Dennis R. Stewart

The relationship between hCG and relaxin secretion in normal pregnancies vs peri‐implantation spontaneous abortions

OBJECTIVE We determined the ovarian response to human chorionic gonadotrophin (hCG) in terms of relaxin and progesterone secretion during the peri‐implantation period of normal and failing pregnancies. We wished to test the hypotheses that relaxin production in failing pregnancies is different from that in normal pregnancies, that relaxin is a reliable, quantitative indicator of the biological activity of endogenous hCG, and that relaxin is a useful predictor of peri‐implantation spontaneous abortions.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) modulates function of human luteinizing granulosa cells via cAMP signaling and early reduction of glucose transporting activity

This study examined the changes in cellular glucose uptake, cAMP-dependent protein kinase (PKA), and progesterone production induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in human luteinizing granulosa cells (LGCs) in culture. The role of Ah receptor on TCDD-mediated toxicity in human LGCs was investigated. Treatment of human LGCs with TCDD produced a time- and dose-dependent decrease in the cellular uptake of glucose. The Vmax and the K(m) of glucose transport were decreased by TCDD treatment. Furthermore, cytochalasin B, a specific inhibitor of facilitative glucose transporter proteins, totally abolished the portion of glucose transport activity that is sensitive to TCDD. Pretreatment of the cells with the Ah receptor blockers 4,7-phenanthroline and alpha-naphthoflavone antagonised the effect of TCDD on 3H-Me-glucose uptake. Structure-activity relationship studies with TCDD and three dioxin congeners revealed a rank order for their potency in the inhibition of glucose transport as follows: TCDD > 1,2,3,7,8-PCDD > 1,2,4,7,8-PCDD > 2,7-DCDD. Such a rank order is consistent with the previously determined biological activity of TCDD and the other dioxin congeners. Treatment of cells for 48 h with 10 nM TCDD substantially reduced PKA and progesterone production. The inhibitory effect of TCDD on progesterone production was more pronounced in the presence of insulin (10 micrograms/mL) and D-glucose (13.3 mM). However, cytochalasin B abolished the effect of TCDD on progesterone production. Forskolin (adenylate cyclase activator) abolished the effect of TCDD on glucose uptake and progesterone production but it did not affect the action of TCDD on PKA activity. A relationship between glucose transporting activity and progesterone production in human LGCs treated with TCDD is indicated by several lines of evidence: a) cytochalasin B downregulated glucose transporting activity and progesterone production, b) insulin plus D-glucose downregulated glucose uptake and amplified the negative effect of TCDD on progesterone production, and c) forskolin abolished the negative effect of TCDD on glucose transporting activity and on progesterone production. From the present data we conclude that glucose transporting activity can be used as a sensitive biomarker to detect the very early response to TCDD in human steroid-producing cells and that effect of TCDD on steroid production is mediated through the cAMP-dependent protein kinase.

Mechanism of toxic action of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in cultured human luteinized granulosa cells

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) caused a significant decrease in estradiol (E2) production when it was administered to human luteinized granulosa cells (hLGCs) in culture. We investigated the involvement of the epidermal growth factor receptor (EGFR) and protein tyrosine kinase (PTK) in this TCDD-induced toxicity. Upregulation in 125I-EGF binding to EGFR was measured after 24 h of TCDD treatment, while downregulation in EGFR binding was measured after 72 h of TCDD treatment. Upregulation of EGFR binding was associated with a significant decrease in postnuclear (7000 x g supernatant) PTK activity, but this activity was stimulated after 72 h of TCDD treatment. TCDD altered the level of tyrosine phosphorylation in proteins with molecular weights 35, 40, 43, 45, 60, and > 205 kDa. TCDD caused a significant increase in postnuclear cAMP-dependent protein kinase (PKA) after 24 h of treatment. The actions of TCDD on protein kinases were partially blocked by the protein synthesis inhibitor, cycloheximide. On the other hand, TCDD increased nuclear PTK and decreased nuclear PKA activity. E2 inhibited the postnuclear and nuclear activity of both PTK and PKA in control samples, but did not affect TCDD actions on either postnuclear or nuclear PTK activity. However, E2 abolished the stimulatory effect of TCDD on PKA activity in postnuclear protein. In the presence of insulin, TCDD did not induce any additional changes in postnuclear or nuclear PTK. Forskolin (FK) alone inhibited postnuclear PTK activity and stimulated its nuclear activity. The addition of TCDD 20 min after FK resulted in an increase in postnuclear PTK, but there was little change in nuclear PTK as compared to the effect of FK alone. The stimulatory effect of TCDD on postnuclear PKA activity was enhanced by insulin and TCDD reversed the negative effect of FK, but there was no effect of either insulin or FK on the inhibition by TCDD of nuclear PKA activity. TCDD decreased the activity of MAP2 kinase and reduced the binding activity of AP-1 DNA when given alone, and also blocked the E2 stimulation of MAP2K. These findings suggest that TCDD may interrupt the endocrine function of hLGCs through the blockage of the mitotic signal directly or indirectly through the interaction of PTK/MAP2K and PKA signaling.

