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Biology of Reproduction 66, 1210-1218 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Characterization of a Prolactin-Regulated Gene in Reproductive Tissues Usingthe Prolactin Receptor Knockout Mouse Model1

Nathalie Barana, Paul A. Kellya, and Nadine Binart2,a

a INSERM Unité 344, Endocrinologie Moléculaire, Faculté de Médecine Necker, 75730 Paris Cedex 15, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Prolactin (PRL) exerts pleiotropic physiological effects in various cells and tissues, although it is mainly considered as a regulator of reproduction and cell growth. Null mutation of the prolactin receptor (PRLR) gene leads to female sterility due to a failure of embryo implantation. Using this mouse model and the method of mRNA differential display, we identified PRL target genes that are regulated during the peri-implantation period. We characterized 1 among the 45 isolated genes, UA-3, which is regulated in the uterus as well as in the ovary during early pregnancy. This gene corresponds to a P311 mouse cDNA that was originally identified for its high expression in late-stage embryonic brain and adult cerebellum. We report here that UA-3 is present in numerous tissues as well as in ovary and uterus at the site of blastocyst apposition, and that its expression is hormonally regulated. Moreover, in situ hybridization reveals high expression in ovarian granulosa cells and in uterine epithelium. Recently, it has been suggested that P311 expression is tightly regulated at several levels by mechanisms that control cellular growth, transformation, motility, or a combination of these. Taken together, these results suggest that P311 could be involved in these processes during pregnancy, although its function remains to be clearly established.

implantation, pregnancy, prolactin, prolactin receptor, uterus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Prolactin (PRL) is a pleiotropic hormone whose numerous actions are associated with reproduction, growth and development, water and electrolyte balance, metabolism, behavior, and immunoregulation [1]. Actions related to the process of female reproduction represent the largest group of different functions that have been identified for PRL in rodent: nurturing of young, development and secretory function of ovarian follicles and corpora lutea, or promoting blastocyst implantation [2–5]. PRL is crucial for luteal function and maintenance of pregnancy [6]. For instance, PRL stimulates ovarian progesterone (P4) secretion as well as P4 receptor expression in uterine epithelium [7–9]. PRL and its receptor are expressed in the uterus [5, 10, 11] as well in the placenta, suggesting that this hormone may also have some potential for a paracrine/autocrine effect, such as stimulation of cell proliferation, although the mechanism or mechanisms by which PRL regulates early pregnancy are not clearly understood.

In rodent, the process of implantation involves complex interactions and requires a precise coordination between the establishment of uterine receptivity and blastocyst activation [12–17]. This process is primarily dependent on the coordinated effects of steroids [12, 16, 18–22]. On Days 1.5 and 2.5 of pregnancy, preovulatory ovarian estradiol (E2) directs epithelial cell proliferation. On Day 3.5, P4 from newly formed corpora lutea initiates stromal cell proliferation, which is further potentiated by preimplantation ovarian E2 secretion involving increased endometrial vascular permeability at the sites of blastocyst apposition, the first conspicuous sign of implantation [12, 16, 18]. On Day 4.5, blastocysts implant [23, 24], and spontaneously, luminal epithelial cells cease to proliferate and become differentiated to allow decidualization [25]. The decidualization begins when stromal cells, which surround the implanting blastocyst, proliferate and form the primary decidual zone (pdz) late on Day 5.5 [17, 18].

Previous studies have shown the essential role of E2 and P4 in the maintenance of pregnancy [26, 27]. Female PRLR knockout mice are sterile, due to a failure of implantation [28]. Generation of viable PRL knockout and PRLR knockout mice also suggests that embryo-uterine interactions during the preimplantation period are not directly dependent on embryonic PRLR activation [28, 29]. The basis of the sterility of PRLR knockout mice is attributed to the absence of sufficient P4 to support implantation, and subsequently, placental development and maintenance [4]. The primary site of P4 production during pregnancy in the mouse is the corpus luteum of the ovary [9, 30]. The rescue of implantation failure in PRLR knockout mice is made possible by P4 supplementation. Although implantation occurs, the maintenance of full-term pregnancy is not complete, with a major embryonic loss occurring at mid-gestation. We have demonstrated that implantation and decidualization defects in PRLR knockout mice are mediated by ovarian but not by uterine PRLR [5]. Nevertheless, ovarian steroid deficiencies are not sufficient to explain the failure of pregnancy in PRLR knockout mice, but PRL could play a direct or indirect role in maintenance. Overall, these observations indicate that preventing PRL action by disruption of the PRLR gene alters the maternal-decidual transformation in response to the implanting blastocyst, demonstrating an essential role of PRL in reproduction. However, given the complexity of the peri-implantation period and hormone and gene regulation, the precise molecular events during early pregnancy are still not well understood, with many factors still unidentified.

