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Departments of Physiology and of Medicine, Dartmouth Medical School, Lebanon, New Hampshire 03756-0001
Address all correspondence and requests for reprints to: Valerie Anne Galton, Departments of Physiology and of Medicine, Dartmouth Medical School, 1 Medical Center Drive, Borwell Building, Lebanon, New Hampshire 03756-0001. E-mail: val.galton{at}dartmouth.edu.
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Introduction |
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The D2, which converts T4 to T3 by 5'-deiodination (5'D), is present in the subendothelial stromal cells lining the uterine lumen and is increased 3-fold during pregnancy (2). In contrast, the D3 metabolizes T4 and T3 to inactive compounds by 5D and is induced in the uterus more than 200-fold during pregnancy to levels considerably higher than those observed in any other tissue. At gestation (embryonic) d 9 (E9) and E10, the D3 is highly expressed, primarily in the decidual tissue; whereas at later stages, the enzyme is found in the epithelial cells lining the uterine cavity that surrounds the fetal unit (1). We have speculated that these alterations in uterine deiodinase activity play an important role in maternal/fetal thyroid hormone homeostasis and thus may have important implications for fetal development. In particular, the high levels of D3 expressed in the pregnant uterus may serve, together with the placenta, to limit the exposure of the embryo to maternal thyroid hormones.
The factors that regulate D2 and D3 in the uterus (and, in particular, those mechanisms responsible for the marked induction of expression of these enzymes during pregnancy) remain undefined. In preparation for implantation, the mammalian uterus undergoes profound cyclic morphological and physiological changes under the control of estrogen and progesterone (P). For example, estrogen serves as a trophic hormone in the uterus by stimulating growth and differentiation. These changes are accompanied by well-documented increases in protein synthesis, glucose utilization (4), and gene expression (5, 6, 7). P is also required for optimal uterine receptivity (8) and, at the molecular level, this hormone often modifies the actions of estrogen (4). The magnitude of these estrogen- and P-induced changes in gene expression in the human uterus has recently been demonstrated by Kao et al. (9) using DNA array techniques. The implantation process results in additional alterations in gene expression (10).
To characterize further the uterine expression of the deiodinases, we have examined D3 activity in the rat uterus before implantation. In addition, we have determined the effects of estrogen, P, and the implantation stimulus on D2 and D3 expression using ovariectomized (ovx) and pseudopregnant animals. Our results suggest that multiple factors, including ovarian steroid hormones, are involved in the regulation of these enzymes in this organ.
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Materials and Methods |
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Mice in which the gene for the cytokine IL-11 receptor (IL-11R)
had been disrupted (11) were kindly provided by Dr. Petra Bilinski (Jackson Laboratories, Bar Harbor, ME). The shipment consisted of three homozygous females, three heterozygous females, one homozygous male, and one heterozygous male. Mice were shipped at approximately 8 wk of age. To obtain sufficient female animals for study, the heterozygous mice were bred, and the females in the litters were typed by Southern blot analysis of tail DNA using a probe external to the 3'region of the targeted area of the gene as described by Bilinski et al. (11). The resulting homo- and heterozygous females were bred with the corresponding original male; the day that a plug was found in the vagina as designated d1 (E1) of pregnancy.
Studies in nonpregnant female rats
To determine the profiles of deiodinase activity in the uterus and ovaries during the estrous cycle, intact female rats were subjected to a daily vaginal lavage at 0800 h, and the cells obtained were examined microscopically to determine the stage of the estrous cycle (12). Rats were killed for study at two stages of the cycle (proestrus and diestrus).
