| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Female Reproductive Tract |
Department of Veterinary Biosciences,4
Molecular and Integrative Physiology,5 University of Illinois at Urbana-Champaign, Urbana, Illinois 61802
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
estradiol receptor, female reproductive tract, implantation, uterus
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Estrogen (E) profoundly influences uterine functions at various phases of the reproductive cycle and pregnancy. In the rat, the preovulatory ovarian E is important for uterine cellular proliferation and differentiation during early stages (Days 1 and 2 postfertilization) of pregnancy [7, 8]. This transformation of the uterus in response to E is important for subsequent uterine preparation for embryonic implantation and successful establishment of pregnancy [7, 8]. After fertilization, the level of E declines and remains low throughout gestation, except a transitory rise in E level that occurs on Day 4 of pregnancy [9]. Although E is essential to create the receptive state of the uterus that allows implantation on Day 5, the molecular basis of this hormonal effect remains unclear.
The cellular actions of E are mediated through estrogen receptors (ERs), which function as ligand-inducible transcription factors [10–12]. The uterotropic hormonal responses to E are believed to be mediated through the expression of specific E-regulated genes in the uterus [13– 23]. Studies in rodents employing immature and ovariectomized model systems have demonstrated that, in the uterus, E modulates the expression of several genes that are likely to be involved in the regulation of cell growth and division. These include the genes encoding protooncogenes, such as c-fos and c-myc; growth factors, such as epidermal growth factor (EGF); and insulin-like growth factor-I (IGF-I), and their receptors [13–23]. However, the relationship of these gene activation events to the complex E-regulated physiological processes, such as uterine receptivity and embryonic implantation, has not been clearly established.
To investigate the molecular basis of the E regulation of implantation, we sought to identify the genes the expression of which, in the preimplantation endometrium, is induced by E. For this purpose, we used a delayed implantation model system in which embryo attachment to endometrium is dependent on E administration [24, 25]. Using a polymerase chain reaction-based subtractive hybridization method, we isolated a number of cDNAs representing mRNAs the expression of which is either turned on or turned off in response to an implantation-inducing dose of E. Here we report the identification of one of these cDNAs as that encoding secretory leukocyte protease inhibitor (SLPI), an epithelial cell-derived inhibitor of serine proteases including elastase, chymotrypsin, trypsin, and cathepsin G [26, 27]. In this study, we analyzed the steroid hormone regulation and cell-type-specific expression of SLPI in pregnant rat uterus during implantation.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Progesterone and 17-ß estradiol were purchased from Sigma Chemical Co., (St. Louis, MO). ICI 182,780 was purchased from Tocris Cookson Inc. (Ellisville, MO).
Animals
All experiments involving animals were approved by the Animal Care Committee at the University of Illinois at Urbana-Champaign, and the studies were conducted in accordance with the National Institutes of Health standards for the use and care of animals. Virgin female rats (Sprague-Dawley, from Charles River, Wilmington, MA; 60–75 days of age), in proestrus, were mated with adult males. The different stages of the cycle in the nonpregnant rats were ascertained by examining vaginal smears. The presence of a vaginal plug after mating was designated as Day 1 of pregnancy. The animals were killed at various stages of gestation (n = 1 at each stage) and the uteri collected. In some experiments, animals were ovariectomized and 2 wk later were injected subcutaneously with either E (2 µg/kg body weight), progesterone (P) (40 mg/kg body weight), or a combination of both hormones or vehicle (sesame oil) (n = 2 at each stage) as described in the Results section. The rats were killed 16 h after final injection.
Gene Expression Screen
Rats were ovariectomized on Day 4 of pregnancy and injected daily with P (10 mg) during Days 5–7 (n = 2). On Day 8, the delayed animals were divided into two groups. One group was treated with P (10 mg) (n = 2), while the other group was treated with P (10 mg) plus an implantation-initiating dose of E (0.25 µg) (n = 2). The animals were killed at 24 h after the E injection. We isolated RNAs from uteri of both groups of animals and synthesized corresponding cDNAs. Using these two cDNA pools, we carried out differential gene screening.
