complementary to rabbit bax, rat
bcl-x, or rat Mn-SOD mRNA coding sequences were synthesized
by in vitro transcription from linearized plasmid templates
using RNA polymerase, [
-32P]CTP (3000 Ci/mmol;
Amersham), and the Gemini II Riboprobe Core System (Promega Corp., Madison, WI) (34, 35, 40). The probe for 18S rRNA was
radiolabeled with [-32P]deoxy-CTP (3000 Ci/mmol;
Amersham) using the random priming method (46) and then purified from
unincorporated radionucleotides by nick column Sephadex G-50
(Pharmacia Biotech, Uppsala, Sweden).
Extraction of RNA and Northern blot analysis
Total RNA was isolated from frozen CL using the single step
guanidinium thiocyanate method (47), resuspended in
diethylpyrocarbonate-treated water, and quantitated by reading the
absorbance at 260 nm using a spectrophotometer. Total RNA (10
µg/sample) was fractionated by electrophoresis through denaturing
formaldehyde-agarose gels, transferred to nitrocellulose membranes, and
UV cross-linked. The membranes were hybridized to the radiolabeled cRNA
probe of interest and subsequently washed under highly stringent
conditions at 65 C, as detailed previously for these probes (34, 35, 40). After cRNA probe hybridization analysis, the radioactivity on the
blots was allowed to decay, and the blots were then rehybridized with
the radiolabeled 18S rRNA probe at 42 C under conditions detailed
previously (34). All data on changes in bcl-x,
bax, and Mn-SOD mRNA levels were then normalized relative to
18S rRNA levels in each sample. In all cases, hybridization signal
intensities were obtained from Northern blots by exposing the
hybridized membranes to Fuji Photo Film Co., Ltd. imaging
plates (Bas-IIs, Fuji Photo Film Co., Ltd., Tokyo, Japan),
and the resultant images were scanned using a Fuji Photo Film Co., Ltd. Bioimager.
Statistical analysis and data presentation
All experiments were repeated at least three times, with
separate rabbits for each experiment. A representative autoradiogram is
presented where appropriate for qualitative analysis (DNA cleavage,
Northern blot), whereas quantitative results obtained from ß counting
of radiolabeled low mol wt DNA fragments (apoptosis analysis) or image
analysis of hybridization signal intensities (mRNA analysis) represent
the mean ± SEM of combined data from replicate
experiments. Statistical differences (P < 0.05)
between mean values were analyzed by one-way ANOVA and least
significant difference tests.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). By comparison, bax mRNA
levels significantly declined (43 ± 5%; P <
0.05) from day 1 to day 11 of pseudopregnancy and then markedly
increased (208 ± 14%; P < 0.05) on day 18
compared to those on day 11 (Fig. 1B). Lastly, levels of Mn-SOD mRNA
were significantly increased (159 ± 12%; P <
0.05) on day 11 vs. day 1 and decreased (41 ± 6%;
P < 0.05) on day 18 compared with those on day 11
(Fig. 1C).
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). This inhibition
of apoptosis was mimicked by treatment with AA, albeit a significant
inhibition of DNA cleavage was obtained only with the highest
concentration of this free radical scavenger (Fig. 3). Incubation of CL with NALC also
inhibited apoptotic DNA breakdown, with both concentrations of NALC
providing a significant and dose-related suppression of
internucleosomal cleavage (Fig. 4).
Lastly, inclusion of catalase in the incubation medium suppressed
apoptosis in a dose-dependent manner, with maximal suppression at 100
U/ml (Fig. 5).
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). The slight increase in
bcl-x mRNA levels noted with hCG treatment (122 ± 6%;
P < 0.05) was also significant (Fig. 6A). As corpora
lutea exposed to gonadotropin expressed significantly higher levels
(223 ± 6%; P < 0.05) of Mn-SOD mRNA
vs. untreated controls (Fig. 6C), we next tested the
possibility the SOD itself was a direct regulator of, and thus a
possible mediator of the actions of hCG on, bcl-x or
bax expression in the CL. Corpora lutea treated with SOD for
2 h possessed significantly higher levels of bcl-x mRNA
(259 ± 12%; P < 0.05) and significantly lower
levels of bax mRNA (27 ± 3%; P <
0.05) than untreated controls (Fig. 7, A
and B, respectively).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, we sought to confirm and extend these observations suggesting the involvement of oxidative stress as a trigger for apoptosis during luteal regression using the pseudopregnant rabbit as a model. In the first series of experiments, Northern blot analysis of changes in the levels of bcl-x, bax, and Mn-SOD in the rabbit CL during pseudopregnancy revealed an inverse relationship between expression of the bcl-x and bax genes. Levels of bcl-x mRNA were highest, whereas levels of bax mRNA were lowest, on day 11, corresponding to the time of maximal luteal function. However, by day 18, upon the initiation of apoptosis and luteolysis (19), bcl-x mRNA levels had declined, and bax mRNA levels had dramatically increased. These data support published studies with the bovine CL regarding elevated bax expression during luteolysis (39) and are in agreement with the known anti- and proapoptotic functions of Bcl-x and Bax, respectively (23). We also noted that there occurred a slight but significant decline in luteal expression of Mn-SOD from days 11–18, data that are again consistent with reported changes in SOD activity in the rat and rabbit CL during luteolysis (48, 49) as well as in Mn-SOD mRNA levels in the regressing CL of the cow (20).
