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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.

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.

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. 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.

Summary. Pregnancy-associated endometrial α₂-globulin (α₂-PEG), the major secretory protein of the human uterine endometrium during the luteal phase of the menstrual cycle and early first trimester of pregnancy, has been detected by immunochemical techniques in seminal plasma. Biochemical analysis and immunoblotting has verified that immunoreactive α₂-PEG in seminal plasma exhibits properties identical to those of endometrial α₂-PEG, i.e. Concanavalin A-binding dimeric glycoprotein of native M _r 56000, subunit M _r 28 000, average pI 4·7 and of α₂-mobility. Concentration of α₂-PEG in seminal plasma was 22·13 ± 2·82 μg/ml (mean ± s.e.m., n = 110) which compared to 12·02 ± 1 ·65 μg/ml (mean ± s.e.m., n = 48) found in amniotic fluid at 11–20 weeks of pregnancy, to 4·29 ± 1·66 μg/ml (mean ± s.e.m., n = 15) in uterine luminal fluid in women during the luteal phase and to 0·245 ± 0·025 μg/ml (mean ± s.e.m., n = 10) in sera at 10 weeks of pregnancy. This distribution is very different from that observed for pregnancy-associated placentally-derived serum proteins detected in seminal plasma. The source of α₂-PEG in seminal plasma is unknown but is unlikely to be the testis because of the normal levels observed in vasectomized men. In the endometrium α₂-PEG synthesis and secretion appears to be related to progesterone-dependent differentiation of the glandular epithelium. Therefore these observations suggest that a different mechanism of regulation of the gene for α₂-PEG operates in the male reproductive tract.

Summary. Monoclonal and polyclonal antibodies to pregnancy-associated endometrial α₂-globulin (α₂-PEG), a glycosylated human β-lactoglobulin homologue, were used in an immunohistological technique to determine the cellular localization of this protein in the decidua and placental tissues during pregnancy. During the first trimester the protein was principally localized to the glandular epithelium of the decidua spongiosa region of the endometrium with only weak staining associated with glands of the decidualized decidua compacta region. No significant cellular staining was detected in the decidua capsularis. At term in the decidua of the amniochorion and the placental bed weak staining for α₂-PEG was only associated with the epithelium of attenuated glands. No significant staining was detected in the placenta during pregnancy. These results suggest that the epithelium of glands associated with non-decidualized stroma represents the primary source of α₂-PEG during the first trimester and that a function of the decidua spongiosa in early pregnancy may be related to production of α₂-PEG. The decline in production of α₂-PEG during pregnancy is suggested to result from involution of the decidua spongiosa and at term the attenuated glands of the decidua represents the source of α₂-PEG.

Keywords: endometrium; decidua; placenta; pregnancy-associated proteins; β-lactoglobulin homologue; man

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.