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Biology of Reproduction 64, 284-292 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Increased Expression of a Novel Heat Shock Protein Transcript in the Mouse Uterus During Decidualization and in Response to Progesterone1

Brent M. Bany2,a, and Gilbert A. Schultza

a Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada T2N 4N1

ABSTRACT

The objective of the present study was to identify and characterize transcripts whose levels are increased in the mouse uterus during decidualization. Using the method of suppression subtractive hybridization, we identified a novel transcript. This transcript contained a potential open reading frame that coded for a 196-amino-acid protein that shows homologies to the heat shock protein 20 family of genes. This transcript was expressed in several adult tissues and in the embryo. Its steady-state level was significantly greater in implantation segments of the uterus compared to nonimplantation segments. Furthermore, the steady-state levels of this novel transcript were significantly greater in uterine horns undergoing artificially induced decidualization compared to control contralateral horns. Using in situ hybridization methods, signals for the transcript were localized to the endometrial stromal cells that were undergoing decidualization. Finally, we found that progesterone caused a significant increase in the steady-state level of this novel transcript in the uterus when administered to ovariectomized mice. In the presence of estradiol-17ß, this effect was significantly reduced. In conclusion, we have identified a novel transcript of a potential heat shock protein whose level is significantly increased in the uterus during decidualization and in response to progesterone.

decidua, female reproductive tract, implantation, placenta, uterus

INTRODUCTION

In rodents, the decidual cell reaction (i.e., decidualization) begins in the endometrium just after the onset of blastocyst implantation [1–3]. This process involves the proliferation and differentiation of the fibroblast-like endometrial stromal cells into large, polyploid decidual cells that eventually form the maternal component of the placenta. Decidualization can also occur in pseudopregnant and nonpregnant uteri in response to an artificial deciduogenic stimulus if the uterus has been properly primed hormonally [4]. To distinguish it from the decidua that is formed during pregnancy, the resulting endometrial tissue of this artificially induced decidualization is called a deciduoma. Artificially induced decidualization has been extensively studied and used as a model of decidualization in mice and rats, because it provides a response in the absence of an embryo and results in formation of the deciduoma that appears to be morphologically similar to the decidua [4].

The molecular interactions that occur during the process of decidualization are complex and remain to be completely elucidated. Early decidualization is accompanied by metabolic changes, extensive tissue remodeling, and cellular differentiation [1, 5–7]. To further explore the molecular basis of decidualization, we used an artificially induced decidualization model in the mouse to identify novel transcripts whose steady-state levels are increased in the uterus during the decidual cell reaction. This paper reports the sequence of one such transcript and describes its expression in the uterus during decidualization and in response to progesterone. This transcript was predicted to encode a heat shock 20-like protein.

MATERIALS AND METHODS

Materials

Estradiol-17ß, progesterone, sesame oil, Evans blue, and nuclear fast red were purchased from Sigma (St. Louis, MO). Guanidine isothiocyanate, competent DH5{alpha} bacteria, Random Primer Labeling kit, EcoRV, T4 DNA polymerase, Platinum Taq polymerase, 0.24–9.5-kilobase (kb) RNA ladder, and dNTPs were purchased from Life Technologies (Burlington, ON, Canada). Hybond-N, Hybond-N+, {alpha}32P-deoxy-CTP, Nick columns, X-OMAT AR film, and BioMax MS film were purchased from Amersham Pharmacia Biotech (Baie d'Urfe, PQ, Canada). The ZAP cDNA synthesis and library construction kit, PCR-Script cloning kit, and pBluescript SK vector were purchased from Stratagene (La Jolla, CA). The PCR-Select cDNA Subtraction and Advantage cDNA PCR kits were purchased from Clontech (Palo Alto, CA). The RNA labeling mix, T7 RNA polymerase, T3 RNA polymerase, antidigoxigenin alkaline phosphatase-conjugated antibody, BCIP, and NBT were purchased from Roche Molecular Biochemicals (Laval, PQ, Canada). The polymerase chain reaction (PCR) primers were prepared by the UCDNA Synthesis Lab (University of Calgary, AB, Canada). The Oligotex mRNA kit was purchased from Qiagen (Mississauga, ON, Canada). All other chemicals were purchased from VWR Canada (Mississauga, ON, Canada).

