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T. G. KENNEDY

Mirskaia & Crew (1930) have reported that the first vaginal oestrus in young female mice was frequently not associated with mating. In addition, pregnancy followed first mating in only 24% of cases whereas in the same mice at 3 to 6 months of age, 80 to 90 % of females became pregnant following mating. Since it seemed not unlikely that genetic and/or environmental factors may have contributed to this poor reproductive performance, it was decided to reexamine the fertility of young mice.

Mice of the Quackenbush (QS) strain were used in this study. They were housed under light- and temperature-controlled conditions and given free access to a commercial pelleted mouse food and tap water. The 'young' female mice were 21 days old at the start of the experiment while the 'mature' females were virgin mice approximately

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T. G. KENNEDY and J. P. KENNEDY

Summary.

The effects of age and of parity on reproductive capacity were estimated by comparing old virgin with young virgin mice, and old parous with old virgin mice, respectively.

Increased age was associated with a greater body weight at `joining', a smaller proportion of mice pregnant, a greater number of cl of pregnancy, a greater number of implantation sites at Day 5 or 6 of pregnancy, a greater number of viable fetuses at Day 18 of pregnancy, and a smaller proportion of cl represented as implantation sites at Day 5 or 6 of pregnancy.

Parity significantly increased body weight at `joining' and the number of cl of pregnancy. The number of implantation sites at Day 5 or 6 of pregnancy and the number of viable fetuses at Day 18 of pregnancy were non-significantly greater for old parous than old virgin mice.

It was concluded from the relationships between body weight at `joining' and the number of cl of pregnancy that the age and parity effects on the numbers of cl, implantation sites and viable fetuses were due to differences between groups in body weight at mating.

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B. M. Bany and T. G. Kennedy

Interleukin 1α (IL-1α) stimulates prostaglandin production and cyclooxygenase activity in endometrial stromal cells isolated from the uteri of ovariectomized rats that have been sensitized for the decidual cell reaction. The aim of the present study was to examine the effect of IL-1α on the amount of cyclooxygenase mRNA and protein in these cells. Treatment with IL-1α (20 ng ml−1) for 24 h significantly increased steady-state concentrations of cyclooxygenase 2 (COX-2) mRNA and protein in the cells, as determined by northern and western blot analyses, respectively. Cyclooxygenase 1 (COX-1) mRNA and protein were not detected. Dexamethasone (5 μmol l−1) prevented the IL-1α-induced increase in COX-2 steady-state mRNA. Immunocytochemical staining of COX-2 in the treated cells indicated that IL-1α increased staining, while dexamethasone inhibited this increase. Furthermore, the changes in staining were generalized and not confined to a small subpopulation of cells. These data demonstrate that IL-1α increases steady-state concentrations of COX-2 mRNA and protein in endometrial stromal cells isolated from the uteri of rats that have been sensitized for decidualization.

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T. G. Kennedy and H. E. Ross

The ability of endometrial stromal cells from nonsensitized rat uteri to undergo decidualization in vitro was investigated. Cells were obtained by enzymatic dispersion from uteri of ovariectomized, steroid-treated rats at the equivalent of day 4, 5 or 6 of pseudopregnancy, or on day 5 from rats treated with 0, 0.3 or 1.0 μg oestradiol (low, intermediate or high doses of oestradiol, respectively) on day 4, and cultured for 24, 48 or 72 h. Decidualization in vivo, as assessed by uterine mass 5 days after the unilateral intrauterine injection of 100 μl sesame oil, was maximal for rats receiving the deciduogenic stimulus on day 5 and treated with the intermediate dose of oestradiol. Under control conditions in vitro, alkaline phosphatase (ALP) activity, the increase in ALP activity with time, and prostaglandin E2 (PGE2) accumulation in the medium were greatest for cells from maximally sensitized uteri. Indomethacin, an inhibitor of PG synthesis, reduced PGE2 accumulation to barely detectable amounts, and decreased ALP activity, especially in cells from maximally sensitized uteri, indicating that endogenous PG production contributed to the increase in ALP activity in these cells. The addition of PGE2 with indomethacin increased ALP activities. However, ALP activities were lower for cells derived from nonsensitized uteri when compared with cells from maximally sensitized uteri. These results suggest that endometrial stromal cells from nonsensitized uteri have a reduced capacity to undergo decidualization in vitro, and that this reduced capacity is not explained by differences in PGE2 production.

