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Regular Article |
a Institute for Research in Reproduction, Indian Council of Medical Research, Parel, Mumbai 400 012, India
ABSTRACT
The expression profiles of leukemia inhibitory factor (LIF), transforming growth factor ß2 (TGFß2), and transforming growth factor ß2 receptor (TGFß2R) were analyzed during the peri-implantation period in regularly menstruating, fertile bonnet monkeys and in animals in which endometrial nonreceptivity was induced by administering an antiprogestin, onapristone. Based on our previous experiences, a dose of 2.5 or 5 mg of onapristone was administered s.c. every third day during the menstrual cycle, because these dosages impair endometrial development without upsetting the normal gonadal endocrine profiles. Endometrial biopsy specimens were collected during the proliferative phase (estradiol levels about 200 pg/ml, n = 5) and peri-implantation period (Day 8 after midcycle peak in estradiol levels, n = 5) from normal ovulatory animals and during the peri-implantation period from onapristone-treated animals (n = 10). The biopsy specimens were processed to determine the expression patterns of LIF, TGFß2, and TGFß2R by immunohistochemical and reverse transcription-polymerase chain reaction (RT-PCR) methods. Levels of both protein and mRNA for LIF, TGFß2, and TGFß2R (analyzed by immunohistochemistry and RT-PCR, respectively) were greater in the endometrial samples collected during the peri-implantation period compared to samples collected during the proliferative phase in control animals. Treatment with either of the two doses (2.5 or 5 mg) of onapristone caused a significant (P < 0.05) down-regulation in the expression of LIF in the peri-implantation endometria. The endometrial expressions of TGFß2 and TGFß2R mRNAs were reduced significantly in animals treated with 5 mg of onapristone, but not in those treated with the lower dose. However, immunoreactive TGFß2 and TGFß2R proteins were significantly (P < 0.05) down-regulated in the endometrial samples from both the 2.5- and 5-mg-treated groups. The alterations observed in the expression patterns of LIF, TGFß2, and TGFß2R were specific, because the expression levels of epidermal growth factor receptor remained unaffected in the endometria from the treated groups. The present study demonstrates derangement in the expression profiles of LIF, TGFß2, and TGFß2R during the peri-implantation period in infertile bonnet monkeys. It may be hypothesized that TGFß2 function is one of the early steps in the regulation of the progesterone-driven cascade of events leading to endometrial receptivity, and that any aberration in this step may adversely affect the subsequent molecular events (i.e., expression of LIF). These data also suggest that potential aberrations in the functional network of locally produced cytokines and growth factors even may occur in an endometrium exposed to the optimal peripheral hormonal levels.
cytokines, implantation, uterus
INTRODUCTION
The endometrial factors dictating success or failure of implantation in primates are not fully understood. This could be one of the major reasons for low success rates of in vitro-assisted technologies, lack of clinical solutions for unexplained infertility, recurrent miscarriages, and other reproductive disorders in women.
Data are emerging that suggest potential roles of various local regulators (i.e., cytokines and growth factors) in endometrial growth and development during the menstrual cycle and pregnancy [1–4]. Furthermore, several reports suggest that expression of at least some of these factors is regulated by progesterone [5, 6]. These progesterone-mediated effects result from a series of sequential events involving binding of progesterone to the progesterone receptor (PR), dimerization of PR, interaction of progesterone-PR complexes with specific elements in DNA, and subsequent transcriptional activation or repression of target genes [7].
The crucial role of progesterone in endowing the endometrium with receptivity has been substantiated in several reports demonstrating retardation in endometrial development and subsequent implantation failure when progesterone actions were neutralized using antiprogestins such as RU 486 (mifepristone) [8, 9], ZK 98.299 (onapristone) [10–12], or ZK 98.734 (lilopristone) [13] in primates. These antiprogestins compete with progesterone for its binding site on PR and, once bound, make the receptor nonfunctional by inducing conformational changes. Although these antiprogestins are 19-nor steroids with a p-dimethylaminophenyl group at the 11ß position, onapristone has a distinctly different molecular shape. It has a 3-hydroxypropyl chain at the C-17 position, whereas mifepristone has one propyl chain at the same position [14].