Animals and Treatments

All procedures involving animals were carried out in accordance with the Guidelines of the Canadian Council on Animal Care and were approved by the University of Calgary Animal Care Committee. The CD1 mice (6–8 wk old, 22–25 g) were obtained from Charles River Breeding Laboratories (Lachine, PQ, Canada) and were housed under temperature- and light-controlled conditions (lights-on from 0700–1900 h) with free access to food and water.

To obtain uteri sensitized for a deciduogenic stimulus, mice were ovariectomized under methoxyflurane (2,2-dichloro-1,1-difluoroethyl-methyl-ether) anesthesia (Metofane; Janssen, Toronto, ON, Canada) and allowed at least 6 days to recover. Estradiol and/or progesterone in 0.1 ml of sesame oil were then administered s.c. at 0900 h over 8 days, essentially as described elsewhere [8]. Briefly, the animals received 100 ng of estradiol on Days 1 to 3, then 1 mg of progesterone plus 10 ng of estradiol on Days 6 to 8. On the morning of Day 8, when the uteri were optimally sensitized for a deciduogenic stimulus, 15 µl of sesame oil were injected into the lumen of one uterine horn (stimulated horn) between 1100 and 1300 h. The other uterine horn (nonstimulated horn) was not injected with oil and served as a control. From Day 9 onward, the mice were injected with 1 mg of progesterone. At 24, 48, or 72 h after the unilateral intraluminal oil injection, the mice were killed and the uterine horns dissected.

To obtain pregnant mice, females were placed with fertile males, and the day that a vaginal plug was observed was considered to be Day 1 of pregnancy. In one set of experiments, embryos were dissected from the uterus on Days 10, 14, 16, and 18 of pregnancy and newborn pups collected. Under a dissecting microscope, organs were dissected from the Day 14–18 embryos and newborn pups. In another set of experiments, mice were killed by cervical dislocation and their uteri dissected at 1000 h on Days 4 to 8 of pregnancy. On Days 5 to 8 of pregnancy, segments (containing both endometrium and myometrium) of the uterus containing implanting embryos (implantation segments) were dissected from those that did not (nonimplantation segments). To identify implantation segments of the uterus on Day 5 of pregnancy, 0.1 ml of Evans blue dye solution (2% [w/v] in 0.9% [w/v] sodium chloride) was injected i.v. (tail vein) 5 min before killing the mice.

In one set of experiments, ovariectomized mice were injected s.c. with vehicle (0.1 ml of sesame oil), 10 ng of estradiol, 1 mg of progesterone, or 10 ng of estradiol plus 1 mg of progesterone at 1100 h for 3 days. At 24 h after the last injection, the animals were killed and the uterine horns dissected.

RNA Isolation

Tissue samples were homogenized into a guanidine isothiocyanate solution, and total RNA was isolated using methods previously described [9]. Total RNA was quantified by absorbance at 260 nm, and its quality was assessed by ethidium bromide staining after electrophoresis through agarose gels. In the cases where poly(A)+ RNA was required, an Oligotex mRNA kit was employed, using the methods suggested by the manufacturer.

Suppression Subtractive Hybridization and Screening for Differentially Expressed Clones

Transcripts whose levels are potentially up-regulated in the uterus during artificially induced decidualization were identified using the method of suppression subtractive hybridization as previously described [10] with the use of PCR-Select cDNA Subtraction, PCR-Select differential screening, and Advantage cDNA PCR kits. This method allows the detection of differentially expressed genes without some of the technical limitations of traditional subtractive hybridization methods [10–12]. It is a suppression PCR-based subtraction hybridization method that selectively amplifies differentially expressed sequences and simultaneously suppresses amplification of those sequences that are not. Unlike conventional subtractive hybridization methods, it is simple, rapid, and results in the equalization of cDNAs that represent rare and abundantly expressed transcripts.