To investigate how the dynamic nature of gonadal steroids during the estrous cycle influence D2 and D3 activities in the uterus, rats that had been ovx for a minimum of 7 d were treated with 17ß-estradiol (E2) and/or P (Calbiochem, La Jolla, CA) according to two protocols. In both, E2 and P (2 µg and 2 mg/injection, respectively) were injected sc in 0.1 ml sesame seed oil. In the first, five groups of rats (four rats/group) received a daily injection for 4 consecutive days and were killed on the fifth day. Group I received vehicle only, group II received E2 alone, group III received P alone, group IV received E2 + P, and group V received E2 + P for 2 d then P alone for 2 d. In the second, six groups of rats (four/group) received one of the following treatments: vehicle only for 4 d; E2 + P for 4 d; E2 + P on d 1 and 2, P alone on d 3 and 4; E2 + P on d 1 and 2, E2 alone on d 3 and 4; E2 on d 1 and 2, P on d 3 and 4; or P on d 1 and 2, E2 on d 3 and 4. Uteri were harvested on the fifth day.
Studies in pregnant rats
For studies of uterine D3 and D2 activities during pregnancy, female and male rats were mated in our animal facility. The morning that sperm were detected in the vaginal smear was designated as d1 (E1) of pregnancy.
Studies in uterine deciduomas
Two techniques were employed to initiate the formation of uterine deciduomas. The first was based on the method of Kaiser et al. (13). Female rats were subjected to a daily vaginal lavage, and the cells obtained were examined microscopically to determine the stage of the estrous cycle (12). Pseudopregnancy was induced by mating them with vasectomized male rats on the night of proestrus. Day 1 of pseudopregnancy was defined as the day on which a vaginal plug was obtained. Decidualization was induced in one uterine horn on d 5 of pseudopregnancy in the following manner. The rat was lightly anesthetized with ether, and a small incision was made in the abdominal wall just ventral to the cervix. The decidualization process was initiated in one uterine horn by inserting a plastic catheter into the base of the left uterine horn and gently scratching the entire antimesometrial surface of the horn three times. The right horn was not manipulated; it provided corresponding nondecidualized uterine tissue. The incision was then closed, and the animal was allowed to recover. This technique resulted in the formation of large deciduoma in some, but not all, rats. A second method, in which ovx rats were given a schedule of hormone treatments to sensitize the uterus for decidualization, was subsequently found to support decidualization in almost every rat (14). Beginning at least 6 d after ovariectomy, E2 and P were injected sc in sesame seed oil (maximum of 0.2 ml/d) according to the following schedule: d 1–3, 0800 h, 0.2 µg E2; d 3, 1600 h, 0.2 µg E2 + 1.0 mg P; d 5 and 6, 1600 h, 4 mg P; d 7, 1600 h, 0.3 µg E2 + 4 mg P; d 8 (until the day of death), 0800 h, 0.1 µg E2 + 4 mg P. On the morning of d 8, the rats were lightly anesthetized with ether, and decidualization was initiated by making a small incision in the abdominal wall, inserting a needle attached to a 1-ml syringe into the upper end of the left uterine horn and injecting 0.1 ml sesame seed oil into the lumen. The horn was ligated above the entrance point of the needle to prevent loss of the oil or uterine fluids from the uterus. Control ovx animals were injected with vehicle and were not subjected to the decidualization process.
All animal protocols were approved by the Animal Research Review Committee of Dartmouth Medical School.
Preparation of tissues
All rats and mice were killed by decapitation and exsanguination, and the uteri were rapidly removed and dissected free of the surrounding adipose tissue. Each uterine horn was divided into several pieces, some of which were processed for deiodinase assay and others for RNA analysis. In the experiments in which one uterine horn had been decidualized, the weights of both horns were obtained. In the experiment that used intact nonpregnant rats, the ovaries were also processed for deiodinase assay.
