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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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Lisa M. Williams, M. Hollingsworth, and J. S. Dixon

Summary. The isolated cervix from non-pregnant, early Day 22 pregnant (late pregnant) and parturient rats was studied. Cyclic loading and unloading tensile tests showed that the creep properties of the cervix were greater in late pregnant than in non-pregnant rats. This change in tensile properties, or softening, was associated with a marked rise in % of water and a fall of collagen concentration (as % of wet but not dry weight). These findings, plus electron micrographs showing a marked increase in extracellular matrix, separation of bundles of collagen fibrils and active fibroblasts, suggest that softening is related to controlled tissue hydration. Cervices were also removed from 3 groups of rats killed at different times during the 3-h parturient period and creep rate measured by continuous loading. A nearly 3-fold increase in creep rate over this period indicates that there is a second stage of cervical softening in pregnancy which precedes dilatation at parturition.

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Samples of pooled cervical mucus from groups of animals fed melengestrol acetate (MGA) for 14 days were taken 3 and 4 days after cessation of treatment. The mucus samples were separated into protein and glycoprotein portions and the epithelial glycoprotein was isolated. The protein fraction was examined by disc electrophoresis and the epithelial glycoprotein was characterized by chemical analysis and determination of the molecular weight. The cervical mucus of one of the treated groups yielded a glycoprotein which had a reduced sulphate content when compared with that of the controls and did not show the lowered neuraminic acid content characteristic of the glycoprotein from untreated animals at oestrus. Two other groups of MGA-treated animals which received HCG 3 days after withdrawal of the progestagen did not show these anomalies.

The protein in the MGA-treated samples of mucus contained considerably larger amounts of material migrating in the transferrin region of the electrophoretogram than did comparable controls.

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N. C. Jackson, H. Jackson, J. H. Shanks, J. S. Dixon, and R. G. Lendon

Summary. Gonadotrophin binding to rat Leydig cells after a single administration of ethylene dimethanesulphonate (EDS) (75mg/kg i.p.) was followed by using intratesticular microdoses of 125I-labelled hCG, whilst corresponding morphological changes in the testicular interstitium were studied with light and electron microscopy. No discernible effect on 125I-labelled hCG binding compared with controls was observed until 24 h after treatment. Between 24 and 32 h a sharp decline in binding occurred which was correlated with extensive Leydig cell destruction. By 48 h the 125I-labelled hCG binding was negligible and no morphologically recognizable Leydig cells were found at this time. The specific binding remained low until 21 days after treatment and then a marked increase occurred to give nearly normal levels by 49 days. This was consistent with a generalized repopulation of the interstitium with Leydig cells, seemingly the result of differentiation of fibroblast-like precursor cells.

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A. Zaidi, R. G. Lendon, J. S. Dixon, and I. D. Morris

Summary. Male rats were injected with 50 mg ethylene-1,2-dimethanesulphonate/kg from Day 5 to Day 16 after birth and control rats received injections of the same volume of vehicle. Testes were studied at various times from Day 6 to Day 108 using histochemistry, light and electron microscopy. Fine structural degenerative changes were observed in the Leydig cells and seminiferous tubules of EDS-treated animals as early as Day 6. By Day 11 no Leydig cells could be detected and the interstitia of EDStreated testes contained large numbers of fibroblast-like cells which formed peritubular collars 3–5 cells thick; the tubules contained Sertoli cells with heterogeneous inclusions and large numbers of lipid droplets. A small number of Leydig cells was found at Day 14 and their numbers increased so that, in animals of 28 days and older, large clusters of Leydig cells were present between severely atrophic tubules. These tubules contained Sertoli cells with few organelles; germinal cells were not observed after 28 days in EDStreated animals.

These results show that EDS destroys the fetal population of Leydig cells postnatally and this mimics the well documented effect of EDS on adult Leydig cells. The seminiferous tubules were permanently damaged by EDS in the present experiments. Tubular damage could have been due to a direct cytotoxic effect of multiple injections of EDS on the tubule before the blood–testis barrier develops or due to withdrawal of androgen support secondary to Leydig cell destruction.

Keywords: testis; Leydig cell; development; EDS; cytotoxicity; rat

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S C Town, C T Putman, N J Turchinsky, W T Dixon, and G R Foxcroft

Unmodified, third parity, control sows (CTR; n = 30) or sows subjected to unilateral oviduct ligation before breeding (LIG; n = 30), were slaughtered at either day 30 or day 90 of gestation and used to determine the effects of numbers of conceptuses in utero on prenatal, and particularly muscle fibre, development. Ovulation rate, number of conceptuses in utero, placental and fetal size, and (day 90 sows) fetal organ and semitendinosus muscle development were recorded. Tubal ligation reduced (P < 0.05) the number of viable embryos at day 30 and fetuses at day 90. Placental weight at day 30 and day 90, and fetal weight at day 90, were lower (P < 0.05) in CTR sows. All body organs except the brain were lighter, and the brain:liver weight ratio was higher in CTR fetuses (P < 0.05), indicative of brain sparing and intrauterine growth restriction in fetuses from CTR sows. Muscle weight, muscle cross-sectional area and the total number of secondary fibres were also lower (P < 0.05) in CTR fetuses. The number of primary fibres, the secondary:primary muscle fibre ratio, and the distribution of myosin heavy chain-Iβ, -IIa, fetal and embryonic isoforms did not differ between groups. Thus, even the relatively modest uterine crowding occurring naturally in CTR sows negatively affected placental and fetal development and the number of secondary muscle fibres. Consequences of more extreme crowding in utero on fetal and postnatal development, resulting from changing patterns of early embryonic survival, merit further investigation.