The sources of poly(A)+ RNA for cDNA synthesis was from nonstimulated and stimulated uterine horns 24 h after the unilateral injection of sesame oil into one horn of uteri sensitized for a deciduogenic stimulus. Forward (cDNA from nonstimulated horns used as a driver)-subtracted PCR products were cloned into a vector using a PCR-Script Cloning kit and the methods suggested by the manufacturer. The cloned PCR products were then used to transform DH5{alpha} bacterial cells using the methods suggested by the manufacturer. Southern blots containing samples of 30 cDNA clones were probed with cDNA from four different sources: 1) forward-nonsubtracted, 2) forward-subtracted, 3) reverse-nonsubtracted, or 4) reverse-subtracted cDNA pools. Preparation of the Southern blot and hybridizations were carried out as previously described [13]. A clone with strong hybridization signals using pools 1 and 2 and weak to absent signals using pools 3 and 4 as probes was considered to be a strong candidate for a transcript whose levels were increased in the uterus during artificially induced decidualization.

cDNA Library Construction and Screening

Poly(A)+ RNA was isolated from deciduomata at 24 h after the induction of decidualization. A cDNA library using a lambda ZAP cDNA Synthesis and Library Construction kit was used to build a library from this poly(A)+ RNA using the protocol suggested by the manufacturer. The phage library was screened using Hybond-N+ membranes using methods suggested by the manufacturer. Potential full-length clones were isolated and sequenced by the UCDNA Services Facility (University of Calgary). To obtain more sequence data from the 5'-end, we designed an internal antisense primer (5'-TCCACGTATCCATCCTTGGTC-3') and a forward primer within the lambda ZAP sequence near the 5'-end of the cloned cDNA insert. The PCR reactions were carried out using 1 µl of the cDNA library, 1 mM MgCl, 100 mM dNTPs, 1x buffer, 1 µM of each primer, and 0.5 U of platinum Taq polymerase in a 50-µl reaction. The PCR products were blunt-ended using T4 DNA polymerase and cloned into the EcoRV site of the pBluescript vector (Stratagene) using methods suggested by the manufacturer. The cloned PCR products were then sequenced by the UCDA Services Facility (University of Calgary).

Sequences obtained were analyzed using the basic local alignment search tool (BLAST) program of the National Center for Biotechnology Information web server, the FASTA3 program at the European Bioinformatics Institute web server, and several programs at the ExPAsy Molecular Biology Server (Canadian Bioinformatics Resource, Halifax).

Northern Blot Analysis

Samples of total RNA (10 µg) were subjected to denaturing agarose gel electrophoresis and then transferred to Hybond-N membranes by capillary transfer as previously described [13]. The RNA was crosslinked to the membranes by ultraviolet irradiation (0.12 J/cm2). Fragments of the mouse heat shock protein (HSP) 20-like mRNA (nucleotides 579–1360; Fig. 1) and mouse 18 S rRNA (nucleotides 1300–1504; kindly provided by Dr. Dylan Edwards, University of East Anglia, UK) cDNA (25 ng) were labeled using a Random Primer DNA Labeling kit in the presence of 32P-dCTP, then purified using Nick columns. Membranes were incubated in prehybridization buffer (1 M sodium phosphate [pH 7.2], 7% SDS, and 1% BSA) for 3 h at 65°C. Labeled probe (25 ng; specific activity ~1 Ci/mg), denatured by placement in boiling water for 5 min followed by snap-cooling on ice, was then added and hybridization carried out overnight at 65°C. After hybridization, the membranes were washed for 15 min twice in wash buffer 1 (0.1% SDS, 0.3 M sodium chloride, and 30 mM sodium citrate [pH 7.2]) and then twice in wash buffer 2 (0.1% SDS, 30 mM sodium chloride, and 3 mM sodium citrate [pH 7.2]) at 65°C. After autoradiography at -70°C with autoradiography film and intensifying screens (Eastman Kodak, Rochester, NY), probes were removed from the membranes as described previously [13].



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  		FIG. 1. The cDNA and predicted amino acid sequences of HSP20-like mRNA. A HSP20 family domain (Prosite accession no. PS01031; amino acids 94–170; arrows) and a nuclear localization signal (amino acids 13–19; boxed) were found in the protein. The polyadenylation signal at the 3'-end of the cDNA sequence (AATAAA) is underlined. Circled amino acids in the predicted protein denote four potential serine or tyrosine phosphorylation sites

The relative amounts of RNA loaded into each lane and transferred to the membrane were determined by probing the blots with 32P-labeled cDNA for mouse 18 S rRNA. The HSP20-like mRNA along with 18 S rRNA signals were quantified by densitometry. The relative levels of the signals for the mRNAs on the autoradiographs were calculated as the ratios of mRNA signal to 18 S rRNA signal. In some cases, an RNA ladder was used to determine the approximate size of HSP20-like mRNA.