Relaxin secretion by human granulosa cell culture is predictive of in-vitro fertilization–embryo transfer success*

We have developed a cell culture system for human luteinizing granulosa cells which supports the timely and dynamic secretion of oestrogen, progesterone and relaxin in patterns that mimic serum concentrations of these hormones during the luteal phase of the menstrual cycle. There was a wide variation in the amount of relaxin secreted by the cultured cells for the 69 patients studied. As relaxin production was generally maximal by day 10 of culture, comparisons were made at this time point. It was observed that most of the conceptions occurred in patients with higher relaxin secretion in vitro. All cycles with relaxin > 800 pg/ml on day 10 had a term pregnancy while only 13% of cycles with relaxin < 200 pg/ml had term pregnancies. A limited number of cycles from donor/recipient cycles did not show similar results. Steroid concentrations were not predictive of conception. These results demonstrated that in-vitro production of relaxin is predictive of implantation success in in-vitro fertilization (IVF)-embryo transfer cycles. This supports the hypothesis that relaxin may be involved in implantation and that lowered relaxin concentrations may be a partial cause of poor pregnancy rates after IVF.

Effects of Progesterone Receptor Blockers on Human Granulosa-Luteal Cell Culture Secretion of Progesterone, Estradiol, and Relaxin

Abstract We have developed culture methods for human luteinizing granulosa cells (GLC) that support the timely and dynamic secretion of estrogen (estradiol-17β; E2), progesterone (P4), and relaxin (Rlx) in patterns that mimic serum hormone concentrations during the luteal phase of the menstrual cycle. Additional hCG, to simulate rescue of the corpus luteum, prevented the normal decline in GLC hormone production. To test the importance of the P4 receptor in P4 production, GLC were treated in vitro with two P4 receptor antagonists. Human GLC received one of two hCG support protocols: a Baseline group simulating the normal luteal phase or a Rescue group simulating early pregnancy. Baseline and Rescue groups were treated with either RU-486 or HRP2000 either early or late in the cell culture period. The effects of treatments or control on ovarian steroid and peptide hormone production were determined (significant difference was P < 0.05). In the Rescue group, late treatment resulted in an immediate and dramatic decline in E2, P4, and Rlx secretion to nearly nondetectable levels within 1 day after treatment, and hormones remained depressed for the remaining 10 days of culture. In contrast, early treatment resulted in a decline in steroid hormone secretion that returned to control levels within 5 days of cessation of treatment, and Rlx secretion was delayed for approximately 5 days more than in controls. The data support the hypothesis that P4 may be a required autocrine factor, not only for its own production but also for the maintenance of full endocrine function of the corpus luteum.

Purification and sequence determination of canine relaxin

Relaxin immunological activity has been observed in the plasma of pregnant bitches, and preliminary studies in our laboratory indicated that the highest relaxin concentrations were found in placentas. Therefore, canine placentas were collected at term and also from spay and relaxin was purified by methods developed for equine relaxin. Tissue was prepared by homogenization and purification on a C18 column. The preparation was further purified by stepwise elution ion-exchange chromatography, gel filtration, and gradient elution ion-exchange chromatography. One predominant peak in relaxin immunoactivity was collected. Canine relaxin was found to be larger than either porcine or equine relaxin as determined by SDS-PAGE. It migrated faster under reducing conditions, indicating a subunit structure. Purified canine relaxin was used for tracer and standard in a canine radioimmunoassay (RIA) using an antiporcine relaxin antibody. Concentrations of relaxin immunoactivity using the canine assay were up to 300-fold higher in placental preparations than those measured in the porcine relaxin assay. Sequence analysis of canine relaxin revealed a structure similar to other relaxins in the presence and placement of cystine residues.