For the studies in the pregnant rat uterus, animal groups included nonpregnant rats and pregnant rats at E4, E6–E10, E15, and E21. In the nonpregnant rats and pregnant rats at E4, which is before implantation of the blastocyst, each uterine horn was divided into two pieces. In rats at E6–E21, the uterus was cross-sectioned to obtain individual implantation sites. To facilitate identification of the implantation sites at E6 and E7, these rats were injected iv with 0.3 ml of 1% Chicago blue (Sigma, St. Louis, MO), under light ether anesthesia, 15 min before decapitation. The sites take up more dye than the nonimplanted parts of the uterus because of their increased vascularity and vascular permeability and thus can be clearly identified by the naked eye. At E15 and E21, a longitudinal cut was made along the antimesometrial side of the uterine wall, and the uterus was obtained by folding it back over the amniotic sac and gently peeling it free from the placenta. The following tissues were taken for assay of 5'D and 5D activities: nonpregnant rats and rats at E4, pieces of whole uterus; rats at E6-E10, individual implantation sites (uterus plus embryo); and E15 and E21, uterus alone. In one experiment, uteri were obtained from four pregnant rats at E4 and four nonpregnant rats. One horn of each uterus from the pregnant rats was gently perfused with approximately 3 ml saline to flush out the embryos. The fluid was collected, and at least two embryos were detected in each when examined under a microscope, confirming that pregnancy had indeed occurred.
For studies in the decidualized uterus, rats were killed from 2–12 d after decidualization. In some experiments, pieces of the entire deciduoma (uterus plus contents) were taken for assay; in others, the uterus and its contents were also taken separately by cutting open the uterus and gently scraping the decidual tissue from the uterine wall. Pieces of the nondecidualized horns and of nondecidualized uteri from ovx vehicle-injected rats were also obtained.
For studies in the IL-11R knockout mice, nonpregnant uterus and E8 implantation sites were obtained.
Determination of 5D and 5'D activities
Tissues were homogenized in 0.25 mM sucrose, 20 mM Tris-HCl (pH 7.6), as previously described (15), using a Tissumizer (Tekmar Co., Cincinnati, OH), and sufficient buffer to yield approximately a 1:5 homogenate (wt/vol). The homogenates were centrifuged at 1000 x g for 15 min, and the supernatants were stored at -20 C for subsequent assay of 5D and 5'D activities using our published methods (16, 17). For the 5D assay, the reaction mixture (total vol, 50 µl) contained between 1–100 µg of tissue protein, adjusted to ensure that deiodination was less than 20%; 1 nM [125I]T3 was used as substrate and 50 mM dithiothreitol as cofactor. The assay incubation time was 1 h. Activity is expressed as picomoles of 3,3'-diiodothyronine generated/h·mg protein. For assay of 5'D activity, the reaction mixture (total vol, 50 µl) contained between 25–100 µg tissue protein and 1.2 mM EDTA. The substrate was 1.0 nM [125I]rT3, and the cofactor was 20 mM dithiothreitol. Incubation was carried out for 1 h at either 37 or 0 C. The percent deiodination of substrate that occurred at 37 C was corrected for any nonenzymatic deiodination that took place during the same time period at 0 C. Activity is expressed as femtomoles iodide generated/h·mg protein. In determining 5'D activity, the percent iodide generated was multiplied by 2 because the specific activities of the labeled products were only half that of the substrate. D1 and D2 5'D activities were distinguished, respectively, by the inclusion of 1 mM 6-n-propyl-2-thiouracil and/or 100 nM nonradioactive T4 in the incubation medium. [125I]T3 (
2200 Ci/mmol) and [125I]rT3 (specific activity 959 µCi/µg) were obtained from Perkin-Elmer (Norwalk, CT) and were purified by chromatography using Sephadex LH-20 (Sigma) before use. Protein concentrations of all samples were determined according to the method of Comings and Tack (18) using BSA as the standard.
In situ hybridization
In situ hybridization for D2 and D3 mRNA was performed on cross-sections of oil-induced deciduoma from pseudopregnant rats, as previously described, using specific antisense and sense RNA probes labeled with [35S]uridine 5'-triphosphate (1, 2).
