Biochemical evidence for autocrine/paracrine regulation of apoptosis in cultured uterine epithelial cells during mouse embryo implantation in vitro.

During embryo implantation, apoptosis is observed morphologically at the implantation site of endometrium. The objectives of this study were to demonstrate biochemical evidence of apoptosis and quantitative assessment of DNA fragmentation in uterine epithelial cells using a mouse implantation model, and to investigate the autocrine/paracrine regulation of apoptosis in uterine epithelial cells during blastocyst outgrowth. Blastocysts from day 4 pregnant mice were cultured on uterine epithelial cells for 96 h. Uterine epithelial cells dislodged by trophoblasts in endometrium-trophoblast unit demonstrated morphological features of apoptosis by Acridine Orange staining. Electrophoresis demonstrated DNA ladder and DNA fragmentation by enzyme-linked immunosorbent assay markedly increased after 48 h period of incubation. Apoptosis increased in an exponential way in accordance with trophoblast outgrowth. In addition, DNA fragmentation was shown in the epithelial cells by adding embryo-conditioned medium (CM) and the effect of embryo CM on apoptosis was significantly inhibited by anti-transforming growth factor (TGF)-beta antibody. Delayed outgrowth was observed after 48 h of incubation in the blastocysts cultured with anti-TGF-beta antibody. These results suggest there is autocrine/paracrine regulation of apoptosis in uterine epithelial cells at mouse embryo implantation and that TGF-beta might play an important role in the occurrence of apoptosis in the endometrium-trophoblast unit.

Relaxin stimulates interstitial collagenase activity in cultured uterine cervical cells from nonpregnant and pregnant but not immature guinea pigs; estradiol-17 beta restores relaxin's effect in immature cervical cells.

Relaxin has been implicated in dilatation of the cervix at parturition. Dilatation of the guinea pig cervix at parturition is mediated by an estrogen-induced degradation of type I collagen by interstitial collagenase (matrix metalloproteinase I, MMPI). This study was designed to test the hypothesis that human recombinant relaxin (hrR) stimulates MMPI activity in cultured guinea pig cervical cells. Primary cervical monolayer cultures from immature, adult nonpregnant, and 50-day-pregnant Hartley guinea pigs were exposed to hrR (1-1000 ng/ml) daily for 3 days in serum-free Dulbecco's Modified Eagle's Medium (DMEM). The effect of priming immature cells with estradiol-17 beta (E2, 10(-6) M) daily for 3 days prior to treatment with hrR was also determined. Tissue inhibitors of metalloproteinases were inactivated by reduction, alkylation, and dialysis. MMPI activity at 96 h was assayed via a highly sensitive and specific assay that utilizes [3H]telopeptide-free type I collagen as a substrate. Aminophenylmercuric acetate (0.5 mM) was used to activate latent MMPI. Phenanthroline-1,10 (1 mM), a known inhibitor of metalloproteinases, was used as a blank control. Phorbol-12-myristate-13-acetate (PMA, 10(-8) M), a known stimulator of MMPI biosynthesis, was used as a positive control. The hrR in serum-free DMEM had no significant effect on cell number in nonpregnant, 50-day-pregnant, E2-primed, or nonprimed immature animals. It stimulated MMPI activity in a dose-dependent manner with a maximum response at 10 ng/ml in nonpregnant (2-fold), 50-day-pregnant (3-fold), and E2-primed immature (3-fold) animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Phospholipase A2 activity is increased in guinea pig uterine cervix in late pregnancy and at parturition.

Release of arachidonic acid from phospholipids by phospholipases A2 (PLA2) is the rate-limiting step in prostaglandin (PG) synthesis. PGE2 and PGF2 alpha are essential intermediates in interstitial collagenase-mediated degradation of type I collagen, a key step in cervical dilatation at parturition. We demonstrate that PLA2 is present in cytosolic fractions of guinea pig cervix and PLA2 activity is increased during cervical dilatation at parturition. In the cervix of nonpregnant animals, PLA2 activity against phosphatidylcholine (PC) and phosphatidylethanolamine (PE) was 13 +/- 6 and 49 +/- 27 pmol.min-1.mg protein-1, respectively. Levels were similar at day 25 of pregnancy. At day 50, PLA2-PC increased to 190 +/- 54 pmol.min-1.mg-1, and PLA2-PE rose to 592 +/- 127 pmol.min-1.mg-1. At parturition (68 +/- 2 days) there were further increases of 49-67%. PLA2 activity declined toward basal levels 2 days postpartum. Almost 50% of the enhanced cytosolic PLA2 (cPLA2) activity at day 50 or at parturition was of high molecular mass and was identified as the "85-kDa cPLA2." Increases in cPLA2 activity at these time points were not, however, associated with increases in cPLA2 protein or phosphorylation of cPLA2, compared with nonpregnant animals. This study suggests that multiple PLA2 enzymes, including cPLA2, are involved in cervical dilatation at parturition.