Mifepristone and onapristone also differ in their modes of action employed in neutralizing various progesterone-dependent functions (i.e., ovulation, corpus luteum function, endometrial development, and pregnancy maintenance). Mifepristone promotes the formation of PR dimers and may not always interfere with binding of PR complexes to chromatin structure, but onapristone does not facilitate PR dimerization and impairs interaction of the PR complexes with progesterone-responsive elements of the DNA. Thus, onapristone is considered to be a pure progesterone antagonist [15].
Our previous studies have demonstrated differential sensitivities of the hypothalamus, pituitary, ovary, and endometrium to onapristone [16]. Inferences drawn from these studies led us to focus our attention on the endometrium, which could be rendered inhospitable for embryo implantation without adversely affecting ovulation, hormonal levels, and menstrual cyclicity in nonhuman primates [12]. That no significant aberration was detected in the expression of PRs in these infertile animals [17] suggested the possibility of a postreceptor defect in the endometria. This prompted us to look into the expression of certain endometrial factors, such as leukemia inhibitory factor (LIF), transforming growth factor ß2 (TGFß2), and transforming growth factor ß2 receptor (TGFß2R), in a nonreceptive endometrium exposed to normal levels of circulatory progesterone.
Various studies have demonstrated increased expression of LIF, TGFß2, and TGFß2R during the peri-implantation period compared to that during the proliferative phase of the menstrual cycle in primates. These observations suggest that the expression of these factors is dependent on progesterone [6, 18, 19].
In endometrial stromal cells, TGFß2 induced the expression of LIF [20]. Under similar culture conditions, progesterone alone did not stimulate LIF production [20]. However, the inferences drawn from in vitro studies may not reflect the actual relationship between progesterone and these factors. To our knowledge, no information exists concerning the in vivo comparative expressions of LIF, TGFß2, and TGFß2R during the peri-implantation period in primates. Such studies should provide a better insight regarding the mechanism regulating endometrial receptivity in vivo.
Induction of endometrial nonreceptivity in a subhuman primate without affecting the normal endocrine profile offers an ideal experimental model for identification of endometrial factors that facilitate implantation. In the present study, we have used nonreceptive endometria from antiprogestin-treated animals as a model to analyze in vivo expressions of LIF, TGFß2, and TGFß2R.
MATERIALS AND METHODS
Chemicals and Radioactive Materials
3-Aminopropylethoxysilane, bovine serum albumin (BSA), 17ß-estradiol, and progesterone were obtained from Sigma (St. Louis, MO). Vectastain ABC-AP kit and Vectastain Elite ABC kit were obtained from Vector Laboratories (Burlingame, CA). Primary antibodies for LIF were obtained from R & D Systems (Minneapolis, MN). Primary antibodies for TGFß2, TGFßR type II, and the ABC staining system were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Onapristone (ZK 98.299) was a gift from Schering AG (Berlin, Germany).
Custom primers for actin, TGFß2, TGFß2R, and epidermal growth factor receptor (EGFR) were supplied by Gibco BRL Life Technologies (Grand Island, NY). Primers for LIF were a gift from Dr. W.C. Okulicz (Umass School of Medicine, MA). RNase-free DNase I and the Single-Step Titan Reverse Transcription-Polymerase Chain Reaction (RT-PCR) system were purchased from Roche Diagnostics GmbH (Mannheim, Germany). RNeasy minikit columns were purchased from Qiagen (Santa Clarita, CA).
Antisera for estradiol and progesterone were procured from ICN Diagnostics (Costa Mesa, CA). 1,2,6,7-3H-Progesterone (93 Ci/mmol) and 2,4,6,7-3H-estradiol (99 Ci/mmol) were obtained from Amersham (Buckinghamshire, UK). All other chemicals used in this study were high-grade, commercially available products.
Animals
Healthy and proven fertile female bonnet monkeys (Macaca radiata) weighing between 3.5 and 4.5 kg and showing regular cyclicity were used. The monkeys were individually caged in the institute's primate facility in an environment with controlled photoperiod (14L:10D) and temperature (23°C). The present study was approved by the Ethics Committee for Care and Use of Laboratory Animals for Biomedical Research of the Institute for Research in Reproduction.
