SUMMARY

1. 216 mice, spayed on the 14th day of their first pregnancies, were injected daily thereafter with progesterone, either alone, with oestradiol or relaxin extracts or both; no injections were given after parturition. Extracts of relaxin obtained from pregnant rabbit serum and pregnant sow ovaries were used.

2. When given alone, 1 mg/day progesterone maintained pregnancy to full term in 83%, and 0·5 mg/day in only 30% of mice. Parturition was often delayed, prolonged and difficult, with consequent death of the foetus in utero, even when injections were stopped on the 18th day. A high proportion of young were stillborn, and none were reared. The symphysis pubis remained closed and cartilaginous.

3. 1·5 μg/day oestradiol did not synergize the action of progesterone in maintaining pregnancy, but there were fewer delayed deliveries and a higher incidence of live births; a small number of mice reared some of their young. The innominate bones did not separate, but the symphysis was less rigid than when progesterone was given alone.

4. Relaxin extracts had a synergizing action when all three hormones were given together, 0·5 mg/day (but not 0·25 mg) of progesterone then being enough for maintenance of pregnancy in over 80% of mice. The experiments have not established whether oestradiol was necessary for this synergism. There was a positive relation between punctual and normal delivery of live young and potency of the relaxin extracts in producing full pregnancy changes in the pelvis. More mice reared some of their young. None of the hormone combinations used completely compensated for the absence of the ovaries.

SUMMARY

1. In uninjected ovariectomized mice, the matrix of the symphysial cartilage, like that outside the symphysis, is strongly metachromatic. Following nine daily injections of 1·5 μg oestradio, metachromasia disappeared from the caudal part of the symphysis and decreased in the cranial part, accompanied by swelling of the matrix. Cartilage outside the symphysis was not affected. Hyaluronidase banished metachromasia from both symphysial and extrasymphy. sial cartilage but did not cause swelling of the matrix. Disappearance of metachromasia is attributed to depolymerization or breakdown of the molecules of chondroitin sulphate, resulting in a more pliable ground substance. Swelling is tentatively attributed to uptake of water.

2. One injection of relaxin at the end of such pretreatment produced, by lateral displacement of the two halves of the pelvis, a gap in the middle of the cartilage. Subsequent injections increased this displacement which, it is suggested, may be an indirect effect of relaxin acting through tensile forces mediated in some way as yet unknown.

3. When large doses of progesterone were given simultaneously, oestradiol was less effective in abolishing metachromasia.

4. It is suggested that during pregnancy, oestrogens, perhaps through local liberation of an enzyme, break down the polysaccharides of the symphysial cartilage and make the matrix sufficiently pliable to respond to tensions set up by relaxin during the last week of pregnancy.

5. Separation of the innominates, produced by prolonged treatment with oestradiol alone, is a different process brought about by resorption of bone on either side of the symphysis and not involving lateral displacement of the two bones.

SUMMARY

A re-investigation has been made of the histological changes in the tissues of the symphysis pubis of the mouse during the first pregnancy and after parturition, using the techniques of metachromatic staining with toluidine blue, the McManus-Hotchkiss periodic acid-Schiff (p.a.s.) reaction, and basophilic staining with haematoxylin.

During the last week of pregnancy two maj or changes take place: (1) resorption of the anteromedial bony walls of the innominates leading to shortening of the symphysis, and (2) transformation of the symphysial cartilage, manifested by loss of metachromasia and basophilia and ability to react with p.a.s., leading to ultimate disappearance of stainable chondroid matrix and its replacement first by argyrophil and then by collagenous connective tissue. Further resorption of bone from the symphysial walls of the innominates and, probably, retention of fluid in the newly formed connective tissue helps to widen the interpubic gap.

After parturition, the collagenous connective tissue which had replaced the cartilage and resorbed bone reverts to a mesenchyme-like tissue, the cells of which differentiate into osteoblasts or chondroblasts or haemopoietic cells. Cartilage differentiates by deposition of metachromatic chondroid matrix around chondroblasts, the matrix becoming more and more metachromatic, p.a.s.-positive and basophilic. Bone differentiates, first in connective tissue, later by endochondral ossification. Marrow begins to differentiate even before the bone which will confine it is laid down.

The effect of hyaluronidase on metachromasia, basophilia and reaction to p.a.s. of the symphysial tissues is described, and its significance discussed in relation to the pregnancy and post-partum changes.

1. The effect on the symphysis pubis of oöphorectomized mice of oestrone (1·5 μg./day) and progesterone (1·0 mg./day) injected separately and simultaneously for 8–20 days has been investigated histologically.

2. A brief course of treatment with oestrone had a definite effect on the pelvis, greater than appears on X-ray photographs, but not comparable in intensity with that which follows the addition of relaxin.

