Extensive evidence demonstrates pronounced effects of relaxin on the differentiation of human endometrial cells in vitro. In vivo data in rhesus monkeys suggest a role for relaxin in the development of endometrial vascular architecture. In women, pregnancy can be established and maintained in the absence of circulating relaxin. Thus, local synthesis by the endometrium is necessary if relaxin plays a physiological role in human endometrial function. Although relaxin protein and the prorelaxin C peptide have been localized to human endometrium, no data for relaxin synthesis have been provided to date. We therefore assessed relaxin mRNA and protein levels in cultured, defined human endometrial cells. Reverse transcriptase-polymerase chain reaction (RT-PCR) techniques were used to demonstrate the presence of relaxin mRNA in human stromal and glandular epithelial cells. Secretion of the protein into the media of cultured cells of both types was also detected. Relaxin stimulated the expression of vascular endothelial growth factor in glandular epithelial and stromal cells that were isolated from tissue that had been taken during the secretory phase of the cycle. Relaxin inhibited the expression of procollagenase from both glandular epithelial cells, with a more marked inhibition demonstrated from cells that were isolated from tissue that had been taken during the secretory phase, and from stromal cells. These data demonstrate that human endometrial cells synthesize relaxin, and they support the concept that relaxin fosters endometrial conditions that are required for implantation in women.

Prolactin (PRL) and insulin-like growth factor-binding protein (IGFBP-1) are two major secretory proteins of human endometrial/decidual cells. We have characterized the mRNA of PRL and IGFBP-1 and studied the effect of progestin, medroxyprogesterone acetate (MPA), anti-progestin (RU486), and relaxin (RLX) on the levels of these two mRNA transcripts in a long-term culture of human endometrial stromal cells. Northern blot analysis showed that the size of PRL mRNA was 1.15 kb and that of IGFBP-1 mRNA, 1.6 kb. Primer extension of endometrial/decidual IGFBP-1 mRNA showed two transcription initiation sites identical to those found in HepG2 human hepatoma cell line. The levels of mRNA in control samples remained low, approximately 2 pg PRL and approximately 5 pg IGFBP-1/microgram RNA at various times of culture. When stromal cells were treated with MPA for 28 days, PRL mRNA gradually increased 100-fold whereas IGFBP-1 mRNA exponentially increased approximately 1000-fold compared to control values and leveled after 25 days in culture. The timing of maximal stimulation was shortened by withdrawing MPA or by replacing MPA with RU486. After removal of MPA, levels of both mRNAs increased and each peaked after approximately 10 days, with PRL showing a 2-fold and IGFBP-1 a 20-fold increase compared to cells treated with MPA continuously. Replacing MPA by RU486 caused a rapid increase of PRL mRNA (2-3-fold) in 2-3 days followed by a gradual reduction to less than 20% of peak levels over the next 3 days. IGFBP-1 mRNA levels increased 30- and 100-fold in 1-2 days followed by a reduction to less than 20% of peak levels over the next 24 h. The reduction of mRNA levels by RU486 was reversed when cells were rechallenged with MPA. Relaxin alone caused a transient stimulation of PRL and IGFBP-1 mRNA. Maximal stimulation occurred between 10 and 20 days of culture and was 100-fold for PRL and 1000-fold for IGFBP-1 relative to control values. Cells treated with MPA and RLX in sequence had higher mRNA levels than cells treated with MPA continuously or cells subjected to MPA withdrawal. Maximal mRNA levels reached 0.4 ng PRL and approximately 8 ng IGFBP-1/microgram total RNA, approximately 0.04% and 0.8% of cellular RNA. The mRNA levels under various hormonal manipulations were similar to the previously published synthesis and secretion patterns of PRL and IGFBP-1 proteins in this system.(ABSTRACT TRUNCATED AT 400 WORDS)

Estradiol-17beta dehydrogenase activity in proliferative human endometrium (average of 1.5 nmole of estrone formed from estradiol/mg protein/h) was stimulated as much as as 6-fold during incubations of tissue slices in culture medium containing progesterone. Stimulation was already detectable at 7 h and the highest activity values were reached at 48-72 h of incubation in the presence of excess progesterone. Maximal stimulation was achieved with concentrations of the hormone of 0.25 mug/ml or higher. At concentrations approximately equal to midluteal plasma levels (20 ng/ml) more than 50% of the maximal response was observed. Norgestrel (17alpha-ethynyl-18-methyl-19-nortestosterone) was also effective in inducing enzymatic activity. The similarity of the effects obtained with progesterone (a possible substrate for estradiol dehydrogenase) and the synthetic progestin indicates that the stimulation of enzymatic activity was not due to substrate induction. Addition of estradiol to the culture medium had no influence on the activity of the enzyme. The induction of estradiol dehydrogenase by progesterone was inhibited by puromycin or actinomycin D. These observations indicate that progestational agents increase the rate of de novo synthesis of the enzyme. Stimulation of endometrial estradiol dehydrogenase was also observed after 2-3 day oral administration of medroxyprogesterone acetate to women in the follicular phase. In contrast, the enzymatic activity in endometrium obtained from women taking estrogens was found to be as low as in normal proliferative tissue. These in vitro and in vivo results point to progesterone as the agent responsible for the 10-fold increase in endometrial estradiol dehydrogenase activity observed during the luteal phase in menstruating women. Data obtained from superfusion studies of estrogen dynamics in endometrium indicate that changes in enzyme concentrations may play a physiologic role in the regulation of tissue levels of estradiol.