In this study, we used the PRLR knockout mouse as a model to identify PRL target genes at the peri-implantation period. During embryo implantation, the uterus undergoes dynamic changes, which are associated with up-regulation and down-regulation of several genes at the sites of blastocyst attachment, many of which are still unknown. To identify such genes during early pregnancy, the mRNA differential display technique [31] was used to compare the mRNA expression patterns in uteri of wild type, P4-treated PRLR knockout, and PRLR knockout mice on Day 5.5 of pregnancy. This report describes the isolation and characterization of one of these genes, UA-3, during pregnancy.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice

The disruption of the PRLR gene was previously performed in our laboratory by introducing a TK-neo cassette in place of a 1.5-kilobase (kb) EcoRV genomic fragment housing exon 5 and surrounding sequences [28]. Polymerase chain reaction (PCR) of tail DNA determined the genotypes as described previously [5]. Mice were kept two per cage at 25°C and 80% relative humidity with a 12L:12D cycle, and were fed a pelleted diet ad libitum. Heterozygous females were mated with fertile males in a pure 129/Sv background. The morning of a vaginal plug detection was designated as Day 0.5 of pregnancy. A subgroup of PRLR knockout females received a P4 pellet (25 mg; Innovative Research of America, Toledo, OH) supplementation from Day 0.5 to rescue the implantation process and subsequent pregnancy as previously described [4].

In a second study, wild-type and PRLR knockout mice were ovariectomized and allowed to recover for 2 wk. Mice were treated with steroids or PRL. The treatment schedule was the following: ovine PRL (NIAMMD-oPRL-16, 0.5 mg; a kind gift of the National Hormone and Pituitary Program, National Institutes of Health, Bethesda, MD) or P4 (1 mg; Sigma Chemical Company, St. Louis, MO) or 100 ng estradiol-17ß (Sigma), or a combination of both steroids (1 mg P4 and 100 ng E2) diluted in sesame oil. All mice were killed 8 or 24 h after s.c. injection. A daily injection of 1 mg P4 was performed during 3 consecutive days, resulting in a treatment for 72 h.

All experimental designs and procedures were performed in agreement with the guidelines of the animal ethics committee of the Ministère de l'Agriculture of France.

Tissue Preparations

Wild-type, PRLR knockout, and P4-treated PRLR knockout uteri were collected on Day 5.5 of pregnancy and fresh frozen for mRNA differential display. Whole uteri were collected from Day 0.5 to Day 12.5 of pregnancy. Pregnancy on Days 5.5–12.5 was confirmed by recovering embryos from the reproductive tracts. On Day 5.5, implantation sites were localized by i.v. injections of 0.1 ml of Chicago Blue dye solution (1% in saline) and were separated from interimplantation sites and frozen separately. On Days 9.5 and 12.5, implantation and interimplantation sites were distinct without special manipulation and collected separately. Ovaries were also collected at these stages.

Differential Display of mRNA

Differential display was performed as described previously [31]. Total RNA was extracted from tissues by the standard guanidine-isothiocyanate-phenol-chloroform procedure [32]. Reverse transcription of mRNAs and polymerase chain reaction (PCR) amplification were performed using 35S-labeled nucleotides and primers from an RNA image Kit 1 (GenHunter Corporation, Nashville, TN), given 24 primer combinations for each sample. After extraction and reamplification of the differentially expressed cDNAs, a standard Northern blot analysis was performed to confirm the gene-specific expression using reamplified PCR products as probes. The bands giving differential expression patterns were subcloned into pCR 2.1 vector (Invitrogen, Carlsbad, CA) and sequenced with a dye terminator kit using the ABI Prism System (Perkin-Elmer, Norwalk, CT).