3. The action of oestrone affected the dorsal bony walls of the two innominates, and started at the anterior symphysial borders. Simultaneous resorption of the outer or periosteal surfaces, and deposition of new bone on the endosteal surfaces occurred, until the marrow cavities were obliterated at least in the medial parts of the two innominates. Resorption then continued so that the solid symphysial borders of the bones gradually disappeared. No ligament proliferation occurred.

4. A vascular, richly nucleated tissue replaced the resorbed dorsal surfaces of the bones, and was thought to be derived mainly from the marrow. Many osteoclasts were present along the eroding surfaces but the origin and function of these enigmatic cells is still undecided.

5. When progesterone was given for periods up to 20 days the histological structure of the symphysis pubis was similar to that of untreated control animals.

6. When oestrone and progesterone were given simultaneously for periods up to 20 days changes were produced of the same type as, but much less pronounced than, those produced by oestrone alone.

7. The results of histological investigation support the conclusion that progesterone is not only ineffective in producing relaxation of the pelvis in the mouse but that, at least in certain dose ratios, it inhibits the action of oestrone on the symphysis.

The experiments described are part of an attempt to discover why, in the response of the mouse pelvis to oestrogens, the anterior border and dorsal aspect of the symphysis pubis are always affected first, and in particular whether the reproductive tract (part of which lies in close apposition to the dorsal surface of the symphysis) plays a role in the reaction.

Ninety-one ovariectomized albino mice were used in five experiments. Oestrone in doses of 3–30 μg./day was injected subcutaneously for 8–24 days into animals with the reproductive tract intact, or from which uterine horns, or cervix, or cervix and vagina had been removed. The pelves were examined by means of X-ray photographs, macroscopically at autopsy, and by histological investigation.

The results showed that the action of oestrone on the symphysis pubis is not affected by removal of the uterine horns, cervix or vagina, and it is therefore unlikely that these structures play any role in the reaction.

SUMMARY

The distribution of histochemically demonstrable 5′-nucleotidase activity in the uterus of mice around the time of nidation, is compared with those of specific and non-specific acid phosphatases, alkaline phosphatase, phosphorylase, and with deposition of glycogen. The sequences of changes in distribution were similar during normal (first pregnancy) or delayed (by lactation) implantation, or when implantation was induced in lactating mice by administration of hormones.

5′-Nucleotidase activity was located at the free border of the luminal epithelium and at surfaces of stromal cells and muscle fibres up to the time of implantation, and in non-decidualized areas after implantation, but disappeared from differentiated decidual cells. The disappearance of 5′-nucleotidase activity from decidual cells occurred at the time when their mitotic activity was waning and when alkaline phosphatase activity had appeared at their surfaces, and lagged slightly behind both glycogen deposition and increased phosphorylase activity in these cells. There was parallel distribution of phosphorylase activity and glycogen distribution within decidua.

At the time of implantation, a reaction appeared in stromal cells in the immediate vicinity of the blastocyst after incubation with either adenosine-5′-monophosphate or glycerophosphate at acid pH, and it spread in area during the next 24 h. It was believed to indicate activity of a non-specific acid hydrolase, possibly lysosomal.

In all uteri examined, epithelial cells of glands showed specific acid phosphatase, but no 5′-nucleotidase activity.

SUMMARY

Lipids in the uterine endometrium of mice during early pregnancy were investigated by histochemical techniques. Three groups of animals were studied: (1) on days 1–8 inclusive of the first pregnancy, (2) on days 1–9 after mating at the post-partum oestrus and suckling seven or eight young from their first pregnancy, (3) on days 1–6 after delivery of the first litter but with no access to males. Day 1 was that on which a seminal plug appeared in the vagina or (group 3) after birth of a litter during the previous night. In post-partum mice, only the areas between the former implantation sites were studied.

Epithelial lipids are sparse during the first 24 h. On day 2 and early day 3 in nulliparous mice, and for a longer period in post-partum mice, the presence of lipid in epithelium and stroma seems mainly a degenerative phenomenon associated with cell breakdown and consequent tissue disruption. During late day 3 and on day 4 the increase in lipid droplets giving staining reactions characteristic for triglycerides may represent the storage in this form of fatty acids no longer needed for phospholipid synthesis or as energy sources, once the intense mitotic proliferation of these cells dies down. Triglycerides also predominated in the necks of the glands, but in the deeper parts, the sparse droplets often gave staining reactions for acidic lipids. In differentiating decidual cells during day 5 and early day 6 in nulliparous mice, histochemical reactions suggest that fatty acids accumulate. Some of these are probably utilized in synthesis of the increasing amounts of phospholipids which were particularly prominent later on day 6 and during day 7, while others appeared to be temporarily stored during that period as triglycerides. The phased deposition and removal of triglyceride proceeds centrifugally through the decidua and is slightly in advance of glycogen deposition and removal in the same cells; its time-course correlated well with that which other workers have described from chemical assay. Histochemical tests appeared to reveal activity of a lipase-esterase (fatty acid ester hydrolase) which strengthened and spread through the decidua in parallel with the disappearance of lipid droplets. In lactating mice, as long as the blastocysts remained in diapause, epithelial lipid resembled that on day 4; in the stromal cells, lipid droplets were plentiful in areas close to the former placental sites but were few or absent elsewhere. Once the delayed implantation had started no abnormalities were detected. Alterations in lipids are discussed in the context of the known changes in the levels of ovarian hormones. Lipids within uterine macrophages are also discussed.