Estimation of Hormone Levels
Estradiol and progesterone concentrations were estimated in serum samples by specific radioimmunoassay methods as described previously [12]. The inter- and intraassay coefficients of variation were 9.2% and 6.8%, respectively, for estradiol (n = 26 assays) and 11.1% and 6.7%, respectively, for progesterone (n = 17 assays).
Treatment with Onapristone
Twenty female bonnet monkeys showing at least two consecutive ovulatory menstrual cycles of normal duration were selected. Average length of the menstrual cycle was 27.7 ± 2.0 days, and average duration of the menses was 2.7 ± 1.2 days in these animals. Female monkeys were randomly distributed into four groups (n = 5 per group). The animals in groups 1 and 2 were injected s.c. with 0.5 ml of vehicle (1:15 benzyl benzoate:castor oil v/v). The animals in groups 3 and 4 were injected s.c. with 2.5 and 5.0 mg onapristone, respectively, in the same volume of vehicle. Treatment was initiated on Day 1 of the menstrual cycle and repeated every third day thereafter until the day of the biopsy. Our previous study has shown the 100% efficacy of this treatment regimen for inhibiting implantation in bonnet monkeys [12].
Endometrial Biopsies
Endometrial biopsy specimens from animals in group 1 were collected during the proliferative phase (generally between Days 8 and 10 of the cycle) when the circulating levels of estradiol were approximately 200 pg/ml. The biopsy specimens from animals in groups 2, 3, and 4 were collected on the eighth after the midcycle peak in estradiol levels. Part of the endometrial tissue from each sample was fixed in 4% buffered formalin and embedded in paraffin by the routine procedure. Serial paraffin sections (5 µm) were cut and mounted on 3-aminopropylethoxysilane-coated glass slides. Some of the sections were stained with hematoxylin-and-eosin for histopathological evaluation. The remaining biopsies were processed for RNA extraction.
Immunohistochemical Localization
Leukemia inhibitory factor Immunohistochemical localization of LIF was carried out as described previously [21, 22]. The slides were incubated with goat antirecombinant human LIF antibody diluted 1:20 in 1x PBS. The sections were then incubated with biotinylated secondary antibody diluted in PBS, followed by addition of a complex of avidin and biotinylated alkaline phosphatase according to the manufacturer's protocol (Vectastain ABC-AP kit). Endogenous alkaline phosphatase activity was inhibited with levamisole. The sections were incubated with freshly prepared substrate p-nitro-phenyl phosphate for 5 min to complete the reaction. No counterstaining was done, because the immunoprecipitants were pink in color. The sections were dehydrated, cleared in xylene, mounted, and viewed under the Zeiss Ultraphot microscope (Carl Zeiss, Inc., Thornwood, NY) at 120x magnification.
TGFß2 and TGFß2R The sections were deparaffinized in xylene and rehydrated through grades of methanol in distilled water. They were washed twice with 1x PBS for 10 min each. Endogenous peroxidase was inactivated by incubating the sections in 0.3% H2O2 in methanol for 30 min, followed by rehydration and washing in PBS. Sections were blocked with 1% BSA and 1% normal goat serum in PBS for 30 min. These sections were then incubated in respective primary antibody overnight at 4°C after a wash in PBS. Primary antibodies against TGFß2 and TGFß2R type II raised in rabbit were used at 1:50 and 1:30 dilution, respectively. This was followed by two washes with 1x PBS for 10 min each. Sections were then incubated for 2 h with goat biotin-conjugated secondary antibody diluted 1:200 in PBS. After washing for 10 min in PBS, the sections were incubated in substrate diaminobenzidine for 3–5 min until the color developed. Counterstaining was done by incubating the sections for 30–60 sec in Delafield hematoxylin (1:5 dilution) stain. After brief washes in distilled water, the slides were dehydrated in methanol, cleared for 2 h in xylene, and mounted in DPX. Negative control had rabbit sera and plain PBS in place of the primary antibodies; the rest of the procedure was as followed for the sections treated with primary antibodies.