Northern analysis of RNA
Total RNA was prepared from uteri using a commercial RNA isolation reagent (TRIzol Reagent; Life Technologies, Inc., Gaithersburg, MD), according to the manufacturer’s instructions. Isolation of Poly (A)+ RNA from the total RNA and Northern analysis were carried out as previously described (19). Between 10 and 20 µg poly(A)+ mRNA were loaded into each lane. The blot was probed sequentially with the NS43–1 rat D3 cDNA (20) and a rat D2 cDNA (21). To document the relative amounts of RNA in each lane, the blot was also probed with a cDNA for the unregulated gene, cyclophilin. Hybridizations were conducted at 42 C and washings at 55 C. Hybridization signals were analyzed using a PhosphorImager 445 SI (Molecular Dynamics, Sunnyvale, CA), quantified by ImageQuant (version 1.2) and normalized for variations in mRNA content using the cyclophilin signal.
Statistics
Student’s t test was used to compare differences between the mean of values obtained in two groups of animals (22). Statistical analyses involving multiple groups were performed using the GB-STAT software for Macintosh. The data were subjected to randomized ANOVA, and the means were compared using Fisher’s LSD (protected t test). Statistical significance was defined as P < 0.05. All reported findings have been confirmed in two or more experiments.
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. In the six rats studied at E6–E8, D3 activity in the implantation site was invariably higher than that in the nonpregnant uterus. However, this increase was small compared with the very marked increase that began after E8, was clearly evident at E9, and further increased by E10. As previously noted (1), D3 activity in uterus was lower at E15 than in the implantation site at E10. It was also noted that D3 activity in uterus at E4 was significantly lower than that in the nonpregnant uterus. This observation was confirmed in a second experiment, where D3 activity in the nonpregnant uterus was compared with that in the perfused and nonperfused E4 uterus. Levels of D3 activity in the perfused and nonperfused d-E4 uterus were 0.009 ± 0.0015 (±SE) and 0.0087 ± 0.0018 pmol/h·mg protein, respectively. D3 activity in the nonpregnant uterus was 0.038 ± 0.0098, a value that was significantly higher than that in either the perfused or nonperfused uterus (P < 0.025).
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. Groups of 3 rats were killed at 2, 5, 8, and 12 d post decidualization. Deciduomas were well developed in all 12 rats; and, as indicated by the relative weights of the decidualized and nondecidualized uterine horns, formation of decidual tissue was maximal after 5 d. At 2, 5, 8, and 12 d, the mean weights of the decidualized horns were, respectively, 5, 15, 11, and 2 times that of their corresponding control horns. At 2 and 5 d post decidualization, levels of D3 activity in the decidualized uterus were 30- to 40-fold higher than those in the corresponding nondecidualized horn, and the measured velocities were at least as high as the maximum levels seen in implantation sites in normal pregnancy (Fig. 1A
) (1). By d 8, the activity had started to decrease; but on d 12, it was still higher that that in the corresponding nondecidualized horn. It was also noted that D3 activity in all 12 of the nondecidualized horns was higher than that observed in the uterine tissue from the two control (ovx, vehicle-injected, P < 0.05) rats. At 2, 5, 8, and 12 d, levels of D3 activity in the nondecidualized uterus were, respectively, 0.89 ± 0.073, 0.78 ± 0.089, 0.463 ± 0.031, and 0.932 ± 0.295 pmol/h·mg protein. D3 activity in the control rats was 0.127 ± 0.045. These findings suggest that D3 activity in the nonpregnant uterus is influenced either by one or both of the administered steroid hormones, or by a factor(s) originating in the decidual tissue.
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To localize the D3 activity in the decidualized uterus, large deciduomas were induced in pseudopregnant rats by scratching the antimesometrial surface of the uterus with a plastic catheter. The uteri were obtained at 4, 5, and 8 d after this procedure. Homogenates were made of the entire uterus (including decidual tissue), of decidual tissue alone, and of the uterus minus the decidual tissue. The majority of the D3 activity was located in the decidual tissue (Fig. 3). However, significant D3 activity was also found in the nondecidualized part of the uterus. This could be attributable either to activity present in some remaining decidual cells or to activity present in the uterine epithelial cells, where it has been shown to be present during the second half of pregnancy (1).
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, the spatial pattern of D2 and D3 expression differed markedly. D3 mRNA was seen prominently in the decidual tissue present in the central portion of the deciduoma. In contrast, D2 mRNA was expressed in the periphery of the specimen in a nearly circular pattern that surrounded the D3-expressing decidualized cells.