SUMMARY

Incidence and patterns of mitoses and histochemical localization of alkaline phosphatase (APase) and adenosine triphosphatase (ATPase) were studied from days 5 to 20 in pseudopregnant mice in which deciduomata had been induced in the left uterine horns by intrauterine injection of oil on day 4, the right horns serving as controls.

In stromal cells, mitoses were very numerous throughout the endometrium of the left (but not the right) horn on days 5 and 6, in the basal, non-decidualized stroma until day 8 or day 9, and were not seen in stromal cells of either horn thereafter. In glandular epithelium, mitoses were absent from days 5 to 10, and numerous from day 11 or 12 until at least day 17 in both horns. Mitoses were present in capillaries within developing deciduomata on days 5 and 6, then seldom seen until day 12 and during the next 3 days were numerous in endothelial and pericapillary cells in the mesometrial quadrant of the left horn and around glands in both horns.

The deciduoma cells reacted strongly with AP and ATP substrates from days 5 to 10, after which the intensity of the reaction weakened and had usually disappeared by day 13. ATPase activity disappeared from vascular endothelium within the deciduoma a few hours after APase had appeared within the deciduoma cells on day 5. It reappeared in the vessel walls on day 9 and thereafter was usually present until the deciduoma was shed. In the basal, non-decidualized stroma, APase was absent until about day 10, then appeared in the stroma cells nearest to the myometrium, extending gradually into the densely packed cells nearest to the regressing deciduoma. The possible role of this enzyme in reparative growth of the endometrium after regression of the deciduoma is discussed.

SUMMARY

(1) The effects of combinations of oestrogen, progesterone and relaxin on glycogen content, and on amylophosphorylase, transglycosylase and uridine diphosphate glucose-glycogen glucosyl transferase activities in the corpus uteri of intact and ovariectomized virgin mice were investigated by histochemical techniques.

(2) Glycogen and the enzyme activities were localized to myometrial and arterial muscle fibres, mobilized leucocytes when present, and luminal and glandular epithelium. Transglycosylase activity was not found in glandular epithelium and no information was obtained about UDPG-glycogen synthesis in epithelium or in leucocytes; otherwise the distribution of the three activities appeared to be similar.

(3) In untreated ovariectomized mice no glycogen was detected in vivo and phosphorylase activity was low. In untreated intact mice little histochemically detectable glycogen was found in myometrial muscle at any stage of the cycle and almost no UDPG-synthesized glycogen; amylophosphorylase activity appeared to be increased during pro-oestrus and oestrus.

(4) Oestrogen produced increased amounts of glycogen in vivo and stimulated phosphorylase activity in both muscle layers in intact and ovariectomized mice; UDPG-glycogen synthesis was probably also increased.

(5) Relaxin had no detectable effect on myometrial glycogen or on phosphorylase activity in non-primed ovariectomized mice, but both were increased when relaxin was given to oestrogen-primed ovariectomized mice or to intact mice at pro-oestrus or oestrus. Only small increases were detected in UDPG-glycogen synthesis.

(6) In both intact and ovariectomized oestrogen-primed mice progesterone had a differential action on the two layers of the myometrium: it increased both glycogenolysis and phosphorylase activity in the longitudinal fibres, but inhibited phosphorylase activity in the circular fibres without resulting in glycogen synthesis in vivo. Results on UDPG-glycogen synthesis were inconclusive. Simultaneous administration of relaxin prevented the inhibitory action of progesterone on glycogen and phosphorylase activity in the circular muscle layer and UDPG-glycogen synthesis was also high in these mice.

(7) No consistent effects of the hormones were detected on glycogen or enzyme activities in arterial muscle.

(8) The histochemical tests visualized total phosphorylase activity but gave no information about hormonal influence on phosphorylase a and b ratios.

This investigation stems from two papers by Rudzik & Miller (1962a, b) on the mechanism by which relaxin effects uterine myometrial inhibition. From the results of experiments in vitro, the authors concluded that, in the rat, the uterine inhibitory action of relaxin is mediated indirectly through the release of catecholamines, mainly adrenaline. Relaxin also inhibits uterine contractions in the mouse. A number of experiments on the mouse uterus in vitro have been carried out in this laboratory by Mr P. A. Judd (unpublished). Although more work needs to be done before Rudzik & Miller's hypothesis can be accepted for this species, the preliminary results are, in the main, similar to those reported for the rat.

The experiments of Rudzik & Miller give no clue to the site of storage and release of adrenaline in the uterus, but the authors point out that Adams-Ray, Nordenstam & Rhodin (1958) reported the