Northern Blot Analysis

Total RNA (7 µg) was electrophoresed on a denaturing 1% agarose gel and transferred to a nylon membrane (Hybond N+; Amersham, Piscataway, NJ) then UV cross-linked. Membrane prehybridization was performed at 42°C for 3 h in a formamide/SSPE (180 mM NaCl, 10 mM NaH2PO4, 1 mM EDTA, pH 7.4) buffer, then hybridized with the labeled probe overnight at 42°C. Probes were synthesized from specific cDNA (25 ng) using the Rediprime II kit and [{alpha}-32P]dCTP (50 µCi/reaction, Amersham). Unincorporated nucleotides were removed with a microspin column. Moreover, membranes were hybridized to ribosomal protein L-7 (RPL-7) probe to confirm integrity, equal loading, and blotting of the RNA samples. This housekeeping gene was used to normalize the expression between samples. Membranes were washed 3 times for 10 min each at 42°C with 5x SSPE/0.5% SDS for 10 min at 42°C with 0.5x SSPE/0.1% SDS, then for 30 min at 50°C with 0.1x SSPE/0.1% SDS. Results were visualized by autoradiography and analyzed on Storm 840 (Molecular Dynamics, Inc., Watertown, MA).

Semiquantitative Reverse Transcriptase-PCR Analysis

Analysis of gene expression levels in ovariectomized mice was performed by semiquantitative reverse transcriptase (RT)-PCR. Two micrograms of total RNA were reverse transcribed using oligo-poly(dT) reverse primer and Moloney murine leukemia virus RT (Gibco BRL, Rockville, MD). The amplification of UA-3 was performed with specific upper and lower primers (5'-GTACTTATGAGAGGACCACGTT-3', 5'-GCATGCACTGTCCCTCTTC-3'), using Taq polymerase (Gibco BRL) on 1 µl of the RT reaction. An increasing number of cycles was tested to assess the best conditions to achieve linear amplification. The thermal cycling parameters consisted of 22 cycles of denaturing (45 sec at 94°C), annealing (1 min at 62°C), and extension (45 sec at 72°C). The reaction product was separated on 1.5% TBE agarose gels and stained with ethidium bromide. Results were analyzed with a DC digital camera (Kodak, Rochester, NY) coupled with Kodak ID 2.02 software. In parallel, two primers (5'-GAGGGATCTCGCTCCTGGAAGA-3' and 5'-GGTGAAGGTCGGAGTCAACGGA-3') corresponding to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were included in a separate PCR reaction for each experiment as an internal control. The intensity of each UA-3 band was reported to the intensity of GAPDH.

Statistical Analysis

Densitometric analysis of the intensity of the signals from the Northern blots was determined by Image quaNT (Molecular Dynamics, Sunnyvale, CA). Because experimental variability exists in vivo, an arbitrary value of 10 was given to the average of all values corresponding to samples at wild-type Day 5.5 (the common point between all membranes) either in ovaries or uteri. All other values were reported to this reference. The different levels of expression between samples were normalized according to the housekeeping gene RPL-7.

Densitometric analysis of the signal intensity from the semiquantitative RT-PCR products was determined by Kodak 2.1. Values of each band were normalized to GAPDH for the same sample.

The data were reported as means ± SEM and were subjected to a one-way ANOVA followed by pairwise comparison procedures (the Student t-test) or multiple comparison (the Newman-Keuhl method) to determine differences between groups, using the Statview package.

Bioinformatics

To identify the sequenced cDNA, BLAST, and Unigene from NCBI were used. To characterize the clones, Infobiogene, ProfineScan, PSORT, pfam, and ProDom were used.

In Situ Hybridization

The original UA-3 clone was removed from pCR2.1 by restriction digestion using EcoRI, purified, and subcloned into pGEM3Z-vector. This vector was used to prepare sense or antisense RNA probes with T7 or SP6 polymerase (Promega, Madison, WI). The single-stranded RNA transcripts were labeled with [{alpha}-35S]UTP (Amersham), resulting in probes with a specific activity of 2 x 109 dpm/µg.