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knockout mice form only small amounts of decidual tissue which degenerates before the formation of placenta (11). Thus, female IL-11R knockout mice are infertile. The consequences of this impairment in decidual formation on deiodinase expression during early pregnancy were investigated in these mice. A minimum of three implantation sites was obtained from three homozygous and five heterozygous pregnant mice. Samples of nonpregnant uteri from two homozygous mice were also obtained. Mice were studied on d E8 of pregnancy because this has previously been shown to be the stage when the greatest amount of decidual tissue is present in the IL-11R knockout animals. The implantation sites in the heterozygotes seemed normal morphologically; it has been shown previously (11), and confirmed in the present study, that reproductive capacity is not impaired in these heterozygous mice. The implantation sites in the knockout mice appeared smaller, some were hemorrhagic, and others had been resorbed. However, levels of D3 activity in the implantation sites of the knockout mice, when expressed on a per-milligram protein basis, were not significantly different from those in the healthy sites obtained from the heterozygous dams (Fig. 5). D2 activity in the implantation sites from the homo- and heterozygous IL-11R mice was also found to be comparable and not significantly different from that in the nonpregnant mouse uterus [-/-, 48.9 ± 9.19; +/-, 37.2 ± 8.69, P not significant (NS)].
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Uteri were obtained from rats in either diestrus or proestrus. The differences in the gross appearance of the uteri at the two stages were evident. The uteri obtained at proestrus were thick, well-vascularized, and filled with fluid. In contrast, the uteri obtained at diestrus were very thin and less vascularized.
The rat uterus has been shown previously to exhibit both D2 and D3 activities but no significant D1 activity (23). Levels of activity of both D2 and D3 were much higher in uteri from rats in proestrus than in rats in diestrus (Fig. 6, A and B). D2 activity was increased more than 3-fold and D3 activity more than 8-fold, and both differences were highly significant (P < 0.001).
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). In contrast, and similar to the uterus, ovarian D3 activity was almost 5-fold greater at proestrus than during diestrus (Fig. 7B, P < 0.001).
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Uterine D2 activity was increased by gonadal steroid hormones (Fig. 8A); activity was increased 9-fold by E2 alone (P < 0.01) and 4-fold by P alone (P < 0.05). However, when the two hormones were given together, the increase in D2 activity was not significantly different from that observed after E2 alone. Furthermore, in the group given the combination of E2 and P followed by P alone, D2 activity was not significantly different from that in the ovx control group.
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). When administered together, the two hormones acted synergistically to induce a 70-fold increase in D3 activity (P < 0.01). However, when the E2 and P were administered together for only 2 d, followed by only P for 2 d, the increase in D3 activity, although highly significant (P < 0.01), was considerably less (only 16-fold).
The effects of E2 and P on levels of uterine deiodinase activities were explored further in an experiment in which rats were either injected with E2 and P together for 2 d, followed by either E2 or P separately for 2 d, or they were given either E2 or P for 2 d, followed by the other hormone for the next 2 d. For comparison, other rats received either vehicle or E2 + P for all 4 d. Uteri were harvested 24 h after the last injection. A substantial increase in D2 activity was obtained after administration of both E2 and P for 4 d, as noted previously. A comparable increase was also observed in rats given E2 + P for 2 d but only E2 on d 3 and 4 (Fig. 9A). However, no increase in D2 activity occurred in uteri from rats given E2 and P for 2 d, followed by P only on d 3 and 4. Furthermore, uterine D2 activity was enhanced in the rats given P followed by E, but not when E2 was followed by P.
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, P < 0.01). However, D3 activity was not increased if, on d 3 and 4, only P was given, and only modestly increased if only E2 was given (P < 0.01). When E2 was given alone, followed by P alone, no increase in D3 activity was observed; whereas a small, but significant, increase occurred when P was followed by E2 (P < 0.01).