Intensity of staining was graded as absent (-), weak (+), moderate (++), or strong (+++) under low (10x) and high (40x) magnification in a Zeiss Ultraphot microscope. Photomicrographs were taken using Kodak Gold Max 100 ASA film (Eastman Kodak Company, Rochester, NY) at 120x magnification.
Intensity of immunohistochemical staining for LIF, TGFß2, and TGFß2R in both endometrial glands and stroma for each animal in the control and treated groups was evaluated by two independent observers using the semiquantitative HSCORE method:
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2 test and Mann-Whitney rank sum test. The data were considered to be significant when P 
0.05. Reverse Transcription-Polymerase Chain Reaction
Preparation of total RNA Total RNA was extracted from endometrial biopsies using RNeasy Minikit according to the manufacturer's protocol. The RNA samples were incubated with RNase-free DNase I at 37°C for 30 min. Samples were repurified through RNeasy minikit columns.
RT-PCR for actin, LIF, TGFß2, TGFß2R, and EGFR Nucleotide sequences of the forward and reverse primers used in RT-PCR experiments for detection of actin (a housekeeping gene), LIF, TGFß2, TGFß2R, and EGFR are given in Table 1.
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Total RNA (150 ng) was amplified in a 25-µl reaction containing 0.4 µM primers, 200 µM dNTPs (deoxynucleotide mix), 1x buffer, and 1 U of Titan enzyme mix using the Single-Step Titan RT-PCR system in a thermal cycler (50°C for 30 min, 94°C for 2 min for cDNA synthesis, and 94°C for 30 sec, 50–58°C for 30 sec, and 68°C for 2 min) for 30–35 cycles. Each experiment was repeated three times to ensure reproducibility. The PCR products were analyzed by ethidium bromide-stained, 1% agarose gel electrophoresis. Comparative quantitation of RT-PCR products was performed by densitometric analysis of the photographed gels using Gel-Pro analyzer software (Gel-Pro, Image Database for Windows, Silver Spring, MD).
RESULTS
Hormone Levels
Peripheral levels of estradiol and progesterone before and after treatment with vehicle and onapristone in bonnet monkeys are shown in Figure 1. No significant change was observed in the levels of estradiol and progesterone after treatment with vehicle and 2.5 or 5.0 mg of onapristone, thereby indicating that folliculogenesis, ovulation, and gonadal profiles remain unaltered even after such treatment.
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Leukemia Inhibitory Factor
The endometrial RNA samples subjected to RT-PCR using LIF-specific primers revealed a band of the expected size (153 base pairs [bp]), as shown in Figure 2. To rule out the possibility of using an unequal amount of target RNA from each sample, the same amount (150 ng) of total RNA was subjected to RT-PCR for the expression of actin, a housekeeping gene expected to demonstrate equivalent expression in the endometrium during different phases of the menstrual cycle. Densitometric analysis was carried out to compare the amounts of 280-bp actin product in each sample using Gel-Pro analyzer software. No change was found in the intensities of actin product in the control endometrial samples collected during the late proliferative and midsecretory phases and in samples from the treated animals collected during the midsecretory phase (Fig. 2, bottom). However, significant down-regulation of LIF expression was observed, as revealed by a significant decline in the intensities of LIF product in the endometrial samples from bonnet monkeys treated with 2.5 (Fig. 2, top, lanes 4 and 5) and 5.0 mg (lanes 6 and 7) of onapristone as compared to samples from the control animals in the midsecretory phase (lanes 2 and 3).
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Immunohistochemical localization of LIF in the proliferative- and secretory-phase endometria from control animals and in the secretory-phase endometria from treated animals is shown in the Figure 3, A–E. Immunoreactive LIF protein was localized in the cytoplasm of endometrial epithelium and stromal cells as well as in the glandular lumen. Immunostaining for LIF protein was weak in the stroma as well as in the glands of the endometrium during the proliferative phase (Fig. 3A). However, during the secretory phase (8 days following the estradiol peak), an intense localization was observed for LIF in the cytoplasm of endometrial glandular epithelial cells, with moderate localization in the stromal cells (Fig. 3B).