Uteri and ovaries at Day 12.5 were embedded in OCT and frozen in cold isopentane. Frozen sections (8 µm) were placed onto superfrost gold slides, then fixed in 4% paraformaldehyde in PBS for 10 min at room temperature. Following prehybridization, sections were hybridized to cRNA probes for 18 h at 53°C in a humidified box for 16–18 h in 50% deionized formamide, 0.3 M NaCl, 20 mM Tris-HCl pH 8.0, 5 mM EDTA, 10 mM NaPO4 pH 8.0, 10% dextran sulfate, 1x and Denhardt 0.5 mg/ml yeast RNA. After hybridization and washing, the sections were incubated with ribonuclease A (10 µg/ml) at 37°C for 15 min, then dehydrated and exposed to NTB-2 liquid Kodak emulsion for 1–3 wk. Finally, slides were poststained with hematoxylin.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Messenger RNA Differential Display and Analysis

To identify PRL-regulated genes in uterus during the peri-implantation period, we applied the mRNA differential display technique to three experimental conditions. We compared gene expression patterns on Day 5.5 of pregnancy uteri from wild-type mice, PRLR knockout mice, and P4-treated PRLR knockout mice. Several mRNA species were obtained; they exhibited differential profiles at an early implantation step. We isolated the bands, which were repressed or activated by PRL between wild-type and knockout animals. Among 148 selected bands, 45 were confirmed by Northern blot analysis as potential transcripts involved in early events of pregnancy. Most were predominantly expressed in PRLR knockout mice. The bands were sequenced, and the corresponding genes were distributed into three divisions: genes already known and well described, such as pregnancy-specific genes DAF (i.e., decay accelerating factor), or crry in the mouse [33]; second, genes already known but for which expression during pregnancy has not been reported, such as the decysin gene [34]; and third, novel genes for which no known function has been described in pregnancy.

Among the genes isolated, 29 cDNAs corresponded to known mouse expressed sequence tags (ESTs), 4 were unknown mouse ESTs, and 12 were entirely unknown. Most of the former cDNAs shared significant homology with several mouse and human ESTs that had been isolated from various libraries, suggesting that the corresponding transcripts were ubiquitously expressed. The known genes could be classified to five different categories depending on their functions: immunity, DNA and RNA machinery, cell components, cell cycle, and metabolism. Details of these genes, including putative identification, specific expression, and arbitrary functional groupings are shown in Table 1.


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  		TABLE 1. Differentially expressed transcripts found in uterus at Day 5.5 of pregnancy

We selected one gene, UA-3, for study on the basis of the strong signal appearing on the polyacrylamide gel and of its potential function in proliferation (Fig. 1A). This band was isolated from transcripts of PRLR knockout mouse. To analyze its pattern, the extracted band was labeled and used as a probe on a Northern blot containing the initial RNA populations used for the mRNA DD (Fig. 1B, left). UA-3 hybridized to a transcript of ~2–2.2 kb from PRLR knockout mouse. The expression of RNAs from 9 to 12 individual mice is shown on the right in Figure 1B. The band of 308 base pairs (bp) was sequenced and analyzed in BLAST/UniGene, and was found to be homologous (99%) to a Mus musculus P311 mRNA (accession numbers X70398 and D45203), a putative neuronal protein. This fragment revealed homology in the 3' extremity (1726–2016), corresponding to a clone previously described [35, 36]. The coding region was very short (from 118 to 324) and exhibited a long 3' untranslated region (UTR) with three polyA signals (1596–1601, 1859–1864, and 1997–2002; Fig. 1C). Assuming that UA-3 was part of P311 mRNA, the full-length cDNA provided by M. Levi-Strauss was also used to confirm that the uterine mRNA was the same as in brain.



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  		FIG. 1. Isolation of a PRL UA-3 regulated gene at Day 5.5 in uterus. A) Differential display of uterine mRNAs at Day 5.5 of pregnancy. Three different RNA samples from wild-type, PRLR knockout, and P4-treated knockout (knockout + P4) mice on Day 5.5 of pregnancy were compared by differential display analysis using the anchored primer HT11A and the arbitrary primer HAP2. The arrow indicates position of the UA-3 band. B) The PCR-amplified cDNA fragment UA-3 was subsequently cloned. Northern blot hybridization of the uterine RNA samples (7 µg) used for differential display was performed with a cDNA probe corresponding to UA-3 clone (left). The histogram (right) represents a mean of 9 to 12 specific signals of Northern blot, normalized to RPL-7 from independent uteri at Day 5.5 of pregnancy (means ± SEM). C) Nucleotide sequence of UA-3 cDNA (308 bp). Below, consensus sequences for polyadenylation sites are represented as white boxes. Location of clone UA-3 is shown with respect to P311 cDNA (X70398). Homology of UA-3 (308 bp, black box) is 99% identical to P311, which displays a short coding DNA sequence (206 bp, black arrow) and a long 3'-untranslated DNA sequence (1707 bp)

