A progesterone-induced endometrial homolog of a new candidate tumor suppressor, DMBT1.

We have previously prepared and characterized a subtracted library enriched for endometrial progesterone (P)-dependent genes in the rhesus monkey. One of the fragment clones (H3) that we selected for sequencing from this library was found to be homologous to human DMBT1, a recently isolated member of the scavenger receptor cysteine-rich superfamily and a new putative tumor suppressor. In this report, we provide evidence that H3 is the rhesus monkey homolog of DMBT1. Additional sequence data of H3 (1071 bp) showed a striking homology with DMBT1 (92% identical). Semiquantitative kinetic PCR of estrogen-dominant vs. P-dominant endometrial complementary DNA populations showed that the H3 gene was up-regulated 5-fold by normal secretory P levels. In situ hybridization with unique probes to H3 confirmed the up-regulation by P in the endometrium and a restricted expression in the stromal compartment. Another recent report suggested the presence of an endometrial tumor suppressor in the same chromosomal region as DMBT1 (10q23-26); deletions in this region were associated with endometrial cancers. Together, these studies potentially provide a molecular link to the protective effect of the action of P on unopposed estrogen exposure in reproductive tract cancers in women.

Analysis of differential gene regulation in adequate versus inadequate secretory-phase endometrial complementary deoxyribonucleic acid populations from the rhesus monkey.

The ability to create artificial menstrual cycles in the rhesus monkey provides a model for studies on the regulation of genes and gene networks by estradiol or progesterone (P) in the primate endometrium. This model allowed us to create both a normal level of secretory phase P or an inadequate level of secretory phase P, i.e. endometria that cannot support implantation. The objective of our present study focused on PCR analyses of genes for several factors that are believed to be important in the proper maturation of the endometrium. Complementary DNA (cDNA) populations were prepared from endometria harvested on day 13 (peak E level), days 21-23 of an adequate secretory phase (PcDNA) and days 21-23 of an inadequate secretory phase (IcDNA). Although placental protein 14, leukemia inhibitory factor and 17-beta hydroxysteroid dehydrogenase displayed highly upregulated levels in PcDNA (P-activated genes), there was little or no up-regulation in IcDNA. Transforming growth factor-beta 2 and its receptor and insulin growth factor-I and its receptor were up-regulated in PcDNA, whereas little or no expression was observed in IcDNA. Regulators of the cell cycle and transcription, such as retinoblastoma, c-fos, and c-jun genes, were also greatly underexpressed in IcDNA compared with PcDNA. Interestingly, one gene that we studied, keratinocyte growth factor, that was up-regulated by P (peak E levels vs. PcDNA) was more highly expressed in IcDNA. This latter result suggests that low levels of circulating P are sufficient for expression of this gene, whereas high sustained P levels result in an autologous down-regulation. These data show that the regulation of genes that may play pivotal roles in endometrial maturation are differentially expressed in IcDNA vs. PcDNA and may, in part, characterize improper endometrial maturation.

Isolation of progesterone-dependent complementary deoxyribonucleic acid fragments from rhesus monkey endometrium by sequential subtractive hybridization and polymerase chain reaction amplification.

The steroid sex hormone progesterone (P) induces the expression of a variety of genes through a signal transduction pathway mediated by the P receptor, a DNA-binding regulator of transcription. To identify genes and gene networks that are P dependent in the rhesus endometrium, we used a powerful technique employing subtractive hybridization coupled with polymerase chain reaction (PCR). Poly(A)+ RNA was isolated from both P-dominant (days 21-23 of artificial menstrual cycles) and estrogen (E)-dominant (days 9-13) endometrium. The two classes of RNA were converted to cDNA, ligated to EcoRI adaptors, and amplified by PCR using an adaptor-complimentary primer. E-dominant cDNA was labeled with biotin, hybridized in excess to P-dominant cDNA (PcDNA), and complexed with streptavidin. Labeled cross-hybrid sequences common to both populations were subtracted by phenol-chloroform extraction. The remaining cDNA fragments were amplified by PCR. After four rounds of hybridization/amplification, the subtracted PcDNA was analyzed for P-dependent sequences by semiquantitive PCR. Initial analysis revealed that housekeeping genes were undetectable in subtracted cDNA, but a previously characterized P-dependent gene was retained. Three of five clones sequenced at random from the subtracted library exhibited P-inducibility/dependency by PCR analysis of E and PcDNA. One of these, an 835-basepair fragment designated H5, may represent a novel P-dependent gene, as no comparable homology could be found with existing sequences in GenBank and Swissprot databases. We estimate that the procedure described here resulted in highly significant enrichment of up-regulated cDNA fragments from P-dominant tissue.

Estradiol increases prolactin synthesis and prolactin messenger ribonucleic acid in selected brain regions in the hypophysectomized female rat.

Immunoreactive PRL which is not of pituitary origin, has been identified in many regions of the rat brain. We have previously demonstrated that estradiol increases hypothalamic immunoreactive PRL content in hypophysectomized female rats. To determine if estradiol stimulates PRL synthesis, we examined the effect of estradiol on the in vivo production of PRL, and on the expression of PRL messenger RNA (mRNA) in the hypothalamus, pons, and cerebral cortex. To examine the effect of estradiol on the in vivo production of PRL, [35S] methionine was injected into the lateral ventricle and its incorporation into immunoprecipitable PRL was determined by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In estradiol, but not vehicle-treated hypophysectomized rats, a 24,000 M(r) immunoprecipitable PRL protein was detected in the hypothalamus and pons-medulla, 2 and 4 h after methionine administration. No immunoprecipitable PRL proteins were detected in the amygdala, hippocampus, cortex, or serum at either time point. In addition, in the hypothalamus, but not the pons-medulla, a second PRL band was detected with an apparent mol wt of 16,000K. To determine if estradiol increased the expression of PRL mRNA, copy DNA was obtained by reverse transcription of poly(A+) mRNA prepared from intact and vehicle or estradiol-treated hypophysectomized rats and analyzed by polymerase chain reaction amplification. In tissues from hypophysectomized rats, there was little, or no, detectable levels of PRL mRNA. In contrast, in estradiol-treated hypophysectomized rats PRL mRNA was easily detected in the hypothalamus and pons-medulla by polymerase chain reaction amplification. These data suggest that estradiol increases the PRL content in the hypothalamus and pons-medulla by increasing PRL gene expression, in a manner similar to that reported in the pituitary.