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Department of Gynecological Oncology (N.G., A.de.F.), Westmead Hospital; and Department of Medicine (K.B.) and Westmead Institute for Cancer Research (N.G., C.L.C., R.L.A.-M., A.de.F.), University of Sydney at Westmead Millennium Institute, Westmead, New South Wales 2145, Australia
Address all correspondence and requests for reprints to: Anna deFazio, Department of Gynecological Oncology, Westmead Hospital, Westmead, New South Wales 2145, Australia. E-mail: anna_defazio{at}wmi.usyd.edu.au.
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Introduction |
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PR is expressed as two isoforms, PRA and PRB (7, 8), that are encoded by a single gene, each under the control of a separate promoter (9). The expression of two protein isoforms from the one gene is conserved in several species, including rodents (10, 11, 12, 13). The human PRA isoform differs from PRB by lacking 164 amino acids from its N terminus. Even though PRA and PRB are coexpressed in the same cells in humans (14), in vivo and in vitro data suggest that the two isoforms function differently (15). Selectively ablating the expression of either PRA (16) or PRB (17) in mice by mutating the respective internal ATG codon has illustrated the differential actions of each PR isoform in vivo. Ovaries from mice lacking PRA (PRAKO) contain several mature anovulatory follicles arrested from further development and display a reduction in the number of oocytes that have undergone ovulation (16). Comparatively, mice lacking PRB (PRBKO) develop normal functioning ovaries, which ovulate in response to the surge of gonadotrophins and produce normal-size litters (17). These studies illustrate the different functions of PRA and PRB in the ovary, with neither being crucial for folliculogenesis and only PRA being essential for ovulation.
In progesterone target tissues such as the mammary gland and uterus, expression of PR is induced by estradiol and down-regulated in response to progesterone (18). However, in the rodent ovary, PRA and PRB mRNA and protein are expressed in the granulosa cells of preovulatory follicles in direct response to the ovulatory surge of pituitary gonadotrophins, both in vivo (4, 19, 20, 21) and in vitro (4, 5, 19). Unlike other target tissues, estradiol alone is not sufficient to induce PR mRNA or activate PR promoter-reporter constructs in cultured rat ovarian granulosa cells (22). Rather, PR expression occurs only in granulosa cells that have differentiated into a preovulatory phenotype in response to estradiol and FSH, followed by an ovulatory dose of gonadotrophins (22). The onset of PRA and PRB expression in rat preovulatory granulosa cells is rapid, but several hours after the LH surge or administration of human chorionic gonadotrophin, the expression of both isoforms decreases and neither is detectable once the corpus luteum forms (5, 23, 24). In humans and other primates, PRA and PRB are similarly expressed in the granulosa cells of preovulatory follicles (25, 26, 27, 28); however, both isoforms continue to be expressed in corpora lutea throughout the luteal phase of the cycle (28, 29, 30, 31), during which progestin levels are high. In humans, corpora lutea PR mRNA and protein expression have been shown to correlate with serum progesterone levels, with the highest levels of PR observed in the midluteal phase and lowest levels in the late luteal phase (25, 31). In contrast, PRA protein levels decline over the course of the luteal phase and PRB levels remain relatively unchanged in the rhesus monkey corpora lutea (29). Overall these studies clearly illustrate that the cellspecific localization and regulation of PR in the ovary varies among species and indicate that in the ovary, increased PR expression is LH regulated, whereas down-regulation of PR, if it occurs, is associated with increased progestin levels.
Although PRA and PRB protein expression has been examined in the ovary during the menstrual cycle of primates, few studies have examined the localization and relative expression of each PR isoform in the ovary of mature cycling mice. Like the ovary and other tissues of the reproductive tract, functions of the oviduct are also believed to be regulated by progesterone. However, in rodents PR expression in the oviduct has been examined only in neonatal mice (32) and rats (33). Furthermore, there has been no examination of the distribution of PR isoforms in the oviduct of cycling mice and whether these compare with results previously observed in the uterus. Knowledge of the expression of the PR isoforms during the estrous cycle may lead to a better understanding of the diverse functions of PRA and PRB and have important implications in understanding the mechanisms of progesterone action in the mouse ovary and oviduct.
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Materials and Methods |
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Formalin-fixed, paraffin-embedded sections were cut at 4 µm and mounted onto SuperFrost Plus slides (Menzel-Glaser, Braunschweig, Germany), which had been coated with Mayers albumin adhesive (beaten egg white mixed 1:1 with glycerin). Slides were air dried at 37 C for 72 h and stored at 4 C until use.
