ABSTRACT
Throughout the normal reproductive cycle in Xenopus laevis the serum calcium level is higher in females than in males.
The reproductive cycle in females is associated with changes in the serum calcium level and in ovarian activity. In male toads the serum calcium remains remarkably constant except for a slight rise during the breeding season.
Captivity first causes a rise and then a steady decline in the serum calcium level of females. Both phases are accompanied by a progressive involution of the ovaries. The serum calcium of males is unaffected by captivity.
It is suggested that seasonal variations in the amount of solar radiation and inadequate illumination during captivity are the main agencies influencing serum calcium (through the pituitary and parathyroid glands) and ovarian activity (through the anterior lobe of the pituitary).
I. INTRODUCTION
In a previous paper (Shapiro and Zwarenstein, 1933) it was shown that castration in Xenopus laevis results after 6 months in a persistent fall in serum calcium in males and females. During the course of the investigation it was observed that the serum calcium of the females used as controls, i.e. animals maintained in captivity, was significantly lower than that of animals brought in fresh from the ponds. The ovaries of captive females were severely atrophied after 6 months’ captivity and often presented the appearance of gelatinous masses in which individual ova were no longer discernible on naked-eye examination. On the other hand, the normal ovaries of pond animals at the height of the breeding season are filled with large ova easily visible macroscopically. An account of the histological and other changes in the ovaries associated with captivity and hypophysectomy will be published later.
In striking contrast to the females, the serum calcium of males kept in captivity for 6 months did not differ significantly from that of pond male toads examined at the same time. No macroscopic changes were observed in testes of the captive males.
The correlation between ovarian retrogression and decreased serum calcium of animals kept in captivity and the seasonal changes in serum calcium and ovarian activity of animals in their natural environment were more fully investigated. The results of this investigation, extending over 12 months, form the subject of the present paper.
II. EXPERIMENTAL METHODS AND DATA
The serum calcium was estimated by the modified micro-method described previously (Shapiro and Zwarenstein, 1933).
Several hundred toads were collected from a pond in the Cape Peninsula in January, 1932, and kept in a large tank in the animal house. The water in the tank was changed thrice weekly and the animals were fed on raw minced meat twice a week. Animals from this stock were killed at monthly intervals for a year and their serum calcium estimated. On each such occasion the serum calcium of animals which had been brought in fresh from the pond was also determined.
In Table I each figure represents, unless otherwise indicated, the mean of the serum calcium values of eight animals in milligrams per 100 c.c.
The above data are graphically represented in Fig. 1.
III. DISCUSSION
Serum calcium of males and females
From Table I and Fig. I it is seen that there is throughout the year a striking and significant difference in the serum calcium of male and female toads. Charles (1931) recorded this fact for the first time in the case of Xenopus, but the highest figure she gives, the mean of nine determinations made in the hot season, i.e. in February, is 9.82 ± 0.13 mg. Ca per 100 c.c. This figure represents actually the lower limit of the range of variation exhibited by female toads under natural conditions. The greatest difference occurs in August, soon after the beginning of the breeding season, with a serum calcium for females 46 per cent, higher than that of males.
Seasonal changes in pond animals
The serum calcium of female toads is lowest in autumn and early winter and highest in late winter and early spring, i.e. during the rainy and breeding seasons.
The serum calcium remains at a high level throughout the spring and summer months. The figures for the ovary weight : body weight ratios included in Figs. 2 and 3 have been taken from data which will be published in detail later. It is seen that up to the beginning of the breeding season there is a striking correlation between the state of the ovaries and the serum calcium (Fig. 2). There is a gradual increase in both ovary weight and serum calcium, the highest level for both being reached in July, the beginning of the breeding season. After this the ovaries decline and lose weight, as the ova are extruded in large numbers during the breeding season, and this loss of weight continues during the summer months. The serum calcium, on the other hand, after a slight fall is maintained at a high level during this time. This association of a high calcium level and a low ovary weight is analogous to the relationship, described in a previous paper, between the high serum calcium and the completely involuted ovary of animals with regenerated pars tuberalis. These observations support the hypothesis that the influence of the pituitary on ovarian activity and on calcium metabolism are concomitant but independent activities referable to hormones produced in different parts of the pituitary. Previous evidence suggests that the pars tuberalis plays an important part in regulating calcium metabolism (Shapiro and Zwarenstein) and that the pars anterior is the main agent controlling ovarian growth (Hogben, Charles and Slome) and ovulation (Bellerby).
