Protocollagen proline hydroxylase of the mouse uterus during pregnancy and post-partum involution

1. The protocollagen proline hydroxylase in mouse uterus was found to be similar to that in other animal sources in its subcellular distribution and cofactor requirements. 2. The activities of this enzyme in uterine tissue from non-pregnant mice were comparable with those in various embryonic tissues. 3. In the second half of pregnancy the protocollagen proline hydroxylase activity increased markedly. 4. After parturition the activity of this enzyme decreased rapidly, reaching normal non-pregnant values at 24h post partum. The results suggest a good correlation between the synthesis of collagen and the activity of protocollagen proline hydroxylase.     

Oestradiol inhibits collagen breakdown in the involuting rat uterus

1. The earlier observation (Woessner, 1969) of oestradiol inhibition of collagen breakdown is confirmed and extended. Administration of 100mug of oestradiol-17beta/day to parturient rats strongly inhibits the loss of collagen from the involuting uterus. Three experiments show that this effect is due to an inhibition of collagen degradation rather than to a stimulation of collagen synthesis. 2. Uterine collagen was labelled with hydroxy[(14)C]-proline by the administration of [(14)C]proline near the end of pregnancy. By 3 days post partum, control uteri lost 83% of their collagen and 90% of their hydroxy[(14)C]proline. Uteri from oestradiol-treated rats lost only 50% of both total and labelled hydroxyproline, with no decrease in the specific radioactivity of the hydroxyproline. 3. Incorporation of [(14)C]proline into uterine collagen hydroxyproline in vivo was not affected by oestradiol treatment. 4. Urinary excretion of hydroxyproline was increased in post-partum control rats and decreased in oestradiol-treated rats. 5. An enzyme capable of cleaving 4-phenylazobenzyloxycarbonyl-l-prolyl-l-leucylglycyl- l-prolyl-d-arginine (a substrate for clostridial collagenase) increased in activity in the post-partum uterus and was unaffected by oestradiol treatment. 6. Uterine homogenates digested uterine collagen extensively at pH3.2. This digestion was unaffected by the oestradiol treatment. 7. Lysosomal fractions prepared by density-gradient centrifugation of uterine homogenates contained coincident peaks of cathepsin D activity and peptide-bound hydroxyproline. The cathepsin D and hydroxyproline contents of this peak were unaffected by oestradiol treatment.     

An electron microscopic study of the post-partum involution of the rat uterus. With a note on apparent crinophagy of collagen

During pregnancy the increase in mass of the rat uterus has been accounted for mainly by hypertrophy rather than hyperplasia of the smooth muscle cells and by an increase in collagen content. Following parturition the rat uterus regains its non-pregnant weight within little more than a week. Autophagy is a likely mechanism for the reduction in size of the smooth muscle cells. However, many previous investigations have failed to demonstrate an extensive lysosomal apparatus in uterine smooth muscle cells, a morphologically detectable prerequisite for augmented autophagy. The superfluous collagen has been assumed to be digested by macrophages. In the present electron microscopical study the involution of the rat uterus was followed at various time points post-partum. The results indicated that three pathways were involved namely, autophagocytosis, heterophagocytosis and crinophagy. Autophagocytosis was noted in smooth muscle cells--apparently a means to reduce their size. In some instances extensive autophagy appeared to lead to cell death. Macrophages and also to some extent fibroblasts have the ability to endocytose collagen and cell debris including split off portions of smooth muscle cell cytoplasm. The evidence obtained suggested that at the time of parturition the (pro)collagen synthesized but not yet secreted was retained within the fibroblasts and degraded by means of crinophagy.     

Inhibition by oestrogen of collagen breakdown in the involuting rat uterus

1. Both the post-partum involution of the rat uterus and the rapid breakdown of collagen that accompanies it are extensively inhibited by oestrogenic hormones. In the normal rat, 85% of the uterine collagen is degraded within 4 days after parturition; in rats treated with 100μg. of 17β-oestradiol/day, only 35% of uterine collagen is broken down in the same period. 2. Similar effects are produced by diethylstilboestrol if the dose is increased tenfold. 3. Collagen breakdown is inhibited to a greater extent than is the loss of wet weight by oestradiol but not by diethylstilboestrol. 4. The oestrogens appear to act by blocking the breakdown of collagen. There is a greatly decreased concentration of free hydroxyproline in the uterus of treated animals. 5. Acid hydrolase concentrations (β-glucuronidase, β-galactosidase, cathepsin D and acid phosphatase) in the uterus are decreased by oestrogen treatment compared with controls, but the total amounts of these enzymes in the uterus are somewhat elevated. Oestrogens do not appear to inhibit collagen breakdown by altering the concentration and total amount of acid hydrolases.     

Ca2+-activated proteinase in the rat. Quantification by immunoassay in the uterus during pregnancy and involution, and in other tissues.

