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Identification of Progesterone-Dependent Messenger Ribonucleic Acid Regulatory Patterns in the Rhesus Monkey Endometrium by Differential-Display Reverse Transcription-Polymerase Chain Reaction¹

^a Departments of OB/GYN and Physiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION NOTE ADDED IN PROOF REFERENCES

 
We used differential-display reverse transcription-polymerase chain reaction (DDRT-PCR) to identify different patterns of progesterone (P₄)-dependent gene regulation in rhesus monkey endometria. Complementary DNA populations representing the proliferative phase (estrogen dominant, EcDNA) and an inadequate secretory phase (low level of P₄, IcDNA) were compared with a cDNA population representing an adequate secretory phase (normal level of P₄, PcDNA). We were able to distinguish four different levels of mRNA regulation: 1) up-regulation by P₄ during an adequate secretory phase, 2) autologous down-regulation (IcDNA versus PcDNA), 3) lower abundance in IcDNA compared to PcDNA, and 4) P₄-dependent inhibition of EcDNA gene expression. We isolated and sequenced 16 fragments representing these different levels of P₄ regulation. The sequence of three fragments that were autologously down-regulated (I1, I2, I4) matched previously entered GenBank mRNAs: I1 encodes serine/threonine protein phosphatase A; I2 encodes oxobutanoate dehydrogenase E1b-beta; and I4 encodes line-1 reverse transcriptase homologue. Six other fragments exhibited homology to uncharacterized expressed sequence tags, sequence site tags, and cosmid clones. The remaining seven fragments exhibited no significant homology to GenBank entries at this time. The various patterns of P₄-dependent gene regulation identified in the present study are likely to play roles in the temporal orchestration of events that lead to proper maturation of the endometrium.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION NOTE ADDED IN PROOF REFERENCES

 
The sequential secretion of ovarian estrogen (E) and progesterone (P₄) during the primate menstrual cycle is essential for the orderly regulation of normal endometrial growth and differentiation. Moreover, the action of P₄ is essential for normal development and maturation of the endometrium in preparation for embryo implantation and the maintenance of pregnancy [1–3]. P₄ is known to be necessary for correct endometrial maturation during the menstrual cycle because inadequate endometria (with a low circulating P₄ level) do not support blastocyst implantation in the human and nonhuman primate [1–3].

The molecular mechanisms, which act to achieve temporal and spatial control of differential growth in the nonhuman primate endometrium, are largely unknown. It is evident, however, that target tissues for steroid hormones contain high-affinity binding proteins that mediate the hormonal response. The evolving model of P₄ action in normal tissues requires the binding of hormone to the P₄ receptor (PR), which increases the affinity of the steroid-receptor complex to P₄ response elements that function as DNA enhancer and repressor elements to control transcription of specific genes and gene networks [4–7]. Thus, it is envisaged that a cascade of gene expression initiated by PR, involving up- and down-regulation of ligands, growth factors, receptors, extracellular matrix proteins, and enzymes controlling cellular metabolism, is required for correct endometrial maturation. Endometria that cannot support implantation due to an inadequate P₄ level are expected to show differences in gene expression of factors required for normal endometrial differentiation.

We have constructed cDNA populations from rhesus monkey endometrial tissue in artificial menstrual cycles (AMCs) under normal (adequate) and inadequate secretory phases and have used differential-display reverse transcription-polymerase chain reactions (DDRT-PCR) to identify genes regulated by P₄ in vivo. These cDNA populations represent mRNA expression in 1) the E-dominant proliferative phase (EcDNA), 2) the P₄-dominant secretory phase (PcDNA), and 3) inadequate secretory phases (IcDNA). In addition, we have analyzed by DDRT-PCR two P₄-enriched cDNA populations created by subtractive hybridization. Using this approach we have identified several different levels of P₄-dependent regulation of endometrial genes in the normal secretory phase. These studies are a further step toward the identification of irregularities of gene expression that may result in infertile, inadequate endometria.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION NOTE ADDED IN PROOF REFERENCES

 
Animals

The animal model and the AMCs used in this study have been described in detail previously [8, 9]. Briefly, the approach uses ovariectomized female rhesus monkeys in which the menstrual cycle is simulated artificially by controlled administration of E- followed by P₄-releasing silicone elastomer implants [8, 9]. Peak levels of E occur on Day 13 and mimic the natural proliferative phase [8]. Similarly, the secretory phase of the menstrual cycle is induced by P₄ administration starting on Day 13, with peak P₄ levels occurring between Days 20 and 23. Creating low levels of P₄ (2–3 ng/ml) during the secretory phase that are within ranges determined to be inadequate in the rhesus monkey [3, 9] simulates the inadequate menstrual cycle. All procedures were approved by the Institutional Animal Care and Use Committee at UMass Medical Center.