Antibodies
The antibodies used to detect PR in this study were raised against human PR (35) and recognize PRA (83 kDa) and PRB (115 kDa) in mouse tissue by immunoblot analysis (12, 36, 37, 38). The mouse antihuman PR monoclonal antibody hPRa6 detects only PRB by immunoblot (35) and immunoperoxidase staining (14, 39). The mouse antihuman PR monoclonal antibody hPRa7 detects PRA and PRB by immunoblot (35, 37) but only PRA by immunoperoxidase staining of formalin-fixed tissue due to hindrance of the PRB epitope on formalin-fixation (39). The selectivity of hPRa7 for PRA in formalin-fixed tissue has also been demonstrated by dual immunofluorescence (14).
Dual immunofluorescent staining
Sections were stained for PRB and then PRA as described previously (14). To detect PRB, sections were incubated with hPRa6 diluted 1:20 in PBS/0.5% Triton X-100 (PBT), with a biotinylated goat antimouse antibody (Dako Corp., Carpinteria, CA), and Texas red (TXR)-avidin (Vector Laboratories, Burlingame, CA). To block sites of potential cross-reactivity between the two staining sequences, sections were incubated overnight with goat antimouse Ig Fab (50 µg/ml in 1% BSA/PBS; Cappel Antibodies, ICN Biomedical, Aurora, CA). To detect PRA, sections were incubated with hPRa7 diluted 1:40 in PBT, with a biotinylated goat antimouse antibody (Dako Corp.) and fluorescein isothiocyanate (FITC)-avidin (Calbiochem, Sydney, New South Wales, Australia). Sections were mounted with Vectashield mountant for fluorescence (Vector Laboratories) and stored in the dark at 4 C.
The dual immunofluorescent technique used in this study is selective for PRA and PRB (39) as mentioned above and reflects relative levels of the two isoforms (14). Under dual fluorescent excitation, PRB proteins that were labeled with TXR, appeared orange/red; PRA proteins, labeled with FITC, appeared green, and nuclei expressing equivalent levels of PRA and PRB were yellow. To control for nonspecific staining, adjacent sections were stained as above, except the primary antibody was replaced with PBT: 1) in place of both primary antibodies to control for nonspecific staining and 2) to replace the second sequence primary antibody to ensure no cross-reactivity between the two staining sequences. Human myometrium served as a positive control and was stained as above for both PRA and PRB.
Analysis of PR expression by fluorescence microscopy
PR staining was examined using a BX 40 microscope (Olympus, Tokyo, Japan) fitted with filters to detect both TXR (BP 545–580) and FITC (BP 450–480) fluorescence simultaneously and each of the two fluorochromes separately. The whole section was examined in detail under both individual fluorochrome excitations and also using the dual filter to identify the distribution of each PR isoform.
Immunoperoxidase staining
Sections were stained as previously described (39). After antigen retrieval, endogenous biotin was blocked by incubation with Dako Biotin Blocking system according to manufacturer’s instructions (Dako Corp.) and then washed. To minimize nonspecific background staining, slides were incubated for 1 h with goat Fab fragment to mouse IgG [50 µg/ml in 1% BSA/PBS (Cappel Antibodies, ICN Biomedical)]. After a brief rinse, sections were then incubated for 30 min with normal goat serum (Hunter Antisera, Jesmond, New South Wales, Australia), diluted 1:1 in PBS. Excess normal goat serum was removed before incubation with the primary antibody. To detect PRA, sections were incubated with hPRa7 diluted 1:10 in PBT. To detect PRB, adjacent sections were incubated with hPRa6 diluted 1:5 in PBT. After rinsing, biotinylated goat antimouse antibody (10.8 µg/ml in PBT; Biosource International, Camarillo, CA) was added to each section. Sections were then incubated with streptavidin-biotinylated peroxidase prepared according to the manufacturer’s instructions (Zymed Laboratories, Inc., San Francisco, CA) and then rinsed. Both PR isoform proteins were visualized using diaminobenzidine (Dako Corp.), and the reaction was stopped in distilled water. Finally, sections were counterstained with hematoxylin (Amber Scientific, Belmont, WA) and allowed to air dry before mounting with histolene and normount (Fronine, North Rocks, New South Wales, Australia). To control for nonspecific staining, adjacent sections were stained as above, except the primary antibody was replaced with PBT. Human myometrium served as a positive control and was stained as above for both PRA and PRB.
Analysis of stained sections
The developmental stages of follicles, corpora lutea, and oviduct segments were classified as previously described for the mouse (40). Similar staining patterns were observed in both ovaries and oviducts from each mouse, and therefore only one ovary and oviduct per mouse (chosen at random) were analyzed in detail.