In Situ Hybridization

Uterine segments were fixed by immersion in 4% paraformaldehyde for 30 h and embedded in paraffin. Sections (6 µm) were mounted onto aminopropyltriethoxysilane-coated glass slides. The template for the transcription of digoxigenin (DIG)-labeled riboprobes was a linearized vector containing a 781-base pair (bp) fragment (nucleotides 579–1360) of the HSP20-like cDNA. The sense and antisense RNA probes were prepared using DIG-labeling mix and RNA polymerase (T3 or T7) using the methods suggested by the manufacturer. The DIG-labeled RNA was treated with mild alkaline hydrolysis to fragment the RNA into an average size of 150 bases before in situ hybridization. The in situ hybridization was carried out exactly as previously described [14]. Briefly, after hybridization and posthybridization washes, the DIG-labeled RNA hybridized to the sections was detected using an anti-DIG alkaline phosphatase-conjugated antibody and BCIP/NBT substrate. Using this procedure, positive staining was indicated by a blue to black color. Sections were lightly counterstained in nuclear fast red (0.1%) for 2 min.

Statistical Analysis

The significance of the effects of day of pregnancy or time after stimulation and the site of sampling on the relative mRNA levels was determined by repeated-measures ANOVA, with tissue site as a repeated measure. The significance of the effects of estradiol-17ß and progesterone on HSP20-like mRNA levels was also determined by ANOVA. Where appropriate, paired t-tests or Duncan's multiple range tests were used to determine differences between means. Heterogeneity of variance was reduced by logarithmically transforming the data before statistical analysis. All statistical analyses were carried out using SAS Software (Statistical Analysis Systems Institute, Cary, NC).

RESULTS

Of the 30 subtracted cDNA clones screened, 10 were identified as being strong candidates representing up-regulated transcripts. At present, only one of these clones has been sequenced and further characterized. This 781-bp cDNA clone did not show any significant sequence identity to any known mouse genes. It did contain some sequence identity with many mouse-expressed sequence tags as determined by a BLAST search of the mouse EST database. Furthermore, it contained a polyadenylation signal and a poly-A stretch, suggesting that the 3'-end of the transcript was within the cloned sequence.

Because it appeared to represent a novel gene, we next sought to obtain its full-length cDNA sequence. A larger, 1412-bp clone was obtained by screening the mouse deciduomal cDNA library, and an additional 334 bp of the cDNA sequence at the 5'-end was obtained by PCR. The combined 1746 bp of cDNA sequence obtained (GenBank accession no. AF273453, 30 May 2000; Fig. 1) predicted an open reading frame that codes for a protein of 196 amino acids. A search of the Prosite database indicated that the putative protein contains stretches of amino acids similar to that of the HSP20 protein family (called the HSP20 or {alpha}-crystallin domain; signature PS01031). The protein also contains potential serine and tyrosine phosphorylation sites. During the preparation of this paper, a BLAST search revealed that a region of the sequence was identical to that of a very recent, 685-bp GenBank submission (accession no. AF250139). The cDNA and predicted protein sequence found in this study also showed a high degree of homology to a few unpublished human genes (accession no. NM014365, AF250138, AF191017, and AF133207). Due to these homologies, we have named the transcript HSP20-like mRNA. Alignment of the HSP20-like protein and four other mouse HSP20 proteins is shown in Figure 2. The protein sequence shares 18%–32% identity with the sequence of these other HSP20 proteins. In the alignment of the HSP20 domains within this sequence, identity was higher at 35%–56% (Fig. 2).