For the remaining experiments, we switched to an in vitro model system of incubating individual CL as a means to directly study the potential involvement of oxidative stress in the process of luteal cell apoptosis under defined conditions. As previously reported (19), luteal cells in healthy CL collected from day 11 pseudopregnant rabbits exhibited extensive apoptosis after 2 h of incubation in the absence of tropic hormone support. Moreover, in this model, we have already shown that provision of hCG effectively and dose-dependently inhibits apoptosis (19). From the present studies, we observed that the ability of hCG to inhibit apoptosis was reproduced by treatment of CL with SOD, suggesting that accumulation of superoxide anion and/or its downstream reactive metabolites in tropic factor-deprived CL is involved in triggering luteal cell death. This conclusion is supported by the fact that AA, which functions as a general free radical scavenger, also suppressed apoptosis in incubated CL. Furthermore, inclusion of NALC, a free radical scavenger and putative stimulator of endogenous glutathione peroxidase activity, blocked apoptotic DNA cleavage in incubated CL. Lastly, inclusion of catalase in the medium also effectively suppressed apoptosis, collectively indicating that an inhibition of oxidative stress, through a variety of experimental treatments, mimics the ability of hCG to inhibit apoptosis in CL incubated in vitro.
Northern blot analysis of changes in bcl-x, bax, and Mn-SOD mRNA levels using the in vitro organ culture model provided additional novel insight into the possible mechanisms underlying the involvement of antioxidant molecules in hCG-mediated luteal cell survival. In vitro treatment of CL with gonadotropin for 2 h significantly reduced the levels of bax mRNA compared with those levels present in untreated CL while concomitantly slightly increasing bcl-x mRNA levels. We also observed that hCG treatment elevated Mn-SOD levels over the 2-h incubation period, providing additional evidence of a functional link between a gene induced by hCG (i.e. Mn-SOD) and the ability of that gene product to mimic the effects of hCG on apoptosis in this model system. These findings, however, raised the intriguing possibility that the actions of hCG on luteal bcl-x and/or bax expression may also be mediated by SOD. Indeed, Northern blot analysis of RNA isolated from CL incubated for 2 h with SOD revealed that bcl-x and bax mRNA levels were up- and down-regulated, respectively, vs. those levels in untreated CL. Consequently, these data indicate a novel role for SOD in protecting cells from oxidant-induced damage and death, that being direct regulation of expression of Bcl-2 family members that, in turn, are known to influence cell fate via changes in cellular redox state or susceptibility to ROS-induced apoptosis (28, 51).
The present findings may offer additional insight into why luteal cell apoptosis is initiated only in CL deprived of luteotropic support. During development of the CL, luteotropic agents, such as hCG or estradiol (in the case of rabbit), maintain high levels of steroidogenesis with increased cytochrome P-450 enzymatic activities as well as with overall cellular metabolic activity. At the same time, the consequences of luteotropic actions, namely mitochondrial generation of oxygen free radicals associated with electron transport needed for steroid and ATP synthesis, must be handled quickly and efficiently by the luteal cell. This defense mechanism is in all likelihood accomplished by the induction of antioxidant molecules (SOD, catalase, and peroxidases) and suppression of prooxidant factors (Bax). Upon diminishing levels of luteotropic agents, luteal cells fail to sustain a balanced redox state, and thus luteal cell death may be initiated at least in part as a consequence of the damaging effects of accumulating ROS. In conclusion, the data presented in this report provide important new functional links among luteotropins, antioxidant enzymes, Bcl-2 family members, and apoptosis as primary determinants of the life span of the CL.
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Footnotes |
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Received October 7, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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receptor in the rat ovary.
Prostaglandins Leukotrienes Essential Fatty Acids 46:223–229[CrossRef][Medline]
-dihydroprogesterone, and estradiol-17ß in
pregnant and pseudopregnant rabbits: evidence for post-implantation
recognition of pregnancy. Biol Reprod 23:1014–1019[Abstract]
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