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Summary.

The histochemical localization of acetylcholinesterase (AChE)-containing nerves has been determined in the parametrial arterial supplies of dog, cat, rat, rabbit, sheep, pig and cow.

The main uterine arteries of dog and pig contained a plexus of nerves which stained for AChE and which were closely applied to the surface of the media. This plexus was absent from the secondary arterial branches. In contrast, the equivalent arteries of rat, cat, rabbit, sheep and cow were devoid of an AChE-positive nerve plexus.

In the light of other evidence, it is suggested that the uterine hyperaemia of pregnancy in dog and pig may be partly due to the action of cholinergic vasodilator nerves. Such a mechanism is unlikely to be involved in the other species examined.

Summary. The synthesis of a soluble protein (referred to as 'decidualization-associated protein', DAP), has been examined in uterine and placental tissues of rats during pregnancy by means of polyacrylamide gel electrophoretic analysis of [³H]leucine-labelled soluble proteins. No synthesis of the protein was detected in non-implantation regions of the uterus. In implantation site tissue, no synthesis was detected on Days 6 or 7 of pregnancy. Only slight synthesis was present in the endometrium on Day 8, but synthesis rose rapidly from Days 9 to 12 in both the endometrium and myometrium although differences in the rates of increase were observed. Synthesis fell from Day 12 to 14 in both tissues. Synthesis by the myometrium was entirely localized in the mesometrial region, which contains the metrial gland.

After Day 12, when the endometrium is represented by the chorioallantoic placenta, synthesis was examined in the labyrinthine and the decidua basalis/basal zone placenta tissues. No synthesis of 'DAP' was detected in the labyrinthine placenta from Day 16 of pregnancy. Synthesis observed in the decidua basalis/basal zone placenta fell dramatically from Day 14 to 20. The pattern of synthesis of 'DAP' during pregnancy suggests a role in the establishment of the chorioallantoic placenta and metrial gland in the rat.

Summary. The guinea-pig uterine artery responded to acetylcholine (ACh) with vasodilatation only during pregnancy or after oestrogen treatment. Even with high doses (1 mg/day) oestradiol-17β esters had to be administered for several days to effect sensitization to ACh, but oestradiol-17β itself was active within a few hours. Oestriol was equipotent with oestradiol. Sensitization was prevented when protein synthesis was inhibited over the period of oestrogen administration, but was not dependent on the integrity of the cholinergic vasodilator nerve supply to the artery.

Summary. During unilateral pregnancy in the guinea-pig there is loss of formaldehyde-induced fluorescence from the adrenergic nerves supplying the uterus and its vasculature. This loss occurs initially near the site of implantation at about Day 20 of gestation and spreads progressively. Implantation of wax pellets containing progesterone into the uterine lumen or the gastrocnemius muscle of virgin guinea-pigs for 7 days produced loss of fluorescence from all local adrenergic nerves. No diminution of fluorescence was seen when pellets containing oestradiol were substituted. Chronic denervation studies showed that the adrenergic axons supplying the uterus and its arteries originated from both the ovarian artery and the pelvic region. Our results suggest that loss of adrenergic fluorescence within the uterus during pregnancy is due to an effect of placental progesterone which is localized to the uterus because the high concentration of progesterone necessary to cause fluorescence loss is not attained in the systemic circulation.

Summary.

The autonomic innervation of the rat testicular capsule has been investigated pharmacologically and histochemically. Electrical field stimulation of isolated capsules produced contractile responses which were abolished, or largely depressed, in the presence of bretylium (2 to 4× 10⁻⁶ g/ml). Any residual response was abolished in the presence of hyoscine (5 × 10⁻⁷ g/ml). No evidence for the presence of a relaxatory innervation was obtained. Fluorescence histochemistry demonstrated the existence of a dense plexus of fine varicose fibres exhibiting catecholamine fluorescence in the mediastinal region, and of more scattered fluorescent fibres in other areas. The blood vessels in the region of the rete testis also received a dense innervation by fluorescent fibres. Fibres which stained heavily for acetylcholinesterase were sparsely distributed in the mediastinal and non-mediastinal regions of the capsule, but were absent from the blood vessels. It is concluded that the capsule receives both adrenergic and cholinergic motor nerves, the adrenergic supply predominating, and that acetylcholinesterase positivity can be used as a criterion of cholinergic autonomic nerves in the rat, at least with the preparative conditions employed by us.

Summary.

Retrograde arteriography has been used to examine the effect of pregnancy on the diameters of the main uterine, internal iliac and inferior epigastric arteries in the guinea-pig. The diameters of the internal iliac and inferior epigastric arteries were not altered during gestation. By contrast, the diameter of the main uterine artery supplying a pregnant horn of the uterus increased in diameter to a mean value of 167% of its diameter in non-pregnancy. In cases of unilateral pregnancy, the uterine artery supplying the non-pregnant horn of the uterus was not increased in diameter. Administration of atropine (0·1 to 0·5 mg/kg) to animals in late pregnancy caused constriction of the enlarged uterine arteries to a mean value of 137% of their pre-pregnant diameter, but the diameters of the internal iliac and inferior epigastric arteries were not changed. In non-pregnant animals, the diameter of the uterine arteries was not affected by atropine. The results support previous evidence for an effector rôle of cholinergic vasodilator nerves in the production of uterine hyperaemia during pregnancy in the guinea-pig.