Cell origin and paracrine control of interstitial collagenase in the guinea pig uterine cervix--evidence for a low molecular weight epithelial cell-derived collagenase stimulator.

Dilatation of the uterine cervix at parturition is achieved by degradation of type I collagen, the main structural protein in the cervix, by interstitial collagenase. In order to determine the cell origin of interstitial collagenase in the cervix, separate stromal and epithelial cell cultures were established. Using a highly sensitive and specific assay for collagenase that utilizes [3H]telopeptide-free type I collagen as a substrate, we determined that the cells of origin of collagenase were cervical stromal cells and not cervical epithelial cells. Cells of cervical epithelium produced factors in culture that stimulated stromal cell collagenase production. The addition of epithelial cells or epithelial cell-conditioned culture medium to stromal cells resulted in a dose-dependent stimulation of stromal cell collagenase production with a maximum increase of 3- or 6-fold, respectively. To characterize the collagenase-stimulatory activity produced by epithelial cells, epithelial cell-conditioned culture medium was extracted under conditions that optimized the recovery of peptides and was subjected to ion-exchange batch extraction as well as reverse-phase and size-exclusion HPLC. Collagenase-stimulatory activities were mainly recovered in neutral extracts of epithelial cell-conditioned medium with an apparent molecular mass of 6 kDa. In conclusion, interstitial collagenase is produced by cervical stromal cells and not by cervical epithelial cells. Epithelial cells, however, secrete factors of low molecular size that stimulate stromal cell collagenase production.

Activation of protein kinase C stimulates collagenase production by cultured cells of the cervix of the pregnant guinea pig.

OBJECTIVE: Dilatation of the uterine cervix at parturition is achieved by an estrogen-induced, collagenase-mediated degradation of type I collagen in the cervix. The objective was to test the hypothesis that collagenase production in the cervix of pregnant guinea pig in culture is mediated by activation of protein kinase C. STUDY DESIGN: The effects of 17 beta-estradiol, prostaglandin F2 alpha, or activation of protein kinase C by phorbol-12-myristate-13-acetate, 1-stearoyl-2-arachidonyl-sn-glycerol and phospholipase C on collagenase production was studied with primary monolayer cultures of cervical cells from Hartley guinea pigs at 50 days' gestation. Results are analyzed for statistical significance with analysis of variance. RESULTS: Collagenase production is increased twofold to threefold by 17 beta-estradiol, prostaglandin F2 alpha, or activation of protein kinase C. The observed stimulation of collagenase production by 17 beta-estradiol and prostaglandin F2 alpha was blocked by the protein kinase C inhibitor 1-(5-isoquinoline sulfonyl) 2-methylpiperazine dihydrochloride. CONCLUSION: Collagenase production in cultured cervical cells of pregnant guinea pig is stimulated by activation of protein kinase C.

Circulating hyaluronic acid in nonpregnant, pregnant, and postpartum guinea pigs: elevated levels observed at parturition.

OBJECTIVE: Dilatation of the uterine cervix at parturition is associated with an increase in cervical hyaluronic acid content. The objective is to test the hypothesis that circulating hyaluronic acid is increased at parturition. STUDY DESIGN: Serum hyaluronic acid levels from nonpregnant (n = 5), pregnant (n = 13), and postpartum (n = 4) adult Hartley guinea pigs were determined with a radiometric assay that utilizes iodine 125-labeled hyaluronic acid-binding protein. Results were analyzed for statistical significance with Student's paired t test and regression analysis. RESULTS: The serum hyaluronic acid level in nonpregnant animals was 238 +/- 88 ng/ml (mean +/- SEM). During pregnancy, serum hyaluronic acid levels were 127 +/- 12 and 126 +/- 34 ng/ml at 25 and 50 to 63 days' gestation, respectively. At parturition, hyaluronic acid levels increased fivefold to 765 +/- 111 ng/ml (p less than 0.001). Hyaluronic acid levels returned to antepartum values 2 days post partum (153 +/- 27 ng/ml). There was no significant difference between arterial and venous levels. CONCLUSION: Circulating hyaluronic acid levels increase significantly at parturition in the guinea pig.