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  		FIG. 2. Alignment of the deduced amino acid sequence of HSP20-like protein (HSP20-like) and four other mouse HSP20 proteins. Amino acid sequences for {alpha}A-crystallin (alphaA), {alpha}B-crystallin (alphaB), HSP25, and cardiovascular HSP (cvHSP) were obtained from GenBank (accession no. J00376, M63170, X14687, and AF155909). The HSP20 domain is indicated by the double arrow. The table shows the percentage amino acid identity among these HSP20 proteins. Identity for the whole protein (total % identity) and alignment of the HSP20 domain (HSP20 signature % identity) are shown

To determine the distribution of HSP20-like mRNA, tissues were obtained from intact female mice, except for testis, which was collected from mature male mice. The size of HSP20-like mRNA on agarose gels was estimated to be 2.1 kb, and this mRNA was found in abundance in skeletal muscle, heart, uterus, liver, lung, and ovary (Fig. 3). Low, but detectable, signal was observed in the stomach and brain. The HSP20-like mRNA was not detectable in the small intestine, large intestine, kidney, spleen, and testis. The HSP20-like mRNA was, however, also detected in whole-mouse embryos from Days 10 and 18 of pregnancy (Fig. 3) and in the developing heart and liver (Fig. 4). Although HSP20-like mRNA was detected in the developing heart on each day examined, it was only detected in the liver near term (Day 18) and in the newborn (Fig. 4).



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  		FIG. 3. Distribution of HSP20-like transcripts in adult mouse tissues and whole embryos. A) Representative autoradiograph of a Northern blot probed with a HSP20-like cDNA probe. Positions of the RNA size markers are shown on the left by arrows. B) Autoradiograph of a subsequent hybridization using a 18 S rRNA probe to show relatively equal RNA loading and transfer



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  		FIG. 4. Distribution of HSP20-like transcripts in select tissues during development. Representative autoradiograph of a Northern blot probed with a HSP20-like cDNA probe. Tissues were from embryos at Days 14, 16, and 18 of pregnancy and from newborn pups (NB)

We also assessed if HSP20-like transcripts were up-regulated in the uterus during artificially induced decidualization and early pregnancy. As determined by Northern blot analysis, steady-state HSP20-like mRNA levels were significantly (P < 0.05) greater in stimulated compared to nonstimulated uterine horns at 24, 48, and 72 h during oil-induced decidualization (Fig. 5). The HSP20-like transcripts were also detected in the uterus on Days 4 (preimplantation) and 5–8 (during implantation) of pregnancy (Fig. 6A). Steady-state HSP20-like mRNA levels were significantly (P < 0.05) greater in implantation compared to nonimplantation segments of the uterus on Days 5 to 8 of pregnancy (Fig. 6B).



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  		FIG. 5. Northern blot analyses of steady-state HSP20-like mRNA levels in nonstimulated (N) and stimulated (S) mouse uterine horns at 24, 48, and 72 h after the initiation of oil-induced decidualization. A) Representative autoradiographs probed with a HSP20-like cDNA probe and subsequently with a 18 S rRNA probe. B) Mean (± SEM; n = 5) ratios of HSP20-like mRNA to 18 S rRNA signals, as determined by image analysis, for nonstimulated (solid bar) and stimulated (open bar) uterine horns at 24, 48, and 72 h after the initiation of oil-induced decidualization. *P < 0.05 and **P < 0.01 compared to nonstimulated uterine horn



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  		FIG. 6. Northern blot analyses of steady-state HSP20-like mRNA levels in the mouse uterus on Day 4 of pregnancy and nonimplantation (N) and implantation (I) uterine segments on Days 5 to 8 of pregnancy. A) Representative autoradiographs probed with a HSP20-like cDNA probe and subsequently with a 18 S rRNA probe. B) Mean (± SEM; n = 3–4) ratios of HSP20-like mRNA to 18 S rRNA signals, relative to that of Day 4 pregnant uteri, for nonimplantation (solid bar) and implantation (open bar) sites on Days 5 to 8 of pregnancy. *P < 0.05 and **P < 0.01 compared to nonimplantation segment

Next, we determined the localization of HSP20-like mRNA in the uterus during artificially induced decidualization. In situ hybridization studies were carried out on sections from nonstimulated and stimulated uterine horns 48 h after the induction of decidualization (Fig. 7). No positive staining (blue/black color) was detected in sections from nonstimulated or stimulated uterine horns using DIG-labeled sense riboprobes (Fig. 7, A and B). Positive staining was, however, seen in the endometrium of sections from nonstimulated or stimulated uterine horns using DIG-labeled antisense riboprobes (Fig. 7, C–H). This hybridization signal was localized to the endometrial stroma in sections from nonstimulated (Fig. 7, C and D) and stimulated (Fig. 7, E–H) uterine horns. No signal was detected in the luminal or glandular epithelia. The hybridization signal present in the endometrium was stronger in the decidualizing endometrial stromal cells of the stimulated uterine horn compared to endometrial stromal cells not undergoing decidualization in the nonstimulated horn. In addition to the endometrium, positive HSP20-like mRNA hybridization signals were also detected in the connective tissue between the circular and longitudinal muscle layers of the myometrium in sections from both nonstimulated and stimulated uterine horns.



