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Reproductive Endocrinology |
Vincent Center for Reproductive Biology (X.-J.T., K.I.T., D.V.M., J.L.S., J.L.T., K.B.I.) and Division of Reproductive Endocrinology and Infertility (J.L.S., K.B.I.), Department of Obstetrics and Gynecology, Massachusetts General Hospital/Harvard Medical School, Boston, Massachusetts 02114; and,The Burnham Institute (S.K., J.C.R.), La Jolla, California 92037
Address all correspondence and requests for reprints to: Keith B. Isaacson, M.D., Vincent Memorial Obstetrics and Gynecology Service, WACC II, Massachusetts General Hospital, Boston, Massachusetts 02114.
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Abstract |
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Introduction |
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The sequential changes in the architecture of the human endometrium during the menstrual cycle are controlled in large part by ovarian hormones, and thus, cyclic episodes of apoptosis in the endometrium suggest its regulation by steroid hormones. However, little is known of the molecular mechanisms by which cells of the endometrium undergo cyclic and localized apoptosis. Progress toward understanding the intracellular regulatory events that determine apoptosis susceptibility in cells has been greatly advanced by the identification of a unique set of genes that have been conserved both structurally and functionally through evolution (6, 7, 8, 9). The protein encoded by the bcl-2 (B cell lymphoma/leukemia-2) gene is probably the most well characterized of these genes, and data now unequivocally support a role for the BCL-2 protein as a cell death repressor (6, 7). Overexpression of bcl-2 has been shown to prevent apoptotic cell death induced by an impressive array of physiological, pathological, and experimental stimuli in a variety of cell types (7, 10, 11, 12). However, in some circumstances bcl-2 overexpression fails to protect cells from apoptosis (13, 14, 15), suggesting the existence of a bcl-2-independent pathway in the regulation of physiological cell death. In keeping with this proposal, recent studies have revealed the existence of a family of bcl-2-related genes that are expressed in most eukaryotic cells. At least 12 cellular genes have been discovered that encode proteins sharing significant amino acid sequence homology with BCL-2 (6, 7, 8, 9).
For example, BAX is a BCL-2 family member that promotes cell death susceptibility, possibly by countering the effect of BCL-2 on cellular survival through heterodimer interaction (16). Another member of this family of genes, bcl-x, provides an interesting example of a single gene that, via alternate splicing mechanisms, encodes both a positive and a negative regulator of apoptosis (17). The long form of BCL-X (BCL-Xlong) contains an open reading frame of 233 amino acids with 2 domains homologous to BCL-2, whereas BCL-Xshort is a 170-amino acid truncated form of BCL-Xlong in which the region of highest homology to BCL-2 has been deleted (17). These 2 forms of BCL-X have opposing functions, in that BCL-Xlong renders cells resistant to apoptotic cell death upon deprivation of growth factors, whereas BCL-Xshort counters the resistance to apoptotic cell death provided by BCL-2 (17, 18). Based on these observations, a dueling dimer hypothesis has been proposed that identifies the ratio of BCL-2/BCL-Xlong to BAX/BCL-Xshort as a homeostatic mechanism for determination of cellular death susceptibility (16). Furthermore, as BAX interacts with both BCL-2 and BCL-Xlong (19), it has been suggested that BAX may be a common partner involved in heterodimerization and regulation of the function of other family members (19, 20, 21).
Cyclic changes in the level of BCL-2 protein in the human endometrium have been reported (22, 23, 24). Immunoreactive staining for BCL-2 predominates in proliferative glandular epithelial cells and peaks in the late proliferative phase. Interestingly, BCL-2 disappears at the onset of secretory activity (24), possibly serving to make these cells susceptible to the signals that lead to apoptotic cell death in the endometrium at the end of the secretory phase. As it is known that BCL-2 interacts with other family members, such as BAX and BCL-Xshort, to precisely regulate apoptosis, we designed the present studies to determine the patterns of BAX, BCL-2, and BCL-Xlong/short expression relative to the occurrence of apoptosis in human endometrium during the proliferative and secretory phases of the normal menstrual cycle.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Included in this study were 11 proliferative endometrial samples [early proliferative phase (days 5–7), n = 2; midproliferative phase (days 8–10), n = 5; late proliferative phase (days 11–14), n = 4] and 11 secretory endometrial samples [early secretory phase (days 15–20), n = 2; midsecretory phase (days 21–24), n = 5; late secretory phase (days 25–28), n = 4] from premenopausal women who underwent hysterectomy or endometrial biopsy for benign conditions. Paraffin sections of endometrial samples from 14 patients were obtained from the Department of Pathology at Massachusetts General Hospital. Four proliferative and four secretory endometrial samples from different patients were frozen and used for extracting protein, ribonucleic acid (RNA), or genomic DNA. The utilization of human residual tissues was approved by the Massachusetts General Hospital committee on human studies.
