ABSTRACT
It is generally recognized, on the basis of electrophoretic studies, that the serum proteins of individuals in the perinatal period of development are somewhat different than those of the adult (Kekwick, 1959; Lehrer & Toben, 1965). It is also known that the protein content of the serum of perinatal mammals is low, and gradually rises to the adult level over a period of weeks or months (McMurray, Roe & Sweet, 1948; Knapp & Routh, 1949; Ewerbeck & Levens, 1950; Potter, 1952; Halliday & Kekwick, 1957). Virtually nothing, however, is known about the serum proteins of very young vertebrate embryos, presumably because of technical difficulties involved in obtaining samples suitable for quantitative study.
In the present study the protein levels of embryonic serum were assayed in samples obtained from the earliest age possible, shortly after circulation began, to birth. Ontogenetic patterns of changes in concentration were compared between the chick, which develops as a cleidoic egg, and the rat, a mammalian embryo which develops in intimate relationship with the maternal blood stream.
The study was prompted not only by interest in the normal physiology of embryos, but also by teratological considerations. Abnormal serum proteins have been described in human mothers who subsequently had abnormal births (Langman, Van Drunen & Bouman, 1959) and in pregnant rabbits treated with teratogenic doses of Trypan blue (Langman & Van Drunen, 1959). Qualitative and quantitative changes in yolk-sac proteins, which apparently reflect changes in maternal proteins, have been described after exposing mothers to Trypan blue (Beaudoin & Ferm, 1961). Such observations indicate that the plasma proteins of the young embryo may mediate or reflect abnormal development, and additional information is desirable.
MATERIALS AND METHODS
The chick embryos used were of the Kimber strain of White Leghorn. Blood samples were obtained from 3- and 5-day embryos from vitelline arteries with the aid of glass microneedles. At 10 and 15 days, samples were obtained from chorio-allantoic vessels; at 20 and 25 days, from the heart of lightly anesthetized chicks. Adult samples were obtained from young roosters, either from the wing vein or the heart. The rats were of the Holtzman strain. Mothers were quickly anesthetized with chloroform and maternal blood samples obtained from the heart. Uterine segments were excised as needed, placed under a stereoscopic microscope and embryonic blood samples withdrawn from the umbilical arteries with glass needles, Samples were drawn form newborn rats by heart puncture of lightly anesthetized individuals. Rabbits were killed by air injection and samples obtained in the same manner as for rats.
All samples were drawn as rapidly as possible and centrifuged within 2–5 min. Assays were performed on 5 μl samples with the Beckman Ultramicro Analytic System, utilizing a modification of the method of Kingsley (1939) and Gornall, Bardawill & David (1949). Since this is a micromethod, only samples obtained from the very youngest embryos were pooled. All other assays were performed on individually drawn samples.
RESULTS
Chick embryos
The protein content of serum of chick embryos between 3 and 10 days old ranged from 1·1 to l·2 g/100 ml, approximately one-fourth of the adult level (Table 1; Fig. 1). From 15 days on, there is a simple, gradual increase in protein levels. At 25 days (4 days after hatching) the concentration has not quite reached the adult level.
Rat embryos
The youngest rat embryos from which blood samples were obtained were days old. This is about 2 days after circulation begins. The protein concentration at this stage is 0·9 g/100 ml, approximately one-eighth the adult level of 7 · 2 g/100 ml (Table 2; Fig. 1). The concentration rises very slowly to 2 · 1 g/100 ml at 18 days and then increases sharply before birth. In the perinatal period the protein level is still approximately one-half the adult concentration. After birth, the rise becomes more gradual (see also Halliday & Kekwick (1957) who report an increase in plasma proteins of young rats from 3 · 92 g/100 ml at 12 days to 5 · 85 g/100 ml at 90 days).
Ontogenetic changes in serum protein concentration of chick and rat embryos.
Other mammals
Several protein determinations were made on the serum obtained from 22-day rabbit embryos, which is approximately the end of the second trimester. At this time the serum contained 2 · 5 g/100 ml of protein, compared to a maternal level of 7 · 1 g/100 ml. Single determinations of serum proteins of relatively young human fetuses have been reported as follows: 2 · 86 g/100 ml at months (Potter, 1952); 1 · 55 g/100 ml at 4 months and 1 · 95 g/100 ml at 5 months (E werbeck & Levens, 1950). It would appear that the low serum protein levels reported here for rat embryos are a characteristic of mammalian development.