Analysis of PR expression by light microscopy
Stained sections were analyzed using an Olympus BX-40 microscope at x400 and x600 magnification and assisted by Image Pro-Plus software (Media Cybernetic Inc., Silver Spring, MD). Cells were scored as negative or positively stained for PR and assigned an intensity score of low (1), moderate (2), or high (3) expression. To combine the percentage of positivity and intensity of expression, a histoscore value was calculated: [(number of high cells x 3) + (number of medium cells x 2) + (number of low cells x 1)]/(total number of cells).
Statistical analysis
For each ovary and oviduct section per mouse, the mean histoscore was calculated for each cell/follicle type. Preliminary analyses of these aggregated data revealed no departures from the assumptions of approximate normality and homogeneity of variance across cell/follicle type and estrous cycle stage. The aggregated data were analyzed using two-way ANOVA. These analyses determined whether there was any significant difference in PR expression throughout the estrous cycle or between different ovarian or oviduct structures. Pairwise comparisons between stages were adjusted for multiple comparisons using the Bonferroni method. Inspection of the normal probability plots of the standardized residuals from the final fitted model revealed no systematic departures from normality. P < 0.05 was considered statistically significant. The statistical software package SPSS for Windows (version 10.0, SPSS Inc., Chicago, IL) was used for all analyses.
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Results |
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In this study, the PRA-specific antibody hPRa7 stains predominantly the stromal cells of the oviduct ampulla (Fig. 1E
), whereas the PRB antibody hPRa6 stains only the epithelial cells (Fig. 1F). Dual-immunofluorescence staining further confirms the PR isoform selectivity of each antibody in formalin-fixed mouse tissue (Fig. 1, A–C). Dual-fluorescent staining of adjacent areas of the oviduct ampulla detected PRA (green) in the stromal cells of the oviduct (Fig. 1, A and C), whereas the majority of epithelial cells were positive for only PRB (red; Fig. 1, B and C).
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, E–T). Adjacent sections in which the primary antibody was omitted showed no detectable nuclear staining (Fig. 1D).
The patterns of expression of PRA and PRB in the ovary throughout the estrous cycle were distinctive. Throughout the estrous cycle, PRA staining was restricted to the theca cells of preantral and antral follicles, with no detectable expression observed in the granulosa cells during the early stages of follicle development (Figs. 1, G and I, and 2A). However, on the evening of proestrus (2200 h) just before ovulation, PRA was detected in both the granulosa and theca cells of the preovulatory follicles (Figs. 1K and 2A). Furthermore, on the morning of estrus (1000 h), PRA was also observed during corpora lutea formation (Fig. 1O); however, it was not detectable in the corpus luteum at any other cycle stage examined (Fig. 1S). In contrast, PRB expression was widespread in the ovary, detected in both the granulosa and theca cells at all stages of folliculogenesis throughout the estrous cycle (Figs. 1, H, J, and L, and 2B). PRB was also detected during corpora lutea formation (Fig. 1P) as well as in the corpora lutea from previous cycles (Figs. 1T and 2B). Interestingly, PRB was also detected in the germinal vesicle of primordial, primary, and preantral follicles (Fig. 1H). No staining of either PR isoform was detected in the surface epithelial cells (Fig. 1, G–J, S, and T), atretic follicles or blood vessels (data not shown).
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). Throughout the estrous cycle, PRA expression in the theca cells of antral follicles was consistently higher than in the preantral theca cells (P < 0.001; Fig. 3A), whereas PRB levels were similar in both follicle types (Fig. 3B). The highest level of PR isoform expression in the theca cells was seen on the evening of proestrus (2200 h; Figs. 1, M and N, and 3, A and B); this was significantly higher, compared with all other stages examined (P < 0.001 for PRA; P < 0.01 for PRB), coinciding with the previously described surge of LH, estradiol, and progesterone (41, 42, 43). However, a second increase in PRB expression was observed during metestrus (Figs. 1R and 3B; P < 0.01 between estrus and metestrus and metestrus and diestrus), correlating with the known increase in serum progesterone levels (43), whereas PRA expression remained low in the antral theca cells (Figs. 1Q and 3A) and almost undetectable in the majority of preantral theca cells until the next cycle (Fig. 3A).
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). The pattern of PRB expression in the granulosa cells of primary and preantral follicles was similar to that observed in the theca cells, with increased levels observed on the evening of proestrus (2200 h) and morning of metestrus (1000 h), compared with all other stages of the cycle examined (P < 0.01; Fig. 2B). The pattern of PRB expression in the granulosa cells of primordial and antral follicles was significantly different (P < 0.001). In the primordial follicles, the level of PRB expression was comparable at all stages of the cycle examined (Fig. 2B). In the antral granulosa cells, PRB expression was similar at all stages of the cycle except on the morning of metestrus (Fig. 1R), during which levels were significantly higher, compared with proestrus evening, estrus, and diestrus (P < 0.05).