The mouse full-length nucleotide sequence (Mn 4919) was surprisingly well-conserved between human (99.0%, Hs 142827), rat (99.1%, Rn 8180), and zebrafish (88.5%, Dr 5000). The deduced open reading frame (ORF) encoded a putative protein of 68 amino acids (PD019364), which appeared to be cytoplasmic (PSORT II), and is derived from an ancestral gene (ProDom) of human and mouse.

Tissue Distribution of UA-3 mRNA

To investigate whether or not the expression of UA-3 was limited to uterus, multi-tissue Northern blot analysis was performed on a variety of tissues from nonpregnant female and male wild-type and PRLR knockout adult mice.

The UA-3 mRNA was detected in all tissues examined (Fig. 2). The 2.2-kb band was predominant in brain and muscle for both sexes. The UA-3 expression was markedly decreased in muscle and ovary of female knockout mice, and was increased in uterus and liver of knockout mice (Fig. 2A). Moreover, because UA-3 corresponded to a neuronal protein, male brain was dissected and its expression appeared mainly localized in cerebellum (Fig. 2B). The profile of UA-3 is overall similar to that reported in all female tissues.



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  		FIG. 2. Expression of UA-3 mRNA in different tissues from adult mice. A) Total RNA (7 µg) of tissues from wild-type (white boxes) and PRLR knockout (black boxes) female mice were separated by agarose formaldehyde gel electrophoresis and hybridized sequentially to 32P-labeled UA-3 and RPL-7 cDNA probes. Results are expressed as a relative expression (specific signal/RPL-7) by histograms. B) Total RNA (7 µg) of tissues from wild-type and PRLR knockout male mice were separated and hybridized as described in A

Uterine Expression of UA-3 During Pregnancy

Steady-state levels of UA-3 mRNA in prepubertal, pubertal, and pregnant uteri from Day 0.5 to Day 12.5 were examined by Northern blot analysis. No difference of UA-3 mRNA expression appeared between prepubertal and pubertal wild-type mice and knockout mice (Fig. 3, A and B). PRL probably limited this gene expression in absence of pregnancy. Moreover, UA-3 expression was measured in whole uteri (Fig. 3, A–C) as well as in embryo implantation sites and interimplantation sites during pregnancy (inserts A and C). During early pregnancy, on Day 0.5 the UA-3 expression was repressed, then on Day 1.5 when preovulatory ovarian estrogen directed epithelial cell proliferation [37], an increase was observed in both wild-type and PRLR knockout uteri. Thus, a progressive decrease of UA-3 expression was noted until Day 12.5. In untreated knockout mice in which implantation did not occur, we measured UA-3 expression only until Day 3.5. However, after supplementation by P4, the same pattern of expression was observed, except on Day 1.5, when a 2-fold reduction of UA-3 level was seen (Fig. 3C). UA-3 expression of pregnancy (Day 1.5) appeared to be independent of the PRL receptor and was reduced in the presence of progesterone. Moreover at Day 5.5, the UA-3 expression was as abundant as it was on Day 1.5 in P4-treated knockout uteri, whereas its expression progressively decreased in wild-type uteri. It is interesting that expression was different between implantation sites and interimplantation sites from Day 5.5 to mid-gestation stages (Fig. 3, A and C). The overall expression was clearly more abundant in implantation sites.