Total RNAs were extracted from delayed rats before and after E treatment using a RNAgents isolation system (Promega, Madison, WI). RNA samples were subjected to polymerase chain reaction (PCR)-based subtractive hybridization reactions using PCR-Select cDNA subtraction kit as per manufacturer's protocol (Clontech, Palo Alto, CA). At the completion of the procedure, populations enriched in genes, which were either upregulated or downregulated during E-induced delayed implantation, were obtained. The enriched cDNA fragments were then subcloned into pTAdV vector (Clontech) and subjected to nucleotide sequence analysis. To confirm that the isolated clones were differentially regulated in the uteri of delayed rats before and after E treatment, we performed Northern blot analysis using 20 distinct cDNAs. We observed that 4 out of 20 cDNAs were indeed differentially expressed in the uteri of delayed rats following E injection. The differential gene expression methodology, therefore, is about 20% efficient.
Northern Blot Analysis
For Northern analysis, 20 µg of total RNA (isolated from individual rats) was separated by formaldehyde agarose gel electrophoresis and transferred to Duralon membrane (Stratagene, La Jolla, CA). Blots were prehybridized in 50 mM NaPO4 (pH 6.5), 5x SSC, 5x Denhardt, 50% formamide, 0.1% SDS, and 100 µg/ml salmon sperm DNA for 4 h at 42°C. Hybridization was carried out overnight in the same buffer containing 106 cpm/ml of a 32P-labeled SLPI cDNA fragment. The filters were washed twice for 15 min in 1x SSC/0.1% SDS at room temperature, then twice for 20 min in 0.2x SSC/0.1% SDS at 55°C and the filters were exposed to x-ray films for 24–72 h. The intensities of signals on the autoradiogram were estimated by densitometric scanning. To correct for RNA loading, the obtained signals were normalized with respect to the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) signal in the same blot. For this, the filters were stripped of the radioactive probe by washing for 10 min in 0.5% SDS at 95°C. The blots were then reprobed with a 32P-labeled GAPDH probe as described above.
In Situ Hybridization
Uterine tissue from pregnant animals was collected and frozen. Tissues were fixed in 4% paraformaldehyde at 4°C. Cryostat sections were cut at 8 µm and attached to 3-aminopropyl triethyl silane coated slides (Sigma). In situ hybridization was performed with 280-base pair (bp) digoxygenin (DIG)-labeled sense or antisense RNA probes complimentary to rat SLPI gene. DIG-labeled RNA probes were synthesized from SLPI cDNA using T3 or T7 RNA polymerase and DIG-labeled nucleotides according to manufacturer's specifications (Roche Diagnostics Corp, Indianapolis, IN). Prehybridization was carried out in a damp chamber at 37°C for 60 min in hybridization buffer (50% formamide, 5x SSC, 2% blocking reagent, 0.02% SDS, 0.1% N-laurylsarcosine). Hybridization was carried out at 42°C overnight in a damp, humidified chamber. To develop the substrate, sections were sequentially washed in 2x SSC, 1x SSC, and 0.1x SSC for 15 min in each buffer at 37°C. Sections were then incubated with anti-DIG alkaline phosphatase-conjugated antibody. Excess antibody was washed away and the color substrate (Nitroblue tetrazolium salt and 5-bromo-4-chloro-3-indoylphosphate) was added. Slides were allowed to develop in the dark and the color was visualized under light microscopy until maximum levels of staining were achieved (
3 h). The reaction was stopped with water and the slides counterstained in Nuclear Fast Red for 5 min. The slides were washed in water, dehydrated, and coverslipped. Control incubations used a DIG-labeled RNA sense strand and were performed under identical conditions. Six sections from the uterus of an animal were processed at each time point.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
To isolate the uterine genes that are regulated by E during delayed implantation, we employed the differential gene expression screen method as described in the Materials and Methods. Our studies led to the isolation of several cDNA clones representing genes the expression of which altered in the uteri of delayed rats within 24 h of E treatment (unpublished observation). Nucleotide sequence analysis revealed that one of the isolated cDNAs is a homologue of mouse secretory leukocyte protease inhibitor (SLPI) gene [28]. Using this cDNA fragment as a probe, we then isolated a full-length cDNA (GenBank accession number AF151982) from a rat uterus cDNA library that we had previously synthesized in our laboratory [29]. The rat SLPI cDNA codes for a protein of 130 amino acids and exhibits 80% identity to the mouse SLPI.