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The low-dose onapristone regimen (2.5 and 5.0 mg) resulted in a significant (P < 0.05), dose-dependent decrease (Fig. 4) in the localization of LIF protein in glandular epithelial cells of the endometrium (Fig. 3, C and D).
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Transforming Growth Factor ß2
The RT-PCR studies used to analyze TGFß2 expression in endometrial RNA samples revealed a 408-bp product (Fig. 5). The expression of TGFß2, as determined by densitometric analysis of RT-PCR products, was fourfold higher during the peri-implantation period (Fig. 5, lane 2) as compared to the proliferative phase in normally cycling bonnet monkeys (Fig. 5, lane 1). No change in the expression of TGFß2 was observed in the endometria from 2.5-mg-treated animals as compared to that in control animals (Fig. 5, lanes 3 and 4). Expression of TGFß2 was 1.2-fold less in endometria from 5-mg-treated bonnet monkeys (Fig. 5, lanes 5 and 6).
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Immunohistochemical localization of TGFß2 in proliferative- and secretory-phase endometria of control animals and in secretory-phase endometria of treated animals is shown in Figure 3, F–J. Immunoreactive TGFß2 protein was localized in the cytoplasm of glandular epithelium and lumen. The TGFß2 protein was also localized in the luminal epithelium of the endometrium (not shown in the Fig. 3). Immunostaining for TGFß2 protein was weak in the endometrium during the proliferative phase (Fig. 3F). However, during the secretory phase, an intense localization was observed for TGFß2 in the cytoplasm of the glandular epithelium (Fig. 3G). Furthermore, a significant (P < 0.05), dose-dependent decrease (Fig. 4) in the localization of TGFß2 protein was found in the glandular epithelia of endometria from 2.5- and 5-mg-treated animals (Fig. 3, H and I).
Transforming Growth Factor ß2 Receptor
The RT-PCR studies of TGFß2R expression in endometrial RNA samples showed a 700-bp product. In addition, TGFß2R was up-regulated during the peri-implantation period (Fig. 6, lane 2) as compared to the proliferative phase in normally cycling bonnet monkeys (Fig. 6, lane 1). Expression of TGFß2R was not affected in the endometrial samples from 2.5-mg-treated animals (Fig. 6, lanes 3 and 4), whereas the expression of TGFß2R was 1.5-fold less in endometria from 5-mg-treated bonnet monkeys as compared to that in control animals (Fig. 6, lanes 5 and 6).
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Immunohistochemical localization of TGFß2R in proliferative- and secretory-phase endometria of control animals and in secretory-phase endometria of treated animals is shown in Figure 3, K–O. Immunoreactive TGFß2R type II was localized in the cytoplasm of glandular epithelial cells. Immunostaining for TGFß2R was stronger in the endometria during the midsecretory phase as compared to that during the proliferative phase (Fig. 3, K and L). A significant (P < 0.05), dose-dependent decrease (Fig. 4) was found in the localization of TGFß2R type II protein in the glandular epithelia of endometria from 2.5- and 5-mg-treated animals (Fig. 3, M and N).
Epidermal Growth Factor Receptor
The RT-PCR studies of EGFR expression by endometrial RNA samples revealed a 269-bp product. In the endometrial samples, EGFR revealed no difference in its expression between the proliferative and midsecretory phases in control animals or between control and treated animals during the midsecretory phase (Fig. 7).
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DISCUSSION
Various endometrial events, such as growth, differentiation, shedding, and receptivity to the blastocyst, are regulated by a precisely controlled, changing pattern of steroids during the menstrual cycle [5]. Progesterone is an absolute requirement for implantation to succeed in primates. This has led to the development of antiprogestins as a fertility-regulating strategy.
Our studies have shown that onapristone, an antiprogestin, at selected dosages retards endometrial development and renders it nonreceptive without creating hormonal imbalance [12]. Antiprogestin-induced endometrial nonreceptivity may arise either because of insufficient levels of circulatory progesterone or because of nonresponsiveness of the endometrium to progesterone as a result of a potential receptor defect or some block in the cascade of progesterone-driven events mediated via cytokines, growth factors, and their receptors.