The pattern of PRB expression in the corpora lutea from previous cycles was similar to that observed in primary and preantral follicles, with increased levels observed on the evening of proestrus (2200 h; Fig. 2B) and morning of metestrus (1000 h; Fig. 2B), compared with all other stages of the cycle examined (P < 0.01). Overall, the level of PRB expression in the corpora lutea was higher, compared with the follicular theca cells (P < 0.001), but was lower than the expression in the granulosa cells of preantral and antral follicles (P < 0.001; Fig. 2B).
Oviduct expression of PRA and PRB during the estrous cycle
In the oviduct both PRA and PRB were expressed (Fig. 1, A–C, E, and F); however, the pattern of PRA expression was significantly different from PRB, and the level of expression was dependent on both the oviduct cell type and estrous cycle stage (Fig. 4). PRA was the predominant isoform observed in the oviduct, observed within both the epithelial and stromal cells of the oviduct ampulla and isthmus (Figs. 1E and 4A). The pattern of PRA expression in the ampulla stroma and isthmus epithelium and stroma was similar throughout the estrous cycle (Fig. 4A), with relatively high expression observed throughout the cycle except on the morning of metestrus (1000 h) during which levels were significantly decreased (P < 0.001). However, the pattern of PRA expression was significantly different in the ampulla epithelium (P < 0.05), remaining consistently low throughout the cycle (Fig. 4A).
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). The pattern of PRB expression was similar in both the ampulla and isthmus epithelium throughout the estrous cycle (Fig. 4B). PRB expression remained relatively low throughout the cycle, except on the evening of proestrus (2200 h), during which levels significantly increased (Fig. 4B; P < 0.05, compared with all other stages examined). |
Discussion |
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In the ovary, PR expression was present throughout the estrous cycle during follicle development and ovulation and in the corpus luteum. In agreement with previous reports (5, 19), both PRA and PRB proteins were detected in the mural granulosa cells of preovulatory follicles on the evening of proestrus. PRA expression was greater than that of PRB in agreement with LH-stimulated granulosa cells in culture, which showed a 3-fold increase in PRA over PRB (5). It has been suggested that changes in the ratio of PR isoform expression may play a role in the differential responses to progesterone as well as other steroid hormones (15, 29). Previous reports have shown that unlike in other target tissues, estradiol alone is not sufficient to induce PR mRNA (5) or activate PR promoter-reporter constructs (22, 44) in cultured rat ovarian granulosa cells, but rather PR is induced in response to ovulatory levels of LH (5). Transfection studies in rat granulosa cells using the intact mouse PR promoter have identified regions within the proximal but not within the distal promoter region as being LH responsive (44). This is supported by our observations that PRA was detected in the granulosa cells of preovulatory follicles and not at any other stage of follicle development. Taken together with results from PRAKO mice (16), this clearly indicates that induction of PRA within the granulosa cells of preovulatory follicles by the LH surge is critical for ovulation to occur, whereas the PRB protein, although present in the granulosa cells of preovulatory follicles, is not obligatory for ovulation (17). However, because ovulation is completely ablated in mice null for both PRA and PRB (6), some interaction between PRA and PRB is still required for ovulation to occur efficiently.
For the first time in preovulatory follicles, we have been able to show similar expression of both isoforms in the theca cells. The presence of PR has only previously been reported in the theca cells of preantral and antral follicles in rabbits (45) and primates (25, 26, 46, 47) and in the granulosa cells of primordial and primary follicles in primates (46, 47). The presence of PRA in the theca cells of preantral and antral follicles in addition to the preovulatory follicles suggests that PRA expression is cell type specific and not only induced by LH. Furthermore, the presence of PRB in both the theca and granulosa cells throughout folliculogenesis clearly indicates that PRA and PRB are differentially regulated in the ovary. The pattern of expression in the theca cells of PRA was significantly different from the expression of PRB and the expression of both isoforms was dependent on the stage of the estrous cycle. The increase in expression of both isoforms on the evening of proestrus, correlates with the preovulatory surge of steroid hormones and gonadotrophins, suggesting that the surge in hormones associated with ovulation is able to up-regulate PRA and PRB in the theca cells in preantral and antral follicles. Interestingly a second increase in PRB expression was observed on the morning of metestrus, possibly under the influence of rising serum progesterone and estradiol levels that occurs during metestrus (43). However, it is unknown whether gonadotrophins or ovarian steroid hormones induce PR in the theca cells because PR regulation has been analyzed only in granulosa cells (5, 22, 44).