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B. M. Bany and T. G. Kennedy

The effect of epidermal growth factor on prostaglandin production and cyclooxygenase activity in endometrial stromal cells isolated from the uteri of ovariectomized rats sensitized for the decidual cell reaction was examined. Treatment with epidermal growth factor (40 ng ml−1) for 24 h caused approximately 6.9-, 3.4-, and 4.1-fold increases in prostaglandin E2, prostaglandin F and 13,14-dihydro-15-keto-prostaglandin F accumulation in the media of cultured cells. The increase in prostaglandin E2 accumulation induced by epidermal growth factor was inhibited by α-amanitin (2 μg ml−1), cycloheximide (0.5 μg ml−1) and dexamethasone (5 μmol l−1). Epidermal growth factor increased cyclooxygenase activity in the stromal cells in a time-dependent fashion and this increase in activity was also inhibited by α-amanitin, cycloheximide and dexamethasone. These results provide evidence that epidermal growth factor stimulates prostaglandin production in rat endometrial stromal cells from uteri sensitized for the decidual cell reaction through a mechanism that involves an increase in cyclooxygenase activity.

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P. M. Squires and T. G. Kennedy

Summary. Experiments were performed in vivo and in vitro to determine the effects of enalaprilat, a specific inhibitor of angiotensin-converting enzyme, on various aspects of the decidual cell reaction in rats. Ovariectomized, adult female rats were sensitized for the decidual cell reaction with steroid treatments. For in vivo experiments, intrauterine infusions of enalaprilat alone, and in combination with angiotensin II and prostaglandin E2 (PGE2), were initiated on the day of uterine sensitivity. Enalaprilat inhibited the increases in uterine PG concentrations, endometrial vascular permeability, alkaline phosphatase activity and uterine weight that occurred sequentially following infusion of vehicle. Concurrent infusion of angiotensin II did not reverse any of these inhibitory effects; PGE2 infusion partially, but not completely, reversed the inhibition of increase in uterine weight, although it did not alter the inhibition of endometrial vascular permeability. For in vitro experiments, endometrial stromal cells were obtained from uteri on the day of sensitivity and cultured for up to 3 days in the presence of enalaprilat and angiotensin II. Enalaprilat inhibited in a dose-dependent manner the increases in stromal cell alkaline phosphatase activity and media PGE concentration that occurred in the control cultures; these effects were fully reversed by concurrent treatment with angiotensin II. The inhibition of stromal alkaline phosphatase activity was also reversed by PGE2; conversely, the ability of angiotensin II to reverse the effect of enalaprilat was lost in the presence of indomethacin. These studies provide evidence of a requirement for angiotensin II during the decidual cell reaction in rats and suggest that it acts, at least in part, through a PG-dependent mechanism.

Keywords: angiotensin; decidualization; prostaglandins; uterus; rat

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C. A. Evans and T. G. Kennedy

Summary. In hamsters, localized areas of increased uptake of Evans Blue dye, representing the first uterine sign of blastocyst implantation, had an increased concentration of PGE and their appearance on Day 4 was prevented by treatment with indomethacin, an inhibitor of PG synthesis. Indomethacin treatment did not terminate pregnancy: the proportion of animals pregnant on Days 5, 7, 10 and 16 was not affected, although fetal mortality was slightly greater in these animals. Indomethacin treatment caused a decrease in the weights of Evans Blue sites on Day 5 and implantation swellings on Day 10, and the duration of gestation was slightly increased, indicating that implantation had been delayed. This delay did not result from changes of circulating progesterone levels or uterine blood supply. The PG synthesis inhibitors, indomethacin and meclofenamic acid reduced uterine PG concentrations and prevented the appearance of Evans Blue sites in ovariectomized pregnant hamsters treated with progesterone. It is suggested that PGs may be mediators in the uterine Evans Blue response which precedes implantation in the hamster.