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  		FIG. 7. In situ localization of HSP20-like transcripts in the mouse uterus during oil-induced decidualization. Control sections (6 mm) from nonstimulated (A) and stimulated (B) uterine segments probed with DIG-labeled sense riboprobe showed no positive staining (blue/black color). In situ hybridization of antisense riboprobe to sections from nonstimulated (C and D) and stimulated (E–H) uterine horns. C, Circular muscle layers of the myometrium; DESC, endometrial stromal cells undergoing decidualization of the endometrium; ESC, fibroblast-like stromal cells; GE, glandular epithelium; L, longitudinal muscle; LE, luminal epithelium. Bars = 0.5 mm (A–C and E) and 120 µm (D and F–H)

To test whether estrogen or progesterone regulates uterine HSP20-like mRNA levels, ovariectomized animals were injected with vehicle or the steroids. Total RNA isolated from the uterus of these animals was subjected to Northern blot analyses (Fig. 8). Whereas estrogen had no effect, progesterone caused a significant (P < 0.01) increase in the steady-state HSP20-like mRNA levels in the uterus. In the presence of estradiol, the effect of progesterone was reduced by approximately 69% compared to its effect in the absence of estradiol.



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  		FIG. 8. Northern blot analyses of steady-state HSP20-like mRNA levels in the uteri of ovariectomized mice after exposure to vehicle (V), estradiol-17ß (E), progesterone (P), or estradiol-17ß plus progesterone (B). A) Representative autoradiographs probed with a HSP20-like cDNA probe and subsequently with a 18 S rRNA probe. B) Mean (± SEM; n = 4) ratios of HSP20-like mRNA to 18 S rRNA signals, as determined by image analysis. **P < 0.01 compared to vehicle alone

DISCUSSION

Using the method of suppression subtractive hybridization [10, 11], we identified a novel transcript that is expressed in the uterus and several other tissues. Designated as HSP20-like mRNA in the present paper, this transcript contains a predicted open reading frame that encodes a 196-amino-acid protein. Analysis of the sequence of the predicted HSP20-like protein reveals that it belongs to the HSP20 family. The steady-state level of HSP20-like mRNA were significantly greater in uterine segments undergoing the decidual cell reaction compared to segments that were not. This increase in HSP20-like mRNA was localized to the endometrial stromal cells undergoing the decidual cell reaction in the deciduoma. Finally, we showed that the HSP20-like mRNA is a progesterone-inducible transcript in the uterus.

The HSP20-like mRNA found in the present study is likely a member of the HSP20 family of genes. Like all members of the HSP20 gene family, the predicted protein contains a HSP20/{alpha}-crystallin domain. In previously described HSP20 proteins, this HSP20 domain and remaining carboxy-terminal amino acids are believed to be involved in chaperone activity and subunit assembly, respectively [15]. Members of the HSP20 family are also kinase substrates, and their phosphorylation may be involved in controlling multimer breakdown [16], multimer formation [17, 18], and HSP function [19–21]. Notably, the predicted HSP20-like protein in the present study has potential serine and tyrosine phosphorylation sites.

Although the transcript in the present study is predicted to be a member of the HSP20 family, its precise function remains to be elucidated. The HSP20 genes are expressed in virtually all organisms examined, and they exhibit monomeric relative molecular masses in the 20-kDa range. They form multimeric complexes that can reach a size of 1 MDa [22] and may interact with members of other HSP families [23]. Although their functions remain incompletely understood, they appear to be expressed and to function under both physiological and pathological conditions. For example, they have been shown to act as chaperones [24], to be essential for actin remodeling in vivo [25, 26], and to play a role in maintaining intermediate filament networks [27, 28]. They have also been implicated in mRNA stabilization [29] and stress resistance [30–32].