We then performed Northern blotting to confirm that rat SLPI mRNA is induced during delayed implantation. RNA samples were obtained from uteri of delayed rats on Day 8 of pregnancy before (lane 1) and after (lane 2) administration of E and subjected to Northern blotting as described in the Materials and Methods (Fig. 1). The SLPI cDNA fragment was labeled with 32P and used to probe the blot. A strong signal corresponding to 800-bp SLPI mRNA was observed in lane 2, while no detectable signal was seen in lane 1, indicating that the expression of rat SLPI gene is indeed upregulated in delayed uteri of rats during E-induced implantation.
|
Profile of SLPI mRNA Expression in Rat Uterus During Estrous Cycle and Pregnancy
To examine the pattern of SLPI gene expression in rat uterus during the estrous cycle, we performed Northern blot analysis of total RNA isolated from uteri of nonpregnant animals in proestrous, estrous, and diestrous stages. As shown in Figure 2A, when the blot was hybridized with a 32P-labeled SLPI cDNA probe, a weak signal emerged in the RNA sample at the proestrous stage of the cycle (lane 1). The signal corresponding to SLPI mRNA increased markedly at the estrous stage (lane 2), but declined sharply at the diestrous stage of the cycle (lane 3).
|
We then investigated the pattern of SLPI gene expression in the rat uterus during Days 1–6 of gestation by employing Northern blot analysis. As shown in Figure 2B, a high level of SLPI mRNA was detected on Day 1 of pregnancy. The level of this mRNA then declined on Day 2 of gestation and remained low on Day 3. It rose again on Day 4, immediately preceding implantation, and then declined by Day 6 of pregnancy.
SLPI mRNA Is Localized in the Luminal Epithelium
The site of expression of SLPI mRNA in the pregnant rat uterus was investigated by employing in situ hybridization. Uterine sections from Days 1, 3, 4, 5, and 6 pregnant animals were hybridized with a 280-bp digoxygenin-labeled antisense or sense RNA probe containing sequences from SLPI cDNA. A strong signal corresponding to SLPI mRNA was observed in the uterine sections of rat at Day 1 of pregnancy (Fig. 3, panel D1). The signal was localized exclusively in the luminal epithelial cells. Consistent with the Northern blot data, no SLPI mRNA was detected on Day 3 of gestation (panel D3). However, on Days 4 and 5 of pregnancy, there was a rise in SLPI expression (panels D4 and D5, respectively), which declined on Day 6 of pregnancy (panel D6). Control uterine sections hybridized with the sense RNA probe did not exhibit any significant signal (panel D1/S), indicating the specificity of the hybridization reaction.
|
Analysis of Steroid Regulation of SLPI mRNA Expression in the Uterus
Because expression of SLPI is stimulated during E-induced delayed implantation in the presence of P, we further analyzed the steroid hormone regulation of SLPI expression in the uterus. For this purpose, we administered steroid hormones to ovariectomized rats. A week after ovariectomy, the animals were treated with E (2 µg/kg body weight), P (40 mg/kg body weight), or a combination of both. Uteri were collected from animals 16 h after the last injection and mRNAs were isolated from these tissues for Northern blot analysis. As shown in Figure 4, SLPI mRNA was undetectable in the uterus of an ovariectomized rat (lane ovex). P treatment for 4 days (lane 4P) did not induce SLPI mRNA. In contrast, SLPI transcript was markedly induced after treatment with E for 4 consecutive days (lane 4E). Simultaneous administration of E and P (lane E+3[P+E]) elicited the same response as E alone, indicating that P does not significantly affect SLPI expression.