Synthesis and Amplification of cDNA Populations

We and our colleagues have previously described the detailed procedure for the isolation of EcDNA, PcDNA, and IcDNA populations [9, 10]. To obtain cDNA populations representing proliferative, secretory, and inadequate phases, poly(A)⁺ RNA was prepared from surgically removed biopsies of rhesus monkey endometria pooled from 4–6 animals on Days 9, 11, and 13 of normal AMCs (E dominant), Days 21 and 23 of normal AMCs (P₄ dominant), and Days 21 and 23 of AMCs. All procedures were as previously described [9–11]. The mRNA populations were then converted to double-stranded cDNA. The cDNA populations were end-ligated with EcoRI adaptors and amplified by the polymerase chain reaction (PCR) using adaptor-complementary primers [10]. Two libraries enriched for P₄-dependent fragments were constructed by subtractive hybridization of PcDNA (target) from EcDNA (driver) to yield P₄-enriched cDNA [10], and PcDNA from IcDNA to yield adequate-enriched cDNA.

DDRT-PCR

Twenty nanograms of cDNA was amplified in 100-µl reactions containing 2.5 µM dNTPs, 1.5 mM magnesium chloride, single-strength buffer, 12.5 µCi [-³⁵S]dATP, 2 U Tfl polymerase, and 0.5 µM arbitrary primers (10-mers, listed below). Reactions were carried out at 94°C/1 min, 40°C/2 min, and 72°C/30 sec for 40 cycles and analyzed by denaturing polyacrylamide gel electrophoresis, omitting the fixing stage. Experiments were duplicated to ensure reproducibility and to avoid isolating false-positive bands. The autoradiogram and gel were aligned by needle punctures, and individual bands were carefully excised from the gel with a razor blade. Gel slices attached to filter paper were boiled in 100 µl water and spun to remove debris, and the supernatant was precipitated with glycogen. DNA fragments thus isolated were reamplified as described above except with 250 µM dNTPs and no radiolabel. Products were blunt-ended using T4 DNA polymerase and cloned into the plasmid vector pCDNAII (Invitrogen, San Diego, CA) and then sequenced. Complementary DNA fragments isolated in this fashion are flanked by sequences homologous to the arbitrary primers. Arbitrary primers were ARB-1: CTGATCCATG; ARB-2: CTTGATTGCC; ARB-4: GTTGCGATCC; ARB-5: GACCGCTTGT; ARB-7: CTTTGGTCAG; ARB-8: CAAGCGAGGT; ARB-9: AACGCGCAAC.

Semiquantitative PCR Analysis

Twenty nanograms of template cDNA was amplified in 100-µl reactions containing 0.5 µM primers, 0.25 mM dNTPs, 1.5 mM magnesium chloride, single-strength PCR buffer (Sigma Chemical Co., St. Louis, MO), and 2 U of Tfl polymerase for 20–50 cycles. Each experiment was repeated three times to ensure reproducibility. Products were analyzed by 0.5–1.0% ethidium bromide-stained agarose gel electrophoresis, and comparative quantitation was performed by densitometric analysis of photographed gels. Representative gels of PCR products determined to be within the linear range of amplification are shown. The primers used and the expected product sizes are as follows: I1 upper: 5'-AAAGTATGGGAATGCCAACG, I1 lower: 5'-CTATGGATGGAGAGAGGCCA, product: 117 bp; I2 upper: 5'-GTCCATGGCAAAAGAAAAGC, I2 lower: 5'-TGATGTTGGTTGCTGCAAAT, product: 137 bp; I4 upper: 5'-GCGATCCTGGTCTCTGAAAA, I4 lower: 5'-ACCGCTTGTTGAATTGATCC, product: 100 bp.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION NOTE ADDED IN PROOF REFERENCES

 
When EcDNA, IcDNA, and PcDNA were compared by DDRT-PCR, we were able to identify at least four levels of mRNA regulation, as illustrated by the pattern of cDNA fragments in Figure 1. First, we observed fragments up-regulated by a high level of circulating P₄ during the adequate secretory phase (indicated by fragment 1 in Fig. 1). Second, there were fragments up-regulated by a low level of P₄ in inadequate secretory phases but apparently down-regulated by an adequate level of P₄ (e.g., fragment 2). Third, some fragments were underrepresented in inadequate versus adequate secretory phases (e.g., fragment 3). Last, it was seen that fragment 4 in Figure 1 was down-regulated by P₄ in the secretory phase as compared to the proliferative phase. It should be noted that Figure 1 is a small, convenient part of one gel showing an example of each level of regulation and that examination of Figures 2–4 (see below) demonstrates that many fragments exhibited the different kinds of regulation noted above.