Alteration in canine cardiac basal levels of cAMP and cGMP, and elevated tissue Pco2 levels, induced by coronary ligation

Abstract Forty dogs were subjected to coronary ligation by placing snares around the two consecutive diagonal branches of the left anterior descending coronary arteries. Both acute and chronic studies were made of intramyocardial gas tensions using the mass spectrometer. Other studies such as levels of cyclic nucleotides and myosin ATPase activity were made at the chronic periods. Following coronary ligation in dogs there was a significantly elevated tissue P co 2 level in the ischemic region and left ventricular border zone as late as 5 weeks post-ligation. There was a severe decrease in cAMP, whereas cGMP was either the same as normal values or in some cases significantly elevated, in the ischemic area during this post-ligation time. These data were reflected in the border area of the left ventricle, but to a lesser extent. There was a minor, but significant decrease in myosin enzymatic V max values in the border area of the left ventricle during the early post-ligation period when tissue P co 2 levels were significantly elevated.

Primate preimplantation embryo is a target for relaxin during early pregnancy

OBJECTIVE To determine whether preimplantation embryos are targets for relaxin secreted from the corpus luteum of the menstrual cycle. DESIGN Rhesus monkey oocytes obtained from females undergoing controlled ovarian hyperstimulation were inseminated, and the resulting embryos were cultured in medium with or without recombinant human relaxin (20 ng/mL) for 8 days. SETTING Research laboratory. ANIMAL(S) Rhesus monkey. INTERVENTION(S) Controlled ovarian stimulation to obtain oocytes for in vitro-produced embryos that were cultured with or without human recombinant relaxin. MAIN OUTCOME MEASURE(S) Rate of blastocyst development, percentage of blastocysts, and inner cell mass/trophectoderm cell ratio were measured on day 8 of culture. The presence of relaxin receptor (RXFP1) messenger RNA in eight-cell embryos was observed by array hybridization. RESULT(S) RXFP1 receptor expression was localized to the inner cell mass of blastocysts, as shown by immunohistochemistry. The percentage of embryos that developed to blastocyst and the inner cell mass/trophectoderm cell ratio was unchanged with relaxin supplementation; however, the relaxin-treated embryos developed into blastocysts significantly sooner than untreated embryos. CONCLUSION(S) These results are the first evidence that the preimplantation primate embryo is a target for relaxin and that the addition of relaxin to in vitro culture medium enhances rhesus monkey embryo development.

Serum relaxin concentrations in patients with out-of-phase endometrial biopsies*†

When luteal phase relaxin concentrations were summed to give an integrated measure (pg/mL per cycle), relaxin was found to be significantly lower in those cycles with an out-of-phase endometrial biopsy. In addition, peak relaxin concentrations were lower in out-of-phase cycles compared with normal cycles. These data indicate that relaxin secretion may be related to normal luteal function and suggest that shortening of the luteal phase results in reduced relaxin production. Measurement of circulating relaxin may prove to be useful in making the diagnosis of out-of-phase biopsy and needs to be assessed for its usefulness in diagnosing abnormal luteal function.

Changes in cAMP concentrations during chronic cardiac hypertrophy

SummaryMild pulmonic stenosis in the dog, where right ventricular peak systolic pressure was increased approximately 150% at the time of sacrifice, induced 100% or more increase in right ventricular free wall weight by 3 weeks postoperative. Accompanying cardiac hypertrophy at these postoperative times, there was a decrease in both tissue PO2 levels and cAMP concentrations in the hemodynamically stressed ventricle, the right ventricle. Myosin ATPase activity was elevated as well as the velocity of contractile element shortening. The hemodynamically nonstressed left ventricle did not hypertrophy at these early postoperative times.Zusammenfassung3 Wochen nach Operation verursachte eine milde Stenosierung der Pulmonalarterie mit Steigerung der systolischen Spitzendrucke im rechten Ventrikel um 150% beim Hund einen 100% igen oder stärkeren Anstieg im Gewicht der freien Wand des rechten Ventrikels. Als Begleiterscheinung der Herzhypertrophie ergab sich um diese Zeit ein Rückgang sowohl des Gewebe-Sauerstoffpartialdruckes als auch der Konzentration von cAMP im hämodynamisch mehrbeanspruchten (rechten) Ventrikel. Die ATPase-Aktivität von Myosin war erhöht, desgleichen die Verkürzungsgeschwindigkeit des kontraktilen Elements. Der hämodynamisch nicht mehrbeanspruchte linke Ventrikel zeigte in diesem frühen postoperativen Stadium keine Hypertrophie.