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  		FIG. 3. UA-3 mRNA expression in uterus. A) Results of Northern blot analysis from wild-type uterus. Seven micrograms of total RNA were isolated from whole uterus at the different periods of pregnancy, separated, transferred to nylon membrane, and hybridized sequentially to 32P-labeled UA-3 and RPL-7 probes. The lower panel represents values from Northern blot analysis of embryo implantation sites (S) and interimplantation sites (I) (Day 5.5 to Day 12.5). B) Results of Northern blot analysis from PRLR knockout uterus. C) Results of Northern blot analysis from P4-treated PRLR knockout uterus. The lower panel is as described in A. All results are means ± SEM of 3 to 29 individual experiments and normalized to RPL-7. An arbitrary value of 10 was attributed to the average of specific signals at Day 5.5 and results were expressed according to this value. *P < 0.05; **P < 0.03; ***P < 0.01

Ovarian Expression of UA-3 During Pregnancy

Because differential expression of UA-3 was observed in the ovary (Fig. 2A), we examined whether UA-3 mRNA expression was modified during pregnancy by Northern blot analysis. UA-3 expression was not different in prepubertal and pubertal virgin mice for both genotypes (Fig. 4, A and B). During early pregnancy, in wild-type mice the expression of UA-3 decreased progressively from Day 0.5 to Day 12.5, whereas in untreated knockout mice in which pregnancy failed, the expression remained low and unchanged. In P4-treated knockout mice in which pregnancy was rescued, the values were comparable to those obtained in wild-type mice until Day 9.5 (Fig. 4C). UA-3 expression changed during early stages of pregnancy, when luteinization process takes place.



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  		FIG. 4. UA-3 mRNA expression in ovary. A) Northern blots were performed and results are expressed as means ± SEM of three to seven individual wild-type mice and relative to the RPL-7 control. B) UA-3 expression in PRLR knockout mice. C) UA-3 expression in P4-treated PRLR knockout mice. All results are mean ± SEM of three to seven individual experiments. *P < 0.05; ***P < 0.01

Regulation of Uterine UA-3 mRNA by PRL and Steroid Hormones

Because steroid hormones are essential for the preparation and maintenance of pregnancy, we examined whether uterine UA-3 expression was modulated by steroids and prolactin (Fig. 5). Therefore, wild-type and PRLR knockout female mice were ovariectomized, and after 2 wk were treated with PRL or steroids (E2, P4, or both). UA-3 mRNA expression was analyzed by semiquantitative RT-PCR. Ovariectomized mice treated with oil alone served as controls. PRL administration repressed the UA-3 expression in wild-type mice, whereas no change appeared in PRLR knockout mice, as was expected. The addition of E2 repressed UA-3 expression in wild-type and knockout mice. A single P4 administration for 8 h was sufficient to diminish the UA-3 level in both genotypes. A 3-day treatment of P4 partially repressed the expression of this gene. A significant difference was visible only at this time between both genotypes but not at the intermediate times. The response to a combined treatment of P4 and E2 (24 h) was similar to P4 alone, suggesting that expression of the gene is controlled primarily by P4. This response is also significantly modified by the presence of PRLR. These results indicate that UA-3 is a PRL-regulated gene and a steroid-responsive gene as well. Moreover, these results suggest that PRL exerts direct or indirect actions on uterine UA-3 levels through uterine PRLR.



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  		FIG. 5. Effects of PRL and steroids on uterine expression of UA-3 in ovariectomized mice. Both ovariectomized adult wild-type and PRLR knockout mice were given a single injection of E2, P4, or both; or oPRL, and were killed 24 h later. Mice injected with P4 were killed 8 and 24 h later. Daily injections of P4 were performed for 72 h. Mice injected with oil served as a control. Uteri were removed and RNA was extracted. Two micrograms of total RNA were used for a reverse transcription and the PCR reactions were performed using specific UA-3 primers. Data are expressed relative to the value for mRNA expression for PRLR knockout uteri (fixed at 100%). *P < 0.05; **P < 0.03

In Situ Hybridization of Uterus and Ovary on Day 12.5 of Pregnancy

To determine the expression of UA-3 in a cell-type specific manner, in situ hybridization was performed. The cell-type specific localization of UA-3 mRNA in uterus was detected in the embryo and to a lesser level in uterine epithelium (decidua; Fig. 6). In the ovary, the accumulation of UA-3 mRNA was detected mainly in follicles (granulosa cells) and interstitial tissue (stroma) but not in corpora lutea at mid-gestation (Fig. 7). In the absence of PRLR and maintenance of pregnancy by P4 treatment, accumulation of UA-3 mRNA was also present in the same compartments (data not shown). This result was consistent with Northern blot experiments. No specific autoradiographic signal was detected when uterine and ovarian sections were hybridized with the sense probe as control.