Reagents
All restriction enzymes, ribonuclease (RNase)-free
deoxyribonuclease, RNase inhibitor, SP6 and T7 RNA polymerases, NTPs,
and avian myeloblastosis virus reverse transcriptase were obtained from
Promega Corp. (Madison, WI). Random hexamer primers, proteinase K,
protein gel mix, and terminal deoxynucleotidyl transferase were
purchased from Boehringer Mannheim Corp. (Indianapolis, IN). The PCR
cloning vector, PCR II, was obtained from Invitrogen (San Diego, CA).
Biotin-16-deoxy-UTP was purchased from Life Technologies (Gaithersburg,
MD). RNase A was purchased from ICN Biochemicals (Costa Mesa, CA).
[
-32P]Dideoxy-ATP and [-32P]CTP were
obtained from Amersham Life Science Inc. (Arlington Heights, IL). The
avidin-biotin-peroxidase kit was purchased from Vector Laboratories
(Burlingame, CA), a Micro-BCA protein assay kit was obtained from
Pierce Chemical Co. (Rockford, IL). Pure nitrocellulose membranes were
purchased from Schleicher and Schuell (Keene, NH). The enhanced
chemiluminescence (ECL) Western blotting detection reagents were
obtained from Amersham. All other chemicals were purchased from Sigma
Chemical Co. (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA).
Polyclonal antihuman BAX, BCL-2, and BCL-X antibodies were generated in
rabbits and were described in detail previously (25, 26, 27). A rabbit
polyclonal antiserum against human BCL-Xshort/long (SC-634)
was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
In situ DNA labeling for apoptosis
The extent of DNA breakdown in endometrial sections from four mid- to late proliferative endometria and four mid- to late secretory endometria was assessed using an in situ terminal transferase reaction to label free 3'-ends of the DNA, as described previously (28). Briefly, paraffin sections were deparaffinized and rehydrated, then incubated in 2% H2O2 in 95% methanol for 1 min and deproteinated by incubation with 10 µg/mL proteinase K for 30 min at 37 C. After labeling with 50 µmol/L biotin-16-deoxy-UTP using 125 U terminal deoxynucleotidyl transferase for 15 min at 37 C, sections were blocked for 30 min with 3% (wt/vol) BSA and subsequently incubated with avidin-biotinylated horseradish peroxidase complex at room temperature for 15 min. Localization of broken DNA was detected by incubating slides with 3,3'-diaminobenzidine for 5 min. Colorimetric reactions were terminated by 10 mmol/L Tris-HCl and 1 mmol/L ethylenediamine tetraacetate (pH 8.0). Negative controls, conducted by omitting terminal deoxynucleotidyl transferase, yielded a completely negative result (data not shown). Slides were analyzed by Nikon light microscopy after counterstaining with hematoxylin.
Autoradiographic analysis of internucleosomal DNA cleavage during apoptosis
Genomic DNA was extracted from frozen samples (three mid- to late proliferative and two late secretory endometria) as previously described (29). Genomic DNA was 3'-end labeled (1 µg/reaction) with [32P]dideoxy-ATP using the terminal transferase reaction, and then analyzed by autoradiography after fractionation through 2% agarose gels as previously described (28, 29).