DISCUSSION
The physiological relationships of chick and mammalian embryos to their surrounding environments are different. The chick embryo develops within a closed system except for gaseous interchange. Furthermore, the extra-embryonic capillaries are surrounded by fluids which are of markedly different composition from the serum itself (Grabowski, 1963, 1966). The rat embryo develops in juxtaposition to a maternal environment which presumably is similar to that of the embryo. A comparison of any characteristic of the embryonic blood of the two groups is therefore of interest.
With respect to total protein content, we find that both curves start at a surprisingly low figure of about 1 ·0 g/100 c.c. Both curves rise very slowly during the early stages of development, then more rapidly nearing birth. At birth the concentration of protein in chick serum approaches the adult level more nearly than it does in the newborn rat. However, protein concentration in the adult chick is substantially lower than in the adult mammal. In summary, the ontogenetic patterns of changes in protein concentration of the serum of both chick and rat embryos are similar, despite different adult levels and the different physiological circumstances of development.
What is the functional significance of these observations? Little can be said at the present, but one speculation is offered here. Proteins make an important contribution to the osmotic pressure of serum and, by virtue of their ion-binding capacities, help regulate ionic concentrations. In the early stages of mammalian development, the embryonic serum is in juxtaposition to maternal serum containing 4 – 6 times more protein. Such a concentration differential can be expected, therefore, to affect fluid and ionic interchange between the two blood streams. Fluid disturbances are readily induced in embryos of the chick (Grabowski, 1964, 1966), rabbit (Jost, 1951), rat (Giroud, Lefebvres, Prost & Dupuis, 1955), and mouse (Waddington & Carter, 1953) by a variety of teratogenic agents. These disturbances, in turn, can lead to abnormal development. Is it possible that the plasma proteins are somehow involved in these manifest fluid problems? What is the relationship of the observed quantitative and qualitative changes in serum proteins to the abnormal development mentioned in the introduction? Additional studies of the serum proteins of young embryos, in normal and abnormal circumstances, are needed before these questions can be more intelligently phrased.
SUMMARY
The concentration of serum proteins was determined in chick embryos between 3 and 25 days old and rat embryos between
and days old. In both species, the earliest determinations were made on embryos about 2 days after circulation started.
In the chicken the adult level of serum proteins is 4·2 g/100 ml; at 3 days it is 1·2 g/100 ml and gradually rises to around 3 g/100 ml at hatching. In the rat the adult level is 7·2 g/100 ml; at
days it is 0·9 g/100 ml and gradually rises to about 4 g/100 ml at birth. Comparison with a few scattered observations of others suggests that the very low level reported here for the young rat embryos may be a general mammalian characteristic.
The physiological and teratological significance of these data is briefly discussed.
RÉSUMÉ
Changements dans la concentration des protéines sériques durant le développement des embryons de poulet et de Mammifères
La concentration en protéines du sérum est mesurée chez des embryons de poulet âgés de 3 à 25 jours et chez des embryons de rat âgés de 12 et demi à 23 jours et demi. Chez des deux espèces, les déterminations les plus précoces sont pratiquées sur les embryons environ 2 jours après le début de la circulation.
Chez les poulets adultes le taux de protéines sériques est de 4,2 g/100 ml; à 3 jours, il est de 1,2 g/100 ml et augmente graduellement jusqu’à 3 g/100 ml environ à l’éclosion. Chez le rat adulte le taux est de 7,2 g/100 ml; à 12 jours et demi il est de 0,9 g/100 ml et augmente graduellement jusqu’à 4 g/100 m environ à la naissance. Une comparaison avec des observations non systématiques d’autres auteurs suggère que le niveau très faible trouvé ici chez les jeunes embryons de rat est un caractère général des Mammifères.
La signification physiologique et tératologique de ces faits est discutée brièvement.
ACKNOWLEDGEMENTS
This investigation was supported by grant No. CRMS 197 from The National Foundation-March of Dimes and grant No. HD 00641 from the National Institutes of Child Health and Human Development, National Institutes of Health.