View larger version (85K): [in this window] [in a new window]   FIG. 1. DDRT-PCR of EcDNA, IcDNA, and PcDNA populations that demonstrates four different levels of gene regulation, identified numerically in the figure (see Results).

View larger version (50K): [in this window] [in a new window]   FIG. 2. a) DDRT-PCR comparing expressed fragments from proliferative phase (E), secretory phase (P4 [P on figure]), inadequate secretory phase (I), and P4-enriched and adequate-enriched subtracted cDNA populations (sP and sA, respectively). Different combinations of arbitrary primers used were 1+2 (a and d), 5+9 (b and f), 5+8 (c), and 7+8+9 (e). P4-dependent fragments 1–8 are indicated.

View larger version (28K): [in this window] [in a new window]   FIG. 3. DDRT-PCR comparing EcDNA, PcDNA, and IcDNA populations using arbitrary primers 4+5. I-specific fragments are indicated.

View larger version (134K): [in this window] [in a new window]   FIG. 4. DDRT-PCR comparing EcDNA and PcDNA populations (in duplicate) using arbitrary primers 1+2 (lanes 1–4), 1+3 (lanes 5–8), and 1+4 (lanes 9–12). Fragments down-regulated during E to P4 transition are indicated.

We first compared EcDNA, PcDNA, and P₄-enriched cDNA by differential display to identify P₄-dependent fragments. Our criteria for selection of these fragments required that the fragment be absent in EcDNA and strongly expressed in PcDNA. Fragments P1 and P2 met these criteria as shown in Figure 2a and were subsequently isolated. These two P₄-dependent fragments were also more abundant in a subtracted cDNA population enriched for P₄-dependent genes, reinforcing our assumption that these are genuine P₄-induced sequences and not artifacts of the technique. This pattern of fragments in the cDNA populations was reproducible in several repeat experiments using the same set of arbitrary primers (data not shown).

Figure 2, b–f, shows additional gels using different primer sets in which IcDNA and the other P₄-enriched subtracted library, adequate-enriched cDNA, were analyzed. From these gels the P₄-dependent fragments P3, P4, P5, P6, P7, and P8 (shown in Fig. 2) were isolated. It should also be noted that in panels b–f of Figure 2 the expression of fragments P3–P8 in IcDNA was absent or very low, providing additional confidence that these fragments required normal levels of serum P₄ for proper expression. In addition to identification of genes that are up-regulated during an adequate secretory phase (PcDNA), Figure 2 also shows that there is most often a coincident reduction in mRNA abundance in IcDNA.

All the fragments (P1–P8) were between approximately 50 and 400 base pairs (bp) in length. In some cases we were able to identify the genes that these fragments represent by DNA and predicted protein homology with the GenBank databases (Table 1). P1 is 103 bp, and its DNA sequence matched an as yet uncharacterized human P1 (bacteriophage) artificial chromosome clone (PAC) from chromosome 6. Fragments P2, P3, and P4 yielded no significant sequence homologies to GenBank entries. P5 is 91 bp and showed limited, gapped DNA homology to an uncharacterized human bacterial artificial chromosome (BAC) clone from chromosome 7. P6 is 261 bp, of which 71 bp exhibited limited homology to an uncharacterized human expressed sequence tag (EST) from fetal brain. P7 and P8 showed no homology to database entries. Since fragments P1–P8 have, to date, yielded no significant gene matches, we searched for potential encoded protein domains using the Prosite (Swiss Institute for Bioinformatics, Geneva, Switzerland) database, but again no significant homologies were found.

In addition to identification of fragments up-regulated by P₄ in the normal secretory phase, we also identified several fragments that were highly expressed in IcDNA but showed low or absent expression in PcDNA and EcDNA (Fig. 3). The rationale for the isolation of inadequate-specific fragments is based on the hypothesis that some genes are rapidly turned on by low levels of P₄ and subsequently turned off during the midsecretory phase during elevated serum P₄ levels (autologous down-regulation). Indeed, Figure 3 shows that there are numerous gene fragments that are up-regulated in inadequate endometrium (low P₄) and repressed during an adequate secretory phase. These genes do not appear to be E-dependent, because their expression is similar in E- and P₄-dominant endometria.