In the present study, retarded endometrial development was observed in the animals treated with both dosages of onapristone (2.5 and 5 mg). These dosages were efficacious in preventing pregnancy without disturbing endocrine profiles [12]. The possibility of insufficient circulating progesterone as a cause of endometrial nonreceptivity was ruled out, because the treatment had no effect on ovulation or corpus luteum function, as also was shown during our previous studies [12]. Furthermore, no aberrations were detected in PR expression in the endometria from treated animals [17]. This ruled out the possibility of endometrial nonreceptivity occurring because of defects in the PR, which justified looking into the expression of cytokines, growth factors, and their receptors during the peri-implantation period in treated animals.
Several lines of evidence suggest that LIF may be an active participant in the process of endometrial receptivity. Studies demonstrating increased expression of LIF during the midsecretory phase of the menstrual cycle in women [18], implantation failure in LIF knock-out mice [24], and reduced LIF concentrations in uterine flushings from women with unexplained infertility [25, 26] clearly indicate its potential relevance in implantation. However, these studies have not examined the expression of its potential regulators (i.e., steroids or other cytokines/growth factors).
Our immunohistochemical studies demonstrated a significant increase in the expression of LIF during the peri-implantation period in the control group. This confirmed the findings of previous reports that indicated increased expression of LIF during the midsecretory phase in the primate endometrium [6, 18], suggesting that LIF is dependent on progesterone for its expression.
In contrast to Ghosh et al. [22], who did not find any change in the levels of endometrial LIF following mifepristone (RU 486) treatment, we found a significant decline in LIF expression during the peri-implantation period in nonreceptive endometrium. This discrepancy can be attributed to Ghosh et al. having analyzed the LIF expression in endometrial biopsy specimens from RU 486-treated, mated animals, in which embryonic factors may play a role in stimulating endometrial LIF expression. A single dose of RU 486 (2 mg/kg body weight) administered during the early luteal phase of mated rhesus monkeys [22] also likely does not lead to similar changes in the endometrial cytokine profile as those induced by the onapristone treatment (2.5 or 5 mg) on every third day from Day 1 of the nonmated cycle in bonnet monkeys, as seen in the present study. Disagreement in the results of these two studies may also have resulted from different modes of action by RU 486 and onapristone. However, the results of our study support a previous observation that indicated reduced immunostaining for LIF in endometrial biopsy specimens from women administered a single dose of onapristone or RU 486 during the early luteal phase [21].
A significant down-regulation of LIF in antiprogestin-treated bonnet monkeys with no luteal insufficiency suggested that the expression of LIF is not directly dependent on progesterone. This confirmed the findings of previous investigations that demonstrated an inability of progesterone to directly stimulate LIF production by endometrial cultures in vitro [20, 25]. It has been reported that the promoter of the gene for LIF lacks progesterone-responsive elements [27]. Therefore, LIF production in vivo does not seem to be under the direct control of progesterone. Expression of LIF during the peri-implantation period is probably regulated by the effect of progesterone on other endometrial factors.
Arici et al. [20] have shown TGFß2 to be one of the potent inducers of LIF expression. Furthermore, TGFß polypeptides exert a strong influence on cellular proliferation, differentiation, angiogenesis, and immunomodulation; therefore, these have been speculated to be potential modulators of many endometrial functions [19].
A significant increase in TGFß2 staining was observed in the glandular epithelium of endometrium from the proliferative to the secretory phase of the menstrual cycle in regularly cycling bonnet monkeys during the present study. Furthermore, endometrial luminal and glandular epithelial cell types were found to be the major cell types showing expression of immunoreactive TGFß2 protein, whereas stromal cells demonstrated lesser expression. These results confirm the findings of a previous investigation in humans [19].
