ABSTRACT
A comparative study of the distribution of placental scars from the first and second pregnancies in laboratory rats indicated that the most posterior scar of first pregnancy tends to be nearer to the cervix than the most posterior second pregnancy scar (Momberg & Conaway, 1956). Frazer (1955) in an earlier study observed that more embryos implanted in the caudal half of the uterus of the rat than in the cranial half when the embryo number was four or less. Momberg & Conaway (1956) found this difference to be greater when the embryo number was high.
The reasons for such distributions are not known, but these findings suggest that the uterus varies along its length and does not provide a uniform environment for the implanting embryos. It would further appear that analyses of the various regions of the uterus might furnish information about conditions offering favourable and less favourable environments for the implantation of the blastocysts. Conspicuous alterations in total mass and composition of the rat uterus during the estrous cycle are well established. However, relatively little is known about regional differences in the uterus. An observation regarding differences between regions of the same uterus has been made by Hayashi et al. (1957). Their histochemical studies revealed that the B-glucuronidase activity varied considerably, not only among individuals, but among blocks cut from different portions of the same uterus.
Even such physiologically important enzyme systems as the phosphatases have not been studied in a manner that would determine local differences in various regions of the same uterus. Phosphatases have been identified in all animal tissues and are now known to be concerned in many important processes such as carbohydrate metabolism, nucleotide metabolism, and calcium deposition (Sumner & Somers, 1943). It has been shown that as uterine weight, average embryonic weight, number of implantations, or number of viable embryos increases, there is also an increase in the total phosphatase activity of the uterus (Bredeck & Mayer, 1955). The vast amount of work on alkaline phosphatase indicates its probable role in glycogen synthesis and the transport of secretory substances across cell membranes (Wislocki & Dempsey, 1945; Dempsey & Wislocki, 1946; Pritchard, 1947; Moog, 1946; and Stafford et al., 1947). The effect of estrogens and progesterones on these enzymes has been reviewed by Miner (1951), and Roberts & Szego (1953).
This study was initiated in order to determine (a) if regional differences in phosphatases exist in the uterus of the rat, and (b) if these differences correlate with embryo distribution patterns.
MATERIALS AND METHODS
Albino rats of the Wistar strain whose previous history was known were used in this study. The immature animals were 6-7 weeks old when killed. Virgin and primiparous rats were approximately 4-5 months old when killed. Vaginal smears were examined to select rats from different stages of the estrous cycle. All rats used were followed through two consecutive estrous cycles before being killed.
Pseudopregnant rats were obtained by placing an estrous female with a vasectomized male. If the animals had a vaginal plug and showed a diestrous type of smear the following morning when they were separated from the males, and continued to exhibit the same type of smear for 5 days, they were considered to be pseudopregnant. The females thus obtained were killed on the sixth day of pseudopregnancy; the first day being counted from the day prior to the finding of a diestrous type of smear.
Primiparous rats were obtained in the following manner: young virgin females were placed in breeding cages with mature males in the ratio of 5 to 1. From time to time the visibly pregnant females were separated and each one was kept in a separate cage. They were allowed to go to term. The number of young and the date of birth of the litters were recorded. The young were weaned at 23 to 24 days of age. After an additional 25 days each female was examined through two consecutive estrous cycles to check the uniformity of the cycles. The females having uniform cycles were killed at the required stage.
The rats were weighed before killing. As soon as each rat was stunned by a blow on the head, the abdomen was opened by a median incision. The reproductive tract was cut at the cervix and released from the surrounding tissue. It was then lifted and the uterine cornua were stretched in a manner which would produce as much uniformity along the length of each uterus and also between different uteri as possible. The uterus was pinned to a board covered with a cold wet paper towel. The mesometrium was then cut as close to the uterus as possible. The location of any placental scars along the uterine horns was recorded. Each uterine horn was then divided into three equal portions—the cranial, middle, and caudal segments. The segments were placed in separate glass, or rubber-stoppered, numbered vials and stored in ice. Mesometrial and antimesometrial halves were obtained by dividing the uterus approximately into two longitudinal halves. Each segment thus obtained was then weighed on a Roller-Smith torsion balance. Efforts were made to process each rat as rapidly as possible to prevent loss of the enzymes. If the material was not used immediately, it was stored at or below -20° C.
The phosphatases were determined by the ortho-nitrophenal phosphate method (Sigma Chemical Company, 1957). The uterine segments were homogenized in a Pyrex tissue grinder and the volume of the homogenate made to equal 2 c.c. Phosphatase activity was determined colorimetrically by a Bausch and Lomb ‘Spectronic 20’. Phosphatase activity is reported in ‘Sigma’ units (Sigma Chemical Company, 1957).
Uterine phosphatase activity of the different segments was also estimated in two groups of estrogen- or progesterone-treated 30-to 32-day-old immature rats. One group was given each day for 6 days a subcutaneous injection of 0.1 c.c. of Mazola oil containing 0.55 mg. of estradiol benzoate per c.c. of oil. Another group was similarly treated with the same level of progesterone. A probability level of 0.05 or less was considered to be significant.