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  		FIG. 6. Expression of UA-3 mRNA in uteri at Day 12.5 of pregnancy. In situ hybridization was performed with the UA-3 probe at the site of implantation on wild-type uteri at Day 12.5 of pregnancy. The bright field micrograph shows the site of embryo implantation. Dark field exposure shows UA-3 transcripts highly expressed in brain and embryo, at a lesser extent in decidua. De, Decidua; E, embryo; M, metencephalon; D, diencephalon; T, telencephalon



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  		FIG. 7. Expression of UA-3 mRNA in ovary at D12.5 of pregnancy. In situ hybridization was performed to detect UA-3 expression. A representative wild-type section of ovary at Day 12.5 of pregnancy is shown in bright field and dark field to demonstrate the histology and to highlight the hybridization signal. The main sources of expression are theca cells forming the outer perimeter of each follicle and the interstitial cells between follicles. Expression does not occur in corpora lutea. F, Follicle; CL, corpus luteum; OS, ovarian stroma; TF, theca folliculi; O, oocyte


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Female PRLR knockout mice are sterile due to an absence of blastocyst implantation [28]. P4 supplementation is able to rescue the implantation process [4]. Whereas this hormone is indispensable to prepare the uterus during the peri-implantation stage to allow placental development and maintenance, alone it is insufficient to recover full-term pregnancy.

In the present study, we used differential display screening [31] to identify genes that could be regulated by PRL in early pregnancy. We found among the 45 isolated clones a new UA-3 mRNA that was down-regulated by PRL in several tissues. We characterized UA-3 during pregnancy as an up-regulated uterine cDNA in PRLR knockout mice. UA-3 expression was repressed during the peri-implantation stage in wild-type and P4 rescued animals. The cDNA was homologous (99%) to P311, which was previously cloned in rat [35] and human brain [36], but its role remains to be defined. UA-3 expression is abundant in brain and muscle, and it is also differentially expressed in ovary as opposed to uterus in knockout and wild-type adult mice. Recently, this cDNA was characterized as a 2036-bp mRNA encoding a 68-amino acid polypeptide with a short half-life [38]. A role has been attributed to P311 in activating glioma invasion though enhanced glioma cell motility [39].

We determined whether UA-3 could be regulated by hormones during pregnancy. Pregnancy is a complex process involving in synergy a series of growth factors, hormones, and cytokines. Furthermore, the uterus is composed of heterogeneous cell types that undergo dynamic changes to support embryo development and implantation, requiring the coordination of steroid hormones [40]. First, in prepubertal and pubertal mice, UA-3 expression is more abundant in the absence of PRL signaling. Second, in the present study, we noticed a high expression of UA-3 at Day 1.5 of pregnancy, both in wild-type and knockout mice, but not in P4-treated knockout mice. Consequently, the early states of pregnancy (Day 1.5) appear to be independent of PRL. At this stage, E2 acts alone on epithelial cell proliferation to prepare blastocyst uterine receptivity. Third, UA-3 expression is repressed by P4 administration. P4 is known to inhibit E2-mediated proliferation of the luminal and glandular epithelial cells and could prevent the action of E2 on UA-3 gene regulation. Thus at Day 5.5, when a large proliferation of the secondary decidual zone of uterus occurred, the overall expression of UA-3 was clearly more abundant at implantation sites than interimplantation sites, as was seen also at Day 12.5. At this stage of pregnancy, P4 treatment resulted in the same pattern of expression in knockout mice as in wild-type mice. P4 probably regulates UA-3 gene expression at the latter stages.

Moreover, we noted a large increase of UA-3 expression in the ovary at the early stages of pregnancy both in a wild-type and in P4-rescued knockout mice, and a progressive, mild decrease in rescued mice, suggesting that P4 was not the only regulator of UA-3 expression. Moreover, because the level of UA-3 is high at Days 0.5–1.5 of pregnancy, it also could be regulated by E2, which is known to be mitogenic during folliculogenesis, implantation, and decidualization processes. E2 is produced in the rodent ovary, secreted mainly by the corpus luteum [41]. Because PRLR knockout mice were unable to maintain corpus luteum function, PRL could alter E2 synthesis, and mediates its regulation [4]. Serum E2 in PRLR knockout mice was 33% lower than in wild-type animals [4]. These serum level differences between wild-type and knockout mice could be ascribed to a weak variation of E2, PRL, or both because UA-3 expression at Days 2.5–3.5 in ovary was similar between knockout and wild-type mice. P4 is not sufficient to restore its expression (Day 3.5) and could not be considered as a major regulator at this stage.