Northern analysis of D2 and D3 transcripts in nonpregnant uterus
A Northern blot containing four samples of poly(A)+ RNA, two prepared from uteri of ovx rats and two from uteri of ovx rats treated with E2 + P for 4 d, was probed sequentially with cDNAs for D2, D3, and cyclophilin (Fig. 10). Transcripts of the appropriate size for D2 (>7.0 kb) and D3 (2.2 kb) were evident in all the lanes containing uterine mRNA. However, the signal in the lanes containing mRNA from the hormone-treated rats was at least four times that in the lanes containing mRNA from the ovx control rats. These data suggest that the effects of E2 and P occur, at least in part, at a pretranslational level.
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The levels of uterine activity of both D2 and D3 were severalfold higher in proestrus, compared with diestrus. Given that both E2 and P levels are highest during this phase of the cycle (24, 25, 26, 27), these results suggest that the expression of these enzymes is regulated by gonadal steroids. Studies of D2 and D3, at both the activity and mRNA levels, in ovx rats administered E2 and P singly, sequentially, or in combination confirmed that this is the case, and that regulation occurs (at least in part) at the pretranslational level, as would be expected based on the known genomic actions of these hormones.
It was noted that the effects of E2 and P on the expression of the two deiodinases differed in some potentially important aspects. Although injections of P alone did significantly increase D2 activity in the ovx rat uterus, several findings suggest that the regulation of this enzyme is dependent primarily on E2. First, the level of D2 activity achieved after the injection of E2 alone was twice that observed after P alone and was equal to that obtained with the administration of both hormones together. Second, the increase achieved after administration of both hormones for 2 d was maintained after withdrawal of P but not maintained when E2 was withdrawn. Third, when P was given for 2 d followed by E, D2 activity was increased, but no increase was seen when the order of the hormones was reversed. Thus, uterine D2 activity seems to respond primarily to the circulating level of estrogen.
In contrast, the regulation of uterine D3 activity is clearly dependent on the presence of both E2 and P, because they have a marked synergistic effect on expression. This synergism was underscored by the finding that the withdrawal of either E2 or P after 2 d of combined treatment resulted in a significant fall in D3 activity; only limited stimulation was achieved if either hormone was administered alone, or if they were given sequentially with E2 followed by P, each for 2 d, or vice versa.
In early pregnancy, before implantation, uterine D2 and D3 activities fall to levels below that found in the nonpregnant uterus (Fig. 1) (2). Given the preeminent role of E2 in D2 regulation and its importance in D3 regulation, this decrease in activity may reflect the fact that E2 levels are relatively low during this phase of gestation (26). After implantation, however, D2 and D3 levels increase, with D3 expression being induced 200-fold over a period of 48–72 h, to levels much greater than those observed during any phase of the estrous cycle or after the administration of hormones to ovx animals. As indicated from our findings in pseudopregnant rats using artificial decidualization stimuli, the increase in D3 activity was a consequence of the implantation process per se. Thus, within 48 h of a decidualization stimulus, D3 activity is markedly enhanced in the decidualized uterine horn; whereas in the nondecidualized horn, activity is only slightly higher than that in the untreated ovx rats. Given our findings that the activities of D2 and D3 are enhanced by administration of E and P, the small (but significant) increase in the nondecidualized horn is most likely attributable to the hormones administered to render the rats pseudopregnant. The localization of D3 activity in the decidualized horn to the decidual tissue itself was demonstrated using both in situ hybridization and by the measurement of activity in dissected uterine specimens. These findings are compatible with our prior report of the expression of D3 in the decidual cells during native pregnancies (1) and clearly indicate that an induction of D3 mRNA is a fundamental part of the decidualization reaction.