Immunohistochemistry
The specificity of the antibodies for human BAX, BCL-2, and BCL-Xlong/short has been established in previous studies (25, 26, 27). Paraffin sections from seven proliferative and seven secretory endometria were warmed to 75 C for 1 h, then deparaffinized and rehydrated. Endogenous peroxidase activity was exhausted by incubating the sections with 2% H2O2 for 45 s, and antigen retrieval was performed by microwaving for 5 min in 10 mmol/L sodium acetate, as previously described (25, 26). After blocking nonspecific binding with 2% BSA and 1% normal goat serum, sections were incubated with a 1:500 (vol/vol) dilution of anti-BAX, BCL-2, or BCL-X antibodies at 4 C overnight. After washing in PBS, sections were exposed to 1 µg/mL biotinylated goat anti-rabbit antibody, then incubated with streptavidin horseradish peroxidase complex for 45 min. Immunodetection was performed by incubating the sections with 3,3'-diaminobenzidine. The sections were counterstained with hematoxylin and examined under a Nikon microscope. The presence and intensity of staining were evaluated in five random fields in each section. The presence of BAX, BCL-2, and BCL-X immunoreactivity was expressed as a percentage of the cells exhibiting specific staining. The intensities of staining were assessed blindly and independently by two different investigators as negative staining (-), positive staining (+), or intense positive staining (++). Negative controls were performed by replacing the primary antibody with the same dilution of preimmune rabbit serum, and no immunoreaction product was observed (data not shown). Additionally, previous studies confirmed the specificity of these antibodies with either primary antibody deletion or peptide preabsorption (25, 26, 27).
Western blot analysis of BCL-X proteins
Approximately 100 mg of each endometrial sample, including three proliferative (ranging from the early to late proliferative phase) and three secretory (ranging from the early to late secretory phase) endometria were homogenized in 300 µL protein extraction buffer [0.1 mol/L NaCl, 0.01 mol/L Tris-HCL (pH 7.6), 1 mmol/L ethylenediamine tetraacetate (pH 8.0), 100 µg/mL phenylmethylsulfonylfluoride, 1 µg/mL aprotinin, and 250 µmol/L leupeptin]. Homogenates were boiled for 10 min and sonicated for 30 s (Sonic Dismembrator 60, setting 3, Fisher Scientific). The suspension was centrifuged at 10,000 x g for 10 min, and the supernatant was collected. A 2-µL aliquot of the supernatant was removed for subsequent protein quantitation. The remaining homogenate was combined with an equal volume of 2-fold concentrated loading buffer [0.1 mol/L Tris-HCl (pH 7.6), 4% SDS, 200 mmol/L dithiothreitol, 20% glycerol, and 0.2% bromophenol blue]. Equivalent amounts of protein (50 µg) from each sample were subjected to 15% SDS-PAGE for 1 h at 200 V. Resolved proteins were then electrophoretically transferred to pure nitrocellulose membranes at 4 C for 45 min at 100 V. After blocking nonspecific binding, blots were incubated with 0.1% (vol/vol) rabbit anti-BCL-X antibody at 4 C overnight. Specific binding was detected by incubating the membrane with 2 µg/mL biotinylated goat antirabbit IgG for 30 min and then with streptavidin-horseradish peroxidase complex for 45 min. Membranes were incubated with ECL reagents for 1 min and exposed to ECL Hyperfilm (Amersham Life Science Inc.).
Extraction of RNA
Total RNA was extracted using the guanidinium thiocyanate-phenol-chloroform single step procedure, as previously described (30). The quantity and purity of each nucleic acid sample were assessed by measuring the optical density at A260 vs. A280 nm.