The decidualized endometrium during the first trimester of pregnancy synthesizes and secretes a 32-kDa insulin-like growth factor-binding protein (termed hIGFBP-1) at high levels. IGFBP-1 is the major soluble protein product of this tissue and is principally localized to the differentiated endometrial stromal cell, the decidual cell. In the present study long term culture of stromal cells from the nonpregnant endometrium have been employed to elucidate the hormonal requirements for IGFBP-1 production. Immunoreactive IGFBP-1 was undetectable in control cultures. However, inclusion of medroxyprogesterone acetate (MPA) induced rates of 0.35 +/- 0.09 microgram/0.1 mg cell DNA.day (mean +/- SEM; n = 5) after 20-30 days. In these cultures cells exhibited morphological changes consistent with decidual cell differentiation. In all cultures removal of MPA after exposure for 10-16 days, with or without subsequent inclusion of relaxin (RLX), increased production of IGFBP-1 450- to 4600-fold to rates of 150-710 micrograms/0.1 mg cell DNA.day or 26-131 micrograms/10(6) cells.day on days 24-26. The rates tended to be higher with the inclusion of RLX and were sustained in contrast to cultures without RLX, where rates fell by day 30. Individual cultures responded differently to RLX when added from the initiation of culture, with either a response similar to MPA alone or a cyclical change in production, achieving maximal rates of 190-290 micrograms/0.1 mg cell DNA.day. Cultures in which RLX alone induced high IGFBP-1 high production were obtained from endometrium during the progesterone-dominated luteal phase. In cultures exhibiting high rates of immunoreactive IGFBP-1 production, the protein represented their major secretory protein product. This was confirmed by [35S]methionine incorporation and the presence of IGFBP-1 as the predominant protein in serum-free culture medium. The immunoreactive IGFBP-1 isolated from culture medium was found to be identical, by a number of criteria, with IGFBP-1 derived from decidual tissue. These results were consistent with a primary role of progestin exposure, whether in vivo or in vitro, in converting endometrial stromal cells to cells potentially able to exhibit the high rates of IGFBP-1 production typical of the decidualized endometrium of pregnancy.

Estradiol dehydrogenase (E 2 -DH) and20

Decidual cells arise by proliferation and differentiation of endometrial stromal cells of the uterus after appropriate stimulation by ovarian hormones. Previously we have shown that progestin and relaxin stimulate the secretion of several decidual-cell-specific secretory proteins in a long-term primary cell culture system. We now report the effects of progestin and relaxin on the morphology of stromal cells in association with the production rate of two major decidual secretory proteins, prolactin and insulin-like growth factor binding protein-1 (IGFBP-1). Stromal cells were cultured in RPMI 1640 and 2% fetal bovine serum for 22 days under control conditions (no hormone), with relaxin or medroxyprogesterone acetate (MPA), or MPA plus relaxin. Cells treated with MPA alone or MPA plus porcine relaxin grew to a high density with many areas of heaping while control cells and cells grown in medium containing relaxin alone formed discontinuous layers. The cytoplasm was distinguished by aggregates of rough endoplasmic reticulum and secretory granules. Surfaces of cells treated with MPA plus relaxin had clusters of short blunt processes containing secretory granules. The processes were rarely seen in cells exposed to MPA alone and completely absent in control cells or cells exposed to relaxin alone. Intercellular space was greatly widened in cells treated with MPA alone or MPA plus relaxin. There were many extended gap junctions in MPA- and relaxin-treated cells in contrast to controls.(ABSTRACT TRUNCATED AT 250 WORDS)

The relative contributions of type I and type II insulinlike growth factor (IGF) receptors and IGF carrier proteins to the binding of IGF

The present study showed that in undifferentiated endometrial stromal cells, progestin increases the RLX receptor content to enhance the effect of RLX on the target gene (IGFBP-1). In decidual cells, RLX alone up-regulates its receptor, resulting in a large scale induction of IGFBP-1. TGFbeta1 has an inhibitory effect on LGR7 and IGFBP-1.