|
An Antagonist of ER Blocks SLPI mRNA Expression in the Uterus
We next investigated whether the E regulation of SLPI mRNA expression is mediated by the estrogen receptors. We therefore employed an antiestrogen, ICI 182,780, which is known to severely impair the transcriptional activity of ERs [30]. Animals were ovariectomized and injected with vehicle or a single dose of E (2 µg/kg body weight) or a combination of E (2 µg/kg body weight) and ICI 182,780 (1 mg/kg body weight). As depicted in Figure 5A, SLPI expression was not observed in vehicle-treated ovariectomized animals (lane 1). Consistent with our earlier results, administration of E induced SLPI in the uterus (lane 2). Treatment of animals with E in combination with ICI 182,780 (lane 3), however, abolished SLPI mRNA expression but did not affect the expression of GAPDH mRNA.
|
We also investigated the E regulation of SLPI mRNA expression in the uterus during early pregnancy. Animals were injected on Day 3 of pregnancy either with a vehicle or a single dose of ICI 182,780 (1 mg/kg body weight). Uteri were isolated on Day 4 and analyzed for SLPI expression by Northern blot analysis (Fig. 5B). As shown in this figure, treatment with the antiestrogen caused a drastic decline in the level of SLPI mRNA without affecting the expression of GAPDH mRNA. Collectively, these results strongly support our conclusion that E acting via ERs controls SLPI gene expression in the pregnant uterus during the implantation window.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
in these cells (Fig. 4 [31]). A direct regulatory role for ER in SLPI expression, however, remains to be established. To explore this possibility, a detailed analysis of the 5'-flanking regulatory region of SLPI gene will be required. Previous studies indicated that SLPI is associated with mucosal surfaces of lung and cervix and functions as a potent serine protease inhibitor [32]. It is capable of inhibiting the activity of a wide array of serine proteases, including neutrophil elastase, cathepsin G, mast cell chymase, and chymotrypsin. The neutrophil elastase cleaves elastin and also degrades collagen, fibronectin, laminin, and proteoglycans, leading to a reduction in matrix deposition [26, 33]. By neutralizing elastase, SLPI regulates inflammation and promotes wound healing. Recent evidence indicates that SLPI exhibits antibacterial properties and antagonizes lipopolysaccharide-induced inflammatory events induced by monocytes and macrophages [34, 35]. SLPI also inhibits the production and activity of prostaglandin H synthase-2 and matrix metalloproteinases (MMPs) in monocytes and macrophages [36]. Collectively, these reports are consistent with a role of SLPI in the regulation of proteolytic cascades and inflammatory events. These predictions were confirmed by the recent development of the SLPI null mouse. This model shows that the absence of SLPI indeed leads to an enhanced elastase activity and increased activation of MMPs, which in turn leads to reduced matrix deposition and impaired cutaneous wound healing [37]. The SLPI null mice also exhibit an increased and prolonged inflammatory response [37]. These results support a role of SLPI as an endogenous factor protecting against inflammatory insults, infection, and tissue damage. Although the SLPI null mice are reported to be fertile, no detailed analysis of their reproductive function has been performed [37].
The functional role of SLPI in rat uterus is presently unclear. It is possible that SLPI regulates uterine inflammatory response, which occurs in response to mating and sperm deposition during estrus or on Day 1 of pregnancy. It is also possible that SLPI functions in a similar capacity at the time of implantation. The embryo implantation in rodents begins in a way similar to an inflammatory process. Initially, there is an increased permeability of the subepithelial capillaries at the endometrial sites where the blastocyst is located [1, 38]. The attachment of the trophoblast to the uterine epithelium is followed by the degeneration and sloughing of uterine epithelial cells. The differentiating trophoblast then begins to penetrate the decidualizing stromal compartment [38]. It is generally believed that the trophoblast produces several proteases, which function by degrading the maternal extracellular matrix [39–43]. The endometrium, on the other hand, plays an active role in limiting this invasion. It does so by several mechanisms, including synthesis of protease inhibitors, which counteract the trophoblast-derived proteases. Because SLPI expression during early pregnancy occurs on Days 4 and 5 of gestation, it is conceivable that the function of SLPI in rat endometrium is to control protease activity during early stages of implantation. SLPI, therefore, could play a role as a regulator of improper or excessive proteolysis and inflammatory responses during implantation.