Isolation and characterization of human complementary DNAs (cDNAs)
Total RNA isolated was reverse transcribed into first strand cDNA using random hexamer primers and avian myeloblastosis virus reverse transcriptase. Primers were synthesized (DNA International, Lake Oswego, OR) based on human bcl-x (forward, 5'-TTG-GAC-AAT-GGA-CTG-GTT-GA-3', bases -39 through -20 of the 5'-untranslated region; reverse, 5'-GTA-GAG-TGG-ATG-GTC-AGT-G-3', bases 6–24 of the 3'-untranslated region) (17) or human bax (forward, 5'-GGT-TTC-ATC-CAG-GAT-CGA-GAC-GG-3', bases 85–106 of the coding region; reverse, 5'-ACA-AAG-ATG-GTC-ACG-GTC-TGC-C-3', bases 530–509 of the coding region) (16) sequence. The first strand cDNA was subjected to 35 cycles of PCR amplification using human bax or bcl-x primer sets (1-min denaturation at 94 C, 1-min annealing at 50 C and 2-min extension at 72 C). The amplified products were resolved through 1.5% agarose gels, isolated, and subcloned into the PCR II vector for large scale plasmid preparation and automated DNA sequence analysis. We were not successful in the isolation of a bcl-2 cDNA from human endometrial RNA by reverse transcription-PCR amplification (data not shown).
Preparation of radiolabeled probes and Northern blot analysis
RNA probes complementary (antisense) to human bax or
bcl-x messenger RNA (mRNA)-coding sequences were synthesized
by in vitro transcription from linearized bax
(466 bp) and bcl-x (800 bp) plasmid templates using RNA
polymerase, [-32P]CTP (3000 Ci/mmol), and the Gemini
II Riboprobe Core System (Promega), as previously described (31). Total
RNA samples prepared from midsecretory human endometrium were
fractionated through a 1.2% denaturing agarose gel (5 µg RNA/lane),
visualized with ethidium bromide staining and UV transillumination
to confirm RNA integrity and sample loading equality, and blotted to
pure nitrocellulose membranes by overnight capillary transfer using
20-fold concentrated sodium chloride-sodium citrate solution (3 mol/L
sodium chloride and 0.3 mol/L sodium citrate) as the transfer buffer.
The RNA samples were UV cross-linked to the membranes and hybridized to
radiolabeled antisense RNA probes (3 x 106 cpm/mL
hybridization buffer) under highly stringent conditions at 65 C for
18–20 h (32). After a 10-min wash at 20 C in 2-fold concentrated
SSC-0.1% SDS and extensive washing (20–40 min) at 65 C in 0.1-fold
concentrated SSC-0.1% SDS, autoradiography was carried out by exposure
of membranes to Amersham Hyperfilm for 2–4 days at -80 C.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Apoptosis in the human endometrium was confirmed by
autoradiographic analysis of internucleosomal DNA cleavage. Genomic DNA
collected from proliferative endometrium showed no evidence of
fragmentation, whereas DNA extracted from the late secretory
endometrium contained oligonucleosomal fragments characteristic of cell
death via apoptosis (Fig. 1
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, A and B). In contrast,
late secretory endometrium exhibited extensive and widespread apoptosis
in the glandular epithelial cells in both the functionalis and basalis
layers (Fig. 2, C and D); however, a few apoptotic cells were also
detected in stroma. Characteristically, apoptosis was restricted to
single cells surrounded by a group of viable cells.
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Figures 2
and 3 depict the spatial
distribution of BAX, BCL-2, and BCL-X, as assessed by
immunohistochemistry in the human endometrium during the proliferative
and secretory phases of the menstrual cycle. The intensity of
immunostaining and the percentage of cells that demonstrated the
immunoreactivity are summarized in Tables 1–3.
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, E and F). By comparison, in
secretory endometrium there was a dramatic increase in the expression
of BAX, especially in glandular epithelial cells of the functionalis
layer (Fig. 2, G and H). The majority of glandular epithelial cells in
the functionalis layer of secretory endometrium showed intense BAX
immunostaining (Fig. 2H). BAX immunostaining was also observed in some
stromal cells (Fig. 2H).
In contrast to the faint staining of BCL-2 in secretory endometrium,
there was stronger staining for BCL-2 in most of the glandular
epithelial cells and in some stromal cells in proliferative endometrium
(Fig. 3, A and B). Furthermore, BCL-2 expression demonstrated more
intense immunostaining in the basalis layer than in the functionalis
layer. Myometrium also showed BCL-2 immunoreactivity (Fig. 3A). After
ovulation, there was a marked decrease in the proportion of
BCL-2-immunopositive cells and a reduced intensity of BCL-2 staining in
the glandular epithelial cells of the human endometrium (Fig. 3, C and
D). However, even in the late secretory endometrium, a low level of
immunoreactive staining for BCL-2 remained in the basalis layer (Fig. 3C).