Interestingly, down-regulation of TGFß2 mRNA during the peri-implantation period was seen only in those animals treated with 5 mg of onapristone. This initially suggested that the expression of TGFß2 may not be a determinant of the endometrial receptivity, because TGFß2 mRNA levels remained unaffected in 2.5-mg-treated, infertile animals. However, a significant decline was seen in the levels of immunoreactive TGFß2 in the animals treated with 2.5 as well as with 5 mg of onapristone. To explain this discordant expression of TGFß2 mRNA and protein in the endometria from 2.5-mg-treated animals, it may be hypothesized that onapristone, at a lower dose, does not directly affect the transcription of TGFß2. The factors regulating TGFß2 protein synthesis also likely are progesterone dependent. Even slight perturbations in the progesterone-driven cascade may influence the activity of these factors, which, in turn, may affect TGFß2 protein synthesis. The effects on TGFß2 expression (i.e., mRNA) at the higher dosage (5 mg) of onapristone could be a result of more pronounced structural retardation induced in the endometrium.
Antibodies used for the immunohistochemical localization of TGFß2 protein in the present study were directed toward an epitope of the precursor or latent form of TGFß2. Conversion of the biologically inactive or latent form of TGFß2 into active TGFß2 requires plasmin, which is synthesized from plasminogen in the presence of plasminogen activator [28, 29]. Progesterone regulates the expression of plasminogen activator [30]. Therefore, in the endometrium rendered nonreceptive using antiprogestin treatment, plasminogen-activator synthesis likely is deregulated, which, in turn, interferes with the events required for activation of the TGFß2 protein.
The biological activities of TGFß2 are mediated through specific cell-surface receptors. The activation of the signaling cascade by TGFß2 involves interaction of TGFß2 with two transmembrane receptors, called receptors I and II. Endometrial glandular epithelial and stromal cells have been shown to express TGFß2R [19]. The expression of TGFß2 as well as TGFß2R in endometrium indicates that TGFß2 exerts local influence.
The pattern of expression for TGFß2R was found to be similar to that seen for TGFß2 in the present study. In regularly cycling, fertile bonnet monkeys, TGFß2R showed enhanced expression during the midsecretory phase. Furthermore, it was down-regulated in the endometria from animals treated with 5 mg of onapristone but remained unaltered in animals treated with 2.5 mg of onapristone. Decrease in TGFß2R expression is, again, an indicator of aberrant biological activity of TGFß2 during the peri-implantation period in infertile animals.
We also found that declines in the levels of TGFß2, TGFß2R, and LIF were not caused by any nonspecific damage to general transcription or translation machinery in the nonreceptive endometrium. No change was found in the expression of actin, a housekeeping gene in the endometrial samples from both treated and control animals. To further rule out the possibility of antiprogestin-induced, nonspecific alterations in endometrial function, expression of endometrial EGFR was analyzed in the treated and control animals. In estrogen- and progesterone-dominated phases of the menstrual cycle in primates, EGFR has been reported to demonstrate equivalent expression [5]. In the present study, the expression levels of EGFR were equivalent in the endometrial samples from the control and treated groups, thereby suggesting that the endometrial nonreceptivity induced in bonnet monkeys results from dysfunction of specific factors.
Although the present study does not provide direct evidence of the link between the expressions of LIF and TGFß2, it supports the inferences drawn from in vitro culture studies that indicate TGFß2 is a potent inducer of LIF expression. Expression of these factors during the peri-implantation period likely is of crucial significance in determining the endometrial response to an embryo during the implantation window. This study also suggests that even with normal levels of circulating progesterone, dramatic changes may occur in the endometrium that may contribute to endometrial nonreceptivity.
ACKNOWLEDGMENTS
The secretarial assistance of Ms. Shobha Waradkar and Mr. Prasanna Chavan is greatly appreciated. The authors also thank Mr. Hemant Karekar for the artwork.
FOOTNOTES
First decision: 16 September 2000.
1 Funded by research grants from the Indian Council of Medical Research and the Indo-U.S. Programme on Contraceptive and Reproductive Health Research, Department of Biotechnology, Government of India. 
2 Correspondence: Chander Puri, Deputy Director, Institute for Research in Reproduction, Indian Council of Medical Research, Jehangir Merwanji Street, Parel, Mumbai 400 012, India. FAX: 91 22 4964853, 4139412; vichin{at}bom4.vsnl.net.in
Accepted: February 7, 2001.
Received: July 27, 2000.
REFERENCES
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