SLPI expression in pregnant uteri of species other than rat has been reported previously, although its hormonal regulation was not described. Studies by Simmen and coworkers revealed high levels of SLPI expression in pig uterus during mid- to late pregnancy [44, 45]. These investigators also found SLPI mRNA in the pregnant endometrium of horse and cow, which, like pig, exhibit epitheliochorial placentation. Based on these studies, they proposed that SLPI acts to maintain the uterine-placental border in species with epitheliochorial placentation. It is, however, evident from our results that SLPI expression is not restricted to the animals exhibiting epitheliochorial placentation. As described in this paper, abundant uterine SLPI expression is seen in the rat, which exhibits hemochorial rather than epitheliochorial placentation. The SLPI expression in the rat uterus, however, occurs in a stage-specific manner during the reproductive cycle and pregnancy. We found that SLPI mRNA is virtually undetectable in rat uterus during midpregnancy (unpublished observation).
SLPI is also expressed in the rhesus monkey and human endometrium during the menstrual cycle, further confirming that its expression is not limited to the uteri of species with epitheliochorial placentation [46, 47]. In the human endometrium, SLPI secretion increases during the progesterone-dominated mid- to late secretory phase, overlapping the putative window of implantation [47]. A significant amount of SLPI was also detected in first-trimester decidua. It has been speculated that SLPI expression in the human endometrium provides protection against infection of the uterus at the time of implantation and during early pregnancy. Recent in vitro studies have indicated that SLPI possesses growth regulatory roles and positively modulates the expression of cyclin D1 and negatively modulates the expression of TGFß1 in Ishikawa cells [48]. Whether SLPI regulates cell growth in nonpregnant or pregnant uterine epithelium in vivo, however, remains to be determined.
In summary, SLPI has emerged as a novel downstream target of E action in the cycling and pregnant rat endometrium. While the regulation of this gene may differ among species, it is important to note that SLPI is one of the few molecules that is expressed in the pregnant uterus of diverse species, ranging from rats, pigs, ruminants, and equines, and is also induced in the monkey and human endometrium during a critical phase in the menstrual cycle. It is, therefore, predicted that SLPI performs a critical function in the pregnant or cycling uterus that is conserved among species. Further in-depth analysis of SLPI null mice is likely to provide insights into the precise function of this molecule in the uterine tissue.
|
FOOTNOTES |
|---|
2 Correspondence: FAX: 217 244 1652; ibagchi{at}uiuc.edu
3 Current address: Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas
Received: 30 October 2003.
First decision: 18 November 2003.
Accepted: 16 March 2004.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  M.H. Hassan, N. Khatoon, D.T. Curiel, F.M. Hamada, H.M. Arafa, and A. Al-Hendy Toward gene therapy of uterine fibroids: targeting modified adenovirus to human leiomyoma cells Hum. Reprod., March 1, 2008; 23(3): 514 - 524. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Moral, R. Wang, I. H Russo, C. A Lamartiniere, J. Pereira, and J. Russo Effect of prenatal exposure to the endocrine disruptor bisphenol A on mammary gland morphology and gene expression signature J. Endocrinol., January 1, 2008; 196(1): 101 - 112. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. V. Fahey, T. M. Schaefer, and C. R. Wira Sex hormone modulation of human uterine epithelial cell immune responses Integr. Comp. Biol., December 1, 2006; 46(6): 1082 - 1087. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