The fragments I1–I4 were thus identified (Fig. 3), cut from the gel, reamplified, and cloned into a prokaryotic vector. Sequence homology analysis against GenBank entries was then performed (Table 1). Fragment I1 contained an open reading frame (ORF) that matched (100%) human serine/threonine protein phosphatase 2A. Fragment I2 contained an ORF that showed significant homology (83%) to human oxobutanoate dehydrogenase. The DNA sequence of fragment I3 was homologous to an uncharacterized human EST clone, and fragment I4 contained an ORF that was 88% identical to primate line-1 reverse transcriptase homologue.

Using differential display we were also able to identify E-dependent genes that are inhibited by P₄ (Fig. 4). We isolated, cloned, and sequenced four of these fragments (E1–E4) as noted in Figure 4 and Table 1. Fragments E2 and E9 contained no ORFs and showed no significant homology to GenBank entries. These fragments represent undefined noncoding regions. E3 was significantly homologous (probability = e^-36) to an as yet uncharacterized human sequence site tag (STS) clone (see Table 1). Fragment E4 was also homologous (e-17) to uncharacterized human genomic DNA near the Down syndrome region on chromosome 21.

For the three fragments that correspond to previously characterized mRNAs (I1, I2, and I4), we designed specific primers for semiquantitative PCR in order to confirm their regulation by P₄. Figure 5 shows that I1 and I2 were up-regulated (5- and 3-fold, respectively) in IcDNA compared to EcDNA and PcDNA, as expected. However, I4 appears to be a false positive from DDRT-PCR, since its expression was greatest in PcDNA (i.e., up-regulated by P₄ in the normal secretory phase). This observation shows the necessity for confirming the regulation of differential-display fragments of known genes and avoiding false positives.

View larger version (41K): [in this window] [in a new window]   FIG. 5. Semiquantitative PCR of fragments I1 (20 cycles), I2 (26 cycles), and I4 (28 cycles) in EcDNA, PcDNA, and IcDNA. Expected product sizes are 117 bp (I1), 137 bp (I2), and 100 bp (I4).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION NOTE ADDED IN PROOF REFERENCES

 
DDRT-PCR is a recent approach for the identification of mRNAs that show differences in expression level between two or more populations or that are unique to a cell type, tissue, or developmental stage [12]. We used a modified method that allows random cDNAs to be amplified by a pair of short arbitrary (10-mer) PCR primers [13]. The sequence of the primers dictates which panel of cDNA fragments of the total will be amplified: only those gene fragments containing sequences complementary to the primers will be amplified. Use of different primer sets will result in different patterns (gene fragments) that are amplified, and we have utilized this method to broaden the scope of genes to be analyzed. Because the fragments are small (50–400 bp), they can be quickly sequenced and compared by homology to GenBank database entries.

We used three primary endometrial cDNA populations and two P₄-enriched populations, created by subtractive hybridization, as templates for analysis by differential gene display. The primary cDNAs represent mRNA populations from the proliferative (E dominant), secretory (P₄ dominant), and inadequate secretory (low P₄) phases of the menstrual cycle (EcDNA, PcDNA, and IcDNA, respectively). The subtracted cDNAs were made by subtracting E/P-common fragments from PcDNA to yield P₄-enriched cDNA, and I/P-common fragments from adequate (P₄) cDNA to yield adequate-enriched cDNA, respectively [10]. In this study we focused on the different levels of P₄-dependent regulation identified by differential display and isolated 16 gene fragments for cloning and sequencing.

Seven fragments (P2, P3, P4, P7, P8, E2, and E9) appeared to represent previously undescribed transcripts since they showed no match to GenBank entries. Six fragments (P1, P5, P6, I3, E3, and E4) exhibited homology to uncharacterized sequences on cosmid and ESTs, for which there is no further genetic information. Three fragments (I1, I2, and I4) corresponded to previously characterized mRNA sequence entries.

We have identified two gene fragments (I1,I2) that are overexpressed in IcDNA (inadequate) compared to PcDNA (adequate). These fragments are also underrepresented or absent in EcDNA, suggesting that an inadequate level of P₄ can induce P₄-dependent gene expression but that an adequate level of P₄ appears to exert an autologous down-regulation of these early secretory phase response genes. Homology searches for fragments I1 and I2, which are induced by a low P₄ level, resulted in virtually identical, extensive matches (95–100%) to previously known genes. I1 encodes protein phosphatase 2A (PP2A), a heterotrimeric serine/threonine phosphatase present in most tissue and cell types that regulates such diverse cellular processes as DNA replication, cell cycle progression, signal transduction, and intermediary metabolism [14]. PP2A can both positively and negatively regulate cellular processes in conjunction with different catalytic subunits and protein kinases by reversible phosphorylation of factors that function at different steps of signaling cascades [15].