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P. M. Squires, S. J. Dixon and T. G. Kennedy

Studies were performed to determine whether the inhibition of the decidual cell reaction induced by intrauterine infusion of the angiotensin converting enzyme inhibitor enalaprilat in rats is reversed by activation of Ca2+ influx. Influx of Ca2+ was shown to be stimulated by angiotensin II in endometrial cells in this study. Ovariectomized, adult female rats were sensitized for the decidual cell reaction with steroid treatments. For experiments in vivo, intrauterine infusions of enalaprilat alone, or in combination with the Ca2+ ionophore A23187, a synthetic diacylglycerol, and dioctanoyl-sn-glycerol (diC8), and PGE2 were initiated on the day of uterine sensitivity. Enalaprilat inhibited the increases in uterine PG concentrations and uterine weight that occur following infusion of the vehicle. Concurrent infusion of A23187 partially, but not completely, reversed the inhibition of uterine weight increase; diC8 did not affect the inhibition of enalaprilat. A23187 did not reverse the effects of enalaprilat on uterine PG concentrations. Concurrent infusion of A23187 and PGE2 fully reversed the inhibitory effect of enalaprilat on uterine weight. For experiments in vitro, endometrial stromal and epithelial cells were obtained from uteri on the day of sensitivity and maintained in suspension. Cytosolic free calcium concentration ([Ca2+]i) was monitored in cell suspensions by fluorescence spectrophotometry using the Ca2+-sensitive probe, indo-1. Angiotensin II induced a transient increase in [Ca2+]i of endometrial stromal cell suspensions, but not of epithelial cells; PGE2 did not increase [Ca2+]i in stromal or epithelial cells. The effect of angiotensin II on stromal cell [Ca2+]i occurred in the absence of extracellular Ca2+ and was blocked by pretreatment of the suspensions with the angiotensin II antagonist, saralasin. Maximal decidualization in rats appears to require the interaction of separate Ca2+-dependent and PG-dependent processes, both of which may involve angiotensin II.

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D. Martel, T. G. Kennedy, M. N. Monier and A. Psychoyos

Summary. Membrane preparations from endometria of rats in different physiological states (e.g. pseudopregnancy, ovariectomized animals receiving progesterone + oestradiol or oestradiol alone) were studied for [3H]PGF-2α binding by methods which detected PGF-2α binding in ovary preparations and PGE binding in the same endometrial preparations. There was no evidence of high-affinity binding sites for [3H]PGF-2α Saturable [3H]PGF-2α binding that increased with the onset of uterine sensitivity was detected but this binding does not fulfil all the criteria required for a PGF-2α receptor and is probably due to binding to PG metabolizing enzymes in our preparations, or to binding of [3H]PGF-2α to PGE binding sites.

The failure to detect specific PGF-2α binding sites seems to reflect a true absence of these sites in the rat endometrium.

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G. H. STABENFELDT, J. P. HUGHES, J. D. WHEAT, J. W. EVANS, P. C. KENNEDY and P. T. CUPPS

Summary.

The effect of hysterectomy on ovarian activity was studied in four mares. The cyclic secretion pattern of plasma progestins normally observed in the intact mare was interrupted by hysterectomy. Follicular activity was observed in all four hysterectomized mares, in spite of prolonged luteal activity, with a large number of follicles attaining ovulatory size without the occurrence of ovulation. Some ovulations were observed at irregular intervals in two out of four hysterectomized mares in spite of plasma progestin levels which ranged from 2 to 6 ng/ml. While all ovulations which occurred in the hysterectomized mares were followed by the formation of CL as determined by palpation per rectum, the rise of progestins in the peripheral plasma was variable as significant increases followed CL formation in some instances but not in others. Four mares had CL with functional life spans of 48, 74, 75 and 77 days at the time the experiment was terminated. There was no difference in the ability of the CL formed following these ovulations to convert pregnenolone to progesterone (per unit weight) as compared to CL of 9 days duration. The longest estimated CL life span in any hysterectomized mare was 137 days. Sexual receptivity did not occur in conjunction with follicular growth and ovulation in the hysterectomized mares in the presence of an active CL.