Biochemical evidence of collagenase-mediated collagenolysis as a mechanism of cervical dilatation at parturition in the guinea pig.

Dilatation of the uterine cervix at parturition is accompanied by a remarkable alteration in its biomechanical characteristics leading to a significant reduction in its strength and stiffness. Our previous studies and those of others have suggested the involvement of collagenolysis leading to cervical dilatation. This study provides further evidence for the occurrence of collagenolysis in the dilated guinea pig cervix at birth. The changes in collagenase and collagenase inhibitory activity in vivo in cervices of nonpregnant, pregnant, and postpartum guinea pigs were determined. There were no significant changes in procollagenase, collagenase inhibitory activity, and net procollagenase in animals at 25 and 50 days of gestation compared with nonpregnant animals. At parturition (68 +/- 2 days), there was a 6-fold increase in procollagenase, a 26-fold increase in collagenase inhibitory activity, and a 2-fold increase in net procollagenase activity. Cervices in organ culture obtained at birth produced 2.9-fold more procollagenase, 1.6-fold more collagenase inhibitory activity, and a 10-fold increase in net procollagenase activity when compared to nonpregnant or 25-day pregnant animals. These studies indicate that dilatation of the guinea pig cervix at parturition involves collagenase-mediated degradation of collagen in the cervix.

Immunochemical and immunohistochemical evidence of estrogen-mediated collagenolysis as a mechanism of cervical dilatation in the guinea pig at parturition.

The mechanism of dilatation of the uterine cervix at birth is poorly understood. Several indirect lines of evidence have suggested that cervical ripening is accompanied by collagen degradation. In this study, immunochemical methods have been developed to identify and analyze type I collage degradation in the cervix of the pregnant guinea pig. Using cyanogen bromide-derived peptides of purified guinea pig type I collagen as an immunogen, a polyclonal rabbit antiserum was prepared that recognizes epitopes on the denatured and degraded alpha 2 chain of type I collagen as demonstrated by enzyme-linked immunosorbant assay and immunoblotting. This antibody was used to demonstrate degradation of type I collagen in the extracellular matrix of the dilated cervix at parturition. Moreover, physiological concentrations of 17 beta-estradiol stimulated degradation of type I collagen in the nonpregnant cervix in organ culture. Collagenase degradation products were detected in the extracellular matrix and in the culture media. The effect of 17 beta-estradiol (10(-6) M) was completely blocked by progesterone (10(-4) M). These studies suggest that dilatation of the guinea pig cervix at parturition may be associated with estrogen-mediated degradation of type I collagen.

Changes in active and latent collagenase in human placenta around the time of parturition.

High levels of collagenase are present in cervical extracts and in the circulation in women at parturition. This study examines the content of collagenase in human placentas at term, the possible contribution to circulating enzyme, and the changes that occur at parturition. Active and latent forms of collagenase are detectable in placentas with apparent relative molecular mass of 60,000 and 65,000 d, respectively. Gel-filtration chromatography was used to identify the presence of excess tissue inhibitor of metalloproteinase as the major collagenase inhibitor in the extracellular matrix of human placentas. After the onset of labor, there was a significant increase in total collagenase activity. Inactivation of the tissue inhibitor of metalloproteinase by reduction of placental extracts with dithiothreitol and alkylation with iodoacetamide resulted in a twelvefold to seventeenfold increase in collagenase activity. Umbilical cord collagenase levels were significantly lower than those in maternal circulation. The possibility of circulating collagenase originating from placenta in labor is discussed.

Elevated tissue levels of collagenase during dilation of uterine cervix in human parturition.

Seven biopsy specimens from the cervix and 17 from the lower uterine segment were obtained in 24 women at term (37 to 42 weeks). Collagenase was extracted and assayed on telopeptide-free [3H]collagen; typical collagen cleavage products were found on sodium dodecyl sulfate-gel electrophoresis. There was no significant difference between collagenase levels in the cervix and in the lower uterine segment in women not in labor and with the cervix closed. Levels of active and latent collagenase in 11 such specimens were 0.14 +/- 0.03 and 0.64 +/- 0.90 U/gm wet weight, respectively (mean +/- SEM; 1 U = 1 micrograms collagen digested per minute at 30 degrees C). Thirteen women at term in active labor with cervical dilation of 4 to 8 cm exhibited a thirteenfold increase in mean collagenase activity in the lower uterine segment. Active and latent collagenase increased to 2.06 +/- 0.92 and 8.64 +/- 2.87 U/gm, respectively. This is the first direct evidence that interstitial collagenase increases markedly during cervical dilation in human parturition.