In proliferative endometrium, BCL-X immunostaining was observed mostly
in glandular epithelium and was more concentrated in the basalis layer
(Fig. 3, E and F). In secretory endometrium, the level of BCL-X
immunostaining increased, particularly in the glandular epithelial
cells of the functionalis layer (Fig. 3, G and H). Some stromal
cells also showed BCL-X immunostaining. The pattern of immmunostaining
of BCL-X using rabbit anti-BCL-Xlong/short IgG (Santa Cruz
Biotechnology) was identical to that of the rabbit polyclonal IgG (27)
(data not shown).
Overall, BAX immunoreactivity, in terms of both proportion of cells
stained and intensity of staining, predominated in secretory
endometrium. By comparison, BCL-2 immunoreactivity predominated in
proliferative endometrium. Immunoreactive staining of BCL-X increased
slightly in secretory endometrium compared to the proliferative
endo-metrium (Tables 1–3).
Western blot analysis of BCL-Xlong vs. BCL-Xshort
As neither of the antisera used can distinguish between the long
(antiapoptosis) vs. short (proapoptosis) form of BCL-X
in situ, expression of this protein in human endometrium was
further characterized by Western blot analysis (Fig. 4).
Both BCL-Xlong and BCL-Xshort were present in
all three proliferative and three secretory endometrial samples from
different patients, with BCL-Xlong being the predominant
form. No obvious difference in the ratio of BCL-Xlong to
BCL-Xshort was observed between the samples prepared from
proliferative and secretory endometria.
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As shown in Fig. 5, Northern blot hybridization
with the bax or bcl-x antisense RNA probe and
total RNA prepared from secretory endometrium yielded a specific
hybridization signal with the expected size transcript
[bax, 1.0 kilobase (kb); bcl-x, 2.7 kb], thus
confirming expression of these genes in the human endometrium.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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With the role of apoptosis in endometrial cell turnover now relatively well established, our next effort is focused on determining the relationship between apoptosis and cell death regulatory genes in the human endometrium. Expression of the protein product of one of these genes, bcl-2, in the human endometrium has been described in recent studies (22, 23, 24). However, bcl-2 is only one member of this multigene family consisting of numerous proteins homologous to BCL-2 that interact by forming homo- and heterodimers (16, 17, 18, 19, 20). As such, without examining the expression of other members of the BCL-2 family, it is difficult to interpret the role of a single member of this family in the regulation of endometrial cell apoptosis. Therefore, we studied the expression of BCL-2, BAX, and BCL-X in the human endometrium as well as their relationship to apoptosis.
In addition to confirming previous reports that BCL-2 immunostaining predominates in the proliferative phase of the cycle (22, 23, 24), our results demonstrated that BCL-2 immunoreactivity was most intense in the basalis layer. This region of the uterus is known to show the highest growth activity during the proliferative phase and is maintained during menses despite the massive tissue loss associated with turnover of the adjacent functionalis region at the end of the secretory phase (33, 34). Interestingly, BCL-2 immunoreactivity in the functionalis layer decreased after ovulation, contrasting the maintenance of BCL-2 expression in the basalis layer even during the late secretory phase. These data support the contention that the death repressor activity of BCL-2 is important for promoting survival of uterine epithelial cells within the basalis layer throughout the cycle, thus possibly maintaining a progenitor population of cells for regrowth of the functionalis layer during the proliferative phase.