To investigate the regulation of UA-3 expression, we treated ovariectomized mice with hormones. These results demonstrate for the first time that E2 and P4, as well as PRL, down-regulate UA-3. We have shown that UA-3 mRNA was localized in proliferative tissues as well as in the embryo and ovarian follicles. Moreover, high expression was observed in growing muscle (data not shown). Such localization suggests at least a putative role for P311 in proliferation events, because the homologue of UA-3 is the P311 gene. Colocalization of P311 with vinculin at focal adhesion points in normal human astrocytes has been reported in vitro [38], although others demonstrated that its localization was diffuse in human gliomal cell cytoplasm [39], suggesting a role in gliomal migration. During pregnancy many events regulated by hormones modify the uterus, first for the reception of embryos, and then decidual development, following by placental development. Our results suggest that the UA-3 gene could be involved in mechanisms of proliferation.

P311 first was described as an overexpressed transcript by neurons belonging to the late migration wave from the germinal to the cortical layers [35]. Because high neuronal plasticity is known in cortex, hippocampus, and the olfactory bulb, a role for P311 was hypothesized in this context. In humans, UA-3 has been reported to be an important factor for inward calcium currents, which are closely connected to pentylenetetrazol-induced seizure activity, observed in epilepsy [36]. It was also considered as a novel tumor marker in androgen-dependent prostate [42], and recently was found to be highly expressed in intestinal smooth cells, normal astrocytes in culture, and the leiomyosarcoma cell line SK-LMS [38]. Expression of P311 was reduced in cells that were modified to have a high c-Met-HGF/SF signaling, and were able to induce motility, invasiveness, and angiogenesis [39].

It has been proposed that the UA-3 sequence lacking a large ORF may be noncoding, however, the ORF has a good Kozak sequence [35]. Recently, the 2031-bp mRNA sequence was shown to encode a small protein (68 amino acids) with a long 3' untranslated sequence (1707 bp). The protein is proposed to turn over rapidly, which is believed to be due to degradation by the proteasome-ubiquitin system, and an unidentified metalloproteinase [38]. The first of the three ORFs of P311 is well conserved among species, arguing for a fundamental function of the gene product. Its long 3' UTR sequence exhibited AT-rich elements (AREs) such as ATTTA repeats and U-rich sequences, which confer a destabilizing effect and lead to mRNA degradation. This rapid turnover is often involved in the control of expression of proteins that play a key regulatory role when a particular concentration may be critical (for example c-fos and c-myc) [43].

The UA-3 sequence represents the P311 gene, however, its expression is regulated by steroid hormones during pregnancy in the ovary and uterus. Because its mRNA expression is highly expressed in the embryo, we suggest a potential role in proliferation, but it could also affect motility, which is known to be important in development of the female reproductive tract during pregnancy, such as the establishment of equidistant embryos in uterus [44, 45]. Therefore, P311 represents a protein that has links to cellular transformation and differentiation, and it turns over rapidly in the proteasome [38]. The function of this protein, however, remains to be clearly established. We have clearly established localization in the uterine epithelium, granulosa cells of ovarian follicles, and neuronal compartments of the embryo. Future work should address the potential role of UA-3 in cellular differentiation, transformation, and motility.


   ACKNOWLEDGMENTS
 
We are grateful to Christine Helloco and Prune Imbert-Bolloré for suggestions, and Cécile Kedzia for advice with in situ hybridization. We thank Matthieu Lévi-Straus for helpful discussions and for providing the full-length P311 cDNA.


   FOOTNOTES
 
First decision: 18 October 2001.

1 This work was supported in part by grants from INSERM, la Fondation pour la Recherche Médicale, Organon FARO 61/subv.99, and ARC 9952. Back

2 Correspondence: Nadine Binart, INSERM U-344, Faculté de Médecine Necker Enfants malades, 156 rue de Vaugirard, 75730 Paris Cedex 15, France. FAX: 33 1 43 06 04 43; binart{at}necker.fr Back

Accepted: November 19, 2001.

Received: October 3, 2001.


   REFERENCES
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 RESULTS
 DISCUSSION
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