RESULTS
Alkaline phosphatase activity in the uterus of immature, virgin, pseudopregnant and uniparous rats
The average number of units (per mg. of wet weight of tissue) of alkaline phosphatase found in the cranial, middle, and caudal segments of the uterine horns from the various groups of rats studied is indicated in Text-figs. 1 & 2. The significance of the disparity between the segments in the mean number of units of alkaline phosphatase activity was estimated by t-test (Fisher, 1950) within the immature, virgin, pseudopregnant, and postpartum groups. The virgin and postpartum groups were not subdivided into stages of the estrous cycle for this analysis. In the immature group there is no significant difference between any two segments. In the virgin and in the pseudopregnant animals, the cranial segment is significantly higher than either the caudal or middle segment, but the middle and caudal segments do not differ significantly. In the postpartum animals, however, the cranial and caudal segments differ significantly from the middle segment, but the difference between the cranial and caudal segments is not significant.
Alkaline phosphatase activity in segments of uterine horns from immature, virgin diestrous, virgin proestrous, virgin estrous, and pseudopregnant animals. The most anterior segment is indicated by 1, the middle by 2, and the most posterior by 3. One half of each black bar plus the white bar at either end denotes one standard deviation on either side of the mean. The white portion comprises two standard errors on either side of the mean. The middle vertical line shows the range with the figure at the upper end being the sample size.
Alkaline phosphatase activity in segments of uterine horns from immature, virgin diestrous, virgin proestrous, virgin estrous, and pseudopregnant animals. The most anterior segment is indicated by 1, the middle by 2, and the most posterior by 3. One half of each black bar plus the white bar at either end denotes one standard deviation on either side of the mean. The white portion comprises two standard errors on either side of the mean. The middle vertical line shows the range with the figure at the upper end being the sample size.
Alkaline phosphatase activity in segments of uterine horns from diestrous, proestrous, and estrous postpartum animals. Phosphatase activity for mesometrial and antimesometrial halves of virgin diestrous uterine horns is also given. Symbols as in Text-fig. 1.
The estrogen- and progesterone-treated rats, in addition to having a higher alkaline phosphatase activity in all the segments (Text-fig. 3), display a different pattern of alkaline phosphatase distribution. In these animals the middle segment has a higher phosphatase activity than either of the other two segments. The phosphatase in the cranial uterine segment from estrogen-treated animals is significantly lower than that of the other two segments, while the caudal segment in the progesterone-treated animals has a significantly lower phosphatase activity.
Alkaline phosphatase activity in segments of uterine horns from estrogen- and progesterone-treated immature animals. Symbols as in Text-fig. 1.
Alkaline phosphatase activity of the mesometrial and the antimesometrial halves of the uterus
The enzyme activity of the mesometrial half of the uterus is significantly higher than that of the antimesometrial half. The values are shown in Textfig. 2.
Alkaline phosphatase activity in relation to the number of placental scars in the postpartum uteri
With an increasing number of placental scars and hence the number of implantation sites, the level of alkaline phosphatase activity in the uterus tends to decline (Text-fig. 4). The phosphatase activity of the segments having two, three, or four scars is significantly lower than that of segments having no placental scars.
Alkaline phosphatase activity in segments of uterine horns grouped by numbers of placental scars in each segment. Symbols as in Text-fig. 1.
Acid phosphatase
The pattern of distribution of the uterine acid phosphatase is not very different between segments within most of the groups studied (Table 1). No correlation was found between the acid phosphatase activity in the different segments and the varying number of placental scars. No significant variation in the acid phosphatase activity was found between the mesometrial and antimesometrial halves.
DISCUSSION
Previous studies on the effects of circulating estrogens and progesterones on uterine alkaline phosphatase have involved chiefly histochemical methods. The results obtained from such studies are difficult to compare with quantitative measurements of total enzyme activity such as those obtained in this study. The results in the histochemical studies are expressed in relative reactivity of the specific tissues in the uterus of experimental and control animals. Pritchard (1949) has stated that estrogens cause a concentration of the enzyme in the uterine epithelium whereas progesterones cause it to disappear. Atkinson & Elftman (1947) and Hayashi et al. (1955) obtained essentially similar results in mice and rats respectively. On the basis of such studies it might be concluded that the total uterine alkaline phosphatase also would be highest when estrogens are high in the body.
The evidence presented in this study indicates a variable relationship between alkaline phosphatase activity of the uterus and circulating estrogen and progesterone levels, especially in the groups of untreated animals. The total alkaline phosphatase activity of the uterus in the immature rats is significantly higher than that of the virgin animals in all stages. Within the virgin group, the diestrous rats have the highest phosphatase activity, estrous the lowest, and, proestrous an intermediate activity. In the postpartum group all stages except proestrus are higher in uterine phosphatase than are the virgin animals. Within the postpartum group the uterine alkaline phosphatase activity at estrus is significantly higher than at proestrus. During pseudopregnancy the phosphatase activity is the lowest recorded. In contrast the estrogen- and progesterone-treated animals showed a higher uterine phosphatase activity than any untreated group.