The present study further demonstrated a strikingly different expression pattern of another member of the BCL-2 family, namely BAX, during the proliferative and secretory phases of the human menstrual cycle. In proliferative endometrium, only a few scattered glandular epithelial cells were found to be immunopositive for BAX protein. By comparison, BAX immunostaining was increased in secretory endometrium. Importantly, BAX expression was predominantly localized to glandular epithelial cells within the functionalis layer of the endometrium, consistent with the function of the protein in conveying increased apoptosis susceptibility to this population of cells at the end of the secretory phase. As discussed previously, the opposing actions of BCL-2 and BAX suggest the possibility that one dimer partner acts as a dominant inhibitor of the other (35). In fact, changes in the BAX to BCL-2 ratio in glandular epithelial cells during the human menstrual cycle parallel the cycle-related changes in endometrial cell apoptosis. These results provide the first evidence that the BAX to BCL-2 "rheostat" may be a critical factor in regulating apoptosis in the human endometrium during the normal menstrual cycle. These findings are in agreement with those of Tilly et al., who reported that the ratio of bcl-2 to bax expression is probably a critical determinant of cell fate in ovarian granulosa cells during follicular maturation and atresia (32). Moreover, a role for increased bax expression in apoptosis associated with other aspects of female reproductive function (e.g. luteolysis) has recently been reported (36).
The BCL-2:BAX rheostat, however, does not completely account for the regulation of apoptosis in the human endometrium. For instance, although almost all of the glandular epithelial cells in the functionalis layer of the secretory endometrium expressed BAX with little or no expression of BCL-2, only a small percentage of these cells exhibited evidence of apoptosis. As such, other members of the bcl-2 gene family, such as the bcl-x gene product, may also play important roles in controlling apoptosis by mechanisms that are independent of or complementary to the actions of BCL-2 (17, 37). It has also been proposed that BCL-Xlong can be mutated to a form unable to heterodimerize with BAX, yet it can still protect cells from apoptosis in a BAX-independent mechanism in some cell systems (18). Our results demonstrated that in proliferative endometrium, BCL-X immunostaining was primarily observed in glandular epithelial cells and was more concentrated in the basalis layer. However, in secretory endometrium, BCL-X was expressed in glandular epithelial cells in the functionalis layer. By immunoblot analysis we confirmed that BCL-Xlong was the predominant isoform expressed in both proliferative and secretory endometrium. These results suggested that BCL-Xlong together with BCL-2 are important for promoting survival of glandular epithelial cells within the basalis layer of proliferative endometrium. By comparison, BCL-Xlong in the functionalis layer of secretory endometrium may provide some localized resistance to cell death triggered by BAX-dependent or BAX-independent mechanisms. This may also partly explain the phenomenon that only a small percentage of BAX-positive glandular epithelial cells in the functionalis layer of the secretory endometrium undergo apoptosis at any one time.
These data collectively suggest that BCL-2 and BCL-Xlong are important antiapoptosis factors in the human endometrium, whereas BAX induced in the secretory phase facilitates the increased endometrial cell turnover that may be associated with menses. Of final note, the menstrual cycle-related changes in the levels of BAX, BCL-X, and BCL-2 in the human endometrium suggest that the expression of the genes encoding these apoptosis-related proteins may be directly regulated by ovarian hormones. In support of this hypothesis, apoptosis in hamster and rodent uterine epithelia is known to be controlled by estrogen and progesterone (38, 39, 40, 41). Furthermore, Otsuki et al. reported that cyclic changes in BCL-2 in glandular epithelial cells of the human endometrium correlated with changes in the expression of receptors for estrogen and progesterone (24). Recently, Matsuo and colleagues (42) reported that expression of BCL-2 protein in human uterine leiomyoma cell in vitro was up-regulated by progesterone, providing the first evidence that ovarian steroid hormones directly regulate the expression of BCL-2. Our finding that the levels of BAX, BCL-2, and BCL-X change coordinately in the human endometrium during the menstrual cycle supports this proposal and provides a foundation for further studies on the ovarian hormone regulation of expression of bcl-2 and related genes in the human uterus.
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Footnotes |
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Received January 31, 1997.
Revised April 17, 1997.
Accepted May 1, 1997.
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References |
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in human
endometrium. Hum Reprod. 10:277–286.. Endocr J. 2:87–95.
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Y. Morita, T. F. Manganaro, X.-J. Tao, S. Martimbeau, P. K. Donahoe, and J. L. Tilly Requirement for Phosphatidylinositol-3'-Kinase in Cytokine-Mediated Germ Cell Survival during Fetal Oogenesis in the Mouse Endocrinology, February 1, 1999; 140(2): 941 - 949. [Abstract] [Full Text]
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