The distribution of the α₁, α₃–α₆, β₁, β₃ and β₄ integrin subunits in fetal membranes at term was examined using an indirect immunofluorescence technique and confocal laser scanning microscopy. In the amniotic epithelium, β₄ integrin (α₆β₄) exhibited distinct basal localization, whereas β₁ integrins (α₃β₁, α₅β₁) were localized basolaterally. This finding suggests that integrins, especially α₆β₄ which is a structural component of the hemidesmosomes, may function as basement membrane receptors. Integrins localized laterally may play a role in cell–cell interactions. β₁ (α₁β₁, α₅β₁) integrins are probably involved in cell–matrix interactions in the connective tissue layers which are rich in collagens and fibronectin. Cytotrophoblasts, located predominantly towards the chorionic basement membrane, mainly expressed α₆β₄, while those located predominantly in the vicinity of decidua expressed α₅β₁, α₃β₁ and α₁β₁. Decidual cells expressed α₃β₁ and α₁β₁, whereas α₁, α₅, α₆, β₁ and β₄ were expressed in blood vessels. This pattern of integrin expression reflects the reported difference in composition of the extracellular matrix at these locations and obviates an important role for α₅β₁ at the chorio–decidual interface. The differential integrin expression at the cell–basement membrane interfaces demonstrated in this study (at amniotic epithelium, cytotrophoblasts, decidual cells and blood vessels) indicated a differential recognition of basement membranes by these cells. β₄ may have a specialized function at the blood vessels, and possibly in cytotrophoblasts, that is distinct from its role in hemidesmosome-mediated attachment. This study suggests that integrins may be involved in cell–matrix and cell–cell adhesions in fetal membranes and may therefore be important for maintaining their normal structural and functional integrity.

Summary. A murine pregnancy-associated protein (α₁-PAP) with α₁-electrophoretic mobility and an estimated molecular weight of 150 000 was present in serum from pregnant C57BL/10 mice but could not be detected in serum of mature non-pregnant females and males. During pregnancy α₁-PAP was first detected on Day 7, rose to maximum levels between Days 12 and 14, and thereafter declined during the remainder of pregnancy and was undetectable by Day 8 post partum. The protein was also detected in the serum of females, but not males, subjected to an inflammatory stimulus. Examination of the α₁-PAP levels during an acute-phase response in females demonstrated that the protein behaved as a typical classical acute-phase reactant, although the levels were only 10% of those observed during Days 12 to 14 of pregnancy. α₁-PAP therefore appears to represent a hitherto undescribed female-specific acute phase reactant in the mouse.

Summary. Intrauterine instillation of heat-deaggregated glycogen in virgin mice induced a marked but transient luminal infiltration of polymorphonuclear leucocytes (PMNL). Similar injections of glycogen 2 days post coitum resulted in termination of pregnancy in the majority of treated females. Embryos recovered on Day 4 of pregnancy from the glycogen-treated females, before the expected time of implantation, had developed to the cavitated blastocyst stage, and were capable of undergoing characteristic outgrowth when cultured in vitro. A small proportion, however, appeared abnormal and did not develop in vitro. Cavitated blastocysts obtained from control mated females did not undergo outgrowth in vitro in the presence of intact PMNL, homogenates of these cells or a low molecular weight (<1500) fraction of the homogenates and after culture appeared similar to the abnormal blastocysts recovered from glycogen-treated uteri. It is proposed that the anti-fertility effect of PMNL infiltrates is due to a low molecular weight component of these cells which acts in utero and is cytotoxic to preimplantation blastocysts before their hatching from the zona pellucida.

Summary. Mouse uterine cells were obtained by trypsinization of uteri at timed intervals after the induction of a decidual reaction by intraluminal instillation of arachis oil on Day 4 of normal pregnancy. Cells were also obtained from ovariectomized mouse uteri, some of which had received a progesterone–oestradiol sequence to sensitize the uterus to a decidual stimulus. The differentiation of decidual cells was followed in cultures of these cells. The morphology of the cells obtained after 6 days in culture was dependent upon the seeding density employed. At low seeding density (plating densities of 75–100 cells/mm²) no net increase in cell number was observed, but large mononucleated stellate cells were present, with cytoplasmic and nuclear areas increased by 4-fold. At higher seeding densities (plating densities of up to 709 cells/mm²), a prolongation of cell survival and the appearance of substantial numbers of binucleated cells were observed. However, both cell types were characterized by the accumulation of filamentous material in the cytoplasm. Even at optimal seeding density the life-span of the decidualized cells could not be prolonged beyond 9 days. Uterine cells from hormone-treated ovariectomized animals underwent similar transformations but those from untreated ovariectomized mice gave only isolated islets of epithelial cells and scattered fibroblast-like cells in culture. These observations suggest that discrepancies in previous reports of in-vitro decidualization of rat uterine cells result from differences in the seeding densities employed.