It thus appears that the administration of exogenous estrogens or progesterone elevates the total phosphatase activity. In the untreated animal a much more complex relationship obtains. The second highest activity was recorded in the immature group in which presumably endogenous hormones are minimal while the lowest activity occurs in pseudopregnancy when the uterus is receiving hormonal stimulation.
A comparison of the various segments of the uterus in untreated and hormone-treated animals also indicates a variation in the relationships between alkaline phosphatase and hormonal levels. At estrus, when oestrogens are high, the uterine alkaline phosphatase was found to be highest in the cranial segment in both virgin and primiparous animals. In the virgin animals the middle and caudal segments were almost equal in their phosphatase activity, but in the postpartum rats the middle segment had a lower activity than the caudal segment. In the estrogen-treated immature rats the cranial segment had the lowest phosphatase level while the middle segment had the highest.
The pseudopregnant rats which are assumed to have a high endogenous progesterone do not coincide with the progesterone-treated immature rats in the alkaline phosphatase distribution in the different segments of the uterine horns. In the pseudopregnant rats the cranial segment had the highest phosphatase level while in the progesterone-treated animals the middle segment had the highest. In the pseudopregnant rats the middle and caudal segments were almost equal in their phosphatase activity. The caudal segment had a lower phosphatase value in the progesterone-treated rats than either the cranial or the middle segment, both of which were almost equal. Thus, at those stages when estrogens and progesterones are probably high in the untreated animals, the alkaline phosphatase distribution in the different segments of the uterine horns is not at all similar to that of estrogen- and progesterone-treated immature rats. Perhaps the results are attributable to unphysiological doses of injected hormones.
The pattern of alkaline phosphatase activity in the uterine segments is seen to change after the first pregnancy. In the virgin animals the mean number of units of alkaline phosphatase activity of the middle and caudal segments does not differ significantly, but the cranial segment differs from both in its phosphatase activity. This is similar to the pattern of embryo distribution since the cephalic segment has the lowest number of embryos in the first pregnancy (Momberg & Conaway, 1956). In postpartum rats the cranial and caudal segments are similar, whereas the middle segment differs from both of these segments in its lower phosphatase distribution after the first pregnancy. This seems to coincide with the shift in the embryo implantation sites in the second pregnancy which results in greater numbers of implantations in the middle segment (Momberg & Conaway, 1956), suggesting a possible relationship between low alkaline phosphatase and the increased frequency in implantation. Such a relationship between the enzyme and implantation site is supported by the lower alkaline phosphatase activity found in the antimesometrial half of the uterus wherein implantation always takes place (Parkes, 1952). The uterine segments having a high number of placental scars show a low phosphatase activity. This inverse relationship between the phosphatase activity and the number of placental scars lends an added support to the suggestion that alkaline phosphatase may be related to embryo implantation.
SUMMARY
The uterine horns were divided into three regions (i.e. the cranial, middle, and caudal). Uteri from immature, virgin, pseudopregnant, primiparous, and from estrogen- and progesterone-treated immature rats were used in this study.
In the majority of the groups the cranial segment had the highest enzymatic activity, whereas the middle segment had lowest. The cranial and caudal segments of the rat uterus differed significantly in alkaline phosphatase activity in the virgin and pseudopregnant animals but not in the primiparous animals. Such a change in the phosphatase activity of segments after the first pregnancy seems to coincide with the reported anterior shift in implantation sites in the second pregnancy.
No significant variation was found in the concentration of acid phosphatase in the different segments that might correlate with embryo distribution patterns.
The phosphatase pattern in normal rats was not similar to that of immature rats treated with exogenous estrogen or progesterone. The dissimilarity may be due to unphysiological doses.
RÉSUMÉ
Activité phosphatasique alcaline dans l’utérus de Ratte
Les cornes utérines ont été divisées en trois régions (c’est-à-dire craniale, moyenne et caudale). On a utilisé dans ces recherches des utérus de rattes immatures, vierges, pseudo-gravides, primipares, et immatures traitées aux œstrogènes et à la progestérone.
Dans la majorité des groupes, le segment cranial possédait l’activité enzymatique la plus élevée, tandis que le segment moyen possédait la plus basse. Les segments cranial et caudal de l’utérus de ratte montraient une différence significative dans l’activité phosphatasique alcaline chez les animaux vierges et pseudogravides, mais pas chez les individus primipares. Une telle modification de l’activité phosphatasique des segments après la première grossesse semble co ïncider avec le changement, signalé antérieurement, des points d’implantation lors de la seconde grossesse.
On n’a pas trouvé de variation significative dans la concentration en phosphatase acide des divers segments, qui pourrait être en corrélation avec le mode de répartition des embryons.
Le type phosphatasique des rattes normales était différent de celui de rattes immatures traitées aux œstrogènes exogènes ou à la progestérone. Cette dissemblance est peut-être due aux doses extra-physiologiques employées.