The molecular interactions that occur in the endometrium during the decidual cell reaction are complex and involve multiple gene products. The expression of several genes increases in the endometrial stroma during decidualization [1, 6], and this provides correlative evidence that they play a role in the decidual cell reaction. In the present study, we have identified a novel transcript of a potential HSP20 gene that can be added to this list of genes. Notably, the increased level of the HSP20-like transcripts occurred in the presence and absence of an embryo during pregnancy and artificially induced decidualization, respectively. This suggests that a signal from the embryo is not required for the increase to occur.

During pregnancy, HSP20 genes are expressed in endometrial stromal cells undergoing the decidual cell reaction. Heat shock protein 25 is expressed in endothelial and luminal epithelial cells at the onset of implantation in the rat [33]. This is followed by its expression in the endometrial stromal cells undergoing the decidual cell reaction. Expression of HSP27 increases in human cultured endometrial stromal cells during in vitro decidualization [34, 35]. Heat shock protein 27 is expressed in the decidual stromal cells throughout pregnancy in humans [36]. Therefore, HSP20 proteins may play a role in the process of decidualization and decidual function. In the present study, increased levels of HSP20-like transcripts were localized to the endometrial stromal cells undergoing the decidual cell reaction, suggesting that it too may play a role in the process. However, because HSP20-like transcripts are expressed in many other tissues, including uterine segments not undergoing decidualization, its role does not appear to be restricted to endometrium undergoing decidualization. Clearly, more work is required to determine the function of the HSP20-like and HSP20 genes before their reproductive and physiological significance can become fully realized.

Recently, a great deal of attention has been focused on genes that make the uterus become receptive to an implanting embryo or a deciduogenic stimulus. Gruidl et al. [37] found a distinct pattern of {alpha}-crystallin B (a HSP20 protein) in the human endometrium during the menstrual cycle, which suggests that it is an important factor that makes the endometrium receptive to implantation. In another recent study, Simmons and Kennedy [38] found a transient increase in the expression of the glucose-regulated protein 78 gene (a member of the HSP70 family) in the glandular epithelium of the rat endometrium during the time of optimal receptivity to a deciduogenic stimulus. Therefore, these two HSPs potentially play a role in making the endometrium receptive to implantation. Whether the HSP20-like gene is involved in the establishment of a receptive endometrium remains to be determined.

Progesterone and estrogen have pleiotrophic effects on the endometrium and are involved in preparing the endometrium to become receptive to implantation and decidualization [3, 39–43]. In human endometrium, the expression of HSP27 increases progressively during the late proliferative to early secretory phases of the menstrual cycle, then decreases during the rest of the cycle [44]. {alpha}-Crystallin B gene expression, on the other hand, is low during the proliferative phase and then progressively increases during the secretory phase [37]. These menstrual cycle-dependent changes in the expression of HSP20 genes provide evidence that steroid hormones may modulate their expression in the uterus. In the present study, HSP20-like transcript levels were increased in the uterus in response to progesterone. Therefore, it appears to be a progesterone-inducible gene in the mouse uterus. Notably, progesterone receptor expression dramatically increases in the endometrial stroma during decidualization [43]. Therefore, the increased HSP20-like mRNA in the endometrium during decidualization found in the present study can be correlated with increased progesterone receptor expression.

In conclusion, we have identified a novel transcript (i.e., HSP20-like mRNA) in the mouse that may encode a protein of the HSP20 family. An increased level of this transcript occurs during artificially induced decidualization and is localized to the decidualizing endometrial stromal cells. It is up-regulated in implantation segments of the uterus and in response to progesterone. Expression of the HSP20-like gene may play a role in decidualization of mouse endometrial stromal cells.

FOOTNOTES

First decision: 20 July 2000.

1 Supported by the Canadian Institutes of Health Research (G.A.S.) and the Lalor Foundation (B.B.). B.B. was the recipient of Lalor Foundation and the Alberta Heritage Foundation for Medical Research postdoctoral fellowships during this work. Back

2 Correspondence: Brent M. Bany, Department of Biochemistry and Molecular Biology, University of Calgary, 3330 Hospital Drive N.W., Calgary, AB, Canada T2N 4N1. FAX: 403 270 0737; bany{at}ucalgary.ca Back

Accepted: August 28, 2000.

Received: June 15, 2000.

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