The expression of both isoforms in the theca cells of preantral and antral follicles suggest the continuing need for progesterone action in the theca cells throughout the estrous cycle. Although gene targets of each PR isoform in the ovary have yet to be described, the differential regulation during the estrous cycle suggests that PRA and PRB are able to mediate distinct pathways of progesterone action.
The pattern of PRB expression during the estrous cycle in primary and preantral follicles and the corpora lutea was similar to that observed in the theca cells. Interestingly, PRB expression in the antral granulosa cells was significantly different, with only a significant increase in expression on the morning of metestrus. The presence of PRB, but not PRA, in the granulosa cells during folliculogenesis indicates the possibility that PRB may play a different role from PRA in follicle growth and development.
Previous reports in rats have not observed PR mRNA or protein in the corpora lutea, not even in response to elevated hormone levels (5) or during pregnancy (23, 24). After gonadotrophin stimulation of cultured granulosa cells to induce PR expression, PRA protein persists longer than PRB and has been detected until 24 h later (5). In this study we found that after ovulation both isoforms were present in the developing corpora lutea on the morning of estrus. However, only PRB protein persisted in the corpora lutea on the morning of metestrus and in corpora lutea from previous cycles. Whereas both isoforms have been detected in the primate corpora lutea, the expression of PRB only in the mouse corpora lutea clearly illustrates species specific differences in PR expression and regulation in the corpora lutea, possibly due to differences in steroid hormone levels and corpus luteum function. However, PRB expression is not critical for the development and functioning of normal corpora lutea because PRB null mice are fertile and able to maintain pregnancies to full term (16).
The mechanisms that regulate luteal PR mRNA and protein expression are currently unknown, but progesterone has been implicated (28, 30, 48, 49). In humans and other primates, PRA and PRB are both present in the corpora lutea throughout the luteal phase, with total PR content correlating with serum progestin levels (29, 30, 31, 46). Even though we did not measure steroid hormone serum levels in this study, PRB expression in the corpora lutea correlates with the previous reports of progesterone serum levels (43), with significantly higher protein levels observed during proestrus and metestrus.
Elevated levels of progestins has been postulated to be protective against the development of epithelial ovarian carcinomas (50, 51, 52), with epidemiological studies finding that oral contraceptives with a high progestin content (53) and pregnancy are associated with a reduced risk in developing ovarian cancer. Progesterone has also been shown to increase apoptosis in ovarian epithelial cells in primates (52) and thus may underlie the protective effect of progestins in epithelial ovarian cancer. Studies in rat ovarian cells have indicated that progesterone acting directly through PR may be involved in regulating apoptosis, although other receptors and progesterone-binding proteins have also been implicated (reviewed in Refs. 54 and 55). However, it is unknown whether these effects are mediated via only one PR isoform or both. Whereas PR mRNA (48, 56) and protein (47) are expressed in the ovarian epithelial cells of primates, PR was undetectable in the ovarian epithelial cells in this study at all stages of the estrous cycle examined, indicating further variation in PR expression among species. In addition to this 7,12dimethylbenz(a)anthracene/hormone-treated PRKO mice are more prone to developing granulosa cell tumors than wild-type mice (50); therefore, PR may mediate protection against carcinogenesis in granulosa cells.
Compared with the ovary, PRA was the predominant isoform present in all oviductal cells, except the ampulla epithelium. The predominance of PRA has previously been observed in other murine reproductive tissues, such as the uterus and vagina (38) and the rat oviduct (33). The predominance of PRA in the majority of cells in the oviduct throughout the cycle suggests that this isoform is the principal isoform involved in mediating progesterone effects in the oviduct. The physiological role of PR in the mouse oviduct is unknown; however, progesterone has been suggested to regulate ovum transport via ciliogenesis (33).
Results from this study provide significant new information in understanding the mechanisms of progesterone action in the mouse ovary and oviduct. Our data clearly illustrate the differential expression of PRA and PRB in the mouse ovary and oviduct during the estrous cycle, and the cell-type specificity of PRA and PRB expression may reflect the differential regulation of PRA and PRB and/or different isoform function.
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Acknowledgments |
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Footnotes |
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Abbreviations: FITC, Fluorescein isothiocyanate; PBT, PBS/0.5% Triton X-100; PR, progesterone receptor; PRA and PRB, PR isoforms; PRAKO, PRA knockout; PRBKO, PRB knockout; PRKO, PR knockout; TXR, Texas red.
Received February 18, 2004.
Accepted for publication March 11, 2004.
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