The changes in the serum calcium level in Xenopus are, except for the time relations, generally similar to the changes which occur in birds at the time of egg-laying. Riddle and Reinhart (1926) found that the serum calcium of female pigeons rose during the pre-ovulation phase to a value double that of the resting period. This high level was maintained during the greater part of the ovulation period and was succeeded by a post-ovulation fall to the normal resting level. These changes occupied in all about 10 days of the reproductive cycle. Sun and Macowan (1930) showed that the serum calcium of fowls rises considerably when egg-laying commences, maintains a high average level during the laying period, to fall again when moulting commences. In Xenopus the eggs are not enclosed in a lime shell, but Riddle and Reinhart found that the increase in serum calcium in pigeons begins days before egg shell formation begins and came to the conclusion that it is in no wise correlated with the large calcium need for shell formation. They conclude, further, that the very high calcium values accompanying each ovulation period is an expression of increased parathyroid activity. The rise and fall in calcium level in the fowl was found by Macowan (1932) to be associated with distinct histological changes in the parathyroids. Changes in the parathyroid, and thus presumably in serum calcium, as the result of differences in solar radiation are described below. An important factor in determining the seasonal variations in serum calcium in Xenopus in natural surroundings may thus be the different amounts of sunlight at different times of the year. The changes in the parathyroid glands and serum calcium as the result of differences in solar radiation are probably effected through the intermediation of the pituitary gland. The seasonal changes in ovarian activity are probably also due to variations in the amount of light. Bissonnette (1932) has shown that in birds and ferrets the sexual cycle is conditional to a great degree by the daily light ration from the visible region of the spectrum. Experiments of numerous workers on anterior lobe pituitary implants, injections of extracts etc., suggest that “light-induced sexual cycle reactions in both birds and mammals are bound up, at least in part with anterior lobe hypophysis activity.” The work of Hogben, Charles and Slome (1931), described below, and of Bellerby (1933) indicates that in Xenopus light and anterior lobe activity are also important factors controlling the normal ovarian cycle.
The serum calcium of males in their natural surroundings remains remarkably constant except for a slight rise in late spring and early summer. The serum calcium of males is on the whole more resistant to changes in the environment than that of females. Captivity has no significant effect and the fall in calcium after castration occurs much later than in females.
Changes in the serum calcium in captivity
From Fig. 1 it is seen that the calcium values for captive and pond animals during the first two months follow each other closely. This is followed by a period of two months during which the serum calcium of the captive animals rises. The difference between the calcium values for captive and pond animals during this period is probably significant. After this a steady and progressive decrease in the serum calcium of captive toads occurs and, after 10 months’ captivity, has dropped to the typical low level for males. These changes in serum calcium are accompanied throughout the period of captivity by a progressive involution of the ovaries (Fig. 3). The ovaries after 10 months’ captivity are gelatinous masses in which no individual ova can be seen macroscopically.
Males, in captivity, show no macroscopic gonadal changes nor any significant decrease in the serum calcium level. In striking contrast to the females, which have become very emaciated and weak by the 9th or 10th month of captivity despite regular feeding and change of water, the males appear healthy and in good condition. All the deaths recorded in the latter months of captivity were of females.
A probable explanation of the changes in serum calcium during captivity is suggested by the results obtained by Higgins and Sheard (1928) on the effect of light on the parathyroids of chicks. They find that both the longer and shorter wave-lengths are essential to the maintenance of normal parathyroid glands. In the absence of light of certain wave-length hyperplastic parathyroid glands develop within the first few weeks. The cells in the hyperplastic glands do not differ from those of normal glands and they conclude that the cells are functioning and that secretory activity is accentuated so that hormonal output may be increased. At the conclusion of the third week regressive changes within the enlarged glands become evident. This involves destruction of the cords, shrinking of the cells and the development of cysts. They find further that the initial hyperplasia may be partially obviated by the addition to the diet of a small amount of cod-liver oil.
The toads used for the investigation of the effects of captivity were kept in deep slate-lined tanks covered with fine meshed wire netting. They were thus exposed to a dim light on a light-absorbing background. Lack of sufficient solar radiation would presumably have had the same effects as those described above. First an initial hyperplasia of the parathyroid glands leading to increased hormonal output and a rise in serum calcium, and secondly a progressive cystic degeneration of the glands, which would account for the steady decrease in serum calcium observed during the later months of captivity. The absence of vitamin D in the diet was probably a contributory factor.
In regard to the effect of captivity on the ovaries light seems to be the important factor. This suggestion is supported by the following facts. Hogben, Charles and Slome (1931) recorded the effects of hypophysectomy on the ovary body weight ratio. As controls they used normal and eyeless toads which had been kept in the laboratory for the same length of time as the operated animals. An examination of their figures shows that no retrogressive changes had occurred in the ovaries of normal toads even after five months’ captivity. Prof. Hogben informs us that his animals were kept in a room in which the lights were kept on day and night. The conditions of illumination were thus in striking contrast to those described above. Their figures show, further, that quite a considerable degree of retrogression had occurred in the ovaries of eyeless animals. The ovary body weight ratios were intermediate between those of normal toads and of hypophy-sectionised toads in which the anterior lobe only had been removed.
In the field mouse Baker and Ranson (1932) have shown that reducing the daily hours of light from 15 to 9 almost prevents reproduction, the females being mainly affected. Bissonnette (1932) draws attention to the fact that in birds, the males, and in mammals, the females, i.e. the homogametic sex is the more susceptible to the influence of light. The evidence presented in this paper suggests the applicability of this generalisation to amphibia.
The toads in captivity were subjected to temperature variations which were on the whole less extreme than the variations in pond temperatures. This difference must be taken into account as an additional factor which may affect ovarian activity. On the other hand, unpublished experiments by the authors showed that a difference of 20° C. had no significant effect on the calcium content of the serum.