I2 encodes branched-chain alpha-keto acid dehydrogenase (BCAD), a multienzyme complex occupying a key position in intermediary metabolism. BCAD catalyses an irreversible step in the catabolism of several essential amino acids, including the branched-chain amino acids. BCAD is located in mitochondria and is under stringent control by hormones and dietary factors. The promoter region of BCAD contains a progesterone/glucocorticoid response element [16] that is consistent with regulation by P₄ [7]. Interestingly, BCAD is active only when dephosphorylated [17].

I4 was shown to be a false positive in that it was up-regulated by an adequate rather than inadequate level of P₄. I4 has significant homology to a human reverse transcriptase homologue similar to RNA-dependent DNA polymerases found in transposable elements and retroviruses [18]. Primates and rodents have been shown to contain L1 transposable element-like repeats thought to be derived from a reverse transcriptase-related protein [18]. Although the fragments I1, I2, and I4 are considered identified, their roles in the endometrium remain unknown.

The homology search results presented above are all statistically significant at the 99% confidence level, but biological knowledge should temper the conclusions for these findings. For instance, the fragments P5 and P6 exhibit only limited, gapped DNA homology (75–85%) to their respective genes/gene products listed in Table 1. No direct biological conclusions can be drawn from these homologies at this time, and we are currently obtaining longer sequences of each fragment from cDNA libraries in order to help resolve the significance of the results.

We envisage that coordinated, steroid-induced activation and repression of many genes during the changeover from E to P₄ dominance is important for correct endometrial maturation. On the basis of the P₄-dependent endometrial regulation identified in the present study and other studies [9, 11], we propose that waves of P₄-dependent gene regulation, a cascade, are central to proper maturation of the primate endometrium. During a normal secretory phase, the early phase of P₄ regulation likely involves both inhibition of E-dependent genes and induction of P₄-dependent genes that are sensitive to the initial rise in serum P₄ level. As the secretory phase proceeds, rising titers of serum P₄ lead to further inhibition of E-dependent genes and continued induction of P₄-dependent genes. Superimposed on these two regulatory actions of P₄ is an autologous down-regulation of some but not all P₄-dependent genes. In addition, several laboratories have shown that some genes are activated only during a sharply restricted window, just prior to and during potential implantation in the human and other animal models [19–25]. Subsequently, P₄-dependent endometrial gene regulation necessary for proper maturation subsides with falling titers of P₄ in the late secretory phase, signaling a decline in receptivity of the endometrium.

The use of differential display with our hormonally distinct cDNA populations has allowed us to identify different P₄-dependent mRNA regulatory patterns in the normal secretory phase of the rhesus monkey. Although these data provide evidence for the relative changes in mRNA abundance, other potential effects of steroid hormones, such as alterations in degradation of mRNAs and proteins, need to be addressed by other approaches. In addition, approaches such as immunohistochemistry and in situ hybridization will be necessary to address important issues of cell type-specific expression in hormonally distinct microenvironments within the primate endometrium [26, 27].

	NOTE ADDED IN PROOF

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION NOTE ADDED IN PROOF REFERENCES

 
PP2Aß (I1 fragment) has very recently been shown to be a human candidate tumor suppressor in the lung and colon (Wang SS, Esplin ED, Li Jl, Huang AG, Minna J, Evans GA. Alterations of the PPP2R1B gene in human lung and colon cancer. Science 1998; 282:284–287). This is the second human candidate tumor suppressor that our laboratory has shown to be progesterone-dependent in the primate endometrium (Ace CI, Okulicz WC. A progesterone-induced endometrial homolog of a new candidate tumor suppressor, DMBT1. J Clin Endocrinol Metab 1998; 83:3569–3573).

	ACKNOWLEDGMENTS

 
The authors thank Eric Merithew and Drs. C. Longcope and J. Tast for their help and support of this work.

	FOOTNOTES

 
¹ This work was supported in part by a grant from the NICHD (HD 31620) to W.C.O.

² Correspondence: William C. Okulicz, Dept. of OB/GYN, Umass Medical School, 55 Lake Ave. N., Worcester, MA 01655. FAX: 508 856 5016; william.okulicz{at}banyan.ummed.edu

Accepted: November 30, 1998.

Received: July 8, 1998.

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