Rat uteri were taken at various stages of pregnancy and involution post partum, and several other tissues were taken from pregnant and non-pregnant animals. Portions of each tissue were homogenized in the presence of proteinase inhibitors, and the amounts of the high-Ca2+-requiring Ca2+-activated proteinase in the supernatants were measured by a two-site immunoradiometric assay using 125I-immunoglobulin G. The proteinase was shown, by protein blotting, to be immunologically identical in all tissues. The amounts in the various tissues, expressed in units of proteinase activity/g wet wt., were: lung, 95; kidney and small intestine, 42; liver, 20; brain, heart and skeletal muscle, 13. Uterine wet weight increased at the end of pregnancy by about 8-fold, but the amounts of proteinase per uterus increased by about 22-fold; alternatively, expressed in units of proteinase activity/g wet wt., the mean uterine values were: non-pregnant, 28.6; term-pregnant, 77.0. As the wet weight of the uterus fell rapidly during involution, the amounts of proteinase activity remained relatively high. The data suggest that the Ca2+-activated proteinase may have some role in tissue resorption during uterine involution, but the high proteinase activity present before parturition must be regulated in ways which are not yet clear.     

Decidualization in the post-partum uterus of the mouse

Unilaterally ovariectomized mice were allowed to complete one pregnancy before the second ovary was removed and sensitivity to decidualization was induced by hormone administration. The virgin uterine horn and the post-partum horn were stimulated to decidualize by the intraluminal injection of arachis oil. A greater decidual response was found in post-partum horns than in virgin horns. The foci of decidual induction in post-partum horns were regularly spaced reflecting the regular spacing of used and unused areas of the uterus. A focus of decidual induction occurred in 68% of the recently used uterine areas observed, i.e. areas associated with post-partum nodules. When compared with foci of decidualization in adjacent unused areas of the uterus, none of the decidualized used zones showed more decidual tissue, about half showed less decidual tissue and half showed the same amount of decidual tissue. The remaining 32% of used zones were not associated with foci of decidual induction. The results indicate that decidualization can be induced in recently used zones of the uterus using a non-traumatic method of induction, but that used zones are associated with less decidual tissue than unused zones of the post-partum uterus.     

Extracellular breakdown of collagen as affected by lysosomal enzymes during the involution of liver cirrhosis

Electron microscopy and electron histochemistry (exposure to acid phosphatase) were used to study the mechanisms of extracellular degradation of collagen in the liver during involution of experimental cirrhosis. The following results were obtained: extracellular secretion of lysosomal enzymes from hepatocytes and connective tissue cells takes place in liver cirrhosis and its involution; partial hepatectomy during liver cirrhosis stimulates the activity of acid phosphatase in the liver cells; the lysosomal enzymes, excreted from hepatocytes and connective tissue cells by means of exocytosis take an active part in collagen extracellular degradation in vivo; at initial stages of cirrhosis involution extracellular degradation of collagen in the liver occurs at the expense of lysosomal enzymes from hepatocytes and connective tissue cells. Subsequently, as cirrhosis regresses, the principal role in the lysis of collagen gradually passes to lysosomal enzymes of hepatocytes.     

The collagen substrate specificity of rat uterus collagenase

The collagen substrate specificity of rat uterus collagenase was studied as a function of both collagen type and species of substrate origin. For each collagen examined, values for the basic kinetic parameters, Km and Vmax (kcat), were determined on collagen in solution at 25 degrees C. In all cases, Lineweaver-Burk plots were linear and rat uterus collagenase behaved as a normal Michaelis-Menten enzyme. Collagen types I, II, and III of all species tested were degraded by rat uterus collagenase. Collagen types IV and V were resistant to enzymatic attack. Both enzyme-substrate affinity and catalytic rates were very similar for all susceptible collagens (types I-III). Values for Km ranged from 0.9 to 2.5 X 10(-6) M. Values for kcat varied from 10.7 to 28.1 h-1. The homologous rat type I collagen was no better a substrate than the other animal species type I collagens. The ability of rat uterus collagenase to degrade collagen types I, II, and III with essentially the same catalytic efficiency is unlike the action of human skin fibroblast collagenase or any other interstitial collagenase reported to date. The action of rat uterus collagenase on type I collagen was compared to that of human skin fibroblast collagenase, with regard to their capacity to cleave collagen as solution monomers versus insoluble fibrils. Both enzymes had essentially equal values for kcat on monomeric collagen, yet the specific activity of the rat uterus collagenase was 3- to 6-fold greater on collagen fibrils than the skin fibroblast enzyme. Thus, in spite of their similar activity on collagen monomers in solution, the rat uterus collagenase can degrade collagen aggregated into fibrils considerably more readily than can human skin fibroblast collagenase.     

Bacteriological investigation of the postpartum uterus: Relationship to involution and histopathological findings

A bacteriological investigation of the postpartum uteri of suckled and nonsuckled Bunaji (White Fulani) cows was undertaken to determine the microflora, the effect of suckling on the flora and the influence of the flora on uterine involution and histopathology. Uterine contamination by bacteria was highest between days 10 and 21 postpartum with Staphylococcus aureus being the commonest organism isolated. There was no consistent isolation of one type of bacterial organism from a cow throughout the sampling period. Suckling did not affect the incidence of uterine contamination by bacteria. Foci of leucocytes (mainly lymphocytes) were found in uterine sections in an increasing frequency with days postpartum. Bacteria were isolated from only 3 of the 12 uteri that had the leucocytic foci. Uterine involution was normal and complete by 25 days after calving irrespective of the presence or absence of bacteria or leucocytic foci. We concluded that postpartum uterine contamination by bacteria was not influenced by suckling and that puerperal uterine contamination by bacteria did not interfere with the normal process and duration of uterine involution.