The factors responsible for the stimulation of D3 in decidual tissue are uncertain; but clearly, the presence of the blastocyst itself is not required based on the present studies. Numerous growth factors, cytokines, and other factors have been implicated in the decidualization stimulus, and some of these (e.g. epidermal growth factor and TGFs) are known to be potent stimulants of D3 activity in cell culture systems (29, 30). To investigate further the possible triggering factors for D3 induction, the potential role of IL-11 was investigated in the present studies using IL-11R-deficient mice. The process of decidualization is known to be impaired in these mice, rendering them infertile (11, 31). Consistent with these prior reports, we did observe a diminished decidual response in the homozygous knockout mice. However, the level of D3 activity in the implantation sites, when expressed on a per-milligram-of-protein basis, was not impaired. This suggests that expression of D3 in decidual tissue is not directly dependent on IL-11 signaling.
The pattern of D2 expression in the artificially decidualized uterus was markedly different from that of D3. Only a minimal increase in D2 activity was noted, and this seemed to occur over a more protracted time course than that observed for D3 activity. In marked contrast to the finding regarding D3 activity, there was no difference between the level of D2 activity in the decidualized vs. nondecidualized horn when expressed on a per-milligram-of-protein basis; though clearly, a greater total level of D2 expression in the decidualized horn was present, given its marked increase in size. However, these results seem to make clear that factors other than the direct implantation stimulus seem to mediate the increase in uterine D2 activity during pregnancy. P could be a factor in this modest increase, because P levels are high during pregnancy, and this hormone does have a small inductive effect on D2 activity as shown herein.
An additional finding of interest in these studies relates to the ovary, which expresses D1 and D3 activities at relatively low levels (23). As in the uterus, the level of D3 activity in the ovary was considerably higher during proestrus than in diestrus. In contrast, the level of D1 activity was unchanged. This demonstrates a fundamental difference in the regulation of D1 and D2 activities in reproductive organs.
The physiological implications of these dramatic patterns of deiodinase expression in the uterus remain a matter of speculation. Based on these and our previous studies, it is apparent that the uterus is programmed to surround completely the blastocyst, and then the developing embryo, with high levels of D3 activity beginning almost immediately after implantation. In effect, this allows the embryo to develop initially in an environment that contains minimal, but appropriate, levels of thyroid hormone; one that is devoid of most, but not all, maternal thyroid hormone (32).
Studies are currently in progress to demonstrate that indeed the implantation site is a tissue with low levels of T4 and T3, relative to maternal serum and other tissues. The recent availability of D2 and D3 knockout mice in our laboratory (33, 34) will facilitate defining the exact role of these enzymes in the regulation of uterine thyroid hormone levels, and the physiological consequences of their deficiency. In this regard, it needs to be borne in mind that these patterns of deiodinase expression and their consequences in terms of uterine T4 and T3 levels may affect uterine processes as well as fetal development, to the extent that the uterine tissues are responsive to thyroid hormones.
Understanding the spatial pattern of D2 expression in the uterus is also of interest. As observed by in situ hybridization, D2 is expressed in cells surrounding the decidual tissue and away from the immediate site of implantation. This suggests that the T3 formed by the actions of D2 could be available to regulate uterine processes rather than have effects on the developing fetus. An alternative explanation derives from the fact that the Michaelis-Menten constant (Km) of the D3 for T3 is significantly lower than that for T4, suggesting that T3 is used more efficiently than T4 by this enzyme as a substrate for 5D (35). Thus, by converting T4 to T3 on the edge of the decidual tissue, the D2 may actually facilitate the inactivation of maternal thyroid hormones.
In summary, the studies described herein indicate that the expression of both D2 and D3 in the uterus vary significantly during the estrous cycle and during the initial part of pregnancy in response, at least in part, to gonadal steroid hormone levels. Furthermore, the implantation process results in a profound induction of D3, which seems to be an integral part of the decidualization process designed to provide the embryo with an optimal TH environment for development. Further studies aimed at examining the functional consequences of these events are planned.
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Footnotes |
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Abbreviations: D2, Deiodinase type 2; 5D, 5-deiodination; E2, 17ß-estradiol; E4, embryonic d 4; IL-11R, IL-11 receptor; ovx, ovariectomized; P, progesterone.
Received April 18, 2003.
Accepted for publication June 30, 2003.
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